Vietnamese Pharmacopoeia V - Dược Điển Việt Nam V (English version) [5 ed.]

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SOCIALIST REPUBLIC OF VIETNAM

MINISTRY OF HEALTH

VIETNAMESE PHARMACOPOEIA Fifth edition Volume 1 ENGLISH VERSION

MEDICAL PUBLISHING HOUSE HANOI - 2019

Copyright by the Vietnamese Pharmacopoeia Commission registered with the copyright Department. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, without prior written permission of the copyright holder. Vietnamese Pharmacopoeia Commission, Vietnamese Pharmacopoeia and Formulary Center Address : 48 - Hai Ba Trung Str., Hoan Kiem Dist., Hanoi, Vietnam Telephone : (84 - 4) 38256905 Fax : (84 - 4) 39343547

PHARMACOPOEIA VIETNAMICA EDITIO V Tomus 1

MINISTRY OF HEALTH

SOCIALIST REPUBLIC OF VIETNAM Independence - Freedom - Happiness

Số: 5358/QĐ-BYT

Hanoi, 28 November 2017

DECISION

In reference to the promulgation of the fifth edition of Vietnamese Pharmacopoeia THE MINISTER OF HEALTH Based on the Law on Pharmacy No 105/2016/QH13 dated 06 April 2016; Based on the Law on Standards and Technical regulations No 68/2006/QH11 dated 29 June 2006; Based on the Law on Quality of products, commodities No 05/2007/QH12 dated 21 November 2007; Based on Governmental Decree No 75/2017/NĐ-CP dated 20 June 2017 defining functions, powers and organizational structure of the Ministry of Heath; Based on Governmental Decree No127/2007/NĐ-CP dated 1 August 2007 prescribing in details the implementation of some articles in the Law on Standards and Technical regulations; Based on Governmental Decree No 132/2008/NĐ-CP dated 31 December 2008 prescribing in details the implementation of some articles in the Law on Quality of products, commodities; Governmental Decree No 67/2009/NĐ-CP correcting some articles of Governmental Decree No127/2007/ NĐ-CP dated 1 August 2007 prescribing in details the implementation of some articles in the Law on Standards and Technical regulations and Governmental Decree No 132/2008/NĐ-CP dated 31 December 2008 prescribing in details the implementation of some articles in the Law on Quality of products, commodities; Based on the inter-ministerial Joint Circular No 11/2008/TTLT/BYT-BKHCN, by the Ministry of Science and Technology and the Ministry of Health, dated 29 December 2008 regarding the directions for establishment, consideration and approval, proclamation of the Set of National Standards for medicines, and the promulgation and publication of the Vietnamese Pharmacopoeia; Based on Decisions by the Ministry of Science and Technology, No 2702/QĐ-BKHCN dated 08 October 2012 in reference to the proclamation of the Set of national standards for medicines TCVN II:2012; No 59/QĐ-BKHCN dated 22 April 2014 in reference to the proclamation of the Set of national standards for medicines TCVN III:2014; No 746/ QĐ-BKHCN dated 15 April 2015 in reference to the proclamation of the Set of national standards for medicines TCVN IV:2015; No 968/QĐ-BKHCN dated 28 April 2017 in reference to the proclamation of the Set of national standards for medicines TCVN V:2017; No 2760/QĐ-BKHCN dated 13 October 2017 in reference to the proclamation of the Set of national standards for medicines TCVN I:2017 and TCVN VI:2017; Considering the proposal of the Chairman of Vietnamese Pharmacopoeia Commission; Considering the proposal of the Director General of Drug Administration of Vietnam,

DECIDES: Article 1. To publish the fifth edition of Vietnamese Pharmacopoeia including 1519 national standards for medicines: 485 standards for chemico-pharmaceutical substances; 385 standards for formulated preparations; 372 standard for materia medica and traditional medicines; 41 standards for immunological products;

8 standards on containers and materials used for the manufacture of containers 228 standards on general testing methods for medicines and materials used for the manufacture of medicines. Article 2. The fifth edition of Vietnamese Pharmacopoeia comes into effect from 1 July 2018. Abrogating any previous regulation which is different from that in the edition. Article 3. The Chiefs of Drug Administration of Vietnam, Administration of Science Technology and Training, the Chairman of Vietnamese Pharmacopoeia, the Director of Vietnamese Pharmacopoeia and Formulary Center are responsible for guiding the application of the fifth edition of Vietnamese Pharmacopoeia. Article 4. The Chiefs of Health Ministry Secretariat, Administration of Science Technology and Training, Drug Administration of Vietnam, Department of Planning and Finance, Departments/Administrations, and Health Services reporting directly to the Health Ministry, the Directors of provincial Health Services, Health Services of cities reporting directly to the Government, the Director of National Institute of Drug Quality Control, the Chairman of Vietnamese Pharmacopoeia Commission and the Director of Vietnamese Pharmacopoeia and Formulary Center are responsible for the implementation of this Decision.

MINISTER OF HEALTH Nguyen Thi Kim Tien

CONTENTS Page

VOLUME 1 Preface xi History of the Pharmacopoeia of the Socialist Republic of Vietnam xiii Membership of the fifth Vietnamese Pharmacopoeia Commission xix Collaborators xxi Organizations participating in the compilation of Vietnamese Pharmacopoeia xxiii List of monographs xxv Additions xxxix Omissions xlv General notices xlvii Abbreviations li Monographs Chemico-pharmaceutical substances and formulated preparations 1 Index I-1 VOLUME 2 General notices xlvii Abbreviations li Monographs Immunological products 1027 Materia medica 1101 Extracts, oils, volatile oils 1415 Traditional medicines 1435 Infrared reference spectra S-1 Appendices A-1 Index I-1

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PREFACE After four editions, the Vietnamese Pharmacopoeia (VP) has become an important technical regulation document regarding the standardization and the quality control of medicines in pharmaceutical manufacture, import, export and distribution area in Viet Nam. The previous Vietnamese Pharmacopoeia, VP IV, had been promulgated by the Ministry of Health and has been effective since 1 January 2010. Being an official standard technical documentation of the Ministry of Health, Vietnamese Pharmacopoeia IV is used as reference to all activities of training and scientific studies related to medicines in healthcare service establishments. The VP IV has made a positive contribution to the development and modernization of pharmaceutical industry as well as to the quality assurance and quality improvement of medicines to serve the community’s health care. Right after the VP IV publication, the Minister of Health established the Standing Committee of the fifth Vietnamese Pharmacopoeia Commission and assigned the Commission to compile the VP V, as well as to deliver the guidance for applications of the current VP IV, under Decision No 3931/BYT-QĐ dated 14 October 2009 and Decision No 5322/BYT-QĐ dated 31 December 2010. After 7 years of hardworking efforts and careful considerations, the Commission completed the compilation of the Set of National Standards for medicines which was formally proclaimed by the Minister of Science and Technology. Based on this achievement, on 28 November 2017, the Decision No 5358/ QĐ-BYT was signed by the Minister of Health for the Vietnamese Pharmacopoeia V promulgation with effective date from 1 July 2018. In this publication, the Vietnamese Pharmacopoeia has made a significant progress both in terms of quality and quantity with 361 new monographs and 357 updated monographs from VP IV. These monographs have been established with the more comprehensives evaluation criteria such as drug release, impurities, safety etc. The Vietnamese Pharmacopoeia V has included tests with higher reliability level. More effective and modern analytical methods have been also introduced while still applicable to the majority of domestic laboratories, in accordance with the international regulations as well as harmonized with others Pharmacopoeias. The Vietnamese Pharmacopoeia V contains as large as 1519 monographs, including 485 monographs of chemicopharmaceutical substances and 385 monographs of formulated preparations, 372 monographs of crude drugs and traditional medicines, 41 monographs of immunological products, 8 monographs of containers and materials used for the manufacture of containers, 138 infrared reference spectra, 228 general monographs of quality control methods with more than 650 reagents. With the renewed content and the number of pages of 2200, the Vietnamese Pharmacopoeia V is published into two volumes, the first one covering chemico-pharmaceutical substances and formulated preparations, the second volume includes crude drugs, traditional medicines, immunological products and appendices. Vietnamese Pharmacopoeia V has been constructed and compiled under a scientific and consistent procedure with consultations from the latest versions of other pharmacopoeias and with consideration of the practical situation of pharmaceutical manufacturing and analytical laboratories in the country at present and with vision for the next five years. In order to assure feasibility of application, the majority of analytical procedures in relevant monographs was validated in various drug quality control laboratories (National Institute of Drug Quality Control, Institute of Drug Quality Control - Ho Chi Minh city, National Institute for Control of Vaccines and Biologicals, Hanoi University of Pharmacy, Provincial Centers of Drug Quality Control, several pharmaceutical manufacturers…). Vietnamese Pharmacopoeia V is defined as the Set of National Standards for medicines stipulated in the Law on Pharmacy and the Law on Standards and Technical Regulations. That confirms the technical regulatory traits of pharmacopoeial standards. The implementation and application of Vietnamese Pharmacopoeia V, therefore, are compulsory in pharmaceutical manufacture, distribution and quality control in order to make significant contribution to quality assurance of medicines. Having achieved the compilation and publication of Vietnamese Pharmacopoeia V, the Vietnamese Pharmacopoeia Commission would like to express sincere thanks to the leaders of the Ministry of Health and the Ministry of Science and Technology for their great concern and effective concrete guidance regarding the development and compilation of the Pharmacopoeia. The Commission highly appreciates the contributions of intellect and effort from many experts and xi

officials inside and outside the pharmaceutical field, of members and collaborators of the Vietnamese Pharmacopoeia Commission. Heartfelt thanks also go to the pharmaceutical establishments, manufacturers, businesses … for their positive contribution to setting up the Pharmacopoeia. The Commission would like to express sincere gratitude to the Medical Publishing House for their valuable contribution to the printing and publishing Vietnamese Pharmacopoeia V with accurate contents and fine presentation. Although Vietnamese Pharmacopoeia V has been compiled following a very closely coordinated and elaborate working procedure, shortcomings and errors are inevitable. Any suggestions and comments related to the contents of Vietnamese Pharmacopoeia V are welcome and will be taken into consideration for further amendments, making the Pharmacopoeia increasingly improved so as to meet practical requirements on drug quality control and supervision, to enhance drug quality and to serve the cause of people's health care and protection. VIETNAMESE PHARMACOPOEIA COMMISSION CHAIRMAN Associate Prof. Dr. Trinh Van Lau

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HISTORY OF THE PHARMACOPOEIA OF THE SOCIALIST REPUBLIC OF VIETNAM

HISTORY OF THE PHARMACOPOEIA OF THE SOCIALIST REPUBLIC OF VIETNAM The Democratic Republic of Vietnam was founded on 2 September 1945, now it is the Socialist Republic of Vietnam. During the times of French colonization and resistance war against French colonialists, standards in French Pharmacopoeia were applied for drug quality control and management in Vietnam. After the reestablishment of peace in the North in 1954, the mission of setting up the Vietnamese Pharmacopoeia that relevant to our own situations and practices was essentially arise, especially when the medicine standardization was a great concern of the Government. On 28 March 1963, Governmental Decision No 123/CP relating the work of standardization was issued by the Government. To implement the Decision, BSc Pharm Vu Cong Thuyet, Vice-Minister of Health, was appointed by the Ministry of Health to take up the responsibility for organizing Vietnamese Pharmacopoeia Commission. Prof. Truong Cong Quyen, one of the first five Vietnamese professors in medical field, was nominated by the Ministry of Health as the direct organizer of the Vietnamese Pharmacopoeia Commission at the proposal of BSc Pharm Vu Cong Thuyet. On 27 February 1964, the Decision No 183/BYT/QĐ signed by Minister of Health MD Pham Ngoc Thach had determined the organizational structure of the Vietnamese Pharmacopoeia Commission, in which Prof.Truong Cong Quyen was appointed as a Chairman and BSc Pharm Vu Cong Thuyet, BSc Pharm Tran Van Luan, MD Nguyen Ngoc Doan, MD Nguyen Van Huong as Vice-Chairmen.The Commission Secretariat was composed of BSc Pharm Ngo Gia Truc, Head of Secretariat, and BSc Pharm Nguyen Huu Bay, Deputy Head. There were 16 members (12 BSc Pharm and 4 medical doctors) in the Vietnamese Pharmacopoeia Commission. The Vietnamese Pharmacopoeia I Commission (from 1963 to 1984) was composed of six committees, including that of chemico-pharmaceutical substances, formulated preparations, materia medica, pharmacology, vaccines and sera and general methods of quality control. There were total 92 experts participating in the VPI Volume 1’s construction, with only 2 full-time staffs and the others were part-time members. As the first elaboration of Vietnamese Pharmacopoeia, the guiding principles put forward by the Ministry of Health for the Pharmacopoeia’s compilation were as follows: The Vietnamese Pharmacopoeia should be based on the practical situation in Vietnam. The Pharmacopoeia should promote and improve the technical standards to a higher level. The Pharmacopoeia should have the right balance of traditional and western medicine combination. Several seminars were held in Hanoi by the VP Standing Executive Committee to discuss the principles for the monograph selection as well as to determine the monograph list to be presented in the Pharmacopoeia. The Pharmacopoeia Commission has approved that the medicines to be appeared in the Pharmacopoeia must be those being of common use in the country, defined of prophylactic and therapeutic value, and promising to be used in a 5 - 10 year period. Special attention should be focused on the domestic medicinal substances, materials and the traditional pharmaceutical products, after that the foreign medicines circulated in Vietnam should also be taken into consideration. The principle for the selection and elaboration of methods for quality control was that they should be suitable for the equipment capability in central laboratories of quality control, taking into consideration the situation at provincial levels. The methods introduced in the VP are acted as the legislation and arbitration approach. After the 6-year constant efforts under the wartime conditions, Volume 1 of the first edition of Vietnamese Pharmacopoeia was published under Decision No 1008/BYT-QĐ of the Ministry of Health on 20 November 1970. The Pharmacopoeia contained 572 monographs of medicines including 269 pharmaceutical chemicals, 120 materia medica, 167 formulated preparations, 16 biological products, and monographs of quality control methods, reference and standard substances, chemicals and reagents for the application concerning production, distribution, quality control and utilization of drugs. In March 1977, the Ministry of Health signed a Decision to publish the supplement part to the Volume 1 of VP I, which included the corrigenda and addition section of 136 Vietnamese standards on quality control methods of medicines and medicinal forms, 7 criteria on chemicals and reagents, and the list of Toxic Medicines Schedule A or B with their doses. Further publication of 44 standards on medicines and medicinal substances was carried out at the same time. After the country’s reunification, 4000 copies of the VP I with the latest amendments and additions in 1977 were reprinted in order to satisfy the drug standardization requirements and quality assurance for the pharmaceutical industry in the whole country. Although being constructed during the severe wartime, the VP I had satisfied the requirements of a Set of National Standards on medicines and drug testing methods to promote the mission of drug quality control for serving the branch’s duties in drug production and supply, in the people’s health care during the wartime as well as in the economic development in a peaceful and reunified nation. Since the Vietnamese Traditional Medicine has evolved for thousand years and has promising perspective to develop in combination with modern techniques, the government wants to promote our Oriental Medicine practice for wider usage and application. Therefore, it is an urgent task to build the Volume 2 of VP I for the pharmacognosy of Oriental Medicine to meet the standardization xiii

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requirements in the pharmaceutical industry. On 16 September 1968, the Ministry of Health decided to establish the Pharmacognosy Committee of Vietnamese Pharmacopoeia Commission with Prof Truong Cong Quyen, the Commission Chairman, acting concurrently as Committee Head, Prof Do Tat Loi, Doctor of Traditional Medicine Le Van Loi, Traditional Healers La Van Quynh and Pho Duc Thanh as Deputy-Heads. Three subcommittees were involved in the committee in respective specialized fields, namely materia medica, traditional-medical principle and traditionalmedicinal formulation. The members participating in Volume 2 of VP I (pharmacognosy part) consisted of 5 specialized and responsible officials among the totality of 8 officials working as office staff at that time, 23 members, 11 collaborators and 7 national pharmaceutical or medical establishments.The process of setting up Volume 2 of Vietnamese Pharmacopoeia I (Pharmacognosy part) included the following steps: 1. Compilation and publication of the “Vietnamese materia medica” (1969- 1972) with 341 domestic materia medica, 74 imported ones, 3 general monographs on medicinal plant cultivation, drug processing, formulation and storage. In this step, the selection of official names, scientific names and other names of medicinal plants or plant parts for medicinal use, and the determination of appropriate description, pictures of plant parts for medicinal use, characters, action and use, dosage and administration... of traditional drugs were implemented. Making use of the knowledge and know-how from experience pharmacists and healers, the Pharmacognosy Committee had discussed and arrived to common perspective of the specialized issues. The “Vietnamese materia medica” was published in two times, 5,000 copies in the first edition and 12,000 in the second. 2. Proposal of monograph list, agreement on contents, arrangement of monographs and compilation of the draft text of VP I, pharmacognosy part (1970 - 1975). The contents of Volume 2 of VP I (pharmacognosy part) were required to be compiled with higher quality than that of the monographs included in the “Vietnamese materia medica”. Apart from the methods of identification, tests for purity and assays, the book was added with specifications of processing, storage and other matters necessary for traditional healers such as characters, channel tropism, actions, indications, administration, dosage, and abstinence. 3. Printing the draft version of and sending for comments on Volume 2 of VP I (pharmacognosy part) to local organizations concerned (1975 - 1981). 4. Revising the draft version and submitting to the Ministry of Health for approval and publication. In April 1983, the first edition of Volume 2 of Vietnamese Pharmacopoeia I (national traditional medicines) was published with a circulation of 4,000 copies, including 244 monographs. Thus, up to 1983, VP I was fully worked out to present both western medicines and traditional medicines. From the middle of the eighties until now, the country has undergone a dramatic transformation by the Economic Reform. The drug quality has been required to the higher level and the international integration has been increased, the domestic pharmaceutical industry had engaged in the new stage of development. At the same time, many new editions of Pharmacopoeias from other countries have been available for reference. The qualification of Vietnamese pharmacists has been upgraded, the country has got a significant increase of Prof, DSc Pharm, PhD Pharm and BSc Pharm, Specialists. The drug quality control system of the state as well as in businesses has been gradually modernized in accordance with the requirements of quality management and services to medicinal production and trading. Therefore, many points in the contents of VP I were no more suitable until then. The compilation of the second edition of Vietnamese Pharmacopoeia (VP II) became a practical need. On 12 May 1984, Decision No 328-BYT/QĐ was promulgated by the Ministry of Health to restructure the Vietnamese Pharmacopoeia Commission for compiling VP II. The Standing Executive Committee was consisted of the Chairman, Prof. Truong Cong Quyen, and 3 Vice-Chairmen and 1 Vice-Chairwoman undertaken by Prof Nguyen Van Dan, Prof Nguyen Kim Hung, Assoc Prof Ngo Gia Truc, Assoc Prof Tran Thi Hoang Ba. The Secretariat Committee was composed of Assoc Prof Doan Huy Khac as Head of Secretariat and BSc Pharm Phan Van Tin, PhD Pharm Nguyen Ba Hiep and BSc Pharm Nguyen Van Thi as Deputy-Heads. The office of the Vietnamese Pharmacopoeia Commission was a professional administrative unit involved in the staff of the National Institute of Drug Quality Control, working as assistant for the Standing Executive Committee and the Secretariat Committee. The members participating in setting up Vietnamese VP II totally amounted to 221, including Professors, Doctors of Science in Pharmacy, BSc Pharm, Specialists, in which there were 5 medical doctors, 4 traditional healers and 9 officials of other fields.The VP II was published in 3 volumes. Volume 1 composed of western medicines was published in 1990 with a circulation of 1,500 copies, including 89 monographs, in which 39 chemico-pharmaceutical substances, 12 formulated preparations, 8 materia medica, 29 general methods for quality control and 1 general stipulation were involved. Volume 2 (traditional medicines) was published in 1991 including 63 monographs of materia medica and 1 general monograph on the method of processing materia medica. Volume 3 was published in December 1994 including 84 monographs of chemico-pharmaceutical substances, 70 monographs of materia medica, 62 monographs of general methods for quality control and 90 infrared spectra. In this volume, many entries were newly added; the others were thoroughly reviewed and updated in accordance with some advanced testing methods such as gas chromatography, high performance liquid chromatography. After 10 years of renovation, considerable changes have been seen in domestic pharmaceutical industry. In the middle of the nineties, the drug market in Vietnam was increased 6 times in comparison with that in the late eighties. Home pharmaceutical industry has developed with xiv

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new steps. Nearly 5000 pharmaceutical products have been in production. About 200 foreign pharmaceutical companies have been licensed their business operation in Vietnam. About 4,000 foreign pharmaceutical products from various regions in the world have been registered for circulation in Vietnam. The drug standardization and the assurance of domestic and imported drug quality have been increasingly improved. In 1995, Vietnam became the 7th member of the Association of South-East Asian Nations and actively engaged in the international integration process. The compilation and publication of the third edition of Vietnamese Pharmacopoeia were an important measure to ensure the government control task over the medicine quality at the new situations in the country and in the world. On 25 July 1994, the member list of the Standing Executive Committee of the Vietnamese Pharmacopoeia III Commission were proclaimed by the Minister of Health in Decision No 519/BYT/QĐ which constituted of 11 members with Prof Truong Cong Quyen acting as Chairman of the Commission, 6 Vice-Chairmen and 1 Vice-Chairwoman undertaken by Assoc Prof PhD Pharm Le Van Truyen, Assoc Prof Doan Huy Khac (being concurrently Head of the secretariat), Prof DSc Pharm Nguyen Van Dan, Prof DSc Med Dang Duc Trach, Assoc Prof Vu Khanh, Assoc Prof Ngo Gia Truc, and Assoc Prof Tran Thi Hoang Ba, and 3 Deputy-Heads of the Secretariat acted by PhD Pharm Nguyen Vi Ninh, Assoc Prof PhD Pharm Trinh van Quy and PhD Pharm Nguyen Van Thi. Late in 1997, Assoc Prof PhD Pharm Le Van Truyen, Vice-Minister of Ministry of Health, was appointed by the Minister of Health in Decision No 2678/1997/QĐ-BYT on 23 December 1997 to be as Chairman of Vietnamese Pharmacopoeia III Commission, to take the responsibility which had been assumed by Prof Truong Cong Quyen for over 3 decades. The committees involved in the Commission were generally kept unchanged in terms of organization, except the committee of general methods for quality control was split into 2 committees: one concerned with analytical methods, reagents and reference substances and the other with biological methods. The members engaged in setting up VP III included 4 full - time staffs of the Commission, 176 experts and collaborators and many state agencies in the pharmaceutical and medical areas. After 5 years of working efforts (1995 - 2000), the Vietnamese Pharmacopoeia Commission had completed the VP III for draft version printing. The draft version of VP III (traditional medicines) was composed of 154 monographs and printed in 1998. The draft version of VP III (western medicines and materia medica) was printed in 2000. They were both sent for comments to the various units in the whole industry. In 2001, based on the constructive comments from experts and agencies, the Vietnamese Pharmacopoeia Commission had organized the assessment, revision and additional studies to improve the draft versions and to get the VP III officially printed in early 2002. In comparison with the previous publications, the VP III has got many new monographs, has added some advanced techniques and methods for drug testing, has raised the standards of medicinal substances and products to the high levels equivalent to those in the regional and global scope in order to satisfy the practical requirements of the country and the international integration process. Being aware of the importance of pharmacopoeial work in the period of global economic integration, the Ministry of Health continued to strengthen the Vietnamese Pharmacopoeia Commission for implementation guidance of VP III and for further improvement and preparation of the next edition of pharmacopoeia, the VP IV. Under Decision 689/BYT-QĐ dated 28 February 2003, the Minister of Health had appointed Assoc Prof PhD Pharm Trinh Van Quy, Director of the National Institute of Drug Quality Control, to hold the office as Chairman of the Vietnamese Pharmacopoeia Commission in place of Assoc Prof PhD Pharm Le Van Truyen for retirement. The Standing Executive Committee was then strengthened with the Vice-Chairmen who have been holding in office as Directors of Drug Administration of Vietnam, National Institute of Drug Quality Control, National Institute of Quality Control for Vaccines and Medical Bio-products, Department for Science and Training, Ministry of Health. So far, the Standing Executive Committee of Vietnamese Pharmacopoeia Commission IV composed of 9 members with Assoc Prof PhD Pharm Trinh Van Quy as chairman of the Commission; 6 Vice-Chairmen: Prof PhD Pharm Pham Thanh Ky, PhD Pharm Tran Cong Ky, Assoc Prof PhD Pharm Trinh Van Lau, PhD Pharm Cao Minh Quang, Prof PhD Med Nguyen Dinh Bang and PhD Pharm Pham Quoc Bao. Due to the changes in leading positions of the pharmaceutical administrative agencies, in 2007 the vicechairmen of the Vietnamese Pharmacopoeia Commission were 6 persons, who are Prof PhD Pharm Pham Thanh Ky, PhD Pharm Truong Quoc Cuong, Assoc Prof PhD Pharm Trinh Van Lau, BSc Pharm Nguyen Van Mo, Assoc Prof PhD Pharm Le Van Phung and PhD Pharm Pham Quoc Bao. The Commission Secretariat was composed of Assoc Prof PhD Pharm Trinh van Lau (Director of National Institute of Drug Quality Control) as Head of the Secretariat and MSc Public Health Cao Thi Mai Phuong (Head of the Vietnamese Pharmacopoeia Commission Office) and BSc Pharm Nguyen Van Vien (Staff of Drug Administration of Vietnam) as Deputy-Heads. On 31 March 2008, the Minister of Health had appointed PhD Pharm Nguyen Van Tuu, Vice-Director of National Institute of Drug Quality Control, to hold position as Chairman of the Vietnamese Pharmacopoeia Commission in place of Assoc Prof PhD Pharm Trinh Van Quy for retirement, to continue the management of activities of the Vietnamese Pharmacopoeia Commission. Next, on 2 January 2009, the Minister of Health signed a decision to establish the Vietnamese Pharmacopoeia and Formulary Center based on the previous Vietnamese Pharmacopoeia Commission office as an agency directly under the National Institute of Drug Quality Control, which is a legal entity with its own seal and financial account. The Vietnamese Pharmacopoeia and Formulary xv

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Center is a permanent unit of the Vietnamese Pharmacopoeia Commission. After the re-enforcement of the Standing Executive Committee, the Expert Committees have also been reorganized by some decrease in number of units, the Committee of Materia Medica and Committee of Traditional Medicines were combined to form the Committee of Materia Medica and Traditional Medicines; the Committee of Analytical Methods, Reagents and Reference Substances and Committee of Biological Methods were combined to form the Committee of General Quality Control Methods. Members of the Standing Executive Committee were assigned to have the responsibility for respective expert committees; the staff of the Commission Office were appointed as secretaries of committees. Along with the personnel arrangement, the Commission has setting up immediately its pharmacopoeial activities. In the years 2004 - 2005, the Commission activities were focused on completing and editing the English version of the VP III. In April 2005, Vietnamese Pharmacopoeia III, the English version was published and dispatched mainly for the foreign partners, the import-export pharmaceutical companies and the international organizations. This was the first time the Vietnamese Pharmacopoeia got its English version that helps the foreign experts to access the Set of National Pharmaceutical Standards of the country which has a strong developing pharmaceutical industry and is deeply integrating with the world economy. Also in the years 2004 - 2005 some corrigenda and addenda to VP III were made. In early 2006 the Supplement of VP III was published. The contents of the Supplement were compiled as a constitutive part of VP IV to be published later on.The elaboration of VP IV was carried out following a comprehensive work plan approved by the Ministry of Health. At that time the Law on Pharmacy was issued by the National Assembly XI, the 7th Session on 14 June 2005. In the Law on Pharmacy, the Vietnamese Pharmacopoeia is defined to be the Set of National Standards concerning the quality of medicines and the methods for their quality control. The Law on Standards and Technical Regulations issued by the XI National Assembly, the 9th Session on 29 June 2006 also regulated the process of setting up and elaborating the Pharmacopoeia which is required to comply adequately and closely with the operating procedures and protocols. On 29 December 2008 interministerial joint Circular No 11/2008/TTLT/BYT-BKHCN was signed by the Ministry of Health and the Ministry of Science and Technology to give Directions for the elaboration, consideration and proclamation of the Set of National Standards for medicines and the promulgation, publication of Vietnamese Pharmacopoeia. The standardization of medicines therefore has been brought up to a new stage of development, giving assurance for the elaboration of the national standards for medicines - the Vietnamese Pharmacopoeia in compliance with the Law on Pharmacy and the Law on Standards and Technical Regulations, suitable to specific characteristics of national standards for medicines and international rules; satisfying the requirements of international pharmaceutical integration. By this time, Vietnamese Pharmacopoeia IV was basically completed and was checked, taken over by the Ministry of Health at ministerial level. To meet the regulations under the joint circular 11/2008/TTLB/BYT-BKHCN, the national standards for medicines in VP IV were sent to the Vietnam Drug Administration for professional verification and to the Directorate for Standards, Metrology and Quality for consideration and approval. On 16 July 2009, the Minister of Science and Technology signed a Decision proclaiming the first edition of the Set of National Standards for medicines (TCVN I:2009) including the following codes TCVN I-1:2009, Set of National Standards for medicines - part 1: Drug quality control methods; TCVN I-2:2009, Set of National Standards for medicines - part 2: Chemico-pharmaceutical substances; TCVN I-3:2009, Set of National Standards for medicines - part 3: Formulated preparations; TCVN I-4:2009, Set of National Standards for medicines - part 4: Materia medica and Traditional medicines; TCVN I-5:2009, Set of National Standards for medicines - part 5: Vaccines and Biologicals. Based on the proclamation of the Set of National Standards for medicines TCVN I:2009, on 1 September 2009, the Minister of Health signed the Decision No 3195/QĐ-BYT for the promulgation of VP IV with effective date on 1 January 2010. Along with the promulgation of Vietnamese Pharmacopoeia IV, on 14 October 2009 Decision No 3931/QĐ-BYT was signed by the Minister of Health, to establish the Vietnamese Pharmacopoeia Commission V which includes the Chairman, Vice-Chairmen, the Standing Executive Committee and seven expert committees. According to this Decision, Dr.Nguyen Van Tuu was appointed as the chairman of the Vietnamese Pharmacopoeia Commission, and Assoc.Prof. Trinh Van Lau, Dr.Truong Quoc Cuong, Assoc.Prof. Tran Thi Oanh, Assoc.Prof. Nguyen Ngoc Vinh, Assoc.Prof. Le Van Phung, M.Econ. Nguyen Quang An as Vice-chairmen. The Standing Executive Committee was composed of members who are heads of regulatory agencies, research centers, institutes and experience experts with profound impact and vision to develop the Vietnamse Pharmacopoeia. By the end of the year 2010, after the retirement of Dr.Nguyen Van Tuu, Assoc.Prof. Trinh Van Lau was designated as a Chairman by Decision No 5322/QĐ-BYT signed by the Minister of Health on 31 December 2010. Following that Decision, the Minister of Health has also appointed Assoc.Prof. Doan Cao Son, the Director of the National Institute of Drug Quality Control, as a vice-Chairman to strengthen the personnel of the Commission. xvi

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HISTORY OF THE PHARMACOPOEIA OF THE SOCIALIST REPUBLIC OF VIETNAM

Right after the establishment, Vietnamese Pharmacopoeia Commission with the assistance of Vietnamese Pharmacopoeia and Formulary Center has defined the comprehensive plan for the compilation of the Set of National Standards for Medicines and for the Vietnamese Pharmacopoeia V promulgation, according to the above mentioned Decisions. Since the year 2012, annual Set of National Standards on Medicines is proclaimed TCVN II:2012 includes 79 standards by Decision No 2702/ QĐ-BKHCN dated 8 October 2012; TCVN III:2014 includes 51 standards by Decision 759/ QĐBKHCN dated 22 April 2014; TCVN IV:2015 includes 58 standards by Decision No 746/ QĐ-BKHCN dated 15 April 2015; TCVN V:2017 includes 109 standards by Decision No 968/ QĐ-BKHCN dated 28 April 2017; TCVN VI:2017 includes 64 standards by Decision No 2760/QĐ-BKHCN dated 13 October 2017. The National Standards for Medicines from TCVN II, III, and IV were already gathered up into the Supplement to Vietnamese Pharmacopoeia IV and were promulgated by the Minister of Health in June 2015. On 28 November 2017, the Minister of Health signed the Decision No 5358/QĐ-BYT for the promulgation of Vietnamese Pharmacopoeia V which comes into effect on 1 July 2018. The period to compile Vietnamese Pharmacopoeia V is the era in which the information technology and advanced techniques are available to help reaching much better performance in drug quality control, to raise up the Vietnam’s level to keep pace with other countries in the region and in the world. Actively engage in the global integration process, the Vietnamese Pharmacopoeia Commission advocates many efforts to expand the international collaboration, to participate and contribute in the forums and exchange programs for drug testing, quality control and standard harmonization with other countries, both in the area of Western and traditional medicines. The fifth edition of Vietnamese Pharmacopoeia shows a great progress with as many as 1519 monographs, including 228 general monographs, 485 monographs of chemico-pharmaceutical substances and 385 monographs of formulated preparations, 372 monographs of crude drugs and traditional medicines, 41 monographs of immunological products, 8 monographs of containers and materials used for the manufacture of containers. With nearly 2200 pages of content, Vietnamese Pharmacopoeia V is printed in two volumes, the first one covering chemico-pharmaceutical substances and formulated preparations; the second volume includes crude drugs and traditional medicines, immunological products and appendices. With experience built up over 50 years and five editions, the Vietnamese Pharmacopoeia becomes indispensable technical reference documentation in the pharmaceutical industry which plays very important role to ensure the drug quality to serve the cause of communities’ health care while contributing to the country’s socio-economical development.

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VP V

MEMBERSHIP OF THE FIFTH VIETNAMESE PHARMACOPOEIA COMMISSION

MEMBERSHIP OF THE FIFTH VIETNAMESE PHARMACOPOEIA COMMISSION (According to Decision No 3931/QĐ-BYT dated 14/10/2009, Decision No 5322/QĐ-BYT dated 31/12/2010, Decision No 1032/QĐ-BYT dated 03/4/2012) Chairman of the Vietnamese Pharmacopoeia Commission Nguyen Van Tuu, PhD Pharm (10/2009 - 2010) Trinh Van Lau, PhD Pharm, Assoc Prof (from 12/2010) Vice-Chairmen Director General of Drug Administration of Vietnam: Truong Quoc Cuong, PhD Pharm. Director of National Institute of Drug Quality Control: Doan Cao Son, PhD Pharm, Assoc Prof (from 04/2012). Director of Institute of Drug Quality Control Ho Chi Minh city: Nguyen Ngoc Vinh, PhD Pharm, Assoc Prof. Director of National Institute for Control of Vaccines and Biologicals: Le Van Phung, PhD Med, Assoc Prof (until 12/2014). Vice-Director of Administration of Science Technology and Training MOH: Tran Thi Oanh, PhD Pharm, Assoc Prof. Department of Planning and Finance MOH: Nguyen Quang An, M.Econ. Secretaries: Nguyen Van Loi, PhD Pharm. Cao Thi Mai Phuong MSc PH. Executive Standing Committee Chair: Nguyen Van Tuu, PhD Pharm. Vice Chairs: Trinh Van Lau, PhD Pharm, Assoc Prof. Nguyen Van Thanh, BSc Pharm. Members: Trinh Van Quy, PhD Pharm, Assoc Prof; Pham Thanh Ky, PhD Pharm, Prof; Vo Xuan Minh PhD Pharm, Prof; Tran Thi Oanh, PhD Pharm, Assoc Prof; Nguyen Ngoc Vinh, PhD Pharm, Assoc Prof; Le Van Phung, PhD Med, Assoc Prof; Nguyen Quang An, M.Econ; Le Viet Hung PhD Pharm, Assoc Prof; Le Quan Nghiem, PhD Pharm, Prof; Pham Quoc Bao, PhD Pharm; Nguyen Minh Khoi, DSc Pharm, Assoc Prof; Nguyen Quy Son, BSc Pharm; Le Thanh Hai, PhD Med; Nguyen Van Loi, PhD Pharm; Cao Thi Mai Phuong, MSc PH. Expert Committees: Nomenclature and Regulatory information Chair: Truong Quoc Cuong, PhD Pharm. Vice Chairs: Pham Quoc Bao, PhD Pharm. Nguyen Van Thanh, BSc Pharm. Secretary: Nguyen Van Loi, PhD Pharm. Members: Dinh Thi Thanh Hai, PhD Pharm, Assoc Prof; Luc Thi Thu Hang, MSc Pharm; Nguyen Thi Phuong Mai, MSc Pharm; Cao Thi Mai Phuong, MSc PH; Chu Dang Trung, MSc Pharm; Nguyen Van Vien, BSc Pharm. Chemico - Pharmaceutical Substances Chair: Trinh Van Lau, PhD Pharm, Assoc Prof. Vice Chairs: Phan Thi Thuy Chi, MSc Pharm. Truong Phuong, PhD Pharm, Assoc Prof. Secretary: Luc Thi Thu Hang, MSc Pharm. Members: Tran Hong Anh, PhD Pharm; Pham Thi Duyen, MSc Pharm; Tran Thanh Dao, PhD Pharm, Assoc Prof;

Nguyen Thanh Ha, MSc Pharm; Nguyen Thi Thu Ha, MSc Pharm; Tran Thuy Hanh, MSc Pharm; Tran Duc Hau, PhD Pharm, Assoc Prof; Le Thi Thien Huong, MSc Pharm; Chuong Ngoc Nai, PhD Pharm; Nguyen Hai Nam, PhD Pharm, Prof; Nguyen Thi Kim Thanh, MSc Pharm; Nguyen Thi Thanh Thao, PhD Pharm; Le Thi Thu, MSc Pharm; Le Minh Tri, PhD Pharm, Assoc Prof; Tran Thi Bich Van, MSc Pharm; Nguyen Thi Hong Yen, BSc Pharm. Formulated Preparations Chair: Vo Xuan Minh, PhD Pharm, Prof. Vice Chairs: Doan Cao Son, PhD Pharm, Assoc Prof. Nguyen Ngoc Vinh, PhD Pharm, Assoc Prof. Secretary: Luc Thi Thu Hang, MSc Pharm. Members: Tran Thi Quynh Chi, MSc Pharm; Ha Minh Hien, PhD Pharm; Le Thi Huong Hoa, PhD Pharm; Nguyen Dang Hoa, PhD Pharm, Assoc Prof; Bui Thi Hoa, MSc Pharm; Huynh Van Hoa, PhD Pharm, Assoc Prof; Pham Thi Minh Hue, PhD Pharm, Assoc Prof, Le Van Lang, MSc Pharm; Nguyen Dang Lam, MSc Pharm; Nguyen Tran Linh, PhD Pharm; Nguyen Van Long, PhD Pharm, Assoc Prof; Ha Dieu Ly, PhD Pharm, Assoc Prof; Duong Cong Minh, BSc Pharm, Degree II Specialist; Pham Thi Hong Nhung, MSc Pharm; Pham Hong Phuong, BSc Pharm; Cao Thi Mai Phuong MSc PH; Bui Van Thanh, BSc Pharm, Degree I Specialist; Luc Thi Van, MSc Pharm. Materia Medica and Traditional Medicines Chair: Pham Thanh Ky, PhD Pharm, Prof. Vice Chairs: Tran Hung, PhD Pharm, Assoc Prof. Nguyen Minh Khoi, DSc Pharm, Assoc Prof. Secretary: Nguyen Thi Phuong Mai, MSc Pharm. xix

MEMBERSHIP OF THE FIFTH VIETNAMESE PHARMACOPOEIA COMMISSION

Members: Vuong Van Anh, MSc Pharm; Nguyen Thi Minh Chau, BSc Pharm; Nguyen Thi Kim Danh, MSc Pharm; Huynh Ngoc Duy, MSc Pharm; Pham Thi Giang, MSc Pharm; Nguyen Thu Hang, PhD Pharm, Assoc Prof; Nguyen Viet Kinh, PhD Pharm; Vo Van Leo, PhD Pharm; Bui My Linh, PhD Pharm; Tran Cong Luan, PhD Pharm; Pham Dong Phuong, PhD Pharm; Tran Thi Hong Phuong, PhD Pharm, Assoc Prof; Trinh Thi Quy, MSc Pharm; Do Quyen, PhD Pharm, Assoc Prof; Pham Xuan Sinh, PhD Pharm, Prof; Le Thi Minh Thao, MSc Pharm; Nguyen Viet Than, PhD Pharm, Assoc Prof; Nguyen Thi Bich Thu, PhD Pharm, Assoc Prof; Huynh Ngoc Thuy, PhD Pharm, Assoc Prof; Nguyen Duc Toan, PhD Pharm; Pham Xuan Truong, PhD Pharm; Nguyen Van Tuu, PhD Pharm; Nguyen Tien Vung, PhD Pharm, Assoc Prof . Vaccines and Biologicals Chair: Le Van Phung, PhD Med, Assoc Prof (until 12/2014). Vice Chairs: Nguyen Thi Hong Linh, PhD Med, Assoc Prof . Phan Thi Nga, PhD Med, Prof. Secretaries: Do Thuy Ngan, PhD Med. Nguyen Thi Phuong Mai, MSc Pharm. Members: Nguyen Thi Kieu Anh, MSc; Le Kim Hoa, PhD Med, Do Diep Lan, M.D; Truong Xuan Lien, PhD Med, Assoc Prof; Le Thi Luan, PhD Med, Prof; Luu Anh Thu MSc; Nguyen Thu Van, DSc, Prof; Le Thi Hai Yen, MSc. General Methods for Quality Control Chair: Trinh Van Quy, PhD Pharm, Assoc Prof. Vice Chairs: Phung Thi Vinh, PhD Pharm. Cao Thi Mai Phuong, MSc PH. Secretary: Nguyen Thi Phuong Mai, MSc Pharm.

xx

VP V

Members: Nguyen Tuan Anh, MSc Pharm; Le Dinh Chi, PhD Pharm; Do Trung Dam, DSc Pharm, Assoc Prof; Pham Thi Thanh Ha, PhD Pharm, Assoc Prof; Luc Thi Thu Hang, MSc Pharm; Dang Tran Phuong Hong, MSc Pharm; Nguyen Thi Vinh Hong, MSc Pharm; Ta Manh Hung, PhD Pharm; Tran Viet Hung, PhD Pharm, Assoc Prof; Nguyen Thi Kim Huong, PhD Biology; Truong Thi Thu Lan, MSc Pharm; Thai Nguyen Hung Thu, PhD Pharm, Prof; Nguyen Duc Tuan, PhD Pharm, Assoc Prof. Editorship Chair: Nguyen Van Tuu, PhD Pharm. Vice Chairs: Luc Thi Thu Hang, MSc Pharm. Cao Thi Mai Phuong, MSc PH. Secretary: Nguyen Thi Phuong Mai, MSc Pharm. Members: Phan Thi Thuy Chi, MSc Pharm; Do Trung Dam, DSc Pharm, Assoc Prof; Le Thi Thu Hien, MSc Pharm; Dang Tran Phuong Hong, MSc Pharm;Nguyen Thi Huong, MSc Pharm; Pham Thanh Ky, PhD Pharm, Prof; Do Thuy Ngan, PhD Med; Trinh Van Quy, PhD Pharm, Assoc Prof; Tran Thi Le Sung, BSc Pharm, Degree II Specialist; Phung Thi Vinh, PhD Pharm. Vietnamese Pharmacopoeia and Formulary Center Head: Luc Thi Thu Hang, MSc Pharm. Deputy Head: Nguyen Thi Phuong Mai, MSc Pharm. Staffs: Le Thi Thu Hien, MSc Pharm; Nguyen Thi Huong, MSc Pharm; Pham Binh Minh, Tech; Nguyen Thanh Thao, MSc Pharm; Ha Thi Thuy, BAcc; Nguyen Thi Trang, BSc Pharm.

VP V

COLLABORATORS

COLLABORATORS Nguyen Kim Anh, BSc Pharm

Nguyen Thi Lien, PhD Pharm

Cao Ngoc Anh, MSc Pharm

Nguyen Ngoc Lieu, BSc Pharm

Nguyen Thi Van Anh, BSc Pharm

Do Khanh Linh, BSc Biology

Pham Huy Bach, MSc Pharm

Ngo Thi Lu, BSc Pharm, Degree I Specialist

Pham Thi Kieu Dung, MSc Pharm

Nguyen Thi Ly, MSc Biology

Le Viet Dung, PhD Pharm

Doan Tuyet Mai, MSc Med

Tran Kieu Duyen, MSc Pharm

Pham Hong Minh, BSc Pharm

Do Tuan Dat, PhD Med

Le Thi Quynh Nga, MSc Pharm

Nguyen Thanh Dat, MSc Pharm

Tran Minh Ngoc, PhD Pharm

Mai Thanh Ha, MSc Pharm

Ha Thi Van Oanh, PhD Pharm

Pham Thi Ha, MSc Pharm

Bui Viet Phuong, MSc Pharm

Tran Thi Thu Ha, MSc Pharm

Nguyen Thi Phuong, PhD Pharm

Nguyen Thi Thu Hang, MSc Pharm

Nguyen Thi Lan Phuong, PhD Biology

Nguyen Thi Minh Hien, PhD Biology

Nguyen Thi Lan Phuong, MSc Pharm

Nguyen Thi Thu Hien, BSc Pharm

Nguyen Thi Thanh Phuong, MSc Pharm

Tran Hoang, MSc Pharm

Hoang Thi Tam, MSc Med

Pham Gia Hue, Prof

Nguyen Thi Minh Tam, MSc Pharm

Tran Thi Thanh Hue, MSc Pharm

Pham Thi Minh Tam, MSc Pharm

Le Thi Thu Huyen, MSc Pharm

Nguyen Thi Phuong Thao, MSc Pharm

Le Thi Cam Huong, BSc Pharm

Doan Huu Thien, PhD Med

Hoang Thi Huong, MSc Pharm

Do Bich Thuan, MSc Pharm

Nguyen Thi Thu Huong, MSc Pharm

Nguyen Thi Ngoc Tram, BSc Pharm, Degree

Tran Dinh Khai, MSc Pharm

I

Dang Van Khanh, PhD Pharm

Tran Thi Thu Trang, MSc Pharm

Pham Van Kien, MSc Pharm

Le Van Truyen, PhD Pharm, Assoc Prof

Nguyen Thi Kieu, PhD Med

Nguyen Hoang Tuan, PhD Pharm

Le Thanh Lam, BSc Pharm

Nguyen Tuong Vi, PhD Pharm, Assoc Prof

Specialist,

xxi

xxii

VP V

ORGANIZATIONS PARTICIPATING IN THE COMPILATION OF VIETNAMESE PHARMACOPOEIA

ORGANIZATIONS PARTICIPATING IN THE COMPILATION OF VIETNAMESE PHARMACOPOEIA Drug Administration of Vietnam - Ministry of Health Administration of Science Technology and Training - Ministry of Health Directorate for Standards, Metrology and Quality - Ministry of Science and Technology National Institute of Drug Quality Control - Ministry of Health Institute of Drug Quality Control Ho Chi Minh City - Ministry of Health Hanoi University of Pharmacy Faculty of Pharmacy, University of Medicine and Pharmacy in Ho Chi Minh City National Institute of Medicinal Materials - Ministry of Health National Institute for Control of Vaccines and Biologicals - Ministry of Health National Hospital of Traditional Medicine - Ministry of Health Centre for the Quality Control of Food, Drugs and Cosmetics in Hanoi Centre for the Quality Control of Food, Drugs and Cosmetics in Hue City Centre for the Quality Control of Food, Drugs and Cosmetics in Ho Chi Minh City Military Institute for Research, Quality Control of Drugs and Medicinal devices Vietnamese Pharmaceutical Manufacturers

xxiii

xxiv

VP V

LIST OF MONOGRAPHS

LIST OF MONOGRAPHS 1. Chemico-pharmaceutical substances and formulated preparations 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46.

Abacavir sulfate Acebutolol hydrochloride Acebutolol tablets Acenocoumarol Acenocoumarol tablets Acetazolamide Acetazolamide tablets Acetylcysteine Acetylcysteine capsules Acetylcysteine powder for oral suspension  Acetylsalicylic acid Acetylsalicylic acid tablets Acetylsalicylic acid, gastro-resistant tablets Aspirin and caffeine tablets Aciclovir Aciclovir cream Aciclovir tablets Adrenaline Adrenaline acid tartrate Adrenaline injection Albendazole Albendazole tablets Alimemazine tartrate Alimemazine syrup Alimemazine tablets Allopurinol Allopurinol tablets Aluminium hydroxide, dried Aluminium phosphate, dried Alverine citrate Alverine capsules Ambroxol hydrochloride Ambroxol hydrochloride capsules Ambroxol hydrochloride tablets Amikacin Amikacin injection Aminocaproic acid Aminophylline Aminophylline injection Aminophylline tablets Amiodarone hydrochloride Amiodarone tablets Amitriptyline hydrochloride Amitriptyline tablets Amlodipine besilate Amlodipine tablets

47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91.

Ammonium chloride Amodiaquine hydrochloride Amodiaquine hydrochloride tablets Amoxicillin sodium Amoxicillin for injection Amoxicillin and clavulanic acid for injection Amoxicillin trihydrate Amoxicillin powder for suspension Amoxicillin capsules Amoxicillin tablets Amoxicillin and clavulanic acid powder for oral suspension Amoxicillin and clavulanic acid tablets Amoxicillin and cloxacillin capsules Amphotericin B Amphotericin lozenges Ampicillin Ampicillin sodium Ampicillin for injection Ampicillin and sulbactam powder for injection Ampicillin trihydrate Ampicillin capsules Arginine Arginine aspartate Arginine hydrochloride Arginine capsules Artemether Artemether capsules Artemether tablets Artemether and lumefantrin tablets Artemisinin Artesunate Artesunate for injection Ascorbic acid Ascorbic acid injection Ascorbic acid tablets Aspartame Aspartame powder Atenolol Atenolol tablets Atorvastatin calcium trihydrate Atorvastatin tablets Atropine sulfate Atropine sulfate injection Atropine sulfate tablets Attapulgite xxv

LIST OF MONOGRAPHS

92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 122. 123. 124. 125. 126. 127. 128. 129. 130. 131. 132. 133. 134. 135. 136. 137. 138. 139. 140. 141. 142. xxvi

Azithromycin Azithromycin capsules Azithromycin powder for oral suspension Bacitracin Barium sulfate Barium sulfate for oral suspension Benzalkonium chloride Benzathine benzylpenicillin Benzathine benzylpenicillin for injection Benzoic acid Benzosali ointment Benzylpenicillin potassium Benzylpenicillin sodium Benzylpenicillin for injection Berberine chloride Berberine chloride tablets Betamethasone Betamethasone tablets Betamethasone dipropionate Bethamethasone sodium phosphate Betamethasone eye drops Betamethasone valerate Biotin Biotin tablets Bisacodyl Bisacodyl, gastro-resistant tablets Bisoprolol fumarate Boric acid Boric acid ointment (10%) Boric acid solution (3%) Bromhexine hydrochloride Bromhexine hydrochloride tablets Bupivacaine hydrochloride Caffeine Caffeine and sodium benzoate injection Calcitriol Calcitriol soft capsules Calcium carbonate Calcium and vitamin D tablets Calcium chloride dihydrate Calcium chloride injection (10%) Calcium gluconate Calcium gluconate, effervescent tablets Calcium gluconate for injection Calcium gluconate injection Calcium glycerophosphate Calcium hydroxide Calcium lactate pentahydrate Calcium lactate trihydrate Calcium pantothenate Calcium phosphate

VP V

143. 144. 145. 146. 147. 148. 149. 150. 151. 152. 153. 154. 155. 156. 157. 158. 159. 160. 161. 162. 163. 164. 165. 166. 167. 168. 169. 170. 171. 172. 173. 174. 175. 176. 177. 178. 179. 180. 181. 182. 183. 184. 185. 186. 187. 188. 189. 190. 191. 192. 193.

Calcium sulfate, dried Camphor, natural Camphor, racemic Captopril Captopril tablets Carbamazepine Carbamazepine tablets Carbidopa Carbomers Carmellose calcium Carmellose sodium Cefaclor Cefaclor capsules Cefaclor powder for oral suspension Cefadroxil monohydrate Cefadroxil capsules Cefadroxil powder for suspension Cefadroxil tablets Cefalotin sodium Cefamandole nafate Cefazolin sodium Cefazolin for injection Cefdinir Cefdinir capsules Cefdinir powder for oral suspension Cefepime hydrochloride monohydrate Cefixime Cefixime capsules Cefixime powder for oral suspension Cefixime tablets Cefoperazone sodium Cefoperazone for injection Cefoperazone and sulbactam for injection Cefotaxime sodium Cefotaxime for injection Cefpodoxime proxetil Cefpodoxime capsules Cefpodoxime powder for oral suspension Cefpodoxime tablets Cefradine Cefradine capsules Ceftazidime pentahydrate Ceftazidime for injection Ceftriaxone sodium Ceftriaxone for injection Cefuroxime axetil Cefuroxime powder for oral suspension Cefuroxime tablets Cefuroxime sodium Cefuroxime for injection Celecoxib

VP V

194. 195. 196. 197. 198. 199. 200. 201. 202. 203. 204. 205. 206. 207. 208. 209. 210. 211. 212. 213. 214. 215. 216. 217. 218. 219. 220. 221. 222. 223. 224. 225. 226. 227. 228. 229. 230. 231. 232. 233. 234. 235. 236. 237. 238. 239. 240. 241. 242.

LIST OF MONOGRAPHS

Cellulose acetate Cellulose, microcrystalline Cephalexin Cephalexin capsules Cephalexin powder for oral suspension Cephalexin tablets Cetirizine dihydrochloride Cetirizine tablets Cetostearyl alcohol Cetyl alcohol Charcoal, activated Chloral hydrate Chloramphenicol Chloramphenicol capsules Chloramphenicol ear drops Chloramphenicol eye drops Chloramphenicol tablets Chloramphenicol and dexamethasone sodium phosphate cream Chloramphenicol and dexamethasone sodium phosphate eye drops Chloramphenicol palmitate Chloramphenicol sodium succinate Chloramphenicol for injection Chlorhexidine gluconate solution Chloroform Chloroquine phosphate Chloroquine phosphate tablets Chlorpheniramine maleate Chlorpheniramine tablets Chlorpromazine hydrochloride Chlorpromazine hydrochloride injection Chlorpromazine hydrochloride tablets Chymotrypsin Chymotrypsin tablets Cilastatin sodium Cimetidine Cimetidine tablets Cineole Cinnarizine Cinnarizine tablets Ciprofloxacin hydrochloride Ciprofloxacin eye drops Ciprofloxacin tablets Citric acid monohydrate Clarithromycin Clarithromycin capsules Clarithromycin tablets Clindamycin hydrochloride Clindamycin capsules Clofazimine

243. 244. 245. 246. 247. 248. 249. 250. 251. 252. 253. 254. 255. 256. 257. 258. 259. 260. 261. 262. 263. 264. 265. 266. 267. 268. 269. 270. 271. 272. 273. 274. 275. 276. 277. 278. 279. 280. 281. 282. 283. 284. 285. 286. 287. 288. 289. 290. 291. 292. 293.

Clofazimine capsules Clopidogrel hydrogen sulfate Clopidogrel tablets Clotrimazole Clotrimazole cream Clotrimazole pessaries Cloxacillin sodium Cloxacillin capsules Cocaine hydrochloride Codeine Codeine phosphate Codeine phosphate tablets Colchicine Colchicine tablets Colecalciferol Colecalciferol tablets Copper sulfate Copper sulfate, anhydrous Cortisone acetate Cortisone tablets Cotton (absorbent) Cotton (sterilised absorbent) Cyanocobalamin Cyanocobalamin injection Cyproheptadine hydrochloride Cyproheptadine hydrochloride tablets Dapsone Dapsone tablets Dexamethasone Dexamethasone tablets Dexamethasone acetate Dexamethasone sodium phosphate Dexamethasone injection Dexchlorpheniramine maleate Dexchlorpheniramine tablets Dexpanthenol Dexpanthenol tablets Dextromethorphan hydrobromide Dextromethorphan hydrobromide tablets Diazepam Diazepam injection Diazepam tablets Diclofenac diethylamine Diclofenac sodium Diclofenac sodium injection Diclofenac, gastro-resistant tablets Dicloxacillin sodium Diethyl phtalate Diltiazem hydrochloride Diltiazem tablets Dimenhydrinate xxvii

LIST OF MONOGRAPHS

294. 295. 296. 297. 298. 299. 300. 301. 302. 303. 304. 305. 306. 307. 308. 309. 310. 311. 312. 313. 314. 315. 316. 317. 318. 319. 320. 321. 322. 323. 324. 325. 326. 327. 328. 329. 330. 331. 332. 333. 334. 335. 336. 337. 338. 339. 340. 341. 342. 343. 344. xxviii

Dimenhydrinate tablets Dimercaprol Dimercaprol injection Diphenhydramine hydrochloride Diphenhydramine oral solution Diphenhydramine tablets Domperidone maleate Domperidone tablets Doxycycline hydrochloride Doxycycline capsules Efavirenz Efavirenz capsules Emetine hydrochloride Enalapril maleate Enalapril tablets Ephedrine hydrochloride Ephedrine hydrochloride injection Ephedrine hydrochloride tablets Ergocalciferol Ergocalciferol tablets Erythromycin Erythromycin tablets Erythromycin ethyl succinate Erythromycin stearate Erythromycin stearate capsules Erythromycin stearate tablets Erythrosine Esomeprazole magnesium trihydrate Esomeprazole, gastro-resistant capsules Esomeprazole, gastro-resistant tablets Ethambutol hydrochloride Ethambutol tablets Ethambutol and isoniazid tablets Ethanol Ethanol (96%) Ethanols, dilute Ether Ether, anaesthetic Ethinylestradiol Ethinylestradiol tablets Ethylcellulose Eugenol Famotidine Famotidine tablets Felodipine Fenofibrate Fenofibrate capsules Ferric oxide Ferrous fumarate Ferrous fumarate and folic acid tablets Ferrous sulfate

VP V

345. 346. 347. 348. 349. 350. 351. 352. 353. 354. 355. 356. 357. 358. 359. 360. 361. 362. 363. 364. 365. 366. 367. 368. 369. 370. 371. 372. 373. 374. 375. 376. 377. 378. 379. 380. 381. 382. 383. 384. 385. 386. 387. 388. 389. 390. 391. 392. 393. 394. 395.

Ferrous sulfate tablets Ferrous sulfate, dried Fexofenadine hydrochloride Fexofenadine tablets Flucloxacillin sodium Flucloxacillin capsules Fluconazole Fluconazole capsules Fluocinolone acetonide Fluocinolone acetonide dihydrate Fluocinolone cream Folic acid Folic acid tablets Formaldehyde solution Furosemide Furosemide tablets Gabapentin Gabapentin capsules Gabapentin tablets Gelatin Gelatin, hard capsule shells Gentamicin sulfate Gentamicin eye drops Gentamicin injection Glibenclamide Glibenclamide tablets Glibenclamide and metformin tablets Gliclazide Gliclazide tablets Glimepiride Glimepiride tablets Glimepiride and metformin tablets Glipizide Glipizide tablets Glipizide and metformin tablets Glucosamine hydrochloride Glucosamine sulfate potassium chloride Glucosamine sulfate sodium chloride Glucosamine tablets Glucose monohydrate Glucose, anhydrous Glucose injection Glucose intravenous infusion Glutathione Glycerol Glycerol monostearat 40 - 55 Glyceryl trinitrate solution Glyceryl trinitrate tablets Griseofulvin Griseofulvin tablets Guaifenesin

VP V

396. 397. 398. 399. 400. 401. 402. 403. 404. 405. 406. 407. 408. 409. 410. 411. 412. 413. 414. 415. 416. 417. 418. 419. 420. 421. 422. 423. 424. 425. 426. 427. 428. 429. 430. 431. 432. 433. 434. 435. 436. 437. 438. 439. 440. 441. 442. 443. 444. 445. 446.

LIST OF MONOGRAPHS

Haloperidol Haloperidol tablets Halothane Heptaminol hydrochloride Heptaminol tablets Histidine Histidine hydrochloride monohydrate Hydrochloric acid Hydrochloric acid, dilute Hydrochlorothiazide Hydrochlorothiazide tablets Hydrocortisone acetate Hydrocortisone acetate injection Hydrocortisone acetate ointment Hydrocortisone and neomycin eye drops Hydrogen peroxide, concentrated Hydrogen peroxide solution dilute (10%) Hydrogen peroxide solution dilute (3%) Hydroxocobalamin acetate Hydroxocobalamin chloride Hydroxocobalamin sulfate Hydroxocobalamin injection Hydroxyethylcellulose Hydroxyethylmethylcellulose Hydroxypropylcellulose Hyoscine butylbromide Hyoscine butylbromide tablets Ibuprofen Ibuprofen tablets Imipenem Imipenem and cilastatin for injection Imipramine hydrochloride Imipramine tablets Indapamide Indapamide tablets Indinavir sulfate Indinavir capsules Indomethacin Indomethacin capsules Indomethacin tablets Iodine Iodine solution (1%) Irbesartan Irbesartan tablets Isoleucine Isoniazid Isoniazid tablets Isosorbide dinitrate, diluted Isosorbide dinitrat tablets Isosorbide mononitrate, diluted Isosorbide mononitrate tablets

447. 448. 449. 450. 451. 452. 453. 454. 455. 456. 457. 458. 459. 460. 461. 462. 463. 464. 465. 466. 467. 468. 469. 470. 471. 472. 473. 474. 475. 476. 477. 478. 479. 480. 481. 482. 483. 484. 485. 486. 487. 488. 489. 490. 491. 492. 493. 494. 495. 496. 497.

Itraconazole Itraconazole capsules Kanamycin sulfate Kanamycin injection Kaolin, heavy Kaolin, light Kaolin, light (natural) Ketoconazole Ketoconazole cream Ketoconazole tablets Ketoconazole and neomycin cream Ketoprofen Ketoprofen capsules Lactose Lamivudine Lamivudine oral solution Lamivudine tablets Lamivudine and zidovudine tablets Lanolin, anhydrous Lansoprazole Lansoprazole, gastro-resistant capsules Levamisole hydrochloride Levodopa Levodopa tablets Levodopa and carbidopa tablets Levofloxacin Levofloxacin tablets Levomenthol Levomepromazine maleate Levomepromazine tablets Levonorgestrel Levonorgestrel tablets Levothyroxine sodium Levothyroxine tablets Lidocaine hydrochloride Lidocaine injection Lincomycin hydrochloride Lincomycin capsules Lincomycin injection Loperamide hydrochloride Loperamide capsules Loperamide tablets Lopinavir Loratadine Loratadine tablets Losartan potassium Losartan potassium tablets Lovastatin Lovastatin tablets Lumefantrine Lysine acetate xxix

LIST OF MONOGRAPHS

498. 499. 500. 501. 502. 503. 504. 505. 506. 507. 508. 509. 510. 511. 512. 513. 514. 515. 516. 517. 518. 519. 520. 521. 522. 523. 524. 525. 526. 527. 528. 529. 530. 531. 532. 533. 534. 535. 536. 537. 538. 539. 540. 541. 542. 543. 544. 545. xxx

Macrogols Magnesium carbonate, heavy Magnesium carbonate, light Magnesium chloride Magnesium hydroxide Magnesium - aluminium hydroxide tablets Magnesium lactate dihydrate Magnesium - B6 tablets Magnesium oxide, heavy Magnesium oxide, light Magnesium stearate Magnesium sulfate Magnesium trisilicate Mangiferine Mannitol Mebendazole Mebendazole tablets Mefenamic acid Mefenamic acid tablets Mefloquine hydrochloride Mefloquine tablets Meloxicam Meloxicam tablets Menthol, racemic Meprobamate Mercurochrome Metformin hydrochloride Metformin tablets Methacrylic acid and ethyl acrylate copolymer (1:1) Methacrylic acid and ethyl acrylate copolymer (1:1), dispersion 30 per cent Methacrylic acid and methyl methacrylate copolymer (1:1) Methacrylic acid and methyl methacrylate copolymer (1:2) Methadone hydrochloride Methadone, oral concentrate solution DL-methionine Methionine tablets Methyl parahydroxybenzoate Methyl salicylate Methylcellulose Methyldopa Methyldopa tablets Methylprednisolone Methylprednisolone tablets Methylprednisolone acetate Methylprednisolone acetate injection Metoclopramide Metoclopramide hydrochloride Metoclopramide injection

VP V

546. 547. 548. 549. 550. 551. 552. 553. 554. 555. 556. 557. 558. 559. 560. 561. 562. 563. 564. 565. 566. 567. 568. 569. 570. 571. 572. 573. 574. 575. 576. 577. 578. 579. 580. 581. 582. 583. 584. 585. 586. 587. 588. 589. 590. 591. 592. 593. 594. 595.

Metoclopramide tablets Metronidazole Metronidazole intravenous infusion Metronidazole tablets Metronidazole and nystatin tablets Metronidazole and spiramicin tablets Miconazole Morphine hydrochloride Morphine hydrochloride injection Nalidixic acid Nalidixic acid tablets Naloxone hydrochloride Naphazoline nitrate Neomycin sulfate Neomycin eye drops Nevirapine, anhydrous Nevirapine tablets Niclosamide anhydrous Niclosamide monohydrate Niclosamide tablets Nicotinamide Nicotinamide tablets Nicotinic acid Nifedipine Nifedipine tablets Nifuroxazide Nikethamide Nikethamide solution Nitrazepam Nitrofurantoin Nitrofurantoin tablets Norfloxacin Norfloxacin tablets Nystatin Nystatin ointment Nystatin tablets Nystatin vaginal tablets Ofloxacin Ofloxacin capsules Ofloxacin eye drops Ofloxacin tablets Omeprazole Omeprazole, gastro-resistant capsules Oresol Oseltamivir phosphate Oseltamivir capsules Ouabain Oxacillin sodium monohydrate Oxygen Oxymetazoline hydrochloride

VP V

596. 597. 598. 599. 600. 601. 602. 603. 604. 605. 606. 607. 608. 609. 610. 611. 612. 613. 614. 615. 616. 617. 618. 619. 620. 621. 622. 623. 624. 625. 626. 627. 628. 629. 630. 631. 632. 633. 634. 635. 636. 637. 638. 639. 640. 641. 642. 643. 644. 645. 646.

LIST OF MONOGRAPHS

Oxymetazoline nasal drops Oxytetracycline dihydrate Oxytetracycline hydrochloride Oxytetracycline capsules Pantoprazole sodium sesquihydrate Pantoprazole, gastro-resistant tablets Papaverine hydrochloride Papaverine hydrochloride tablets Paracetamol Paracetamol capsules Paracetamol effervescent tablets Paracetamol intravenous infusion Paracetamol suppositories Paracetamol tablets Paracetamol and caffeine tablets Paracetamol and chlorpheniramine tablets Paracetamol and codeine tablets Paracetamol and ibuprofen tablets Liquid paraffin Pefloxacin mesilate Pefloxacin mesylate tablets Penicillamine Pepsin Perindopril tert-butylamine Perindopril tert-butylamine tablets Pethidine hydrochloride Phenobarbital Phenobarbital tablets Phenol Phenoxymethylpenicillin Penicillin V tablets Phenoxymethylpenicillin potassium Penicillin V potassium tablets Phenylpropanolamine hydrochloride Phenytoin Phenytoin tablets Phthalylsulfathiazole Phthalylsulfathiazole tablets Phytomenadione Phytomenadione tablets Pilocarpine nitrate Piperacillin sodium Piperazine adipate Piperazine citrate Piperazine hydrate Piperazine phosphate Piperazine phosphate tablets Piracetam Piracetam capsules Piracetam injection Piroxicam

647. 648. 649. 650. 651. 652. 653. 654. 655. 656. 657. 658. 659. 660. 661. 662. 663. 664. 665. 666. 667. 668. 669. 670. 671. 672. 673. 674. 675. 676. 677. 678. 679. 680. 681. 682. 683. 684. 685. 686. 687. 688. 689. 690. 691. 692. 693. 694. 695. 696. 697.

Piroxicam capsules Piroxicam tablets Polymyxin B sulfate Polysorbate 20 Polysorbate 60 Polysorbate 80 Potassium bromide Potassium chloride Potassium chloride for injection concentrate Potassium chloride tablets Potassium clavulanate Potassium iodide Potassium permanganate Povidone Povidone, iodinated Povidone-iodine solution Praziquantel Praziquantel tablets Prednisolone Prednisolone tablets Prednisone Primaquine diphosphate Primaquine diphosphate tablets Procainamide hydrochloride Procaine hydrochloride Procaine hydrochloride injection Progesterone Progesterone injection Progesterone soft capsules Promethazine hydrochloride Promethazine hydrochloride cream Promethazine hydrochloride syrup Promethazine hydrochloride tablets Propranolol hydrochloride Propranolol tablets Propyl parahydroxybenzoate Propylene glycol Propylthiouracil Propylthiouracil tablets Pyrantel pamoate Pyrantel pamoate tablets Pyrazinamide Pyrazinamide tablets Pyridoxine hydrochloride Pyridoxine hydrochloride injection Pyridoxine hydrochloride tablets Pyrimethamine Quinapril hydrochloride Quinidine bisulfate Quinine dihydrochloride Quinine dihydrochloride injection xxxi

LIST OF MONOGRAPHS

698. 699. 700. 701. 702. 703. 704. 705. 706. 707. 708. 709. 710. 711. 712. 713. 714. 715. 716. 717. 718. 719. 720. 721. 722. 723. 724. 725. 726. 727. 728. 729. 730. 731. 732. 733. 734. 735. 736. 737. 738. 739. 740. 741. 742. 743. 744. 745. 746. 747. 748. xxxii

Quinine hydrochloride Quinine sulfate Quinine sulfate tablets Ramipril Ramipril tablets Ranitidine hydrochloride Ranitidine tablets Riboflavin Riboflavin tablets Riboflavin sodium phosphate Rifampicin Rifampicin capsules Rifampicin tablets Rifampicin and isoniazid capsules Rifampicin, isoniazid and pyrazineamide tablets Ringer-lactate infusion Ritonavir Rotundine Rotundine tablets Roxithromycin Roxithromycin powder for suppension Roxithromycin tablets Rutin Rutin tablets Rutin and ascorbic acid tablets Salbutamol Salbutamol sulfate Salbutamol tablets Salicylic acid Silver nitrate Silver vitellinate Simvastatin Simvastatin tablets Sodium benzoate Sodium bromide Sodium calcium edetate Sodium camphosulfonate Sodium chloride Sodium chloride eye drops (0.9%) Sodium chloride injection Sodium chloride, isotonic infusion Sodium citrate Sodium hydrogen carbonate Sodium hydrogen carbonate injection Sodium hydrogen carbonate powder Sodium salicylate Sodium starch glycolate (type A) Sodium starch glycolate (type B) Sodium starch glycolate (type C) Sodium sulfate Sodium sulfate, anhydrous

VP V

749. 750. 751. 752. 753. 754. 755. 756. 757. 758. 759. 760. 761. 762. 763. 764. 765. 766. 767. 768. 769. 770. 771. 772. 773. 774. 775. 776. 777. 778. 779. 780. 781. 782. 783. 784. 785. 786. 787. 788. 789. 790. 791. 792. 793. 794. 795. 796. 797. 798. 799.

Sodium thiosulfate Sodium thiosulfate tablets Sodium valproate Sodium valproate tablets Sorbitol Sorbitol powder Sparteine sulfate Sparteine sulfate injection Spectinomycin hydrochloride Spiramycin Spiramycin tablets Pregelatinised starch Maize starch Potato starch Rice starch Tapioca starch Wheat starch Stavudine Stavudine tablets Stearyl alcohol Streptomycin sulfate Streptomycin for injection Strychnine sulfate Sucralfate Sucrose Sulbactam sodium Sulfacetamide sodium Sulfadiazine Sulfadimidine Sulfadoxine Sulfadoxine and pyrimethamine tablets Sulfaguanidine Sulfaguanidine tablets Sulfamethoxazole Sulfamethoxazole tablets Sulfamethoxypyridazine Sulfasalazine Sulfathiazole Sulpiride Sulpiride capsules Sultamicillin Sultamicillin tosilate dihydrate Talc Tamoxifen citrate Tartrazine Telmisartan Telmisartan tablets Tenoxicam Tenoxicam tablets Terbutaline sulfate Terfenadine

VP V

800. 801. 802. 803. 804. 805. 806. 807. 808. 809. 810. 811. 812. 813. 814. 815. 816. 817. 818. 819. 820. 821. 822. 823. 824. 825. 826. 827. 828. 829. 830. 831. 832. 833. 834. 835. 836. 837. 838. 839. 840. 841. 842. 843. 844. 845. 846. 847. 848. 849. 850.

LIST OF MONOGRAPHS

Terfenadine tablets Terpin hydrate Tetracaine hydrochloride Tetracycline hydrochloride Tetracycline hydrochloride capsules Tetracycline hydrochloride eye ointment Tetracycline hydrochloride tablets Theophylline Theophylline tablets Thiamine hydrochloride Thiamine hydrochloride injection Thiamine nitrate Thiamine tablets Vitamin B1, B6 and B12 tablets Thiamphenicol Thiopental sodium and sodium carbonate Ticarcillin sodium Timolol maleate Timolol tablets Tinidazole Tinidazole tablets Titanum dioxide Tobramycin Tobramycin eye drops Tobramycin injection Tocopherol (all-rac-alpha) Tocopheryl acetate (all-rac-alpha) Vitamin E soft capsules Tolbutamide Tolbutamide tablets Tramadol hydrochloride Tranexamic acid Tranexamic acid capsules Tranexamic acid tablets Triamcinolone acetonide Triamcinolone acetonide cream Triglycerides, medium-chain Trihexyphenidyl hydrochloride Trihexyphenidyl tablets Trimetazidine hydrochloride Trimetazidine tablets Trimethoprim Cotrimoxazole tablets Vancomycin hydrochloride Vancomycin powder for injection Vanillin Vaseline Verapamil hydrochloride Vinblastine sulfate Vinblastine sulfate for injection Vincristin sulfate

851. 852. 853. 854. 855. 856. 857. 858. 859. 860. 861. 862. 863. 864. 865. 866. 867. 868. 869. 870.

Vincristin sulfate for injection Vinpocetine Vinpocetine tablets Vitamin A concentrate (oily form), synthetic Vitamin A concentrate (powder form), synthetic Vitamin A soft capsules Vitamin A and D soft capsules Water for injections Water, distilled Water, purified Water, sterilised for injections Xylometazoline hydrochloride Xylometazoline nasal drops Zidovudine Zidovudine oral solution Zidovudine tablets Zinc oxide Zinc oxide ointment Zinc sulfate Zinc sulfate eye drops

2. Immunological products 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

Human normal immunoglobulin Immunosera for human use Anti-rabies serum Antivenom sera Diphtheria antitoxin Human hepatitis B immunoglobulin Interferon alpha-2 Tetanus antitoxin Tuberculin PPD Vaccines for human use BCG vaccine Oral inactivated cholera vaccine Diphtheria and tetanus vaccine (adsorbed) for adults and adolescents Diphtheria, tetanus and pertussis (acellular, component) vaccine (adsorbed) Diphtheria - tetanus - pertussis - hepatitis B heamophilus influenza type B (DTwP - HeB - Hib) combined vaccine Adsorbed diphtheria, tetanus and pertussis vaccine (DTwP) Adsorbed diphtheria vaccine Haemophilus influenzae type B conjugate vaccine Hepatitis A vaccine (live, attenuated) Hepatitis A vaccine (inactivated, absorbed) Hepatitis A vaccine (inactivated, virosome) Hepatitis A (inactivated) and hepatitis B (rDNA) vaccine (adsorbed) Recombinat hepatitis B vaccine (rADN) xxxiii

LIST OF MONOGRAPHS

24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41.

Japanese encephalitis vaccine Influenza vaccine (inactivated) Measles vaccine (live) Measles, mumps, rubella vaccine (MMR vaccine - live) Meningococcal polysaccharide vaccine Mumps vaccine Human papillomavirus vaccine (recombination) Poliomyelitis vaccine (inactivated) Poliomyelitis vaccine, live (oral) Pneumococcal polysaccharide conjugate vaccine (adsorbed) Pneumococcal polysaccharide vaccine Rabies vaccine for human use Rotavirus vaccine – live attenuated, oral Rubella vaccine Adsorbed tetanus vaccine Typhoid vaccine (oral) Typhoid vi polysaccharide vaccine Varicella vaccine (live)

3. Materia medica 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. xxxiv

Aloe Arillus Longan Arillus Momordicae cochinchinensis Benzoinum Bombyx Botryticatus Bulbus Allii sativi Bulbus Eleutherinis subaphyllae Bulbus Fritillariae Bulbus Lilii Cacumen Platycladi Calyx Kaki Carapax et Plastrum Testudinis Carapax Trionycis Caulis Coscinii fenestrati Caulis cum folium Lonicerae Caulis et folium Gymnematis sylvestris Caulis et Radix Fibraureae Caulis Perillae frutescensis Caulis Spatholobi suberecti Caulis Tinosporae sinensis Cera alba Cera flava Colla Cornus Cervi Concha Ostreae Concretio Silicae Bambusae Cornu Cervi Cornu Cervi degelatinatum Cornu Cervi Pantotrichum Cortex Acanthopanacis gracilistyli

VP V

30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80.

Cortex Acanthopanacis trifoliati Cortex Cinnamomi Cortex Eucommiae Cortex Holarrhenae Cortex Magnoliae officinalis Cortex Mori albae radicis Cortex Oroxyli Cortex Periplocae Cortex Phellodendri Cortex Radicis Lycii Cortex Radicis Paeoniae suffruticosae Cortex Schefflerae heptaphyllae Cortex Strychni wallichianae Cortex Terminaliae nigrovenulosae Embryo Nelumbinis nuciferae Endothelium Corneum Gigeriae Galli Flos Carthami tinctorii Flos Chrysanthemi indici Flos Cleistocalysis operculati Flos Daturae metelis Flos Eriocauli Flos Lonicerae Flos Plumeriae rubrae Flos Sambuci javanicae Flos Styphnolobii japonici imaturus Flos Syzygii aromatici Flos Tussilaginis farfarae Folium Ampelopsis Folium Ardisiae Folium Artemisiae annuae Folium Calotropis Folium Catharanthi rosei Folium Cleistocalysis operculati Folium Clerodendri chinense Folium Cratoxyli pruniflori Folium Crini asiatici Folium Crini latifolii Folium Cynarae scolymi Folium Daturae metelis Folium Eriobotryae Folium Erythrinae variegatae Folium et Ramulus Crotonis tonkinensis Folium Excoecariae Folium Ilexi kaushii Folium Jasmini subtriplinervis Folium Lawsoniae Folium Maclurae cochinchinensis Folium Mori albae Folium Nelumbinis nuciferae Folium Perillae frutescensis Folium Plantaginis

VP V

81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 122. 123. 124. 125. 126. 127. 128. 129. 130. 131.

LIST OF MONOGRAPHS

Folium Plectranthi amboinici Folium Plucheae pteropodae Folium Premnae corymbosae Folium Psidii guajavae Folium Sambuci javanicae Folium Senna alatae Folium Solani erianthi Folium Steviae rebaudianae Fructus Alpiniae oxyphyllae Fructus Amomi Fructus Amomi compacti Fructus Amomi aromatici Fructus Apii graveolens Fructus Arctii lappae Fructus Armeniacae praeparatus Fructus Aurantii Fructus Aurantii immaturus Fructus Bruceae javanicae Fructus Chaenomelis Fructus Cnidii Fructus Corni officinalis Fructus Evodiae rutaecarpae Fructus Foeniculi Fructus Forsythiae suspensae Fructus Gardeniae Fructus Gleditsiae australis Fructus Hordei germinatus Fructus Illicii veri Fructus Lycii Fructus Mali Fructus Momordicae charantiae Fructus Mori albae Fructus Morindae citrifoliae Fructus Perillae frutescensis Fructus Piperis longi Fructus Piperis nigri Fructus Psoraleae corylifoliae Fructus Rosae laevigatae Fructus Rubi Fructus Schisandrae chinensis Fructus Silybi Fructus Terminaliae chebulae Fructus Tribuli terrestris Fructus Trichosanthis Fructus Viticis trifoliae Fructus Xanthii strumarii Fructus Zanthoxyli Fructus Ziziphi jujubae Galla chinensis Ganoderma Gekko

132. 133. 134. 135. 136. 137. 138. 139. 140. 141. 142. 143. 144. 145. 146. 147. 148. 149. 150. 151. 152. 153. 154. 155. 156. 157. 158. 159. 160. 161. 162. 163. 164. 165. 166. 167. 168. 169. 170. 171. 172. 173. 174. 175. 176. 177. 178. 179. 180. 181. 182.

Gummi resina Olibanum Gypsum fibrosum Herba Abutili indici Herba Adenosmatis bracteosi Herba Adenosmatis caerulei Herba Adenosmatis indiani Herba Agerati conyzoides Herba Agrimoniae Herba Andrographii Herba Apii graveolens Herba Artemisiae apiaceae Herba Artemisiae vulgaris Herba Bidensis pilosae Herba Centellae asiaticae Herba Centipedae minimae Herba Cistanches Herba Clerodendri philippini Herba Dendrobii Herba Desmodii styracifolii Herba Ecliptae Herba Eleusinis indicae Herba Elsholtziae ciliatae Herba Ephedrae Herba Epimedii Herba Equiseti debilis Herba et Radix Scopariae Herba Glini oppositifolii Herba Gynostemmatis pentaphylli Herba Hedyotidis capitellatae Herba Hedyotidis diffusae Herba Houttuyniae cordatae Herba Lactucae indicae Herba Leonuri japonici Herba Lobeliae chinensis Herba Lophatheri Herba Loranthi Herba Loranthi paracitici Herba Menthae Herba Ocimi gratissimi Herba Ocimi tenuiflori Herba Orthosiphonis spiralis Herba Passiflorae foetidae Herba Phyllanthi amari Herba Phyllanthi urinariae Herba Piperis lolot Herba Pistiae Herba Pogostemonis Herba Portulacae Herba Sarcandrae glabrae Herba Scutellariae barbatae Herba Siegesbeckiae xxxv

LIST OF MONOGRAPHS

183. 184. 185. 186. 187. 188. 189. 190. 191. 192. 193. 194. 195. 196. 197. 198. 199. 200. 201. 202. 203. 204. 205. 206. 207. 208. 209. 210. 211. 212. 213. 214. 215. 216. 217. 218. 219. 220. 221. 222. 223. 224. 225. 226. 227. 228. 229. 230. 231. 232. 233. xxxvi

Herba Solani procumbensis Herba Spirodelae polyrrhizae Herba Wedeliae Hippocampus Lignum Dracaenae Lignum Sappan Medulla Junci effusi Medulla Tetrapanacis papyriferae Mel Myrrha Os Sepiae Pericarpium Arecae catechi Pericarpium Citri reticulatae perenne Pericarpium Citri reticulatae viride Pericarpium Garciniae mangostanae Periostracum Cicadae Pheretima Polyporus Poria Radix Abelmoschi sagittifolii Radix Achyranthis asperae Radix Achyranthis bidentatae Radix Aconiti Radix Aconiti lateralis Radix Angelicae acutilobae Radix Angelicae dahuricae Radix Angelicae pubescentis Radix Angelicae sinensis Radix Asparagi cochinchinensis Radix Astragali membranacei Radix Boehmeriae niveae Radix Bupleuri chinensis Radix Catharanthi rosei Radix Clerodendri japonici Radix Codonopsis Radix Codonopsis javanicae Radix Codonopsis javanicae praeparata Radix Dipsaci Radix et Rhizoma Asari Radix et Rhizoma Asteris tatarici Radix et rhizoma Clematidis Radix et Rhizoma Gentianae Radix et Rhizoma Ginseng Radix et Rhizoma Glycyrrhizae Radix et Rhizoma Salviae miltiorrhizae Radix Eurycomae longifoliae Radix Fallopiae multiflorae Radix Gentianae Radix Glehniae Radix Linderae Radix Millettiae speciosae

VP V

234. 235. 236. 237. 238. 239. 240. 241. 242. 243. 244. 245. 246. 247. 248. 249. 250. 251. 252. 253. 254. 255. 256. 257. 258. 259. 260. 261. 262. 263. 264. 265. 266. 267. 268. 269. 270. 271. 272. 273. 274. 275. 276. 277. 278. 279. 280. 281. 282. 283. 284.

Radix Morindae citrifoliae Radix Morindae officinalis Radix Nymphaeae stellatae Radix Ophiopogonis japonici Radix Paeoniae rubra Radix Paeoniae lactiflorae (Radix Paeoniae alba) Radix Panasis notoginseng Radix Peucedani Radix Phytolaccae Radix Platycodi grandiflori Radix Plucheae pteropodae Radix Polygalae Radix Polygoni cuspidati Radix Polysciacis Radix Puerariae thomsonii Radix Rehmanniae glutinosae Radix Rehmanniae glutinosae praeparata Radix Sanguisorbae Radix Saposhnikoviae divaricatae Radix Saussureae lappae Radix Scrophulariae Radix Scutellariae Radix Stemonae tuberosae Radix Stephaniae tetrandrae Radix Streptocauli Ramulus Cinnamomi Ramulus cum folio Melaleucae Ramulus cum Unco Uncariae Ramulus Mori albae Rhizoma Acori Rhizoma Alismatis Rhizoma Alpiniae officinari Rhizoma Anemarrhenae Rhizoma Atractylodis Rhizoma Atractylodis macrocephalae Rhizoma Belamcandae chinensis Rhizoma Bletillae striatae Rhizoma Cibotii Rhizoma Cimicifugae Rhizoma Coptidis Rhizoma Curculiginis Rhizoma Curcumae longae Rhizoma Curcumae zedoariae Rhizoma Cyperi Rhizoma Dioscoreae Rhizoma Dioscoreae collettii Rhizoma Drynariae Rhizoma et Radix Notopterygii Rhizoma et Radix Panacis vietnamensis Rhizoma Gastrodiae elatae Rhizoma Homalomenae occultae

VP V

285. 286. 287. 288. 289. 290. 291. 292. 293. 294. 295. 296. 297. 298. 299. 300. 301. 302. 303. 304. 305. 306. 307. 308. 309. 310. 311. 312. 313. 314. 315. 316. 317. 318. 319. 320. 321. 322. 323. 324. 325. 326. 327. 328. 329. 330.

LIST OF MONOGRAPHS

Rhizoma Imperatae cylindricae Rhizoma Kaempferiae galangae Rhizoma Ligustici wallichii Rhizoma Pinelliae Rhizoma Polygonati Rhizoma Polygonati odorati Rhizoma Rhei Rhizoma Smilacis glabrae Rhizoma Thalictri foliolosi Rhizoma Typhonii trilobati Rhizoma Zingiberis Sargassum Scolopendra Scorpio Semen Lablab Semen Arecae catechi Semen Armeniacae amarum Semen Coicis Semen Cuscutae Semen Euryales Semen Momordicae cochinchinensis Semen Myristicae Semen Nelumbinis nuciferae Semen Pharbitidis Semen Plantaginis Semen Platycladi orientalis Semen Pruni Semen Quisqualis Semen Raphani sativi Semen Sennae torae Semen Sesami Nigrum Semen Sinapis albae Semen Strychni Semen Trichosanthis Semen Vignae cylindricae Semen Vignae radiatae Semen Ziziphi mauritianae Spica Prunellae Spina Gleditsiae australis Squama Manis Styli et stigmata Maydis Talcum Thallus Laminariae Tuber Corydalis Tuber Dioscoreae persimilis Tuber Stephaniae.

4. Extracts, oils and volatile oils 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Aetheroleum Anisi stellati Aetheroleum Cajuputi Aetheroleum Cinnamomi Aetheroleum Cinnamomi camphorae Aetheroleum Curcumae Aetheroleum Eucalypti Aetheroleum Menthae arvensis Aetheroleum Ocimi gratissimi Aetheroleum Plectranthi amboinici Aetheroleum Zingiberis Extractum Ampelopsis siccusum Extractum Cynarae spissum Extractum Dracaenae siccusum Extractum Folii Ginkgo siccusum Extractum Leonuri japonici spissum Extractum Phyllanthi amari spissum Extractum Polysciacis fruticosae spissum Oleum Calophylli inophylli Oleum Momordicae

5. Traditional medicines 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

An Thai pills Bat Tran pills Bat Vi pills Binh Vi powder Bo Phoi extract Bo Trung Ich Khi pills Doc Hoat Ky Sinh mixture for decoction Hoac Huong Chinh Khi liquid extract Hy Thiem extract Ich Mau extract Luc Vi pills Minh Muc Dia Hoang pills Ngan Kieu Giai Doc pills Nhi Tran pills Ninh Khon pills Phi Nhi pills Qui Ty pills Sam Nhung Bo Than pills Tang Cuc extract Thap Toan Dai Bo pills Thien Vuong Bo Tam pills Tieu Dao pills Tu Nghich extract

xxxvii

xxxviii

VP V

ADDITIONS

ADDITIONS 1. Chemico-pharmaceutical substances 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45.

Abacavir sulfate Acebutolol hydrochloride Acenocoumarol Ambroxol hydrochloride Aminocaproic acid Amiodarone hydrochloride Amodiaquine hydrochloride Amoxicillin sodium Arginine Arginine aspartate Arginine hydrochloride Atorvastatin calcium trihydrate Attapulgite Bethamethasone sodium phosphate Bisoprolol fumarate Bupivacaine hydrochloride Calcitriol Carbidopa Carbomers Carmellose calcium Carmellose sodium Cefalotin sodium Cefamandole nafate Cefdinir Cefepime hydrochloride monohydrate Cefoperazone sodium Cefpodoxime proxetil Ceftazidime pentahydrate Celecoxib Cellulose acetate Cellulose, microcrystalline Cilastatin sodium Clopidogrel hydrosulfate Colchicine Diclofenac diethylamine Diltiazem hydrochloride Dimenhydrinate Efavirenz Emetine hydrochloride Esomeprazole magnesium trihydrate Ethylcellulose Felodipine Fexofenadine hydrochloride Gabapentin Glimepiride

46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88.

Glipizide Glutathione Heptaminol hydrochloride Histidine Histidine hydrochloride monohydrate Imipenem Imipramine hydrochloride Indapamide Indinavir sulfate Irbesartan Isoleucin Isosorbide dinitrate, diluted Isosorbide mononitrate, diluted Itraconazole Kanamycin sulfate Lansoprazole Levamisole hydrochloride Levodopa Levofloxacin Lopinavir Loratadine Losartan potassium Lovastatin Lumefantrine Lycine acetate Magnesium lactate dihydrate Methacrylic acid and ethyl acrylate copolymer (1:1) Methacrylic acid and ethyl acrylate copolymer (1:1), dispersion 30 per cent Methacrylic acid and methyl methacrylate copolymer (1:1) Methacrylic acid and methyl methacrylate copolymer (1:2) Methadone hydrochloride Methylcellulose Metoclopramide hydrochloride Naloxone hydrochloride Nevirapin, anhydrous Niclosamide, anhydrous Nifuroxazide Nitrazepam Oseltamivir phosphate Ouabain Oxacillin sodium monohydrate Oxytetracycline hydrochloride Pantoprazole sodium sesquihydrate xxxix

ADDITIONS

89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 122. 123.

Liquid paraffin Pefloxacin mesilate Penicillamine Perindopril tert-butylamine Piperacillin sodium Polymyxin B sulfate Pregelatinised Starch Propylen glycol Quinapril hydrochloride Ramipril Ritonavir Salbutamol sulfate Simvastatin Sodium Starch glycolate (type A) Sodium Starch glycolate (type B) Sodium Starch glycolate (type C) Sodium valproate Stavudine Sucralfate Sulbactam sodium Sulfasalazine Sultamicillin Sultamicillin tosilate dihydrate Tamoxifen citrate Telmisartan Terbutaline sulfate Thiamphenicol Ticarcillin sodium Tramadol hydrochloride Tranexamic acid Triamcinolone acetonide Triglycerides, medium-chain Trimetazidine hydrochloride Verapamil hydrochloride Vinpocetine

2. Formulated preparations 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

xl

Acebutolol tablets Acenocoumarol tablets Acetylsalicylic acid, gastro-resistant tablets Alimemazine syrup Ambroxol hydrochloride capsules Ambroxol hydrochloride tablets Amikacin injection Amiodarone tablets Amitriptyline tablets Amlodipine tablets Amodiaquine hydrochloride tablets Amoxicillin and clavulanic acid for injection Amoxicillin and clavulanic acid powder for oral suspension

VP V

14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62.

Amoxicillin and cloxacillin capsules Amoxicillin powder for oral suspension Amphotericin lozenges Ampicillin and sulbactam powder for injection Arginine capsules Artemether and lumefantrin tablets Aspirin and caffeine tablets Atorvastatin tablets Betamethasone eye drops Boric acid solution (3%) Calcitriol soft capsules Calcium gluconate effervescent tablets Cefaclor powder for oral suspension Cefdinir capsules Cefdinir for oral suspension Cefixime capsules Cefixime powder for oral suspension Cefoperazone and sulbactam for injection Cefoperazone for injection Cefpodoxime capsules Cefpodoxime powder for oral suspension Cefpodoxime tablets Ceftazidime for injection Cefuroxime for injection Cefuroxime powder for oral suspension Chloramphenicol and dexamethasone sodium phosphat cream Chloramphenicol and dexamethasone sodium phosphat eye drops Chloramphenicol ear drops Clofazimine capsules Clopidogrel tablets Codeine phosphate tablets Colchicine tablets Colecalciferol tablets Cortisone tablets Dapsone tablets Diazepam injection Diltiazem tablets Dimenhydrinate tablets Dimercaprol injection Diphenhydramine oral solution Diphenhydramine tablets Efavirenz capsules Ergocalciferol tablets Esomeprazole, gastro-resistant capsules Esomeprazole, gastro-resistant tablets Ethinylestradiol tablets Fenofibrate capsules Fexofenadine tablets Flucloxacillin capsules

VP V

63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113.

ADDITIONS

Gabapentin capsules Gabapentin tablets Gelatin, hard capsule shells Glibenclamide tablets Glibenclamide and metformin tablets Glimepiride tablets Glimepiride and metformin tablets Glipizide tablets Glipizide and metformin tablets Glyceryl trinitrate tablets Heptaminol tablets Hyoscine butylbromide tablets Imipenem and cilastatin for injection Imipramine tablets Indapamide tablets Indinavir capsules Iodine solution (1%) Irbesartan tablets Isosorbide dinitrat tablets Isosorbide mononitrate tablets Itraconazole capsules Kanamycin injection Lamivudine and zidovudine tablets Lamivudine oral solution Lansoprazole, gastro-resistant capsules Levodopa and carbidopa tablets Levodopa tablets Levofloxacin tablets Loratadine tablets Losartan potassium tablets Lovastatin tablets Methadone, oral concentrate sulution Metoclopramide injection Metoclopramide tablets Metronidazole and spiramicin tablets Nevirapin tablets Nitrofurantoin tablets Oseltamivir capsules Oxytetracycline capsules Pantoprazole, gastro-resistant tablets Paracetamol and ibuprofen tablets Paracetamol intravenous infusion Pefloxacin mesylate tablets Perindopril tert-butylamine tablets Piperazine phosphate tablets Piracetam injection Promethazine hydrochloride cream Promethazine hydrochloride syrup Ramipril tablets Rifampicin, isoniazid and pyrazineamide tablets Simvastatin tablets

114. 115. 116. 117. 118. 119. 120. 121. 122. 123. 124. 125. 126. 127. 128.

Sodium valproate tablets Stavudine tablets Telmisartan tablets Terfenadine tablets Timolol tablets Tobramycin eye drops Tobramycin injection Tranexamic acid capsules Tranexamic acid tablets Triamcinolone acetonide cream Trimetazidine tablets Vancomycin powder for injection Vinpocetine tablets Zidovudine oral solution Zidovudine tablets

3. Immunological products 1. Human hepatitis B immunoglobulin 2. Diphtheria, tetanus and pertussis (acellular, component) vaccine (adsorbed) 3. Diphtheria - tetanus - pertussis - hepatitis B heamophilus influenza type B (DTwP - HeB - Hib) combined vaccine. 4. Haemophilus influenzae type B conjugate vaccine 5. Hepatitis A vaccine (live, attenuated) 6. Hepatitis A vaccine (inactivated, absorbed) 7. Hepatitis A vaccine (inactivated, virosome) 8. Hepatitis A (inactivated) and hepatitis B (rDNA) vaccine (adsorbed) 9. Influenza vaccine (inactivated) 10. Measles, mumps, rubella vaccine (MMR vaccine - live) 11. Meningococcal polysaccharide vaccine 12. Mumps vaccine 13. Human papillomavirus vaccine (recombination) 14. Pneumococcal polysaccharide conjugate vaccine (adsorbed) 15. Pneumococcal polysaccharide vaccine 16. Rotavirus vaccine – live attenuated, oral 17. Rubella vaccine 18. Varicella vaccine (live) 4. Materia medica 1. 2. 3. 4. 5. 6. 7. 8. 9.

Caulis et folium Gymnemae sylvestris Colla Cornus Cervi Cornu Cervi Cornu Cervi degelatinatum Cornu Cervi Pantotrichum Cortex Acanthopanacis gracilistyli Flos Cleistocalysis operculati Flos Sambuci javanicae Folium Ardisiae xli

ADDITIONS

10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47.

Folium Clerodendri chinense Folium Cratoxyli pruniflori Folium Crini asiatici Folium Crini latifolii Folium Ilexi kaushii Folium Lawsoniae Folium Maclurae cochinchinensis Folium Premnae corymbosae Folium Psidii guajavae Folium Solani erianthi Fructus Apii graveolens Fructus Gleditsiae australis Fructus Momordicae charantiae Fructus Silybi Ganoderma Herba Agerati conyzoides Herba Agrimoniae Herba Andrographii Herba Apii graveolens Herba Bidensis pilosae Herba Eleusinis indicae Herba Glini oppositifolii Herba Gynostemmae Herba Lobeliae chinensis Herba Sarcandrae glabrae Herba Scutellariae barbatae Herba Solani procumbensis Herba Spirodelae polyrrhizae Pericarpium Citri reticulatae viride Polyporus Radix Clerodendri japonici Radix Eurycomae longifoliae Rhizoma Dioscoreae collettii Semen Quisqualis Semen Sesami Nigrum Spina Gleditsiae australis Styli et stigmata Maydis Thallus Laminariae

5. Extracts, oils, volatile oils 1. Aetheroleum Curcumae 2. Aetheroleum Zingiberis 3. Extractum Ampelopsis siccusum 4. Extractum Dracaenae siccusum 5. Extractum Folii Ginkgo siccusum 6. Extractum Leonuri japonici spissum 7. Extractum Phyllanthi amari spissum 8. Extractum Polysciacis fruticosae spissum 9. Oleum Calophylli inophylli 10. Oleum Momordicae

xlii

VP V

6. Appendices APPENDIX 1 1.25 1.26 1.27

Preparation for irrigation General requirements for Probiotics Terms relating to drug release in formulated preparations

APPENDIX 4 4.5 4.6 4.7 4.8

Mass spectrometry Inductively coupled plasma-mass spectrometry (ICP-MS) X-ray fluorescence spectrometry Raman spectrometry

APPENDIX 6 6.12 6.13

Thermal analysis Bulk density and tapped density of powders

APPENDIX 10 10.20 Determination of antimicrobial agents 10.21 Determination of omega 3-acids in fish oil 10.22 Vitamin D assay APPENDIX 11 11.9 11.10

Uniformity of dosage units Determination of drug realease of transdermal systems

APPENDIX 12 12.21 Determination of aflatoxin B1 in herbal drugs 12.22 Test for aristolochic acids I in herbal drugs 12.23 Guideline for the gas chromatographic and high-performancce liquid chromatographic fingerprinting 12.24 Stomata and stomatal index APPENDIX 13 13.11

Vitamin B12 activity assay

APPENDIX 15 15.42 Determination of total polysaccharide content by orcinol method 15.43 Potency procedure (invivo) of recombinant hepatitis B vaccine 15.44 Immunologcal methods used in vaccine quality control 15.45 Determination of residual BSA in vaccine 15.46 Elisa techniques (enzyme linked immunosorbent assay methods, elisa methods) Solution instructions for the problems usually occurs in elisa test 15.47 Test for mycoplasas in vaccines/biologicals (Culture and indicator cell culture method)

VP V

ADDITIONS

APPENDIX 17 17.8 Containers for blood and blood components 17.9.1 Plastic additives 17.9.2 Materials for containers for human blood and blood components 17.9.3 Polyethylene 17.9.4 Polyethylene terephtalate for containers for preparations not for parenteral use 17.9.5 Poly(ethylen-vinyl acetate) for containers and tubing for total parenteral 17.9.6 Polyolefines 17.9.7 Polypropylene for containers and closures for parenteral preparations and closures for parenteral preparations and ophthalmic preparations APPENDIX 19 Alcoholimetric table

xliii

xliv

VP V

OMISSION

OMISSIONS Formulated preparations Artemisinin tablets Artemisinin capsules Artesunate tablets Immunological products Biosubtyl Hepatitis B vaccine, human plasma derived Appendices Appendix 15.30 Determination of the potency of rabies vaccine by Habel test

xlv

xlvi

VP V

GENERAL NOTICES

GENERAL NOTICES 1. In the Vietnamese version of the fìfth edition of Vietnamese Pharmacopoeia (abbreviated VP V), the Vietnamese title of a monograph is used as the official designation accompanied with Latin name and other commonly used names if available. For monographs of crude drugs, their titles are usually the names of their medicinal plants, animals, followed by words given in parentheses, for example (Leaf), (Fruit)... showing the plant part for medicinal use. In this English version, the titles of monographs are stated as follows: For the monographs of pharmaceutical chemicals and their formulated preparations, the title is stated in English accompanied with Latin name. For the monographs of crude drugs, the title is stated in Latin name wich is included the part (s) of plant or animal followed by the binomial nomenclature without author and accompanied with conventional Vietnamese names. For the monographs of oils, volatile oils, extracts, their title is stated in Latin name. For the monographs of traditional medicines, the title is stated in Vietnamese traditional name with formulated form translated into English and followed by the Vietnamese full name. 2. The relative atomic masses adopted in VP V are the values given in the Appendix 18. Names, symbols and atomic masses of elements. 3. The units of measurement used in VP V are defined according to the Law on Metrology promulgated on 11 November 2011 and Decree No 86/2012/ NĐ-CP dated 19 October 2012 prescribing in details the implementation of some articles in the Law on Metrology 4. The following abbreviations are used for the measurement units: Metre: m Second: s Decimetre: dm Kilopascal: kPa Centimetre: cm Pascal: Pa Millimetre: mm Pascal second: Pa·s Micrometre: μm Ampere: A Nanometre: nm Milliampere: mA Litre: l or L Volt: V Millilitre: ml or mL Milivolt: mV Microlitre: μl or μL Degree Celsious: °C Kilogram: kg Mole: mol Gram: g Mole per litre: mol/l, Mol/L, M Centigram: cg Becquerel: Bq Microgram: μg International Unit: IU Nanogram: ng Percent: % Hour: h Part per million: ppm Minute: min 5. Temperature in the VP V is expressed in degrees centigrade °C (degree Celsius), unless otherwise specified. 6. Standard temperature and ordinary temperature of laboratory (room temperature) are defined as 20 °C and 20 °C to 30 °C, respectively. Unless otherwise specified, all tests of the drugs shall be performed at ordinary temperature (20 °C to 30 °C) and observations of the results shall follow immediately after the operations. However, the consideration for a test affected by temperature should be given on the conditions at standard temperature (20 °C). The temperature in a test for “loss on drying”, where defined at a given temperature, implies a deviation of ±2 °C from the stated value (for example: 100 °C means that 100 °C ± 2 °C). 7. Water temperature: The temperature of a water bath is 98 °C to 100 °C, unless otherwise specified; the statement “in a water bath” means that the object is heated in a bath of boiling water, the statement “on a water bath” means that the object is heated with a steam of boiling water. Hot water: 70 °C to 80 °C. Warm water: 40 °C to 50 °C. Cold water: 2 °C to 10 °C. Ice water: 0 °C. xlvii

GENERAL NOTICES

VP V

8. Temperature for storage places: In a deep-freeze : below -10 °C. In a cold place: 2 oC to 10 oC. In a cool place: 10 oC to 20 oC. Room temperature: 20 °C to 30 °C (the temperature prevailing in a working area). Conditioned room temperature: 20 °C to 25 °C (the temperature is conditioned by an air conditioner). On a warm place: 35 °C to 40 °C. On an excessive heat place: above 40 °C. 9. Pressure is expressed in terms of kilopascal (kPa). 1 kPa = 7.5006 Torrs 1 Torr is the pressure of 1 mm Hg. The term “in vacuum” indicates, unless otherwise specified, a pressure not exceeding 2.0 kPa (15 mm Hg). 10. The term “weigh accurately” means to weigh down to the degree of 0.1 mg, 0.01 mg or 0.001 mg according to the sensitivity in the balance to be used, provided that weighing error does not exceed 0.1%. Quantities are weighed with an accuracy commensurate with the indicated precision. The precision corresponds to plus or minus 5 units after the last figure stated; for example, 0.25 g is to be interpreted as 0.245 g to 0.255 g. The term “weigh” means that the weighing is carried out with an error smaller than 1%. The term “weigh about” indicates that the weighed quantity should not exceed ± 10% of the specified quantity. The terms “drying to constant mass” and “ignition to constant mass” mean that two consecutive weighings do not differ by more than 0.5 mg. The second weighing is carried out following an additional period of drying or ignition respectively according to the nature and quantity of the residue (1 hour is usually suitable). The term “previously weighed” (concerning devices such as crucibles, flasks, capsules, ...) means that these devices have been processed to constant mass. In monographs, if it is specified to weigh a residue or precipitate (dried, ignited, evaporated) in such a device that means the device has been dried or ignited to constant mass. The term “negligible residue” or “unweighable residue” means that the residue mass does not exceed 0.5 mg. 11. For the measurement of volumes, if the figure after the decimal point of volume is a zero or ends in a zero (for example, 10.0 ml or 0.50 ml), the volume should be measured accurately. The term “measure accurately” a solution or liquid volume means to measure the volume being measured with a standard pipette, volumetric flash or burette being used for the requirement of required volume. “Measure” indicates that the use of a graduated measuring cylinder, graduated pipette or other measuring apparatus suitable for measurement of the volume. To measure the number of drops, a standard dropping device that delivers 20 drops of purified water weighing 0.90 - 1.10 g at 20 °C shall be used. 12. “Water” used in the analytical procedure or for the preparation of reageant refers to purified water, except some test requires water of a higher quality (e.g bacterial endotoxins or microbial contamination). 13. Where the name of the solvent is not stated, the term "solution" implies a solution in water. 14. The terms “immediately” and “at once” used in a test mean that the operation is to be performed within 30 seconds after the preceding operation. 15. Identification is the test necessary to identify the drug or the main ingredients of the drug based upon a specific physical or chemical character. The identification of a crude drug or a traditional medicine is based on its macroscopical and microscopical descriptions and other physic-chemical characteristic tests. It is usually assumed that infrared absorption spectrophotometry is a fully accurate method for identification, because there is only one given fingerprint region for a drug substance to be detected by spectrum. The characteristics of infrared spectra can be used as the test of first identification. The infrared spectra test usually provides by itself the verification of the article identity and needs no other tests. Nevertheless, when the drug is a salt it is necessary to apply an additional test of “specific ion” for identification. The followed identification tests in each monograph are to check the previous identification by infrared spectrophotometry. When necessary, carry out an additional determination of the drug melting point; if the prescribed temperature is not reproduced, an approximate value can be used with the limit of errors prescribed in the individual monograph. 16. Purity tests are those collected for detecting contaminants in drugs, and it, as well as other requirements in each monograph, specifies the purity of the drug usually by limiting the kind and quantity of the contaminants. The contaminants which are considered to be the subject of the test are those supposed to contaminate the drug during the course of the manufacturing process or storage, and hazardous contaminants such as heavy metals, arsenic, etc... The contaminants to be detected in the Vietnamese Pharmacopoeia are only those being commonly seen. This does not imply the acceptance of other contaminants contained in the drug though they are not involved in the requirement of the pharmacopoeia. If xlviii

VP V

GENERAL NOTICES

substitution by foreign substances or addition of such materials is expected, the corresponding tests are necessary. The concentration of impurity is given either in parts per million by mass (ppm) or as a percentage where the limit exceeds 500 ppm. The figures are approximate only in conformity with the requirements determined on the basis of compliance with the stated test. 17. In antibiotic monographs where the assay specifies concurrently two methods, namely, microbiological method and another method, both are applicable. The method of microbiological assay is considered to be the suggested method, which is optional; the other method of assay can be employed but its precision must be not less than that required for the microbiological method. If the results obtained from both methods deviate widely, only the result obtained from the microbiological assay is conclusive (unless otherwise specified). 18. The test methods of the Vietnamese Pharmacopoeia can be replaced by alternative methods that give better accuracy and precision. However, where a difference is suspected, only the result obtained by the procedure given in this Pharmacopoeia is effective for the final judgment. 19. In monographs where the determination is to be carried out using a standard or a reference substance for comparison, it will have to use those issued under the direction of the Ministry of Health. 20. In assays and tests (unless otherwise specified) where the substance being examined is specified to be compared with a blank determination, the determination is to be treated in the same manner as the substance being examined, but omitting the substance under test or assay and to be done concurrently under the same conditions as the substance being examined. 21. The results obtained from assay should be calculated to one decimal place more than the significant figures stated and then rounded up or down as follows: If the last figure calculated is 5 to 9, the preceding figure is increased by 1. If it is 4 or less, the preceding figure is left unchanged. The results obtained from other tests, for example, from standardizing volumetric solutions, should be calculated in the same manner. For example: 8.2758 is rounded to 8.276. 1.2634 is rounded to 1.263. 22. Standards for content: Where the standard for the content of a substance in a monograph is expressed in terms of the chemical formula for that substance an upper limit exceeding 100% may be stated. Such an upper limit applies to the result of the assay calculated in terms of the equivalent content of the specified chemical formula. For example, the statement contains not less than 98.5% and not more than 102.0% of C12H22CaO14,H2O implies that the result of the assay is not less than 98.5% and not more than 102.0%, calculated in terms of the equivalent content of C12H22CaO14,H2O. In individual monographs where the upper limit is not stated, the upper limit is implied not more than 101.0%. 23. In monographs, in the section under the heading Description or Characters, the term “white” is used to indicate white or practically white; “colourless” denotes colourless or practically colourless; “odourless” indicates odourless or practically odourless. Unless otherwise specified, the tests are carried out as follows: a) Colour: Solid drug: Place 1 g of the solid drug on a sheet of white paper or in a watch glass placed on white paper for observation. Liquid drug: The liquid drug is put into a colourless test tube of 15 mm inside diameter and is horizontally observed in front of a white background by a distance of 30 mm from the tube, in daylight. b) Odour: Solid drug: Spread 0.5 - 2.0 g of the solid drug to form a thin layer onto a watch glass of 6 - 8 cm diameter, discern the odour by smelling after 15 minutes. Liquid drug: Place 2 ml of the liquid drug onto a watch glass as mentioned above, discern the odour by smelling. 24. In the expression of concentrations of solution, unless otherwise specified, the concentration of a solution is expressed as percentage (%) mass in volume (m/v), expressing the number of grams of solute in 100 millilitres of solution, symbolized by % (m/v) or %. Other symbols are used for the expression of other circumstances: Percentage mass in mass % (m/m) expresses the number of grams of solute in 100 grams of solution. Percentage volume in volume % (v/v) expresses the number of millilitres of solute in 100 millilitres of solution. Percentage volume in mass % (v/m) expresses the number of millilitres of solute in 100 grams of solution. 25. The term “ethanol” without qualification means absolute ethanol. The term “alcohol” without qualification means ethanol 96 (% v/v) of ethanol (C2H6O). Other dilutions of ethanol are indicated by the term “ethanol” followed by xlix

GENERAL NOTICES

VP V

a statement of the percentage v/v or m/m of ethanol (C2H6O) required. If percentage without v/v or m/m it means percentage by volume, for example, ethanol (70%) means ethanol (70%v/v). 26. The term “ether” means diethyl ether. 27. The solvent mixtures are usually expressed as (10 : 1) or (50 : 9 : 1), etc... indicate the ratio by volume of the solvents in the mixture, respectively. For example: Chloroform - methanol - ammonia (50 : 9 : 1) denotes mixing in turn 50 ml of chloroform, 9 ml of methanol and 1 ml of ammonia to form a mixture. In individual monographs, names of those liquids in a solvent mixture are in italics and not followed by letter R. 28. In monographs, the names of chemicals, reagents, test solutions, indicators, volumetric solutions, standard (reference) solutions, buffer solutions are all represented in italics and followed by letter R if those are present in Appendix 2.1 (General reagents), Appendix 2.3 (Buffer solutions) and Appendix 2.4 (Standard solutions), or by letter VS if those are present in the Appendix 2.2 (Volumetric solutions). 29. Definitions of solubility are given as follows: Solubility means that the substance being examined (previously powdered in case of a solid) is dissolved in a solvent to form a clear, homogeneous solution in which no particle is left. The quantity of solvent is added to the substance being examined to dissolve within 30 minutes at the temperature of 25 °C ± 2 °C, by shaking for 30 seconds each time at 5 minutes intervals. The term “parts” indicates the number of millilitres of solvent to dissolve 1 gram or 1 millilitre of the substance being examined. The following table indicates the meanings of the terms used in statements of approximate solubilities.

Descriptive term very soluble freely soluble soluble sparingly soluble slightly soluble very slightly soluble practically insoluble

Approximate volume of solvent in millilitres per gram of solute less than 1 from 1 to 10 from 10 to 30 from 30 to 100 from 100 to 1000 from 1000 to 10 000 more than 10 000

30. The acidity or alkalinity of a solution, unless otherwise specified, is determined by litmus paper. To indicate these properties more accurately, its pH values should be measured by pH meter. 31. Desiccator: The term “desiccator” means a tightly-closed container of suitable size in which an atmosphere of low moisture is maintained by means of indicator silica gel or other suitable desiccant. Desiccator in vacuum is a desiccator treated by means of a suitable desiccant and in a pressure not exceeding 2.0 kPa (15 mm Hg), unless otherwise specified. 32. The botanical or zoological nomenclature of a medicinal plant or animal includes its name, family name and the part used for pharmaceutical purposes given following the monograph title. 33. The part used for pharmaceutical purposes refers to the commercially available crude drug, which is free from extraneous matters. The collection of crude drug on the spot treatment involve that particular part only. 34. The specification of a crude drug is described on the basis of the dried substance in general. The quality standard of fresh material is also specified if it is to be used in fresh. 35. The description of a crude drug refers to the part intended for medicinal use only, not to the original plant or animal. 36. The drying of crude drugs, on-the-spot or during their collection, is described as follows: When only the word “dry’’ is used, it means that drying can be done either by drying at a high temperature, baking, exposure to sunlight or standing in the shade (drying in the air). The term “dry in the sun” or “dry at a low temperature (not exceeding 60 °C)” is used for crude drugs, which are undesirable to be dried at a high temperature. The term “dry in the shade” or “dry in the air” is used for those unsuitable to be dried by baking or by exposure to sunlight. In several cases, the term “dry fast in strong sunlight” or “ dry time” is used for those preferable to be dried within a short time. The statement “calculated with reference to the dried drug” means the weight of crude drug is adjusted by subtracting the amount of water content which is determined by test for Loss on drying or by distillation method. l

VP V

GENERAL NOTICES

37. The microscopical examination of the transverse section and powder of a crude drug are the characteristics of the prepared sample observed under a microscope. 38. Crude drugs, drug substances, excipients and additives used in a preparation must comply with the requirements of this Pharmacopoeia. For those not stated in this Pharmacopoeia, the specifications stipulated by the Ministry of Health or the health authorities of provinces, autonomous regions and municipalities must be complied with. The excipients and additives should not harm the safety and reduce the efficacy of the medicines. Care should be taken to avoid the interference on the analytical procedures specified in the monographs of the Pharmacopoeia. 39. Crude drugs used for the manufacture of preparations (traditional medicines) should comply with the specifications stated in the Pharmacpoeia. If processing is required, they must be processed with the method specified in the monographs of the Pharmacopoeia, unless otherwise indicated. 40. The quantity specified for each ingredient of a preparation refers to the quantity of pulverized clean crude drug or its processed product. 41. Crude drugs for oral use are usually administered in the form of a decoction, unless otherwise indicated. 42. The dose specified in the Pharmacopoeia is the usual dose for adults. Appropriate adjustment may be necessary depending on the condition of individual patients. The maximum dose of a drug refers to the highest tolerable dose for adults. Overdose is prohibited, except special cases. 43. Water being used for the processing of traditional medicines refers to clean, drinking water in compliance with the medical hygienic standard. 44. Wine being used for the processing of crude drugs, traditional medicines refers to liquors made from rice, corn, cassava… containing about 30% to 40% of ethanol, unless otherwise indicated in the individual monograph. 45. Basic requirements for the preservation of a drug are stated under the heading “Storage”. The container is the device that holds drug. The requirement of a container also includes requirement of its constituent parts such as the stopper or cap. Containers should be tight and do not affect the drug quality held inside as well as protect it from the effect of the outside environment. The temperature of storage place should comply with the required temperature for storage. Statement “Protected from light” means that the drug should be kept in an amber coloured glass container, a glass container wrapped with black paper or any light-resistant container. Statement “tightly closed” means that the container should be able to protect the contents from efflorescence, deliquescence, volatilization or interference of extraneous matters. Statement “Well closed” means that the container is able to protect the contents from the invasion of soil, dust and extraneous matters. Statement “Hermetically sealed” means that the container should be impervious to air, gas and able to protect the contents from moisture and microorganisms. 46. Labelling must comply with the current regulations.

ABBREVIATIONS VP V M. R RS VS BCG Lf/mg PN Lf/mg N Kf Lf L+ L+/10

The fifth edition of Vietnamese Pharmacopoeia Molecular mass Reagent Reference substance Volumetric solution Bacillus Calmette-Guérin Flocculation units per milligram of protein nitrogen Flocculation units per milligram of total nitrogen Time for flocculation (in minutes) The quantity of toxin or toxoid that, when mixed with 1 IU of antitoxin flocculates in the shortest time The smallest quantity of a toxin that, when mixed with 1 IU of antitoxin causes the death of an animal with given mass within 4 days (the L+ dose varies depending on the kind of test animals) The smallest quantity of a toxin that, when mixed with 0.1 IU of antitoxin causes the death of a test animal with given mass within 4 days. li

GENERAL NOTICES

Lr LD50 MLD ED50 ABV CPE MEM FCS CCID50 PSS EU Water BET Me Et Pri Prn Bui Bus Bun But Ph Ac

lii

VP V

The smallest quantity of a toxin that, when mixed with a fixed quantity of antitoxin (usually 0.002 IU of antitoxin) in 0.2 ml causes a local skin reaction at the site of injection that is just visible (only for leukocyte). The quantity of toxin that causes the death of 50 per cent of a group of test animals within 4 days (LD 50 varies depending on the kind of test animals). Minimum lethal dose is the quantity of toxin that causes the death of test animals within 4 days (MLD varies depending on the kind of test animals). MLD is generally replaced by LD50. The quantity of a vaccine that may be expected to induce specific antibodies in 50 per cent of the animals against a challenge dose of the toxic micro-organisms or toxins. Antitoxin Binding Value. It is the defined value of toxin to add to antitoxin to make a mixture (determined in the test animals). Cytopathic effect Minimum Essential Medium Foetal Calf Sera The quantity of virus that may be expected to infect 50 per cent of the cell cultures to which it is added. Physiological Saline Solution Endotoxin Unit Water for bacterial endotoxin test - CH3 (methyl) - CH2CH3 (ethyl) - CH (CH3)2 (iso-propyl) - CH2 CH2CH3 (n-propyl) - CH2 CH (CH3)2 (iso-butyl) - CH (CH3) CH2 CH3 (sec-butyl) - CH2CH2CH2CH3 (n-butyl) - C(CH3)3 (tert-butyl) - C6H5 (phenyl) - COCH3 (acetyl)

Monographs CHEMICO - PHARMACEUTICAL SUBSTANCES AND FORMULATED PREPARATIONS

1

2

VP V

ABACAVIR SULFATE Abacaviri sulfas

ABACAVIR SULFATE

Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with solution B. Dilute 1.0 ml of this solution to 10.0 ml with solution B. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase amylose derivative of silica gel for chiral separation R (10 µm). Column temperature: 30 °C. Detector: A spectrophotometer set at 286 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions:

Time Mobile phase A Mobile phase B C28H38N12O6S   M. 671 (min) (% v/v) (% v/v) 0 - 25 100 0 Abacavirsulfateisbis[[(1S,4R)-4-[2-amino-6-(cyclopropylamino)9H-purin-9-yl]cyclopent-2-enyl]methanol] sulfate. It contains 25 - 27 100 → 0 0 → 100 not less than 99.0% and not more than 101.0% of C28H38N12O6S, 27 - 37 0 100 calculated with reference to the anhydrous substance. Identification of impurities: Use the chromatogram supplied with abacavir for system suitability RS and the Characters chromatogram obtained with reference solution (1) to White or almost white powder. Soluble in water, practically insoluble in ethanol (96%) identify the peaks due to impurities A and D. Relative retention with reference to abacavir (retention time and methylene chloride. = about 17 min): Impurity D = about 0.8; impurity A = about 0.9. System suitability: In the chromatogram obtained with Identification reference solution (1), the resolution between the peaks due to Apply one of the two following identifications: impurity D and impurity A is at least 1.5; the resolution between First identification: A, B and D. the peaks due to impurity A and abacavir is at least 1.5. Second identification: A, C and D. A. The infrared absorption spectrum (Appendix 4.2) of the Limits: In the chromatogram obtained with the test solution: substance to be examined is concordant with the spectrum The area of the peak due to impurity A is not more than 3 times the area of the principal peak in the chromatogram of abacavir sulfate RS. B. Specific optical rotation: -58.0° to -54.0° (Appendix 6.4). obtained with reference solution (2) (0.3%). Note: Determined on solution S. Solution S: Dissolve 0.250 g of the substance to be examined Impurity A: [(1R,4S)-4-[2-amino-6-(cyclopropylamino)-9Hpurin-9-yl]cyclopent-2-enyl]methanol. in water and dilute to 25.0 ml with the same solvent. Impurity D: [(1R,4R)-4-[2-amino-6-(cyclopropylamino)-9HC. It complies with the test for Enantiomeric purity. D. Solution S gives reaction (A) of sulfates (Appendix 8.1). purin-9-yl]cyclopent-2-enyl]methanol.

Enantiomeric purity Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Diethylamine - 2-propanol - heptane (0.1 : 15 : 85). Mobile phase B: Heptane - 2-propanol (50 : 50). Solution A: Trifluoroacetic acid - methanol (0.5 : 100). Solution B: Methanol - 2-propanol - heptane (30 : 30 : 40). Test solution: Dissolve 40 mg of the substance to be examined in 30 ml of solution A, sonicate until dissolution is complete, add 30 ml of 2-propanol R and dilute to 100.0 ml with heptane R. Reference solution (1): Dissolve 2 mg of abacavir for system suitability RS (containing impurities A and D) in 1.5 ml of solution A. Sonicate until dissolution is complete, add 1.5 ml of 2-propanol R and dilute to 5.0 ml with heptane R.

Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use and transfer them to brown, neutral glass vials. Mobile phase A: Dilute 0.5 ml of trifluoroacetic acid R in 1000 ml of water. Mobile phase B: Water - methanol (15 : 85). Test solution: Dissolve 25 mg of the substance to be examined in water and dilute to 100.0 ml with the same solvent. Sonicate until dissolution is complete. Reference solution (1): Dissolve 2.5 mg of abacavir for peak identification RS (containing impurities B and D) in 10.0 ml of water. Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with water. Dilute 1.0 ml of this solution to 10.0 ml with water. 3

VP V

ACEBUTOLOL HYDROCHLORIDE

Chromatographic system: A column (15 cm × 3.9 mm) packed with stationary phase endcapped octadecylsilyl silica gel for chromatography R (5 µm). Column temperature: 30 °C. Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0-5

95

5

5 - 25

95 → 70

5 → 30

25 - 40

70 → 10

30 → 90

Identification of impurities: Use the chromatogram supplied with abacavir for peak identification RS and the chromatogram obtained with reference solution (1) to identify the peaks due to impurities B and D. Relative retention with reference to abacavir (retention time = about 22 min): Impurity D = about 1.04; impurity B = about 1.3. System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to abacavir and impurity D is at least 1.5. Limits: In the chromatogram obtained with the test solution: The area of the peak due to impurity B is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (2) (0.2%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.10%). The sum of the peak areas of all impurities is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.5%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%).

Note: Impurity B: 6-(cyclopropylamino)-9-[(1R,4S)-4-[[(2,5-diamino6-chloropyrimidin-4-yl)oxy]methyl]cyclopent-2-enyl]-9Hpurine-2-amine.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 12 ml of solution S with limit test for heavy metals, method 1. Prepare the standard using 2 ml of lead standard solution (1 ppm Pb) R. Water Not more than 0.5% (Appendix 10.3). Determined on 60.0 mg. 4

Sulfated ash Not more than 0.2% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.300 g of the substance to be examined in 50 ml of water. Titrate with 0.1 N sodium hydroxide VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N sodium hydroxide VS is equivalent to 33.54 mg of C28H38N12O6S. Storage Store in an airtight container, protected from light. Action and use Antiviral (HIV). Preparations Tablets, oral solution. ACEBUTOLOL HYDROCHLORIDE Acebutolol hydrochloridum

C18H28N2O4,HCl

M: 372.89

Acebutolol hydrochloride is N-[3-acetyl-4-[(2RS)-2hydroxy-3-[(1-methylethyl)amino]propoxy]phenyl] butanamide hydrochloride. It contains not less than 99.0% and not more than 101.0% of C18H29ClN2O4, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder. Freely soluble in water and in ethanol (96%), very slightly soluble in acetone and in methylene chloride. Melting point: About 143 °C. Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of acebutolol hydrochloride RS. Examine the substances prepared as discs. B. Dissolve 20.0 mg of the substance to be examined in a 0.1% v/v solution of hydrochloric acid R and dilute to 100.0 ml with the same solution. Dilute 5.0 ml of this solution to 100.0 ml with a 0.1% v/v solution of hydrochloric acid R. The ultraviolet absorptiom spectrum (Appendix 4.1) of

VP V

this solution, in the range between 220 nm and 350 nm, exhibits two absorption maxima at 233 nm and at 322 nm. Specific absorbance at 233 nm is 555 to 605. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Perchloric acid - methanol - water (5 : 395 : 600). Test solution: Dissolve 20 mg of the substance to be examined in methanol R and dilute to 20 ml with the same solvent. Reference solution (1): Dissolve 20 mg of acebutolol hydrochloride RS in methanol R and dilute to 20 ml with the same solvent. Reference solution (2): Dissolve 20 mg of pindolol RS in methanol R and dilute to 20 ml with the same solvent. Add 1 ml of this solution to 1 ml of reference solution (1). Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 3/4 of the plate. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) shows two clearly separated spots. D. It gives reaction (A) of chlorides (Appendix 8.1).

Appearance of solution Dissolve 0.5 g in water and dilute to 10 ml with the same solvent. The solution is not more opalescent than reference suspension II (Appendix 9.2) and not more intensely coloured than reference solution BY5 (Appendix 9.3, method 2). pH Dissolve 0.20 g in carbon dioxide-free water and dilute to 20 ml with the same solvent. pH of the resulting solution is from 5.0 to 7.0 (Appendix 6.2). Related substances Examine by liquid chromatography (Appendix 5.3) Mobile phase A: Mix 2.0 ml of phosphoric acid R, and 3.0 ml of triethylamine R and dilute to 1000 ml with water. Mobile phase B: Acetonitrile - mobile phase A (50 : 50). Test solution: Dissolve 0.100 g of the substance to be examined in mobile phase A and dilute to 50.0 ml with the same solution. Reference solution (1): Dissolve 20.0 mg of the substance to be examined in mobile phase A and dilute to 100.0 ml with the same solution. Dilute 0.5 ml of this solution to 50.0 ml with mobile phase A. Reference solution (2): Dissolve the contents of a vial of acebutolol impurity I RS in 1.0 ml of mobile phase A. Reference solution (3): Mix 2.0 ml of reference solution (1) and 1.0 ml of reference solution (2) and dilute to 10.0 ml with mobile phase A. Reference solution (4): Dissolve 5.0 mg of acebutolol impurity C RS in 10 ml of acetonitrile R and dilute to 25.0 ml with mobile phase A. Dilute 0.5 ml of this solution to 50.0 ml with mobile phase A.

ACEBUTOLOL HYDROCHLORIDE

Reference solution (5): Dissolve 5.0 mg of acebutolol impurity B RS in 10.0 ml of acetonitrile R and dilute to 25.0 ml with mobile phase A. Dilute 1.0 ml of this solution to 50.0 ml with mobile phase A. Chromatographic system: A column (12.5 cm × 4 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (5 µm). Column temperature: 40 °C. Detector: A spectrophotometer set at 240 nm. Flow rate: 1.2 ml/min. Volume of injection: 25 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0-2

98

2

2 - 30.5

98 → 10

2 → 90

30.5 - 41

10

90

System suitability: In the chromatogram obtained with reference solution (3), the resolution between the peaks due to impurity I and acebutolol is at least 7.0. Limits: Impurity B: The area of the peak due to impurity B is not more than the area of the principal peak in the chromatogram obtained with reference solution (5) (0.2%). Impurity C: The area of the peak due to impurity C is not more than the area of the principal peak in the chromatogram obtained with reference solution (4) (0.1%). Impurity I: The area of the peak due to impurity I is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%). The sum of the peak areas of all impurities is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%).

Note: Impurity A: N-[3-acetyl-4-[(2RS)-oxiran-2-ylmethoxy]phenyl] butanamide. Impurity B: N-[3-acetyl-4-[(2RS)-2-hydroxy-3-[(1-methylethyl) amino]propoxy]phenyl]acetamide (diacetolol). Impurity C: N-(3-acetyl-4-hydroxyphenyl)butanamide. Impurity D: 1-[5-amino-2-[(2RS)-2-hydroxy-3-[(1-methylethyl) amino]propoxy]phenyl]ethanone. Impurity E: N-[4-[(2RS)-2-hydroxy-3-[(1-methylethyl)amino] propoxy]phenyl]butanamide.

5

ACEBUTOLOL TABLETS Impurity F: N-[3-acetyl-4-[(2RS)-2,3-dihydroxypropoxy]phenyl] butanamide. Impurity I: N-[3-acetyl-4-[(2RS)-3-(ethylamino)-2- hydroxypropoxy] phenyl]butanamide. Impurity J: N-[3-acetyl-4-[(2RS)-2-hydroxy-3-[(1- methylethyl) amino]propoxy]phenyl]propanamide. Impurity G: N,N’-[[(1-methylethyl)imino]bis[(2-hydroxypropane1,3-diyl)oxy(3-acetyl-1,4- phenylene)]] dibutanamide (biamine). Impurity H: N,N’-[(2-hydroxypropane-1,3-diyl)bis[oxy(3-acetyl-1,4phenylene)]]dibutanamide. Impurity K: N-[3-butanoyl-4-[(2RS)-2-hydroxy-3-[(1- methylethyl) amino]propoxy]phenyl]butanamide.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). Dissolve 0.50 g in 20.0 ml of water. The solution complies with limit test for heavy metals, method 5. Prepare the standard by diluting 10.0 ml of lead standard solution (1 ppm Pb) R to 20.0 ml with water. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C; 3 h). Sulfated ash Not more than 0.1% (Appendix 9.9). Determined on 1.0 g. Assay Dissolve 0.300 g of the substance to be examined in 50 ml of ethanol (96%) R and add 1 ml of 0.1 M hydrochloric acid R. Titrate with 0.1 N sodium hydroxide VS. Determine the end-point potentiometrically (Appendix 10.2). Read the volume of 0.1 N sodium hydroxide VS added between the two points of inflexion. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 37.29 mg of C18H29ClN2O4. Storage Protected from light. Action and use Beta-adrenoceptor antagonist. Preparations Tablets, capsules. ACEBUTOLOL TABLETS Tabellae acebutololi Acebutolol tablets contain acetobutolol hydrochloride. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements:

Content of acebutolol, C18H28N2O4, 95.0% to 105.0% of the stated amount. 6

VP V

Identification In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of the acebutolol peak in the chromatogram obtained with the reference solution. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of water. Rotation speed: 50 rpm. Time: 30 min. Procedure: Test solution: After the specified time, withdraw a sample of the medium, and filter. Dilute with water if necessary. Reference solution: Weigh accurately a quantity of acebutolol hydrochloride, dissolve in water to obtain a solution having a similar concentration of acebutolol hydrochloride to that expected in the test solution. Measure the absorbances of the reference solution and the test solution at 232 nm (Appendix 4.1). Calculate the dissolved content of acebutolol, C18H28N2O4, using the absorbances of the reference solution, the test solution and the declared content of C18H28N2O4 in acebutolol hydrochloride RS. Tolerance: Not less than 80% (Q) of the labeled amount of acebutolol, C18H28N2O4, is dissolved in 30 min.

Related substances Examine by thin layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Chloroform - dimethylformamide - glacial acetic acid (60 : 20 : 20). Test solution: Shake a quantity of powdered tablets containing about 0.4 g of acebutolol with 20 ml of a mixture of equal volumes of chloroform R and methanol R for 2 min, centrifuge and use the clear solution. Reference solution (1): Dilute 3 ml of the test solution to 100 ml with a mixture of equal volumes of chloroform R and methanol R. Further dilute 1 ml of the obtained solution to 10 ml with the same solvent. Reference solution (2): Dilute 1 ml of the test solution to 100 ml with a mixture of equal volumes of chloroform R and methanol R. Further dilute 1 ml of the obtained solution to 10 ml with the same solvent. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of about 15 cm. Remove the plate, allow it to dry in air. Examine under ultraviolet light (254 nm). In the chromatogram obtained with the test solution, any secondary spot is not more intense than the principal spot in the chromatogram obtained with reference solution (1) (0.3%) and not more than two such spots are more intense than the principal spot in the chromatogram obtained with reference solution (2) (0.1%). Disregard any spot remaining on the line of application. Assay Examine by liquid chromatography (Appendix 5.3). Buffer solution: Dissolve 2.4 g of sodium decanesulfonate R in 1000 ml water, adjust the pH to 3.5 with glacial acetic acid R.

VP V

ACENOCOUMAROL

Mobile phase: Methanol - buffer solution (60 : 40). Make adjustments if necessary. Reference solution: Weigh accurately a quantity of acebutolol hydrochloride RS and dissolve in methanol R to obtain a solution having a known concentration of 0.22 mg/ml (equivalent to about 0.2 mg/ml of acebutolol). Test solution: Weigh 20 tablets, finely powder. Weigh accurately a quantity of the powdered tablets containing about 200 mg of acebutolol. Tranfer into a 200-ml volumetric flask, add 180 ml of methanol R, shake for 30 min and dilute to volume with the same solvent, shake and filter. Dilute 5.0 ml of the filtrate to 25.0 ml with methanol R. Chromatographic system: A column (15 cm × 3.9 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution, in the chromatogram obtained, the symmetry factor of the principal peak is not more than 1.5. The relative standard deviation of the peak areas from six replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of acebutolol, C18H28N2O4, in the tablets using the peak areas in the chromatograms obtained with the reference solution, the test solution and the declared content of C18H28N2O4 in acebutolol hydrochloride RS.

Storage Store in a well-closed container, in a cool place, protected from light. Action and use Beta-adrenoceptor antagonist. Usual strength 200 mg, 400 mg. ACENOCOUMAROL Acenocoumarolum

C19H15NO6  

and enantiomer

M.353.3 

Acenocoumarol is (RS)-4-hydroxy-3-(1-p-nitrophenyl-3oxobutyl) coumarin. It contains not less than 98.5% and not more than 100.5% of C19H15NO6, calculated with reference to the dried substance.

Characters Polymorphism powder, almost white to dark yellow. Practically insoluble in water and in ether, slightly soluble in ethanol (96%). It dissolves in aqueous solutions of the alkali hydroxides. Identification The infrared absorption spectrophotometry (Appendix 4.2) of the substance to be examined is concordant with the reference spectrum of acenocoumarol RS. If the spectra are not concordant, dissolve 0.1 g of the substance being examined in 10 ml of acetone R and add water dropwise until the solution becomes turbid. Heat on a water bath until the solution is clear and allow to stand. Filter, wash the crystals obtained with a mixture of equal volumes of acetone R and water. Dry at 100 °C at a pressure of 2 kPa for 30 min. Measure spectrum of the residue obtained. Appearance of solution A. A 2.0% solution of the substance to be examined in acetone R is clear (Appendix 9.2). B. The absorbance (Appendix 4.1) of a 4-cm layer of a 2.0% solution of the substance to be examined in acetone R at 460 nm is not more than 0.12. C. A 2.0% solution of the substance to be examined in 0.1 M sodium hydroxide R is clear (Appendix 9.2), and yellow. Light absorption Absorbance (Appendix 4.1) of a 0.001% solution of the substance to be examined in a mixture of 1 M hydrochloric acid solution and methanol (1 : 9) at the maximum at 306 nm, 0.50 to 0.54, calculated with reference to the dried substance. Related substances Not more than 0.1%. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Dichloromethane - cyclohexane - glacial acetic acid (50 : 50 : 20). Test solution: A 2.0% solution of the substance to be examined in acetone R. Reference solution: A 0.0020% solution of the substance to be examined in acetone R. Procedure: Apply separately to the plate 20 µl of each solution. Develop over a path of 15 cm. Remove the plate, allow it to dry in air and immediately examine under ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution . Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g, 105 °C). 7

VP V

ACENOCOUMAROL TABLETS

Sulphated ash Not more than 0.1% (Appendix 9.9, method 1). Determined on 1.0 g. Assay Dissolve 0.6 g of the substance to be examined in 50 ml of acetone R and titrate with 0.1 N sodium hydroxide VS using bromothymol blue solution R3 as indicator. Carry out a blank titration. Each ml of 0.1 N sodium hydroxide VS is equivalent to 35.33 mg of C19H15NO6. Action and use Anticoagulant. Preparation Tablets. ACENOCOUMAROL TABLETS Tabellae Acenocoumaroli Acenocoumarol tablets contain acenocoumarol. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements: Content of acenocoumarol, C19H15NO6, 92.5% to 107.5% of the stated amount.

Identification A. Heat a quantity of the powdered tablets containing 50 mg of acenocoumarol with 30 ml of acetone R under a reflux condenser for 5 min, filter and wash the residue with two 10 ml quantities of acetone R. Evaporate the combined filtrate and washings to 5 ml, add water drop wise until the solution becomes turbid, heat on a water bath until the solution is clear and allow to stand. Filter, wash the crystals with a mixture of equal volumes of acetone R and water and dry at 100 °C at a pressure of 2 kPa for 30 minutes. The infrared absorption spectrum (Appendix 4.2) of the residue, is concordant with the reference spectrum of acenocoumarol RS . B. The absorbance (Appendix 4.1) of the final solution obtained in the Assay exhibits maxima at 283 nm and 306 nm. C. Heat 25 mg of the residue obtained in test A with 2.5 ml of glacial acetic acid R, 0.5 ml of hydrochloric acid R and 0.2 g of zinc powder R on a water bath for 5 min, cool and filter. To the filtrate add 0.05 ml of a 10% solution of sodium nitrite R and add the mixture to 10 ml of a 1% solution of 2-naphthol R containing 3 ml of 5 M sodium hydroxide R. A bright red precipitate is produced. Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Glacial acetic acid - chloroform - cyclohexane (20 : 50 : 50). 8

Test solution: Shake a quantity of the powdered tablets containing 20 mg of acenocoumarol with 5 ml of acetone R, centrifuge and use the supernatant liquid. Reference solution: Dilute 1 volume of the test solution to 200 volumes with acetone R. Procedure: Apply separately to the plate 20 µl of each solution. Develop over a path of 15 cm. After removal of the plate, allow it to dry in air and examine under ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the principal spot in the chromatogram obtained with the reference solution (0.5%).

Uniformity of content The requirements for tablets containing 4 mg or less of acenocoumarol. Finely crush one tablet, add 30 ml of methanol R, stir the mixture for 30 minutes and filter through sintered glass, washing the residue with three 15 ml quantities of methanol R. To the combined filtrate and washings add 10 ml of 1 M hydrochloric acid R and sufficient methanol R to produce 100.0 ml. If necessary dilute further with a solvent prepared by diluting 1 volume of 1 M hydrochloric acid R to 10 volumes with methanol R to produce a solution containing about 0.001% of acenocoumarol. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum at 306 nm, in a 1-cm cell and use the mixture of 1 M hydrochloric acid R and methanol R (1 : 9) as a blank. Calculate the content of C19H15NO6, taking 521 as the value of A (1%, 1 cm) of acenocoumarol at the maximum at 306 nm. Assay Weigh 20 tablets and powder finely. Weigh accurately a quantity of the powdered tablets containing the equivalent of 1 mg acenocoumarol add 30 ml of methanol R, stir the mixture for 30 minutes and filter through sintered glass, washing the residue with three 15 ml quantities of methanol R. To the combined filtrate and washings add 10 ml of 1 M hydrochloric acid R and sufficient methanol R to produce 100.0 ml. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum at 306 nm, in a 1-cm cell and use the mixture of 1 M hydrochloric acid R and methanol R (1 : 9) as a blank. Calculate the content of C19H15NO6 taking 521 as the value of A (1%, 1 cm) of acenocoumarol at the maximum at 306 nm. Storage Store in a cool and dry place, protected from light. Action and use Anticoagulant - Vitamin K antagonist. Usual strength 4 mg.

VP V

ACETAZOLAMIDE

ACETAZOLAMIDE Acetazolamidum O H 2N

O H N

S

S N

C4H6N4O3S2  

N

CH3 O

M. 222.2

Acetazolamide is N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl) acetamide. It contains not less than 98.5% and not more than 101.0% of C4H6N4O3S2, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder. It shows polymorphism. Very slightly soluble in water, slightly soluble in ethanol (96%). It dissolves in dilute solutions of alkali hydroxides. Identification Apply one of the two following identifications: First identification: A, B. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of acetazolamide RS. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in ethanol (96%) R, evaporate to dryness and record new spectra using the residues prepared as discs. B. Dissolve 30.0 mg in 0.01 M sodium hydroxide R and dilute to 100.0 ml with the same solvent. Dilute 10.0 ml of the solution to 100.0 ml with 0.01 M sodium hydroxide R (solution A). The ultraviolet absorptiom spectrum (Appendix 4.1) of the solution A, in the range between 230 nm and 260 nm, exhibits absorption maximum at 240 nm. Specific absorbance at the absorption maximum is 162 to 176. Dilute 25.0 ml of solution A to 100.0 ml with 0.01 M sodium hydroxide R. The ultraviolet absorptiom spectrum (Appendix 4.1) of the solution, in the range between 260 nm and 350 nm, exhibits absorption maximum at 292 nm. Specific absorbance at the absorption maximum is 570 to 620. C. Introduce about 20 mg into a test-tube and add 4 ml of dilute hydrochloric acid R and 0.2 g of zinc powder R. Immediately place a piece of lead acetate paper R over the mouth of the tube. The paper shows a brownish-black colour. D. Dissolve about 25 mg in a mixture of 0.1 ml of dilute sodium hydroxide solution R and 5 ml of water. Add 0.1 ml of 10% solution of copper sulfate R. A greenish-blue precipitate is formed. Appearance of solution Dissolve 1.0 g in 10 ml of 1 M sodium hydroxide R. The solution is not more opalescent than reference suspension II (Appendix 9.2) and not more intensely coloured than reference solution Y5 or BY5 (Appendix 9.3, method 2).

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile for chromatography - 0.68% solution of potassium dihydrogen phosphate (10 : 90). Test solution: Dissolve 40 mg of the substance to be examined in the mobile phase and dilute to 100.0 ml with the mobile phase. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (2): Dissolve the contents of a vial of acetazolamide for system suitability RS (containing impurities A, B, C, D, E and F) in 1.0 ml of the mobile phase. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase end-capped propoxybenzene silica gel for chromatography R (4 µm). Detector: A spectrophotometer set at 265 nm. Flow rate: 1.0 ml/min. Volume of injection: 25 µl. Procedure: The run time is 3.5 times the retention time of acetazolamide. Relative retention with reference to acetazolamide (retention time = about 8 min): Impurity E = about 0.3; impurity D = about 0.4; impurity B = about 0.6; impurity C = about 1.4; impurity A = about 2.1; impurity F = about 2.6. Identification of impurities: Use the chromatogram supplied with acetazolamide for system suitability RS and the chromatogram obtained with reference solution (2) to identify the peaks due to impurities A, B, C, D, E and F. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurities D and E is at least 2.0. Limits: Correction factors: For the calculation of content, multiply the peak areas of the following impurities by the corresponding correction factor: Impurity B = 2.3; impurity C = 2.6; impurity D = 1.6. Impurities A, B, C, D, E, F, G: For each impurity, the area is not more than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.15%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%). The sum of the peak areas of all impurities is not more than 6 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.6%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A: N-(5-chloro-1,3,4-thiadiazol-2-yl)acetamide. Impurity B: N-(1,3,4-thiadiazol-2-yl)acetamide. Impurity C: N-(5-sulfanyl-1,3,4-thiadiazol-2-yl)acetamide.

9

ACETAZOLAMIDE TABLETS

VP V

Impurity D: 5-amino-1,3,4-thiadiazole-2-sulfonamide. Impurity E: 5-acetamido-1,3,4-thiadiazole-2-sulfonic acid. Impurity F: N-[5-[(5-acetamido-1,3,4-thiadiazol-2-yl)sulfonyl] sulfamoyl-1,3,4-thiadiazol-2- yl]acetamide. Impurity G: 5-amino-1,3,4-thiadiazole-2-thiol.

and filter. Add gradually n-hexan R to form precipitation. Filter and collect the precipitate, dry the residue at 105°C to dryness. The infrared absorption spectrum of the residue (Appendix 4.2) is concordant with the reference spectrum of acetazolamide.

Sulfates Not more than 0.05% (Appendix 9.4.14). Add 20 ml of distilled water to 0.4 g of the substance to be examined and dissolve by heating to boiling. Allow to cool with frequent shaking and filter. Determined on 15 ml of the filtrate.

Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Propan-2-ol - ethyl acetate - ammonia (50 : 30 : 20). Prepare the mobile phase immediately before use, saturate the chromatographic chamber for 1 h before developing without covering the inside wall by filter paper. Test solution: Weigh accurately a quantity of the powdered tablets containing 50 mg of acetazolamide, shake with 10 ml of a mixture of equal volumes of ethanol (96%) R and ethyl acetate R for 20 min and filter. Reference solution: Dilute 1 ml of the test solution to 100 ml with the same solvent mixture. Procedure: Apply separately to the plate 20 µl of each solution. Develop over a path of 15 cm. Dry the plate in air. Examine under ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution (1%).

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2.0 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.200 g of the substance to be examined in 25 ml of dimethylformamide R. Titrate with 0.1 N ethanolic sodium hydroxide VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N ethanolic sodium hydroxide VS is equivalent to 22.22 mg of C4H6N4O3S2. Storage Store in an airtight container, protected from light. Action and use Treatment of glaucoma. Preparations Injection, tablets. ACETAZOLAMIDE TABLETS Tabellae Acetazolamidi Acetazolamide tablets contain acetazolamide. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements. Content of acetazolamide, C4H6N4O3S2, 95.0% to 105.0% of the stated amount.

Identification Weigh a quantity of the powdered tablets containing about 0.5 g of acetazolamide, add 50 ml of acetone R, shake well 10

Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 900 ml of 0.01 M hydrochloric acid R. Rotation speed: 100 rpm. Time: 60 min. Procedure: After the specified time, withdraw a sample of the medium and filter, discard the first portion of the filtrate. Dilute the filtrate with 0.01 M hydrochloric acid R if necessary. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum wavelength at about 265 nm, in comparison with the reference solution having the same concentration of acetazolamide in 0.01 M hydrochloric acid R, in a 1-cm cell, using 0.01 M hydrochloric acid R as the blank. Calculate the content of acetazolamide, C4H6N4O3S2, using the absorbance of the test solution and the reference solution and the declared content of C4H6N4O3S2 in acetazolamide RS. Tolerance: Not less than 75% (Q) the content of acetazolamide, C4H6N4O3S2, of the stated amount is dissolved in 60 min. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 4.1 g of anhydrous sodium acetate R in 950 ml of water, add 20 ml of methanol R, 30 ml of

VP V

acetonitrile R and mix. Adjust the pH to 4.0 ± 0.05 with glacial acetic acid R. Internal reference solution: Weigh accurately about 100 mg of sulfadiazine RS and transfer into a 100-ml volumetric flask. Add 10 ml of 0.5M sodium hydroxide R and shake to dissolve. Dilute to volume with water, mix. Reference solution: Weigh accurately about 25 mg of acetazolamide RS, transfer into a 25-ml volumetric flask, add 2.5 ml of 0.5 M sodium hydroxide R, shake to dissolve. Add sufficient water and mix. Transfer 10.0 ml of this solution into a 100-ml volumetric flask, add 10.0 ml of the internal reference solution, 10 ml of 0.5 M sodium hydroxide R, dilute to volume with water and mix. Test solution: Weigh 20 tablets, calculate the average weight and finely powder. Weigh accurately a quantity of powdered tablets containing about 100 mg of acetazolamide and transfer into a 100-ml volumetric flask, add 10 ml of 0.5 M sodium hydroxide R, sonicate for 5 min. Allow to cool, dilute to volume with water, mix and filter. Transfer 10.0 ml of the filtrate to a 100-ml volumetric flask, add 10.0 ml of the internal reference solution, 10 ml of 0.5 M sodium hydroxide R, dilute to volume with water and mix. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 2 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution. The relative retention time of acetazolamide is about 0.7 and of sulfadiazine about 1.0. The resolution factor between acetazolamide and sulfadiazine peaks is not less than 2.0. The relative standard deviation of the ratios between the peak areas due to acetazolamide and sulfadiazine for 6 replicate injections is not more than 1.0%. Inject alternately the reference solution and test solution. Calculate the content of acetazolamide, C4H6N4O3S2, in tablets using the ratios between the peak areas due to acetazolamide and sulfadiazine in the chromatograms obtained with the test solution, the reference solution and the declared content of C4H6N4O3S2 in acetazolamide RS.

Storage Store in a well-closed container, protected from light, at a cool place. Action and use Treatment of glaucoma. Usual strength 125 mg, 250 mg.

ACETYLCYSTEINE

ACETYLCYSTEINE Acetylcysteinum CH3 O H HS

C5H9NO3S  



NH CO2H

M. 163.2  

Acetylcysteine is (2R)-2-(acetylamino)-3-sulfanyl propanoic acid. It contains not less than 98.0% and not more than 101.0% of C5H9NO3S, calculated with reference to the dried substance.

Characters A white, crystalline powder or colourless crystals. Freely soluble in water and in ethanol (96%), practically insoluble in dichloromethane. Identification Apply one of the two following identifications: First identification: A, E. Second identification: B, C, D, E. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of acetylcysteine RS. Examine the substances prepared as discs using potassium bromide R. B. Melting point (Appendix 6.7): 104 °C to 110 °C. C. To 0.5 ml of solution S (see Appearance of solution), add 0.05 ml of a 5% solution of sodium nitroprusside R and 0.05 ml of concentrated ammonia R. A dark violet colour develops. D. In the test for Related substances, the retention time and size of the principal peak in the chromatogram obtained with test solution (2) are approximately the same as those of the principal peak in the chromatogram obtained with reference solution (2). E. It complies with the test for Specific optical rotation. Appearance of solution Solution S: Dissolve 1.0 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2).

pH To 2 ml of solution S add 8 ml of carbon dioxide-free water R and mix. The pH of the solution is 2.0 to 2.8 (Appendix 6.2). Specific optical rotation +21.0º to +27.0º, calculated with reference to the dried substance (Appendix 6.4). Preparation: In a 25 ml volumetric flask, mix well 1.25 g of the substance to be examined with 1 ml of a 1% solution of 11

VP V

ACETYLCYSTEINE

sodium edetate R. Add 7.5 ml of a 4% solution of sodium hydroxide R, shake well to dissolve. Dilute to 25.0 ml with phosphate buffer solution pH 7.0 R2.

Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use, except test solution (3). Mobile phase: Stir 3 volumes of acetonitrile R and 97 volumes of water in a beaker, adjust to pH 3.0 with phosphoric acid R. Test solution (1): Suspend 0.80 g of the substance to be examined in 1 ml of 1 M hydrochloric acid R and dilute to 100.0 ml with water. Test solution (2): Dilute 5.0 ml of test solution (1) to 100.0 ml with water. Dilute 5.0 ml of the solution to 50.0 ml with water. Test solution (3): Use test solution (1) after storage for at least 1 hour. Reference solution (1): Suspend 4.0 mg of acetylcysteine RS, 4.0 mg of L-cystine R, 4.0 mg of L-cysteine R, 4.0 mg of acetylcysteine impurity C RS (N,N’-diacetyl-L-cystine) and 4.0 mg of acetylcysteine impurity D RS (N,S-diacetylL-cysteine) in 1 ml of 1 M hydrochloric acid R and dilute to 100.0 ml with water. Reference solution (2): Suspend 4.0 mg of acetylcysteine RS in 1 ml of 1 M hydrochloric acid and dilute to 100.0 ml with water. Chromatographic system: A column (25 cm × 4 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 220 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Under the prescribed conditions, the retention times are: L-cystine, about 2.2 min; L-cysteine, about 2.4 min; 2-methyl-2-thiazoline-4-carboxylic acid, originating in test solution (3), about 3.3 min; acetylcysteine, about 6.4 min; acetylcysteine impurity C, about 12 min; acetylcysteine impurity D, about 14 min. The test is not valid unless: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to L-cystine and L-cysteine is at least 1.5; the resolution between the peaks due to acetylcysteine impurity C and acetylcysteine impurity D is at least 2.0. Inject 0.01 M hydrochloric acid R as a blank. Inject three times reference solution (1), reference solution (2) and test solutions. Continue the chromatography for five times the retention time of acetylcysteine (about 30 min). From the chromatogram obtained with test solution (1), calculate the percentage content of the known impurities (T1) and the unknown impurities (T2) using the following expressions: 12

T1 =

A1 × m2 × 100 A2 × m1

T2 =

A3 × m3 × 100 A4 × m1

in which: A1: Peak area of individual impurity (L-cystine, L-cysteine, acetylcysteine impurity C and acetylcysteine impurity D) in the chromatogram obtained with test solution (1). A2: Peak area of the corresponding individual impurity (L-cystine, L-cysteine, acetylcysteine impurity C and acetylcysteine impurity D) in the chromatogram obtained with reference solution (1). A3: Peak area of unknown impurity in the chromatogram obtained with test solution (1). A4: Peak area of acetylcysteine in the chromatogram obtained with reference solution (2). m1: Mass of the substance to be examined in test solution (1). m2: Mass of the individual impurity in reference solution (1). m3: Mass of acetylcysteine in reference solution (2). Limits: The percentage content of each known impurity and of each unknown impurity is not greater than 0.5%. The sum of the calculated percentage contents of known and unknown impurities is not greater than 0.5%. Disregard any peak due to the solvent, any peak appearing at retention time of about 3.3 min corresponding to 2-methyl-2-thiazoline-4-carboxylic acid and any peak with an area less than 0.1 times that of the principal peak in the chromatogram obtained with reference solution (2).

Zinc Not more than 10 ppm of Zn. Determined by atomic absorption spectrometry (Appendix 4.4, method 2). Test solution: Dissolve 1.00 g in 0.001 M hydrochloric acid and dilute to 50.0 ml with the same solvent. Reference solution: Prepare the reference solutions using zinc standard solution (5 mg/ml Zn) R, diluted with 0.001 M hydrochloric acid. Measure the absorbance at 213.8 nm using a zinc hollowcathode lamp as the source of radiation, an air-acetylene flame and a correction procedure for non-specific absorption. Heavy metals Not more than 10 ppm (Appendix 9.4.8). 2.0 g complies with limit test 3 for heavy metals. Prepare the reference solution using 2 ml of lead standard (10 ppm Pb) R. Loss on drying Not more than 1.0% (Appendix 9.6). (1.000 g; in vacuo; 70 °C, 3 h). Sulfated ash Not more than 0.2% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.140 g in 60 ml of water and add 10 ml of dilute hydrochloric acid R. After cooling in iced water, add 10 ml

VP V

of potassium iodide solution R and titrate with 0.1 N iodine VS, using 1 ml of starch solution R as indicator. 1 ml of 0.1 N iodine VS is equivalent to 16.32 mg of C5H9NO3S.

Storage Store protected from light. Action and use Antidote for paracetamol poisoning; mucolytic. Preparations Injection, oral powders, capsules. ACETYLCYSTEINE CAPSULES Capsulae Acetylcysteini Acetylcysteine capsules contain acetylcysteine. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of acetylcysteine, C5H9NO3S, 95.0% to 105.0% of the stated amount. Identification A. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. B. Shake a quantity of the capsule contents containing about 1.0 g of acetylcysteine, with 20 ml of water, and filter. To 1 ml of the filtrate add 0.1 ml of a 5% solution of sodium nitroprusside R and 0.1 ml of ammonia R. A dark violet colour develops. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 6.8 g of potassium dihydrogen phosphate R in 1000 ml of water, adjust to pH 3.0 with phosphoric acid R, and filter. Test solution: Weigh 20 capsules, calculate the average weight of the capsule contents and powder finely. Weigh accurately a quantity of the powder, equivalent to about 0.1 g of acetylcysteine, in a 100 ml volumetric flask, dissolve in a 0.05% solution of sodium metabisulphite R, dilute with the same solvent to volume, and mix. Filter, discarding the first 20 ml of the filtrate. Dilute 10.0 ml of the filtrate with a 0.05% solution of sodium metabisulphite R to 100.0 ml, and mix. Reference solution: Weigh accurately about 0.1 g of acetylcysteine RS in a 100 ml volumetric flask, dissolve in a 0.05% solution of sodium metabisulphite R, dilute with the same solvent to volume, and mix. Dilute 10.0 ml of this solution with a 0.05% solution of sodium metabisulphite R to 100.0 ml, and mix.

ACETYLCYSTEINE POWDER FOR ORAL SUSPENSION

Chromatographic system: A column (25 cm × 4 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 214 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution, the relative standard deviation of peak areas for 6 replicate injections is not more than 2.0%. Inject separately the reference solution and the test solution. Calculate the content of acetylcysteine, C5H9NO3S, using the areas of the principal peaks in the chromatograms obtained with the test solution, the reference solution and the declared content of C5H9NO3S in acetylcysteine RS.

Storage Store in an airtight container, in a cool place, protected from light. Action and use Mucolytic. Usual strength 200 mg. ACETYLCYSTEINE POWDER FOR ORAL SUSPENSION  Pulveres Acetylcysteini ad suspensionum per oralum Acetylcysteine powder for oral suspension contains acetylcysteine. Suitable excipients such as flavors, colors, preservatives, stabilizers … may be added. The powder complies with the requirements stated under “Powder” (Appendix 1.7) and with the following requirements.

Content of acetylcysteine, C5H9NO3S, 95.0% to 105.0% of the stated amount. Characters Powder should be loose and dry, not be moist and lumpy, with homogeneous colour. Identification A. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. B. Shake a quantity of the powder containing about 1.0 g of acetylcysteine, with 20 ml of water and filter. To 1 ml of the filtrate add 0.1 ml of a 5% solution of sodium nitroprusside R and 0.1 ml of ammonia R. A dark violet colour develops. Loss on drying Not more than 5.0% (Appendix 9.6). (1.000 g; 75 oC; 4 h; in vacuum). 13

VP V

ACETYLSALICYLIC ACID

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 6.8 g of potassium dihydrogen phosphate R in 1000 ml of water, adjust to pH 3.0 with phosphoric acid R, and filter. Test solution: Weigh accurately a quantity of the fine powders obtained in the test for Uniformity of mass, equivalent to about 0.1 g of acetylcysteine, in a 100 ml volumetric flask, dissolve and dilute with a 0.05% solution of sodium metabisulphite R to volume, and mix well and filter. Dilute 10.0 ml of the filtrate with a 0.05% solution of sodium metabisulphite R to 100.0 ml, and mix. Reference solution: Weigh accurately about 0.1 g of acetylcysteine RS in a 100 ml volumetric flask, dissolve in a 0.05% solution of sodium metabisulphite R, dilute with the same solvent to volume, and mix. Dilute 10.0 ml of this solution with a 0.05% solution of sodium metabisulphite R to 100.0 ml, and mix. Chromatographic system: A column (25 cm × 4 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 214 nm. Flow rate: 1.5 ml/min. Volume of injection: 20  µl. Procedure: System suitability: Inject the reference solution, the relative standard deviation for 6 replicate injections is not more than 2.0% Inject separately the reference solution and the test solution. Calculate the content of acetylcysteine, C5H9NO3S, using the areas of the principal peaks in the chromatograms obtained with the test solution, the reference solution and the declared content of C5H9NO3S in acetylcysteine RS. Storage Store in an airtight container, in a cool place, protected from light. Action and use Mucolytic. Usual strength 200 mg. ACETYLSALICYLIC ACID Acidum acetylsalicylicum Aspirin CO2H O O

C9H8O4   14



CH3

M. 180.2

Acetylsalicylic acid is 2-(acetyloxy) benzoic acid. It contains not less than 99.5% and not more than 101.0% of C9H8O4, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder or colourless crystals. Slightly soluble in water, freely soluble in ethanol (96%). Melting point is about 143°C (Appendix 6.7, method 3). Identification Apply one of the two following identifications: First identification: A, B. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of acetylsalicylic acid RS. B. Boil 0.2 g with 4 ml of dilute sodium hydroxide solution R for 3 min. Cool and add 5 ml of dilute sulfuric acid R. A crystalline precipitate is formed. Filter, wash the precipitate with water and dry at 100 °C to 105 °C. The melting point is 156 °C to 161 °C (Appendix 6.7). C. In a test tube, mix 0.1 g with 0.5 g of calcium hydroxide R. Heat the mixture and expose to the fumes produced a piece of filter paper impregnated with 0.05 ml of nitrobenzaldehyde solution R. A greenish-blue or greenishyellow colour develops on the paper. Moisten the paper with dilute hydrochloric acid R. The colour becomes blue. D. Dissolve with heating about 20 mg of the precipitate obtained in Identification test B in 10 ml of water and cool. The solution gives reaction (A) of salicylates (Appendix 8.1). Appearance of solution Dissolve 1.0 g in 9 ml of ethanol (96%) R. The solution is clear (Appendix 9.2). and colourless (Appendix 9.3, method 2). Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use. Mobile phase: Phosphoric acid - acetonitrile for chromatography - water (2 : 400 : 600). Test solution: Dissolve 0.100 g of the substance to be examined in acetonitrile for chromatography R and dilute to 10.0 ml with the same solvent. Reference solution (1): Dissolve 50.0 mg of salicylic acid R (impurity C) in the mobile phase and dilute to 50.0 ml with the mobile phase. Dilute 1.0 ml of the solution to 100.0 ml with the mobile phase. Reference solution (2): Dissolve 10 mg of salicylic acid R (impurity C) in the mobile phase and dilute to 10.0 ml with the mobile phase. To 1.0 ml of the solution add 0.2 ml of the test solution and dilute to 100.0 ml with the mobile phase. Reference solution (3): Dissolve with the aid of ultrasound the contents of a vial of acetylsalicylic acid for peak identification RS (containing impurities A, B, D, E and F) in 1.0 ml of acetonitrile R.

VP V

Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 237 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: The run time is 7 times the retention time of acetylsalicylic acid. Identification of impurities: Use the chromatogram obtained with reference solution (1) to identify the peak due to impurity C; use the chromatogram supplied with acetylsalicylic acid for peak identification RS and the chromatogram obtained with reference solution (3) to identify the peaks due to impurities A, B, D, E and F. Relative retention with reference to acetylsalicylic acid (retention time = about 5 min): impurity A = about 0.7; impurity B = about 0.8; impurity C = about 1.3; impurity D = about 2.3; impurity E = about 3.2; impurity F = about 6.0. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to acetylsalicylic acid and impurity C is at least 6.0. Limits: Impurities A, B, C, D, E, F: For each impurity, the area is not more than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.15%). Any other impurity: For each impurity, the area is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). The sum of the peak areas of all impurities is not more than 2.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.25%). Disregard any peak with an area less than 0.3 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.03%). Note: Impurity A: 4-hydroxybenzoic acid. Impurity B: 4-hydroxybenzene-1,3-dicarboxylic acid (4-hydroxyisophthalic acid). Impurity C: 2-hydroxybenzenecarboxylic acid (salicylic acid). Impurity D: 2-[[2-(acetyloxy)benzoyl]oxy]benzoic acid (acetylsalicylsalicylic acid). Impurity E: 2-[(2-hydroxybenzoyl)oxy]benzoic acid (salsalate, salicylsalicylic acid). Impurity F: 2-(acetyloxy)benzoic anhydride (acetylsalicylic anhydride).

Heavy metals

Not more than 20 ppm (Appendix 9.4.8). Dissolve 1.0 g in 12 ml of acetone R and dilute to 20 ml with water. 12 ml of the solution complies with limit test for heavy metals, method 2. Prepare the reference solution using lead standard solution (1 ppm Pb) obtained by diluting lead standard solution (100 ppm Pb) R with a mixture of acetone R and water (9 : 6).

ACETYLSALICYLIC ACID TABLETS

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; in vacuo). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay In a flask with a ground-glass stopper, dissolve 1.000 g of the substance to be examined in 10 ml of ethanol (96%) R. Add 50.0 ml of 0.5 N sodium hydroxide VS. Close the flask and allow to stand for 1 h. Using 0.2 ml of phenolphthalein solution R as indicator, titrate with 0.5 N hydrochloric acid VS. Carry out a blank titration. 1 ml of 0.5 N sodium hydroxide VS is equivalent to 45.04 mg of C9H8O4. Storage Store in an airtight container, protected from light. Action and use Antipyretic; analgesic; anti-inflammatory. Preparations Tablets, gastro-resistant tablets. ACETYLSALICYLIC ACID TABLETS Tabellae Acidi acetylsalicylici Aspirin tablets Acetylsalicylic acid tablets contain acetylsalicylic acid. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of acetylsalicylic acid, C9H8O4, 95.0% to 105.0% of the stated amount.

Identification

Boil 0.5 g of the powdered tablets for 2 to 3 min with 10 ml of 10% solution of sodium hydroxide R, cool and add an excess of 10% solution of sulphuric acid R; a crystalline precipitate is produced. Filter, and dissolve the precipitate in some ml of water, add 2 drops of a 0.5% solution of iron (III) chloride R; a deep violet colour is produced.

Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 500 ml of buffer solution pH 4.5. Buffer solution pH 4.5: Dissolve 29.9 g of sodium acetate R in water, add 16.6 ml of glacial acetic acid R and sufficient water to produce 10 L. Rotation speed: 50 rpm. Time: 45 min. 15

GASTRO-RESISTANT ACETYLSALICYLIC ACID TABLETS

Procedure: After the specified time, withdraw a sample of the medium and filter. Discard the first 10 ml of the filtrate. Immediately measure the absorbance of the filtrate, suitably diluted if necessary, at the maximum at 265 nm (Appendix 4.1), using the medium as a blank. Measure the absorbance of a suitable solution of acetylsalicylic acid RS in the dissolution medium. Calculate the total content of acetylsalicylic acid, C9H8O4, in the medium using the declared content of C9H8O4 in acetylsalicylic acid RS. Tolerances: Not less than 70% (Q) of the labelled amount of acetylsalicylic acid, C9H8O4, is dissolved in 45 min.

Salicylic acid Not more than 3.0%. Shake a quantity of the powdered tablets containing 0.20 g of acetylsalicylic acid with 4 ml of ethanol (96%) R and dilute to 100 ml with water at a temperature not exceeding 10 °C. Filter through a filter paper immediately, transfer 50 ml of the filtrate to a Nessler cylinder, add 1 ml of a freshly prepared 0.2% solution of ammonium iron (III) sulphate R, mix and allow to stand for 1 minute. Any violet colour produced is not more intense than that obtained by adding 1 ml of a freshly prepared 0.2% solution of ammonium iron (III) sulphate R to a mixture of 3.0 ml of a freshly prepared 0.10% solution of salicylic acid R, 2 ml of ethanol (96%) R and sufficient water to produce 50 ml contained in a second Nessler cylinder. Assay Weigh 20 tablets, calculate the average mass, and powder finely. To an accurately weighed quantity of the powder containing 0.5 g of acetylsalicylic acid add 30 ml of 0.5 N sodium hydroxide VS, boil gently for 10 min and titrate the excess of alkali with 0.5 N hydrochloric acid VS using phenol red solution R as indicator. Repeat the operation without the substance being examined. The difference between the titrations represents the amount of 0.5 N sodium hydroxide VS required. Each ml of 0.5 N sodium hydroxide VS is equivalent to 45.04 mg of C9H8O4. Storage Store in an airtight container, in a cool place, protected from light. Action and use Analgesic, antipyretic, nonsteroidal anti-inflammatory, antiplatelet drugs. Usual strength 100 mg; 300 mg; 500 mg.

16

VP V

GASTRO-RESISTANT ACETYLSALICYLIC ACID TABLETS Tabellae Acidi acetysalicylici Enteric-coated aspirin tablets Gastro-resistant acetylsalicylic acid tablets contain acetylsalicylic acid. The tablets comply with the requirements stated under “Tablets”, “Gastro-resistant tablets” (Appendix 1.20) and with the following requirements:

Content of acetylsalicylic acid, C9H8O4, 93.0% to 107.0% of the stated amount. Identification A. Boil a quantity of the powdered tablets containing 300 mg of acetylsalicylic acid for 2 to 3 minutes with 10 ml of a 10% solution of sodium hydroxide R, cool and add an excess of a 10% solution of sulfuric acid R; a crystalline precipitate is produced and an odour of acetic acid gas is detectable. Filter and dissolve the precipitate in water, add several drops of a 10.5% solution of iron (III) chloride R; a deep violet colour is produced. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that of acetylsalicylic acid peak in the chromatogram obtained with the reference solution. Dissolution (Appendix 11.4) Acid stage Apparatus: Basket. Medium: 1000 ml of 0.1 M hydrochloric acid R. Rotation speed: 100 rpm. Time: 2 h. Procedure: After the specified time (2 h), withdraw a sample of the medium, filter. Dilute the filtrate with the medium (if necessary). Measure the absorbance (Appendix 4.1) of the resulting solution at 276 nm, using the medium as a blank, in comparison with a reference solution having a known concentration of acetylsalicylic acid equivalent to that in the test solution prepared by dissolving an accurately weighed quantity of acetylsalicylic acid RS in the medium. Calculate the content of acetylsalicylic acid, C9H8O4, dissolved using the absorbance of the test solution and the reference solution and the content of C9H8O4 in acetylsalicylic acid RS. Tolerance: Not more than 10% of the stated amount of acetylsalicylic acid, C9H8O4, is dissolved in 2 hours. Buffer stage Continue the test immediately with the same tablets used in the acid stage. Apparatus: Basket. Medium: Mixed phosphate buffer pH 6.8 R. Rotation speed: 100 rpm. Time: 45 min.

VP V

Procedure: Replace the 0.1 M hydrochloric acid in the vessel with 900 ml of mixed phosphate buffer pH 6.8 R, previously held at 37 °C ± 0.5 °C. After the specified time (45 min), withdraw a sample of the medium, filter. Dilute the filtrate with the medium (if necessary). Immediately measure the absorbance (Appendix 4.1) of the resulting solution (test solution) at 265 nm, using mixed phosphate buffer pH 6.8 R as the blank solution, compared to a reference solution of acetylsalicylic acid RS having the same concentration in the same medium. Calculate the content of acetylsalicylic acid, C9H8O4, dissolved using the absorbance of the test solution and the reference solution and the content of C9H8O4 in acetylsalicylic acid RS. Tolerance: Not less than 70% (Q) of the stated amount of acetylsalicylic acid, C9H8O4, is dissolved in 45 minutes.

Free salicylic acid Not more than 3.0%. Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - 0.05 M sodium dihydrogen phosphate adjusted to pH 2.0 with phosphoric acid (1 : 3). Solvent mixture: Acetonitrile - formic acid (99 : 1). Test solution: Transfer an accurately weighed quantity of the powdered tablets containing the equivalent of about 300 mg of acetylsalicylic acid to a 100 ml volumetric flask, add 60 ml of acetonitrile R and 1 ml of formic acid R, shake well for 15 min and dilute to volume with acetonitrile R. Mix and filter. Reference solution: A 0.009% solution of salicylic acid RS in the solvent mixture. Resolution solution: A solution containing 0.009% of salicylic acid RS and 0.3% of acetylsalicylic acid in the solvent mixture. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 μm). Detector: A spectrophotometer set at 280 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 μl. Procedure: System suitability: Inject the resolution solution. The test is not valid unless, the resolution factor between two principal peaks is at least 3. Inject alternately the reference solution and the test solution. Limits: In the chromatogram obtained with the test solution, the area of any peak corresponding to salicylic acid peak is not greater than the area of salicylic acid peak in the chromatogram obtained with the reference solution (3.0%). Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase, solvent mixture and chromatographic system: Proceed as directed in Free salicylic acid.

ASPIRIN AND CAFFEINE TABLETS

Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Transfer an accurately weighed quantity of the powdered tablets containing the equivalent of about 300 mg of acetylsalicylic acid to a 100 ml volumetric flask, add 60 ml of acetonitrile R and 1 ml of formic acid R, shake for 15 min and dilute to volume with acetonitrile R. Mix and filter. Dilute 5.0 ml of the filtrate to 20.0 ml with the solvent mixture. Reference solution: A 0.075% solution of acetylsalicylic acid RS in the solvent mixture. Resolution solution: A solution containing 0.0015% of salicylic acid RS and 0.075% of acetylsalicylic acid RS in the solvent mixture. Procedure: System suitability: Inject the resolution solution. The test is not valid unless, the resolution factor between two principal peaks is not less than 3. Inject the reference solution, the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Chromatograph alternately the reference solution and the test solution. Calculate the content of acetylsalicylic acid, C9H8O4, in the tablets using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C9H8O4 in acetylsalicylic acid RS.

Storage Store in an airtight container, in a cool and dry place, protected from light. Action and use Non-steroidal analgesic, antipyretic, anti-inflammatory drug. Inhibitor of aggregation. Usual strength 81 mg, 100 mg, 150 mg. ASPIRIN AND CAFFEINE TABLETS Tabellae Aspirini et Caffeini Aspirin and caffeine tablets contain aspirin and caffeine. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of aspirin, C9H8O4, 90.0% to 110.0% of the stated amount. Content of caffeine, C8H10N4O2, 90.0% to 110.0% of the stated amount. Identification In the Assay, the retention times of the two principal peaks in the chromatogram obtained with the test solution correspond to those in the chromatogram obtained with the reference solution. 17

VP V

ACICLOVIR

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 500 ml of 0.05 M acetate buffer pH 4.5 (prepared by mixing 2.99 g of sodium acetate R and 1.66 ml of glacial acetic acid R with water to obtain 1000 ml). Rotation speed: 50 rpm. Time: 45 min. Procedure: After the specified time, withdraw a sample of the medium and filter, discard the first 20 ml of the filtrate. Carry out the assay of aspirin and caffeine dissolved by liquid chromatography (Appendix 5.3) with mobile phase and chromatographic system proceed as directed in the Assay. Prepare the reference solution by dissolving aspirin RS and caffeine RS in the medium to obtain a solution having the same concentration of aspirin and caffeine as those in the test solution. Tolerance: Not less than 75% (Q) of the stated amount of aspirin, C9H8O4 is dissolved in 45 minutes. Not less than 75% (Q) of the stated amount of caffeine, C8H10N4O2 is dissolved in 45 minutes. Assay and limit of salicylic acid Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 1.0 g of sodium pentanesulfonate R and 2.3 g of ammonium dihydrogen phosphate R in 850 ml of water. Add 150 ml of acetonitrile R and adjust with phosphoric acid R to pH 2.5. Diluent: Prepare a mixture of water and acetonitrile R (53 : 46), and adjust with phosphoric acid R to pH 2.5. Reference solution: Dissolve an accurately weighed quantity of aspirin RS and caffeine RS in the diluent to obtain a solution having a concentration of about 0.001×A mg aspirin and 0.001×C mg caffeine per 1 ml. (A and C being the labeled amounts, in mg, of aspirin and caffeine, respectively, in each tablet). Salicylic acid reference solution: Dissolve an accurately weighed quantity of salicylic acid RS in the diluent to obtain a solution having a concentration of about 0.005×A mg salicylic acid per ml (A being the labeled amount, in mg, of aspirin per tablet). Resolution solution: Dissolve an accurately weighed quantity of salicylic acid RS in the reference solution to obtain a solution having a concentration of about 0.0001 × A mg salicylic acid per ml (A being the labeled amount, in mg, of aspirin per tablet). Test solution: Weigh 20 tablets and powder finely. Weigh accurately a quantity of powdered tablets equivalent to 1 tablet, transfer into a 100 ml volumetric flask, add about 70 ml of the diluent, shake well and dilute to volume with the diluent. Filter and discard the first 20 ml of the filtrate. Transfer 5.0 m of the filtrate to a 50-ml volumetric flask, dilute with the diluent to volume. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase B (3 - 10 µm). 18

Detector: A spectrophotometer set at 215 nm. Flow rate: 2.0 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject the resolution solution: The relative retention times are about 0.2 for caffeine, 0.7 for aspirin, and 1.0 for salicylic acid. The test is not valid unless the resolution factor between the peaks due to aspirin and salicylic acid is at least 1.5. Inject the reference solution 6 times. The test is not valid unless the the relative standard deviation for replicate of each peaks of aspirin and caffeine is not more than 2.0%. Inject alternately the reference solutions and the test solution. Calculate the content of salicylic acid, C7H6O3, in tablets using the peak areas in the chromatograms obtained with the test solution and the salicylic acid reference solution and the declared content of C7H6O3 in salicylic acid RS. Content of salicylic acid, C7H6O3, not more than 3.0% of the stated amount of aspirin. Calculate the content of aspirin, C9H8O4, in one tablet using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C9H8O4 in aspirin RS. Calculate the content of caffeine, C8H10N4O2, in one tablet using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C8H10N4O2 in caffeine RS.

Storage Protected from light. Action and use Analgesic and antipyretic. Usual strength Aspirin 350 mg and caffeine 30 mg. Aspirin 200 mg and caffeine 20 mg. ACICLOVIR Aciclovirum

C8H11N5O3  

M. 225.2

Aciclovir is 2-amino-9-[(2-hydroxyethoxy)methyl]-1,9dihydro-6H-purin-6-one. It contains not less than 98.5% and not more than 101.0% of C8H11N5O3, calculated with reference to the anhydrous substance.

VP V

Characters White or almost white, crystalline powder. Slightly soluble in water, freely soluble in dimethyl sulfoxide, very slightly soluble in ethanol (96%). It dissolves in dilute solutions of mineral acids and alkali hydroxides. Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of aciclovir RS. Appearance of solution Dissolve 0.25 g in 0.1 M sodium hydroxide R and dilute to 25 ml with the same solvent. The solution is clear (Appendix 9.2) and not more intensely coloured than reference solution Y7 (Appendix 9.3, method 2). Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Acetonitrile - phosphate buffer solution pH 3.1 (1 : 99). Mobile phase B: Acetonitrile - phosphate buffer solution pH 2.5 (50 : 50). Phosphate buffer solution pH 2.5: Dissolve 3.48 g of dipotassium hydrogen phosphate R in 1000 ml of water and adjust to pH 2.5 with phosphoric acid R. Phosphate buffer solution pH 3.1: Dissolve 3.48 g of dipotassium hydrogen phosphate R in 1000 ml of water and adjust to pH 3.1 with phosphoric acid R. Solvent mixture: Dimethyl sulfoxide - water (20 : 80). Test solution: Dissolve 25 mg of the substance to be examined in 5.0 ml of dimethyl sulfoxide R and dilute to 25.0 ml with water. Reference solution (1): Dissolve 5 mg of aciclovir for system suitability RS (containing impurities A, B, J, K, N, O and P) in 1 ml of dimethyl sulfoxide R and dilute to 5.0 ml with water. Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 10.0 ml with the solvent mixture. Reference solution (3): Dissolve the contents of a vial of aciclovir for peak identification 1 RS (containing impurities C and I) in 200 µl of dimethyl sulfoxide R and dilute to 1.0 ml with water. Prepare this solution immediately before use. Reference solution (4): Dissolve the contents of a vial of aciclovir for peak identification 2 RS (containing impurities F and G) in 1.0 ml of reference solution (1). Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl of the test solution and reference solutions (2), (3) and (4). Procedure: Carry out a linear gradient elution using the following conditions:

ACICLOVIR

Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0-5

100

0

5 - 27

100 → 80

0 → 20

27 - 40

80

20

Identification of impurities: Use the chromatogram supplied with aciclovir for peak identification 1 RS and the chromatogram obtained with reference solution (3) to identify the peaks due to impurities C and I; use the chromatogram supplied with aciclovir for peak identification 2 RS and the chromatogram obtained with reference solution (4) to identify the peaks due to impurities A, B, F, G, J, K, N, O and P. Relative retention with reference to aciclovir (retention time = about 13 min): Impurity B = about 0.4; impurity P = about 0.7; impurity C = about 0.9; impurity N = about 1.37; impurity O = about 1.42; impurity I = about 1.57; impurity J = about 1.62; impurity F = about 1.7; impurity A = about 1.8; impurity K = about 2.5; impurity G = about 2.6. System suitability: In the chromatogram obtained with reference solution (3), the resolution between the peaks due to impurity C and aciclovir is at least 1.5. In the chromatogram obtained with reference solution (4), the resolution between the peaks due to impurities F and A is at least 1.5; the resolution between the peaks due to impurities K and G is at least 1.5. Limits: Correction factor: For the calculation of content, multiply the peak area of impurity I by 1.5. Impurity B: The area of the peak due to impurity B is not more than 7 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.7%). Impurity O: The area of the peak due to impurity O is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.3%). Impurities A, G, J, K, N, P: For each impurity, the area is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (2) (0.2%). Impurities C, F, I: For each impurity, the corrected area, if necessary, is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.1%). Any other impurity: For each impurity, the area is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). The sum of the peak areas of all impurities is not more than 15 times the area of the principal peak in the chromatogram obtained with reference solution (2) (1.5%). Disregard any peak with an area less than 0.3 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.03%). Note: Impurity A: 2-[(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl) methoxy]ethyl acetate.

19

VP V

ACICLOVIR CREAM Impurity B: 2-amino-1,7-dihydro-6H-purin-6-one (guanine). Impurity C: 2-amino-7-[(2-hydroxyethoxy)methyl]-1,7-dihydro -6H-purin-6-one. Impurity F: N-[9-[(2-hydroxyethoxy)methyl]-6-oxo-6,9dihydro-1H-purin-2-yl]acetamide. Impurity G: 2-[[2-(acetylamino)-6-oxo-1,6-dihydro-9H-purin9-yl]methoxy]ethyl acetate. Impurity I: 2-amino-7-[[2-[(2-amino-6-oxo-1,6-dihydro-9H-purin -9- yl)methoxy]ethoxy]methyl]-1,7-dihydro-6H-purin-6-one. Impurity J: 9,9’-[ethylenebis(oxymethylene)]bis(2-amino-1,9dihydro-6H-purin-6-one). Impurity K: 2,2’-[methylenediimino]bis[9-[(2-hydroxyethoxy) methyl]1,9-dihydro-6H-purin-6- one]. Impurity L: N-(9-acetyl-6-oxo-6,9-dihydro-1H-purin-2-yl)acetamide (N2,9-diacetylguanine). Impurity M: 2-[[2-(acetylamino)-6-oxo-1,6-dihydro-7H-purin7-yl]methoxy]ethyl acetate. Impurity N: unknown structure. Impurity O: unknown structure. Impurity P: 2-amino-9-(2-hydroxyethyl)1,9-dihydro-6H-purin6-one.

Water Not more than 6.0% (Appendix 10.3). Determined on 0.500 g. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.150 g of the substance to be examined in 60 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). Carry out a blank titration. 1 ml of 0.1 N perchloric acid VS is equivalent to 22.52 mg of C8H11N5O3. Storage Store in an airtight container. Action and use Antiviral drug. Preparations Cream, eye ointment, infusion, oral suspension, tablets. ACICLOVIR CREAM Cremoris Acicloviri Aciclovir cream intended for external use contains aciclovir. The cream complies with the requirements stated under “Topical semi-solid preparations” (Appendix 1.12) and with the following requirements.

Content of aciclovir, C8H11N5O3, 95.0% to 105.0% of the stated amount. 20

Characters A white or off-white cream. Identification A. The ultraviolet absorption spectrum (Appendix 4.1), in the range 230 to 350 nm, of the solution prepared in the Assay exhibits a maximum at 255 nm and a broad shoulder at about 274 nm. B. In the test for Guanine, the principal spot in the chromatogram obtained with solution (2) is similar in position and intensity to that in the chromatogram obtained with solution (3). Guanine Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Cellulose F254. Mobile phase (1): Ethyl acetate. Mobile phase (2): Propan-1-ol - 13.5 M ammonia - 5% solution of ammonium sulfate (10 : 30 : 60). Solution (1): Transfer a quantity of the well-mixed cream containing 30 mg of aciclovir into a 10 ml graduated, stoppered centrifuge tube, add 3 ml of 0.1 M sodium hydroxide R and shake to disperse the cream. Add 5 ml of a mixture of 1 volume of chloroform R and 2 volumes of propan-1-ol R, shake well, centrifuge and dilute the upper aqueous layer to 5 ml with 0.1 M sodium hydroxide R, mix, centrifuge and use the upper aqueous layer. Solution (2): Dilute 1 volume of solution (1) to 10 volumes with 0.1 M sodium hydroxide R. Solution (3): Dissolve 6.0 mg of aciclovir RS in 10 ml of 0.1 M sodium hydroxide R. Solution (4): Dissolve 6.0 mg of guanine in 100 ml of 0.1 M sodium hydroxide R. Procedure: Apply separately to the plate 10 µl of each solution. Develop, using mobile phase (1) but allow the solvent front to ascend to the top of the plate. After removal of the plate, dry it in a current of air and repeat the development in the same direction using mobile phase (2) and allowing the solvent front to ascend 8 cm above the line of application. After removal of the plate, allow it to dry in air and examine under ultraviolet light (254 nm). Any secondary spot corresponding to guanine in the chromatogram obtained with solution (1) is not more intense than the spot in the chromatogram obtained with solution (4) (1.0%). Disregard any spot that appears just below the solvent front. Assay Shake a quantity of the well-mixed cream containing about 7.5 mg of aciclovir with 50 ml of 0.5 M sulfuric acid R. Shake well with 50 ml of ethyl acetate R, allow to separate and collect the clear lower aqueous layer. Wash the organic layer with 20 ml of 0.5 M sulfuric acid R and dilute the combined washings and the aqueous layer to 100.0 ml with 0.5 M sulfuric acid R. Mix well and filter

VP V

(Whatman GF/F is suitable). Discard the first 10 ml of the filtrate. Dilute 10.0 ml of the filtrate with water to produce 50.0 ml. Measure the absorbance of the resulting solution at the maximum at 255 nm (Appendix 4.1) in a 1-cm cell, using a mixture of 0.5 M sulfuric acid R and water (1 : 4) in the reference cell. Calculate the content of C8H11N5O3 taking 562 as the value of A (1%, 1 cm) at the maximum at 255 nm.

Storage Store in an airtight container at cool place, protected from light. Action and use Antiviral. Usual strength Tubes of 2 g or 10 g of 5% aciclovir. ACICLOVIR TABLETS Tabellae Acicloviri Aciclovir tablets contain aciclovir. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of aciclovir, C8H11N5O3, 95.0% to 105.0% of the stated amount. Identification A. The ultraviolet absorption spectrum (Appendix 4.1), in the range 230 to 350 nm, of the solution prepared in the Assay exhibits a maximum at 255 nm and a broad shoulder at about 274 nm. B. In the test for Guanine, the principal spot in the chromatogram obtained with solution (2) is similar in position and intensity to that in the chromatogram obtained with solution (3). Guanine Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Cellulose F254. Mobile phase: Propan-1-ol - 13.5 M ammonia - 5% solution of ammonium sulphate (10 : 30 : 60). Solution (1): Shake a quantity of the powdered tablets containing 0.25 g of aciclovir with 25 ml of 0.1 M sodium hydroxide R for 10 min. Add a sufficient quantity of 0.1 M sodium hydroxide R to produce 50.0 ml, allow to stand and allow any undissolved material to settle before application to the plate. Solution (2): Dilute 1 volume of solution (1) to 10 volumes with 0.1 M sodium hydroxide R. Solution (3): Dissolve 5.0 mg of aciclovir RS in 10 ml of 0.1 M sodium hydroxide R. Solution (4): Dissolve 5.0 mg of guanine in 100 ml of 0.1 M sodium hydroxide R.

ACICLOVIR TABLETS

Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 12 cm. After removal of the plate, allow to dry in air and examine under ultraviolet light (254 nm). Any secondary spot corresponding to guanine in the chromatogram obtained with solution (1) is not more intense than the spot in the chromatogram obtained with solution (4) (1.0%). Disregard any spot that appears just below the solvent front.

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of 0.1 M hydrochloric acid R. Rotation speed: 50 rpm. Time: 45 min. Procedure: After the specified time, withdraw a sample of the medium and filter. Discard the first 20 ml of the filtrate. Measure the absorbance of the filtered solution, diluted if necessary with 0.1 M hydrochloric acid R, at the maximum at 255 nm (Appendix 4.1) in a 1 cm cell, using 0.1 M hydrochloric acid R in the reference cell. Calculate the total content of aciclovir, C8H11N5O3, in the medium taking 560 as the value of A (1%, 1 cm) at the maximum at 255 nm. Tolerance: Not less than 70% (Q) of the labelled amount of aciclovir, C8H11N5O3, is dissolved in 45 min. Related substances Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel GF254. Mobile phase: 13.5 M ammonia - methanol - dichloromethane (2 : 20 : 80). Solution (1): Shake a quantity of the powdered tablets containing 0.25 g of aciclovir with 10 ml of dimethyl sulfoxide R for 15 min and filter. Prepare freshly. Solution (2): Dilute 0.7 ml of solution (1) to 100 ml with dimethyl sulfoxide R. Prepare freshly. Procedure: Apply separately to the plate 2 µl of each solution. Develop over a path of 10 cm. After removal of the plate, allow it to dry in air and examine under ultraviolet light (254 nm). In the chromatogram obtained with solution (1) any secondary spot with an Rf value greater than that of the principal spot is not more intense than the spot in the chromatogram obtained with solution (2) (0.7%). Assay Weigh 20 tablets, calculate the average mass, and powder finely. To an accurately weighed quantity of the powdered tablets containing about 0.1 g of aciclovir add 60 ml of 0.1 M sodium hydroxide R and disperse with the aid of ultrasound for 15 min. Add a sufficient quantity of 0.1 M sodium hydroxide R to produce 100.0 ml, mix well and filter. To 15.0 ml of the filtrate add 50 ml of water and 5.8 ml of 2 M hydrochloric acid R and sufficient water to produce 100.0 ml. To 5.0 ml of the solution add sufficient 21

VP V

ADRENALINE

0.1 M hydrochloric acid R to produce 50.0 ml and mix well. Measure the absorbance of the resulting solution at the maximum at 255 nm (Appendix 4.1) in a 1 cm cell, using 0.1 M hydrochloric acid R in the reference cell. Calculate the content of C8H11N5O3 taking 560 as the value of A (1%, 1 cm) at the maximum at 255 nm.

Storage Store in an airtight container, in a cool place, protected from light. Action and use Antiviral. Usual strength 200 mg; 400 mg; 800 mg. ADRENALINE Adrenalinum Epinephrine

C9H13NO3  

M. 183.2

Adrenaline is 4-[(1R)-1-hydroxy-2-(methylamino)ethyl] benzene-1,2-diol. It contains not less than 99.0% and not more than 101.0% of C9H13NO3, calculated with reference to the dried substance.

Characters White or almost white crystalline powder, it darkens on exposure to air and light. Practically insoluble in water, in ethanol (96%) and in methylene chloride. It dissolves in hydrochloric acid. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of adrenaline RS. B. It complies with the test for Specific optical rotation.

Appearance of solution

Solution S: Dissolve 1.000 g in a 25.75 g/L solution of hydrochloric acid R and dilute to 50.0 ml with the same solvent. Examine the solution immediately. Solution S is not more opalescent than reference suspension II (Appendix 9.2) and not more intensely coloured than reference solution BY5 (Appendix 9.3, method 2).

Specific optical rotation -50° to -54°, calculated with reference to the dried substance (Appendix 6.4). Determined on solution S. 22

Related substances Examine by liquid chromatography (Appendix 6.4). Prepare the solutions protected from light. Mobile phase A: Acetonitrile R1 - solvent mixture A (5 : 95). Mobile phase B: Acetonitrile R1 - solvent mixture A (45 : 55). Solvent mixture A: Dissolve 5.0 g of potassium dihydrogen phosphate R and 2.6 g of sodium octanesulfonate R in water for chromatography R and dilute to 1000 ml with the same solvent (it is usually necessary to stir for at least 30 min to achieve complete dissolution). Adjust to pH 2.8 with phosphoric acid R. Solvent mixture B: Acetonitrile R1 - solvent mixture A (13 : 87). Blank solution: 0.1 M hydrochloric acid - solvent mixture B (1 : 9). Test solution: Dissolve 40 mg of the substance to be examined in 5 ml of 0.1 M hydrochloric acid R and dilute to 50.0 ml with solvent mixture B. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with solvent mixture B. Dilute 1.0 ml of this solution to 10.0 ml with solvent mixture B. Reference solution (2): Dissolve 1.5 mg of noradrenaline tartrate RS (impurity B) and 1.5 mg of adrenalone hydrochloride R (impurity C) in solvent mixture B, add 1.0 ml of the test solution and dilute to 100 ml with solvent mixture B. Reference solution (3): Dissolve the contents of a vial of adrenaline impurity mixture RS (containing impurities D and E) in 1.0 ml of the blank solution. Reference solution (4): Dissolve 4 mg of adrenaline with impurity F RS in 0.5 ml of 0.1 M hydrochloric acid and dilute to 5 ml with solvent mixture B. Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (3 µm). Column temperature: 50 °C. Detector: A spectrophotometer set at 210 nm. Flow rate: 2.0 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 15

92 → 50

8 → 50

15 - 20

50 → 92

50 → 8

20 - 25

92

8

Identification of impurities: Use the chromatogram supplied with adrenaline impurity mixture RS and the chromatogram obtained with reference solution (3) to identify the peaks due to impurities D and E; use the

VP V

chromatogram supplied with adrenaline with impurity F RS and the chromatogram obtained with reference solution (4) to identify the peak due to impurity F. Relative retention with reference to adrenaline (retention time = about 4 min): Impurity F = about 0.2; impurity B = about 0.8; impurity C = about 1.3; impurity D = about 3.3; impurity E = about 3.7. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurity B and adrenaline is at least 3.0. Limits: Correction factors: For the calculation of content, multiply the peak areas of the following impurities by the corresponding correction factor: Impurity D = 0.7; impurity E = 0.6. Impurities B, C, F: For each impurity, the area is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%). Impurities D, E: For each impurity, the corrected area, if necessay, is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%). The sum of the peak areas of all impurities is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity B: (1R)-2-amino-1-(3,4-dihydroxyphenyl)ethanol (noradrenaline). Impurity C: 1-(3,4-dihydroxyphenyl)-2-(methylamino)ethanone (adrenalone). Impurity D: 4-[(1R)-2-(benzylmethylamino)-1-hydroxyethyl] benzene-1,2-diol. Impurity E: 2-(benzylmethylamino)-1-(3,4-dihydroxyphenyl) ethanone. Impurity F: (1R)-1-(3,4-dihydroxyphenyl)-2-(methylamino) ethanesulfonic acid.

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; diphosphorus pentoxide, at a pressure not exceeding 0.7 kPa, 18 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.150 g of the substance to be examined in 50 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2).

ADRENALINE ACID TARTRATE

1 ml of 0.1 N perchloric acid VS is equivalent to 18.32 mg of C9H13NO3.

Storage Store in an airtight container, under nitrogen, protected from light. Action and use Adrenoceptor agonist. Preparations Injection, eye drops. ADRENALINE ACID TARTRATE Adrenaline acidum tartras Adrenaline tartrate

C9H13NO3,C4H6O6  

M. 333.3

Adrenaline acid tartrate is (1R)-1-(3,4-dihydroxyphenyl)2-(methylamino)ethanol hydrogen (2R,3R)-2,3-dihydroxybutanedioate. It contains not less than 98.5% and not more than 101.0% of C9H13NO3,C4H6O6, calculated with reference to the dried substance.

Characters White or greyish-white, crystalline powder. Freely soluble in water, slightly soluble in ethanol (96%). Identification A. The infrared absorption spectrum (Appendix 4.2) of adrenaline base prepared as described under identification test B is concordant with the spectrum of adrenaline base prepared as described from 50 mg of adrenaline tartrate RS dissolved in 5 ml of a 0.5% solution of sodium metabisulfite R. Keep the mixture at room temperature for at least 30 min, filter through a sintered-glass filter. Examine the substances prepared as discs. B. Dissolve 5 g in 50 ml of a 0.5% solution of sodium metabisulfite R and make alkaline by addition of ammonia R. Keep the mixture at room temperature for at least 15 min and filter. Reserve the filtrate for identification test C. Wash the precipitate with 3 quantities, each of 10 ml, of methanol R. Dry at 80 °C. The specific optical rotation (Appendix 6.4) of the residue (adrenaline base) is -50° to -53.5°. Determined using a 2% solution in 0.5 M hydrochloric acid R. C. 0.2 ml of the filtrate obtained in identification test B gives reaction (B) of tartrates (Appendix 8.1).

23

VP V

ADRENALINE ACID TARTRATE

Appearance of solution Dissolve 0.5 g in water and dilute to 10 ml with the same solvent. Examine the solution immediately. The solution is not more opalescent than reference suspension II (Appendix 9.2) and not more intensely coloured than reference solution BY5 (Appendix 9.3, method 2). Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions protected from light. Mobile phase A: Acetonitrile R1 - solvent mixture A (5 : 95). Mobile phase B: Acetonitrile R1 - solvent mixture A (45 : 55). Solvent mixture A: Dissolve 5.0 g of potassium dihydrogen phosphate R and then 2.6 g of sodium octanesulfonate R in water for chromatography R, and dilute to 1000 ml with the same solvent (it is usually necessary to stir for at least 30 min to achieve complete dissolution). Adjust to pH 2.8 with phosphoric acid R. Solvent mixture B: Acetonitrile R1 - solvent mixture A (130 : 870). Blank solution: 0.1 M hydrochloric acid - solvent mixture B (1 : 9). Test solution: Dissolve 75 mg of the substance to be examined in 5 ml of 0.1 M hydrochloric acid R and dilute to 50 ml with solvent mixture B. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with solvent mixture B. Dilute 1.0 ml of this solution to 10.0 ml with solvent mixture B. Reference solution (2): Dissolve 1.5 mg of noradrenaline tartrate RS (impurity B) and 1.5 mg of adrenalone hydrochloride R (impurity C) in solvent mixture B, add 1.0 ml of the test solution and dilute to 100.0 ml with solvent mixture B. Reference solution (3): Dissolve the contents of a vial of adrenaline impurity mixture RS (impurities D and E) in 0.1 ml of 0.1 M hydrochloric acid R and 0.9 ml of solvent mixture B. Reference solution (4): Dissolve 7.5 mg of adrenaline tartrate with impurity A RS in 0.5 ml of 0.1 M hydrochloric acid R and dilute to 5.0 ml with solvent mixture B. Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (3 µm). Column temperature: 50 °C. Detector: A spectrophotometer set at 210 nm. Flow rate: 2.0 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min) 0 - 15 15 - 20 20 - 25

24

Mobile phase A (% v/v) 92 → 50 50 → 92 92

Mobile phase B (% v/v) 8 → 50 50 → 8 8

Identification of impurities: Use the chromatogram supplied with adrenaline impurity mixture RS and the chromatogram obtained with reference solution (3) to identify the peaks due to impurities D and E; use the chromatogram supplied with adrenaline tartrate with impurity A RS and the chromatogram obtained with reference solution (4) to identify the peak due to impurity A. Relative retention with reference to adrenaline (retention time = about 4 min): Impurity B = about 0.8; impurity C = about 1.3; impurity A = about 3.2; impurity D = about 3.3; impurity E = about 3.7. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurity B and adrenaline is at least 3.0. Limits: Correction factors: for the calculation of content, multiply the peak areas of the following impurities by the corresponding correction factor: impurity D = 0.7; impurity E = 0.6. Impurity A: The area of the peak due to impurity A is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.3%). Impurities B, C: For each impurity, the area is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%). Impurities D, E: For each impurity, the corrected area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). The sum of the peak areas of all impurities is not more than 6 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.6%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A: Unknown structure. Impurity B: (1R)-2-amino-1-(3,4-dihydroxyphenyl)ethanol (noradrenaline). Impurity C: 1-(3,4-dihydroxyphenyl)-2-(methylamino)ethanone (adrenalone). Impurity D: 4-[(1R)-2-(benzylmethylamino)-1-hydroxyethyl] benzene-1,2-diol. Impurity E: 2-(benzylmethylamino)-1-(3,4-dihydroxyphenyl) ethanone.

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; in vacuo, 18 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g.

VP V

Assay Dissolve 0.300 g of the substance to be examined in 50 ml of anhydrous acetic acid R, heating gently if necessary. Titrate with 0.1 N perchloric acid VS until a bluish-green colour is obtained, using 0.1 ml of crystal violet solution R as indicator. 1 ml of 0.1 N perchloric acid VS is equivalent to 33.33 mg of C13H19NO9. Storage In an airtight container, or preferably in a sealed tube under vacuum or under an inert gas, protected from light. Action and use Adrenoceptor agonist. Preparation Injection. ADRENALINE INJECTION Injectio Adrenalini Epinephrine injection Adrenaline injection is a sterile, isotonic solution containing 0.18% of adrenaline tartrate in water for injections. The injection complies with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Content of adrenaline, C9H13NO3, 0.09 g to 0.11 g in 100 ml of the injection. Characters A clear, colourless solution. Identification A. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. B. To 1 ml of the injection add a 0.25% solution of iron (III) chloride hexahydrate R dropwise until a green colour is produced. On the gradual addition of a 4.2% solution of sodium hydrogen carbonate R, the solution changes first to blue and then to red. C. To 10 ml of the injection add 2 ml of a 10% solution of disodium hydrogen phosphate R and sufficient iodine iodide solution R to produce a brown colour and remove excess iodine by adding 0.1 M sodium thiosulphate R dropwise. A red colour is produced. pH 2.8 to 3.6 (Appendix 6.2). Noradrenaline Examine by liquid chromatography (Appendix 5.3).

ADRENALINE INJECTION

Mobile phase: A solution containing 4.0 g of tetramethylammonium hydrogen sulphate R, 1.1 g of sodium heptanesulphonate R and 2 ml of 0.1 M Trilon B R in a mixture of 950 ml of water and 50 ml of methanol R and adjusted to pH 3.5 with 1 M sodium hydroxide R. Filter and degas. Test solution: Use the injection. Reference solution: A 0.0018% solution of noradrenaline tartrate in the mobile phase. Resolution solution: A solution containing 0.0018% of adrenaline tartrate RS and 0.0018% of noradrenaline tartrate in the mobile phase. Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase C (5 µm) (Nucleosil C18 is suitable). Detector: A spectrophotometer set at 205 nm. Flow rate: 2 ml/min. Volume of injection: 10 µl. Procedure: The test is not valid unless the resolution factor between the two principal peaks in the chromatogram obtained with the resolution solution is at least 2.0. In the chromatogram obtained with the test solution the area of any peak corresponding to noradrenaline is not greater than the area of the principal peak in the chromatogram obtained with the reference solution.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase and Chromatographic system: Prepare as directed in the test for Noradrenaline. Reference solution: A 0.02% solution of adrenaline tartrate RS in the mobile phase. Test solution: Dilute 1 volume of the injection to 10 volumes with the mobile phase. Resolution solution: A solution containing 0.02% of adrenaline tartrate RS and 0.02% of noradrenaline tartrate in the mobile phase. Procedure: The test is not valid unless the resolution factor between the two principal peaks in the chromatogram obtained with the resolution solution is at least 2.0. Calculate the content of adrenaline, C9H13NO3, in the injection using the areas of the principal peaks in the chromatograms obtained with the test solution, the reference solution, and the content of C9H13NO3 in the reference solution. Storage Store at cool place, protected from light. Action and use Sympathomimetic. Usual strength 1 mg/ml. 25

VP V

ALBENDAZOLE

ALBENDAZOLE Albendazolum

C12H15N3O2S

M. 265.3

Albendazole is methyl [5-(propylsulfanyl)-1H-benzimidazol2-yl] carbamate. It contains not less than 98.0% and not more than 102.0% of C12H15N3O2S, calculated with reference to the dried substance.

Characters A white or faintly yellowish powder. Freely soluble in anhydrous formic acid and in dimethylformamide, sparingly soluble in methanol and chloroform, very slightly soluble in methylene chloride, practically insoluble in water and in ethanol (96%). Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of albendazole RS. Appearance of solution Dissolve 0.10 g in a mixture of anhydrous formic acid R and methylene chloride R (1 : 9) and dilute to 10 ml with the same solvent. The solution is clear (Appendix 9.2) and not more intensely coloured than reference solution BY6 (Appendix 9.3, method 2). Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: A 0.167% solution of ammonium dihydrogen phosphate - methanol (300 : 700). Test solution: Dissolve 25.0 mg of the substance to be examined in 5 ml of methanol R containing 1% v/v of sulfuric acid R and dilute to 50.0 ml with the mobile phase. Reference solution: Dissolve 10.0 mg of the substance to be examined in 10 ml of methanol R containing 1% v/v of sulfuric acid R and dilute to 100.0 ml with the mobile phase. Dilute 0.5 ml of this solution to 20.0 ml with the mobile phase. Resolution solution: Dissolve 50.0 mg of the substance to be examined and 50 mg of oxybendazole RS in 5 ml of methanol R containing 1% v/v of sulfuric acid R and dilute to 100.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with spherical endcapped octadecylsilyl silica gel for chromatography R (5 µm) with a pore size of 10 nm and a carbon loading of 19%. Detector: A spectrophotometer set at 254 nm. 26

Flow rate: 0.7 ml/min. Volume of injection: 20 µl. Procedure: Inject the reference solution. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained is at least 50% of the full scale of the recorder. System suitability: Inject the resolution solution. The test is not valid unless the resolution between the peaks corresponding to albendazole and oxybendazole is at least 3.0. Inject the test solution. Continue the chromatography for 1.5 times the retention time of albendazole. When the chromatograms are recorded in the prescribed conditions, the approximate relative retention times are: 0.40 for impurity D, 0.43 for impurities B and C, 0.47 for impurity E, 0.57 for impurity F, 0.80 for impurity A. Limits: In the chromatogram obtained with the test solution the area of any peak, apart from the principal peak, is not greater than 1.5 times the area of the principal peak in the chromatogram obtained with the reference solution (0.75%). The sum of the areas of any such peaks is not greater than 3 times the area of the principal peak in the chromatogram obtained with the reference solution (1.5%). Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with the reference solution. Notes: Impurity A: 5-(propylsulfanyl)-1H-benzimidazol-2-amine. Impurity B: methyl [5-(propylsulfinyl)-1H-benzimidazol-2-yl] carbamate. Impurity C: methyl [5-(propylsulfonyl)-1H-benzimidazol-2-yl] carbamate. Impurity D: 5-(propylsulfonyl)-1H-benzimidazol-2-amine. Impurity E: methyl (1H-benzimidazol-2-yl)carbamate. Impurity F: methyl [5-(methylsulfanyl)-1H-benzimidazol-2-yl] carbamate.

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 100  °C - 105 °C; 4 h).

Sulfated ash Not more than 0.2% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.250 g of the substance to be examined in 3 ml of anhydrous formic acid R and add 40 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). In order to avoid overheating during the titration, mix thoroughly throughout and stop the titration immediately after the endpoint has been reached. 1 ml of 0.1 N perchloric acid VS is equivalent to 26.53 mg of C12H15N3O2S.

VP V

Storage Store protected from light. Action and use Anthelmintic. Preparation Tablets. ALBENDAZOLE TABLETS Tabellae Albendazoli Albendazole tablets contain albendazole. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of albendazole, C12H15N3O2S, 92.5% to 107.5% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel F254. Mobile phase: Chloroform - glacial acetic acid - ether (60 : 10 : 10). Test solution: Shake a quantity of the powdered tablets, equivalent to about 100 mg of albendazole in 20 ml of glacial acetic acid R, and filter. Reference solution: Dissolve 25 mg of albendazole RS in 5 ml of glacial acetic acid R. Procedure: Apply separately to the plate 10 µl of each solution. After removal of the plate, allow it to dry in air and examine under ultraviolet light (254 nm). The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. B. The ultraviolet absorption spectrum (Appendix 4.1) of the test solution prepared in the Assay exhibits a maximum at about 308 ± 1 nm. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of 0.1 M hydrochloric acid R Rotation speed: 75 rpm. Time: 30 min. Procedure: Acidified methanol: To about 50 ml of methanol R in a 100 ml volumetric flask add 2 ml of hydrochloric acid R, dilute with methanol R to volume, and mix. Reference solution: Weigh accurately about 90 mg of albendazole RS in a 250 ml volumetric flask, add 10 ml of the acidified methanol and shake to dissolve. Dilute with 0.1 M hydrochloric acid R to volume, and mix. Transfer 5.0 ml of this solution to a 200 ml volumetric flask, dilute with 0.1 M sodium hydroxide R to volume, and mix. Test solution: After the specified time, withdraw a sample of the medium and filter, discarding the first 20 ml of the

ALIMEMAZINE TARTRATE

filtrate. Dilute the filtrate with 0.1 M sodium hydroxide R to obtain a solution having a known concentration of albendazole equal to the concentration of albendazole in the reference solution. Measure the absorbances of the test solutions and the reference solution at the maximum at 308 nm and at the minimum at 350 nm (Appendix 4.1) in a 1-cm cell, using 0.1 M sodium hydroxide R as a blank. Calculate the content of albendazole, C12H15N3O3S, dissolved in the medium as described in the test for Assay. Tolerance: Not less than 80% (Q) of the labelled amount of albendazole, C12H15N3O3S, is dissolved in 30 min.

Assay Test solution: Weigh 20 tablets, calculate the average mass, and powder finely. To an accurately weighed quantity of the powdered tablets containing about 0.1 g of albendazole add 150 ml of 0.1 M methanolic hydrochloric acid R, shake for 15 minutes, dilute to 250.0 ml with 0.1 M methanolic hydrochloric acid R, and mix. Filter, discarding the first 20 ml of the filtrate. Dilute 5.0 ml of the filtrate to 250.0 ml with 0.1 M sodium hydroxide R, and mix. Reference solution: Prepare as the test solution using 100 mg of albendazole RS instead of the powdered tablets. Measure the absorbance of the test solution and the reference solution at the maximum at 308 nm and at the minimum at 350 nm (Appendix 4.1) in a 1-cm cell, using 0.1 M sodium hydroxide R as a blank. Calculate the content of albendazole, C12H15N3O2S, using the difference in absorbance betwwen 308 nm and 350 nm obtained with the test solution and the reference solution, respectively, and the declared content of C12H15N3O2S in albendazole RS. Storage Store in an airtight container, protected from light. Action and use Anthelmintic. Usual strength 200 mg; 400 mg. ALIMEMAZINE TARTRATE Alimemazini tartras



and enatiomer

(C18H22N2S)2,C4H6O6  

M. 747.0 27

VP V

ALIMEMAZINE SYRUP

Alimemazine tartrate is (RS)-dimethyl (2-methyl-3phenothiazin-10-ylpropyl)amine (2R,3R)-tartrate. It contains not less than 99.0% and not more than 101.0% of (C18H22N2S)2,C4H6O6, calculated with reference to the dried substance.

1 ml of 0.1 N perchloric acid VS is equivalent to 37.35 mg of (C18H22N2S)2,C4H6O6.

Characters A white or slightly cream powder. It darkens on exposure to light. Freely soluble in water, sparingly soluble in ethanol (96%), very slightly soluble in ether.

Action and use Histamine H1-receptor antagonist; sedative.

Identification A. Dissolve 0.1 g of the substance to be examined in 10 ml of water and add 2 ml of 1 M sodium hydroxide R. Extract with 25 ml of ether R, wash the extract with 5 ml of water, dry over anhydrous sodium sulfate R, evaporate to dryness and dissolve the residue in 1 ml of dichloromethane R. The infrared absorption spectrum (Appendix 4.2) of the solution is concordant with the spectrum of alimemazine RS. B. Melting point: 159 °C to 163 °C (Appendix 6.7). Acidity pH of a 2% solution: 5.0 to 6.5 (Appendix 6.2). Related substances Not more than 0.5%. Examine by thin-layer chromatography (Appendix 5.4). Carry out the test protected from light. Coating substance: Silica gel GF254. Mobile phase: Acetone - diethylamine - cyclohexane (10 : 10 : 80). Test solution: A 2.0% solution of the substance to be examined in methanol - diethylamine (95 : 5). Reference solution: A 0.010% solution of the substance to be examined in methanol - diethylamine (95 : 5). Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 12 cm. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Disregard any spot remaining on the line of application. Unless otherwise specified any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 100 °C; at a pressure not exceeding 0.7 kPa). Sulfated ash Not more than 0.1% (Appendix 9.9, method 1). Determined on 1.0 g. Assay Examine by non-aqueous titration (Appendix 10.6, method 1). Using 1.00 g of the substance to be examined and crystal violet solution R as indicator. 28

Storage Protected from light.

Preparations Tablets, paediatric oral solution. ALIMEMAZINE SYRUP Sirupi Alimemazini Alimemazine syrup contains alimemazine tartrate. The tablets comply with the requirements stated under “Syrups” (Appendix 1.4) and with the following requirements:

Content of alimemazine tartrate, C36H44N4S2,C4H6O6, 90.0% to 110.0% of the stated amount. Identification A. Examine by Thin layer chromatography (Appendix 5.4), protected from light and use the solutions freshly prepared. Coating substance, mobile phase, solvent mixture and procedure are described in the test for Related substances. Test solution: Dilute 1 volume of the test solution in the test for Related substances to 20 volumes with the solvent mixture. Reference solution: Dissolve 20 mg of alimemazine tartrate RS in 15 ml of water and add 2 ml of 1M sodium hydroxide R, mix well. Extract with two 15-ml portions of chloroform R, combine the chloroform extracts and eliminate water with anhydrous sodium sulfate R. Evaporate the extract to dryness on a water bath. Dissolve the residue in 20 ml of the solvent mixture. In the chromatogram obtained with the test solution, the principal spot is similar in position, form and size to that of the spot in the chromatogram obtained with the reference solution. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. Related substances A. Examine by thin layer chromatography (Appendix 5.4), protected from light and use the solutions freshly prepared. Coating substance: Silica gel GF254. Mobile phase: Acetone - diethylamine - cyclohexane (10 : 10 : 80). Solvent mixture: Methanol - diethylamine (95 : 5).

VP V

Test solution: Dilute an accurate volume of the syrup containing about 20 mg of alimemazine tartrate with 15 ml of water and add 2 ml of 1 M sodium hydroxide R, mix well. Extract with two 15 ml portions of chloroform R, combine the chloroform extracts and eliminate water with anhydrous sodium sulfate R. Evaporate the extract to dryness on a water bath. Dissolve the residue in 1 ml of the solvent mixture. Reference solution: Dilute 1 volume of the test solution to 50 volumes with the solvent mixture. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of about 12 cm. Remove the plate, allow it to dry in air and examine under ultraviolet light at 254 nm. In the chromatogram obtained with the test solution, any secondary spot is not more intense than the spot in the chromatogram obtained with the reference solution (2.0%). Disregard any spot remaining on the line of application.

Assay Examine by liquid chromatography (Appendix 5.3), protected from light. Mobile phase: 0.005 M sodium heptane sulfonate R in a mixture of methanol - water - 6 M acetic acid (65 : 34 : 1). Make adjustments if necessary. Test solution: Weigh accurately a quantity of the syrup containing about 5 mg of alimemazine tartrate, transfer into a 100-ml volumetric flask, dilute to volume with the mobile phase, shake well. Filter through a 0.45 µm membrane filter. Reference solution: A 0.005% solution of alimemazine tartrate RS in the mobile phase (0.05 mg/ml). Filter through a 0.45 µm membrane filter. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution, in the chromatogram obtained, the capacity factor, k’, is 2.0 to 5.0; the theoretical plates are not less than 1200; the symmetry factor determined on the alimemazine peak is not more than 3.5. The relative standard deviation of peak areas for 6 replicate injections of the reference solution is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of alimemazine tartrate, C36H44N4S2,C4H6O6, in syrup using the peak areas in the chromatograms obtained with the test solution, the reference solution, the density of the syrup (g/ml) and the declared content of C36H44N4S2,C4H6O6 in alimemazine tartrate RS.

ALIMEMAZINE TABLETS

Storage Store in a tight container, protected from light. Action and use Histamine H1 receptor antagonist; sedative. Usual strength 7.5 mg/5 ml; 30 mg/5 ml. ALIMEMAZINE TABLETS Tabellae Alimemazini Trimeprazine tablets Alimemazine tablets contain alimemazine tartrate. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements. Content of alimemazine tartrate, C36H44N4S2,C4H6O6, 92.5% to 107.5% of the stated amount.

Identification A. To a quantity of the powdered tablets containing 40 mg of alimemazine tartrate add 10 ml of water and 2 ml of 1 M sodium hydroxide R, shake and extract with 15 ml of ether R. Wash the ether layer with 5 ml of water, dry with anhydrous sodium sulfate R and evaporate the ether to dryness on a water bath. Dissolve the residue in 0.4 ml of dichloromethan R. The infrared absorption spectrum of the resulting solution (Appendix 4.2) is concordant with the reference spectrum of alimemazine. B. To a quantity of the powdered tablets containing 1 mg of alimemazine tartrate add 1 ml of a mixture of equal volumes of formaldehyde R and sulfuric acid R. A purple colour is produced. Dissolution Apparatus: Basket. Medium: 500 ml of 0.01 M hydrochloric acid solution R. Rotation speed:100 rpm. Time: 45 min. Procedure: After the specified time, withdraw a sample of the medium and filter. Dilute with the medium if necessary. Measure the absorbance of the solution at the maximum at about 251 nm (Appendix 4.1), in a 1-cm cell, using the medium as the blank, comparing with the reference solution having the same concentration in the medium. Tolerance: Not less than 75% (Q) of alimemazin tartrat, C36H44N4S2,C4H6O6, of the stated amount is dissolved in 45 min. Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Acetone - diethylamine - cyclohexane (10 : 10 : 80). 29

VP V

ALLOPURINOL

Test solution: Extract a quantity of the powdered tablets containing 0.1 g of alimemazine tartrate with 10 ml of a mixture of methanol R and diethylamine R (95 : 5), and filter. Reference solution: Dilute 1 volume of the test solution to 200 volumes with the same solvent mixture. Procedure: Apply separately to the plate 20 µl of each solutions (freshly prepared). Develop over a path of about 12 cm. Remove the plate, allow it to dry in air and examine under ultraviolet light (254 nm). Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution (0.5%). Disregard any spot remaining on the line of application.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: 0.005 M sodium heptanesulfonate R in a mixture of methanol - water - 6 M acetic acid (65 : 34 : 1). Make adjustments if necessary. Test solution: Weigh 20 tablets, calculate the average masse and finely powder. Weigh accurately a quantity of the powdered tablets equivalent to about 5 mg of alimemazine tartrate, transfer into a 100-ml volumetric flask, dissolve in the mobile phase, dilute to volume with the same solvent, mix and filter. Reference solution: A 0.005% solution of alimemazine tartrate RS in the mobile phase (0.05 mg/ml). Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.5 ml/min Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution, the symmetry factor of alimemazin peak is not greater than 3.5; the relative standard deviation of the peak areas for 6 replicate injections is not more than 1.0%. Inject separately the reference solution and the test solution. Calculate the content of alimemazine tartrate, C36H44N4S2,C4H6O6, using the areas of the principal peaks in the chromatograms obtained with the test solution, the reference solution, and the declared content of C36H44N4S2,C4H6O6 in alimemazine tartrate RS. Storage Store in an airtight container, in a cool place, protected from light. Action and use Histamine H1-receptor antagonist; sedative. Usual strength 5 mg. 30

ALLOPURINOL Allopurinolum O N

C5H4N4O  

NH N H

N

M. 136.1

Allopurinol is 1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin4-one. It contains not less than 97.0% and not more than 102.0% of C5H4N4O, calculated with reference to the dried substance.

Characters White or almost white powder. Very slightly soluble in water and in ethanol (96%). It dissolves in dilute solutions of alkali hydroxides. Identification Apply one of the two following identifications: First identification: A. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of allopurinol RS. B. Dissolve 10 mg in 1 ml of 0.1 M sodium hydroxide R and dilute to 100.0 ml with 0.1 M hydrochloric acid R. Dilute 10.0 ml of this solution to 100.0 ml with 0.1 M hydrochloric acid R. The ultraviolet absorption spectrum (Appendix 4.1) exhibits one absorption maximum at 250 nm and one absorption minimum 231 nm. The ratio of the absorbances measured at 231 nm to that measured at 250 nm is 0.52 to 0.62. C. Dissolve 0.3 g in 2.5 ml of dilute sodium hydroxide solution R and add 50 ml of water. Add slowly and with shaking 5 ml of 4.25% solution of silver nitrate R. A white precipitate is formed which does not dissolve on the addition of 5 ml of ammonia R. D. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel F254. Mobile phase: Ethanol - dichloromethane (40 : 60). Test solution: Dissolve 20 mg of the substance to be examined in ammonia R and dilute to 10 ml with the same solvent. Reference solution: Dissolve 20 mg of allopurinol RS in ammonia R and dilute to 10 ml with the same solvent. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution.

VP V

Related substances Examine by liquid chromatography (Appendix 5.3). Use freshly prepared solutions. Store and inject them at 8 °C, using a cooled autosampler. Mobile phase: A 0.125% solution of potassium dihydrogen phosphate R. Test solution (1): Dissolve 25.0 mg of the substance to be examined in 2.5 ml of 0.1 M sodium hydroxide R and dilute immediately to 50.0 ml with the mobile phase. Test solution (2): Dissolve 20.0 mg of the substance to be examined in 5.0 ml of 0.1 M sodium hydroxide R and dilute immediately to 250.0 ml with the mobile phase. Reference solution (1): Dilute 2.0 ml of test solution (1) to 100.0 ml with the mobile phase. Dilute 5.0 ml of this solution to 100.0 ml with the mobile phase. Reference solution (2): Dissolve 5 mg of allopurinol impurity A RS, 5 mg of allopurinol impurity B RS and 5.0 mg of allopurinol impurity C RS in 5.0 ml of 0.1 M sodium hydroxide R and dilute immediately to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 100.0 ml with the mobile phase. Reference solution (3): Dissolve 20.0 mg of allopurinol RS in 5.0 ml of 0.1 M sodium hydroxide R and dilute immediately to 250.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 230 nm. Flow rate: 1.4 ml/min. Volume of injection: 20 µl. Procedure: The run time is twice the retention time of allopurinol. Elution order: Impurity A, impurity B, impurity C, allopurinol. Retention time of allopurinol is about 10 min. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurities B and C is at least 1.1. Limits: Impurity A: The area of the peak due to impurity A is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%). Impurity B: The area of the peak due to impurity B is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%). Impurity C: The area of the peak due to impurity C is not more than the area of the corresponding peak in the chromatogram obtained with reference solution (2) (0.1%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) 0.1%. The sum of the peak areas of all impurities other than impurities A, B and C, is not more than 3 times the area

ALLOPURINOL

of the principal peak in the chromatogram obtained with reference solution (1) (0.3%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A: 5-Amino-1H-pyrazole-4-carboxamide. Impurity B: 5-(formylamino)-1H-pyrazole-4-carboxamide. Impurity C: 5-(4H-1,2,4-triazol-4-yl)-1H-pyrazole-4-carboxamide. Impurity D: Ethyl 5-amino-1H-pyrazole-4-carboxylate. Impurity E: Ethyl 5-(formylamino)-1H-pyrazole-4-carboxylate. Impurity F: Diazane (hydrazine).

Impurities D and E Examine by liquid chromatography (Appendix 5.3). Use freshly prepared solutions. Store and inject them at 8 °C, using a cooled autosampler. Mobile phase: Methanol - solution A (10 : 90). Solution A: A 0.125% solution of potassium dihydrogen phosphate R. Test solution: Dissolve 50.0 mg of the substance to be examined in 5.0 ml of 0.1 M sodium hydroxide R and dilute immediately to 100.0 ml with solution A. Reference solution: Dissolve 5.0 mg of allopurinol impurity D RS and 5.0 mg of allopurinol impurity E RS in 5.0 ml of 0.1 M sodium hydroxide R and dilute immediately to 100.0 ml with solution A. Dilute 1.0 ml of this solution to 100.0 ml with solution A. Chromatographic system: A column (5 cm × 4.6 mm) packed with stationary phase basedeactivated octadecylsilyl silica gel for chromatography R (3 µm). Detector: A spectrophotometer set at 230 nm. Flow rate: 2.0 ml/min. Volume of injection: 20 µl. Procedure: The run time is 1.5 times the retention time of impurity E. Retention times: Impurity D = about 3.6 min; impurity E = about 4.5 min. System suitability: In the chromatogram obtained with the reference solution, the resolution between the peaks due to impurities D and E is at least 2.0. Limits: Impurity D: The area of the peak due to impurity D is not more than the area of the corresponding peak in the chromatogram obtained with the reference solution (0.1%). Impurity E: The area of the peak due to impurity E is not more than the area of the corresponding peak in the chromatogram obtained with the reference solution (0.1%). Impurity F Examine by liquid chromatography (Appendix 5.3). 31

VP V

ALLOPURINOL TABLETS

Under the following conditions, any hydrazine in the sample reacts with benzaldehyde to give benzaldehyde azine. Mobile phase: 2-propanol - hexane (5 : 95). Solvent mixture: Methanol - dilute sodium hydroxide solution (50 : 50). Solution A: Dissolve 2.0 g of benzaldehyde R in the solvent mixture and dilute to 50.0 ml with the solvent mixture. Prepare immediately before use. Test solution: Dissolve 250.0 mg of the substance to be examined in 5 ml of the solvent mixture. Add 4 ml of solution A, mix and allow to stand for 2.5 h at room temperature. Add 5.0 ml of hexane R and shake for 1 min. Allow the layers to separate and use the upper layer. Reference solution: Dissolve 10.0 mg of hydrazine sulfate R in the solvent mixture by sonicating for about 2 min and dilute to 50.0 ml with the solvent mixture. Dilute 1.0 ml to 20.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 20.0 ml with the solvent mixture. To 5.0 ml of the solution obtained, add 4.0 ml of solution A, mix and allow to stand for 2.5 h at room temperature. Add 5.0 ml of hexane R and shake for 1 min. Allow the layers to separate and use the upper layer. Blank solution: To 5.0 ml of the solvent mixture add 4.0 ml of solution A, mix and allow to stand for 2.5 h at room temperature. Add 5.0 ml of hexane R and shake for 1 min. Allow the layers to separate and use the upper layer. Chromatographic system: A column (25 cm × 4.0 mm) packed with stationary phase cyanosilyl silica gel for chromatography R (5 µm) with a pore size of 10 nm. Column temperature: 30 °C. Detector: A spectrophotometer set at 310 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: Inject the blank, the test solution and the reference solution. Relative retention with reference to benzaldehyde (retention time = about 2.8 min): benzaldehyde azine = about 0.8. System suitability: In the chromatogram obtained with the reference solution, the resolution between the peaks due to benzaldehyde azine and benzaldehyde is at least 2.0. The signal-to-noise ratio for the peak due to benzaldehyde azine is not less than 20. Limit: Impurity F: The area of the peak due to benzaldehyde azine in the chromatogram obtained with the test solution is not more than the area of the corresponding peak in the chromatogram obtained with the reference solution (10 ppm of hydrazine sulfate equivalent to 2.5 ppm of hydrazine). 32

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. Inject test solution (2) and reference solution (3). Calculate the content of C5H4N4O using the areas in the chromatograms obtained with test solution (2), reference solution (3) and the declared content of C5H4N4O in allopurinol RS. Storage Store in an airtight container. Action and use Treatment of gout and hyperuricaemia. Preparation Tablets. ALLOPURINOL TABLETS Tabellae Allopurinoli Allopurinol tablets contain allopurinol. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of allopurinol, C5H4N4O, 92.5% to 107.5% of the stated amount. Identification A. To a quantity of powdered tablets containing 50 mg of allopurinol, add 10 ml of 0.1 M sodium hydroxide R, grind, mix and filter. Acidify the filtrate with 1 M acetic acid R and allow to stand for 10 to 15 min. Filter and collect the residue, rinse the residue with 3 ml of ethanol R and then with 4 ml ether R. Allow to dry in air for 15 min and then dry in an oven at 105 °C for 3 h. The infrared absorption spectrum of the residue is concordant with the infrared absorption spectrum of allopurinol RS. B. The ultraviolet absorption spectrum (Appendix 4.1), in the range 230 nm to 350 nm, of the test solution obtained in the Assay exhibits a maximum at about 250 nm.

VP V

C. Shake a quantity of the powdered tablets containing 0.1 g of allopurinol with 5 ml of 1.25 M sodium hydroxide R and add 3 ml of phosphomolybdotungstic solution R and 5 ml of a 20% sodium carbonate solution R. A greyish blue colour is produced.

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of 0.01 M hydrochloric acid R. Rotation speed: 75 rpm. Time: 45 min. Procedure: Test solution: After the specified time, withdraw a sample of the medium and filter, discarding the first portion of filtrate. Dilute the filtrate with 0.01 M hydrochloric acid R to obtain a solution having the same concentration as the reference solution. Reference solution: Weigh accurately about 40 mg of allopurinol RS and transfer into a 200-ml volumetric flask, add 10 ml of 0.1 M sodium hydroxide R and sonicate for 2 min, continue to shake mechanically for about 10 min and add to volume with 0.01 M hydrochloric acid R. Dilute the resulting solution with 0.01 M hydrochloric acid R to obtain a solution having a known concentration of about 8 µg/ml. Tolerance: Not less than 75% (Q) of the labeled amount of allopurinol, C5H4N4O, is dissolved in 45 min. Assay Test solution: Weigh 20 tablets, calculate the average masse and powder finely. Weigh accurately a quantity of the powder containing about 0.1 g of allopurinol and transfer into a 250-ml volumetric flask, add 20 ml of 0.05 M sodium hydroxide R and shake for 20 min, add 80 ml of 0.1 M hydrochloric acid R, shake for 10 min, dilute to volume with 0.1 M hydrochloric acid R. Mix, filter and discard the first portion of filtrate. Dilute 5.0 ml of the filtrate to 250.0 ml with 0.1 M hydrochloric acid R, and mix. Reference solution: Weigh accurately about 100 mg of allopurinol RS and transfer into a 250-ml volumetric flask, add 20 ml of 0.05 M sodium hydroxide R, shake to dissolve and dilute to volume with 0.1 M hydrochloric acid R. Dilute 5.0 ml of the resulting solution to 250.0 ml with 0.1 M hydrochloric acid R, and mix. Measure the absorbance (Appendix 4.1) of the obtained solutions at 250 nm in a 1-cm cell, using 0.1 M hydrochloric acid R as the blank. Calculate the content of allopurinol, C5H4N4O, using the absorbances of the test solution, the reference solution and the declared content of C5H4N4O in allopurinol RS. Storage Store in an airtight container, in a cool place, protected from light.

DRIED ALUMINIUM HYDROXIDE

Action and use Treatment of gout and hyperuricemia. Usual strength 50 mg; 100 mg; 300 mg. DRIED ALUMINIUM HYDROXIDE Aluminii hydroxydum siccum Dried aluminium hydroxide is hydrated aluminium oxide. It contains not less than 47.0% and not more than 60.0% of Al2O3 (M. 102.0).

Characters A white, amorphous powder. Practically insoluble in water, it dissolves in dilute mineral acids and in solutions of alkali hydroxides. Identification Solution S: Dissolve 1.25 g of the substance to be examined in 7.5 ml of hydrochloric acid R by heating on a water bath. Dilute to 50 ml with water. Solution S gives the reaction of aluminium (Appendix 8.1). Appearance of solution Solution S is not more opalescent than reference suspension (II) (Appendix 9.2) and not more intensely coloured than reference solution GY6 (Appendix 9.3, method 2). Alkaline impurities Shake 1.0 g of the substance to be examined with 20 ml of carbon dioxide-free water R for 1 min and filter. To 10 ml of the filtrate add 0.1 ml of phenolphthalein solution R. Any pink colour disappears on the addition of 0.3 ml of 0.1 N hydrochloric acid VS. Neutralising capacity Carry out the test at 37 °C. Dissolve 0.5 g of the substance to be examined in 100 ml of water, heat, add 100.0 ml of 0.1 N hydrochloric acid VS previously heated and stir continuously; the pH of the solution after 10 min, 15 min and 20 min is not less than 1.8, 2.3 and 3.0 respectively and is at no time greater than 4.5. Add 10.0 ml of 0.5 N hydrochloric acid VS previously heated, stir continuously for 1 h and titrate with 0.1 N sodium hydroxide VS to pH 3.5; not more than 35.0 ml of 0.1 N sodium hydroxide VS is required. Chlorides Not more than 1% (Appendix 9.4.5). Dissolve 0.1 g of the substance to be examined with heating in 10 ml of dilute nitric acid R and dilute to 100 ml with water. 5 ml of this solution diluted to 15 ml with water complies with the limit test for Chlorides. 33

VP V

DRIED ALUMINIUM PHOSPHATE

Sulfates Not more than 1% (Appendix 9.4.14). Dilute 4 ml of solution S to 100 ml with water. 15 ml of the resulting solution complies with the limit test for Sulfates. Arsenic Not more than 4 ppm (Appendix 9.4.2). 10 ml of solution S complies with limit test A for Arsenic. Heavy metals Not more than 60 ppm (Appendix 9.4.8). Neutralise 10 ml of solution S with concentrated ammonia R, using metanil yellow solution R as an external indicator. Filter, if necessary, and dilute to 15 ml with water. 12 ml of this solution complies with limit test 1 for heavy metals. Prepare the standard using 10 ml of lead standard solution (1 ppm Pb) R. Microbial contamination Total aerobic microbial count: Not more than 103 CFU/g, determined by plate-count (Appendix 13.6). Absence of Enterobacteria, certain other gram-negative bacteria and Escherichia coli (Appendix 13.6). Assay Dissolve 0.800 g of the substance to be examined in 10 ml of a 25% solution of hydrochloric acid R by heating on a water bath. Allow to cool and dilute to 50.0 ml with water. To 10.0 ml of the resulting solution, add 6 M ammonia until a precipitate begins to appear. Add the smallest quantity of dilute hydrochloric acid R needed to dissolve the precipitate and dilute to 20 ml with water. Carry out the complexometric titration of aluminium (Appendix 10.5). 1 ml of 0.1 M sodium edetate VS is equivalent to 5.098 mg of Al2O3. Storage Store in an airtight container, at a temperature not exceeding 30 ºC. Action and use Antacid.

DRIED ALUMINIUM PHOSPHATE Aluminii phosphas siccum M. 122.0 (Anhydrous)

Dried aluminium phosphate consists mainly of hydrated aluminium phosphate. It contains not less than 94.0% and not more than 102.0% of AlPO4, calculated with reference to the ignited substance. 34

Identification Solution S: Dissolve 2.00 g in 2 M hydrochloric acid R and dilute to 100 ml with the same acid. Solution S gives the reaction of aluminium and reaction B of phosphates (Appendix 8.1). Appearance of solution Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2).

pH The pH of a 4% suspension of the substance to be examined in carbon dioxide-free water R is 5.5 to 7.2 (Appendix 6.2). Neutralising capacity Add 0.50 g to 30 ml of 0.1 N hydrochloric acid VS previously heated to 37 °C and maintain at this temperature for 15 min while stirring. The pH of the mixture after 15 min at 37 °C is 2.0 to 2.5 (Appendix 6.2). Arsenic Not more than 1 ppm (Appendix 9.4.2). 1.0 g complies with limit test for arsenic, method A. Heavy metals Not more than 20 ppm (Appendix 9.4.8). Dissolve 1.0 g in dilute hydrochloric acid R and dilute to 20 ml with the same solvent. 12  ml of the solution complies with limit test 1 for heavy metals. Prepare the standard using lead standard solution (1 ppm Pb) R. Chlorides Not more than 1.3% (Appendix 9.4.5). Dissolve 50.0 mg in 10 ml of dilute nitric acid R and dilute to 200 ml with water. 15  ml of the solution complies with the limit test for Chlorides. Sulfates Not more than 0.6% (Appendix 9.4.14). Dilute 8 ml of solution S to 100 ml with water. 15 ml of the solution complies with the limit test for Sulfates.

Preparations Tablets, suspention.

AlPO4, xH2O

Characters A white or almost white powder. Very slightly soluble in water, practically insoluble in ethanol (96%). It dissolves in dilute solutions of mineral acids and alkali hydroxides.

Soluble phosphate Not more than 1.0%, calculated as PO43-. Test solution: Stir 5.0 g with 150 ml of water for 2 h. Filter and wash the filter with 50 ml of water. Combine the filtrate and the washings and dilute to 250.0 ml with water. Dilute 10.0 ml of this solution to 100.0 ml with water. Reference solution (1): Dissolve 2.86 g of potassium dihydrogen phosphate R in water and dilute to 100 ml with the same solvent. Reference solution (2): Dilute 1 ml of reference solution (1) to 5 ml with water.

VP V

ALVERINE CITRATE

Reference solution (3): Dilute 3 ml of reference solution (1) to 5 ml with water. Procedure: To 5.0 ml of each solution add 4 ml of 1 M sulfuric acid R, 1 ml of ammonium molybdate solution R, 5 ml of water and 2 ml of a solution containing 0.10 g of 4-methylaminophenol sulfate R, 0.5 g of anhydrous sodium sulfite R and 20.0 g of sodium metabisulfite R in 100 ml of water. Mix and allow to stand for 15 min. Dilute to 25.0 ml with water and allow to stand for a further 15 min. Measure the absorbance (Appendix 4.1) at 730 nm. Calculate the content of soluble phosphates from a calibration curve prepared using reference solutions (1), (2) and (3).

Loss on ignition 10.0% to 20.0%. (1.000 g, at 800 °C, ignite to constant mass). Assay Dissolve 0.400 g in 10 ml of 2 M hydrochloric acid R and dilute to 100.0 ml with water. To 10.0 ml of the solution, add 10.0 ml of 0.1 M sodium edetate VS and 30 ml of a mixture of equal volumes of ammonium acetate solution R and dilute acetic acid R. Boil for 3 min, then cool. Add 25 ml of ethanol (96%) R and 1 ml of a freshly prepared 0.025% solution of dithizone R in ethanol (96%) R. Titrate the excess of sodium edetate with 0.1 M zinc sulfate VS until the colour changes to pink. 1 ml of 0.1 M sodium edetate VS is equivalent to 12.20 mg of AlPO4. Storage Store in an airtight container. Action and use Antacid. ALVERINE CITRATE Alverini citras

C20H27N,C6H8O7  

Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of alverine citrate RS. pH 3.5 to 4.5 for a 0.5% solution of the substance to be examined in carbon dioxide-free water R (Appendix 6.2). Related substances Examine by gas chromatography (Appendix 5.2). Use freshly prepared solutions. Test solution: Dissolve 0.250 g of the substance to be examined in water and dilute to 20 ml with the same solvent. Add 2 ml of ammonia R and shake with 3 quantities, each of 15 ml, of methylene chloride R. To the combined lower layers add anhydrous sodium sulfate R, shake, filter, and evaporate the filtrate at a temperature not exceeding 30 °C, using a rotary evaporator. Take up the residue with methylene chloride R and dilute to 10.0 ml with the same solvent. Reference solution (1): Dissolve 5 mg of alverine impurity D RS (impurity D citrate) in 5 ml of water, add 1 ml of ammonia R and shake with 3 quantities, each of 5 ml, of methylene chloride R. To the combined lower layers add anhydrous sodium sulfate R, shake, filter, and evaporate the filtrate at a temperature not exceeding 30 °C, using a rotary evaporator. Take up the residue with methylene chloride R, add 0.2 ml of the test solution and dilute to 2 ml with methylene chloride R. Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with methylene chloride R. Dilute 1.0 ml of this solution to 20.0 ml with methylene chloride R. Reference solution (3): Dissolve the contents of a vial of alverine for peak identification RS (containing impurities C and E) in 1 ml of methylene chloride R. Chromatographic system: A fused-silica capillary column (25 m long and 0.32 mm in internal diameter) coated with poly(dimethyl)(diphenyl) siloxane R (film thickness 0.45 µm). Carrier gas: Helium for chromatography R. Flow rate: 2.2 ml/min. Split ratio: 1 : 11. Temperature programme:

M. 473.6

Alverine citrate is N-ethyl-3-phenyl-N-(3-phenylpropyl) propan-1-amine dihydrogen 2-hydroxypropane-1,2,3tricarboxylate. It contains not less than 99.0% and not more than 101.0% of C20H27N,C6H8O7, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder, melting point is about 104 °C. Slightly soluble in water and in methylene chloride, sparingly soluble in ethanol (96%).

Column

Time (min)

Temperature (°C)

0-7 7 - 13 13 - 21 21 - 24 24 - 39

120 120 → 240 240 240 → 290 290

Injection port

290

Detector

290

35

VP V

ALVERINE CAPSULES

Detector: A flame-ionisation detector. Volume of injection: 1 µl. Procedure: Identification of impurities: Use the chromatogram supplied with alverine for peak identification RS and the chromatogram obtained with reference solution (3) to identify the peaks due to impurities C and E. Relative retention with reference to alverine (retention time = about 16 min): Impurity A = about 0.28; impurity B = about 0.29; impurity C = about 0.46; impurity D = about 0.97; impurity E = about 1.7. System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to impurity D and alverine is at least 3.0. Limits: Impurities A, B: For each impurity, not more than 0.1%. Impurity C: Not more than 0.2%. Impurities D, E: For each impurity, not more than 0.3%. Any other impurity: For each impurity, not more than 0.10%. Total: Not more than 1.0%. Disregard any peak with an area less than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Note: Impurity A: 1-chloro-3-phenylpropane. Impurity B: 3-phenylpropan-1-ol. Impurity C: N-ethyl-3-phenylpropan-1-amine. Impurity D: N-(3-cyclohexylpropyl)-N-ethyl-3-phenylpropan1-amine. Impurity E: 3-phenyl-N,N-bis(3-phenylpropyl)propan-1-amine.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 0.5 g complies with limit test for heavy metals, method 7. Prepare the standard using 1 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 80 °C; 2 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.375 g of the substance to be examined in 50 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 47.36 mg of C20H27N,C6H8O7. Storage Store in an airtight container, protected from light. Action and use Smooth muscle relaxant; antispasmodic. 36

Preparation Capsules. ALVERINE CAPSULES Capsulae Alverini Alverine capsules contain alverine citrate. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements. Content of alverine citrate, C20H27N,C6H8O7, 95.0% to 105.0% of the stated amount.

Identification A. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel GF254. Mobile phase: Chloroform - methanol (90 : 10). Test solution: Shake a quantity of the capsule contents, equivalent to about 100 mg of alverine citrate, with 10 ml of methanol R and filter. Reference solution: A 1% solution of alverine citrate RS in methanol R. Procedure: Apply separately to the plate 20 µl of each solution. After removal of the plate, allow it to dry in air and examine under ultraviolet light (254 nm). The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of 0.1 M hydrochloric acid R Rotation speed: 50 rpm. Time: 45 min. Procedure: Examine by liquid chromatography (Appendix 5.3). Mobile phase and Chromatographic system: Prepare as directed in the Assay. Test solution: After the specified time, withdraw about 20 ml of the medium and filter, discarding the first portion of the filtrate. Dilute the filtrate, if necessary, with 0.1 M hydrochloric acid R to obtain a solution containing about 0.006% of alverine citrat. Reference solution: A 0.006% solution of alverine citrate RS in 0.1 M hydrochloric acid R. Calculate the total content of alverine citrate, C20H27N,C6H8O7, dissolved in the medium using the areas of the principal peaks in the chromatograms obtained with the test solution, the reference solution and the declared content of C20H27N,C6H8O7 in alverine citrate RS.

VP V

AMBROXOL HYDROCHLORIDE

Tolerance: Not less than 70% (Q) of the labelled amount of alverine citrate, C20H27N,C6H8O7, is dissolved in 45 min.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: 0.01 M sodium lauryl sulphate in a mixture of acetonitrile and water (55 : 45), adjusted to pH 3.0 with phosphoric acid R. Test solution: Weigh 20 capsules, calculate the average weight of the capsule contents and powder finely. Weigh accurately a quantity of the powder, equivalent to about 0.300 g of alverine citrate, add 100 ml of methanol R, sonicate for 60 min, dilute with the same solvent to 250.0 ml, and mix for 10 minutes and filter (0.45 µm Cellulose nitrate filters are suitable). Dilute 10.0 ml of the filtrate to 20.0 ml with water, and mix. Reference solution: Dilute 10.0 ml of a 0.12% solution of alverine citrate RS in methanol R to 20.0 ml with water, and mix. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 µm) (Hypersil BDS C18 is suitable). Detector: A spectrophotometer set at 220 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution, the relative standard deviation for 6 replicate injections is not more than 2.0% Separately inject the reference solution and the test solution. Calculate the content of alverine citrate, C20H27N,C6H8O7, using the areas of the principal peaks in the chromatograms obtained with the reference solution, the test solution and the declared content of C20H27N,C6H8O7 in alverine citrate RS. Storage Store in an airtight container, in a cool place, protected from light. Action and use Antispasmodic. Usual strength 40 mg; 60 mg. AMBROXOL HYDROCHLORIDE Ambroxoli hydrochloridum

Ambroxol hydrochloride is trans-4-[(2-amino-3,5dibromobenzyl)amino]cyclohexanol hydrochloride. It contains not less than 99.0% and not more than 101.0% of C13H18Br2N2O,HCl, calculated with reference to the dried substance.

Charaters White or yellowish, crystalline powder. Sparingly soluble in water, soluble in methanol, practically insoluble in methylene chloride. Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of ambroxol hydrochloride RS. B. Dissolve 20.0 mg of the substance to be examined in 0.05 M sulfuric acid R and dilute to 100.0 ml with the same acid. Dilute 2.0 ml of this solution to 10.0 ml with 0.05 M sulfuric acid R. The ultraviolet absorptiom spectrum (Appendix 4.1) of the this solution in the range between 200 nm and 350 nm exhibits two absorption maxima at 245 nm and at 310 nm. The ratio of the absorbances measured at the maxima at 245 nm and 310nm (A245 nm /A310 nm) is 3.2 to 3.4. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Concentrated ammonia - 2-propanol - ethyl acetate - hexane (1 : 10 : 20 : 70). Test solution: Dissolve 50 mg of the substance to be examined in methanol R and dilute to 5 ml with the same solvent. Reference solution: Dissolve 50 mg of ambroxol hydrochloride RS in methanol R and dilute to 5 ml with the same solvent. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 2/3 of the plate. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. D. Dissolve 25 mg in 2.5 ml of water, mix with 1.0 ml of ammonia solution, dilute R and allow to stand for 5 min. Filter and acidify the filtrate with dilute nitric acid R. The filtrate gives reaction (A) of chlorides (Appendix 8.1). Appearance of solution Solution S: Dissolve 0.75 g in methanol R and dilute to 15 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution Y6 (Appendix 9.3, method 2).

C13H18Br2N2O,HCl  

M: 414.6

pH 4.5 to 6.0 (Appendix 6.2). 37

VP V

AMBROXOL HYDROCHLORIDE CAPSULES

Dissolve 0.2 g in carbon dioxide-free water and dilute to 20 ml with the same solvent.

Impurity D: cis-4-[(2-amino-3,5-dibromobenzyl)amino]cyclohexanol. Impurity E: 2-Amino-3,5-dibromobenzaldehyde.

Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use. Mobile phase: A mixture of equal volumes of acetonitrile R and a solution prepared as follows: Dissolve 1.32 g of ammonium phosphate R in 900 ml of water, adjust to pH 7.0 with phosphoric acid R and dilute to 1000 ml with water. Test solution: Dissolve 50 mg of the substance to be examined in water and dilute to 50.0 ml with the same solvent. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with water. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (2): In order to prepare impurity B in situ, dissolve 5 mg of the substance to be examined in 0.2 ml of methanol R, add 0.04 ml of a mixture of 1 volume of formaldehyde solution R and 99 volumes of water. Heat at 60 °C for 5 min. Evaporate to dryness under a current of nitrogen. Dissolve the residue in 5 ml of water and dilute to 20.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.0 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 248 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Procedure: Inject the blank, reference solutions (1), (2) and the test solution. The run time is 3 times the retention time of ambroxol. Relative retention with reference to ambroxol (retention time = about 9 min): Impurity B = about 0.6. Identification of impurities: Use the chromatogram obtained with reference solution (2) to identify the peak due to impurity B. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurity B and ambroxol is at least 4.0. Limits: In the chromatogram obtained with the test solution: Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). The sum of the peak areas of all impurities is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.3%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%).

Heavy metals

Note: Impurity A: (2-Amino-3,5-dibromophenyl)methanol. Impurity B: trans-4-(6,8-dibromo-1,4-dihydroquinazolin3(2H)-yl)cyclohexanol. Impurity C: trans-4-[[(E)-2-amino-3,5-dibromobenzyliden] amino]cyclohexanol.

38

Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R.

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.300 g of the substance to be examined in 70 ml of ethanol (96%) R and add 5 ml of 0.01 N hydrochloric acid VS. Carry out a potentiometric titration (Appendix 10.2), using 0.1 N sodium hydroxide VS. Read the volume of 0.1 N sodium hydroxide VS added between the 2 points of inflexion. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 41.46 mg of C13H19Br2ClN2O. Storage Store in an airtight container, protected from light. Action and use Mucolytic expectorant. Preparations Tablets, capsules. AMBROXOL HYDROCHLORIDE CAPSULES Capsulae Ambroxoli hydrochloridi Ambroxol hydrochloride capsules contain ambroxol hydrochloride. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements:

Content of ambroxol hydrochloride, C13H18Br2N2O, HCl, 95.0% to 105.0% of the stated amount. Identification A. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that of the principal peak in the chromatogram obtained with the reference solution. B. The ultraviolet light absorption spectrum (Appendix 4.1) of the resulting solution obtained in the Dissolution exhibits two absorption maxima at 244 nm and 308 nm.

VP V

Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 900 ml of 0.1 M hydrochloric acid R. Rotation speed: 100 rpm. Time: 30 min. Procedure: After the specified time, withdraw a sample of the medium, filter. Dilute the filtrate with the medium to obtain a solution containing 15 µg of ambroxol hydrochloride per ml. Measure the absorbance of the resulting solution at 244 nm (Appendix 4.1), in a 1 cm cell, using the medium as the blank. Calculate the content of ambroxol hydrochloride, C13H18Br2N2O,HCl, dissolved using the absorbance of a solution of ambroxol hydrochloride RS in the medium having the same concentration as the test solution. Tolerance: Not less than 80% (Q) of the stated amount of ambroxol hydrochloride, C13H18Br2N2O,HCl, is dissolved in 30 min. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase, chromatographic system: Prepare as directed in the Assay. Test solution: Transfer an accurately weighed quantity of the powder containing the equivalent of about 100 mg of ambroxol hydrochloride to a 100 ml volumetric flask, add 70 ml of the mobile phase and sonicate to dissolve. Dilute to volume with the same solvent and mix well, filter. Reference solution: Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Procedure: Inject the reference solution and the test solution. The run time is twice the retention time of the principal peak. In the chromatogram obtained with the test solution, the sum of the areas of any secondary peaks (disregard any peaks corresponding to the peaks in the chromatogram obtained with the blank) is not greater than the area of the principal peak in the chromatogram obtained with the reference solution (1.0%). Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - 0.01 M diammonium hydrogen phosphate adjusted to pH 7.0 with phosphoric acid (50 : 50). Reference solution: Dissolve an accurately weighed quantity of about 30 mg of ambroxol hydrochloride RS in the mobile phase and dilute to 50.0 ml with the mobile phase. Dilute 5.0 ml of the solution to 100.0 ml with the mobile phase. Test solution: Weigh 20 capsules, calculate the average weight of the capsule contents and finely powder. Transfer an accurately weighed quantity of the powder containing the equivalent of about 30 mg of ambroxol hydrochloride to a 50 ml volumetric flask, add 30 ml of the mobile phase and shake thoroughly to dissolve, dilute to volume with the mobile phase, mix well and filter. Dilute 5.0 ml of the filtrate to 100.0 ml with the mobile phase.

AMBROXOL HYDROCHLORIDE TABLETS

Resolution solution: Dissolve 10 mg of ambroxol hydrochloride RS in 0.2 ml of methanol R, add 0.04 ml of a mixture of formaldehyde - water (1 : 99). Heat in a water bath at 60 °C for 30 minutes. Evaporate the solution to dryness under a stream of nitrogen. Dissolve the residue in 5 ml of water and dilute to 20.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 μm). Detector: A spectrophotometer set at 248 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 μl. Procedure: System suitability: Inject the resolution solution, the relative retention time of the peak due to degradation product (Impurity B) with reference to ambroxol peak (retention time = 9 minutes) is about 0.6; the resolution factor between the peaks due to impurity B and ambroxol is at least 4.0. Inject alternately the reference solution and the test solution. Calculate the content of ambroxol hydrochloride, C13H18Br2N2O,HCl, in the capsules using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C13H18Br2N2O,HCl in ambroxol hydrochloride RS.

Storage Store in an airtight container, protected from light. Action and use Mucolytic expectorant. Usual strength 30 mg. AMBROXOL HYDROCHLORIDE TABLETS Tabellae Ambroxoli hydrochloridi Ambroxol hydrochloride tablets contain ambroxol hydrochloride. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements:

Content of ambroxol hydrochloride, C13H18Br2N2O, HCl, 95.0% to 105.0% of the stated amount. Identification A. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that of the pricipal peak in the chromatogram obtained with the reference solution. B. The ultraviolet absorption spectrum (Appendix 4.1) of the resulting solution obtained in the Dissolution exhibits two absorption maxima, at 244 nm and 308 nm. 39

VP V

AMIKACIN

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of 0.1 M hydrochloric acid R. Rotation speed: 75 rpm. Time: 30 min. Procedure: After the specified time, withdraw a sample of the medium, filter. Dilute the filtrate with the medium to obtain a solution containing about 15 µg of ambroxol hydrochloride per ml. Measure the absorbance (Appendix 4.1) of the resulting solution at 244 nm, in a 1 cm cell, using the medium as the blank, Calculate the content of ambroxol hydrochloride, C13H18Br2N2O,HCl, dissolved using the absorbances obtained with a reference solution of ambroxol hydrochloride RS in the medium having the same concentration as the test solution. Tolerances: Not less than 75% (Q) of the stated amount of ambroxol hydrochloride, C13H18Br2N2O,HCl, is dissolved in 30 minutes. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase and Chromatographic system: Prepare as directed in the Assay. Test solution: Transfer an accurately weighed quantity of the powdered tablets containing the equivalent of 100 mg of ambroxol hydrochloride to a 100 ml volumetric flask, add 70 ml of the mobile phase and sonicate to dissolve. Dilute to volume with the mobile phase and mix well, filter. Reference solution: Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Procedure: Inject the reference solution and the test solution. The run time is twice the retention time of the principal peak. In the chromatogram obtained with the test solution, the sum of the areas of any secondary peaks (disregard any peaks corresponding to the peaks in the chromatogram obtained with the blank) is not greater than the area of the principal peak in the chromatogram obtained with the reference solution (1.0%). Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - 0.01 M diammonium hydrogenphosphate adjusted to pH 7.0 with phosphoric acid (50 : 50). Reference solution: Dissolve an accurately weighed quantity of about 30 mg of ambroxol hydrochloride RS in 50.0 ml of the mobile phase. Dilute 5.0 ml of the solution to 100.0 ml with the mobile phase. Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Transfer an accurately weighed quantity of the powdered tablets containing the equivalent of 30 mg of ambroxol hydrochloride to a 50 ml volumetric flask, 40

add 30 ml of the mobile phase, shake to dissolve. Dilute to volume with the mobile phase, mix and filter. Dilute 5.0 ml of the filtrate to 100.0 ml with the mobile phase. Resolution solution: Dissolve 10 mg of ambroxol hydrochloride RS in 0.2 ml methanol R, add 0.04 ml of a mixture of formaldehyde - water (1 : 99). Heat in a water bath at 60 °C for 30 minutes. Evaporate the solution to dryness under a stream of nitrogen. Dissolve the residue in 5 ml of water and dilute to 20.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 μm). Detector: A spectrophotometer set at 248 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 μl. Procedure: System suitability: Inject the resolution solution, the relative retention time of the peak due to degradation product (Impurity B) with reference to ambroxol peak (retention time = 9 minutes) is about 0.6; the resolution factor between the peaks due to impurity B and ambroxol is at least 4.0. Inject alternately the reference solution and the test solution. Calculate the content of ambroxol hydrochloride, C13H18Br2N2O,HCl, in the tablets using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C13H18Br2N2O,HCl in ambroxol hydrochloride RS.

Storage Store in an airtight container, protected from light. Action and use Mucolytic expectorant. Usual strength 30 mg. AMIKACIN Amikacinum

C22H43N5O13 

M. 585.6

VP V

Amikacin is 6-O-(3-amino-3-deoxy-α-D-glucopyranosyl)4-O-(6-amino-6-deoxy-α-D-glucopyranosyl)-1-N-[(2S)4-amino-2-hydroxybutanoyl]-2-deoxy-D-streptamine, an antimicrobial substance obtained from kanamycin A. It contains not less than 96.5% and not more than 102.5% of C22H43N5O13, calculated with reference to the anhydrous substance.

Characters A white or almost white powder. Sparingly soluble in water, slightly soluble in methanol, practically insoluble in acetone and in ethanol (96%). Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of amikacin RS. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel R. Mobile phase: Using the lower layer of a mixture of equal volumes of concentrated ammonia R, methanol R and methylene chloride R (shake well and allow to stand for separation). Test solution: Dissolve 25 mg of the substance to be examined in water and dilute to 10 ml with the same solvent. Reference solution (1): Dissolve 25 mg of amikacin RS in water and dilute to 10 ml with the same solvent. Reference solution (2): Dissolve 5 mg of kanamycin monosulfate RS in 1 ml of the test solution and dilute to 10 ml with water. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in air and spray with ninhydrin solution R. Heat at 110 °C for 5 min. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). The identification test is not valid unless the chromatogram obtained with reference solution (2) shows two clearly separated spots. pH Dissolve 0.1 g in carbon dioxide-free water R and dilute to 10 ml with the same solvent. The pH of the solution is 9.5 to 11.5 (Appendix 6.2). Specific optical rotation +97º to +105º, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.50 g in water and dilute to 25.0 ml with the same solvent. Related substances Examine by liquid chromatography (Appendix 5.3). Chromatographic system are described in the test for Assay. Inject reference solution (1). Adjust the sensitivity of

AMIKACIN

the system so that the height of the principal peak in the chromatogram obtained is at least 50% of the full scale of the recorder. System suitability: In the chromatogram obtained with the resolution solution, the resolution between the peaks corresponding to amikacin and impurity A is at least 3.5. The run time is four times the retention time of amikacin. Limits: In the chromatogram obtained with test solution (1): The area of any peak corresponding to impurity A is not greater than the area of the principal peak in the chromatogram obtained with reference solution (1) (1%). The area of any other impurity peak, is not greater than half the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). The sum of the areas of any secondary peaks (including the peak corresponding to impurity A) is not greater than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (1.5%). Disregard any peak due to the blank and any peak with an area less than 0.1 times that of the principal peak in the chromatogram obtained with reference solution (1).

Water Not more than 8.5% (Appendix 10.3). Determined on 0.20 g. Sulfated ash Not more than 0.5% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: A 0.27% solution of potassium dihydrogen phosphate adjusted to pH 6.5 with a 2.2% solution of potassium hydroxide - methanol (30 : 70) Test solution (1): Dissolve 0.100 g of the substance to be examined in water and dilute to 10.0 ml with the same solvent. In a round-glass-stoppered vial, add 0.2 ml of this solution to 2.0 ml of a 1% solution of 2,4,6-trinitrobenzene sulfonic acid. Then add 3.0 ml of pyridine R and close the vial tightly. Shake vigorously for 30 s and heat in a waterbath at 75 °C for 45 min. Cool in cold water for 2 min and add 2 ml of glacial acetic acid R. Shake vigorously for 30 s. Test solution (2): Dissolve 50.0 mg of the substance to be examined in water and dilute to 50.0 ml with the same solvent. Then prepare as prescribed for test solution (1), from “In a round-glass-stoppered vial”. Reference solution (1): Dissolve 10.0 mg of amikacin impurity A RS in water and dilute to 100.0 ml with the same solvent. Then prepare as prescribed for test solution (1), from “In a round-glass-stoppered vial”. Reference solution (2): Dissolve 50.0 mg of amikacin RS in water and dilute to 50.0 ml with the same solvent. Then prepare as prescribed for test solution (1), from “In a round-glass-stoppered vial”. 41

VP V

AMIKACIN INJECTION

Resolution solution: Dissolve 5 mg of amikacin RS and 5 mg of amikacin impurity A RS in water and dilute to 50 ml with the same solvent. Then prepare as prescribed for test solution (1), from “In a round-glass-stoppered vial”. Blank solution: Prepare as described for test solution (1) using 0.2 ml of water. Maintaining the temperature of the solutions to be examined at 10 °C. Chromatographic system: A column (25 cm × 4.6 mm) packed with octadecylsilyl silica gel for chromatography R (5 µm). Column temperature: 30 °C. Detector: A spectrophotometer set at 340 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Inject reference solution (2). Adjust the sensitivity of the system so that the height of the principal peak is at least 50% of the full scale of the recorder. System suitability: In the chromatogram obtained with reference solution (2), the relative standard deviation of the areas of amikacin peaks for 6 replicate injections is not more than 2.0%. Calculate the content of C22H43N5O13 from the peak areas due to amikacin in the chromatograms obtained with test solution (2) and reference solution (2).

Mobile phase: Chloroform - methanol - ammonia - water (1 : 4 : 2 : 1). Solvent mixture: Methanol - diethylamine (95 : 5). Test solution: Dilute a volume of the injection with water to obtain a solution containing 2.5 mg of amikacin sulfate per ml. Reference solution (1): Dissolve 25 mg of amikacin sulfate RS in water and dilute to obtain 10 ml with the same solvent. Reference solution (2): Dissolve 5 mg of kanamycin monosulfate RS in 1 ml of the test solution and dilute to obtain 10 ml with water. Procedure: Apply separately to the plate 5 µl of the test solution, reference solution (1), reference solution (2). Develop over a path of about 15 cm. Remove the plate, allow it to dry in air and spray with a ninhydrin solution R, heat in an oven at 110 °C for 5 min. In the chromatogram obtained with the test solution, the principal spot is similar in position and size to the principal spot in the chromatogram obtained with reference solution (1). The test is not valid unless in the chromatogram obtained with reference solution (2), two principal spots are completely separated. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of the amikacin peak in the chromatogram obtained with the reference solution. C. The injection yields reaction of sulfate (Appendix 8.1).

Storage Store in an airtight container, protected from light.

pH 3.5 to 5.5 (Appendix 6.2).

Action and use Aminoglycosid antibiotic.

Bacterial endotoxins (Appendix 13.2) Dilute the injection with BET water to obtain a solution having a concentration of amikacin of 10 mg per 1 ml (solution A). The endotoxin limit concentration of solution A is 3.3 EU per 1ml.

Preparation Powder for injection. AMIKACIN INJECTION Injectio Amikacini Amikacin injection is a sterile solution of amikacin sulfate in water for injections. The injection complies with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements:

Content of amikacin, C22H43N5O13, 90.0% to 110.0% of the stated amount. Characters A clear and colourless to yellowish solution. Identification A. Examine by Thin layer chromatography (Appendix 5.4). Coating substance: Silica gel. 42

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Solution A - acetonitrile (98 : 2). Solution A: A mixture prepared in free-carbon dioxide water R containing 0.18% of sodium octanesulfonate R, 2% of anhydrous sodium sulfate R, 5.8% (v/v) of acetonitrile R1 and 5% (v/v) of 0.2 M potassium dihydrogen phosphate R, adjusted the pH to 3.0 with diluted phosphoric acid R. Test solution: Dilute a volume of the injection with the mobile phase to obtain a solution having a concentration of amikacin of 0.5 mg/ml. Reference solution: Weigh accurately about 65 mg of amikacin sulfate RS, transfer into a 100-ml volumetric flask, dissolve and dilute to volume with the mobile phase. Chromatographic system: A column (15 cm × 4.6 mm) packed with end-capped octadecylsylyl silica gel (5 µm). Column temperature is maintained at 40 °C.

VP V

AMINOCAPROIC ACID

Detector: A spectrophotometer set at 200 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Record the chromatogram for 1.3 times the retention time of amikacin. Procedure: System suitability: Inject the reference solution, in the chromatogram obtained, the symmetry factor of the amikacin peak is not more than 2.0. The relative standard deviation of the areas of the peak for six replicate injections is not more than 1.5%. Inject the reference solution and the test solution. Calculate the content of amikacin, C22H43N5O13, in the injection using the areas of the principal peaks in the chromatograms obtained with the reference solution, the test solution and the declared content of C22H43N5O13 in amikacin sulfate RS. 1 mg of C22H43N5O13, 2H2SO4 is equivalent to 0.7488 mg of C22H43N5O13.

B. Examine the chromatograms obtained in the test for ninhydrin-positive substances. The principal spot in the chromatogram obtained with the test solution (2) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). C. Dissolve 0.5 g of the substance to be examined in 4 ml of a mixture of equal volumes of dilute hydrochloric acid R and water. Evaporate the solution obtained on a waterbath to dryness. Dry the residue in a desiccator. Dissolve the residue in about 2 ml of boiling ethanol R. Allow to cool and maintain at 4 °C to 8 °C for 3 h. Filter under reduced pressure. Wash the residue with about 10 ml of acetone R and dry at 60 °C for 30 min. The residue melts (Appendix 6.7) at 131 °C to 133 °C. D. Dissolve about 5 mg of the substance to be examined in 0.5 ml of water. Add 3 ml of dimethylformamide R and 2 ml of ascorbic acid solution R. Heat on a water-bath. An orange colour develops.

Storage Store in a tight container, in a cool place, protected from light.

Appearance of solution Solution S: Dissolve 10.0 g in carbon dioxide-free water R and dilute to 50.0 ml with the same solvent. Solution S is colourless (Appendix 9.3, method 2) and remains clear (Appendix 9.2) on standing for 24 h.

Action and use Aminoglycoside antibacterial.

pH The pH of solution S is 7.5 to 8.0 (Appendix 6.2).

Usual strength Stated in terms of amikacin base: 50 mg/1 ml; 250 mg/ml (2 ml, 4 ml).

Light absorption A. The absorbance (Appendix 4.1) of solution S at 287 nm is not more than 0.10 and at 450 nm is not more than 0.03. B. Place 2.0 g of the substance to be examined in an even layer in a shallow dish 9 cm in diameter, cover and allow to stand at 98 °C to 102 °C for 72 h. Dissolve in water and dilute to 10.0 ml with the same solvent. The absorbance (Appendix 4.1) of the solution obtained at 287 nm is not more than 0.15 and at 450 nm is not more than 0.03.

AMINOCAPROIC ACID Acidum aminocaproicum

C6H13NO2  

M.131.2 

Aminocaproic acid is 6-aminohexanoic acid, it contains not less than 98.5% and not more than 101.0% of C6H13NO2, calculated with reference to the dried substance.

Characters A white, or almost white crystalline powder or colourless crystals. Freely soluble in water, slightly soluble in ethanol (96%). It melts at about 205 °C with decomposition. Identification Apply one of the two following identifications: First identification: A. Second identification: B, C, D. A. The infrared absorption spectrophotometry (Appendix 4.2) of the substance to be examined is concordant with the spectrum of aminocaproic acid RS.

Ninhydrin-positive substances Not more than 0.5%. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel R. Mobile phase: Glacial acetic acid - water - butanol (20 : 20 : 60). Test solution (1): Dissolve 0.10 g of the substance to be examined in water and dilute to 10 ml with the same solvent. Test solution (2): Dilute 1 ml of test solution (1) to 50 ml with water. Reference solution (1): Dissolve 10 mg of aminocaproic acid RS in water and dilute to 50 ml with the same solvent. Reference solution (2): Dilute 5 ml of test solution (2) to 20 ml with water. Reference solution (3): Dissolve 10 mg of aminocaproic acid RS and 10 mg of leucine RS in water and dilute to 25 ml with the same solvent. Procedure: Apply separately to the plate 5 µl of each solution. Allow the plate to dry in air. Develop over a path of 15 cm. Dry the plate in a current of warm air. Spray 43

VP V

AMINOPHYLLINE

with ninhydrin solution R and heat at 100 °C to 105 °C for 15 min. Any spot in the chromatogram obtained with test solution (1), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (2). The test is not valid unless the chromatogram obtained with reference solution (3) shows two clearly separated principal spots.

Heavy metals Not more than 10 ppm (Appendix 9.4.8). 12 ml of solution S complies with limit test for heavy metals, method 1. Prepare the standard using lead standard solution (2 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appedix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.100 g of the substance to be examined in 20 ml of anhydrous acetic acid R. Use 0.1 ml of crystal violet solution R as indicator, titrate with 0.1 N perchloric acid VS until the colour changes from bluish-violet to bluish-green. 1 ml of 0.1 N perchloric acid VS is equivalent to 13.12 mg of C6H13NO2. Action and use Antifibrinolytic; haemostatic. Preparations Injection, tablets.

M. 420.4

Aminophylline is a stable mixxture of theophylline and ethylenediamine. It contains: 84.0% to 87.4% of theophylline (C7H8N4O2) and 13.5% to 15.0% of ethylenediamine (C2H8N2), calculated with reference to the anhydrous substance.

Characters White or slightly yellowish powder, sometimes granular, hygroscopic. Freely soluble in water (the solution becomes 44

Identification Apply one of the two following identifications: First identification: A, C, E. Second identification: B, C, D, E, F. Dissolve 1.0 g of the substance to be examined in 10 ml of water and add 2 ml of dilute hydrochloric acid R dropwise with shaking. Filter. Use the precipitate for identification tests A, B, D and F and the filtrate for identification test C. A. The infrared absorption spectrum (Appendix 4.2) of the precipitate washed with water and dried at 105 °C is concordant with the spectrum of theophylline RS. B. Melting point: 270 °C to 274 °C (Appendix 6.7), determined after washing the precipitate with water and drying at 105 °C. C. To the filtrate add 0.2 ml of benzoyl chloride R, make alkaline with dilute sodium hydroxide solution R and shake vigorously. Filter the precipitate, wash with 10 ml of water, dissolve in 5 ml of hot ethanol (96%) R and add 5 ml of water. A precipitate is formed, which, when washed and dried at 105 °C, melts at 248 °C to 252 °C (Appendix 6.7). D. Heat about 10 mg of the precipitate with 1.0 ml of a 36% solution of potassium hydroxide R in a water-bath at 90 °C for 3 min, then add 1.0 ml of diazotised sulfanilic acid solution R. A red colour slowly develops. Carry out a blank test. E. It complies with the test for Water. F. The precipitate gives the reaction of xanthines (Appendix 8.1). Appearance of solution Dissolve 0.5 g with gentle warming in 10 ml of carbon dioxide-free water R. The solution is not more opalescent than reference suspension II (Appendix 9.2) and not more intensely coloured than reference solution GY6 (Appendix 9.3, method 2).

AMINOPHYLLINE Aminophyllinum Theophylline ethylenediamine

C16H24N10O4  

cloudy through absorption of carbon dioxide), practically insoluble in anhydrous ethanol.

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - a 0.136% solution of sodium acetate R containing 0.50% (v/v) of glacial acetic acid R (7 : 93). Test solution: Dissolve 47 mg (for anhydrous substance) or 50 mg (for hydrate substance) of the substance to be examined in the mobile phase and dilute to 20.0 ml with the mobile phase. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (2): Dissolve 10 mg of theobromine R (impurity G) in the mobile phase, add 5 ml of the test solution and dilute to 100 ml with the mobile phase. Dilute 5 ml of this solution to 50 ml with the mobile phase.

VP V

Chromatographic system: A column (25 cm × 4 mm) packed with stationary phase C (7 µm). Detector: A spectrophotometer set at 272 nm. Flow rate: 2.0 ml/min. Volume of injection: 20 µl. Procedure: The run time is 3.5 times the retention time of theophylline. Relative retention with reference to theophylline (retention time = about 6 min): impurity G = about 0.6. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurity G and theophylline is at least 2.0 Limits: Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). The sum of the peak areas of all impurities is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A: 1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione (caffeine). Impurity B: 3-methyl-3,7-dihydro-1H-purine-2,6-dione. Impurity C: N-(6-amino-1,3-dimethyl-2,4-dioxo-1,2,3,4tetrahydropyrimidin-5-yl)formamide. Impurity D: N-methyl-5-(methylamino)-1H-imidazole-4carboxamide. Impurity E: 1,3-dimethyl-7,9-dihydro-1H-purine-2,6,8(3H)-trione. Impurity F: 7-(2-hydroxyethyl)-1,3-dimethyl-3,7-dihydro-1Hpurine-2,6-dione (etofylline). Impurity G: 3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione (theobromine).

Heavy metals Not more than 20 ppm (Appendix 9.4.8). Use water as the solvent. 0.500 g complies with limit test for heavy metals, method 8. Prepare the standard using 1 ml of lead standard solution (10 ppm Pb) R. The substance precipitates after addition of acetate buffer solution pH 3.5 R. Dilute to 100 ml with water, the substance re-dissolves completely. Water Not more than 1.5% (For anhydrous substance) (Appendix 10.3). 3.0% to 8.0% (For hydrate substance) (Appendix 10.3). Determined on 0.50 g. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g.

AMINOPHYLLINE INJECTION

Assay Ethylenediamine Dissolve 0.250 g of the substance to be examined in 30 ml of water. Add 0.1 ml of bromocresol green solution R. Titrate with 0.1 N hydrochloric acid VS until a green colour is obtained. 1 ml of 0.1 N hydrochloric acid VS is equivalent to 3.005 mg of C2H8N2. Theophylline Heat 0.200 g of the substance to be examined to constant mass in an oven at 135 °C. Dissolve the residue with heating in 100 ml of water, allow to cool, add 20 ml of 0.1 M silver nitrate R and shake. Add 1 ml of bromothymol blue solution R. Titrate with 0.1 N sodium hydroxide VS. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 18.02 mg of C7H8N4O2. Storage Store in an airtight container, protected from light. Action and use Stretch of trachea-bronchi, stretch of blood vessel achalasia, diuretic, treatment of asthma. Preparations Injection, tablets. AMINOPHYLLINE INJECTION Injectio Aminophyllini Aminophylline injection is a sterile solution of aminophylline or aminophylline hydrate in water for injections free from carbon dioxide. The injection complies with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Content of ethylenediamine, C2H8N2, not more than 0.295 g for each g of anhydrous theophylline, C7H8N4O2, determined in the Assay for theophylline. Content of theophylline, C7H8N4O2, 81.4% to 90.0% of the stated amount of aminophylline. Characters A clear solution. Identification A. To a volume containing about 0.1 g of aminophylline add 0.5 ml of 2 M hydrochloric acid R with constant stirring, allow to stand for a few minutes and filter. Wash the residue with small quantities of cold water, and dry at 105 °C. The infrared absorption spectrum of the residue (Appendix 4.2) is concordant with the reference spectrum of theophylline or the spectrum of theophylline RS. 45

AMINOPHYLLINE TABLETS

B. To a volume containing about 0.1 g of aminophylline add 2 ml of a 1% solution of copper (II) sulfate R and shake. A purplish blue colour is produced. C. Evaporate a volume containing about 60 mg of aminophylline to dryness in a porcelain dish. To the residue add 1 ml of hydrochloric acid R and 0.1 g of potassium chlorate R and evaporate to dryness. A reddish residue is produced, which becomes purple on exposure to the vapour of 5 M ammonia R.

pH 8.8 to 10.0 (Appendix 6.2). Assay For theophylline To an accurately measured volume containing 0.1 g of aminophylline add sufficient 0.01 M sodium hydroxide R to produce 250.0 ml, and mix. Dilute 5.0 ml to 250.0 ml with 0.01 M sodium hydroxide R, and mix. Measure the absorbance of the resulting solution at the maximum at 275 nm (Appendix 4.1), using 0.01 M sodium hydroxide R in the reference cell. Calculate the content of theophylline, C7H8N4O2, taking 650 as the value of A (1%, 1 cm) at the maximum at 275 nm. For ethylenediamine To an accurately measured volume containing 0.5 g of aminophylline in a 100 ml conical flask, and if necessary, add sufficient water to produce 20 ml and titrate with 0.05 M sulfuric acid VS, using 0.1 ml of bromocresol green solution R as indicator, until the colour changes from blue to green. Each ml of 0.05 M sulfuric acid VS is equivalent to 3.005 mg of C2H8N2. Calculate the weight of C2H8N2 present for each g of theophylline, C7H8N4O2, found in the Assay of theophylline. Storage Store in an airtight container, in a cool place, protected from light. Action and use Treatment of asthma. Usual strength 0.240 g/10 ml. AMINOPHYLLINE TABLETS Tabellae Aminophyllini Aminophylline tablets contain aminophylline. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of theophylline, C7H8N4O2, 81.4% to 90.0% of the stated amount of aminophylline. 46

VP V

Content of ethylenediamine, C2H8N2, 10.9% to 12,1% of the stated amount of aminophylline. Identification Shake a quantity of the powdered tablets containing 0.5 g of aminophylline with 20 ml of water, filter, add to the filtrate with constant stirring 1 ml of 2 M hydrochloric acid R, allow to stand for a few minutes and again filter. Reserve the residue for test A and test B, and the filtrate for test C. Wash the residue with small quantities of cold water, recrystallise from hot water and dry at 105°C. A. The melting point of the residue is about 271°C (Appendix 6.7). B. The infrared absorption spectrum (Appendix 4.2) of the residue obtained in test A is concordant with the reference spectrum of theophylline or the spectrum of theophylline RS. C. To the filtrate reserved add 0.2 ml of benzoyl chloride R, make alkaline with 5 M sodium hydroxide R and shake vigorously. Filter, wash the residue with cold water and recrystallise from a mixture of 1 volume of water and 3 volumes of ethanol (96%) R. The melting point of the crystals, after drying at 100°C, is about 250°C (Appendix 6.7). D. Shake a quantity of the powdered tablets containing about 0.25 g of aminophylline with 5 ml of water and filter. To 2 ml of the filtrate add 2 ml of a 1% solution of copper (II) sulfate R and shake. A purplish blue colour is produced. Dissolution for theophylline (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of phosphate buffer pH 7.0 R. Rotation speed: 50 rpm. Time: 45 min. Procedure: Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol - water (45 : 55). Test solution: After the specified time, withdraw a sample of the medium and filter, discarding the first 20 ml of the filtrate. Dilute 10.0 ml of the filtrate with phosphate buffer pH 7.0 R, if necessary, to obtain a solution having a same concentration with reference solution. Reference solution: Dissolve about 50 mg of theophylline RS, accurately weighed, in phosphate buffer pH 7.0 R, dilute to 100.0 ml with the same solvent, and mix. Dilute 1.0 ml of this solution to 50.0 ml with phosphate buffer pH 7.0 R, and mix. Chromatographic system: A column (10 cm × 4.6 mm) packed with particles of silica the surface of which has been modified with chemicallybonded phenyl groups (5 µm) (Apex Phenyl is suitable). Detector: A spectrophotometer set at 273 nm. Flow rate: 2.0 ml/min. Volume of injection: 20 µl. Inject separately the reference solution and the test solutions. Calculate the total content of theophylline, C7H8N4O2, in the medium using the areas of the principal peaks in

VP V

AMIODARONE HYDROCHLORIDE

the chromatograms obtained with the reference solution, the test solution and the declared content of C7H8N4O2 in theophylline RS. Tolerance: Not less than 70% (Q) of the amount of theophylline (C7H8N4O2), calculated from the labelled amount of aminophylline, is dissolved in 45 minutes.

Assay Weigh 20 tablets, calculate the average mass and finely powder. For theophylline Shake an accurately weighed quantity of the powder containing about 80 mg of aminophylline with a mixture of 20 ml of 0.1 M sodium hydroxide R and 60 ml of water for 10 min, add sufficient water to produce 200.0 ml, and mix. Filter, discard the first 20 ml of the filtrate. Dilute 5.0 ml of the filtrate to 250.0 ml with 0.01 M sodium hydroxide R, and mix. Measure the absorbance of the resulting solution at the maximum at 275 nm (Appendix 4.1) in a 1-cm cell, using 0.01 M sodium hydroxide R in the reference cell. Calculate the content of theophylline, C7H8N4O2, taking 650 as the value of A (1%, 1 cm) at the maximum at 275 nm. For ethylenediamine Shake an accurately weighed quantity of the powder containing about 0.3 g of aminophylline with 20 ml of water in a 100-ml conical flask, heat to 50 °C for 30 min and titrate with 0.05 M sulfuric acid VS, using 0.1 ml of bromocresol green solution R1 as indicator, until the colour changes from blue to green. Each ml of 0.05 M sulfuric acid VS is equivalent to 3.005 mg of C2H8N2.

Storage Store in an airtight container, in a cool place, protected from light. Action and use Treatment of asthma. Usual strength 100 mg. AMIODARONE HYDROCHLORIDE Amiodaroni hydrochloridum

C25H29I2NO3,HCl

M. 681.8

Amiodarone hydrochloride is (2-butylbenzofuran-3-yl)[4-[2-(diethylamino)ethoxy]-3,5-diiodophenyl] methanone hydrochloride. It contains not less than 98.5% and not more

than 101.0% of C25H29I2NO3,HCl, calculated with reference to the dried substance.

Characters White or almost white, fine, crystalline powder. Very slightly soluble in water, freely soluble in methylene chloride, soluble in methanol, sparingly soluble in ethanol (96%). Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum obtained with amiodarone hydrochloride RS. B. It gives reaction (B) of chlorides (Appendix 8.1). Appearance of solution Dissolve 1.0 g in methanol R and dilute to 20 ml with the same solvent. The solution is clear (Appendix 9.2) and not more intensely coloured than reference solution GY5 or BY5 (Appendix 9.3, method 2). pH 3.2 to 3.8 (Appendix 6.2). Dissolve 1.0 g in carbon dioxide-free water R, heating at 80 °C, cool and dilute to 20 ml with the same solvent. Impurity H Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel F254. Mobile phase: Anhydrous formic acid - methanol methylene chloride (5 : 10 : 85). Prepare the solutions immediately before use and keep protected from bright light. Test solution: Dissolve 0.500 g of the substance to be examined in methylene chloride R and dilute to 5.0 ml with the same solvent. Reference solution (1): Dissolve 10.0 mg of (2-chloroethyl) diethylamine hydrochloride R (impurity H) in methylene chloride R and dilute to 50.0 ml with the same solvent. Dilute 2.0 ml of the solution to 20.0 ml with methylene chloride R. Reference solution (2): Mix 2.0 ml of the test solution and 2.0 ml of reference solution (1). Procedure: Apply separately to the plate 50 µl of the test solution and reference solution (1); 100 µl of reference solution (2). Develop over a path of 15 cm. Allow the plate to dry in air. Spray with potassium iodobismuthate solution R1 and then with dilute hydrogen peroxide solution R; examine immediately in daylight. The test is not valid unless the chromatogram obtained with reference solution (2) shows the spot due to impurity H is clearly visible. In the chromatogram obtained with the test solution, any spot with the same Rf as the spot due to impurity H is not more intense than the spot in the chromatogram obtained with reference solution (1) (0.02%). 47

VP V

AMIODARONE HYDROCHLORIDE

Related substances Examine by liquid chromatography (Appendix 5.3) Mobile phase: Buffer solution pH 4.9 - methanol acetonitrile (30 : 30 : 40). Buffer solution pH 4.9: Add 3.0 ml of glacial acetic acid R to 800 ml of water, adjust to pH 4.9 with dilute ammonia R and dilute to 1000 ml with water. Test solution: Dissolve 0.125 g of the substance to be examined in a mixture of equal volumes of acetonitrile R and water and dilute to 25.0 ml with the same mixture of solvents. Reference solution: Dissolve 5 mg of amiodarone impurity D RS, 5 mg of amiodarone impurity E RS and 5.0 mg of amiodarone hydrochloride RS in methanol R and dilute to 25.0 ml with the same solvent. Dilute 1.0 ml of the solution to 20.0 ml with a mixture of equal volumes of acetonitrile R and water. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 µm). Column temperature: 30 °C. Detector: A spectrophotometer set at 240 nm. Flow rate: 1 ml/min. Volume of injection: 10 µl. Procedure: Run time: Twice the retention time of amiodarone. Relative retention with reference to amiodarone (retention time = about 24 min): impurity A = about 0.26; impurity D = about 0.29; impurity E = about 0.37; impurity B = about 0.49; impurity C = about 0.55; impurity G = about 0.62; impurity F = about 0.69. The test is not valid unless in the chromatogram obtained with the reference solution, resolution between the peaks corresponding to impurities D and E is at least 3.5. Limits: In the chromatogram obtained with the test solution: Impurities A, B, C, D, E, F, G: For each impurity, the area is not more than the area of the peak due to amiodarone in the chromatogram obtained with the reference solution (0.2%). Any other impurity: For each impurity, the area is not more than 0.5 times the area of the peak due to amiodarone in the chromatogram obtained with the reference solution (0.1%). The sum of the peak areas of all impurities is not more than 2.5 times the area of the peak due to amiodarone in the chromatogram obtained with the reference solution (0.5%). Disregard any peak with an area less than 0.25 times the area of the peak due to amiodarone in the chromatogram obtained with the reference solution (0.05%). Note: Impurity A: (2-butylbenzofuran-3-yl)[4-[2-(diethylamino)ethoxy] phenyl]methanone. Impurity B: (2-butylbenzofuran-3-yl)[4-[2-(ethylamino)ethoxy] -3,5-diiodophenyl]methanone.

48

Impurity C: 2-butylbenzofuran-3-yl)[4-[2-(diethylamino)ethoxy] -3-iodophenyl]methanone. Impurity D: (2-butylbenzofuran-3-yl)(4-hydroxy-3,5-diiodophenyl) methanone. Impurity E: (2-butylbenzofuran-3-yl)(4-hydroxyphenyl)methanone. Impurity F: (2-butylbenzofuran-3-yl)(4-hydroxy-3-iodophenyl) methanone. Impurity G: 4-[2-(diethylamino)ethoxy]-3,5-diiodophenyl][2[(1RS) -1methoxybutyl]benzofuran-3-yl]methanone. Impurity H: 2-chloro-N,N-diethylethanamine (2-chlorotriethylamine (2chloroethyl)diethylamine).

Iodides Not more than 0.015%. Prepare the test and reference solutions simultaneously. Solution A: Add 1.50 g of the substance to be examined to 40 ml of water at 80 °C and shake until completely dissolved. Cool and dilute to 50.0 ml with water. Test solution: Add 1.0 ml of 0.1 M hydrochloric acid and 1.0 ml of 0.05 M potassium iodate to 15.0 ml of solution A. Dilute to 20.0 ml with water. Allow to stand protected from light for 4 h. Reference solution: Add 1.0 ml of 0.1 M hydrochloric acid R, 1.0 ml of an 88.2 mg/L solution of potassium iodide R and 1.0 ml of 0.05 M potassium iodate R to 15.0 ml of solution A. Dilute to 20.0 ml with water. Allow to stand protected from light for 4 h. Procedure: Measure the absorbances (Appendix 4.1) of the solutions at 420 nm, using a mixture of 15.0 ml of solution A and 1.0 ml of 0.1 M hydrochloric acid R diluted to 20.0 ml with water as the blank. The absorbance of the test solution is not greater than half the absorbance of the reference solution. Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2.0 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 50 °C; at a pressure not exceeding 0.3 kPa; 4h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.600 g in a mixture of 5.0 ml of 0.01 N hydrochloric acid VS and 75 ml of ethanol (96%) R. Carry out a potentiometric titration (Appendix 10.2) using 0.1 N sodium hydroxide VS. Read the volume added between the 2 points of inflexion. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 68.18 mg of C25H29I2NO3, HCl.

VP V

Storage Store in an airtight container, protected from light, at a temperature not exceeding 30 °C. Action and use Antiarrhythmic. Potassium channel blocker. Preparation Tablets. AMIODARONE TABLETS Tabellae Amiodaroni Amiodarone tablets contain amiodarone hydrochloride. The tablets comply with the requirements stated under “Tablets “(Appendix 1.20) and with the following requirements:

Content of amiodarone hydrochloride, C25H29I2NO3, HCl, from 95.0% to 105.0% of the stated amount. Identification A. Shake a quantity of finely powdered tablets containing about 0.1 g amiodarone hydrochloride in 50 ml of ethanol (96%) R, shake well and filter. Use the filtrate for the following tests: Dilute 0.1 ml of the filtrate with 20 ml of ethanol (96%) R, the ultraviolet absorption spectrum (Appendix 4.1) of the solution in the range 230 nm to 350 nm exhibits a maximum at 242 nm and a minimum at 223 nm. The ratio of the absorbance measured at 242 nm to that at 223 nm is 1.47 to 1.61. To 5 ml of the filtrate add 10% ammonia solution R, a turbidity is produced, filter. Add 10% ammonia solution R until no further turbidity is formed, filter through a 0.45-µm filter. To 2 ml of the filtrate, acidify with a 32% solution of nitric acid R (examine by indicator paper), add 2 ml 0.1 M silver nitrate R, a white precipitate is formed and it is dissolved in 10% ammonia solution R, continue to add a 32% solution of nitric acid R and the precipitate is produced again. B. In the Assay, the principal peak in the chromatogram obtained with the test solution is similar to that of amiodarone hydrochloride peak in the chromatogram obtained with the reference solution. Related substances Examine by liquid chromatography (Appendix 5.3) Mobile phase: Buffer solution pH 4.9 - methanol - acetonitrile (30 : 30 : 40). Buffer solution pH 4.9: To 800 ml of water add 3.0 ml of glacial acetic acid R, adjust the pH to 4.9 with 10% ammonia solution R and add sufficient water to produce 1000.0 ml. Diluent: A mixture of acetonitrile and water (1 : 1).

AMIODARONE TABLETS

Test solution: Weigh accurately a quantity of fine powdered tablets containing about 25 mg of amiodarone hydrochloride and transfer into a 50-ml volumetric flask, add 40 ml of the diluent, sonicate for 15 min, allow to cool, and dilute to volume with the diluent, mix and filter. Reference solution: Transfer accurately 1.0 ml of test solution into a 200-ml volumetric flask, dilute to volume with a mixture of acetonitrile R and water (1 : 1). Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5µm). Detector: A spectrophotometer set at 240 nm. Flow rate: 1.5 ml/min. Volume of injection: 10 µm. Procedure: Inject the diluent. Inject the reference solution, in the chromatogram obtained, the column efficiency determined from the principal peak is not less than 7000 theoretical plates. Inject the test solution, allow the chromatography proceed for 2.5 times the retention time of the peak due to amiodarone hydrochloride. In the chromatogram obtained with the test solution, the sum of the areas of any secondary peaks is not greater than the area of the principal peak in the chromatogram obtained with the reference solution (0.5%).

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 1000 ml of a 0.25% solution of sodium lauryl sulfate R. Rotation speed: 75 rpm. Time: 45 min. Procedure: Test solution: After the specified time, withdraw a sample of the medium and filter, discard the first 20 ml of the filtrate. Dilute a suitable volume of the filtrate to obtain the solution having the same concentration as the reference solution. Reference solution: Weigh accurately about 20 mg of amiodarone hydrochloride RS, transfer into a 200-ml volumetric flask, dissolve and dilute to volume with ethanol (96%) R. Dilute 5 ml of this solution to 50.0 ml with the medium. Measure the absorbances of the reference solution and the test solution (Appendix 4.1) at 243nm, use the medium as the blank. Calculate the content of amiodarone hydrochloride, C25H29I2NO3,HCl dissolved, using the absorbances of the reference solution, the test solution and the declared content of C25H29I2NO3,HCl in amiodarone hydrochloride RS. Tolerance: Not less than 70% (Q) of the labeled amount of amiodarone hydrochloride, C25H29I2NO3,HCl, is dissolved in 45 min. Assay Examine by liquid chromatography (Appendix 5.3). 49

VP V

AMITRIPTYLINE HYDROCHLORIDE

Mobile phase, chromatographic system are described in the test for Related substances. Reference solution: Weigh accurately about 20 mg of amiodarone hydrochloride, transfer into a 200-ml volumetric flask, dissolve and dilute to volume with a mixture of acetonitrile R and water (1 : 1). Test solution: Weigh 20 tablets, calculate the average mass and finely powder. Weigh accurately a quantity of powdered tablets containing about of 20 mg of amiodarone hydrochloride, transfer into a 200-ml volumetric flask, add 170 ml of a mixture of acetonitrile and water (1 : 1), sonicate for 15 min to dissolve, allow it to cool and dilute to volume with the same solvent, shake and filter. Procedure: Inject separately the reference solution and the test solution into chromatographic system. Column efficiency determined on the principal peak is not less than 3000 theoretical plates. Calculate the content of amiodarone hydrochloride, C25H29I2NO3,HCl, using the peak areas in the chromatograms obtained with the reference solution, the test solution and the declared content of amiodarone hydrochloride RS.

Storage Store in a well-closed container, in a cool place, protected from light. Action and use Potassium channel blocker, antiarrhythmic class III. Usual strength 200 mg. AMITRIPTYLINE HYDROCHLORIDE Amitriptylini Hydrochloridum

C20H23N,HCl 

M. 313.9

Amitriptyline hydrochloride is 3-(10,11-dihydro-5Hdibenzo[a,d][7]annulen-5-ylidene)-N,N-dimethylpropan1-amine hydrochloride. It contains not less than 99.0% and not more than 101.0% of C20H23N,HCl, calculated with reference to the dried substance.

Characters White or almost white powder or colourless crystals. Freely soluble in water, in ethanol (96%) and in methylene chloride. 50

Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of amitriptyline hydrochloride RS. B. 20 mg of the substance to be examined gives reaction (A) of chlorides (Appendix 8.1). Appearance of solution Dissolve 1.25 g in water and dilute to 25 ml with the same solvent. The solution is clear (Appendix 9.2) and not more intensely coloured than reference solution B7 (Appendix 9.3, method 2). Acidity or alkalinity Dissolve 0.20 g in carbon dioxide-free water R and dilute to 10 ml with the same solvent. Add 0.1 ml of methyl red solution R and 0.2 ml of 0.01 M sodium hydroxide R. The solution is yellow. Add 0.4 ml of 0.01 M hydrochloric acid R. The solution is red. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - buffer solution 7.0 (35 : 65). Buffer solution 7.0: Dissolve 5.23 g of dipotassium hydrogen phosphate R in 1000 ml water, adjust to pH 7.0 with phosphoric acid R. Test solution: Dissolve 50.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Reference solution (1): Dissolve 5.0 mg of dibenzosuberone RS (impurity A) and 5.0 mg of cyclobenzaprine hydrochloride RS (impurity B) in 5.0 ml of the test solution and dilute to 100.0 ml with the mobile phase. Reference solution (2): Dilute 1.0 ml of reference solution (1) to 50.0 ml with the mobile phase. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase end-capped polar-embedded octadecylsilyl amorphous organosilica polymer R (5 µm). Column temperature: 40 °C. Detector: A spectrophotometer set at 220 nm. Flow rate: 1.2 ml/min. Volume of injection: 10 µl. Procedure: The run time is 3 times the retention time of amitriptyline. Relative retention with reference to amitriptyline (retention time = about 14 min): impurity B = about 0.9; impurity A = about 2.2. System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to impurity B and amitriptyline is at least 2.0. Limits: Impurity B: The area of the peak due to impurity B is not more than the area of the corresponding peak in the chromatogram obtained with reference solution (2) (0.1%).

VP V

Impurity A: The area of the peak due to impurity A is not more than 0.5 times the area of the corresponding peak in the chromatogram obtained with reference solution (2) (0.05%). Any other impurity: For each impurity, the area is not more than the area of the peak due to amitriptyline in the chromatogram obtained with reference solution (2) (0.10%). The sum of the peak areas of all impurities is not more than 3 times the area of the peak due to amitriptyline in the chromatogram obtained with reference solution (2) (0.3%). Disregard any peak with an area less than 0.5 times the area of the peak due to amitriptyline in the chromatogram obtained with reference solution (2) (0.05%). Note: Impurity A: 10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-one (dibenzosuberone). Impurity B: 3-(5H-dibenzo[a,d][7]annulen-5-ylidene)-N,Ndimethylpropan-1-amine (cyclobenzaprine). Impurity C: 3-(10,11-dihydro-5H-dibenzo[a,d][7]annulen-5ylidene)-N-methylpropan-1-amine (nortriptyline). Impurity D: 5-[3-(dimethylamino)propyl]-10,11-dihydro-5Hdibenzo[a,d][7]annulen-5-ol. Impurity E: N,N-dimethyl-3-(1,2,3,4,4a,10,11,11a-octahydro5H-dibenzo[a,d][7]annulen-5-ylidene)propan-1-amine. Impurity F: (5EZ,10RS)-5-[3-(dimethylamino)propylidene]-10, 11-dihydro-5H-dibenzo[a,d][7]annulen-10-ol. Impurity G: 10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-ol (dibenzosuberol).

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 6. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C; 2 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.250 g of the substance to be examined in 30 ml of ethanol (96%) R. Titrate with 0.1 N sodium hydroxide VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N sodium hydroxide VS is equivalent to 31.39 mg of C20H24ClN. Storage Protected from light. Action and use Monoamine reuptake inhibitor; tricyclic antidepressant. Preparation Tablets.

AMITRIPTYLINE TABLETS

AMITRIPTYLINE TABLETS Tabellae Amitriptylini Amitriptyline tablets contain amitriptyline hydrochloride. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of amitriptyline hydrochloride, C20H23N,HCl, 90.0% to 110.0% of the stated amount. Identification A. Shake a quantity of finely powdered tablets, equivalent to about 10 mg of amitriptyline hydrochloride in 100 ml methanol R. Filter a portion of this solution, and dilute 10 ml of the filtrate with methanol R to 100 ml. The UV absorption spectrum of this solution exhibits a maximum at the same wavelength as that of a similar solution of amitriptyline hydrochloride RS, concomitantly measured. B. In the Assay, retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 900 ml of 1 M hydrocloric acid R. Rotation speed: 100 rpm. Time: 45 min. Procedure: Test solution: After the specified time, withdraw a sample of the medium and filter, discard 20 ml of the first portion of the filtrate. Dilute the filtrate with medium to obtain the solution having a suitable concentration, if necessary. Reference solution: A solution of amitriptyline hydrochloride RS in medium has the same concentration as the test solution. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum at 239 nm, in a 1 cm cell, using medium as a blank. Calculate the content of amitriptyline hydrochloride, C20H23N,HCl, dissolved using the absorbances obtained with the test solution and the reference solution and the declared content of C20H23N,HCl in amitriptyline hydrochloride RS. Tolerance: Not less than 75% (Q) of the stated amount of amitriptyline hydrochloride, C20H23N,HCl is dissolved in 45 min. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: A mixture of acetonitrile R and phosphate buffer (42 : 58). Phosphate buffer: Dissolve 11.04 g of sodium dihydrogen phosphat monohydrat R in 900 ml of water, adjust with phosphoric acid R to a pH of 2.5 ± 0.5, dilute with water to make 1000 ml. 51

VP V

AMLODIPINE BESILATE

Reference solution: Dissolve an accurately weighed quantity of amitriptyline hydrochloride RS in mobile phase to obtain a solution having a concentration of about 0.2 mg of amitriptyline hydrochloride per ml. Test solution: Weigh 20 tablets and powder finely. Weigh accurately a quantity of powdered tablets containing the equivalent to about 20 mg amitriptyline hydrochloride, to a 100 ml volumetric flask, add about 70 ml of mobile phase and shake well, dilute to volume with mobile phase, mix and filter. Chromatographic system: A column (30 cm × 4,0 mm) packed with stationary phase C (3 µm or 10 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 2.0 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject 6 times of the reference solution. The tailing factor is not more than 2.0, the column efficiency is not less than 800 theoretical plates, and the relative standard deviation for peak areas of amitriptyline hydrochloride is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of amitriptyline hydrochloride, C20H23N,HCl, in tablets using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C20H23N,HCl in amitriptyline hydrochloride RS.

Storage Protected from light. Action and use Tricyclic antidepressant. Usual strength 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg. AMLODIPINE BESILATE Amlodipini besilas

and enantiomer C20H25ClN2O5,C6H6O3S 

M. 567.1 

Amlodipine besilate is 3-ethyl 5-methyl (4RS)-2-[(2aminoethoxy)methyl]-4-(2-chlorophenyl)-6-methyl-1,4dihydropyridine-3,5-dicarboxylate benzenesulfonate. It 52

contains not less than 97.0% and not more than 102.0% of C20H25ClN2O5,C6H6O3S, calculated with reference to the anhydrous substance.

Characters White or almost white powder. Slightly soluble in water, freely soluble in methanol, sparingly soluble in anhydrous ethanol, slightly soluble in 2-propanol. Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of amlodipine besilate RS. Optical rotation -0.10° to + 0.10° (Appendix 6.4). Dissolve 0.250 g in methanol R and dilute to 25.0 ml with the same solvent. Related substances Examine by liquid chromatography (Appendix 5.3). Carry out the test protected from light. Mobile phase: A 0.23% solution of amonium acetate methanol (30 : 70). Test solution (1): Dissolve 50.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Test solution (2): Dilute 5.0 ml of test solution (1) to 100.0 ml with the mobile phase. Reference solution (1): Dilute 1.0 ml of test solution (1) to 10.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 100.0 ml with the mobile phase. Reference solution (2): Dissolve 2.5 mg of amlodipine impurity B RS and 2.5 mg of amlodipine impurity G RS in the mobile phase and dilute to 25.0 ml with the mobile phase. Dilute 1.0 ml of the solution to 10.0 ml with the mobile phase. Reference solution (3): Dissolve 2.5 mg of amlodipine for peak identification RS (containing impurities D, E and F) in 5 ml of the mobile phase. Reference solution (4): Dissolve 5.0 mg of amlodipine impurity A RS in acetonitrile R and dilute to 5.0 ml with the same solvent. Dilute 1.0 ml of the solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (5): Dissolve 50.0 mg of amlodipine besilate RS in the mobile phase and dilute to 50.0 ml with the mobile phase. Dilute 5.0 ml of the solution to 100.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.0 mm) packed with stationary phase C (5 µm). Column temperature: 30 °C. Detector: A spectrophotometer set at 237 nm. Flow rate: 1.5 ml/min.

VP V

Volume of injection: 20 µl. Procedure: Inject test solution (1) and reference solution (1), (2), (3) and (4). The run time is twice the retention time of amlodipine. Identification of impurities: Use the chromatogram supplied with amlodipine for peak identification RS and the chromatogram obtained with reference solution (3) to identify the peaks due to impurities D, E and F; use the chromatogram obtained with reference solution (4) to identify the peak due to impurity A. Relative retention with reference to amlodipine (retention time = about 20 min): impurity G = about 0.21; impurity B = about 0.25; impurity D = about 0.5; impurity F = about 0.8; impurity E = about 1.3. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurities G and B is at least 2.0. Limits: Correction factors: For the calculation of content, multiply the peak areas of the following impurities by the corresponding correction factor: impurity D = 1.7; impurity F = 0.7. Impurity D: The corrected area of the peak due to impurity D is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.3%). Impurity A: The area of the peak due to impurity A is not more than 1.5 times the area of the corresponding peak in the chromatogram obtained with reference solution (4) (0.15%). Impurities E, F: For each impurity, the corrected area, if necessary, is not more than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.15%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). Sum of all the impurities: Not more than 0.8%. Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%); disregard any peak due to benzene sulfonate (Relative retention with reference to amlodipine = about 0.14). Note: Impurity A: 3-ethyl 5-methyl (4RS)-4-(2-chlorophenyl)-2-[[2(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethoxy]methyl]-6methyl-1,4-dihydropyridine-3,5-dicarboxylate. Impurity B: 3-ethyl 5-methyl (4RS)-4-(2-chlorophenyl)-6methyl-2-[[2-[[2(methylcarbamoyl)benzoyl]amino]ethoxy] methyl]-1,4-dihydropyridine-3,5- dicarboxylate. Impurity D: 3-ethyl 5-methyl 2-[(2-aminoethoxy)methyl]-4-(2chlorophenyl)-6-methylpyridine-3,5-dicarboxylate. Impurity E: diethyl (4RS)-2-[(2-aminoethoxy)methyl]-4-(2chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate.

AMLODIPINE TABLETS Impurity F: dimethyl (4RS)-2-[(2-aminoethoxy)methyl]-4-(2chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate. Impurity G: dimethyl 4-(2-chlorophenyl)-2,6-dimethyl-1,4dihydropyridine-3,5- dicarboxylate. Impurity H: 2-[[2-[[(4RS)-4-(2-chlorophenyl)-3-(ethoxycarbonyl)5-(methoxycarbonyl)-6-methyl-1,4-dihydropyridin-2-yl]methoxy] ethyl]carbamoyl]benzoic acid.

Water Not more than 0.5% (Appendix 10.3). Determined on 1.000 g. Sulfated ash Not more than 0.2% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. Inject test solution (2), reference solution (5). Calculate the content of C26H31ClN2O8S using the areas of the peaks in the chromatograms obtained with test solution (2), reference solution (5) and the declared content of C26H31ClN2O8S in amlodipine besilate RS. Storage Store in an airtight container, protected from light. Action and use Calcium channel blocker. Preparations Tablets, capsules. AMLODIPINE TABLETS Tabellae Amlodipini Amlodipine tablets contain amlodipine besilate. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of amlodipine, C20H25ClN2O5, 90.0% to 110.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: The upper layer of a mixture of glacial acetic acid - water - methyl isobutyl ketone (25 : 25 : 50). Test solution: Shake a quantity of the powdered tablets containing 10 mg of amlodipine with 2 ml of methanol R, centrifuge and use the supernatant liquid. Reference solution: Dissolve amlodipine besilate RS in methanol R to obtain a solution containing 5 mg of amlodipine RS in 1 ml of methanol R. 53

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AMLODIPINE TABLETS

Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm. After removal of the plate, allow to dry in air and examine under ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution corresponds in position, colour and size to that in the chromatogram obtained with the reference solution. B. In the Assay, retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution.

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase and chromatographic system as directed in the Assay. Test solution: Weigh 20 tablets and powder finely. Weigh accurately a quantity of powdered tablets containing the equivalent to 50 mg of amlodipine, dissolve in the mobile phase and dilute to 50.0 ml with the mobile phase. Centrifuge, use a portion of the clear supernatant. Reference solution: Dilute 5.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 5.0 ml of the solution to 50.0 ml with the mobile phase. Resolution solution: Dissolve 5 mg of amlodipine besilate RS in 5 ml of strong hydrogen peroxide solution R. Heat at 70 °C for 45 min. Procedure: The relative retention with reference to amlodipine of impurity D is about 0.5. Inject the resolution solution. The test is not valid unless the resolution between the peaks due to amlodipine and impurity D is at least 4.5. Inject the test solution and the reference solution. Run time of the test solution is 3 times the retention time of amlodipine. Limits: In the chromatogram obtained with the test solution, the area of peak corresponding to impurity D multiplied by 2 is not more than the area of the principal peak in the chromatogram obtained with the reference solution (0.5%). Total peak area of all other impurities is not more than the area of the principal peak in the chromatogram obtained with reference solution (0.5%). Disregard any peak due to benzene sulfonate (relative retention = about 0.2) and any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (0.05%). Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of 0.01 M hydrochloric acid R. Rotation speed: 75 rpm. Time: 45 min. Procedure: 54

Test solution: After the specified time, withdraw a sample of the medium, and filter, discard the first portion of the filtrate. Reference solution: A solution of amlodipine besilate RS in medium has the same concentration as expected in the test solution. Measure the absorbances (Appendix 4.1) of the solutions at the maximum at 239 nm, in a 1 cm cell, using 0.01M hydrochloric acid R as a blank. Calculate the content of amlodipine, C20H25ClN2O5, dissolved using the absorbances obtained with the test solution and the reference solution and the declared content of C20H25ClN2O5 in amlodipine besilate RS. Tolerance: Not less than 70% (Q) of the stated amount of amlodipine, C20H25ClN2O5 is dissolved in 45 minutes.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Triethylamin buffer pH 3.0 - methanol acetonitrile (50 : 35 : 15). Triethylamin buffer pH 3.0: Dissolve 7.0 ml of triethylamine R in 1000 ml of water and adjust to pH 3.0 ± 0.1 with phosphoric acid R. Reference solution: Weigh accurately about 70 mg amlodipine besilate RS, dissolve and dilute to 100.0 ml in mobile phase. Dilute 10.0 ml of the solution to 100.0 ml with the mobile phase. Test solution: Weigh 20 tablets, calculate the average weight and powder finely. Weight accurately a quantity of powdered tablets containing the equivalent of about 50 mg of amlodipine, to a 100 ml volumetric flask, add about 70 ml of mobile phase and sonicate for 10 min, cool, dilute to volume with mobile phase, mix and filter. Dilute 10.0 ml of the solution to 100.0 ml with the mobile phase. Chromatographic system: A column (15 cm × 3.9 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 237 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: Inject separately the reference solution and the test solution. Calculate the content of of amlodipine, C20H25ClN2O5, in the tablets using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C20H25ClN2O5 in amlodipine besilate RS. Storage Store in a cool and dry place, protected from light. Action and use Calcium channel blocker. Usual strength 5 mg; 10 mg.

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AMODIAQUINE HYDROCHLORIDE

AMMONIUM CHLORIDE Ammonii chloridum NH4Cl

M. 53.49

Ammonium chloride contains not less than 99.0% and not more than 100.5% of NH4Cl, calculated with reference to the dried substance.

Characters A white, crystalline powder or colourless crystals, freely soluble in water. Identification A. It gives the reactions of chlorides (Appendix 8.1). B. 10 ml of solution S (see Appearance of solution) gives the reaction of ammonium salts (Appendix 8.1).

Loss on drying Not more than 1.0% (Appendix 9.6) (1.000 g; 100 °C - 105 °C; 2 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 2.0 g. Assay Dissolve 1.000 g in 20 ml of water and add a mixture of 5 ml of formaldehyde solution R, previously neutralised to phenolphthalein solution R, and 20 ml of water. After 1 min to 2 min, titrate slowly with 1 N sodium hydroxide VS, using 0.2  ml of phenolphthalein solution R as indicator. 1 ml of 1 N sodium hydroxide VS is equivalent to 53.49 mg of NH4Cl.

Appearance of solution Solution S: Dissolve 10.0 g in carbon dioxide-free water and dilute to 100 ml with the same solvent. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2).

Storage Store in an airtight container.

Acidity or alkalinity To 10 ml of solution S add 0.05 ml of methyl red solution R. Not more than 0.5 ml of 0.01 N hydrochloric acid VS or 0.01 N sodium hydroxide VS is required to change the colour of the indicator.

Preparation Oral solution.

Bromides and iodides To 10 ml of solution S add 0.1 ml of dilute hydrochloric acid R and 0.05 ml of a 2% solution of chloramine T R. After 1 min, add 2 ml of chloroform R and shake vigorously. The chloroform layer remains colourless. Sulfates Not more than 0.015% (Appendix 9.4.14) 10 ml of solution S diluted to 15 ml with water complies with the limit test for Sulfates. Calcium Not more than 0.02% (Appendix 9.4.3). 5 ml of solution S diluted to 15 ml with water complies with the limit test for Calcium. Heavy metals Not more than 10 ppm (Appendix 9.4.8). 12 ml of solution S complies with limit test for heavy metals, method 1. Prepare the standard using lead standard solution (1 ppm Pb) R. Iron Not more than 20 ppm (Appendix 9.4.13). 5 ml of solution S diluted to 10 ml with water complies with the limit test for Iron.

Action and use Used for the acidification of urine and to correct metabolic alkalosis.

AMODIAQUINE HYDROCHLORIDE Amodiaquini hydrochloridum Cl H N

N

C20H22ClN3O,2HCl,2H2O

N

OH

CH3 CH3

, 2HCl

, 2H2O

M: 464.8

Amodiaquine hydrochloride is 4-[(7-chloro-4-quinolinyl) amino]-2-[(diethylamino)-methyl]phenol dihydrochloride dihydrate or 4-[(7-chloro-4-quinolyl)amino]-α-(diethylamino)o-cresol dihydrochloride dihydrate. It contains not less than 97.0% and not more than 103.0% of C20H22ClN3O,2HCl, calculated with reference to the anhydrous substance.

Characters A yellow, crystalline powder, odourless, sparingly bitter. Soluble in water; sparingly soluble in ethanol (96%), very slightly soluble in benzene, chloroform and ether. Identification A. Dissolve 20 mg of the substance to be examined in 10 ml water in a separating funnel, add 1 ml ammonia R and shake with 25 ml chloroform R. Decant the chloroform extract, evaporate the chloroform layer 55

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AMODIAQUINE HYDROCHLORIDE TABLETS

to dryness and dry the residue at 105 °C for 2 h. The infrared absorption spectrum (Appendix 4.2) of the residue is concordant with the spectrum of amodiaquin hydrochloride RS prepared in the same manner. B. The ultraviolet absorption spectrum (Appendix 4.1) of a 10 µg/ml solution of the substance to be examined in 0.1 M hydrochloride acid R is similar to that of amodiaquine hydrochloride RS in the same content and solve. C. It gives reaction (A) of chloride (Appendix 8.1). D. In the test for Assay, the retention time of principal peak in the chromatogram obtained with the test solution corresponds to that of the peak due to amodiaquine hydrochloride RS in the chromatogram obtained with reference solution.

Appearance of solution A 2% solution of the substance to be examined in water is clear (Appendix 9.2). Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase, reference solution, test solution, system suitability solution and chromatographic system as described in the test for Assay. Procedure: Inject the test solution. Calculate the percentage of each impurity in the chromatogram obtained with the test solution using the normalisation procedure. Limit: For each impurity: Not more than 0.5%.

System suitability solution: Transfer an accurately weighed quantity amodiaquine hydrochloride RS and chloroquine phosphate RS in a suitable volumetric flask, dissolve and dilute to volume with water to obtain a solution having 0.15 mg of amodiaquine hydrochloride RS and 0.15 mg of chloroquine phosphate RS per ml in water. Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase C (5 µm). Column temperature: 25 °C ± 5 °C. Detector: A spectrophotometer set at 224 nm. Flow rate: 1.2 ml/min. Volume of injection: 10 µl. Procedure: Inject the system suitability solution. Retention time of the peak due to amodiaquine hydrochloride is 1.0 and the peak due to chloroquine phosphate is 0.8; the resolution between the peaks due to amodiaquine hydrochloride and chloroquine phosphate is at least 1.5; the symmetry factor of two peaks is not more than 1.5 and the relative standard deviation of 6 replicate injections is not more than 2.0%. Inject the reference solution and the test solution. Calculate the content of C20H22ClN3O,2HCl, using the areas of the amodiaquine hydrochloride peaks in the chromatograms obtained with the reference solution, the test solution and the declared content of C20H22ClN3O,2HCl in amodiaquine hydrochloride RS.

Storage Store in an airtight container.

Water 7.0% to 9.0% (Appendix 10.3).

Action and use Antimalarial.

Sulfated ash Not more than 0.2% (Appendix 9.9, method 2). Determined on 1.0 g.

Preparation Tablets.

Assay Examine by liquid chromatography (Appendix 5.3). Buffer solution: Dissolve 6.8 g of potassium dihydrophosphate R in 1000 ml water. Add 1.0 ml of perchloric acid R, mix well and adjust to a pH of 2.5 ± 0.5 with phosphoric acid R. Pass through a filter of 0.45 µm pore size. Mobile phase: Buffer solution - methanol (78 : 22). If necessary, adjust the proportions of the constituents of the mobile phase to comply with the resolution. Test solution: Transfer an accurately weighed quantity of the substance to be examined in a suitable volumetric flask, dissolve and dilute to volume with water to obtain a 0.15 mg/ml solution of amodiaquine hydrochloride in water. Reference solution: Transfer an accurately weighed quantity amodiaquine hydrochloride RS in a suitable volumetric flask, dissolve and dilute to volume with water to obtain a 0.15 mg/ml solution of amodiaquine hydrochloride RS in water.

AMODIAQUINE HYDROCHLORIDE TABLETS Tabellae Amodiaquini hydrochloridi

56

Amodiaquine hydrochloride tablets contain amodiaquine hydrochloride. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements:

Content of amodiaquine, C20H22ClN3O, 93.0% to 107.0% of the stated amount. Identification A. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that of the amodiaquine hydrochloride peak in the chromatogram obtained with the reference solution. B. Shake a quantity of the powdered tablets containing about 30 mg amodiaquine hydrochloride with 10 ml of

VP V

water, filter. 2 ml of the filtrate yields reaction of chlorides (Reaction A, Appendix 8.1).

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of water. Rotation speed: 50 rpm. Time: 30 min. Procedure: Test solution: After the specified time, withdraw a sample of the medium and filter. Dilute with water to obtain a solution having a suitable concentration if necessary. Measure the absorbances (Appendix 4.1) of the obtained solutions at the maximum wavelength of 342 nm, using water as blank. Calculate the content of amodiaquine, C20H22ClN3O, dissolved of each tablet base on the absorbance of the reference solution having a similar concentration to the test solution. Tolerance: Not less than 75% (Q) of the labeled amount of amodiaquine, C20H22ClN3O, is dissolved in 30 min. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Buffer solution - methanol (78 : 22). Make adjustments if necessary. Buffer solution: Dissolve 6.8 g of potassium dihydrogen phosphate R in 1000 ml of water. Add 1.0 ml of perchloric acid R, mix well and adjust the pH to 2.5 with phosphoric acid R. 1% solution of hydrochloric acid: Dilute 10 ml of hydrochloric acid R to 1000 ml with water. Reference solution: A 0.15 mg/ml solution of amodiaquine hydrochloride RS in water. Test solution: Weigh 20 tablets, calculate the average mass and finely powder. Weigh accurately a quantity of finely powdered tablets containing about 60 mg of amodiaquine hydrochloride. Transfer into a 100-ml volumetric flask, add 60 ml of 1% solution of hydrochloric acid, sonicate for 25 min at 29 °C, dilute to volume with 1% solution of hydrochloric acid, shake and filter. Dilute of 5.0 ml of the filtrate to 20 ml with water. Resolution solution: A solution containing 0.15 mg/ml of amodiaquine hydrochloride RS and 0.15 mg/ml chloroquine phosphate RS in water. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 224 nm. Flow rate: 1.2 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject the resolution solution, record the

AMOXICILLIN SODIUM

chromatogram, in the chromatogram obtained, the relative retention times are 1.0 for amodiaquine hydrochloride peak and 0.8 for chloroquine phosphate peak; the resolution factor between the two peaks is not less than 1.5, the symmetry factors of the two peaks are not more than 1.5. The relative standard deviation of the peak areas for six replicate injections of the reference solution is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of amodiaquine, C20H22ClN3O, in each tablet using the areas of the amodiaquine hydrochloride peaks in the chromatograms obtained with the reference solution, the test solution and the declared content of C20H22ClN3O in amodiaquine hydrochloride RS.

Storage Store in a well-closed container, in a cool place, protected from light. Action and use Antimalarial. Usual strength 200 mg. AMOXICILLIN SODIUM Amoxicillinum natricum

C16H18N3NaO5S

M. 387.4   

Amoxicillin sodium is sodium (2S,5R,6R)-6-[[(2R)-2amino-2-(4-hydroxyphenyl)acetyl]amino]-3,3-dimethyl7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate, it contains not less than 89.0% and not more than 102.0% of C16H18N3NaO5S, calculated with reference to the anhydrous substance. Amoxicillin sodium is a semi-synthetic product from a fermentetion product.

Characters A white or almost white powder, very hygroscopic. Very soluble in water, sparingly soluble in anhydrous ethanol, very slightly soluble in acetone. Identification Apply one of the two following identifications: 57

VP V

AMOXICILLIN SODIUM

First identification: A, D. Second identification: B, C, D. A. Dissolve 0.250 g of the substance to be examined in 5 ml of water, add 0.5 ml of 2 M acetic acid R, stir well and allow to stand for 10 min in iced water. Filter the crystals and wash with 2 ml to 3 ml of a mixture of 1 volume of water and 9 volumes of acetone R, then dry in an oven at 60 °C for 30 min. The infrared absorption spectrophotometry (Appendix 4.2) of the crystals obtained is concordant with the spectrum of amoxicillin trihydrate RS. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silanised silica gel G. Mobile phase: Acetone - 15.4% solution of ammonium acetate adjusted to pH 5.0 with glacial acetic acid (10 : 90). Test solution: Dissolve 25 mg of the substance to be examined in 10 ml of a 4.2% solution of sodium hydrogen carbonate R. Reference solution (1): Dissolve 25 mg of amoxicillin trihydrate RS in 10 ml of a 4.2% solution of sodium hydrogen carbonate R. Reference solution (2): Dissolve 25 mg of amoxicillin trihydrate RS and 25 mg of ampicillin trihydrate RS in 10 ml of a 4.2% solution of sodium hydrogen carbonate R. Procedure: Apply separately to the plate 1 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in air. Expose it to iodine vapour until the spots appear and examine in daylight. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) shows 2 clearly separated spots. C. It gives reaction B described under “ Colour reactions of penicillins and cephalosporins ” (Appendix 8.3). D. It gives reaction of sodium (Appendix 8.1).

Appearance of solution Dissolve 1.0 g of the substance to be examined in water and dilute to 10.0 ml with the same solvent. Immediately examine after dissolution. The solution obtained is not more opalescent than reference suspension II (Appendix 9.2). The solution S may show an initial, but transient, pink colour, after 5 min, the absorbance measured at 430 nm (Appendix 4.1) is not greater than 0.20. pH 8.0 to 10.0 (Appendix 6.2). Dissolve 2.0 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent. Specific optical rotation +240° to +290°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 62.5 mg in a 0.4% solution of potassium hydrogen phthalate R and dilute to 25.0 ml with the same solution. 58

Related substances Examine by liquid chromatography (Appendix 5.3). Phosphate buffer solution pH 5.0: To 250 ml of 0.2 M potassium dihydrogen phosphate R, add dilute sodium hydroxide solution R to adjust pH to 5.0 and dilute to 1000.0 ml with water. Mobile phase A: Acetonitrile - phosphate buffer solution pH 5.0 (1 : 99). Mobile phase B: Acetonitrile - phosphate buffer solution pH 5.0 (20 : 80). Test solution (1): Dissolve 30.0 mg of the substance to be examined in mobile phase A and dilute to 50.0 ml with the same solvent. Test solution (2): Dissolve 30.0 mg of the substance to be examined in mobile phase A and dilute to 20.0 ml with the same solvent. Prepare immediately before use. Reference solution (1): Dissolve 30.0 mg of amoxicillin trihydrate RS in mobile phase A and dilute to 50.0 ml with the same solvent. Reference solution (2): Dissolve 4.0 mg of cefadroxil RS in mobile phase A and dilute to 50.0 ml with the same solvent. To 5.0 ml of this solution add 5.0 ml of reference solution (1) and dilute to 100 ml with mobile phase A. Reference solution (3): Dilute 2.0 ml of reference solution (1) to 20.0 ml with mobile phase A. Dilute 5.0 ml of the solution obtained to 20.0 ml with mobile phase A. Reference solution (4): To 0.20 g of amoxicillin trihydrate R add 1.0 ml of water. Shake and add dropwise dilute sodium hydroxide solution R to obtain a solution. The pH of the solution obtained is about 8.5. Store the solution at room temperature for 4 h. Dilute 0.5 ml of this solution to 50.0 ml with mobile phase A. Chromatograpic system: A stainless steel column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min with gradient elution program as following: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - tR

92

8

tR - (tR + 25)

92 → 0

8 → 100

(tR + 25) - (tR + 40)

0

100

(tR + 40) - (tR + 55)

92

8

(tR is the retention time of amoxicillin determined by reference solution (3)). If the mobile phase composition has been adjusted to achieve the required resolution, the adjusted composition will apply at time zero in the gradient and in the assay. Volume of injection: 50 µl. Procedure: Inject reference solution (2) and (3), elute isocratically with mobile phase composition as at time zero. Inject test

VP V

solution (2) and reference solution (4), elute in gradient program described above. Inject mobile phase A and use the same elution pattern to obtain a blank. Determine impurities: In the chromatogram obtained with reference solution (4), the 3 principal peaks eluted after the peak of amoxicillin correspond to impurity C, amoxicillin dimer (impurity J; n = 1) and amoxicillin trimer (impurity J; n = 2), respectively. The relative retention time, compared to amoxicillin peak of impurities as following: Impurity C is about 3.4; impurity J (n = 1) is about 4.1; impurity J ( n = 2) is about 4.5. System suitability: In the chromatogram obtained with reference solution (2), resolution factor between peak of amoxicillin and peak of cefadroxil is at least 2.0. If necessary, adjust the ratio of mobile phase A and mobile phase B. Limits: Impurity J (n = 1): In the chromatogram obtained with test solution (2), the area of any peak corresponding to impurity J (n = 1) is not greater than 3 times the area of the principal peak in the chromatogram obtained with reference solution (3) (3.0%). Other impurities: In the chromatogram obtained with test solution (2), the area of any peak, apart from the principal peak and any peak corresponding to impurity J (n = 1) is not greater than twice the area of the principal peak in the chromatogram obtained with reference solution (3) (2.0%). Sum of all the impurities: In the chromatogram obtained with test solution (2), the sum of the areas of all the peaks, apart from the principal peak, is not greater than 9 times the area of the principal peak in the chromatogram obtained with reference solution (3) (9.0%). Disregard any peak with an area less than 0.1 times that of the principal peak in the chromatogram obtained with reference solution (3) (0.1%).

N,N-Dimethylaniline Not more than 20 ppm. Examine by gas chromatograpy (Appendix 10.16, method 1 or 2). 2-Ethylhexanoic acid Not more than 0.8% (w/w) (Appendix 10.17). Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Water Not more than 3.0% (Appendix 10.3). Determined on 0.400 g by the semi-micro determination of water. Bacterial endotoxins Less than 0.25 IU/mg (Appendix 13.2).

AMOXICILLIN FOR INJECTION

If intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins.

Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. Mobile phase: A mixture of mobile phase A and mobile phase B in the ratio as at time zero of gradient program stated, adjust the ratio, if necessary. System suitability: Inject reference solution (1) six times. The test is not valid unless the relative standard deviation for the areas of the principal peaks is at most 1.0%. Inject test solution (1) and reference solution (1). Calculate the percentage content of amoxicillin sodium by multiplying the percentage content of amoxicillin by 1.060. Storage Store in an airtight container. If the substance is sterile, store in a sterile, airtight, tamper-proof container. Action and use Beta-lactam antibacterial. Preparation Injection. AMOXICILLIN FOR INJECTION Amoxicillini pro injectione Amoxicillin for injection is a sterile crystalline powder of amoxicillin sodium. It is supplied in a sealed glass container. Dissolve with sterile water for Injections immediately before use. The powder complies with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Content of amoxicillin, C16H19N3O5S, 90.0% to 105.0% of the stated amount. Characters A white or almost white crystalline powder. Identification A. The infrared absorption spectrum of the contents of the sealed container (Appendix 4.2) is concordant with the spectrum of amoxicillin sodium RS. B. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel G. Mobile phase: Acetone - 15.4% solution of ammonium acetate R adjusted to pH 5.0 with glacial acetic acid (10 : 90). Test solution: Dissolve a quantity of the contents of the sealed container in a 4.2% solution of sodium hydrogen carbonate R to produce a solution containing the equivalent of 0.25% of amoxicillin. 59

VP V

AMOXICILLIN AND CLAVULANIC ACID FOR INJECTION

Reference solution (1): A solution containing 0.25% of amoxicillin trihydrate RS in a 4.2% solution of sodium hydrogen carbonate R. Reference solution (2): A solution containing 0.25% of each of amoxicillin trihydrate RS and ampicillin trihydrate RS in a 4.2% solution of sodium hydrogen carbonate R. Procedure: Apply separately to the plate 2 µl of each solution. Develop over a path of 15 cm. After removal of the plate, allow to dry in air, expose to iodine vapour until spots appear and examine in daylight. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to that in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) shows two clearly separated spots. C. Yield the reactions characteristic of sodium salts (Appendix 8.1).

Alkalinity pH of a solution containing the equivalent of 10% of amoxicillin in carbon dioxide-free water R, 8.0 to 10.0 (Appendix 6.2). Water Not more than 4.0% (Appendix 10.3). Use 0.3 g of the substance to be examined. Bacterial endotoxins Dissolve the contents of the sealed container in water BET R to give a solution containing the equivalent of 10 mg of amoxicillin per ml (solution A). The endotoxin limit concentration of solution A is 2.5 EU of endotoxin per ml. Carry out the test using the maximum valid dilution of solution A calculated from the declared sensitivity of the lysate used in the test (Appendix 13.2). Degradation products To an accurately weighed quantity of the contents of the sealed container containing the equivalent of 0.24 g of amoxicillin add 25 ml of boric buffer pH 9.0 R and 0.5 ml of acetic anhydride R, stir for 3 minutes. Add 10 ml of acetate buffer pH 4.6 R and titrate immediately with 0.02 N mercury (II) nitrate VS. Determine the end point potentiometrically (Appendix 10.2). 1 ml of 0.02 N mercury (II) nitrate VS is equivalent to 7.748 mg of degradation products, calculated as amoxicillin sodium, C16H18N3NaO5S. Not more than 9.0% with respect to the content of amoxicillin sodium, C16H18N3NaO5S. Assay Examine by liquid chromatography (Appendix 5.3) Mobile phase: Mobile phase A - mobile phase B (92 : 8). Mobile phase A: Mix 1 volume of acetonitrile R and 99 volumes of a 25% v/v solution of 0.2 M potassium dihydrogen phosphate R adjusted to pH 5.0 with 2 M sodium hydroxide R. 60

Mobile phase B: Mix 20 volumes of acetonitrile R and 80 volumes of a 25% v/v solution of 0.2 M potassium dihydrogen phosphate R adjusted to pH 5.0 with 2 M sodium hydroxide R. Test solution: Weigh 10 containers, calculate the average weight of the contents in a container, and mix. To an accurately weighed quantity of the mixed contents containing the equivalent of about 60 mg of amoxicillin add 80 ml of mobile phase A, and shake for 15 min. Mix with the aid of ultrasound for 1 minute, add sufficient mobile phase A to produce 100.0 ml, mix and filter. Reference solution: A solution containing 0.070% of amoxicillin trihydrate RS in mobile phase A. Resolution solution: A solution containing 0.003% of amoxicillin trihydrate RS and 0.0004% of cefadroxil RS in mobile phase A. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm) (Hypersil 5 ODS is suitable). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 50 µl. Procedure: Inject the resolution solution, the test is not valid unless the resolution factor between the peaks due to amoxicillin and cefadroxil is at least 2.0. If necessary, adjust the composition of the mobile phase to achieve the required resolution. Inject separately the reference solution and the test solution. Calculate the content of C16H19N3O5S in a container from the areas of the principal peaks in the chromatograms obtained with the test solution, the reference solution and the declared content of C16H19N3O5S in amoxicillin trihydrate RS.

Storage Store at a temperature not exceeding 25 °C, protected from moisture. Action and use Beta-lactam antibiotic. Usual strength 250 mg; 500 mg; 1000 mg, calculated as amoxicillin. AMOXICILLIN AND CLAVULANIC ACID FOR INJECTION Amoxicillini et acidi clavulanici pulvis ad injectionem Amoxicillin and clavulanic acid for injection is a sterile powder consisting of amoxicillin sodium and clavulanate potassium. It is supplied in a sealed glass container. The powder complies with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements:

Content of amoxicillin, C16H19N3O5S, 90.0% to 110.0% of the stated amount.

VP V

Content of clavulanic acid, C8H9NO5, 90.0% to 110.0% of the stated amount. Characters A white or almost white crystalline powder. Identification In the Assay, the retention times of the two principal peaks in the chromatogram obtained with the test solution correspond to those in the chromatogram obtained with the reference solution. Acidity or alkalinity pH of a solution containing the equivalent of 10% of amoxicillin in carbon dioxyd-free water R, 8.0 to 10.0 (Appendix 6.2). Water Not more than 3.5% (Appendix 10.3). Determined on 0.5 g. Bacterial endotoxins Carry out the test for “Bacterial endotoxins” (Appendix 13.2). Dissolve a quantity of the substance to be examined in water BET R to give a solution containing the equivalent of 10 mg per ml of amoxicillin (solution A). The endotoxin limit concentration of solution A is 2.5 EU of endotoxin per ml. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: A mixture of 5 volumes of methanol R and 95 volumes of a 0.78% solution of sodium dihydrogen phosphate monohydrate adjusted to pH 4.4 with phosphoric acid R or 10 M sodium hydroxyd R. Reference solution: A solution of amoxicillin trihydrate RS and lithium clavulanate RS (or potassium clavulanate) in water containing the equivalent of 0.5 mg/ml of amoxicillin, C16H19N3O5S, and 0.125 mg/ml of clavulanic acid, C8H9NO5. Test solution: Weigh the contents of 10 containers, determine the average mass and mix. Transfer an accurately weighed quantity of the mixed powder containing the equivalent of about 50 mg of amoxicillin to a 100 ml volumetric flask, add 70 ml of water and shake to dissolve. Dilute to volume with water and mix well. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 μm). Detector: A spectrophotometer set at 220 nm Flow rate: 2.0 ml/min. Volume of injection: 20 μl. Procedure: System suitability: Inject the reference solution. The relative retention times for clavulanic acid and amoxicillin are about 0.5 and 1.0, respectively. The assay is not valid unless, the resolution factor between amoxicillin and

AMOXICILLIN TRIHYDRATE

clavulanic acid peaks is at least 3.5; the tailing factor of the peaks due to amoxicillin and clavulanic acid is not more than 1.5; and the relative standard deviation of the peak areas due to amoxicillin and clavulanic acid for replicate injections is not more than 2.0%. Inject separately the reference solution and the test solution. Calculate the contents of amoxicillin, C16H19N3O5S, and clavulanic acid, C8H9NO5, in the injection using the peak areas of amoxicillin and clavulanic acid in the chromatograms obtained with the test solution and the reference solution, and the declared contents of C16H19N3O5S and C8H9NO5 in amoxicillin trihydrate RS and lithium clavulanate RS (or potassium clavulanate), respectively.

Storage Store in an airtight container, in a cool and dry place, protected from light. Action and use Penicillin antibacterial antibacterial.

+

Beta-lactamase

inhibitor

Usual strength 0.6 g (0.5 g of amoxicillin and 0.1 g of clavulanic acid). 1.2 g (1.0 g of amoxicillin and 0.2 g of clavulanic acid). AMOXICILLIN TRIHYDRATE Amoxicillinum trihydricum

C16H19N3O5S,3H2O  

M. 419.4

Amoxicillin trihydrate is (2S,5R,6R)-6-[[(2R)-2-amino-2-(4hydroxyphenyl)acetyl]amino]-3,3-dimethyl-7-oxo-4-thia1-azabicyclo[3.2.0]heptane-2-carboxylic acid trihydrate. It contains not less than 95.0% and not more than 102.0% of C16H19N3O5S, calculated with reference to the anhydrous substance. Semi-synthetic product derived from a fermentation product.

Characters White or almost white, crystalline powder. Slightly soluble in water, very slightly soluble in ethanol (96%), practically insoluble in fatty oils. It dissolves in dilute acids and dilute solutions of alkali hydroxides. Identification Apply one of the two following identifications: First identification: A. 61

VP V

AMOXICILLIN TRIHYDRATE

Second identification: B, C. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of amoxicillin trihydrate RS. B. It complies with the test for Identification of penicillins (Appendix 8.2), use mobile phase B. C. It complies with the test B for Colour reactions of penicillins and cephalosporins (Appendix 8.3).

Appearance of solution Dissolve 1.0 g in 10 ml of 0.5 M hydrochloric acid R. Dissolve separately 1.0 g in 10 ml of 2 M ammonia R. Examine immediately after dissolution. The solutions are not more opalescent than reference suspension II (Appendix 9.2). pH Solution S: With the aid of ultrasound or gentle heating, dissolve 0.100 g in carbon dioxide-free water R and dilute to 50.0 ml with the same solvent. pH of the solution S is 3.5 to 5.5 (Appendix 6.2). Specific optical rotation +290° to +315°, calculated with reference to the anhydrous substance (Appendix 6.4). Determined on solution S. Related substances Examine by liquid chromatography (Appendix 5.3). Buffer solution pH 5.0: Add dilute sodium hydroxide solution R to 250 ml of 0.2 M potassium dihydrogen phosphate R to pH 5.0 and dilute to 1000.0 ml with water. Mobile phase A: Acetonitrile - buffer solution pH 5.0 (1 : 99). Mobile phase B: Acetonitrile - buffer solution pH 5.0 (20 : 80). Test solution (1): Dissolve 30.0 mg of the substance to be examined in mobile phase A and dilute to 50.0 ml with mobile phase A. Test solution (2): Dissolve 30.0 mg of the substance to be examined in mobile phase A and dilute to 20.0 ml with mobile phase A. Prepare immediately before use. Reference solution (1): Dissolve 30.0 mg of amoxicillin trihydrate RS in mobile phase A and dilute to 50.0 ml with mobile phase A. Reference solution (2): Dissolve 4.0 mg of cefadroxil RS in mobile phase A and dilute to 50 ml with mobile phase A. To 5.0 ml of this solution add 5.0 ml of reference solution (1) and dilute to 100 ml with mobile phase A. Reference solution (3): Dilute 2.0 ml of reference solution (1) to 20.0 ml with mobile phase A. Dilute 5.0 ml of this solution to 20.0 ml with mobile phase A. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 254 nm. 62

Flow rate: 1.0 ml/min. Volume of injection: 50 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - tR

92

8

tR - (tR + 25)

92 → 0

8 → 100

(tR + 25) - (tR + 40)

0

100

(tR + 40) - (tR + 55)

92

8

tR = retention time of amoxicillin determined with the reference solution (3) If the mobile phase composition has been adjusted to achieve the required resolution, the adjusted composition will apply at time zero in the gradient and in the assay. Inject reference solutions (2) and (3) with isocratic elution at the initial mobile phase composition and test solution (2) according to the elution gradient described under Mobile phase; inject mobile phase A as a blank according to the elution gradient described under Mobile phase. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to amoxicillin and cefadroxil is at least 2.0 (if necessary, adjust the ratio A:B of the mobile phase). Limit: Any impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (3) (1%). Note: Impurity A: (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4 -thia1-azabicyclo[3.2.0]heptane-2- carboxylic acid (6-aminopenicillanic acid). Impurity B: (2S,5R,6R)-6-[[(2S)-2-amino-2-(4-hydroxyphenyl) acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0] heptane-2-carboxylic acid (L-amoxicillin). Impurity C: (4S)-2-[5-(4-hydroxyphenyl)-3,6-dioxopiperazin2-yl]-5,5-dimethylthiazolidine-4- carboxylic acid (amoxicillin diketopiperazines). Impurity D: (4S)-2-[[[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl] amino]carboxymethyl]-5,5-dimethylthiazolidine-4-carboxylic acid (penicilloic acids of amoxicillin). Impurity E: (2RS,4S)-2-[[[(2R)-2-amino-2-(4-hydroxyphenyl) acetyl]amino]methyl]-5,5-dimethylthiazolidine-4-carboxylic acid (penilloic acids of amoxicillin). Impurity F: 3-(4-hydroxyphenyl)pyrazin-2-ol. Impurity G: (2S,5R,6R)-6-[[(2R)-2-[[(2R)-2-amino-2-(4hydroxyphenyl)acetyl]amino]-2-(4- hydroxyphenyl) acetyl] amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo [3.2.0]heptane2-carboxylic acid (D-(4-hydroxyphenyl) glycylamoxicillin).

VP V Impurity H: (2R)-2-[(2,2-dimethylpropanoyl)amino]-2-(4hydroxyphenyl)acetic acid. Impurity I: (2R)-2-amino-2-(4-hydroxyphenyl)acetic acid. Impurity J: Co-oligomers of amoxicillin and of penicilloic acids of amoxicillin. Impurity K: Oligomers of penicilloic acids of amoxicillin. Impurity L: (2S,5R,6R)-6-[[(2S,5R,6R)-6-[[(2R)-2-amino-2(4-hydroxyphenyl)acetyl]amino]-3,3-dimethyl-7-oxo-4-thia1-azabicyclo[3.2.0]heptane-2-carbonyl]amino]-3,3-dimethyl7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid (6APA amoxicillin amide).

N,N-Dimethylaniline Not more than 20 ppm (Appendix 10.16, method 1 or 2). Water 11.5% to 14.5% (Appendix 10.3). Determined on 0.100 g. Sulfated ash Not more than 1.0% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. Mobile phase: Initial composition of the mixture of mobile phases A and B, adjusted where applicable. Inject test solution (1) and reference solution (1). System suitability: In the chromatogram obtained with reference solution (1), the relative standard deviation of the peak areas due to amoxicillin trihydrate for 6 replicate injections is not more than 1.0%. Calculate the content of C16H19N3O5S using the areas of the peaks in the chromatograms obtained with test solution (1), reference solution (1) and the declared content of C16H19N3O5S in amoxicillin trihydrate RS. Storage Store in an airtight container. Action and use Beta-lactam antibiotic. Preparations Tablets, capsules, oral suspension. AMOXICILLIN POWDER FOR SUSPENSION Pulveres Amoxicillini ad suspensionum per oralum The powder for suspension contains amoxicillin trihydrate . The powder complies with the requirements under “Powder” (Appendix 1.7) and with the following requirements:

Content of amoxicillin, C16H19N3O5S, 90.0% to 110.0% of the labeled amount.

AMOXICILLIN POWDER FOR SUSPENSION

Description A dry powder with homogeneous colour. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Aceton - water - toluene - glacial acetic acid (65 : 10 : 10 : 2.5) Test solution: Shake a quantity of the powder containing 100 mg of amoxicillin with 20 ml of a mixture of acetone and 0.1 M hydrochloric acid (4 : 1), filter. Reference solution: Dissolve amoxicillin trihydrate RS in a mixture of acetone and 0.1 M hydrochloric acid (4 : 1) to obtain a solution having a known concentration of 0.5% amoxicillin. Procedure: Apply separately 2 µl of each solution into the plate. Develop over a path of about 15 cm, remove the plate and allow it to dry in room temperature. Spray the plate with 0.3% ninhydrin solution in ethanol R, and heat at 90 °C for 15 min. Examine in daylight. The principal spot in the chromatogram obtained with the test solution is similar in colour and position (Rf) to that of the reference solution. A. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of the principal peak in the chromatogram obtained with the reference solution. pH The pH of a reconstituted suspension prepared following manufacturer’s instruction is 5.0 to 7.5 (Appendix 6.2). Assay Examine by liquid chromatography (Appendix 5.3). Solution A: Dissolve 13.6 g of potassium dihydrogene phosphate R in 2000 ml of water, adjust the pH to 5.0 ± 0.1 with a 45% potassium hydroxide solution. Mobile phase: Solution A - acetonitrile (96 : 4). Adjust the acetonitrile content if necessary. Reference solution: Dissolve amoxicillin trihydrate RS in solution A to obtain a concentration of amoxicillin of about 1.2 mg/ml. Filter through a filter membrane having the nominal pore size of 0.45µm (Use within 6 hours). Test solution: Weigh accurately a quantity of the homogeneous powder obtained from the test for Uniformity of weight test containing 120 mg of amoxicillin. Transfer into a 100-ml volumetric flask, add 70 ml of solution A, shake to dissolve and dilute to volume with solution A, mix and filter. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (3 µm to 10 µm). Detector: A spectrophotometer set at 230 nm. Flow rate: 1.5 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject the reference solution, in the chromatogram obtained, the symmetry factor of the 63

VP V

AMOXICILLIN CAPSULES

amoxicillin peak is not more than 2.5 and the relative standard deviation for six replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of amoxicillin, C16H19N3O5S, in the powder using the areas of the principal peaks in the chromatograms obtained with the reference solution, the test solution and the declared content of C16H19N3O5S in amoxicillin trihydrate RS.

Storage Store in a well-closed container, in a cool place, protected from light. Action and use Beta-lactam antibiotic. Usual strength 250 mg, 500 mg, stated in terms of anhydrous amoxicillin. AMOXICILLIN CAPSULES Capsulae Amoxicillini Amoxicillin capsules contain amoxicillin trihydrate. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of amoxicillin, C16H19N3O5S, 90.0% to 110.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Acetone - water - toluene - glacial acetic acid (65 : 10 : 10 : 2.5). Test solution: Shake a quantity of the capsule contents containing the equivalent of 100 mg of amoxicillin with 20 ml of a mixture of acetone R and 0.1 M hydrochloric acid R (4 : 1), and filter. Reference solution: Dissolve a quantity of amoxicillin trihydrate RS in a mixture of acetone R and 0.1 M hydrochloric acid R (4 : 1) to obtain a solution containing 0.5% of amoxicillin. Procedure: Apply separately to the plate 2 µl of each solution. Develop over a path of 15 cm. After removal of the plate, allow it to dry at room temperature. Spray with a 0.3% solution of ninhydrin in ethanol R. Dry at 90 ºC for 15 min and examine in daylight. The principal spot in the chromatogram obtained with the test solution is similar in position and colour to that of the principal spot in the chromatogram obtained with the reference solution. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. 64

Water Not more than 14.5% (Appendix 10.3). Determined on 0.100 g of the capsule contents. Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 900 ml of water. Rotation speed: 100 rpm. Time: 60 min. Procedure: After the specified time, withdraw a sample of the medium and filter, discard the first 10 ml of the filtrate. Measure the absorbance of the filtrate, suitably diluted if necessary, at the maximum at 272 nm (Appendix 4.1) in a 1 cm cell, using water R in the reference cell, in comparision with a reference solution having a same concentration of amoxicillin RS in the same medium. Tolerance: Not less than 80% (Q) of the labelled amount of amoxicillin, C16H19N3O5S, is dissolved in 60 min. Assay Examine by liquid chromatography (Appendix 5.3). Solution A: Dissolve 13.6 g of potassium dihydrogen phosphate R in 2000 ml of water, and adjust to pH 5.0 ± 0.1 with 45% solution of potassium hydroxide R. Mobile phase: A mixture of solution A and acetonitrile R (96 : 4). If necessary, adjust the acetonitrile concentration of the mobile phase to achieve the required resolution. Reference solution: Dissolve an accurately weighed quantity of amoxicillin trihydrate RS in solution A to obtain a solution having a known concentration of about 1.2 mg/ml. Use this solution within 6 hours. Test solution: Weigh 20 capsules, calculate the average weight of the capsule contents and powder finely. Transfer an accurately weighed quantity of the powder, equivalent to about 0.200 g of amoxicillin, to a 200 ml volumetric flask, sonicate if necessary to ensure complete dissolution, add solution A to volume. Mix and filter through a suitable filter of 1 µm or finer porsity. Use this solution within 6 hours. Chromatographic system: A column (25 cm × 4 mm) packed with stationary phase C (3 µm to 10 µm). Detector: A spectrophotometer set at 230 nm. Flow rate: 1.5 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject the reference solution. The capacity factor is from 1.1 to 2.8; the column efficiency is not less than 1700 theoretical plates; the tailing factor is not more than 2.5 and the relative standard deviation for 6 replicate injections is not more than 2.0%. Inject separately the test solution and the reference solution. Calculate the content of amoxicillin, C16H19N3O5S, in the capsules using the areas of the principal peaks in the chromatograms obtained with the test solution and the reference solution, and the declared content of C16H19N3O5S in amoxicillin trihydrate RS.

VP V

Storage Store in an airtight container in a cool place, protected from light. Action and use Beta-lactam antibiotic. Usual strength 250 mg; 500 mg, calculated as anhydrous amoxicillin. AMOXICILLIN TABLETS Tabellae Amoxicillini Amoxicillin tablets contain amoxicillin trihydrate. The capsules comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of amoxicillin, C16H19N3O5S, 90.0% to 120.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel G Mobile phase: Acetone - water - toluene - glacial acetic acid (65 : 10 : 10 : 2.5). Test solution: Shake a quantity of the powdered tablets containing the equivalent of 100 mg of amoxicillin with 20 ml of a mixture of acetone R and 0.1 M hydrochloric acid R (4 : 1), and filter. Reference solution: A solution of amoxicillin trihydrate RS in a mixture of acetone R and 0.1 M hydrochloric acid R (4 : 1), containing the equivalent of 0.5% of amoxicillin. Procedure: Apply separately to the plate 2 µl of each solution. Develop over a path of 15 cm. After removal of the plate, allow it to dry at room temperature. Spray with a 0.3% solution of ninhydrin in ethanol R. Dry at 90 ºC for 15 min and examine in daylight. The principal spot in the chromatogram obtained with the test solution is similar in position and colour to that of the principal spot in the chromatogram obtained with the reference solution. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. Water Not more than 13.0% (Appendix 10.3). Determined on about 0.15 g of the powdered tablets. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of water. Rotation speed: 75 rpm. Time: 60 min. Procedure: After the specified time, withdraw a sample of the medium, filter and discard the first 10 ml of the filtrate.

AMOXICILLIN TABLETS

Measure the absorbance of the filtrate, suitably diluted if necessary, at the maximum at 272 nm (Appendix 4.1) in a 1 cm cell, using water as a blank, in comparision with a reference solution having a same concentration of amoxicillin RS in the same medium. Tolerance: Not less than 80% (Q) of the labelled amount of amoxicillin, C16H19N3O5S, is dissolved in 60 min.

Assay Examine by liquid chromatography (Appendix 5.3). Solution A: Dissolve 13.6 g of potassium dihydrogen phosphate R in 2000 ml of water, and adjust to pH of 5.0 ± 0.1 with a 45% solution of potassium hydroxide. Mobile phase: Solution A - acetonitrile (96 : 4). If necessary, adjust the acetonitrile concentration of the mobile phase to achieve the required resolution. Reference solution: Dissolve an accurately weighed quantity of amoxicillin trihydrate RS in solution A to obtain a solution having a known concentration of about 1.2 mg per ml. Use this solution within 6 hours. Test solution: Weigh 20 tablets, calculate the average mass, and powder finely. Transfer an accurately weighed quantity of the powder, equivalent to about 0.2 g of amoxicillin, to a 200 ml volumetric flask, sonicate if necessary to ensure complete dissolution, add solution A to volume. Mix and filter through a suitable filter of 1 µm or finer porsity. Use this solution within 6 hours. Chromatographic system: A column (25 cm × 4 mm) packed with stationary phase C (3 µm to 10 µm). Detector: A spectrophotometer set at 230 nm. Flow rate: 1.5 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject the reference solution. The capacity factor is from 1.1 to 2.8; the column efficiency is not less than 1700 theoretical plates; the tailing factor is not more than 2.5 and the relative standard deviation for 6 replicate injections is not more than 2.0%. Inject separately the reference solution and the test solution. Calculate the content of amoxicillin, C16H19N3O5S, in the tablets using the areas of the principal peaks in the chromatograms obtained with the test solution and the reference solution, and the declared content of C16H19N3O5S in amoxicillin trihydrate RS. Storage Store in an airtight container, in a cool place, protected from light. Action and use Beta-lactam antibiotic. Usual strength 250 mg; 500 mg, calculated as anhydrous amoxicillin. 65

AMOXICILLIN AND CLAVULANIC ACID POWDER FOR ORAL SUSPENSION

AMOXICILLIN AND CLAVULANIC ACID POWDER FOR ORAL SUSPENSION Pulveres Amoxicillini et Acidi clavulanici ad suspensionum peroralum Amoxicillin and clavulanic acid powder for oral suspension contains amoxicillin trihydrate and clavulanate potassium. The powder complies with the requirements stated under “Powder” (Appendix 1.7) and with the following requirements:

Content of amoxicillin, C16H19N3O5S, 90.0% to 110.0% of the stated amount. Content of clavulanic acid, C8H9NO5, 90.0% to 110.0% of the stated amount. Characters A dry powder. Identification In the Assay, the retention times of the two principal peaks in the chromatogram obtained with the test solution correspond to those in the chromatogram obtained with the reference solution. pH pH of a suspension constituted as directed in the labeling is 3.8 to 6.6 (Appendix 6.2). Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: A mixture of 5 volumes of methanol R and 95 volumes of a 0.78% solution of sodium dihydrogen phosphate monohydrate adjusted to a pH of 4.4 with phosphoric acid R or 10 M sodium hydroxyd R. Reference solution: A solution of amoxicillin trihydrate RS and lithium clavulanate RS (or potassium clavulanate) in water containing the equivalent of 0.5 mg/ml of amoxicillin, C16H19N3O5S, and 0.125 mg/ml of clavulanic acid, C8H9NO5. Test solution: Use the contents in the Uniformity of mass and mix well. Transfer an accurately weighed quantity of the powder, equivalent to about 50 mg of amoxicillin to a 100 ml volumetric flask, add 70 ml of water and dissolve by shaking well, dilute to volume with water, mix well and filter. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 μm). Detector: A spectrophotometer set at 220 nm Flow rate: 2.0 ml/min. Volume of injection: 20 μl Procedure: System suitability: Inject the reference solution. The relative retention times for clavulanic acid and amoxicillin 66

VP V

are about 0.5 and 1.0, respectively. The assay is not valid unless, the resolution factor between the amoxicillin and clavulanic acid peaks is not less than 3.5; the tailing factor of the peak due to amoxicillin and clavulanic acid is not more than 1.5; and the relative standard deviation of the peak area due to amoxicillin and clavulanic acid for replicate injections is not more than 2.0%. Inject separately the reference solution and the test solution. Calculate the contents of amoxicillin, C16H19N3O5S, and clavulanic acid, C8H9NO5, in the powder using the peak areas of amoxicillin and clavulanic acid in the chromatograms obtained with the test solution and the reference solution, and the content of C16H19N3O5S and C8H9NO5 in amoxicillin trihydrate RS and lithium clavulanate RS (or potassium clavulanate RS), respectively. Each mg of lithium clavulanate, C8H8LiNO5, is equivalent to 0.9711 mg of clavulanic acid, C8H9NO5.

Storage Store in an airtight container, in a cool and dry place, protected from light. Action and use Beta-lactamase inhibitor + Penicillin antibacterial. Usual strength 125 mg of amoxicillin and 31.25 mg of clavulanic acid. 250 mg of amoxicillin and 62.5 mg of clavulanic acid. 200 mg of amoxicillin and 28.5 mg of clavulanic acid. 400 mg of amoxicillin and 57 mg of clavulanic acid. 600 mg of amoxicillin and 42.9 mg of clavulanic acid. AMOXICILLIN AND CLAVULANIC ACID TABLETS Tabellae Amoxicillini et Acidi clavulanici Amoxicillin and clavulanic acid tablets contain amoxicillin trihydrate and clavulanate potassium. They are film-coated. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of amoxicillin, C16H19N3O5S, 90.0% to 110.0% of the stated amount. Content of clavulanic acid, C8H9NO5, 90.0% to 110.0% of the stated amount. Identification In the Assay, the retention times of the two principal peaks in the chromatogram obtained with the test solution correspond to those in the chromatogram obtained with the reference solution. Water (Appendix 10.3) Not more than 7.5%, when the labelled amount of amoxicillin in each tablet is 250 mg or less.

VP V

Not more than 10.0%, when the labelled amount of amoxicillin in each tablet is more than 250 mg but less than or equal to 500 mg. Not more than 11.0%, when the labelled amount of amoxicillin in each tablet is more than 500 mg. When tablets are labelled as chewable, not more than 6.0%, when the labelled amount of amoxicillin in each tablet is 125 mg or less, and not more than 8.0%, when the labelled amount of amoxicillin in each tablet is more than 125 mg. Determined on about 0.3 g of the finely powdered tablets.

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of water. Rotation speed: 75 rpm. Time: 45 min. Procedure: After the specified time, withdraw a sample of the medium and filter, discarding the first 20 ml of the filtrate. Dilute the filtrate, if necessary, with medium to obtain a suitable concentration of amoxicillin. Examine content of amoxicillin (C16H19N3O5S) and clavulanic acid (C8H9NO5) in the medium by liquid chromatography (Appendix 5.3). Mobile phase, reference solution and chromatographic system: Prepare as directed in the Assay. Tolerance: Not less than 70% (Q) of the labelled amount of amoxicillin, C16H19N3O5S, is dissolved in 45 min. Not less than 70% (Q) of the labelled amount of clavulanic acid, C8H9NO5, is dissolved in 45 min. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: A mixture of of 5 volumes of methanol R and 95 volumes of a 0.78% solution of sodium dihydrogen phosphate adjusted to pH 4.4 with phosphoric acid R. Reference solution: A solution containing 0.05% of amoxicillin trihydrate RS and 0.02% of clavulanate lithium RS in water. Test solution: Weigh 20 coating-removed tablets, calculate the average mass, and powder finely. Transfer an accurately weighed quantity of the powder, equivalent to about 0.250 g of amoxicillin, to a 500 ml volumetric flask, add 400 ml of water and sonicate for 15 min. Dilute to volume with water, and mix. Filter. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 220 nm. Flow rate: 2.0 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution, the test is not valid unless the resolution factor between the amoxicillin and clavulanic acid peaks is not less than 3.5; the symmetry factor of the peak due to clavulanic acid is not more than 1.5.

AMOXICILLIN AND CLOXACILLIN CAPSULES

Inject separately the reference solution and the test solution. Calculate the contents of amoxicillin, C16H19N3O5S, and clavulanic acid, C8H9NO5, in the tablets using the peak areas of amoxicillin and of clavulanic acid in the chromatograms obtained with the test solution, the reference solution and the concentrations of C16H19N3O5S and C8H9NO5 in the reference solution. 1 mg clavulanat lithi, C8H8LiNO5, equivalent to 0.9711 mg clavulanic acid, C8H9NO5.

Storage Store in an airtight container, in a cool place, protected from light. Action and use Penicillin antibacterial + beta-lactamase inhibitor. Usual strength 500 mg of amoxicillin and 125 mg of clavulanic acid. AMOXICILLIN AND CLOXACILLIN CAPSULES Capsulae Amoxicillini et cloxacillini Amoxicillin and cloxacillin capsules contain amoxicillin trihydrate and cloxacillin sodium. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements:

Content of amoxicillin, C16H19N3O5S, 90.0% to 110.0% of the stated amount. Content of cloxacillin, C19H18ClN3O5S, 90.0% to 110.0% of the stated amount. Identification A. In the Assay, the retention times of the two principal peaks in the chromatogram obtained with the test solution correspond to those in the chromatograms obtained with the reference solutions. B. It gives the reactions of sodium (Appendix 8.1). Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 900 ml of water. Rotation speed: 100 rpm. Time: 60 min. Procedure: Examine by liquid chromatography (Appendix 5.3). Mobile phase and Chromatographic system: Prepare as directed in the Assay. Amoxicillin reference solution: Dissolve an accurately weighed quantity of amoxicillin trihydrate RS in water to obtain a solution having the same concentration of amoxicillin to that in the test solution. 67

VP V

AMPHOTERICIN B

Cloxacillin reference solution: Dissolve an accurately weighed quantity of cloxacillin sodium RS in water to obtain a solution having the same concentration of cloxacillin to that in the test solution. Test solution: After the specified time (60 min) withdraw a sample of the medium, filter. Dilute (if necessary) with water to obtain a solution having a known concentration of about 0.27 mg/ml of amoxicillin. Calculate the total content of amoxicillin, C16H19N3O5S, and cloxacillin, C19H18ClN3O5S, dissolved in the medium from the peak areas of amoxicillin and cloxacillin in the chromatograms obtained with the test solution and the reference solution, and the contents of C16H19N3O5S and C19H18ClN3O5S in amoxicillin trihydrate RS and cloxacillin sodium RS, respectively. Tolerance: Not less than 80% (Q) of the labeled amount of amoxicillin, C16H19N3O5S, is dissolved in 60 minutes. Not less than 80% (Q) of labeled amount of cloxacillin, C19H18ClN3O5S, is dissolved in 60 minutes.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase A: 0.02 M potassium dihydrogen phosphate, adjust with 0.1 M sodium hydroxide to pH 5.0. Mobile phase B: Acetonitrile. Amoxicillin reference solution: Dissolve an accurately weighed quantity of amoxicillin trihydrate RS in mobile phase A to obtain a solution having a known concentration of about 0.25 mg/ml of amoxicillin. Cloxacillin reference solution: Dissolve an accurately weighed quantity of cloxacillin sodium RS in mobile phase A to obtain a solution having a known concentration of about 0.25 mg/ml of cloxacillin. Test solution: Weigh 20 capsules, calculate the average weight of the contents of the capsules, and finely powder. Weigh accurately a quantity of the powder containing the equivalent of 100 mg of amoxicillin to a 100 ml volumetric flask. Add 70 ml of mobile phase A, dissolve by sonicating, dilute to volume with the same solvent. Mix. Filter. Dilute 25.0 ml of the filtrate to 100.0 ml with mobile phase A. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 μm). Detector: A spectrophotometer set at 225 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 μl. Procedure: Carry out a linear gradient elution using the following conditions. 68

Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0-6

96

4

6 - 15

70

30

15 - 20

96

4

Make adjustments if necessary. System suitability: Inject amoxicillin reference solution and cloxacillin reference solution. The tailing factor of the peaks due to amoxicillin and cloxacillin is not more than 2.0. The relative standard deviation for six replicate injections is not more than 2.0%. Inject separately the reference solutions and the test solution. Calculate the content of amoxicillin, C16H19N3O5S, and cloxacillin, C19H18ClN3O5S, in the capsules using the peak areas of amoxicillin and cloxacillin in the chromatograms obtained with the test solution and the reference solution, and the declared content of C16H19N3O5S and C19H18ClN3O5S in amoxicillin trihydrate RS and cloxacillin sodium RS, respectively.

Storage Store in a cool and dry place, protected from light. Action and use Penicillin antibacterial. Usual strength 250 mg of amoxicillin and 250 mg of cloxacillin. AMPHOTERICIN B Amophotericincum B

C47H73NO17  

M. 924.0

Mixture of antifungal polyenes produced by the growth of certain strains of Streptomyces nodosus or obtained by any other means. It consists mainly of amphotericin B which is (1R,3S,5R,6R,9R,11R,15S,16R,17R,18S,19E,21E,23E, 25E,27E,29E,31E,33R,35S,36R,37S)-33-[(3-amino-3,6dideoxy-β-D-mannopyranosyl)oxy]-1,3,5,6,9,11,17,37-

VP V

octah ydr oxy- 15,16,18-trimethyl-13-oxo-1 4,3 9dioxabicyclo[33.3.1]nonatriaconta-19,21,23,25,27,29,31heptaene-36-carboxylic acid. The potency is not less than 750 IU/mg, calculated with reference to the dried substance.

Characters Yellow or orange, hygroscopic powder. Practically insoluble in water, soluble in dimethyl sulfoxide and in propylene glycol, slightly soluble in dimethylformamide, very slightly soluble in methanol, practically insoluble in ethanol (96%). It is sensitive to light in dilute solutions. Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of amphotericin B RS. If the spectra obtained show differences, dry the substance to be examined and reference substance at 60 °C at a pressure not exceeding 0.7 kPa for 1 h and record new spectra. B. Dissolve 25 mg in 5 ml of dimethyl sulfoxide R and dilute to 50 ml with methanol R. Dilute 2 ml of the solution to 200 ml with methanol R. The ultraviolet absorption spectrum (Appendix 4.1) of the solution in the range 300 nm to 450 nm exhibits three absorption maxima at 362 nm, 381 nm and 405 nm. The ratio of the absorbances measured at 362 nm to that measured at 381 nm is 0.57 to 0.61; the ratio of the absorbances measured at 381 nm to that measured at 405 nm is 0.87 to 0.93. C. Add 5 ml of phosphoric acid R to 1 ml of a 0.05% solution in dimethyl sulfoxide R, to form a lower layer, avoiding mixing the 2 liquids. A blue ring is immediately produced at the junction of the liquids. Mix, an intense blue colour is produced. Add 15 ml of water and mix; the solution becomes pale yellow. D. In the test for Related substances, the principal peak in the chromatogram obtained with the test solution at 383 nm is similar in retention time to the principal peak in the chromatogram obtained with reference solution (1). Related substances Examine by liquid chromatography (Appendix 5.3). Protect the solutions from light and use within 24 h of preparation, except for reference solution (3) which should be injected immediately after its preparation. Mobile phase A: Methanol - acetonitrile - buffer solution pH 4.7 (1 : 3 : 6). Mobile phase B: Methanol - buffer solution pH 3.9 acetonitrile (12 : 20 : 68). Buffer solution pH 3.9: A 0.42% solution of citric acid R previously adjusted to pH 3.9 using concentrated ammonia R. Buffer solution pH 4.7: A 0.42% solution of citric acid R previously adjusted to pH 4.7 using concentrated ammonia R.

AMPHOTERICIN B

Solvent mixture: A 1% solution of ammonium acetate R N-methylpyrrolidone - methanol (1 : 1 : 2). Test solution: Dissolve 20.0 mg of the substance to be examined in 15 ml of N- methylpyrrolidone R and within 2 h dilute to 50.0 ml with the solvent mixture. Dilute 5.0 ml of this solution to 25.0 ml with the solvent mixture. Reference solution (1): Dissolve 20.0 mg of amphotericin B RS in 15 ml of N-methylpyrrolidone R and within 2 h dilute to 50.0 ml with the solvent mixture. Dilute 5.0 ml of this solution to 25.0 ml with the solvent mixture. Reference solution (2): Dilute 1.0 ml of reference solution (1) to 100.0 ml with the solvent mixture. Reference solution (3): Dissolve 20.0 mg of nystatin RS in 15 ml of N-methylpyrrolidone R and within 2 h dilute to 50.0 ml with the solvent mixture. Dilute 5.0 ml of the solution to 25.0 ml with reference solution (1). Dilute 2.0 ml of this solution to 100.0 ml with the solvent mixture. Reference solution (4): In order to prepare impurities B and C, dissolve 10 mg of the substance to be examined in 5 ml of N-methylpyrrolidone R and within 2 h add 35 ml of a mixture of methanol R - anhydrous ethanol R (1 : 4). Add 0.10 ml of dilute hydrochloric acid R, mix and incubate at 25 °C for 2.5 h. Add 10 ml of a 1% solution of ammonium acetate R and mix. Reference solution (5): Dissolve 4 mg of amphotericin B for peak identification RS (containing impurities A and B) in 5 ml of N-methylpyrrolidone R and within 2 h dilute to 50 ml with the solvent mixture. Blank solution: The solvent mixture. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase base-deactivated end-capped octadecylsilyl silica gel for chromatography R (3 µm). Column temperature: 20 °C. Detector: A spectrophotometer set at 303 nm to detect tetraenes; at 383 nm to detect heptaenes. Flow rate: 0.8 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0-3

100

0

3 - 23

100 → 70

0 → 30

23 - 33

70 → 0

30 → 100

33 - 40

0

100

Inject the test solution and reference solutions (2), (3), (4) and (5). Identification of impurities: Use the chromatograms supplied with amphotericin B for peak identification RS and the chromatograms obtained with reference solution (5) to identify the peaks due to impurities A and B. 69

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AMPHOTERICIN LOZENGES

Relative retention with reference to amphotericin B (retention time = about 16 min): impurity B = about 0.75; impurity A = about 0.8; nystatin = about 0.85. System suitability at 383 nm: In the chromatogram obtained with reference solution (4), the resolution between the 2 peaks presenting a relative retention of about 0.7 is at least 1.5. Limits: At 303 nm: Impurity A: The area of the peak due to impurity A is not more than 2.5 times the area of the principal peak in the chromatogram obtained with reference solution (3) (5.0%); if intended for use in the manufacture of parenteral preparations: The area of the peak due to impurity A is not more than the area of the principal peak in the chromatogram obtained with reference solution (3) (2.0%). Any other impurity: For each impurity, the area is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (1.0%). Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (3) (0.1%). At 383 nm: Impurity B: The area of the peak due to impurity B is not more than 4 times the area of the principal peak in the chromatogram obtained with reference solution (2) (4.0%). Any other impurity: For each impurity, the area is not more than 2 times the area of the principal peak in the chromatogram obtained with reference solution (2) (2.0%). Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.1%). Total at 303 and 383 nm: Not more than 15.0%. Note: Impurity A: Amphotericin A (28,29-dihydro-amphotericin B). Impurity B: Amphotericin X1 (13-O-methyl-amphotericin B). Impurity C: Amphotericin X2 (13-O-ethyl-amphotericin B).

Loss on drying Not more than 5.0% (Appendix 9.6). (1.000 g; 60 °C; at a pressure not exceeding 0.7 kPa). Sulfated ash Not more than 3.0% and not more than 0.5% if intended for use in the manufacture of parenteral preparations (Appendix 9.9, method 2). Determined on 1.0 g. Bacterial endotoxins Less than 1.0 EU/mg (Appendix 13.2). If intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins. Assay Protect all solutions from light throughout the assay. 70

Dissolve 25.0 mg of the substance to be examined in dimethyl sulfoxide R and dilute, with shaking, to 25.0 ml with the same solvent. Under constant stirring of this stock solution, dilute with dimethyl sulfoxide R to obtain solutions of appropriate concentrations (the following concentrations have been found suitable: 44.4, 66.7 and 100 IU/ml). Prepare final solutions by diluting 1:20 with buffer solution number 14 (Appendix 13.9), so that they all contain 5% v/v of dimethyl sulfoxide R. Prepare the reference and the test solutions simultaneously. Carry out the microbiological assay of antibiotics (Appendix 13.9).

Storage Protected from light, at a temperature of 2 °C to 8 °C in an airtight container. If the substance is sterile, store in a sterile, tamper-proof container. Labelling The label states, where applicable, that the substance is suitable for use in the manufacture of parenteral preparations. Action and use Antifungal. Preparations Lozenges, oral suspension. AMPHOTERICIN LOZENGES Tabellae Amphotericini Amphotericin lozenges are compressed lozenges containing amphotericin B. The lozenges comply with the requirements stated under “Tablets”, under “Tablets for use in the mouth” (Appendix 1.20) and with the following requirements.

Content of amphotericin B, C47H73NO17, 90.0% to 120.0% of the stated amount. Characters Tablets disintegrate slowly in the mouth when sucked, usually have a flavour and sweet taste. Identification Weight a quantity of the powdered lozenges containing the equivalent of 12.5 mg of amphotericin B add 2.5 ml of dimethyl sulphoxide R and shake for 5 minutes. Add 15 ml of methanol R, shake for a further 10 minutes, add sufficient methanol R to produce 25 ml and filter. Dilute 1 ml of the filtrate to 100 ml with methanol R. The ultraviolet absorptiom spectrum (Appendix 4.1) of the resulting solution, in the range 300 nm to 450 nm exhibits three maxima, at 362 nm, 381 nm and 405 nm. The ratio of the absorbance at the maximum at 362 nm to that at the maximum at 381 nm is 0.5 to 0.6. The ratio of the

VP V

AMPICILLIN

absorbance at 381 nm to that at the maximum at 405 nm is about 0.9.

Content of tetraenes Not more than 13.3% of the stated amount of amphotericin B. Test solution: Add to a quantity of the powdered lozenges containing the equivalent of 37.5 mg of amphotericin B 5 ml of dimethyl sulfoxide R and 25 ml of methanol R and shake for 10 minutes, add sufficient methanol R to produce 50 ml, mix thoroughly and filter. Dilute 4 ml of the filtrate to 50 ml with methanol R. Reference solution (1): Dissolve 50 mg of amphotericin B RS in 5 ml of dimethyl sulphoxide R, adding sufficient methanol R to produce 50 ml and diluting 4 ml of the solution to 50 ml with methanol R. Reference solution (2): Dissolve 25 mg of nystatin RS in 25 ml of dimethyl sulphoxide R, dilute to 250 ml with methanol R and dilute 4 ml of the solution to 50 ml with methanol R. Measure the absorbance of the test solution, reference solution (1) and (2) at the maximum at 282 nm and 304 nm (Appendix 4.1), using a 0.8% v/v solution of dimethyl sulphoxide R in methanol R in the reference cell. Calculate the A(1%, 1 cm) of the preparation being examined, at both wavelengths, each with reference to the content obtained in the assay. Calculate the A(1%, 1 cm) of nystatin RS and of amphotericin B RS, at both wavelengths, each with reference to the dried substance. Calculate the percentage content of tetraenes (X) from the expression:

X = F + 100 (B1S2-B2S1) / (N2B1-N1B2)

where: S1 and S2 are the specific absorbance of the preparation being examined at 282 nm and 304 nm respectively, N1 and N2 are the specific absorbance of nystatin RS at 282 nm and 304 nm, respectively. B1 and B2 are the specific absorbance of amphotericin B RS at 282 nm and 304 nm, respectively. F is the declared content of tetraenes in amphotericin B RS.

Assay Weigh and powder 20 lozenges. Weight accurately a quantity of the powdered tablets containing the equivalent of 10 mg of amphotericin B with 10 ml of water, mix, add sufficient dimethyl sulphoxide R to produce 100 ml, shake for 20 minutes and filter. Carry out the micro-biological assay of antibiotics (Appendix 13.9). 1000 IU is equivalent to 1 mg of amphotericin B. Storage Protected from light. Action and use Antifungal. Usual strength 10 mg.

AMPICILLIN Ampicillinum

C16H19N3O4S  

M. 349.4

Ampicillin is (2S,5R,6R)-6-[[(2R)-2-amino-2-phenylacetyl] amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0] heptane-2-carboxylic acid. It contains not less than 96.0% and not more than 102.0% of C16H19N3O4S, calculated with reference to the anhydrous substance. Semi-synthetic product derived from a fermentation product.

Characters White or almost white, crystalline powder, it shows polymorphism. Sparingly soluble in water, practically insoluble in acetone, in ethanol (96%) and in fatty oils. It dissolves in dilute solutions of acids and of alkali hydroxides. Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of ampicillin RS. B. It complies with the test for Identification of penicillins (Appendix 8.2), use mobile phase B. C. It complies with the test B for Colour reactions of penicillins and cephalosporins (Appendix 8.3). D. It complies with the test for Water. Appearance of solution Dissolve 1.0 g in 10 ml of 1 M hydrochloric acid R. Separately dissolve 1.0 g of the substance to be examined in 10 ml of 2 M ammonia R. Examine immediately after dissolution. The solutions are not more opalescent than reference suspension II (Appendix 9.2). pH 3.5 to 5.5 (Appendix 6.2). Dissolve 0.1 g in carbon dioxide-free water R and dilute to 40 ml with the same solvent. Specific optical rotation +280° to +305°, calculated with reference to the anhydrous substance (Appendix 6.4). 71

VP V

AMPICILLIN

Dissolve 62.5 mg in water and dilute to 25.0 ml with the same solvent.

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Mix 0.5 ml of dilute acetic acid R, 50 ml of 0.2 M potassium dihydrogen phosphate R and 50 ml of acetonitrile R, then dilute to 1000 ml with water. Mobile phase B: Mix 0.5 ml of dilute acetic acid R, 50 ml of 0.2 M potassium dihydrogen phosphate R and 400 ml of acetonitrile R, then dilute to 1000 ml with water. Test solution (1): Dissolve 27.0 mg of the substance to be examined in mobile phase A and dilute to 50.0 ml with mobile phase A. Test solution (2): Dissolve 27.0 mg of the substance to be examined in mobile phase A and dilute to 10.0 ml with mobile phase A. Prepare immediately before use. Reference solution (1): Dissolve 27.0 mg of anhydrous ampicillin RS in mobile phase A and dilute to 50.0 ml with mobile phase A. Reference solution (2): Dissolve 2.0 mg of cefradine RS in mobile phase A and dilute to 50 ml with mobile phase A. To 5.0 ml of this solution add 5.0 ml of reference solution (1). Reference solution (3): Dilute 1.0 ml of reference solution (1) to 20.0 ml with mobile phase A. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 50 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - tR

85

15

tR - (tR + 30)

85 → 0

15 → 100

(tR + 30) - (tR + 45)

0

100

(tR + 45) - (tR + 60)

85

15

tR = retention time of ampicilin determined with the reference solution (3)

If the mobile phase composition has been adjusted to achieve the required resolution, the adjusted composition will apply at time zero in the gradient and in the assay. Inject reference solutions (2) and (3) with isocratic elution at the initial mobile phase composition and the test solution (2) according to the elution gradient described under Mobile phase; inject mobile phase A as a blank according to the elution gradient described under Mobile phase. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks 72

due to ampicillin and cefradin is at least 3.0. If necessary, adjust the ratio A : B of the mobile phase. Limit: Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (3) (1.0%).

Note: Impurity A: (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia-1azabicyclo[3.2.0]heptane-2- carboxylic acid (6-aminopenicillanic acid). Impurity B: (2S,5R,6R)-6-[[(2S)-2-amino-2-phenylacetyl]amino]3,3-dimethyl-7-oxo-4-thia- 1-azabicyclo[3.2.0] heptane-2-carboxylic acid (L-ampicillin). Impurity C: (4S)-2-(3,6-dioxo-5-phenylpiperazin-2-yl)-5,5dimethylthiazolidine-4-carboxylic acid (diketopiperazines of ampicillin). Impurity D: (4S)-2-[[[(2R)-2-amino-2-phenylacetyl]amino] carboxymethyl]-5,5- dimethylthiazolidine-4-carboxylic acid (penicilloic acids of ampicillin). Impurity F: (2RS,4S)-2-[[[(2R)-2-amino-2-phenylacetyl]amino] methyl]-5,5-dimethylthiazolidine-4-carboxylic acid (penilloic acids of ampicillin). Impurity E: (2R)-2-[[[(2S,5R,6R)-6-[[(2R)-2-amino-2-phenylacetyl] amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2yl] carbonyl]amino]-2-phenylacetic acid (ampicillinyl - D-phenylglycine). Impurity G: (3R,6R)-3,6-diphenylpiperazine-2,5-dione. Impurity H: 3-phenylpyrazin-2-ol. Impurity I: (2S,5R,6R)-6-[[(2R)-2-[[(2R)-2-amino-2-phenyl -acetyl] amino]-2-phenylacetyl]amino]-3,3-dimethyl-7-oxo-4thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid (D-phenylglycylampicillin). Impurity J: (2S,5R,6R)-6-[(2,2-dimethylpropanoyl)amino]-3,3dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid. Impurity K: (2R)-2-[(2,2-dimethylpropanoyl)amino]-2-phenylacetic acid. Impurity L: (2R)-2-amino-2-phenylacetic acid (D-phenylglycine). Impurity M: co-oligomers of ampicillin and of penicilloic acids of ampicillin.

N,N-Dimethylaniline Not more than 20 ppm (Appendix 10.16, method 2). Water Not more than 2.0% (Appendix 10.3). Determined on 0.300 g. Sulfated ash Not more than 0.5% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances.

VP V

AMPICILLIN SODIUM

Mobile phase: Initial composition of the mixture of mobile phases A and B, adjusted where applicable. Inject test solution (1) and reference solution (1). System suitability: In the chromatogram obtained with reference solution (1), the relative standard deviation of the peak areas for 6 replicate injections is not more than 1.0%. Calculate the content of C16H19N3O4S using the areas in the chromatograms obtained with test solution (1), reference solution (1) and the declared content of C16H19N3O4S in anhydrous ampicillin RS.

wash with 2 ml to 3 ml of a mixture of water and acetone R (1 : 9), then dry in an oven at 60 °C for 30 min. The infrared absorption spectrum (Appendix 4.2) of the residues is concordant with the spectrum of ampicillin trihydrate RS. B. It complies with the test for Identification of penicillins (Appendix 8.2), use mobile phase B. C. It complies with the test B for Colour reactions of penicillins and cephalosporins (Appendix 8.3). D. It gives reaction (A) of sodium (Appendix 8.1).

Storage Store in an airtight container, at a temperature not exceeding 30 °C.

Appearance of solution Place 1.0 g in a conical flask and add slowly with continuous swirling 10 ml of 1 M hydrochloric acid R. Separately dissolve 1.0 g of the substance to be examined in water and dilute to 10.0 ml with the same solvent. Examine immediately after dissolution. The solutions are not more opalescent than reference suspension II (Appendix 9.2) and the absorbance (Appendix 4.1) of solution in water at 430 nm is not greater than 0.15.

Action and use Beta-lactam antibiotic. Preparations Tablets, capsules, oral suspension, powder.

pH 8.0 to 10.0 (Appendix 6.2). Dissolve 2.0 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent. Measure 10 min after dissolution.

AMPICILLIN SODIUM Ampicillinum natricum

C16H18N3NaO4S   

M. 371.4

Ampicillin sodium is sodium (2S,5R,6R)-6-[[(2R)-2-amino2-phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1azabicyclo[3.2.0]heptane-2-carboxylate. It contains not less than 91.0% and not more than 102.0% of C16H18N3NaO4S, calculated with reference to the anhydrous substance. Semi-synthetic product derived from a fermentation product.

Characters White or almost white powder, hygroscopic. Freely soluble in water, sparingly soluble in acetone, practically insoluble in fatty oils and in liquid paraffin. Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D. A. Dissolve 0.250 g of the substance to be examined in 5 ml of water, add 0.5 ml of dilute acetic acid R, swirl and allow to stand for 10 min in iced water. Filter the crystals through a small sintered-glass filter (40), applying suction,

Specific optical rotation +258° to +287°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 62.5 mg in a 0.4% solution of potassium hydrogen phthalate R and dilute to 25.0 ml with the same solvent. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Mix 0.5 ml of dilute acetic acid R, 50 ml of 0.2 M potassium dihydrogen phosphate R and 50 ml of acetonitrile R, then dilute to 1000 ml with water. Mobile phase B: Mix 0.5 ml of dilute acetic acid R, 50 ml of 0.2 M potassium dihydrogen phosphate R and 400 ml of acetonitrile R, then dilute to 1000 ml with water. Test solution (1): Dissolve 31.0 mg of the substance to be examined in mobile phase A and dilute to 50.0 ml with mobile phase A. Test solution (2): Dissolve 31.0 mg of the substance to be examined in mobile phase A and dilute to 10.0 ml with mobile phase A. Prepare immediately before use. Reference solution (1): Dissolve 27.0 mg of anhydrous ampicillin RS in mobile phase A and dilute to 50.0 ml with mobile phase A. Reference solution (2): Dissolve 2.0 mg of cefradine RS in mobile phase A and dilute to 50 ml with mobile phase A. To 5.0 ml of this solution add 5.0 ml of reference solution (1). 73

VP V

AMPICILLIN SODIUM

Reference solution (3): Dilute 1.0 ml of reference solution (1) to 20.0 ml with mobile phase A. Reference solution (4): To 0.20 g of the substance to be examined add 1.0 ml of water. Heat the solution at 60 °C for 1 h. Dilute 0.5 ml of this solution to 50.0 ml with mobile phase A. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 50 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - tR

85

15

tR - (tR + 30)

85 → 0

15 → 100

(tR + 30) - (tR + 45)

0

100

(tR + 45) - (tR + 60)

85

15

tR = retention time of ampicilin determined with the reference solution (3) If the mobile phase composition has been adjusted to achieve the required resolution, the adjusted composition will apply at time zero in the gradient and in the assay. Inject the reference solutions (2) and (3) with isocratic elution at the initial mobile phase composition and the test solution (2) and reference solution (4) according to the elution gradient described under Mobile phase; inject mobile phase A as a blank according to the elution gradient described under Mobile phase. Identification of peaks: Use the chromatogram obtained with reference solution (4) to identify the peaks due to ampicillin and ampicillin dimer. Relative retention with reference to ampicillin: ampicillin dimer = about 2.8. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to ampicillin and cefradin is at least 3.0. If necessary, adjust the ratio A : B of the mobile phase. Limits: Ampicillin dimer: The area of the peak due to ampicillin dimer is not more than 4.5 times the area of the principal peak in the chromatogram obtained with reference solution (3) (4.5%). Any other impurity: For each impurity, the area is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (3) (2%). Note: Impurity A: (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia-1azabicyclo[3.2.0]heptane-2-carboxylic acid (6-aminopenicillanic acid).

74

Impurity B: (2S,5R,6R)-6-[[(2S)-2-amino-2-phenylacetyl]amino] -3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0] heptane-2-carboxylic acid (L-ampicillin). Impurity C: (4S)-2-(3,6-dioxo-5-phenylpiperazin-2-yl)-5,5dimethylthiazolidine-4-carboxylic acid (diketopiperazines of ampicillin). Impurity D: (4S)-2-[[[(2R)-2-amino-2-phenylacetyl]amino] carboxymethyl]-5,5- dimethylthiazolidine-4-carboxylic acid (penicilloic acids of ampicillin). Impurity F: (2RS,4S)-2-[[[(2R)-2-amino-2-phenylacetyl]amino] methyl]-5,5- dimethylthiazolidine-4-carboxylic acid (penilloic acids of ampicillin). Impurity E: (2R)-2-[[[(2S,5R,6R)-6-[[(2R)-2-amino-2-phenylacetyl] amino]-3,3-dimethyl-7- oxo-4-thia-1-azabicyclo[3.2.0]hept-2-yl] carbonyl]amino]-2-phenylacetic acid (ampicillinyl-D-phenylglycine). Impurity G: (3R,6R)-3,6-diphenylpiperazine-2,5-dione. Impurity H: 3-phenylpyrazin-2-ol. Impurity I: (2S,5R,6R)-6-[[(2R)-2-[[(2R)-2-amino-2phenylacetyl]amino]-2phenylacetyl]amino]-3,3-dimethyl7-oxo-4-thia1-azabicyclo[3.2.0]heptane-2- carboxylic acid (D-phenylglycylampicillin). Impurity J: (2S,5R,6R)-6-[(2,2-dimethylpropanoyl)amino]-3,3dimethyl-7-oxo-4-thia-1- azabicyclo[3.2.0] heptane-2-carboxylic acid. Impurity K: (2R)-2-[(2,2-dimethylpropanoyl)amino]-2-phenylacetic acid. Impurity L: (2R)-2-amino-2-phenylacetic acid (D-phenylglycine). Impurity M: co-oligomers of ampicillin and of penicilloic acids of ampicillin. Impurity N: oligomers of penicilloic acids of ampicillin.

N,N-Dimethylaniline Not more than 20 ppm (Appendix 10.16, method 2). 2-Ethylhexanoic acid Not more than 0.8% m/m (Appendix 10.17). Methylene chloride Not more than 0.2% m/m. Examine by gas chromatography (Appendix 5.2). Internal standard solution: Dissolve 1.0 ml of ethylene chloride R in water and dilute to 500.0 ml with the same solvent. Test solution (1): Dissolve 1.0 g of the substance to be examined in water and dilute to 10.0 ml with the same solvent. Test solution (2): Dissolve 1.0 g of the substance to be examined in water, add 1.0 ml of the internal standard solution and dilute to 10.0 ml with water. Reference solution: Dissolve 1.0 ml of methylene chloride R in water and dilute to 500.0 ml with the same solvent. To 1.0 ml of this solution add 1.0 ml of the internal standard solution and dilute to 10.0 ml with water. Chromatographic system: A glass column (1.5 m long and 4 mm in internal diameter) coated with diatomaceous earth for gas chromatography R impregnated with 10 m/m of macrogol 1000 R. Carrier gas: Nitrogen for chromatography R.

VP V

AMPICILLIN FOR INJECTION

Flow rate: 40 ml/min. Detector: A flame-ionisation detector. Temperature: Column: 60 °C; injection port: 100 °C; detector: 150 °C. Calculate the content of methylene chloride taking its density at 20 °C to be 1.325 g/ml.

The powder complies with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R.

Characters A white or almost white crystalline powder, soluble in water.

Water Not more than 2.0% (Appendix 10.3). Determined on 0.300 g. Bacterial endotoxins Less than 0.15 EU/mg (Appendix 13.2). If intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins. Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. Mobile phase: Initial composition of the mixture of mobile phases A and B, adjusted where applicable. Inject test solution (1) and reference solution (1). System suitability: In the chromatogram obtained with reference solution (1), the relative standard deviation of the peak areas for 6 replicate injections is not more than 1.0%. Calculate the content of C16H19N3O4S using the peak areas in the chromatograms obtained with test solution (1), reference solution (1) and the declared content of C16H19N3O4S in anhydrous ampicillin RS. Calculate the percentage content of ampicillin sodium by multiplying the percentage content of ampicillin by 1.063. Storage In an airtight container. If the substance is sterile, store in a sterile, airtight, tamper-proof container. Action and use Beta-lactam antibiotic. Preparation Injection. AMPICILLIN FOR INJECTION Ampicillinum pro injectione Ampicillin for injection is a sterile crystalline powder of ampicillin sodium. It is supplied in a sealed glass container. Dissolve with sterile water for Injections immediately before use.

Content of ampicillin, C16H19N3O4S, 90.0% to 110.0% of the stated amount.

Identification Apply one of the two following identifications: First identification: A and C. Second identification: B,C and D. A. The infrared absorption spectrum of the contents of the sealed container (Appendix 4.2) is concordant with the spectrum of ampicillin sodium RS. If the spectra are not concordant carry out the following procedure. Dissolve a quantity of the contents of the sealed container containing the equivalent of 0.25 g of ampicillin in 5 ml of water, add 0.5 ml of 2 M acetic acid R, mix and allow to stand for 10 minutes in ice. Filter through a sintered-glass filter (porosity No. 3), wash the residue with 2 ml to 3 ml of a mixture of 9 volumes of acetone R and 1 volume of water, dry at 60 °C for 30 min and prepare a new spectrum of the residue. The spectrum of the residue is concordant with the reference spectrum of ampicillin trihydrate. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. C. Yield the reactions characteristic of sodium salts (Appendix 8.1). D. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Acetone - 15.4% solution of ammonium acetate R adjusted to pH 5.0 with glacial acetic acid R (10 : 90). Test solution: Shake a quantity of the capsules contents containing the equivalent of 25 mg of ampicillin with 10 ml of a 4.2% solution of sodium hydrogen carbonate R, and filter. Reference solution (1): A solution containing 0.25% of ampicillin trihydrate RS in a 4.2% solution of sodium hydrogen carbonate R. Reference solution (2): A solution containing 0.25% of each of ampicillin trihydrate RS and amoxicillin trihydrate RS in a 4.2% solution of sodium hydrogen carbonate R. Procedure: Apply separately to the plate 2 µl of each solution. Develop over a path of 15 cm. After removal of the plate, allow it to dry in air, expose it to iodine vapour until spots appear and examine in daylight. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) shows two clearly separated spots. 75

AMPICILLIN AND SULBACTAM POWDER FOR INJECTION

VP V

Appearance of solution Dissolve 1.0 g of the contents of the sealed container in 10 ml of 1 M hydrochloric acid R, shake well (solution A). Dissolve 1.0 g of the contents of the sealed container in 10 ml of water (solution B). Solution A and solution B are clear or not more opalescent than reference suspension II (Appendix 9.2). The absorbance of solution B at 430 nm is not more than 0.15.

a 50 ml volumetric flask, add 30 ml of the mobile phase, shake to dissolve, dilute to volume with the mobile phase, and mix. Dilute 10.0 ml of the solution to 50.0 ml with the mobile phase, and mix. Reference solution: Weigh accurately about 50 mg of anhydrous ampicillin RS in a 50 ml volumetric flask, add 30 ml of the mobile phase, shake to dissolve, dilute to volume with the mobile phase, and mix. Dilute 10.0 ml of the solution to 50.0 ml with the mobile phase, and mix. Resolution solution:Asolution having known concentrations of 200 mg of anhydrous ampicillin RS per ml and 20 mg of cefradine RS per ml in the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm) (Hypersil ODS is suitable). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.2 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the resolution solution. Adjust the sensitivity of the system so that the heights of the peaks in the chromatogram obtained is at least 50% of the full scale of the recorder. The test is not valid unless the resolution factor between the peaks due to ampicillin and cefradine is at least 3.0. If necessary, adjust the composition of the mobile phase to achieve the required resolution. Inject the reference solution, the relative standard deviation for 6 replicate injections is not more than 2.0%. Inject separately the reference solution and the test solution. Calculate the content of ampicillin, C16H19N3O4S, in a container from the areas of the principal peaks in the chromatograms obtained with the test solution, the reference solution and from the declared content of C16H19N3O4S in ampicillin RS.

Alkalinity pH of a solution containing the equivalent of 10% of ampicillin in carbon dioxide free water R, 8.0 to 10.0 (Appendix 6.2), measured within 10 min of preparation. Water Not more than 2.0% (Appendix 10.3). Use 0.3 g of the contents of the sealed container. Iodine-absorbing substances Dissolve a quantity of about 0.100 g of ampicillin in 20 ml of water, add 0.5 ml of 0.1 M hydrochloric acid R and 25.0 ml of 0.02 N iodine VS. Titrate immediately with 0.02 N sodium thiosulphate VS using starch solution R as indicator. Repeat the titration without the preparation being examined. The difference between the titrations represents the amount of iodine-absorbing substances present. 1 ml of 0.02 N sodium thiosulphate VS is equivalent to 0.7392 mg of iodine-absorbing substances. Calculate the percentage of iodine-absorbing substances in the preparation being examined. The sum of the percentage of iodine-absorbing substances and that of ampicillin sodium, both calculated with reference to the anhydrous material, is not less than 97.5%. 1 mg of C16H19N3O4S, as determined in the Assay, is equivalent to 1.063 mg of ampicillin sodium, C16H18N3NaO4S. Bacterial endotoxins Carry out the test for bacterial endotoxins (Appendix 13.2). Dissolve the contents of the sealed container in water BET R to give a solution containing the equivalent of 9.5 mg of ampicillin per ml (solution A). The endotoxin limit concentration of solution A is 1.5 EU per ml. Carry out the test using the maximum valid dilution of solution A calculated from the declared sensitivity of the lysate used in the test. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Water - acetonitrile - 1 M potassium dihydrogen phosphate - 1 M acetic acid (909 : 80 : 10 : 1). Test solution: Weigh the contents of 10 containers, calculate the average weight of the content in a container and mix. Weigh accurately a quantity of the mixed contents containing the equivalent of about 50 mg of ampicillin in 76

Storage Store at a temperature not exceeding 25 ºC. Action and use Beta-lactam antibiotic. Usual strength 500 mg; 1000 mg. AMPICILLIN AND SULBACTAM POWDER FOR INJECTION Ampicillini et Sulbactami pulvis ad injectionem Ampicillin and sulbactam powder for injection is a sterile, dry mixture of ampicillin and sulbactam for preparation of injection. The labeled representing proportions of ampicillin and to sulbactam of 2 : 1. The powder for injection complies with the requirements stated under Injection, Intravenous infusions (Appendix 1.19) and with the following requirements:

VP V

Content of ampicillin, C16H19N3O4S, from 90.0% to 115.0% of the stated amount. Content of sulbactam, C8H11NO4S, from 90.0% to 115.0% of the stated amount. The powder for injection contains not less than 563 µg of ampicillin and not less than 280 µg of sulbactam per mg, calculate on the anhydrous basic. Characters White or almost white powder. Identification In the Assay, the retention times of the two major peaks in the chromatogram obtained with the test solution correspond to those of the peaks due to ampicillin and sulbactam in the chromatogram obtained with the reference solution. pH The pH value of a solution containing 10 mg of ampicillin and 5 mg of sulbactam per ml of carbon dioxide-free water R is 8.0 to 10.0 (Appendix 6.2). Water Not more than 2.0% (Appendix 10.3). Bacterial endotoxins (Appendix 13.2) Not more than 0.17 EU in a portion equivalent to 1 mg of the mixture of ampicillin and sulbactam (equivalent to 0.67 mg of ampicillin and 0,33 mg of sulbactam). Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: 0.005 M tetrabutylammonium hydroxide acetonitrile (1650 : 350), make adjustments if necessary. 0.005 M tetrabutylamonium hydroxide: Dilute 6.6 ml of a 40% solution of tetrabutylammonium hydroxide with water to obtain 1800 ml. Adjust the pH of the solution to 5.0 ± 0.1 with 1 M phosphoric acid R, add sufficient water to 2000 ml. Reference solution: Weigh accurately a quantity of ampicillin RS and sulbactam RS, dissolve in the mobile phase to obtain the solution containing 0.6 mg of ampicillin and 0.3 mg of sulbactam per ml, use the solution immediately. Resolution solution: Prepare a solution containing 0.3 mg of sulbactam RS per ml of 0.01 M sodium hydroxide, allow to stand in 30 min. Adjust the pH to 5.0 ± 0.1 with 1 M phosphoric acid R. Transfer 5 ml of this solution into 25-ml volumetric flask, add 4.25 ml of acetonitrile R and dilute to volume with 0.005 M tetrabutylammonium hydroxide. Transfer 1 ml of the obtained solution into a 25-ml volumetric flask, add 15 mg of ampicillin RS and dilute to volume with the mobile phase, shake well and

AMPICILLIN AND SULBACTAM POWDER FOR INJECTION

use immediately. Test solution: Weigh the contents of 10 containers, calculate the average mass. Weigh accurately a quantity of the powder containing about 60 mg of ampicillin and 30 mg of sulbactam, transfer into a 100-ml volumetric flask. Add 70 ml of the mobile phase, shake to dissolve and dilute to volume with the same solvent. Use the solution immediately. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5µm) Detector: A spectrophotometer set at 230 nm. Flow rate: 2 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject the resolution solution. In the chromatogram obtained, the relative retention time of ampicillin is 0.7 and that of sulbactam alkaline degradation product is 1.0. The resolution factor between the two peaks is at least 4.0. Inject the reference solution. In the chromatogram obtained, the relative retention time of ampicillin is 0.35 and that of sulbactam is 1.0. The column efficiency determined from the sulbactam peak is not less than 3500 theoretical plates, the tailing factor is not more than 1.5 and relative standard deviation of the peak areas obtained from 6 replicate injections is not more than 2.0%. Inject separately the reference solution and the test solution. Calculate the content of ampicillin, C16H19N3O4S and sulbactam C8H11NO5S, in each container using the peak areas in the chromatograms obtained with the reference solution, the test solution and the declared content of C16H19N3O4S and C8H11NO5S in ampicillin RS and sulbactam RS, respectively.

Storage Store in a well-closed container, in a cool place protected from light. Action and use Penicillin antibacterial + beta-lactamase inhibitor. Usual strength 1 g of ampicillin and 0.5 g of sulbactam (1.5 g vial). 2 g of ampicillin and 1 g of sulbactam (3 g vial).

77

VP V

AMPICILLIN TRIHYDRATE

AMPICILLIN TRIHYDRATE Ampicillinum trihydratum

C16H19N3O4S,3H2O  

Dissolve 62.5 mg in water and dilute to 25.0 ml with the same solvent.

M.430.5

Ampicillin trihydrate is (2S,5R,6R)-6-[[(2R)-2-Amino2-phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1azabicyclo[3.2.0]heptane-2-carboxylic acid trihydrate. It contains not less than 96.0% and not more than 102.0% of C16H19N3O4S, calculated with reference to the anhydrous substance. Semi-synthetic product derived from a fermentation product.

Characters White or almost white, crystalline powder. Slightly soluble in water, practically insoluble in ethanol (96%) and in fatty oils. It dissolves in dilute solutions of acids and of alkali hydroxides. Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of ampicillin trihydrate RS. B. It complies with the test for Identification of penicillins (Appendix 8.2), use mobile phase B. C. It complies with the test B for Colour reactions of penicillins and cephalosporins (Appendix 8.3). D. It complies with the test for Water. Appearance of solution Dissolve 1.0 g in 10 ml of 1 M hydrochloric acid R. Separately dissolve 1.0 g of the substance to be examined in 10 ml of 2 M ammonia R. Examine immediately after dissolution. The solutions are not more opalescent than reference suspension II (Appendix 9.2). pH 3.5 to 5.5 (Appendix 6.2). Dissolve 0.1 g in carbon dioxide-free water R and dilute to 40 ml with the same solvent. Specific optical rotation +280° to +305°, calculated with reference to the anhydrous substance (Appendix 6.4). 78

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Mix 0.5 ml of dilute acetic acid R, 50 ml of 0.2 M potassium dihydrogen phosphate R and 50 ml of acetonitrile R, then dilute to 1000 ml with water. Mobile phase B: Mix 0.5 ml of dilute acetic acid R, 50 ml of 0.2 M potassium dihydrogen phosphate R and 400 ml of acetonitrile R, then dilute to 1000 ml with water. Test solution (1): Dissolve 31.0 mg of the substance to be examined in mobile phase A and dilute to 50.0 ml with mobile phase A. Test solution (2): Dissolve 31.0 mg of the substance to be examined in mobile phase A and dilute to 10.0 ml with mobile phase A. Prepare immediately before use. Reference solution (1): Dissolve 27.0 mg of anhydrous ampicillin RS in mobile phase A and dilute to 50.0 ml with mobile phase A. Reference solution (2): Dissolve 2.0 mg of cefradine RS in mobile phase A and dilute to 50 ml with mobile phase A. To 5.0 ml of this solution add 5.0 ml of reference solution (1). Reference solution (3): Dilute 1.0 ml of reference solution (1) to 20.0 ml with mobile phase A. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 50 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - tR

85

15

tR - (tR + 30)

85 → 0

15 → 100

(tR + 30) - (tR + 45)

0

100

(tR + 45) - (tR + 60)

85

15

tR = retention time of ampicillin determined with the reference solution (3) If the mobile phase composition has been adjusted to achieve the required resolution, the adjusted composition will apply at time zero in the gradient and in the assay. Inject the reference solutions (2) and (3) with isocratic elution at the initial mobile phase composition and the test solution (2) according to the elution gradient described under Mobile phase; inject mobile phase A as a blank according to the elution gradient described under Mobile phase. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks

VP V

due to ampicillin and cefradin is at least 3.0. If necessary, adjust the ratio A : B of the mobile phase. Limit: Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (3) (1.0%). Note: Impurity A: (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia-1azabicyclo[3.2.0]heptane-2- carboxylic acid (6-aminopenicillanic acid). Impurity B: (2S,5R,6R)-6-[[(2S)-2-amino-2-phenylacetyl]amino] -3,3-dimethyl-7-oxo-4-thia- 1-azabicyclo[3.2.0] heptane-2carboxylic acid (L-ampicillin). Impurity C: (4S)-2-(3,6-dioxo-5-phenylpiperazin-2-yl)-5,5dimethylthiazolidine-4-carboxylic acid (diketopiperazines of ampicillin). Impurity D: (4S)-2-[[[(2R)-2-amino-2-phenylacetyl]amino] carboxymethyl]-5,5- dimethylthiazolidine-4-carboxylic acid (penicilloic acids of ampicillin). Impurity F: (2RS,4S)-2-[[[(2R)-2-amino-2-phenylacetyl]amino] methyl]-5,5-dimethylthiazolidine-4-carboxylic acid (penilloic acids of ampicillin). Impurity E: (2R)-2-[[[(2S,5R,6R)-6-[[(2R)-2-amino-2-phenylacetyl] amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo [3.2.0]hept2-yl]carbonyl]amino]-2-phenylacetic acid (ampicillinyl-Dphenylglycine). Impurity G: (3R,6R)-3,6-diphenylpiperazine-2,5-dione. Impurity H: 3-phenylpyrazin-2-ol. Impurity I: (2S,5R,6R)-6-[[(2R)-2-[[(2R)-2-amino-2phenylacetyl]amino]-2-phenylacetyl]amino]-3,3-dimethyl7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2- carboxylic acid (D-phenylglycylampicillin). Impurity J: (2S,5R,6R)-6-[(2,2-dimethylpropanoyl)amino]-3,3dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid. Impurity K: (2R)-2-[(2,2-dimethylpropanoyl)amino]-2-phenylacetic acid. Impurity L: (2R)-2-amino-2-phenylacetic acid (D-phenylglycine). Impurity M: co-oligomers of ampicillin and of penicilloic acids of ampicillin.

N,N-Dimethylaniline Not more than 20 ppm (Appendix 10.16, method 2). Water 12.0% to 15.0% (Appendix 10.3). Determined on 0.100 g. Sulfated ash Not more than 0.5% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. Mobile phase: Initial composition of the mixture of mobile phases A and B, adjusted where applicable.

AMPICILLIN CAPSULES

Inject test solution (1) and reference solution (1). System suitability: In the chromatogram obtained with the reference solution (1), the relative standard deviation of the peak areas due to ampicillin for 6 replicate injections is not more than 1.0%. Calculate the content of C16H19N3O4S using the areas in the chromatograms obtained with test solution (1), reference solution (1) and the declared content of C16H19N3O4S in anhydrous ampicillin RS.

Storage Store in an airtight container, at a temperature not exceeding 30 °C. Action and use Penicillin antibacterial. Preparations Tablets, capsules, powder, suspension. AMPICILLIN CAPSULES Capsulae Ampicillini Ampicillin capsules contain ampicillin or ampicillin trihydrate. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of ampicillin, C16H19N3O4S, 90.0% to 110.0% of the stated amount. Characters Hard capsules containing a white or off-white powder; odourless or almost odourless. Identification A. Shake a quantity of the capsule contents containing the equivalent of 10 mg of ampicillin with 10 ml of water, and filter. To the filtrate add 2 ml of Fehling reagent R. A magenta-violet colour is produced immediately. B. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel G. Mobile phase: Acetone - 15.4% solution of ammonium acetate R adjusted to pH 5.0 with glacial acetic acid R (10 : 90). Test solution: Shake a quantity of the capsules contents containing the equivalent of 125 mg of ampicillin with 50 ml of a 4.2% solution of sodium hydrogen carbonate R, and filter. Reference solution (1): A solution containing 0.25% of ampicillin trihydrate RS in a 4.2% solution of sodium hydrogen carbonate R. Reference solution (2): A solution containing 0.25% of each of ampicillin trihydrate RS and amoxicillin trihydrate RS in a 4.2% solution of sodium hydrogen carbonate R. 79

VP V

ARGININE

Procedure: Apply separately to the plate 2 µl of each solution. Develop over a path of 15 cm. After removal of the plate, allow to dry in air, expose to iodine vapour until spots appear and examine in daylight. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to that in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) shows two clearly separated spots.

Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 900 ml of water. Rotation speed: 100 rpm. Time: 60 min. Procedure: Examine by liquid chromatography (Appendix 5.3). Mobile phase, reference solution and chromatographic system: Prepared as directed in the Assay. Test solution: After the specified time, withdraw a sample of the medium, filter, discard the first 20 ml of the filtrate. Dilute an accurately measured volume of the filtrate with mobile phase A to obtain a solution containing about 0.006% of ampicillin. Tolerance: Not less than 80% (Q) of the labelled amount of ampicillin, C16H19N3O4S, is dissolved in 60 minutes. Loss on drying Dry about 500 mg, accurately weighed, of the mixed capsule contents in vacuum at a pressure not exceeding 5 mmHg at 60 ºC for 3 hours. Loss in mass is not more than 4.0% where the capsules contain anhydrous ampicillin, or between 10.0% and 15% where the capsules contain ampicillin trihydrate. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Dilute a mixture of 1 volume of acetic acid (10%) R, 100 volumes of 0.2 M potassium dihydrogen phosphate R and 100 volumes of acetonitrile R to 2000 volumes with water. Mobile phase B: Dilute a mixture of 1 volume of acetic acid (10%) R, 100 volumes of 0.2 M potassium dihydrogen phosphate R and 800 volumes of acetonitrile R to 2000 volumes with water. Mobile phase: Mobile phase A - mobile phase B (85 : 15). Reference solution: A 0.006% solution of ampicillin RS in mobile phase A. Resolution solution: A solution contains 0.025% of ampicillin RS and 0.002% of cefradine RS in mobile phase A. Test solution: Weigh 20 capsules, calculate the average weight of the capsule contents and powder finely. Weigh accurately a quantity of the powder containing the equivalent of 60 mg of ampicillin, add 80 ml of mobile phase A, shake for 15 minutes, dilute to 100.0 ml with the 80

same solvent, and mix. Filter, discarding the first 20 ml of the filtrate. Dilute 5.0 ml of the filtrate to 50.0 ml with mobile phase A. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm) (Nucleosil C18 is suitable). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 50 µl. Procedure: System suitability: Inject the resolution solution, the resolution factor between the peaks due to ampicillin and cefradine is at least 3.0. If necessary, adjust the composition of the mobile phase to achieve the required resolution. Inject separately the reference solution and the test solution. Calculate the content of ampicillin, C16H19N3O4S, in the capsules using the areas of the principal peaks in the chromatograms obtained with the test solution, the reference solution and the declared content of C16H19N3O4S in ampicillin RS.

Storage Store in an airtight container in a cool place, protected from light. Action and use Beta-lactam antibacterial. Usual strength 250 mg; 500 mg (calculated as anhydrous ampicillin). ARGININE Argininum

C6H14N4O2  

M. 174.2

Arginine is (S)-2-amino-5-guanidinopentanoic acid. It contains not less than 98.5% and not more than 101.0% of C6H14N4O2, calculated with reference to the dried substance.

Characters A white or almost white, crystalline powder or colourless crystals. Freely soluble in water, very slightly soluble in ethanol (96%). Identification Apply one of the two following identifications: First identification: A, C. Second identification: B, C, D, E. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of arginine RS. Examine the substances prepared as discs.

VP V

B. In the test for Ninhydrin-positive substances, the principal spot in the chromatogram obtained with test solution (2) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). C. It complies with the test for Specific optical rotation. D. pH of solution S is more than 10 (Appendix 6.2). E. Dissolve about 25 mg of the substance to be examined in 2 ml of water. Add 1 ml of 1-naphthol solution R and 2 ml of a mixture of equal volumes of strong sodium hypochlorite solution R and water. A red colour develops.

Appearance of solution Solution S: Dissolve 2.5 g in distilled water and dilute to 50 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution BY6 (Appendix 9.3, method 2). Specific optical rotation +25.5° to +28.5°, calculated with reference to the dried substance (Appendix 6.4). Dissolve 2.00 g in a 25% solution of hydrochloric acid R and dilute to 25.0 ml with the same solvent. Ninhydrin-positive substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel. Mobile phase: Ammonia - 2-propanol (30 : 70). Test solution (1): Dissolve 0.10 g of the substance to be examined in dilute hydrochloric acid R and dilute to 10 ml with the same solvent. Test solution (2): Dilute 1 ml of test solution (1) to 50 ml with water. Reference solution (1): Dissolve 10 mg of arginine RS in 0.1 M hydrochloric acid R and dilute to 50 ml with the same solvent. Reference solution (2): Dilute 5 ml of test solution (2) to 20 ml with water. Reference solution (3): Dissolve 10 mg of arginine RS and 10 mg of lysine hydrochloride RS in 0.1 M hydrochloric acid R and dilute to 25 ml with the same solvent. Procedure: Apply separately to the plate 5 µl of each solution. Allow the plate to dry in air. Develop over a path of 15 cm. Dry the plate at 100 °C to 105 °C until the ammonia disappears completely. Spray with a 0.2% solution of ninhydrin R and heat at 100 °C to 105 °C for 15 min. In the chromatogram obtained with test solution (1), any spot, apart from the principal spot, is not more intense than the principal spot in the chromatogram obtained with reference solution (2) (0.5%). The test is not valid unless the chromatogram obtained with reference solution (3) shows two clearly separated spots.

ARGININE

Chlorides Not more than 0.02% (Appendix 9.4.5). To 5 ml of solution S, add 0.5 ml of dilute nitric acid R and dilute to 15 ml with water. Sulfates Not more than 0.03% (Appendix 9.4.14). To 10 ml of solution S, add 1.7 ml of dilute hydrochloric acid R and dilute to 15 ml with distilled water. Ammonium Not more than 0.02% (Appendix 9.4.1). 50 mg complies with limit test for ammonium, method B. Prepare the standard using 0.1 ml of ammonium standard solution (100 ppm NH4) R. Iron Not more than 10 ppm (Appendix 9.4.13). Dissolve 1.0 g of the substance to be examined in 10 ml of dilute hydrochloric acid R. Shake with three quantities, each of 10 ml, of methyl isobutyl ketone R1, shaking for 3 min each time. To the combined organic layers add 10 ml of water and shake for 3 min. Use the aqueous layer to determine. Heavy metals Not more than 10 ppm (Appendix 9.4.8). Dissolve 2.0 g of the substance to be examined in water and dilute to 20 ml with the same solvent. 12 ml of the solution complies with limit test for heavy metals, method 1. Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.150 g of the substance to be examined in 50 ml of water and titrate with 1 N hydrochloric acid VS, using 0.2 ml of methyl red mixed solution R as indicator, until the colour changes from green to violet-red. 1 ml of 1 N hydrochloric acid VS is equivalent to 17.42 mg of C6H14N4O2. Storage Store in an airtight container, protected from light. Action and use Amino acid. Preparation Capsules.

81

VP V

ARGININE ASPARTA­­TE

ARGININE ASPARTA­­TE Arginini aspartas

C6H14N4O2,C4H7NO4 

M. 307.3

Arginine aspartate is (2S)-2-amino-5-guanidinopentanoic acid (2S)-2-aminobutanedioate. It contains not less than 99.0% and not more than 101.0% of C6H14N4O2,C4H7NO4, calculated with reference to the dried substance.

Characters White or almost white granules or powder. Very soluble in water, practically insoluble in ethanol (96%) and in methylene chloride. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of arginine aspartate RS. B. It complies with the test for Specific optical rotation. C. In the test for Ninhydrin-positive substances, the two principal spots in the chromatogram obtained with test solution (2) are similar in position, colour and size to the two principal spots in the chromatogram obtained with reference solution (1). Appearance of solution Solution S: Dissolve 5.0 g in carbon dioxide-free water and dilute to 50 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution Y7 (Appendix 9.3, method 2). pH 6.0 to 7.0 (Appendix 6.2). Determined on solution S. Specific optical rotation +25° to +27°, calculated with reference to the dried substance (Appendix 6.4). Dissolve 2.50 g in dilute hydrochloric acid R and dilute to 25.0 ml with the same solvent. Ninhydrin-positive substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Ammonia - propanol (36 : 64). Test solution (1): Dissolve 0.20 g of the substance to be examined in water and dilute to 10 ml with the same solvent. Test solution (2): Dilute 1 ml of test solution (1) to 10 ml with water. Reference solution (1): Dissolve 25 mg of arginine R and 25 mg of aspartic acid R in water and dilute to 25 ml with the same solvent. 82

Reference solution (2): Dilute 2 ml of reference solution (1) to 50 ml with water. Procedure: Apply separately to the plate 5 µl of each solution. Allow the plate to dry in air. Develop over a path of 2/3 of the plate. Heat the plate at 100 °C to 105 °C for 10 min. Spray with a 0.2% solution of ninhydrin R and heat at 100 °C to 105 °C for 10 min. In the chromatogram obtained with test solution (1), any spot, apart from two principal spots, is not more intense than each of the two principal spots in the chromatogram obtained with reference solution (2) (0.2%). The test is not valid unless the chromatogram obtained with reference solution (2) shows two clearly separated spots.

Chlorides Not more than 0.02% (Appendix 9.4.5). To 2.5 ml of solution S and dilute to 15 ml with water. Sulfates Not more than 0.03% (Appendix 9.4.14). To 0.5 g of the substance to be examined, add 2.5 ml of dilute hydrochloric acid R and dilute to 15 ml with distilled water. Examine after 30 min. Ammonium Not more than 0.01% (Appendix 9.4.1). Determined on 100 mg. Heavy metals Not more than 20 ppm (Appendix 9.4.8). 12 ml of solution S complies with limit test for heavy metals, method 1. Prepare the standard using lead standard solution (2 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 60 °C; 24 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 80.0 mg of the substance to be examined in 2 ml of anhydrous formic acid R. Add 50 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 10.24 mg of C10H21N5O6. Storage Store in an airtight container, protected from light. Action and use Amino acid. Preparation Capsules.

VP V

ARGININE HYDROCHLORIDE

ARGININE HYDROCHLORIDE Arginini hydrochloridum

C6H14N4O2,HCl  

M: 210.7

Arginine hydrochloride is hydrochloride of (S)-2-amino5-guanidinopentanoic acid. It contains not less than 98.5% and not more than 101.0% of C6H14N4O2,HCl, calculated with reference to the dried substance.

Characters A white or almost white, crystalline powder or colourless crystals. Freely soluble in water, very slightly soluble in ethanol (96%). Identification Apply one of the two following identifications: First identification: A, B, E. Second identification: B, C, D, E. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of arginine hydrochloride RS. Examine the substances prepared as discs. B. It complies with the test for Specific optical rotation. C. In the test for Ninhydrin-positive substances, the principal spot in the chromatogram obtained with test solution (2) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). D. Dissolve about 25 mg of the substance to be examined in 2 ml of water. Add 1 ml of α-naphthol solution R and 2 ml of a mixture of equal volumes of strong sodium hypochlorite solution (3% Cl) R and water. A red colour develops. E. It gives reaction (A) of chlorides (Appendix 8.1). Appearance of solution Solution S: Dissolve 2.5 g in distilled water and dilute to 50 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution BY6 (Appendix 9.3, method 2). Specific optical rotation +21.0° to +23.5°, calculated with reference to the dried substance (Appendix 6.4). Dissolve 2.00 g in a 25% solution of hydrochloric acid R and dilute to 25.0 ml with the same acid. Ninhydrin-positive substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Ammonia - 2-propanol (30 : 70).

Test solution (1): Dissolve 0.10 g of the substance to be examined in water and dilute to 10 ml with the same solvent. Test solution (2): Dilute 1 ml of test solution (1) to 50 ml with water. Reference solution (1): Dissolve 10 mg of arginine hydrochloride RS in water and dilute to 50 ml with the same solvent. Reference solution (2): Dilute 5 ml of test solution (2) to 20 ml with water. Reference solution (3): Dissolve 10 mg of arginine hydrochloride RS and 10 mg of lysine hydrochloride RS in water and dilute to 25 ml with the same solvent. Procedure: Apply separately to the plate 5 µl of each solution. Dry the plate in air. Develop over a path of 15 cm. Heat at 100 °C to 105 °C until the ammonia disappears completely. Spray with a 0.2% ninhydrin solution R and heat at 100 °C to 105 °C for 15 min. Any spot in the chromatogram obtained with test solution (1), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (2) (0.5%). The test is not valid unless the chromatogram obtained with reference solution (3) shows two clearly separated spots.

Sulfates Not more than 300 ppm (Appendix 9.4.14). Dilute 10 ml of solution S to 15 ml with distilled water. Ammonium Not more than 200 ppm (Appendix 9.4.1). 50 mg of the substance to be examined complies with limit test B for ammonium. Prepare the standard using 0.1 ml of ammonium standard solution (100 ppm NH4) R. Iron Not more than 10 ppm (Appendix 9.4.13). In a separating funnel, dissolve 1.0 g in 10 ml of dilute hydrochloric acid R. Shake with three quantities, each of 10 ml of 4-methylpentan-2-one R1, shaking for 3 min each time. To the combined 4-methylpentan-2-one layers add 10 ml of water and shake for 3 min. The aqueous layer complies with the limit test for iron. Heavy metals Not more than 10 ppm (Appendix 9.4.8). Dissolve 2.0 g in water and dilute to 20 ml with the same solvent. 12 ml of the solution complies with limit test for heavy metals, method 1. Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. 83

ARGININE CAPSULES

Assay Examine by non-aqueous titration (Appendix 10.6). Dissolve 0.180 g of the substance to be examined in 3 ml of anhydrous formic acid R, add 30 ml of anhydrous acetic acid R. Using 0.1 ml of naphtholbenzein solution R as indicator. Titrate with 0.1 N perchloric acid VS until the colour changes from brownish-yellow to green. Carry out a blank titration. 1 ml of 0.1 N perchloric acid VS is equivalent to 21.07 mg of C6H15ClN4O2. Storage Store in an airtight container, protected from light. Action and use Amino acid, nutrient. Preparation Tablets, capsules, infusion, oral suspension. ARGININE CAPSULES Capsulae Arginini Arginine capsules are hard capsules containing arginine or arginine hydrochloride. The capsules comply with the requirements stated under "Capsules" (Appendix 1.13) and with the following requirements.

Content of arginine, C6H14N4O2, 90.0% to 110.0% of the stated amount. Identification A. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of the arginine peak in the chromatogram obtained with the reference solution. B. Examine by thin layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Isopropanol - ammonia (7 : 3). Test solution: To a quantity of the capsule contents containing about 150 mg of arginine, add 80 ml of water and sonicate for 15 min, dilute to 100 ml with water, filter. Reference solution: A solution of arginine RS or arginine hydrochloride RS having a concentration of arginine of 1.5 mg/ml in water. Procedure: Apply separately to the plate 5 µl of each solution. After development, remove the plate, heat at 105 °C to eliminate completely ammonia. Spray the plate with a 0.2% solution of ninhydrin R in a mixture of butanol R and 2 M acetic acid R (95 : 5), continue to heat at 105 °C for 15 min. In the chromatogram obtained with the test solution, the principal spot is similar in position, colour and size to that in the chromatogram obtained with the reference solution. Dissolution (Appendix 11.4) Apparatus: Paddle. 84

VP V

Medium: 900 ml of 0.1 M hydrochloric acid R. Rotation speed: 100 rpm. Time: 60 min. Procedure: Examine by liquid chromatography (Appendix 5.3). Mobile phase, chromatographic system are described in the Assay. Test solution: After the specified time, withdraw a sample of the medium, and filter. Reference solution: Weigh accurately a quantity of arginine or arginine hydrochloride RS, dissolve in 0.1 M hydrochloric acid R to obtain a solution having a concentration of arginine similar to that expected in the test solution. Tolerance: Not less than 75% (Q) of the labeled amount of arginine, C6H14N4O2, is dissolved in 60 min.

Assay Examine by liquid chromatography (Appendix 5.3). Buffer solution: A 0.69% solution of sodium dihydrogen phosphate in water, adjusted to pH 3.5 with phosphoric acid R. Solution A: A 0.05% solution of sodium octanesulfonate in the buffer solution. Mobile phase: Solution A - acetonitrile (95 : 5). Reference solution: Dissolve a quantity of arginine RS or arginine hydrochloride RS in the buffer solution to obtain a solution having a concentration of arginine of about 1.5 mg/ml. Test solution: Weigh 20 capsules, calculate the average mass of the capsule contents and grind to fine powder. Weigh accurately a quantity of the powdered content containing about 150 mg of arginine. Transfer to a 100-ml volumetric flask, add 80 ml of the buffer solution, sonicate for 15 min. Dilute to volume with the buffer solution, shake well and filter. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase B (5 µm). Detector: A spectrophotometer set at 215 nm. Flow rate: 0.8 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject the reference solution, in the chromatogram obtained, the number of theoretical plates determined on the peak due to arginine is not less than 1500. The relative standard deviation of peak areas for six replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of arginine, C6H14N4O2, in each capsule using the areas of the principal peaks in the chromatograms obtained with the reference solution, the test solution and the declared content of C6H14N4O2 in arginine RS or C6H14N4O2,HCl in arginine hydrochloride RS. Conversion factor from arginine hydrochloride to arginine is 0.8269. Storage Store in a well-closed container, in a cool place, protected from light.

VP V

ARTEMETHER

Action and use Amino acid.

D. Dissolve 30 mg in 6 ml of ethanol R. Place a few drops of the mixture on a white porcelain dish and add 1 drop of a 5% solution of vanillin in sulfuric acid R, a pink colour is produced.

Usual strength 200 mg, 400 mg and 500 mg.

Melting range 86.0 °C to 90.0 °C (Appendix 6.7).

ARTEMETHER Artemetherum H3C H

H

H H3C

O H

O

O

Specific optical rotation +166° to +173°, calculated with reference to the dried substance (Appendix 6.4). Use a 10 mg/ml solution in ethanol R.

OCH3 H

H O

CH3

C16H26O5

M. 298.4

Artemether is (3R,5aS,6R,8aS,9R,10S,12R,12aR)-decahydro -10-methoxy-3,6,9-trimethyl-3,12-epoxy-12H-pyrano [4,3-j]-1,2-benzodioxepine. It contains not less than 97.0% and not more than 102.0% of C16H26O5, calculated with reference to the dried substance if assay by HPLC method; and it contains not less than 98.0% and not more than 102.0% of C16H26O5, calculated with reference to the dried substance if assay by specphotometric method.

Characters White crystals or a white, crystalline powder. Freely soluble in ethyl acetate and in ethanol, very soluble in dichloromethane and in acetone, practically insoluble in water. Identification Apply one of the two following identifications: First identification: A, B Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of artemether RS or the reference spectrum of artemether RS. B. In the test for Related substances examined by thin-layer chromatography, the principal spot in the chromatogram obtained with test solution (2) corresponds in position, size, and colour to the principal spot in the chromatogram obtained with reference solution (3). Or in the test for Assay examined by liquid chromatography, the principal peak in the chromatogram obtained with the test solution corresponds in retention time to the principal peak in the chromatogram obtained with the reference solution. C. To 30 mg add about 1 ml of ethanol R and about 0.1 g of potassium iodide R. Heat the mixture on a water-bath, a yellow colour is produced.

Related substances Either test A or test B may be applied. A. Examine by liquid chromatography (Appendix 5.3) as described in the test for Assay, method A. Test solution: Dissolve 100.0 mg of the substance to be examined in the mobile phase and dilute to 10.0 ml with the same solvent. Reference solution: Dilute 1.0 ml of the test solution to 200.0 ml with the mobile phase. Procedure: Inject the test solution and the reference solution. In chromatogram obtained with the test solution, the area of any peaks, apart from the principal peak, is not greater than the area of principal peak in the chromatogram obtained with the reference solution (0.5%). Not more than one peak having area is greater than half the area of the principal peak obtained with the reference solution (0.25%). The sum of the areas of all the peaks, apart from the principal peak, is not more than twice the area of the principal peak obtained with the reference solution (1.0%). Disregard any peak with an area less than 0.1 times that of the principal peak in the chromatogram obtained with the reference solution. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Petroleum ether (40 ºC - 60 ºC) - ethyl acetate (7 : 3). Test solution (1): Dissolve 100.0 mg of the substance to be examined in acetone R and dilute to 10.0 ml with the same solvent. Test solution (2): Dilute 1 ml of test solution (1) to 100 ml with acetone R. Reference solution (1): Dilute 1.0 ml of test solution (1) to 200.0 ml with acetone R. Reference solution (2): Dilute 5.0 ml of reference solution (1) to 10.0 ml with acetone R. Reference solution (3): Dissolve 10 mg of artemether RS in acetone R and dilute to 100 ml with the same solvent. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in air. Spray with a 5% solution of vanillin in sulfuric acid R and examine in daylight. Any spot in the chromatogram obtained with test solution (1), apart from the principal spot is not more intense than the spot in the chromatogram obtained with reference solution (1) (0.5%) and not more than one such spot is more intense than the principal spot in the chromatogram obtained with reference solution (2) (0.25%). 85

VP V

ARTEMETHER CAPSULES

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; at a pressure not exceeding 2.67 kPa; phosphorus pentoxide). Sulfated ash Not more than 0.1% (Appendix 9.9, method 1). Determined on 1.0 g. Assay Either test A or test B may be applied. A. Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - water (62 : 38). Test solution: Dissolve 100.0 mg of the substance to be examined in the mobile phase and dilute to 10.0 ml with the same solvent. Reference solution: Dissolve 100.0 mg of artemether RS in the mobile phase and dilute to 10.0 ml with the same solvent. Chromatographic system: A column (25 cm × 4.0 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 216 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: Inject the reference solution and the test solution. Calculate the content of C16H26O5 using the peak areas in chromatograms obtained with the test solution and the reference solution. B. Examine by ultraviolet and visible absorption spectrophotometry (Appendix 4.1). Weigh accurately about 0.050 g of the substance to be examined, dissolve in ethanol R and dilute to 100 ml with the same solvent. Accurately transfer 2.0 ml of this solution to a 100.0 ml volumetric flask, and add 1 M hydrochloric acid in ethanol R to volume. Stopper the flask and warm at 55 °C in water-bath for 5 h, allow to cool at room temperature. Measure the absorbance of the solution in a 1 - cm cell at the maximum at 254 nm. Calculate the content of artemether, C16H26O5, in the substance to be examined by comparison with the absorbance of a reference solution prepared in the same manner with the test solution. Storage Store in a well-closed container, protected from light, in a cool place. Action and use Antimalarial. Preparations Injection, capsules.

86

ARTEMETHER CAPSULES Capsulae Artemetheri Artemether capsules contain artemether. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of artemether, C16H26O5, 90.0% to 110.0% of the stated amount. Identification A. To a quantity of the capsule contents, equivalent to about 80 mg of artemether, add 10 ml of ethanol R, shake well for 5 min to 10 min, and filter (the filtrate A). To 3 ml of the filtrate A add 0.1 g of potassium iodide R, shake and heat in a water bath. A pale yellow colour is produced. B. Place several drops of the filtrate A on a white porcelain, add 1 drop of a 1% solution of anisaldehyde in sulfuric acid R. A pink colour is produced. C. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. Related substances Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel G. Mobile phase: Petroleum ether (40°C - 60°C) - ethyl acetate (7 : 3). Test solution: To a quantity of the capsule contents, equivalent to about 50 mg of artemether, add 5 ml of acetone R, shake for 10 min, and filter. Use the filtrate. Reference solution (1): Dilute 1 volume of the test solution to 200 volumes with acetone R. Reference solution (2): Dilute 5 volumes of reference solution (1) to 10 volumes with acetone R. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm. After removal of the plate, allow it to dry in air, spray with a 5% solution of vanillin in sulfuric acid R and examine in daylight. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (1) (0.5%). And not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (2) (0.25%). Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 500 ml of water (for strength of 40 mg) or 1000 ml of water (for strength of 100 mg). Rotation speed: 100 rpm. Time: 60 min. Test solution: After the specified time, withdraw a sample of the medium, filter, discard the first portion of the filtrate.

VP V

Dilute 5.0 ml of the filtrate to 25.0 ml with 1 M ethanolic hydrochloric acid R, and mix. Reference solution: Weigh accurately about 40 mg of artemether RS in a 200 ml volumetric flask, dissolve in ethanol R, and dilute to volume with the same solvent, and mix. Transfer 5.0 ml of this solution to a 50 ml volumetric flask, add 5 ml of water, and dilute to volume with 1 M ethanolic hydrochloric acid R, and mix. Immerse the test solutions and the reference solution in a water bath at 70 ± 1 °C for 90 min, and cool to room temperature. Measure the absorbance of the solutions at 254 nm (Appendix 4.1) in a 1-cm cell, using 1 M ethanolic hydrochloric acid R in the reference cell. Calculate the content of artemether, C16H26O5, is dissolved using the measured absorbances of the test solution, the reference solution and the declared content of C16H26O5 in artemether RS. Tolerance: Not less than 65% (Q) of the labelled amount of artemether, C16H26O5, is dissolved in 60 min.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: A mixture of acetonitrile and water (55 : 45). Reference solution: A solution in the mobile phase of artemether RS having a known concentration of about 4 mg/ml. Test solution: Weigh 20 capsules, calculate the average weight of the capsule contents and powder finely. Transfer an accurately weighed quantity of the powder, equivalent to about 100 mg of artemether, to a 25 ml volumetric flask, add 20 ml of the mobile phase, sonicate for 10 min, cool, dilute to volume with the mobile phase, and mix. Filter, discard the first portion of the filtrate. Use the filtrate. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 210 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Inject separately the reference solution and the test solution. Calculate the content of artemether, C16H26O5, in the capsules using the areas (or the height) for the principal peaks in the chromatograms obtained with the test solution, the reference solution and the declared content of C16H26O5 in artemether RS. Storage Store in a cool place, protected from light. Action and use Antimalarial. Usual strength 40 mg; 100 mg.

ARTEMETHER TABLETS

ARTEMETHER TABLETS Tabellae Artemetheri Artemether tablets contain artemether. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of artemether, C16H26O5, 90.0% to 110.0% of the stated amount. Identification A. To a quantity of the powdered tablets containing about 80 mg of artemether, add 10 ml of ethanol R, shake well for 5 to 10 min, and filter (the filtrate A). To 3 ml of the filtrate A add 0.1 g of potassium iodide R, shake and heat in a water bath. A pale yellow colour is produced. B. Place several drops of the filtrate A on a white porcelain, add 1 drop of a 1% solution of anisaldehyde in sulfuric acid R. A pink colour is produced. C. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. Related substances Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel G. Mobile phase: Petroleum ether (40°C - 60°C) - ethyl acetate (7 : 3). Test solution: To a quantity of the powdered tablets containing about 50 mg of artemether, add 5 ml of acetone R, shake for 10 min, and filter. Use the filtrate. Reference solution (1): Dilute 1 volume of the test solution to 200 volumes with acetone R. Reference solution (2): Dilute 5 volumes of reference solution (1) to 10 volumes with acetone R. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm. After removal of the plate, allow it to dry in air, spray with a 5% solution of vanillin in sulfuric acid R and examine in daylight. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (1) (0.5%). And not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (2) (0.25%). Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 500 ml of water (for strength of 40 mg) or 1000 ml of water (for strength of 100 mg). Rotation speed: 100 rpm. Time: 60 min. Test solution: After the specified time, withdraw a sample of the medium, filter and discard the first portion of the 87

ARTEMETHER AND LUMEFANTRINE TABLETS

filtrate. Dilute 5.0 ml of the filtrate to 25.0 ml with 1 M ethanolic hydrochloric acid R, and mix. Reference solution: Weigh accurately about 40 mg of artemether RS in a 200-ml volumetric flask, dissolve in ethanol R, and dilute to volume with the same solvent, and mix. Transfer 5.0 ml of this solution to a 50 ml volumetric flask, add 5 ml of water, and dilute to volume with 1 M ethanolic hydrochloric acid R, and mix. Immerse the test solutions and the reference solution in a water bath at 70 °C ± 1 °C for 90 min, and cool to room temperature. Measure the absorbance of the solutions at 254 nm (Appendix 4.1) in a 1-cm cell, using 1 M ethanolic hydrochloric acid R in the reference cell. Calculate the content of artemether, C16H26O5, is dissolved using the measured absorbances of the test solution, the reference solution and the declared content of C16H26O5 in artemether RS. Tolerance: Not less than 65% (Q) of the labelled amount of artemether, C16H26O5, is dissolved in 60 minutes.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - water (55 : 45). Reference solution: A solution in the mobile phase of artemether RS having a known concentration of about 4 mg/ml. Test solution: Weigh 20 tablets, calculate the average mass, and powder finely. Transfer an accurately weighed quantity of the powder containing the equivalent to about 100 mg of artemether, to a 25 ml volumetric flask, add 20 ml of the mobile phase, sonicate for 10 min, cool, dilute to volume with the mobile phase, and mix. Filter, discarding the first portion of the filtrate. Use the filtrate. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 210 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Inject separately the reference solution and the test solution. Calculate the content of artemether, C16H26O5, in the tablets using the areas (or the height) for the principal peaks in the chromatogram obtained with the test solution and the reference solution, and the declared content of C16H26O5 in artemether RS. Storage Store in a cool place, protected from light. Action and use Antimalarial. Usual strength 40 mg; 100 mg.

88

VP V

ARTEMETHER AND LUMEFANTRINE TABLETS Tabellae Artemetheri et Lumefantrini Artemether and lumefantrine tablets contain artemether and lumefantrine. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of artemether, C16H26O6 and lumefantrine, C30H32Cl3NO, 90.0% to 110.0% of the stated amount. Identification A. Examine by thin layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Petroleum ether (40 °C - 60 °C) - ethyl acetate - glacial acetic acid (40 : 10 : 5). Test solution: Shake a quantity of the powdered tablets containing about 10 mg of artemether (about 60 mg of lumefantrine) in 10 ml of acetone R. Filter. Reference solution: A solution containing 1 mg of artemether RS and 6 mg of lumefantrine RS in 1 ml of acetone R. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of about three-quarters of the plate, dry immediately the plate in a current of cool air. Examine under ultraviolet light at 254 nm. In the chromatogram obtained with the test solution, the principal spot is similar in position, size and colour to the principal spot in the chromatogram obtained with the reference solution (lumefantrine identification). Spray the plate with a 10% solution of sulfuric acid in ethanol R and heat at 140 °C for 10 min, examine in daylight. In the chromatogram obtained with the test solution, the principal spot is similar in position, size and colour with the principal spot in the chromatogram obtained with the reference solution (artemether identification, maybe including a very faint spot of lumefantrine). B. In the Assay, the retention times of two principal peaks in the chromatogram obtained with the test solution are similar to those of the artemether and lumefantrine peaks in the chromatogram obtained with the reference mixture solution. Related substances Examine by thin layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Petroleum ether (40 °C - 60 °C) - ethyle acetate - glacial acetic acid (40 : 10 : 5). Solvent mixture: Water - acetonitrile (1 : 1). Test solution: Weigh a quantity of the powdered tablets containing about 100 mg of artemether, add 20 ml of the solvent mixture, sonicate for 15 min and centrifuge, filter the supernatant liquid through a 0.45 µm membrane filter. Reference solution (1): Dissolve 5 mg of artemether RS, 5 mg of artenimol RS and 5 mg of α-artemether RS in 50 ml of the solvent mixture. Reference solution (2): Dilute 2.0 ml of reference solution (1) to 20 ml with the solvent mixture.

VP V

Reference solution (3): Dilute 3.0 ml of reference solution (1) to 20 ml with the solvent mixture. Reference solution (4): Dilute 5.0 ml of reference solution (1) to 20 ml with the solvent mixture. Reference solution (5): Dilute 1.0 ml of reference solution (1) to 2 ml with the solvent mixture. Reference solution (6): Dilute 3.0 ml of reference solution (1) to 4 ml with the solvent mixture. Procedure: Apply separately to the plate 20 µl of the test solution, reference solution (2), (3), (4), (5) and (6). Allow the spots to dry in a current of cool air. Develop over a path of about 12 cm. Allow it to dry in a current of cool air. Spray the plate with vanillin solution in ethanol (96%) R. Heat the plate at 140 °C for 10 min. Examine in daylight, the Rf value of artemether spot and its impurities are as follow: about 0.25 for impurity A, about 0.3 for impurity B (artenimol), about 0.35 for impurity C, about 0.4 for impurity D (α-artemether) and about 0.55 for artemether. The test is not valid unless the chromatogram obtained with reference solution (2) exhibits 3 completely separated spots. Limits: In the chromatogram obtained with the test solution: Any spot corresponding to the impurity A is not intense than the principal spot in the chromatogram obtained with reference solution (6) (1.5%). Any spot corresponding to the impurity B is not intense than the spot of artenimol in the chromatogram obtained with reference solution (5) (1.0%). Any spot corresponding to the impurity C is not intense than the principal spot in the chromatogram obtained with reference solution (4) (0.5%). Any spot corresponding to the impurity D is not intense than the spot of α-artemether in the chromatogram obtained with reference solution (3) (0.3%). Any other secondary spot is not more intense than the principal spot in the chromatogram obtained with reference solution (2) (0.2%). Disregard any spot remaining on the line of application.

Dissolution (Appendix 11.4) Artemether Apparatus: Paddle. Medium: 1000 ml of water. Rotation speed: 100 rpm. Time: 60 min and 180 min. Procedure: Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - water - propan-1-ol trifluoroacetic acid (500 : 400 : 100 : 1). Test solution: After 60 min, withdraw 10 ml of the dissolved medium and about 20 ml of the dissolved medium after 180 min, filter. After withdraw a sample at 60-min stage, compensate the lost volume. Reference solution: Weigh accurately a quantity about 20 mg of artemether RS, transfer to a 100-ml volumetric flask,

ARTEMETHER AND LUMEFANTRINE TABLETS

add 20 ml of acetonitrile R and shake to dissolve, dilute to volume with water. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 210 nm. Flow rate: 1.0 ml/min. Volume of injection: 100 µl. Procedure: System suitability: Inject the reference solution, the relative standard deviation of the peak areas for six replicate injections is not more than 2.0%. The symmetry factor of the artemether peak in the chromatogram obtained with the reference solution is not more than 2. Inject the test solution. Calculate the content of artemether, C16H26O6, dissolved from each tablet using the peak areas in the chromatogram obtained with the reference solution, the test solution and the declared content of C16H26O6 in artemether RS. Tolerance: Not less than 45% (Q) of the labeled amount of artemether, C16H26O5, is dissolved in 60 min. Not less than 65% (Q) of the labeled amount of artemether, C16H26O5, is dissolved in 180 min. Lumefantrine Apparatus: Paddle. Medium: 1000 ml of a 1% solution of benzalkonium chloride in 0.1M hydrochloric acid R. Rotation speed: 100 rpm. Time: 45 min. Procedure: Test solution: After specified time, withdraw a sample of the medium, filter. Dilute 5.0 ml of the filtrate to 25.0 ml with the medium. Reference solution: Weigh accurately a quantity about 24 mg of lumefantrine RS and transfer into a 200-ml volumetric flask, add 20 ml of a mixture of 2-propanol and tetrahydrofurane (3 : 2), dissolve by heating in a water bath at 60 °C, sonicate if necessary. Allow to cool and dilute to volume with the medium, shake well. Dilute 5.0 ml of the solution to 25.0 ml with the medium. Meassure the absorbance of the reference solution, the test solution at 342 nm (Appendix 4.1), in a 1-cm cell and using the medium as the blank. Calculate the content of lumefantrine, C30H32Cl3NO, dissolved from each tablet using the absorbances of the reference solution, the test solution and the declared content of C30H32Cl3NO in lumefantrine RS. Tolerance: Not less than 60% (Q) of the labeled amount of lumefantrine, C30H32Cl3NO, is dissolved in 45 min.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Ion pair reagent - acetonitrile (700 : 300). 89

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ARTEMISININ

Mobile phase B: Ion pair reagent - acetonitrile (300 : 700). Ion pair reagent: Dissolve 5.65 g of sodium hexanesulfonate R and 2.75 g of sodium dihydrogen phosphate R in about 900 ml of water. Adjust the pH to 2.3 with a 10% solution of phosphoric acid R. Dilute to 1000 ml with water, filter. Diluent: Mix 200 ml of ion pair reagent, 60 ml of water, 200 ml of propanol R, add acetonitrile R to obtain 1000 ml. Artemether reference stock solution: Weigh accurately about 50 mg of artemether RS, transfer to a 50-ml volumetric flask, add the diluent, shake to dissolve, dilute to volume with the same solvent. Reference mixture solution: Weigh accurately about 60 mg of lumefantrine RS, transfer to a 100-ml volumetric flask, add 10.0 ml of the artemether reference stock solution and 75 ml of the diluent, sonicate to dissolve completely. Allow to cool and dilute to volume with the diluent, mix well. Test solution: Weigh 20 tablets, calculate the average mass and finely powder. Weigh accurately a quantity of the powdered tablets containing about 10 mg of artemether (about 60 mg of lumefantrine). Transfer to a 100-ml volumetric flask, add about 85 ml of the diluent, sonicate for 20 min. Allow to cool and dilute to volume with the same solvent, shake and filter. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 210 nm for the first 28 min and then switch to 380 nm. Flow rate: 1.3 ml/min. Volume of injection: 50 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% w/w)

Mobile phase B (% w/w)

Elution condition

0 - 28

60

40

Isocratic

28 -29

60 → 0

40 → 100

Linear gradien

29 - 45

0

100

Isocratic

45 - 46

0 → 60

100 → 40

Linear gradien

46 - 55

60

40

Re-equilibration

With chromatographic gradient above, the retention time of the artemether peak is about 19 min, of the lumefantrine peak is about 34 min. Calculate the content of artemether, C16H26O6 and of lumefantrine, C30H32Cl3NO, in the tablets using the areas of the artemether and lumefantrine peaks in the chromatogram obtained with the reference solution, the test solution and the declared content of C16H26O6 and C30H32Cl3NO in artemether RS and lumefantrine RS, respectively. 90

Storage Store in a well-closed container, in a cool place, protected from light. Action and use Antimalarial. Usual strength 20 mg of artemether and 120 mg of lumefantine. ARTEMISININ Artemisininum

C15H22O5

M. 282.3

Artemisinin is (3R, 5aS, 6R, 8aS, 9R, 12S, 12aR)octahydro-3,6,9-trimethyl-3,12-epoxy-12H-pyrano [4,3-j] -1,2-benzodioxepine-10(3H)-one. It contains not less than 97.0% and not more than 102.0% of C15H22O5, calculated with reference to the dried substance.

Characters Colourless needle crystals or a white, crystalline powder. Practically insoluble in water, very soluble in dichloromethane, freely soluble in acetone and ethyl acetate, soluble in glacial acetic acid, in methanol and in ethanol (96%). Identification Apply one of the two following identifications. Fist identification: A. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of artemisinin RS or with the reference spectrum of artemisinin. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Petrolium ether (40 °C - 60 °C) - ether (1 : 1). Test solution: A solution containing 0.10 mg of the substance to be examined in 1 ml of toluene R. Reference solution: A solution containing 0.10 mg of artemisinin RS in 1 ml of toluene R. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm. Allow the

VP V

plate to dry in air or in a current of cool air. Spray with anisaldehyde solution in sulfuric acid R. Dry the plate at 105 °C for 7 min. Examine the chromatogram in daylight. The principal spot obtained in the chromatogram with the test solution corresponds in position, size and colour with that in the chromatogram obtained with the reference solution. C. Dissolve 5 mg of the substance to be examined in 0.5 ml of ethanol R, add 0.5 ml of hydroxylamine hydrochloride solution R and 0.25 ml of a 8% solution of sodium hydroxide R. Heat the mixture on a water bath to boiling, allow to cool, add 5 drops of 2 M solution of hydrochloric acid R and 2 drops of a 5% solution of ferric chloride R, a deep violet colour is immediately produced. D. Dissolve 5 mg of the substance to be examined in about 0.5 ml of ethanol R, add 1.0 ml of a 8% solution of potassium iodid R, 2.5 ml of a 10% solution of sulfuric acid R, allow to stand for 1 min and add 4 drops of starch solution R, a violet colour is immediately produced.

Specific optical rotation +75º to +78º, calculated with reference to the dried substance (Appendix 6.4). Determined on a 1.0% solution in ethanol R. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - water (50 : 50). Test solution: Dissolve 50 mg of the substance to be examined in 10.0 ml of the mobile phase. Reference solution (1): Dissolve 50 mg of artemisinin RS (containing artemisinin and impurity A) in 10.0 ml of the mobile phase. Reference solution (2): Dilute 1 ml of the test solution to 100 ml with the mobile phase. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 210 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Carry out the chromatography for 1.5 times the retention time of artemisinin. In the chromatogram obtained with reference solution (1), the relative retention time with reference to artemisinin (retention time = about 10 min) of impurity A is about 0.79. In the chromatogram obtained with the test solution, the relative retention time with reference to artemisinin (retention time = about 10 min) of impurity B is about 0.85. System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to artemisinin and impurity A is at least 4. Limits: The correction factor: For the calculation of content, multiply the peak area of impurity A by 0.027. Impurity A: The corrected area of the peak due to impurity A is not greater than 0.15 times the area of the principal

ARTEMISININ

peak in the chromatogram obtained with reference solution (2) (0.15%). Impurity B: The area of the peak due to impurity B is not greater than 0.3 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.3%). Other impurites: The area of each impurity peak is not greater than 0.15 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.15%). The sum of the peak areas of all impurities and the corrected area of peak due to impurity A, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (2) (1.0%). Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Notes: Impurity A: (3R,5aS,6R,8aS,12S,12aR)-3,6-dimethyl-9-methylideneoctahydro-3,12-epoxypyrano[4,3-j ]-1,2-benzodioxepin-10(3H) -one (artemisitene). Impurity B: (3R,5aS,6R,8aS,9S,12S,12aR)-3,6,9-trimethyl-octahy dro-3,12-epoxypyrano[4,3-j ]-1,2- benzodioxepin-10(3H)-one (9-epi-artemisinin).

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g, 80 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 1). Determined on 1.0 g. Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. Inject the test solution and reference solution (1). In the chromatogram obtained with reference solution (1), the relative retention with reference to artemisinin (retention time = about 10 min) of impurity A is about 0.79. System suitability: In the chromatogram obtained with the reference solution (1), the resolution between the peaks due to artemisinin and impurity A is at least 4. Calculate the percentage content of C15H22O5 in the substance to be examined, using the areas of the principal peaks obtained in the chromatograms from the test solution, reference solution (1) and the declared content of C15H22O5 in artemisinin RS. Storage Store in a well-closed container, protected from light, store at cool place. Action and use Antimalarial. Preparations Tablets, suppositories. Usually used in combined with the other antimalarials. 91

VP V

ARTESUNATE

ARTESUNATE Artesunatum

mixture in a water bath to boiling, allow to cool, add 2 drops of 2M solution of hydrochloric acid R and 2 drops of a 5% solution of ferric chloride R, a reddish-brown colour is produced. D. Evaporate the remaining filtrate from test C on a waterbath to a volume of about 5 ml. Place a few drops of the mixture on a white porcelain dish, add one drop of a 5% solution of vanillin in sulfuric acid R, a red colour is produced.

pH

3.5 to 4.5 (Appendix 6.2). Determined on a 10 mg/ml aqueous suspension. C19H28O8

M. 384.4

Artesunate is (3R,5aS,6R,8aS,9R,10S,12R,12aR)-decahy dro-3,6,9-trimethyl-3,12-epoxy-12H-pyrano[4,3-j]-1,2benzodioxepin-10-ol hydrogen succinate. It contains not less than 96.0% and not more than 102.0% of C19H28O8 (Method A), and not less than 98.0% and not more than 102.0% of C19H28O8 (Method B), calculated with reference to the dried substance.

Characters A fine, white, crystalline powder. Very slightly soluble in water, very soluble in dichloromethane, freely soluble in acetone and in ethanol (96%). Identification Apply one of the two following identifications. First identification: A. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of artesunate RS or with the reference spectrum of artesunate. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Ethanol - toluene - ammonia (70 : 30 : 1.5). Test solution: A solution containing 1.0 mg of the substance to be examined in 1 ml of methanol R. Reference solution: A solution containing 1.0 mg of artesunate RS in 1 ml of methanol R. Procedure: Apply separately to the plate 1 μl of each solution. After developing and removal the plate, allow to dry in air. Spray with a anisaldehyde solution in methanol and heat the plate at 120 °C for 5 min. Examine the chromatogram in daylight. The principal spot obtained in the chromatogram with the test solution corresponds in position, size and colour to that obtained in the chromatogram with the reference solution. C. Dissolve 0.1 g of the substance to be examined in 40 ml of ethanol R, shake well and filter. To half of the filtrate (keep the remaining filtrate for test D) add 0.5 ml of hydroxylamine hydrochloride solution in ethanol R and 0.25 ml of a 8% solution of sodium hydroxide R. Heat the 92

Special optical rotation +4.5º to +6.5º, calculated with reference to the anhydrous substance (Appendix 6.4). Determined on a 1.0% solution in dichloromethane R, at 20 °C. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - buffer solution pH 3.0 (44 : 56). Buffer solution pH 3.0: Dissolve 1.36 g of potassium dihydrogen phosphate R in 900 ml of water, adjust the pH to 3.0 with phosphoric acid R and dilute to 1000 ml with water. Test solution: Weigh accurately about 40 mg of the substance to be examined, dissolve in acetonitrile R and dilute to 10 ml with the same solvent. Reference solution (1): Dissolve about 1 mg of artenimol RS, 1 mg of artemisinin RS and 10 mg of artesunate RS in 10 ml of acetonitrile R. Reference solution (2): Dilute 1 ml of the test solution to 100 ml with acetonitrile R. Chromatographic system:A column (10 cm × 4.6 mm) packed with stationary phase C (3 µm). Column temperature: 30 °C. Detector: A spectrophotometer set at 216 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Carry out the chromatography for 4 times the retention time of artesunate. In the chromatogram obtained with reference solution (1), the relative retention time with reference to artesunate (retention time = about 9 min.): 10-epi-artenimol = about 0.58; artenimol = about 0.91; impurity B (artemisinin) = about 1.3. System suitability: The peak-to-valley ratio (Hp/Hv) is at least 5.0, where Hp is the height above the baseline of the peak due to β-artenimol and Hv is the height above the baseline at the lowest point of the curve separating the peak due to artenimol from the peak due to artesunate. In chromatogram obtained with the test solution, relative retention of peak due to impurity C is about 2.7 with reference to artesunate. Limits: The correction factor: For the calculation of content, multiply the peak area of impurity C by 0.07. In the chromatogram obtained with the test solution:

VP V

The sum of areas of all peaks corresponding to 10-epiartenimol and artenimol (impurity A) is not greater than the area of the principal peak in the chromatogram obtained with reference solution (2) (1.0%). The area of any peak corresponding to impurity B (artemisinin) is not greater than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.5%). The area of any peak corresponding to impurity C is not greater than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.2%). The area of any other peak, apart from the principal peak, is not greater than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.2%). The sum of the areas consisting of corrected area of any peak corresponding to impurity C and the areas of all other peaks, apart from the principal peak, is not greater than twice the area of the principal peak in the chromatogram obtained with reference solution (2) (2.0%). Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Notes: Impurity A: Artenimol. Impurity B: Artemisinin. Impurity C: (3R,5aS,6R,8aS,12R,12aR)-3,6,9-trimethyl-3,4,5,5a,6,7,8,8aoctahydro-12H-3,12-epoxy-pyrano[4,3-j ]-1,2-benzodioxepin.

ARTESUNATE FOR INJECTION

Inject the test solution, reference solution (1) and reference solution (3). System suitability as described in the test for Related substances. Calculate the content of C19H28O8 in the substance to be examined, using the areas of the principal peaks in the chromatograms obtained with the test solution, reference solution (3) and the declared content of C19H28O8 in artesunate RS. B. Dissolve about 0.25 g of artesunate, accurately weighed, in 25 ml of neutralized ethanol R and titrate with 0.05 N sodium hydroxide VS, using 2 drops of solution of phenolphthalein R as indicator. Each ml of 0.05 N sodium hydroxide VS is equivalent to 19.22 mg of C19H28O8.

Storage Store in a well-closed container, protected from light, store at cool place. The label states, if the substance not contains bacterial endotoxins or if the substance is sterile. Action and use Antimalarial. Preparation Injections.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). Determined on 1.0 g, method 3.

ARTESUNATE FOR INJECTION Artesunatum pro injectione

Sulphated ash Not more than 0.1% (Appendix 9.9, method 1). Determined on 1.0 g.

Artesunate for injection is a sterile crystalline powder of artesunate. It is supplied in a sterile sealed glass container. The powder for injection complies with the requirements stated under “Injections, infusion” (Appendix 1.19) and with the following requirements.

Water Not more than 0.5% (Appendix 10.3). Determined on 2.000 g. Bacterial endotoxins  Less than 2.5 EU/mg (Appendix 13.2), if intended for use in the manufacture of a parenteral dosage form without a further appropriate procedure for the removal of bacterial endotoxins. Sterility  If intended for use in the manufacture of a parenteral dosage form without a further appropriate sterilization procedure, it complies with the test for Sterility (Appendix 13.7). Assay Apply one of the two following methods: A. Examine by liquid chromatography (Appendix 5.3). Carry out the test as described in the test for Related substances. Reference solution (3): Dissolve 40 mg of artesunate RS in acetonitile R and dilute to 10 ml with the same solvent.

Content of artesunate, C19H28O8, 90.0% to 110.0% of the stated amount. Characters White, crystalline powder. Identification Apply one of the two following identifications. First identification: A. Second identification: B, C, D. A. The infrared absorption spectrum of the powder for injection (Appendix 4.2) is concordant with the reference spectrum of artesunate. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Ethanol - toluene - ammonia (70 : 30 : 1.5). Test solution: Dissolve a quantity of the substance to be examined in methanol R to obtain a solution containing 1 mg/ml of artesunate. 93

ARTESUNATE FOR INJECTION

Reference solution: A solution containing 1mg/ml of artesunate RS in methanol R. Procedure: Apply separately to the plate 1 µl of each solution. Develop over 3/4 of the plate, remove the plate, allow it to dry in air. Spray with anisaldehyde solution R and heat the plate at 120 °C for 5 min. Examine the chromatogram in daylight. The principal spot obtained with the test solution corresponds in position, size and colour to that obtained with the reference solution. C. Dissolve a quantity of the powder containing 0.1 g of artesunate in 40 ml of ethanol R, shake and filter. Add 0.5 ml of hydroxylamine hydrochloride solution in ethanol R into a half of the resulting filtrate (the remaining is used for test D) and 0.25 ml of 2 M sodium hydroxide R. Heat the mixture in a water-bath to boiling, cool, add 2 drops of dilute hydrochloric acid R and 2 drops of 5% solution of ferric (III) chloride R; a light red-violet colour is produced. D. Evaporate the remaining filtrate from test C on a waterbath to a volume of about 5 ml. Place a few drops of the mixture on a white porcelain dish, add one drop of a 5% solution of vanillin in sulfuric acid R, a reddish-brown colour is produced.

Clarity of solution The reconstituted solution prepared following manufacture’s instruction is clear (Appendix 9.2) and there is no visible particulate (Appendix 11.8, part B). pH Shake 0.50 g of the substance to be examined with 50 ml of carbon dioxide-free water R, and filter. The pH of the filtrate is 3.5 to 5.0 (Appendix 6.2). Chlorides Not more than 0.02% (Appendix 9.4). Shake 0.50 g of the powder with 30 ml of water. Filter through a chloride-free filter paper, discarding the first 5 ml of the filtrate. Use 15 ml of the filtrate for the test. Water Not more than 0.5% (Appendix 10.3). Determined on 2.000 g of the powder. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase, chromatographic system, test solution: proceed as directed in the Assay. Reference solution (1): Dilute 1 ml of the test solution to 100 ml with acetonitrile R. Reference solution (2): Dissolve about 1 mg of artenimol RS, 1 mg of artemisinin RS and 10 mg of artesunate RS in 10 ml with acetonitrile R. Procedure: Inject separately the test solution, reference solution (1) and (2). Record the chromatograms for 4 times the retention time of artesunate. 94

VP V

In the chromatogram obtained with reference solution (2), the following peaks are eluted at the following relative retention with reference to artesunate (retention time about 9 min): α-artenimol about 0.58, β-artenimol (impurity A) = about 0.91 and artemisinin (impurity B) = about 1.30. The test is not valid unless the peak-to-valley ratio (Hp/Hv) is at least 5.0, where Hp is the height above the baseline of the peak due to β-artenimol and Hv is the height above the baseline of the lowest point of the curve separating this peak from the peak due to artesunate. The chromatogram obtained with the test solution may show a peak due to impurity C eluting at a relative retention of about 2.7 with reference to artesunate. Limits: In the chromatogram obtained with the test solution: The sum of the areas of any peaks corresponding to α-artenimol and β-artenimol is not greater than the area of the principal peak obtained with reference solution (1) (1.0%). The area of any peak corresponding to impurity B (artemisinin) is not greater than 0.5 times the area of the principal peak obtained with reference solution (1) (0.5%). The area of any peak corresponding to impurity C, when multiplied by a correction factor of 0.07, is not greater than 0.3 times the area of the principal peak obtained with reference solution (1) (0.3%). The area of any other peak, apart from the principal peak, is not greater than 0.3 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.3%). The sum of the corrected area of any peak corresponding to impurity C and the areas of all other peaks, apart from the principal peak, is not greater than twice the area of the principal peak obtained with reference solution (1) (2.0%). Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%).

Bacterial endotoxins Not more than 2.5 EU/mg of artesunate. Carry out the test for bacterial endotoxins (Appendix 13.2). Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - phosphate buffer pH 3.0 (44 : 56). Phosphate buffer pH 3.0: Dissolve 1.36 g of potassium dihydrogen phosphate R in 900 ml of water, adjust the pH to 3.0 with phosphoric acid R and dilute to 1000 ml with water. Test solution: Weigh accurately about 100 mg of the mixture contents from 20 units. Tranfer to a 25-ml volumetric flask, add 15 ml of acetonitrile R, shake to dissolve, and dilute to volume with acetonitrile R, and mix. Reference solution: A solution of artesunate RS in acetonitrile R having a known concentration of about 4 mg per ml. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 µm).

VP V

ASCORBIC ACID

Column temperature: 30 °C. Detector: A spectrophotometer set at 216 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution, the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject separately the reference solution and the test solution. Calculate the content of artesunate, C19H28O8, of the powder for injection in each unit using the areas of the principal peaks in the chromatograms obtained with the test solution, the reference solution and the declared content of C19H28O8 in artesunate RS.

Storage Store in an airtight container, in a cool place, protected from light. Action and use Antimalarial. Usual strength 60 mg. ASCORBIC ACID Acidum ascorbicum Vitamin C, L-ascorbic acid

C6H8O6  

M. 176.1

Ascorbic acid is (5R)-5-[(1S)-1,2-dihydroxyethyl]-3,4dihydroxyfuran-2(5H)-one. It contains not less than 99.0% and not more than 100.5% of C6H8O6.

Characters White or almost white, crystalline powder or colourless crystals, becoming discoloured on exposure to air and moisture. Freely soluble in water, sparingly soluble in ethanol (96%). Melting point: About 190 °C, with decomposition. Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of ascorbic acid RS. B. Dissolve 0.10 g in water and dilute immediately to 100.0 ml with the same solvent. Add 1.0 ml of this solution to 10 ml of 0.1 M hydrochloric acid R and dilute to 100.0 ml with water.

The ultraviolet absorption spectrum (Appendix 4.1) of the solution exhibits only absorption maximum at 243 nm, determined immediately after dissolution. Specific absorbance at 234 nm, A(1%, 1 cm) is 545 to 585. C. Add 0.2 ml of dilute nitric acid R and 0.2 ml of a 2% solution of silver nitrate R to 1 ml of solution S. A grey precipitate is formed. D. pH of solution S: 2.1 to 2.6 (Appendix 6.2).

Appearance of solution Solution S: Dissolve 1.0 g of the substance to be examined in carbon dioxide-free water R and dilute to 20 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution BY7 (Appendix 9.3, method 2). Specific optical rotation +20.5° to +21.5° (Appendix 6.4). Dissolve 2.50 g in water and dilute to 25.0 ml with the same solvent. Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use. Mobile phase: Phosphate buffer solution - acetonitrile R1 (25 : 75). Phosphate buffer solution: Dissolve 6.8 g of potassium dihydrogen phosphate R in water and dilute to about 175 ml with water. Filter through a membrane filter (nominal pore size of 0.45 µm) and dilute to 1000 ml with water. Test solution: Dissolve 0.500 g of the substance to be examined in the mobile phase and dilute to 10.0 ml with the same solvent. Reference solution (1): Dissolve 10.0 mg of ascorbic acid impurity C RS in the mobile phase and dilute to 5.0 ml with the mobile phase. Reference solution (2): Dissolve 5.0 mg of ascorbic acid impurity D RS and 5.0 mg of ascorbic acid RS in the mobile phase, add 2.5 ml of reference solution (1) and dilute to 100.0 ml with the mobile phase. Reference solution (3): Dilute 1.0 ml of the test solution to 200.0 ml with the mobile phase. Mix 1.0 ml of this solution with 1.0 ml of reference solution (1). Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase aminopropylsilyl silica gel for chromatography R (5 µm). Column temperature: 45 °C. Detector: A spectrophotometer set at 210 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Inject the test solution and reference solution (2), (3). 95

VP V

ASCORBIC ACID

The run time is 2.5 times the retention time of ascorbic acid. Identification of impurities: Use the chromatogram obtained with reference solution (2) to identify the peaks due to impurities C and D. Relative retention with reference to ascorbic acid (retention time = about 11 min): Impurity D = about 0.4; impurity C = about 1.7. System suitability: In the chromatogram obtained with reference solution (3), the resolution between the peaks due to ascorbic acid and impurity C is at least 3.0. In the chromatogram obtained with reference solution (2), the signal-to-noise ratio of the peak due to impurity C is not less than 20. Limits: Impurities C, D: For each impurity, the area is not more than 1.5 times the area of the corresponding peak in the chromatogram obtained with reference solution (2) (0.15%). Any other impurity: For each impurity, the area is not more than the area of the peak due to ascorbic acid in the chromatogram obtained with reference solution (2) (0.1%). The sum of the peak areas of all impurities other than C and D is not more than twice the area of the peak due to ascorbic acid in the chromatogram obtained with reference solution (2) (0.2%). Disregard any peak with an area less than 0.5 times the area of the peak due to ascorbic acid in the chromatogram obtained with reference solution (2) (0.05%). Note: Impurity A: 2-furaldehyde. Impurity C: D-xylo-hex-2-ulosonic acid (D-sorbosonic acid). Impurity D: Methyl D-xylo-hex-2-ulosonate (methyl D-sorbosonate). Impurity E: Oxalic acid. Impurity F: (5R)-5-[(1R)-1,2-dihydroxyethyl]-3,4-dihydroxyfuran2(5H)-one. Impurity G: (2R)-2-[(2R)-3,4-dihydroxy-5-oxo-2,5-dihydrofuran2-yl]-2-hydroxyacetic acid. Impurity H: Methyl (2R)-2-[(2R)-3,4-dihydroxy-5-oxo-2,5dihydrofuran-2-yl]-2-hydroxyacetate.

Oxalic acid Not more than 0.2%. Test solution: Dissolve 0.25 g in 5 ml of water. Neutralise using dilute sodium hydroxide solution R. Reference solution: Dissolve 70 mg of oxalic acid R in water and dilute to 500 ml with the same solvent, use 5 ml of this solution to prepare the reference solution. Simultaneously add 1 ml of dilute acetic acid R and 0.5 ml of calcium chloride solution R into the test solution and the reference solution. Allow the solutions to stand for 1 h. Any opalescence in the test solution is not more intense than that in the reference solution. Copper Not more than 5 ppm. 96

Examine by atomic absorption spectrometry (Appendix 4.4, method 1). Test solution: Dissolve 2.0 g in 0.1 M nitric acid R and dilute to 25.0 ml with the same solvent. Reference solutions: Prepare the reference solutions (0.2 ppm, 0.4 ppm and 0.6 ppm) by diluting copper standard solution (10 ppm Cu) R with 0.1 M nitric acid R. Measure the absorbance at 324.8 nm using a copper hollowcathode lamp as a source of radiation and an air-acetylene flame. Adjust the zero of the apparatus using 0.1 M nitric acid R.

Iron  Not more than 2 ppm. Examine by atomic absorption spectrometry (Appendix 4.4, method 1). Test solution: Dissolve 5.0 g in 0.1 M nitric acid R and dilute to 25.0 ml with the same solvent. Reference solutions: Prepare the reference solutions (0.2 ppm, 0.4 ppm and 0.6 ppm) by diluting iron standard solution (20 ppm Fe) R with 0.1 M nitric acid R. Measure the absorbance at 248.3 nm using an iron hollowcathode lamp as a source of radiation and an air-acetylene flame. Adjust the zero of the apparatus using 0.1 M nitric acid R. Heavy metals Not more than 10 ppm (Appendix 9.4.8). Dissolve 2.0 g in water and dilute to 20 ml with the same solvent. 12 ml of the solution complies with limit test for heavy metals, method 1. Prepare the standard using lead standard solution (1 ppm Pb) R. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.150 g of the substance to be examined in a mixture of 80 ml of carbon dioxide-free water R and 10 ml of dilute sulfuric acid R. Add 1 ml of starch solution R. Titrate with 0.1 N iodine VS until a persistent violet-blue colour is obtained. 1 ml of 0.1 N iodine VS is equivalent to 8.81 mg of C6H8O6. Storage Store in an airtight container, in a non-metallic container, protected from light. Action and use Vitamin C, preservative (antioxydant). Preparations Tablets, capsules, injection.

VP V

ASCORBIC ACID INJECTION

ASCORBIC ACID INJECTION Injectio Acidi ascorbici Vitamin C injection

of 2 M acetic acid R and 0.5 ml of a 0.5 M solution of calcium chloride R and allow to stand for 1 hour. Any opalescence produced in the test solution is not more intense than that in the reference solution.

Ascorbic acid injection is a sterile solution of ascorbic acid in water for injections. It may contains prevervatives. The injection complies with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Assay To an accurately measured volume of the injection, equivalent to about 0.20 g to 0.25 g of ascorbic acid, add 0.25 ml of a 1% solution of formaldehyde R, 4 ml of a 2% solution of hydrochloric acid R, 0.5 ml of a 10% solution of potassium iodide R and 2 ml of starch solution R. Titrate with 0.1 N potassium iodate VS until a persistent blue colour is produced. Each ml of 0.1 N potassium iodate VS is equivalent to 8.806 mg of C6H8O6.

Content of ascorbic acid, C6H8O6, 95.0% to 105.0% of the stated amount. Characters A clear, coloueless or yellowish solution. Identification A. To a volume containing about 50 mg of ascorbic acid, add 0.2 ml of 2 M nitric acid R and 0.2 ml of a 2% sulution of silver nitrate R. A greish black precipitate is produced. B. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel GF254. Mobile phase: Ethanol (96%) - water (120 : 20). Test solution: Dilute the injection, if necessary, with water to obtain a 0.5% solution of ascorbic acid. Reference solution: A 0.5% solution of ascorbic acid RS. Procedure: Apply separately to the plate 2 µl of each solution. Develop over a path of about 15 cm. After removal of the plate, allow it to dry in air and examine under ultraviolet light (254 nm). The principal spot in the chromatogram obtained with the test solution is similar in position and colour to that in the chromatogram obtained with the reference solution. pH 5.0 to 6.5 (Appendix 6.2). Colour of solution Dilute an accurately measured volume of the injection with water to obtain a solution having a known concentration of 50 mg of ascorbic acid per ml. Measure the absorbance (Appendix 4.1) of the solution at 420 nm in a 1 cm cell, using water as the blank. The absorbance is not greater than 0.06. Oxalic acid Not more than 0.3%. Prepare the solutions at the same time and in the same conditions. Test solution: Dilute, if necessary, a volume containing 250 mg of ascorbic acid to 5 ml with water, neutralise to litmus paper R with 2 M sodium hydroxide R, add 1 ml of 2 M acetic acid R and 0.5 ml of a 0.5 M solution of calcium chloride R and allow to stand for 1 hour. Reference solution: Dissolve 70 mg of oxalic acid R in 500 ml of water. To 5 ml of the resulting solution add 1 ml

Storage Store 2 °C to 8 °C, protected from light. Action and use Vitamin. Usual strength 100 mg/ml; 250 mg/ml; 500 mg/ml. ASCORBIC ACID TABLETS Tabellae Acidi ascorbici Vitamin C tablets Ascorbic acid tablets contain ascorbic acid. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of ascorbic acid, C6H8O6, 95.0% to 110.0% of the stated amount. Identification A. Shake a quantity of the powdered tablets, equivalent to about 0.10 g of ascorbic acid, with 10 ml of water, shake well, filter. The filtrate is acidic to litmus paper R. To 5 ml of the filtrate add 0.5 ml of a 2% sulution of silver nitrate R. A blackish-gray precipitate is produced. B. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel GF254. Mobile phase: Ethanol (96%) - water (120 : 20). Test solution: Shake a quantity of the powdered tablets, equivalent to about 0.05 g of ascorbic acid, with 10 ml of water, shake well, filter. Reference solution: A 0.5% solution of ascorbic acid RS. Procedure: Apply separately to the plate 2 µl of each solution. Develop over a path of about 15 cm. After removal of the plate, allow to dry in air and examine under ultraviolet light (254 nm). The principal spot in the chromatogram obtained with the test solution is similar in 97

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ASPARTAME

position and colour to that in the chromatogram obtained with the reference solution.

Assay Weigh 20 tablets, calculate the average mass and powder finely. Weigh accurately a quantity of the powder, equivalent to about 0.1 g of ascorbic acid, add 30 ml of a mixture of carbon dioxide-free water R and 1 M acetic acid R (10 : 1), and mix. Add 1 ml of starch solution R, and titrate with 0.1 N iodine VS until a persistent blue colour is produced. Each ml of 0.1 N iodine VS is equivalent to 8.806 mg of C6H8O6. Storage Should be kept free from contact with metal and protected from light and moisture. Action and use Vitamin. Usual strength 100 mg; 500 mg; 1000 mg. ASPARTAME Aspartamum

B. Dissolve 0.1 g in ethanol (96%) R and dilute to 100 ml with the same solvent. The ultraviolet absorption spectrum (Appendix 4.1) of the solution in the range 230 nm to 300 nm exhibits absorption maxima at 247 nm, 252 nm, 258 nm and 264 nm. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Water - anhydrous formic acid - methanol dichloromethane (2 : 4 : 30 : 64). Test solution: Dissolve 15 mg of the substance to be examined in 2.5 ml of water and dilute to 10 ml with acetic acid R. Reference solution: Dissolve 15 mg of aspartame RS in 2.5 ml of water and dilute to 10 ml with acetic acid R. Procedure: Apply to the plate 20 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in air. Spray with ninhydrin solution R and heat at 100 °C to 105 °C for 15 min. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. D. Dissolve about 20 mg in 5 ml of methanol R and add 1 ml of alkaline hydroxylamine solution R1. Heat on a waterbath for 15 min. Allow to cool and adjust to pH 2 with dilute hydrochloric acid R. Add 0.1 ml of 10.5% solution of ferric chloride R. A brownish-red colour is produced.

Appearance of solution Solution S: Dissolve 0.8 g in carbon dioxide-free water R and dilute to 100.0 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution GY6 (Appendix 9.3, method 2). C14H18N2O5

M. 294.3

Aspartame is (3S)-3-amino-4-[[(2S)-1-methoxy-1-oxo-3phenylpropan-2-yl]amino]-4-oxobutanoic acid (methyl α-L-aspartyl-L-phenylalaninate). It contains not less than 98.0% and not more than 102.0% of C14H18N2O5, calculated with reference to the dried substance.

Characters A white, crystalline powder, slightly hygroscopic. Sparingly soluble or slightly soluble in water and in ethanol (96%), practically insoluble in hexane and in dichloromethane. Identification Apply one of the two following identifications: First identification: A. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of aspartame RS. 98

Conductivity Not more than 30 µS.cm-1 (Appendix 6.10). Measure the conductivity of the solution S (C1) and that of the water used for preparing the solution S (C2). The readings must be stable within 1% over a period of 30 s. Calculate the conductivity of the solution of the substance to be examined from the expression: C1 – 0.992 C2

Specific optical rotation +14.5º to +16.5º, calculated with reference to the dried substance (Appendix 6.4). Dissolve 2.00 g in a 69.0% solution of anhydrous formic acid R and dilute to 50.0 ml with the same solvent. Measure optical rotation of preparation. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - 0.68% solution of potassium dihydrogen phosphate previously adjusted to pH 3.7 with phosphoric acid (10 : 90).

VP V

Test solution: Dissolve 0.60 g of the substance to be examined in a mixture of glacial acetic acid R and water (1.5 : 98.5) and dilute to 100.0 ml with the same solvent. Reference solution (1): Dissolve 9.0 mg of diketopiperazine RS in a mixture of glacial acetic acid R and water (1.5 : 98.5) and dilute to 100.0  ml with the same solvent. Reference solution (2): Dissolve 30.0 mg of phenylalanine R in a mixture of glacial acetic acid R and water (15 : 85) and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of the solution to 10.0 ml with water. Reference solution (3): Dilute 5.0 ml of the test solution to 10.0 ml with water. Dilute 3.0 ml of the solution to 100.0 ml with water. Resolution solution: Dissolve 30.0 mg of L-aspartylL-phenylalanine R in a mixture of glacial acetic acid R and water (15 : 85) and dilute to 100.0  ml with the same solvent. Dilute 1.0 ml of the solution to 10.0 ml with water. Mix 1.0 ml of this solution with 1.0 ml of reference solution (2). Chromatographic system: A column (25 cm × 4.0 mm) packed with stationary phase C (5 µm to 10 µm). Detector: A spectrophotometer set at 220 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Carry out the chromatography for twice the retention time of aspartame. Inject reference solution (3). Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained is not less than 50% of the full scale of the recorder. System suitability: In the chromatogram obtained, the resolution between the peaks due to phenylalanine and L-aspartyl-L-phenylalanine is at least 3.5. Limits: In the chromatogram obtained with the test solution the area of any peak corresponding to diketopiperazine is not greater than the area of the principal peak in the chromatogram obtained with reference solution (1) (1.5%); the area of any peak corresponding to phenylalanine is not greater than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.5%); the sum of the areas of any peaks, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (3) (1.5%). Disregard any peak due to the solvent.

Heavy metals Not more than 10 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 1 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 4.5% (Appendix 9.6). (1.000 g; 100 °C to 105 °C).

ASPARTAME POWDER

Sulfated ash Not more than 0.2% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve a bout 0.250 g in a mixture of 1.5 ml of anhydrous formic acid R and 60 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 29.43 mg of C14H18N2O5. Storage Store in an airtight container. Action and use Sweetening agent. Preperations Tablets, powders. ASPARTAME POWDER Pulverecs Aspartami Aspartame powder contain aspartame. The powder complies with the requirements stated under “Powder” (Appendix 1.7) and with the following requirements.

Content of aspartame, C14H18N2O5, 90.0% to 110.0% of the stated amount. Characters A white powder, with a sweet taste. Identification A. Dissolve a quantity of the powder, equivalent to 0.1 g of aspartame, with ethanol R, and dilute to 100 ml with the same solvent, mix and filter. The ultraviolet absorption spectrum (Appendix 4.1) of the filtrate, in the range 230 nm to 300 nm, exhibits absorption maxima at 247 nm, 252 nm, 258 nm and 264 nm. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Water - formic acid - methanol - methylene chloride (2 : 4 : 30 : 64). Test solution: Shake a quantity of the powder, equivalent to 15 mg of aspartame, with 2.5 ml of water, dilute to 10 ml with acetic acid R. Filter. Reference solution: Dissolve 15 mg of aspartame RS in 2.5 ml of water, and dilute to 10 ml with acetic acid R, and mix. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm. After removal of the plate, allow to dry in air. Spray with a 2% solution of ninhydrin R, and heat at 100 °C to 105 °C for 15 min. The 99

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ATENOLOL

principal spot in the chromatogram obtained with the test solution is similar in position and colour to the principal spot in the chromatogram obtained with the reference solution.

Fineness Complies with the requirements of The very fine powder (Appendix 3.5). Loss on drying Not more than 9.0% (Appendix 9.6). (1.0 g; 80 °C). Assay Test solution: Weigh 20 packets, calculate the average mass of the contents, mix and powder finely. Weigh accurately a quantity of the powder, equivalent to about 40 mg of aspartame, in a 100 ml volumetric flask, dissolve with 2 M hydrochloric acid R, dilute to volume with the same solvent, and mix. Filter, discarding the first of the filtrate. Use the filtrate. Reference solution: A 0.040% solution of aspartame RS in 2 M hydrochloric acid R. Measure the absorbances (Appendix 4.1) of the reference solution and the test solution at 258 nm in a 1-cm cell, using 2 M hydrochloric acid R in the reference cell. Calculate the content of aspartame, C14H18N2O5, in the packet using the measured absorbances of the test solution, the reference solution and the declared content of C14H18N2O5 in aspartame RS. Storage Store in an airtight container in a cool place, protected from light. Usual strength 1 g.

ATENOLOL Atenololum

C14H22N2 O3.HCl

M. 266.3

Atenolol is 2-[4-[(2RS)-2-hydroxy-3-[(1-methylethyl) amino]propoxy]phenyl]acetamide, it contains not less than 99.0% and not more than 101.0% of C14H22N2O3, calculated with reference to the dried substance.

Characters A white or almost white crystalline powder. Sparingly soluble in water, soluble in ethanol, slightly soluble in methylene chloride. 100

Identification Apply one of the two following identifications. First identification: A. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of atenolol RS. B. Dissolve 0.100 g of the substance to be examined in methanol R and dilute to 100 ml with the same solvent. Dilute 10.0 ml of this solution to 100 ml with methanol R. Examine the ultraviolet absorption spectrum (Appendix 4.1) of the solution in the range 230 nm to 350 nm. The ultraviolet absorption spectrum exhibits two absorption maxima at 275 nm and 282 nm. The ratio of the absorbance measured at 275 nm to that measured at 282 nm is 1.15 to 1.20. C. Melting point: 152 °C to 155 °C (Appendix 6.7). D. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silanised silica gel F254. Mobile phase: 18 M ammonia - methanol (1 : 99 ). Test solution: Dissolve 10 mg of the substance to be examined in 1 ml of methanol R. Reference solution: Dissolve 10 mg of atenolol RS in 1 ml of methanol R. Procedure: Apply separately to the plate 10 μl of each solution. After developing and removal of the plate, allow to dry in air and examine the chromatogram in ultraviolet light at 254 nm. The principal spot obtained in the chromatogram with the test solution corresponds in position and size to that obtained in the chromatogram with the reference solution. Appearance of solution Solution S: Dissolve 0.10 g in water and dilute to 10 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than degree 6 of the range of reference solutions of the most appropriate colour (Appendix 9.3, method 2). Optical rotation +0.10° to -0.10° (Appendix 6.4). Determined on solution S. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 1.0 g of sodium octanesulfonate R and 0.4 g of tetrabutylammonium hydrogen sulfate R in 1000 ml of a mixture of tetrahydrofuran R - methanol R2 - a 0.34% solution of potassium dihydrogen phosphate R (20 : 180 : 800); adjust to pH 3.0 with phosphoric acid R. Test solution: Dissolve 50.0 mg of the substance to be examined in 20.0 ml of the mobile phase and dilute to 25.0 ml with the same solvent. Reference solution (1): Dissolve 2 mg of atenolol for system suitability RS (containing impurities B, F, G, I and J) in 1.0 ml of the mobile phase.

VP V

Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Chromatographic system: A column (12.5 cm × 4.0 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 226 nm. Flow rate: 0.6 ml/min. Volume of injection: 10 µl. Procedure: The run time is 5 times the retention time of atenolol. Identification of impurities: Use the chromatogram supplied with atenolol for system suitability RS and the chromatogram obtained with reference solution (1) to identify the peaks due to impurities B, F, G, I and J. The relative retention time with reference to atenolol (retention time = about 8 min.): Impurity B = about 0.3; impurity J  =  about 0.7; impurity I  =  about 0.8; impurity F = about 2.0 (pair of peaks); impurity G = about 3.5. System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to impurity J (unidentified impurity) and I is at least 1.4. Limits: The correction factor: For the calculation of content, multiply the peak area of impurity I by 1.5. Impurity B: The peak area of impurity B is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (2) (0.2%). Impurities F, G, I: For each impurity, the peak area is not more than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.15%). Unspecified impurities: The peak area of each impurity is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.10%). The sum of the peak areas of all impurities is more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.5%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Notes: Impurity A: 2-(4-hydroxyphenyl)acetamide. Impurity B: 2-[4-[(2RS)-2,3-dihydroxypropoxy]phenyl]acetamide. Impurity D: 2-[4-[(2RS)-3-chloro-2-hydroxypropoxy]phenyl] acetamide. Impurity E: 2,2′-[(2-hydroxypropane-1,3-diyl)bis(oxy-4,1phenylene)]diacetamide. Impurity F: 2,2′-[[(1-methylethyl)imino]bis[(2-hydroxypropane3,1-diyl)oxy-4,1-phenylene]]diacetamide. Impurity G: 2-[4-[(2RS)-2-hydroxy-3-[(1-methylethyl)amino] propoxy]phenyl]acetic acid. Impurity H: 2-[4-[(2RS)-2-hydroxy-3-[(1-methylethyl)amino] propoxy]phenyl]acetonitrile. Impurity I: 2-[4-[(2RS)-3-(ethylamino)-2-hydroxypropoxy] phenyl]acetamide.

ATENOLOL TABLETS

Chlorides Not more than 0.1%. Dissolve 50  mg of the substance to be examined in a mixture of 1ml of dilute nitric acid R and 15 ml of water. The solution, without further addition of dilute nitric acid R, complies with the test (Appendix 9.4.5). Loss on drying  Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.200 g of the substance to be examined in 80 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). Each ml of 0.1  N perchloric acid  VS is equivalent to 26.63 mg of C14H22N2O3. Storage Store in a well-closed container, protected from light. Action and use Beta-adrenoceptor antagonist. Preparations Tablets, injection, oral solution. ATENOLOL TABLETS Tabellae Atenololi Atenolol tablets contain atenolol. They may be uncoated or coated. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of atenolol, C14H22N2O3, 90.0% to 110.0% of the stated amount. Identification A. In the Assay, the ultraviolet absorption spectrum (Appendix 4.1) of the test solution, in the range 230 nm to 350 nm, exhibits absorption maxima at about 276 nm and 283 nm. B. In the test for Related substances, the retention time of the principal peak in the chromatogram obtained with solution (1) corresponds to that in the chromatogram obtained with solution (3). Related substances Examine by liquid chromatography (Appendix 5.3). 101

VP V

ATORVASTATIN CALCIUM TRIHYDRATE

Phosphate buffer: Dissolve 6.8 g of potassium dihydrogen phosphate R in 1000 ml of water, adjust to pH 3.0 with phosphoric acid R. Mobile phase: Dissolve 1.30 g of sodium octanesulfonate in a mixture of 700 ml of the phosphate buffer and 300 ml of methanol R. Solution (1): Weigh accurately a quantity of the powdered tablets, equivalent to 50 mg of atenolol, add 40 ml of the mobile phase, sonicate for 20 min, dilute to 50.0 ml with the mobile phase, and filter. Use the filtrate. Solution (2): Dilute 1.0 ml of solution (1) to 100.0 ml with the mobile phase, and mix. Solution (3): A 0.1% solution of atenolol RS in the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 275 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Inject solution (2). Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained is about 20 per cent of the full scale of the recorder. Inject solution (1), continue the chromatography for 3 times the retention time of the principal peak. In the chromatogram obtained with solution (1), the sum of the areas of all the secondary peaks is not greater than the area of the principal peak in the chromatogram obtained with solution (2).

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: Dilute 9 ml of hydrochloric acid R to 1000 ml with water. Rotation speed: 50 rpm. Time: 45 min. Procedure: Test solution: After the specified time, withdraw a sample of the medium, and filter, discard the first portion of the filtrate. Dilute an accurately measured volume of the filtrate with the medium to obtain a solution having a known concentration of about 10 mg of atenolol per ml. Reference solution: A solution of atenolol RS in the medium having a known concentration of about 10 mg per ml. Measure the absorbances of the solutions at 224 nm (Appendix 4.1) in a 1-cm cell using the medium in the reference cell. Calculate the total content of atenolol, C14H22N2O3, dissolved using the measured absorbances of the test solution, the reference solution and the declared content of C14H22N2O3 in atenolol RS. Tolerance: Not less than 70% (Q) of the labelled amount of atenolol, C14H22N2O3, is dissolved in 45 min. 102

Assay Test solution: Weigh 20 uncoated or coating-removed tablets, calculate the average mass, and powder finely. Transfer an accurately weighed quantity of the powder, equivalent to about 35 mg of atenolol, to a 50 ml volumetric flask, add 30 ml of ethanol R, heat the resulting suspension to 60 °C and shake for 15 min. Cool, dilute to volume with ethanol R, and mix. Filter, discard the first portion of the filtrate. Dilute 5.0 ml of the filtrate to 50.0 ml with ethanol R, and mix. Reference solution: A solution of atenolol RS in ethanol R having a known concentration of about 0.07 mg/ml. Measure the absorbances of the reference solution and the test solution at the maximum at 276 nm (Appendix 4.1) in a 1 - cm cell using ethanol R in the reference cell. Calculate the content of atenolol, C14H22N2O3, in the tablets using the measured absorbances of the test solution, the reference solution and the declared content of C14H22N2O3 in atenolol RS. Storage Store in a cool place, protected from light. Action and use Antihypertensive drugs. Usual strength 25 mg; 50 mg; 100 mg. ATORVASTATIN CALCIUM TRIHYDRATE Atorvastatinum calcium trihydricum

C66H68CaF2N4O10,3H2O

M: 1209

Atorvastatin calcium trihydrate is calcium (3R,5R)7-[2-(4-fluorophenyl)-5-(1-methylethyl)-3-phenyl-4(phenylcarbamoyl)-1H-pyrrol-1-yl]-3,5-dihydroxyheptanoate trihydrate. It contains not less than 97.0% and not more than 102.0% of C66H68CaF2N4O10, calculated with reference to the anhydrous substance.

Characters White or almost white powder, it shows polymorphism. Very slightly soluble in water, slightly soluble in ethanol (96%), practically insoluble in methylene chloride.

VP V

Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of atorvastatin calcium trihydrate RS. If the spectra obtained with the substance to be examined and the reference substance in the solid state show differences, dissolve separately the substance to be examined and atorvastatin calcium trihydrate RS in methanol R, evaporate to dryness and record new spectra using the residues. B. It complies with the test for Enantiomeric purity. C. It complies with the test for Water. D. Ignite the substance to be examined. The residue gives reaction (A) of calcium (Appendix 8.1). If the residue does not completely dissolve, filter and use the filtrate. Enantiomeric purity Examine by liquid chromatography (Appendix 5.3). Mobile phase: Trifluoroacetic acid - anhydrous ethanol hexane (0.1 : 6 : 94). Solvent mixture: Anhydrous ethanol - methanol (50 : 50). Test solution: Dissolve 10 mg of the substance to be examined in 4 ml of the solvent mixture and dilute to 10.0 ml with hexane R. Reference solution (1): Dissolve 2 mg of atorvastatin impurity E RS in methanol R and dilute to 20.0 ml with the same solvent (solution A). Dissolve 10 mg of the substance to be examined in 1.25 ml of methanol R, add 0.75 ml of solution A and 2 ml of anhydrous ethanol R and dilute to 10.0 ml with hexane R. Reference solution (2): To 2.0 ml of the test solution add 40.0 ml of the solvent mixture and dilute to 100.0 ml with hexane R. To 3.0 ml of this solution add 5 ml of the solvent mixture and dilute to 20.0 ml with hexane R. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase amylose derivative of silica gel for chromatography R (10 µm). Detector: A spectrophotometer set at 244 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: The run time is 1.2 times the retention time of atorvastatin. Relative retention of impurity E with reference to atorvastatin (retention time = about 44 min) is about 0.8. System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to impurity E and atorvastatin is at least 2.0. Limit: In the chromatogram obtained with test solution, the area of the peak due to impurity E is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.3%). Related substances Examine by liquid chromatography (Appendix 5.3).

ATORVASTATIN CALCIUM TRIHYDRATE

Mobile phase A: Tetrahydrofuran - acetonitrile - a 0.39% solution of ammonium acetate adjusted to pH 5.0 with glacial acetic acid (12 : 21 : 67). Mobile phase B: Tetrahydrofuran - acetonitrile - a 0.39% solution of ammonium acetate adjusted to pH 5.0 with glacial acetic acid (12 : 61 : 27). Test solution (1): Dissolve 40.0 mg of the substance to be examined in dimethylformamide R and dilute to 100.0 ml with the same solvent. Test solution (2): Dissolve 50.0 mg of the substance to be examined in dimethylformamide R and dilute to 50.0 ml with the same solvent. Reference solution (1): Dissolve 40.0 mg of atorvastatin calcium trihydrate RS in dimethylformamide R and dilute to 100.0 ml with the same solvent. Reference solution (2): Dilute 1.0 ml of test solution (2) to 100.0 ml with dimethylformamide R. Dilute 1.0 ml of this solution to 10.0 ml with dimethylformamide R. Reference solution (3): Dissolve 2.5 mg of atorvastatin impurity A RS, 2.5 mg of atorvastatin impurity B RS, 2.5 mg of atorvastatin impurity C RS, 2.5 mg of atorvastatin impurity D RS and 2.5 mg of the substance to be examined in dimethylformamide R and dilute to 50.0 ml with the same solvent. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase B (5 µm). Column temperature: 35 °C. Detector: A spectrophotometer set at 244 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl of test solution (2) and reference solutions (2) and (3). Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 40

100

0

40 - 70

100 → 20

0 → 80

70 - 85

20 → 0

80 → 100

Use the chromatogram obtained with reference solution (3) to identify the peaks due to impurities A, B, C, D of atorvastatin. Relative retention with reference to atorvastatin (retention time = about 33 min): Impurity A = about 0.8; impurity B = about 0.9; impurity C = about 1.2; impurity D = about 2.1. If necessary, adjust the mobile phase by increasing or decreasing the percentage of acetonitrile or the pH of the ammonium acetate solution to achieve a retention time of about 33 min for atorvastatin. For example, raising the pH would decrease the retention time of atorvastatin. System suitability: In the chromatogram obtained with reference solution (3), the resolution between the peaks due to impurity B and atorvastatin is at least 1.5. 103

VP V

ATORVASTATIN TABLETS

Limits: In the chromatogram obtained with test solution (2): Impurity A, B: For each impurity , the area is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.3%). Impurity C, D: For each impurity, the area is not more than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.15%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.10%). The sum of the peak areas of all impurities is not more than 15 times the area of the principal peak in the chromatogram obtained with reference solution (2) (1.5%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%) and the peak due to dimethylformamide. Note: Impurity A: (3R,5R)-3,5-dihydroxy-7-[5-(1-methylethyl)-2,3diphenyl-4-(phenylcarbamoyl)-1H-pyrrol-1-yl] heptanoic acid (desfluoroatorvastatin). Impurity B: (3RS,5SR)-7-[2-(4-fluorophenyl)-5-(1-methylethyl)3-phenyl-4-(phenylcarbamoyl)-1H-pyrrol-1-yl]-3,5-dihydroxyheptanoic acid. Impurity C: (3R,5R)-7-[2,3-bis(4-fluorophenyl)-5-(1-methylethyl)4-(phenylcarbamoyl)-1H- pyrrol-1-yl]-3,5-dihydroxyheptanoic acid (fluoroatorvastatin). Impurity D: 3-[(4-fluorophenyl)carbonyl]-2-(2-methylpropanoyl)N,3-diphenyloxirane-2-carboxamide. Impurity E: (3S,5S)-7-[2-(4-fluorophenyl)-5-(1-methylethyl)-3phenyl-4-(phenylcarbamoyl)-1H-pyrrol-1-yl]-3,5-dihydroxyheptanoic acid (ent-atorvastatin). Impurity F: (3R,5R)-7-[[(3R,5R)-7-[2-(4-fluorophenyl)-5-(1methylethyl)-3-phenyl-4- (phenylcarbamoyl)-1H-pyrrol-1-yl]3,5-dihydroxyheptanoyl]amino]-3,5-dihydroxyheptanoic acid. Impurity G: (3R,5R)-7-[2-(4-fluorophenyl)-5-(1-methylethyl)3-phenyl-4-(phenylcarbamoyl)-1H-pyrrol-1-yl]-5-hydroxy-3methoxyheptanoic acid (3-O-methylatorvastatin). Impurity H: (4R,6R)-6-[2-[2-(4-fluorophenyl)-5-(1-methylethyl)3-phenyl-4-(phenylcarbamoyl)-1H-pyrrol-1-yl]ethyl]-4hydroxytetrahydro-2H-pyran-2-one.

Sodium Not more than 0.4% (calculated with reference to the anhydrous substance). Examine by atomic absorption spectrometry (Appendix 4.4, method 1). Solvent mixture: Hydrochloric acid - water - methanol (2 : 25 : 75). Test solution: Dissolve 5.0 mg in the solvent mixture and dilute to 100.0 ml with the solvent mixture. Reference solutions: Prepare the reference solutions using sodium standard solution (50 ppm Na) R, diluting with the solvent mixture. 104

Measure the absorbance at 589.0 nm using a sodium hollow-cathode lamp as a source of radiation and an airacetylene flame.

Heavy metals Not more than 20 ppm (Appendix 9.4.8, method 8). Solvent mixture: Water - methanol (10 : 90). Test solution: Dissolve 0.250 g of the substance to be examined in 30 ml of the solvent mixture. Reference solution: Dilute 0.5 ml of lead standard solution (10 ppm Pb) R to 30 ml with the solvent mixture. Blank solution: 30 ml of the solvent mixture. Water 3.5% to 5.5% (Appendix 10.3). Determined on 0.130 g. Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. Inject test solution (1) and reference solution (1). Calculate the percentage content of atorvastatin calcium, C66H68CaF2N4O10, using the atorvastatin peak areas in the chromatograms obtained with test solution (1), reference solution (1) and the declared content of C66H68CaF2N4O10 in atorvastatin calcium trihydrate RS. Storage Store in an airtight container. Action and use HMG Co-A reductase inhibitor; lipid-regulating drug. Preparations Tablets. ATORVASTATIN TABLETS Tabellae Atorvastatini Atorvastatin tablets contain atorvastatin. They may be coated. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of atorvastatin, C33H35FN2O5, 90.0% to 110.0% of the stated amount. Identification A. Shake a quantity of the powdered tablets containing 10 mg of atorvastatin, with 50 ml of methanol R, centrifuge. Dilute 2.5 ml of the supernatant to 50.0 ml with methanol R. The ultraviolet absorption spectrum (Appendix 4.1) of the resulting solution exhibits a maximum in the range 244 nm to 248 nm. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution

VP V

corresponds to that of the atorvastatin peak in the chromatogram obtained with the reference solution.

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of water. Rotation speed: 75 rpm. Time: 30 min. Procedure: Test slution: After the specified time, withdraw a sample of the medium and filter. Dilute the filtrate with water, if necessary, to obtain a solution containing about 6 μg of atorvastatin per ml. Reference solution: Dissolve an accurately weighed quantity of atorvastatin calcium RS containing the equivalent to 30 mg of atorvastatin, in methanol R to produce 100.0 ml. Dilute 5.0 ml of this solution to 250.0 ml with water. Examine by liquid chromatography (Appendix 5.3). Mobile phase, chromatographic system: Proceed as directed in the Assay. Inject alternately the reference solution and the test solution. Calculate the content of atorvastatin, C33H35FN2O5, dissolved using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C33H35FN2O5 in atorvastatin calcium RS. Tolerance: Not less than 75% (Q) of the stated amount of atorvastatin, C33H35FN2O5, is dissolved in 30 min. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Acetonitrile - tetrahydrofuran (92.5 : 7.5). Mobile phase B: 0.05 M ammonium dihydrogen phosphate - mobile phase A (58 : 42). Mobile phase C: 0.05 M ammonium dihydrogen phosphate - mobile phase A - methanol (20 : 20 : 60). Solvent mixture: Acetonitrile - water (40 : 60). Test solution: Transfer an accurately weighed quantity of the powdered tablets containing 50 mg of atorvastatin to a 100 ml volumetric flask, dissolve in 10 ml of methanol R, add 20 ml of the solvent mixture, sonicate to dissolve, if necessary. Dilute to volume with the solvent mixture. Filter. Reference solution (1): Dissolve an accurately weighed quantity of atorvastatin calcium RS containing 25 mg of atorvastatin in 5 ml of methanol R and dilute to 50.0 ml with the solvent mixture. Reference solution (2): Dilute 1.0 ml of reference solution (1) to 100.0 ml with the solvent mixture. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 μm). Detector: A spectrophotometer set at 246 nm. Volume of injection: 20 μl. Injection delay: 10 min. Procedure: Carry out a linear gradient elution using the following conditions.

ATORVASTATIN TABLETS

Time (min)

Flow rate (ml/min)

Mobile phase B (% v/v)

Mobile phase C (% v/v)

0 - 20

1.8

100

0

20 - 35

1.8

100 → 25

0 → 75

35 - 40

1.5

25

75

40 - 55

1.5

25 → 0

75 → 100

55 - 60

1.8

0 → 100

100 → 0

Inject reference solution (1). The test is not valid unless the column efficiency is not less than 5000 theoretical plates, the tailing factor for atorvastatin peak is not more than 1.5. Inject reference solution (2) and the test solution. In the chromatogram obtained with the test solution, the area of any secondary peak is not greater than the area of the principal peak in the chromatogram obtained with reference solution (2) (1.0%) and the sum of the areas of any secondary peaks is not more than 4 times the area of the principal peak in the chromatogram obtained with reference solution (2) (4.0%). Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%).

Uniformity of content (Appendix 11.2). The test is applied for tablets containing 10 mg or less. Examine by liquid chromatography (Appendix 5.3). Mobile phase, solvent, reference solution and chromatographic system: Prepare as directed in the Assay. Test solution: Transfer 1 tablet to a 50 ml volumetric flask, add 3 ml of water and allow to disperse the tablet. Add 20 ml of methanol R, sonicate and dilute to volume with methanol R, mix and filter. Dilute 10.0 ml of the filtrate to 25.0 ml with methanol R. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Acetonitrile - tetrahydrofuran (92.5 : 7.5). Buffer solution: Dissolve 1.54 g of ammonium acetate R in 1000 ml of water, adjust to pH 4.0 with acetic acid R. Mobile phase: Buffer solution - mobile phase A (50 : 50). Solvent: Dissolve 6.8 g of potassium dihydrogen phosphate R and 0.9 g of sodium hydroxide R in 1000 ml of water, adjust to pH 6.8 with phosphoric acid R or 0.2 M sodium hydroxide R. Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Transfer an accurately weighed quantity of the powder containing 40 mg of atorvastatin to a 50 ml volumetric flask, add 40 ml of methanol R, sonicate to dissolve. Allow to cool and dilute to volume with methanol R, mix and filter. Dilute 5.0 ml of the filtrate to 50.0 ml with methanol R. Reference solution: Transfer an accurately weighed quantity of atorvastatin calcium RS containing 40 mg of atorvastatin to a 50 ml volumetric flask, dissolve in 105

VP V

ATROPINE SULFATE

methanol R and dilute to volume with methanol R. Dilute 5.0 ml of this solution to 100.0 ml with methanol R. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 μm). Detector: A spectrophotometer set at 246 nm. Flow rate: 2.0 ml/min. Volume of injection: 20 μl. Procedure: Inject the reference solution. The test is not valid unless the number of theoretical plates is not less than 2000; the tailing factor is not more than 1.5; the relative standard deviation of the peak areas due to atorvastatin for replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of atorvastatin, C33H35FN2O5, in the tablets using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C33H35FN2O5 in atorvastatin calcium RS.

Storage Store in an airtight container, protected from light, in a cool and dry place or at room temparature. Action and use Lipid-regulating drug. Usual strength 10 mg; 20 mg; 40 mg and 80 mg. ATROPINE SULFATE Atropini sulfas

C34H46N2O6,H2SO4,H2O

M. 695

Atropine sulfate is bis [(1R,3r,5S)-8-methyl-8azabicyclo[3.2.1]oct-3-yl (2RS)-3-hydroxy-2-phenylpropanoate] sulfate monohydrate. It contains not less than 99.0% and not more than 101.0% of C34H46N2O6,H2SO4, calculated with reference to the dried substance.

Characters A white or almost white, crystalline powder or colourless crystals. Very soluble in water, freely soluble in ethanol (96%). Identification Apply one of the two following identifications. First identification: A, B, E. 106

Second identification: C, D, E, F. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of atropine sulfate RS. B. It complies with the test for “Optical rotation” . C. Dissolve about 50 mg in 5 ml of water and add 5 ml of a 1% solution of picric acid solution R. Filter. The precipitate, washed with water and dried at 100 °C to 105 °C for 2 h, melts at 174°C to 179 °C (Appendix 6.7). D. To about 1 mg add 0.2 ml of fuming nitric acid R and evaporate to dryness in a water-bath. Dissolve the residue in 2 ml of acetone R and add 0.1 ml of a 3% solution of potassium hydroxide R in methanol R. A violet colour develops. E. It gives the reactions of sulfates (Appendix 8.1). F. It gives the reaction of alkaloids (Appendix 8.1).

pH Dissolve 0.60 g in carbon dioxide-free water R and dilute to 30 ml with the same solvent. The pH of the solution is 4.5 to 6.2 (Appendix 6.2). Specical optical rotation -0.50° to +0.05° (Appendix 6.4). Weigh accurately about 2.5 g, dissolve in water and dilute to 25.0 ml with the same solvent, measured in a 2-dm tube. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Dissolve 3.5  g of  sodium dodecyl sulfate R in 606 ml of a 0.7% solution of  potassium dihydrogen phosphate  R previously adjusted to pH 3.3 with  0.05  M  phosphoric acid R, and mix with 320 ml of acetonitrile R1. Mobile phase B: Acetonitrile R1. Test solution: Dissolve 24  mg of the substance to be examined in mobile phase A and dilute to 100.0 ml with the same solvent. Reference solution (1): Dilute 1.0  ml of the test solution to 100.0  ml with mobile phase A. Dilute 1.0  ml of this solution to 10.0 ml with mobile phase A. Reference solution (2) Dissolve 5 mg of atropine impurity B RS in the test solution and dilute to 20.0 ml with the same solvent. Dilute 5.0 ml of this solution to 25.0 ml with mobile phase A. Reference solution (3):  Dissolve the contents of a vial of atropine for peak identification RS (containing impurities A, D, E, F, G and H) in 1 ml of mobile phase A. Reference solution (4):  Dissolve 5  mg of  tropic acid  R (impurity C) in mobile phase A and dilute to 10.0 ml with the same solvent. Dilute 1 ml of the obtained solution to 100 ml with mobile phase A. Dilute 1.0 ml of this solution to 10.0 ml with mobile phase A. Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase C (3 µm).

VP V

ATROPINE SULFATE INJECTION

Detector: A spectrophotometer set at 210 nm. Flow rate: 1 ml/min. Volume of injection: 10 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0-2

95

5

2 - 20

95 → 70

5 → 30

Identification of impurities: Use the chromatogram supplied with atropine for peak identification RS and the chromatogram obtained with reference solution (3) to identify the peaks due to impurities A, D, E, F, G and H. Use the chromatogram obtained with reference solution (2) to identify the peak due to impurity B, and use the chromatogram obtained with reference solution (4) to identify the peak due to impurity C. The relative retention time with reference to atropine (retention time = about 11 min): Impurity C = about 0.2; impurity E = about 0.67; impurity D = about 0.73; impurity F = about 0.8; impurity B = about 0.89; impurity H = about 0.93; impurity G = about 1.1; impurity A = about1.7. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurity B and atropine is at least 2.5. Limits: The correction factors: For the calculation of content, multiply peak areas of the following impurities by the corresponding correction factor: impurity A = 0.6; impurity C = 0.6; Impurities E, H: For each impurity, the peak area is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.3%). Impurities A, B, C, D, F, G: For each impurity, the corrected peak area, if necessary, is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%). Any other impurity: For each impurity, the peak area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). The sum of peak areas of all impurities is more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Notes: Impurity A: (1R,3r,5S)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl 2-phenylpropenoate (apoatropine). Impurity B: (1R,3r,5S)-8-azabicyclo[3.2.1]oct-3-yl (2RS)-3-hydroxy -2-phenylpropanoate (noratropine). Impurity C: (2RS)-3-hydroxy-2-phenylpropanoic acid (tropic acid).

Impurity D: (1R,3S,5R,6RS)-6-hydroxy-8-methyl-8-azabicyclo -[3.2.1]oct-3-yl(2S)-3-hydroxy-2-phenylpropanoate 6-hydroxyhyoscyamine). Impurity E: (1S,3R,5S,6RS)-6-hydroxy-8-methyl-8-azabicyclo -[3.2.1]oct-3-yl(2S)-3-hydroxy-2-phenylpropanoate (7-hydroxyhyoscyamine). Impurity F: (1R,2R,4S,5S,7s)-9-methyl-3-oxa-9-azatricyclo[3.3.1.02,4] non-7-yl (2S)-3-hydroxy-2-phenylpropanoate (hyoscine). Impurity G: (1R,3r,5S)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl (2RS)-2-hydroxy-3-phenylpropanoate (littorine). Impurity H: unknown structure.

Water 2.0% to 4.0% (Appendix 10.3). Determined on 0.500 g. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.500 g in 30 ml of anhydrous acetic acid R, warming if necessary. Cool the solution. Titrate with 0.1 N perchloric acid VS and determine the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 67.68 mg of C34H48N2O10S. Storage Store in an airtight container, protected from light. Action and use Anticholinergic.

Preparations

Eye drops, eye ointment, injection, tablets. ATROPINE SULFATE INJECTION Injectio Atropnini sulfatis Atropine sulfate injection is a sterile solution of atropine sulfate in water for injections. The injection complies with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Content of atropine sulfate, (C17H23NO3)2,H2SO4,H2O, 90.0% to 110.0% of the stated amount. Characters A clear, colourless solution. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Chloroform - acetone - diethylamine (50 : 40 : 10). Test solution: Evaporate a volume of the injection containing 5 mg of atropine sulfate to dryness on a water bath, dissolve the residue with 1 ml of ethanol (96%) R. 107

VP V

ATROPINE SULFATE TABLETS

Reference solution: A 0.5% solution of atropine sulfate RS in ethanol (96%) R. Procedure: Apply separately to the plate 5 μl of each solution. Develop over a path of 15 cm. After removal of the plate, dry at 105 ºC for 20 min, cool, and spray with dilute potassium iodobismuthate solution R, and examine in daylight. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution.

Calculate the content of atropine sulfate, (C17H23NO3)2,H2SO4,H2O, in the injection using the areas for the principal peaks in the chromatogram obtained with the test solution, the reference solution and the declared content of (C17H23NO3)2,H2SO4,H2O in atropine sulfate RS.

pH 3.0 to 5.0 (Appendix 6.2).

ATROPINE SULFATE TABLETS Tabellae Atropini sulfatis

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: A solution containing 0.01 M sodium acetate R and 0.005 M dioctyl sodium sulfosuccinate R in methanol (60%) R adjusted to pH 5.5 with glacial acetic acid R. For injections containing less than 0.1% of atropine sulfate Test solution: Use the injection being examined. Reference solution: A solution containing atropine sulfate RS in water at the same concentration as the solution being examined. Resolution solution: A solution contaning atropine sulfate RS and homatropine hydrobromide RS in the mobile phase, both at the same concentration as the solution being examined. Volume of injection: 100 µl. For injections containing 0.1% or more of atropine sulfate Test solution: Dilute the injection, if necessary, to obtain a 0.1% solution of atropine sulfate in water. Reference solution: A 0.1% solution of atropine sulfate RS in water. Resolution solution: A solution containing 0.1% of atropine sulfate RS and 0.1% of homatropine hydrobromide RS in the the mobile phase. Volume of injection: 20 µl. Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 257 nm. Flow rate: 2.0 ml/min. Procedure: System suitability: Inject the resolution solution, the test is not valid unless the resolution factor between the peaks due to atropine sulfate and homatropine hydrobromide is at least 2.5. Inject separately the reference solution and the test solution. 108

Storage Protected from light. Action and use Antispasmodic; antidote to organic phosphorus. Usual strength 0.25 mg/ml; 0.5 mg/ml.

Atropine sulfate tablets contain atropine sulfate. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of atropine sulfate, (C17H23NO3)2,H2SO4,H2O, 90.0% to 110.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G Mobile phase: Chloroform - acetone - diethylamine (50 : 40 : 10). Test solution: To a quantity of the powdered tablets containing 5 mg of atropine sulfate add 10 ml of ethanol (96%) R, shake for 10 to 15 min, and filter. Evaporate the filtrate to dryness on a water bath, dissolve the residue in 1 ml of ethanol (96%) R. Reference solution: A 0.5% solution of atropine sulfate RS in ethanol (96%) R. Procedure: Apply separately to the plate 5 ml of each solution. Develop over a path of 15 cm. Remove the plate, heat at 105 ºC for 20 min, and spray with the dilute potassium iodobismuthate solution R. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. B. In the test for Uniformity of content, the retention time of the principal peak in the chromatography obtained with the test solution is similar to that of the atropine sulfate peak in the chromatography obtained with the reference solution. C. To a quantity of the powdered tablets, equivalent to 5 mg of atropine sulfate, add 5 ml of water, shake for 10 min to 15 min, and filter. To the filtrate add 0.5 ml of 2 M hydrochloric acid R and 0.5 ml of a 5% solution of barium

VP V

chloride R. A white precipitate is formed which is insoluble in acid solutions.

Uniformity of content (Appendix 11.2) Examine by liquid chromatography (Appendix 5.3). Mobile phase: A solution containing 0.01 M sodium acetate R and 0.005 M dioctyl sodium sulfosuccinate R in methanol (60%) R, adjust the pH to 5.5 with glacial acetic acid R. Test solution: To a tablet, add 2.0 ml of the mobile phase and sonicate to disintegrate completely, filter. Reference solution: A solution containing atropine sulfate RS in the mobile phase at the same concentration as the test solution. Resolution solution: A solution containing atropine sulfate RS and homatropine hydrobromide RS in the mobile phase, both at the same concentration of atropine sulfate as the test solution. Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 257 nm. Flow rate: 2 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the resolution solution, the resolution factor between atropine sulfate and homatropine hydrobromide peaks in the chromatogram obtained is not less than 2.5. Inject alternately the reference solution and the test solution. Calculate the content of atropine sulfate, (C17H23NO3)2, H2SO4,H2O, in the tablets using the areas of the peaks obtained with the test solution, the reference solution and the declared content of (C17H23NO3)2,H2SO4,H2O in atropine sulfate RS. Assay Use the average content of 10 tablets obtained in the Uniformity contents. Storage Store in a cool place, protected from light. Action and use Antispasmodic. Usual strength 0.5 mg. ATTAPULGITE Attapulgitum Attapulgite is a purified native hydrated magnesium aluminium silicate essentially consisting of the clay mineral palygorskite.

ATTAPULGITE

Characters A light, cream or buff, very fine powder, free or almost free from gritty particles. Identification A. Ignite 0.5 g of the substance to be examined with 2 g of anhydrous sodium carbonate R for 20 min, cool and extract with 25 ml of boiling water. Cool, filter, wash the residue with water and add the washings to the filtrate. Reserve the residue for test B. Cautiously acidify the combined filtrate and washings with hydrochloric acid R, evaporate to dryness, moisten the residue with 0.2 ml of hydrochloric acid R, add 10 ml of water and stir. A white, gelatinous precipitate is produced. B. Wash the residue reserved in test A with water and dissolve in 10 ml of 2 M hydrochloric acid R. To 2 ml of the solution, add a 10% solution of ammonium thiocyanate R. An intense red colour is produced. C. To 2 ml of the solution obtained in test B, add 1 ml of a 42% solution of sodium hydroxide R and filter. To the filtrate add 3 ml of a 10.7% solution of ammonium chloride R. A gelatinous white precipitate is produced. D. To 2 ml of the solution obtained in test B, add ammonium chloride R and an excess of ammonia R and filter. To the filtrate add 0.15 ml of magneson reagent R and an excess of 5 M sodium hydroxide R. A blue precipitate is produced. pH 7.0 to 9.5 (Appendix 6.2). Shake 5 g in 100 ml of carbon dioxide-free water for 5 min. Determined on the obtained suspension. Adsorptive capacity Moisture adsorption: 5% to 14%. Crush a dried in air and passed through a sieve No. 150 powder of the substance being examined. Spread 0.5 g as a thin layer on a previously weighed piece of aluminium foil (60 mm × 50 mm) of nominal gauge 17.5 µm and transfer to a desiccator containing a dish of sodium chloride crystals partially immersed in saturated brine at 25 °C. After 4 hours, remove from the desiccator and weigh immediately. Then, dry in an oven at 110 °C for 4 h, allow to cool in a desiccator and weigh. The moisture adsorption is the gain in weight of the substance being examined expressed as a percentage of its oven-dried weight. Arsenic Not more than 8 ppm (Appendix 9.4.2, method A). To 0.13 g of the substance being examined, add 5 ml of water, 2 ml of sulfuric acid R and 10 ml of sulfur dioxide solution R and evaporate on a water bath until the sulfur dioxide solution is removed and the volume reduced to about 2 ml. Transfer the solution to the generator flask with the aid of 5 ml of water. 109

VP V

AZITHROMYCIN

Heavy metals A. Not more than 20 ppm (Appendix 9.4.8, method 1). Shake 6.0 g of the substance being examined with 40 ml of 0.5 M hydrochloric acid R at 37 °C for 30 min, cool and filter. Wash the residue with water and dilute the combined filtrate and washings to 50 ml with water. Dissolve 2 g of ammonium chloride R and 2 g of ammonium thiocyanate R in 20 ml of the obtained solution. Shake the solution with 80 ml of a mixture of equal volumes of ether R and isoamyl alcohol R and allow to separate, retaining the aqueous layer. Extract with a further 80 ml of the mixture. To the aqueous layer, add 2 g of citric acid R, neutralise with 13.5 M ammonia R and dilute to 25 ml with water. 12 ml of the resulting solution complies with limit test for heavy metals. Prepare the standard using 2 ml of lead standard solution (2 ppm Pb) R. B. Not more than 10 ppm (Appendix 9.4.8, method 1). Shake 6.0 g of the substance being examined with 40 ml of 0.5 M sodium hydroxid R at 37 °C for 30 min, cool and filter. Wash the residue with water and dilute the combined filtrate and washings to 50 ml with water. Neutralise 20 ml of the solution with hydrochloric acid R and dilute to 25 ml with water. 12 ml of the resulting solution complies with limit test for heavy metals. Prepare the standard using lead standard solution (1 ppm Pb) R. Acid-soluble matter Boil 2 g of the substance being examined with 100 ml of 0.2 M hydrochloric acid R under a reflux condenser for 5 minutes, cool and filter. Evaporate 50 ml of the filtrate to dryness. The residue, after ignition at 600 °C for 30 minutes, weighs not more than 0.25 g. Water-soluble matter Boil 10 g of the substance being examined with 100 ml of water under a reflux condenser for 5 min, cool and filter. Evaporate 50 ml of the filtrate to dryness. The residue, after ignition at 600 °C for 30 min, weighs not more than 50 mg. Loss on drying Not more than 17.0% (Appendix 9.6). (1 g; 105 °C). Loss on ignition 15.0% to 27.0%. Ignited 1 g at 600 °C to constant mass. Storage Store in an airtight container. Action and use Protection of gastric and bowels mucosa. Preparations Powder for suspension.

110

AZITHROMYCIN

Azithromycinum

C38H72N2O12. xH2O (with x = 1 or x = 2) (anhydrous substance)

M. 749.0

Azithromycin is (2R,3S,4R,5R,8R,10R,11R,12S,13S, 14R)-13-[(2,6-Dideoxy-3-C-methyl-3-O-methyl-α-Lribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6-trideoxy-3(dimethylamino)-β-D-xylo-hexopyranosyl]oxy]-1-oxa-6azacyclopentadecan-15-one, the degree of hydration is 1 or 2. Semi-synthetic product derived from a fermentation product, contains not less than 96.0% and not more than 102.0% of C38H72N2O12, calculated with reference to the anhydrous substance.

Characters White or almost white powder. Practically insoluble in water, freely soluble in ethanol and in methylene chloride. Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of azithromycin RS. If the spectra obtained in the solid state show differences, prepare further spectra using 9.0% solutions in methylene chloride R. Appearance of solution Solution S: Dissolve 0.500 g in ethanol R and dilute to 50.0 ml with the same solvent. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). pH 9.0 to 11.0 (Appendix 6.2). Dissolve 0.100 g in 25.0 ml of methanol R and dilute to 50.0 ml with carbon dioxide-free water R. Specific optical rotation -45º to -49º, calculate with reference to the anhydrous substance (Appendix 6.4). Determined on solution S. Related substances Examine by liquid chromatography (Appendix 5.3).

VP V

AZITHROMYCIN

Mobile phase A: Dissolve 1.8 g of anhydrous disodium hydrogen phosphate R in 1000 ml of water, and adjust pH to 8.9 with dilute phosphoric acid R or with dilute sodium hydroxide solution R. Mobile phase B: Methanol R1 - acetonitrile R1 (250 : 750). Solvent mixture: Buffer solution pH 10.0 - acetonitrile R1 - methanol R1 (350 : 300 : 350). Buffer solution pH 10.0: A 0.173% solution of ammonium dihydrogen phosphate R adjusted to pH 10.0 with ammonia R. Test solution: Dissolve 0.200 g of the substance to be examined in the solvent mixture and dilute to 25.0 ml with the same solvent. Reference solution (1): Dilute 1.0 ml of test solution to 100.0 ml with the solvent mixture. Reference solution (2): Dissolve the contents of a vial of  azithromycin for system suitability RS (containing impurities F, H and J) in 1.0 ml of the solvent mixture and sonicate for 5 min. Reference solution (3): Dissolve 8.0 mg of azithromycin for peak identification RS (containing impurities A, B, C, E, F, G, I, J, L, M, N, O and P) in 1.0 ml of the solvent mixture. Chromatographic system: A column (25 cm × 4.6 mm) packed with end-capped octadecylsilyl amorphous organosilica polymer for mass spectrometry R (5 µm). Column temperature: 60 ºC. Detector: A spectrophotometer set at 210 nm. Flow rate: 1.0 ml/min. Volume of injection: 50 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 25

50 → 45

50 → 55

25 - 30

45 → 40

55 → 60

30 - 80

40 → 25

60 → 75

80 - 81

25 → 50

75 → 50

81 - 93

50

50

Identification of impurities:  Use the chromatogram supplied with azithromycin for peak identification RS and the chromatogram obtained with reference solution (3) to identify the peaks due to impurities A, B, C, E, F, G, I, J, L,  M, N, O and P; use the chromatogram supplied with  azithromycin for system suitability RS and the chromatogram obtained with reference solution (2) to identify the peak due to impurity H. Relative retention with reference to azithromycin (retention time = about 45 - 50 min): Impurity L = about 0.29; impurity M = about 0.37; impurity E = about 0.43; impurity F = about 0.51; impurity D = about 0.54; impurity J = about 0.54; impurity I = about 0.61; impurity C = about 0.73; impurity N = about 0.76; impurity H = about 0.79; impurity A = about 0.83; impurity P = about 0.92; impurity O  =  about 1.23; impurity G  =  about 1.26; impurity B = about 1.31.

System suitability: In the chromatogram obtained with reference solution (2), the peak-to-valley ratio (Hp/Hv) is at least 1.4, where Hp is the height above the baseline of the peak due to impurity J and Hv is the height above the baseline of the lowest point of the curve separating this peak from the peak due to impurity F. Limits: The correction factors: For the calculation of content, multiply the peak areas of the following impurities by the corresponding correction factor: Impurity F = 0.3; impurity G = 0.2; impurity H = 0.1; impurity L = 2.3; impurity M = 0.6; impurity N = 0.7. Impurity B: The area of peak due to impurity B is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (1) (2.0%). Impurities A, C, E, F, H, I, L, M, N, O, P: For each impurity, the corrected peak area if necessary is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). The sum of areas of peaks due to impurities D and J is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). Impurity G: The corrected area of peak due to impurity G is not more than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%). Any other impurities: The area of each impurity peak is not more than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%). The sum of the peak areas of all impurities is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (1) (3.0%). Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%); disregard the peaks eluting before impurity L and after impurity B. Notes Impurity A: 6-demethylazithromycin. Impurity B: 3-deoxyazit-hromycin (azithromycin B). Impurity C: 3”-O-demethylazithromycin (azithromycin C). Impurity D: 14-demethyl-14-(hydroxymethyl) azithromycin (azithromycin F). Impurity E: 3’-(N,N-didemethyl)azithromycin (aminoazithromycin). Impurity F: 3’-N-demethyl-3’-N-formylazithromycin. Impurity G: 3’-N-demethyl-3’-N-[(4-methylphenyl) sulphonyl] azithromycin. Impurity H: 3′-N-[[4-(acetylamino)phenyl]sulfonyl]-3′-Ndemethylazithromycin. Impurity I: 3’-N-demethylazithromycin. Impurity J: 13-O-decladinosylazithromycin. Impurity K: C14,1″-epoxya-zithromycin (azithromycin E). Impurity L: Azithromycin 3′-N-oxide. Impurity M: 3′-(N,N-didemethyl)-3′-N-formylazithromycin. Impurity N: 3′-de(dimethylamino)-3′-oxoazithromycin. Impurity O: 2-desethyl-2-propylazithromycin. Impurity P: Unknown structure.

111

VP V

AZITHROMYCIN CAPSULES

Heavy metals Not more than 25 ppm (Appendix 9.4.8). Dissolve 2.0 g in a mixture of water - ethanol (15 : 85) and dilute to 20 ml with the same solvent. 12 ml of the solution complies with test 2. Prepare the reference solution using lead standard solution (2.5 ppm Pb) obtained by diluting lead standard solution (100 ppm Pb) R with a mixture of water - ethanol (15 : 85). Water 1.8% to 6.5% (Appendix 10.3). Determined on 0.200 g. Sulfated ash Not more than 0.2% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile R1 - buffer solution pH 11.0 (60 : 40). Buffer solution pH 11.0: Dissolve 6.7 g of dipotassium hydrogen phosphate R in 1000 ml of water, and adjust pH to 11.0 with a 56% solution of potassium hydroxide R. Solution A: Acetonitrile R1 - a 0.67% solution of dipotassium hydrogen phosphate R adjusted to pH 8.0 with phosphoric acid R (60 : 40). Test solution: Dissolve 53.0 mg of the substance to be examined in 2 ml of acetonitrile R1 and dilute to 100.0 ml with solution A. Reference solution (1): Dissolve 53.0 mg of azithromycin RS in 2 ml of acetonitrile R1 and dilute to 100.0 ml with solution A. Reference solution (2): Dissolve 5.0 mg of the substance to be examined and 5.0 mg of  azithromycin impurity A RS in 0.5 ml of acetonitrile R1 and dilute to 10.0 ml with solution A. Chromatographic system: A column (25 cm × 4.6 mm) packed with octadecylsilyl vinyl polymer for chromatography R (5 µm). Column temperature: 40 ºC. Detector: A spectrophotometer set at 210 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: The run time is 1.5 times the retention time of azithromycin. Retention time of azithromycin is about 10 min. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurity A and azithromycin is at least 3.0. Calculate the percentage content of C38H72N2O12 in the substance to be examined, using the areas of the principal peaks in the chromatogram obtained with the test solution, reference solution (1), and the declared content of C38H72N2O12 in azithromycin RS. Storage In an airtight container. 112

Action and use Antibacterial. Preparations Capsules, tablets, oral suspension. AZITHROMYCIN CAPSULES Capsulae Azithromycini Azithromycin capsules contain azithromycin. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of azithromycin, C38H72N2O12, 90.0% to 110.0% of the stated amount. Identification In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of the principal peak in the chromatogram obtained with the reference solution. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of phosphate buffer solution pH 6.0. Phosphate buffer solution pH 6.0: Prepare 6 L of 0.1 M disodium hydrogen phosphate R, adjust the pH to 6.0 ± 0.1 with about 40 ml of hydrochloric acid R, add 600 mg of trypsin and mix. Rotation speed: 100 rpm. Time: 45 min. Procedure: Determine the content of azithromycin dissolved by liquid chromatography with the chromatographic system and mobile phase as described in the Assay. Test solution: After the specified time, withdraw a sample of the medium and filter. Reference solution: Weigh accurately a quantity of azithromycin RS and dissolve in the medium and dilute step by step if necessary with the medium to obtain a solution having the same concentration of azithromycin as the test solution. Tolerance: Not less than 75% (Q) of the labeled amount of azithromycin, C38H72N2O12, is dissolved in 45 min. Water Not more than 5.0% (Appendix 10.3). Use 0.3 g of the capsule contents. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol - water - ammonia (80 : 19.9 : 0.1). Reference solution: Dissolve an accurately weighed quantity of azithromycin RS in the mobile phase to obtain a solution having a known concentration of about 1.0 mg per ml.

VP V

Test solution: Weigh accurately a quantity of the powdered tablet containing about 50 mg of azithromycin and transfer into a 50-ml volumetric flask, add 30 ml of the mobile phase, sonicate for 15 min, dilute to volume with the mobile phase, mix and filter. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase B (5 mm). Detector: A spectrophotometer set at 215 nm Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: Inject separately the reference solution and the test solution. Calculate the content of azithromycin, C38H72N2O12, in the capsules using the areas of the principal peaks in the chromatograms obtained with the test solution, the reference solution and the declared content of C38H72N2O12 in azithromycin RS.

Storage Store in an airtight container, at a cool place, protected from light. Action and use Macrolide antibacterial. Usual strength 250 mg; 500 mg. AZITHROMYCIN POWDER FOR ORALSUSPENSION Pulveres Azithromycini ad suspensionum peroralum Azithromycin powder for oral suspension contains azithromycin. It may contain suitable flavors, colors, preservatives, stabilizers… The Oral suspension is prepared as directed on the label complies with the requirements stated under "Suspension" (Appendix 1.5). The powder complies with the requirements stated under “Powder” (Appendix 1.7) and with the following requirements.

BACITRACIN

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol - water - ammonia (80 : 19.9 : 0.1). Reference solution: Dissolve an accurately weighed quantity of azithromycin RS in the mobile phase, and quantitatively dilute with the mobile phase to obtain a solution having a known concentration of about 1.0 mg per ml. Test solution: Transfer an accurately weighed quantity of the powder (obtained with the test for Uniformity of mass) containing the equivalent to about 50 mg of azithromycin to a 50-ml volumetric flask, add 30 ml of the mobile phase, sonicate for 15 min, dilute to volume with the mobile phase, and mix. Filter, discarding the first portion of the filtrate. Use the filtrate. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase B (5 µm). Detector: A spectrophotometer set at 215 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: Inject separately the reference solution and the test solution. Calculate the content of azithromycin, C38H72N2O12, using the areas of the principal peaks in the chromatograms obtained with the test solution and the reference solution and the declared content of C38H72N2O12 in azithromycin RS. Storage Store in an airtight container at a cool place, protected from light. Action and use Macrolide antibacterial. Usual strength 125 mg; 250 mg. BACITRACIN Bacitracinum

Content of azithromycin, C38H72N2O12, 90.0% to 110.0% of the stated amount. Characters Powder should be loose and dry, not be moist and lumpy, with homogeneous colour. Identification In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. Water Not more than 1.5% (Appendix 10.3). Determined on 0.5 g of the powder.

Name

Mol. Formula

Bacitracin is mixture of antimicrobial polypeptides produced by certain strains of Bacillus licheniformis or Bacillus subtilis, the main components being bacitracins 113

BACITRACIN

A, B1, B2 and B3. It must contain minimum 60 IU/mg (calculated with reference to the dried substance).

Characters White or almost white powder, hygroscopic. Freely soluble in water and in ethanol (96%). Identification Apply one of the two following identifications: First identification: B, C. Second identification: A, C. A. Thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel. Mobile phase: Glacial acetic acid - water - butanol (1 : 2 : 4). Test solution: Dissolve 10 mg of the substance to be examined in 0.1 M hydrochloric acid R and dilute to 1.0 ml with the same solvent. Reference solution: Dissolve 10 mg of bacitracin zinc RS in 0.1 M hydrochloric acid R and dilute to 1.0 ml with the same solvent. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of over half of the plate. Drying at 100 °C to 105 °C and spray with ninhydrin solution R and heat at 110 °C for 5 min. The spots in the chromatogram obtained with the test solution are similar in position, size and colour to the spots in the chromatogram obtained with the reference solution. B. It complies with the test for Composition. C. Ignite 0.2 g of the substance to be examined. An insignificant residue remains which is not yellow at high temperature. Allow to cool. Dissolve the residue in 0.1 ml of dilute hydrochloric acid R. Add 5 ml of water and 0.2 ml of a 42% solution of sodium hydroxide R. No white precipitate is formed. Clarity of solution Solution S: Dissolve 0.25 g in carbon dioxide-free water R and dilute to 25 ml with the same solvent. Solution S is clear (Appendix 9.2). pH 6.0 to 7.0 for solution S (Appendix 6.2). Composition Examine by liquid chromatography (Appendix 5.3). Use the normalisation procedure. Prepare the solutions immediately before use. Mobile phase: Add 520 volumes of methanol R, 40 volumes of acetonitrile R and 300 volumes of water to 100 volumes of a 3.48% solution of dipotassium hydrogen phosphate R adjusted to pH 6.0 with a 2.72% solution of potassium dihydrogen phosphate R. Test solution: Dissolve 0.100 g of the substance to be examined in 50.0 ml of the mobile phase. Reference solution (1): Suspend 20.0 mg of bacitracin zinc RS in water, add 0.2  ml of dilute hydrochloric acid R and dilute to 10.0 ml with water. 114

VP V

Reference solution (2): Dilute 5.0 ml of the test solution to 100.0 ml with the mobile phase. Reference solution (3): Dilute 1.0 ml of reference solution (2) to 10.0 ml with the mobile phase. Reference solution (4): Dissolve 50.0 mg of the substance to be examined in 25.0  ml of a 4% solution of Trilon B R adjusted to pH 7.0 with dilute sodium hydroxide R. Heat in a boiling water-bath for 30 min. Cool to room temperature. Blank solution: A 4.0% solution of Trilon B R adjusted to pH 7.0 with dilute sodium hydroxide R. Chromatographic system: A column (25 cm × 4.6 mm) packed with end-capped octadecylsilyl silica gel for chromatography R (5 µm). Flow rate: 1.0 ml/min. Detector: A spectrophotometer set at 254 nm. Volume of injection: 100 µl. Procedure: The run time is 3 times the retention time of bacitracin A. Inject the blank, the test solution and reference solutions (1) and (3). Relative retention with reference to bacitracin A (retention time = 15 min to 25 min): bacitracin B1 = about 0.6; bacitracin B3 = about 0.8; impurity E = about 2.5. If necessary, adjust the composition of the mobile phase by changing the amount of organic modifier whilst keeping the ratio constant between methanol and acetonitrile. System suitability: Peak-to-valley ratio: minimum of 1.2, where Hp = height above the baseline of the peak due to bacitracin B1 and Hv = height above the baseline of the lowest point of the curve separating this peak from the peak due to bacitracin B2, in the chromatogram obtained with reference solution (1). Limits: Bacitracin A: minimum 40.0%, sum of bacitracins A, B1, B2 and B3: minimum 70.0%. Disregard any peak observed in the blank run and any peak with an area less than or equal to the area of the peak due to bacitracin A in the chromatogram obtained with reference solution (3) (0.5%).

Related peptides Examine by liquid chromatography (Appendix 5.3) as described in the test for Composition. Limit: Sum of the areas of all peaks eluting before the peak due to bacitracin B1: maximum 20.0%. Impurity E Examine by liquid chromatography (Appendix 5.3) as described in the test for Composition. Detection: A spectrophotometer set at 300 nm for reference solution (4) to determine peak due to impurity E. Injection the test solution and reference solutions (2) and (4). Limit: In the choromatogram obtained with test solution, area of the peak due to impurity E is not more than 1.2 times the area of the principal peak in the chromatogram obtained with reference solution (2) (6.0%).

VP V

BACITRACIN

Loss on drying Maximum 5.0% (Appendix 9.6), determined on 1.000 g by drying at 60 °C over diphosphorus pentoxide R at a pressure not exceeding 0.1 kPa for 3 h.

Assay Carry out the microbiological assay of antibiotics (Appendix 13.9). Use bacitracin zinc RS as the reference substance.

Sulphated ash Maximum 1.0% (Appendix 9.9, method 2). Determine on 1.0 g.

Storage In an airtight container at 2 °C to 8 °C. If the substance is sterile, store in a sterile, airtight, tamper-proof container.

Sterility If intended for the preparation of ophthalmic dosage forms without a further appropriate sterilisation procedure, it complies with the test for sterility (Appendix 13.7).

Action and use Antibacterial.

Bacterial endotoxins Less than 0.8 EU/mg (Appendix 13.2), if intended for use in the manufacture of ophthalmic dosage forms without a further appropriate procedure for the removal of bacterial endotoxins.

Ghi chú: Impurity A: Bacitracin C1, Impurity B: Bacitracin C2, Impurity C: Bacitracin C3, Impurity D: Bacitracin E, Impurity E: Bacitracin F, Impurity F: Bacitracin H1, Impurity G: Bacitracin H2, Impurity H: Bacitracin H3, Impurity I: Bacitracin I1, Impurity J: Bacitracin I2, Impurity K: Bacitracin I3.

The following chromatograms are published for information 100.00 95.00 90.00 85.00 80.00 75.00 70.00 65.00 60.00 55.00

nV

50.00 45.00 40.00 35.00 30.00 25.00 20.00 15.00 10.00 5.00 0.00 -5.00

0.00 0.00

2.00 2.00

4.00 4.00

6.00 6.00

1. impurity A 2. impurity B

8.00 8.00

10.00 10.00

12.00 12.00

14.00 14.00

3. impurity C 4. bacitracin B1

16.00 18.00 16.00 18.00 min min

20.00 20.00

22.00 22.00

5. bacitracin B2 6. bacitracin B3

24.00 24.00

26.00 26.00

28.00 28.00

30.00 30.00

32.00 32.00 34.00 34.00

7. bacitracin A

Chromatogram of the test for composition in bacintracin obtained with the test solution at 254 nm 115

VP V

BARIUM SULFATE 5.00 4.50 4.00 3.50 3.00 2.50 2.00 1.50

nV

1.00 0.50 0.00 -0.50 -1.00 -1.50 -2.00 -2.50 0.00

5.00

1. Bacitracin B1

10.00

15.00

2. Bacitracin B3

20.00

25.00

3. Bacitracin A

30.00

35.00

40.00

4. Impurity E

Chromatogram of the test for impurity E in bacitracin obtained with reference solution (4) at 300 nm

BARIUM SULFATE Barii sulfas BaSO4

M. 233.4

Characters A fine, white or almost white powder, free from gritty particles. Practically insoluble in water and in organic solvents. It is very slightly soluble in acids and in solutions of alkali hydroxides. Identification A. Boil 0.2 g with 5 ml of a 50% solution of sodium carbonate R for 5 min, add 10 ml of water, and filter. Acidify a part of the filtrate with dilute hydrochloric acid R and add several drops of a 5% solution of barium chloride R. A white precipitate is formed. B. Wash the residue collected in the preceding test (test A) with three successive small quantities of water. To the residue add 5 ml of dilute hydrochloric acid R, filter and add to the filtrate 0.3 ml of dilute sulfuric acid R. A white precipitate is formed that is insoluble in dilute sodium hydroxide solution R. 116

Acidity or alkalinity Heat 5.0 g with 20 ml of carbon dioxide-free water R on a water-bath for 5 min and filter. To 10 ml of the filtrate add 2 drops of bromothymol blue solution R. Not more than 0.5 ml of 0.01 N hydrochloric acid VS or 0.01 N sodium hydroxide VS is required to change the colour of the indicator. Soluble barium salts Not more than 10 ppm (Appendix 9.4.8). Solution S: Boil 20.0 g with a mixture of 40 ml of distilled water and 60 ml of 2 M acetic acid R for 5 min. Filter and dilute the cooled filtrate to 100 ml with distilled water. To 2.5  ml of a 0.002% solution of  barium nitrate R in a mixture of ethanol (96%) - water (30 : 70), add 10  ml of dilute sulfuric acid R, shake and allow to stand for 5 min (Solution A). Test solution: To 1 ml of solution A add 10 ml of solution S. Standard solution: To 1  ml of solution A add 10  ml of barium standard solution (2 ppm Ba) R.  After 10 min, any opalescence in the test solution is not more intense than that in the standard solution.

VP V

Acid-soluble substances Not more than 0.3%. Evaporate 25 ml of solution S to dryness on a water-bath and dry to constant mass at 100 °C to 105 °C. The residue weighs not more than 15 mg. Oxidisable sulfur compounds Shake 1.0 g with 5 ml of water for 30 s and filter. To the filtrate add 0.1 ml of starch solution R, dissolve 0.1 g of potassium iodide R in the mixture, add 1.0 ml of a freshly prepared 0.36% solution of potassium iodate R and 1 ml of 1 M hydrochloric acid R and shake well. The colour of the solution is more intense than that of a standard prepared at the same time and in the same manner omitting the potassium iodate. Heavy metals Not more than 10 ppm (Appendix 9.4.8). Dilute 10.0 ml of solution S to 20 ml with water. 12 ml of this solution complies with limit test for Heavy metals, method 1. Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on ignition Not more than 2.0%. (1.0 g; 600 °C ± 50 °C; ignition to constant mass). Storage In an airtight container. Action and use Radio-opaque substance used in the radiodiagnosis. Preparation Barium sulfate for oral suspension. BARIUM SULFATE FOR ORAL SUSPENSION Barii sulfas pro suspensio Barium sulfate for oral suspension is a dry mixture of barium sulfate containing the dispersive agent. It may contain suitable flavour, preservative and antimicrobial agent. The powder complies with the requirements stated under “Powder” (Appendix 1.7) and with the following requirements.

Content of barium sulfate, BaSO4, 90.0% to 110.0% of the stated amount. Characters White or almost white fine powder. Identification A. Heat, and ignite 1 g of the substance to be examined to constant weight. Boil 0.2 g of the residue with 5 ml of a 50% solution of sodium carbonate R for 5 min, add 10 ml

BARIUM SULFATE FOR ORAL SUSPENSION

of water, filter. Reserve the residue for test B. Acidify a portion of the filtrate with dilute hydrochloric acid R and add several drops of a 5% solution of barium chloride R. A white precipitate is formed. B. Wash the residue reserved in test A with three successive small quantities of water. To the residue add 5 ml of dilute hydrochloric acid R, filter and add to the filtrate 0,3 ml of dilute sulfuric acid R. A white precipitate is formed that is insoluble in dilute sodium hydroxide solution R.

pH 3.5 to 8.5 (Appendix 6.2). Use an aqueous suspension containing the equivalent of 60% w/w of barium sulfate or, for lower strengths, the aqueous suspension at the strength of intended use. Soluble barium salts Weigh accurately a quantity of the powder, equivalent to 7.5 g of barium sulfate; add 10 ml of 2 M hydrochloric acid R and 90 ml of water, mix well. Boil the mixture for 10 min, allow to cool and filter. Wash the residue with water, combine the filtrate and the washing and dilute to 100 ml with water. Evaporate 50 ml of the resulting solution, avoid getting burned. Add 0.1 ml of 2 M hydrochloric acid R and 10 ml of hot water to the residue, filter. To the filtrate add 0.5 ml of 1 M sulfuric acid  R. Allow to stand for 30 min, the solution is clear.

Loss on drying Not more than 1.0% (Appendix 9.6). (1.0 g; 105°C; 4 h). Assay To an accurately weighed quantity of the suspension, equivalent to about 0.6 g of barium sulfate, in a platinum dish add 5 g of anhydrous sodium carbonate R and 5 g of potassium carbonate R, and mix. Heat to 1000 °C, and maintain at this temperature for 15 min. Allow to cool and transfer the residue into the 200 ml beaker with 150 ml of water. Wash the dish with 2 ml of 6 M acetic acid R and add the washings to the suspension. Cool in ice and decant the supernatant liquid, transferring the residue to the filter. Wash the residue with successive quantities of a 2% solution of sodium carbonate R until the washings are free from sulfate and discard the washings. Add 5 ml of 2 M hydrochloric acid R to the filter, wash through into the vessel containing the bulk of the solid matter with water, add 5 ml of hydrochloric acid R and dilute to 100.0 ml with water. Add 10 ml of a 40% solution of ammonium acetate R, 25 ml of a 10% solution of potassium dichromate R and 10 g of urea R. Cover and digest in a hot-air oven at 80 °C to 85 °C for 16 h. Filter whilst still hot through a sintered-glass filter (BS porosity No. 4), washing the precipitate initially with a 0.5% solution of potassium dichromate R and finally with 2 ml of water. Dry to constant weight at 105 °C. 117

VP V

BENZALKONIUM CHLORIDE

1 g of the residue is equivalent to 0.9213 g of barium sulfate, BaSO4.

Storage Store in a cool place, protected from light. Action and use Radio-opaque preparation used in the investigation of the gastro-intestinal tract. BENZALKONIUM CHLORIDE Benzalkonii chloridum Benzalkonium chloride is a mixture of alkylbenzyldimethylammonium chlorides, the alkyl groups having chain length of C8 to C18. It contains not less than 95.0% and not more than the equivalent of 104.0% of alkylbenzyldimethylammonium chlorides, calculated as C22H40ClN (M..354.0) with reference to the anhydrous substance.

Characters A white or yellowish-white powder or gelatinous, yellowish-white fragments, hygroscopic, soapy to the touch, very soluble in water and in alcohol. On heating it forms a clear molten mass. An aqueous solution froths copiously when shaken. Identification A. Dissolve 80 mg in water and dilute to 100 ml with the same solvent. The ultravidet absorption spectrum (Appendix 4.1) of the solution in the range 220 nm to 350 nm exhibits three absorption maxima at 257 nm, 263 nm and 269 nm, and a shoulder at about 250 nm. B. To 2 ml of solution S (see Appearance of solution) add 0.1 ml of glacial acetic acid R and dropwise 1 ml of a 1% solution of sodium tetraphenylborate R. A white precipitate is formed. Filter. Dissolve the precipitate in a mixture of 1 ml of acetone R and 5 ml of ethanol (96%) R by heating to not more than 70 °C. Add water dropwise to the warm solution until a slight opalescence forms. Heat gently until the solution is clear and allow to cool. White crystals is formed. Filter, wash with three quantities, each of 10 ml of water and dry in vacuum over diphosphorus pentoxide R or anhydrous silica gel R at a temperature not exceeding 50 °C. The crystals melt at 127 °C to 133 °C (Appendix 6.7). C. To 5 ml of dilute sodium hydroxide solution R add 0.1 ml of bromophenol blue solution R and 5 ml of chloroform R and shake. The chloroform layer is colourless. Add 0.1 ml of solution S and shake. The chloroform layer becomes blue. D. To 2 ml of solution S (see Appearance of solution) add 1 ml of a 12,5% solution of nitric acid R. A white precipitate is formed which dissolves on the addition of 5 ml of ethanol (96%) R. The solution gives reaction (A) of chlorides (Appendix 8.1). 118

Appearance of solution Solution S: Dissolve 1.0 g in carbon dioxide-free water R and dilute to 100 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution Y6 (Appendix 9.3, method 2). Acidity or alkalinity To 50 ml of solution S add 0.1 ml of bromocresol purple solution R. Not more than 0.1 ml of 0.1 N hydrochloric acid VS or 0.1 N sodium hydroxide VS is required to change the colour of the indicator. Amines and amine salts Dissolve 5.0 g of substance to be examined by heating in 20 ml of a mixture of 3 volumes of 1 N hydrochloric acid and 97 volumes of methanol R. Add 100 ml of 2-propanol R. Pass a stream of nitrogen R slowly through the solution. Gradually add 12.0 ml of 0.1 M tetrabutylammonium hydroxide VS and record the potentiometric titration curve (Appendix 10.2). If the curve shows two points of inflexion, the volume of titrant added between the two points is not greater than 5.0 ml. If the curve shows no point of inflexion, the substance to be examined does not comply with the test. If the curve shows one point of inflexion, repeat the test but add 3.0 ml of a 2.5% solution of dimethyldecylamine R in 2-propanol R before the titration. If the titration curve after addition of 12.0 ml of the titrant shows only one point of inflexion, the substance to be examined does not comply with the test. Water Not more than 10% (Appendix 10.3). Determined on 0.300 g. Sulfated ash Not more than 0.1% (Appendix 9.9). Determined on 1.0 g. Assay Dissolve 2.00 g in water and dilute to 100.0 ml with the same solvent. Transfer 25.0 ml of the solution to a separating funnel, add 25 ml of chloroform R, 10 ml of 0.1 N sodium hydroxide and 10.0 ml of a freshly prepared a 5.0% solution of potassium iodide R. Shake well, allow to separate and discard the chloroform layer. Shake the aqueous layer with three quantities, each of 10 ml, of chloroform R and discard the chloroform layers. To the aqueous layer add 40 ml of hydrochloric acid R, allow to cool and titrate with 0.05 M potassium iodate VS until the deep-brown colour is almost discharged. Add 2 ml of chloroform R and continue the titration, shaking vigorously, until the chloroform layer no longer changes colour. Carry out a blank titration on a mixture of 10.0 ml of the freshly prepared a 5.0% solution of potassium iodide R, 20 ml of water and 40ml of hydrochloric acid R.

VP V

BENZATHINE BENZYLPENICILLIN

1ml of 0.05 M potassium iodate VS is equivalent to 35.4 mg of C22H40ClN.

Action and use Antiseptic detergent. BENZATHINE BENZYLPENICILLIN Benzathinum benzylpenicillinum

(C16H18N2O4S)2C16H20N2

M. 909.0

Benzathine benzylpenicillin is N,N’-dibenzylethane-1,2diamine compound with (2S,5R,6R)-3,3-dimethyl-7-oxo -6-[(phenylacetyl)amino]-4-thia-1-azabicyclo[3.2.0] heptane-2-carboxylic acid (1 : 2). It contains not less than 96.0% and not more than 102.0% of benzathine benzylpenicillin, (C16H18N2O4S)2C16H20N2, and not less than 24.0% and not more than 27.0% of N,N’dibenzylethylenediamine (benzathine C16H20N2; M. 240.3), both calculated with reference to the anhydrous substance. It contains a variable quantity of water. Dispersing or suspending agents may be added.

Characters A white powder, very slightly soluble in water, freely soluble in dimethylformamide and in formamide, slightly soluble in ethanol (96%). Identification Apply one of the two following identifications: First identification: A. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) is concordant with the spectrum obtained with benzathine benzylpenicillin RS. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silanised Silica gel. Mobile phase: Acetone - 15.4% solution of ammonium acetate adjusted to pH 7.0 with ammonia R (30 : 70). Test solution: Dissolve 25 mg of the substance to be examined in 5 ml of methanol R. Reference solution: Dissolve 25 mg of benzathine benzylpenicillin RS in 5 ml of methanol R. Procedure: Apply to the plate 1 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in air and expose it to iodine vapour until the spots appear. Examine in daylight. Two principal spots in the chromatogram obtained with the test solution are similar in position, colour and size with two principal spots in the chromatogram obtained with the reference solution. The test is not valid unless the chromatogram obtained with the reference solution shows two clearly separated spots.

C. Place about 2 mg in a test-tube about 150 mm long and 15 mm in diameter. Moisten with 0.05 ml of water and add 2 ml of formaldehyde solution in sulfuric acid R. Mix the contents of the tube by swirling; the solution is practically colourless. Place the test-tube on a water-bath for 1 min; a reddish-brown colour develops. D. To 0.1 g add 2 ml of 1 M sodium hydroxide R and shake for 2 min. Shake the mixture with two quantities, each of 3 ml, of ether R. Collect the ether layer. Evaporate the combined ether layers to dryness and dissolve the residue in 1 ml of ethanol (50%) R. Add 5 ml of picric acid solution R, heat at 90 °C for 5 min and allow to cool slowly. Filter and collect the obtained crystals, recrystallize from ethanol (25%) R containing 1% of picric acid R. The crystals melt at about 214 °C (Appendix 6.7).

Acidity or alkalinity To 0.50 g add 100 ml of carbon dioxide-free water R and shake for 5 min. Filter through a sintered-glass filter. To 20 ml of the filtrate add 0.1 ml of bromothymol blue solution R. The solution is green or yellow. Not more than 0.2 ml of 0.02 N sodium hydroxide VS is required to change the colour of the indicator to blue. Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use, using sonication (for about 2 min) to dissolve the samples. Avoid any over heating during the sample preparation. Mobile phase A: Mixture of a 3.4% solution of potassium dihydrogen phosphate R adjusted to pH 3.5 with phosphoric acid - methanol - water (10 : 30 : 60). Mobile phase B: Mixture of a 3.4% solution of potassium dihydrogen phosphate R adjusted to pH 3.5 with phosphoric acid - water - methanol (10 : 30 : 60). Test solution: Dissolve 70.0 mg of the substance to be examined in 25 ml of methanol R and dilute to 50.0 ml with a solution containing 0.68% of potassium dihydrogen phosphate R and 0.102% of disodium hydrogen phosphate R. Reference solution (1): Dissolve 70.0 mg of benzathine benzylpenicillin RS in 25 ml of methanol R and dilute to 50.0 ml with a solution containing 0.68% of potassium dihydrogen phosphate R and 0.102% of disodium hydrogen phosphate R. Reference solution (2): Dilute 1.0 ml of reference solution (1) to 100.0 ml with mobile phase A. Chromatographic system: Column: A column (25 cm × 4.0 mm) packed with end-capped octadecylsilyl silica gel for chromatography (5 µm). Column temperature: 40 °C. Detector: A spectrophotometer set at 220 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions: 119

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BENZATHINE BENZYLPENICILLIN FOR INJECTION

Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 10

75

25

10 - 20

75 → 0

25 → 100

20 - 55

0

100

55 - 70

75

25

Inject the test solution and the reference solutions. System suitability: In the chromatogram obtained with reference solution (1), the relative retention with reference to benzylpenicillin: benzathine = 0.3 to 0.4; impurity C (benzathid benzylpeniciloic acid)= about 2.4. If necessary, adjust the concentration of methanol in the mobile phase. Limits: In the chromatogram obtained with the test solution, the area of the peak due to impurity C is not more than twice the sum of the areas of the 2 principal peaks in the chromatogram obtained with reference solution (2) (2.0%). The area of any other secondary peak is not more than the sum of the areas of the 2 principal peaks in the chromatogram obtained with reference solution (2) (1.0%). Disregard any peak with an area less than 0.05 times the sum of the areas of the 2 principal peaks in the chromatogram obtained with reference solution (2) (0.05%).

Water 5.0% to 8.0% (Appendix 10.3). Determined on 0.300 g. Sterility If intended for use in the manufacture of parenteral dosage forms without a further appropriate sterilisation procedure, it complies with the test for sterility (Appendix 13.7). Bacterial endotoxins Suspend 20 mg of the substance to be examined in 20 ml of 0.1 N sodium hydroxide R diluted 1 to 100, shake well and centrifuge. Use the supernatant for the test (Appendix 13.2, method E). Bacterial endotoxins are less than 0.13 EU/ml. If intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins, it complies with this test. Assay Examine by liquid chromatography (Appendix 5.3). The test solution, reference solution (1) and column are as described in the test for Related substances. Mobile phase: Phosphate buffer solution pH 3.5 - methanol - water (10 : 35 : 55). Detector: A spectrophotometer set at 220 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Inject the test solution and reference solution (1). 120

Calculate the content of benzathine and of benzathine benzylpenicillin using the areas of the principal peaks in the chromatograms obtained. Calculate the content of benzathine benzylpenicillin by multiplying the percentage content of benzylpenicillin by 1.36.

Storage Store in an airtight container. If the substance is sterile, store in a sterile, airtight, tamper-proof container. Labelling The label states: Expiry date and storage. Where applicable, the name and quantity of any added dispersing or suspending agent, Where applicable, that the substance is free from bacterial endotoxins. Action and use Penicillin atibacterial. Preparation Injection. BENZATHINE BENZYLPENICILLIN FOR INJECTION Benzathinini benzylpenicillini ad injectionem Benzathine benzylpenicillin for injection is a sterile powder of benzathine benzylpenicillin in a sealed glass container. It may contain a suitable amount of buffers, suspension agents and other excipients. The powder complies with the requirements stated under “Injections, intravenous infusion” (Appendix 1.19) and with the following requirements.

Content of benzathin benzylpenicillin, (C16H18N2O4S)2, C16H20N2, 95.0% to 105.0% of the stated amount. Characters White, crystalline powder, almost odorless. Identification A. Weigh about 0.1 g of the substance to be examined, add 1 ml of 1 M sodium hydroxide R, shake well for about 2 min. Add 2 ml of ether R, shake for 1 min and allow it to separate. Evaporate 1 ml of the ether extract to dryness and dissolve the residue in 2 ml of glacial acetic acid R. Add 1 ml of 5% potassium dichromate solution R. A yellow precipitate is formed. B. Weigh about 0.1 g of the substance to be examined, add 2 ml of 1 M sodium hydroxide R, shake well for about 2 min. Extract with two portions of 3 ml of ether R. Combine the extracts, evaporate the extract to dryness. Dissolve the residue in 1 ml of ethanol (50%) R. Add 5 ml of saturated solution of picric acid R, heat at 90 °C for 5 min. Allow it to

VP V

cool, crystals are formed. Recrystallize from ethanol (25%) R containing a small amount of picric acid R. The melting point of the obtained crystals is about 214 °C (Appendix 6.7). C. In the Assay, the retention time of the principal peaks in the chromatogram obtained with the test solution are similar with those of the principal peaks in the chromatogram obtained with the reference solution.

pH 5.0 to 7.5 (Appendix 6.2). Use the reconstituted suspension prepared following manufacturer’s instruction. Water 5.0% to 8.0% (Appendix 10.3). Use 0.3 g of the substance to be examined. Bacterial endotoxins (Appendix 13.2) Not more than 0.13 EU per 1 ml of the suspension prepared as follow: Shake 20 mg of the substance to be examined in 20 ml of 0.1 M sodium hydroxide R diluted 1 to 100. Shake vigorously and centrifuge, use the supernatant liquid. Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the test solution and the reference solution immediately before use. Avoid overheating during the sample preparation. Phosphate buffer pH 3.5: Dissolve 34.0 g of potassium dihydrogen phosphate R in water and dilute to 1000 ml with water. Adjust to pH 3.5 with phosphoric acid R. Diluent: Dissolve 6.8 g of potassium dihydrogen phosphate R and 1.02 g of disodium hydrogen phosphate R in 1000 ml of water. Mobile phase A: Phosphate buffer pH 3.5 - methanol water (10 : 30 : 60). Mobile phase B: Phosphate buffer pH 3.5 - methanol water (10 : 60 : 30). Test solution: Weigh accurately a quantity of the substance to be examined equivalent to about 70 mg of benzathine benzylpenicillin, put into a 50-ml volumetric flask, add 25 ml of methanol R and sonicate for about 2 min to dissolve. Dilute to volume with the diluent, mix. Reference solution (1): Weigh accurately about 70 mg of benzathine benzylpenicillin RS, transfer into a 50-ml volumetric flask, add 25 ml of methanol R and sonicate for about 2 min to dissolve. Dilute to volume with the diluent, mix. Reference solution (2): Dilute 1.0 ml of reference solution (1) to 100 ml with the mobile phase A, mix. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 µm). Column temperature: 40 °C. Detector: A spectrophotometer set at 220 nm.

BENZATHINE BENZYLPENICILLIN FOR INJECTION

Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 10

75

25

10 - 20

75 → 0

25 → 100

20 - 55

0

100

55 - 70

75

25

Inject reference solution (1). In the chromatogram obtained, the relative retention times with reference to benzylpenicillin: Benzanthine = about 0.3-0.4, benzylpeniciloic benzathid acid = about 2.4. Adjust the ratio of methanol in the mobile phase if necessary. Inject the test solution and reference solution (2). Limits: The area of any secondary peak corresponding to benzylpeniciloic benzathid acid is not greater than twice the sum of the areas of two principal peaks obtained with reference solution (2) (2.0%). The area of any other secondary peak is not greater than the sum of the areas of two principal peaks obtained with reference solution (2) (1.0%). Disregard any peak having the area less than 0.05 times the sum of the areas of two principal peaks obtained with reference solution (2) (0.05%).

Assay Examine by liquid chromatography (Appendix 5.3). The chromatographic system, phosphate buffer pH 3.5, test solution, reference solution (1) as described in the Related substances. Mobile phase: Phosphate buffer pH 3.5 - methanol - water (10 : 35 : 55). Procedure: Inject reference solution (1). The relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject alternately the test solution and reference solution (1). Calculate the content of benzylpenicillin in a container using the areas of principal peaks in the chromatograms obtained with the test solution, reference solution (1) and the declared content of benzylpenicillin in benzathine benzylpenicillin RS. The content of benzathine benzylpenicillin is calculated by multiplying the content of benzylpenicillin with 1.36. 1 mg of benzathine benzylpenicillin corresponds to 1330 units of penicillin. Storage Store at a dry and cold place, protected from light. 121

VP V

BENZOIC ACID

Action and use Penicillin antibacterial. Usual strength 300 000 IU; 600 000 IU; 1 200 000 IU. BENZOIC ACID Acidum benzoicum CO2H

C7H6O2

M. 122.1  

Benzoic acid is benzenecarboxylic acid. It contains not less than 99.0% and not more than 100.5% of C7H6O2.

Characters Colourless needle crystals or colourless crystals or a white crystalline powder, odourless or with a very slight characteristic odour. Slightly soluble in water, soluble in boiling water, freely soluble in ethanol (96%), in ether and in fatty oils. Identification Solution S: Dissolve 5.0 g in ethanol (96%) R and dilute to 100 ml with the same solvent. A. Melting point: 121 °C to 124 °C (Appendix 6.7). B. Solution S gives reaction (A) of benzoates (Appendix 8.1). Appearance of solution Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). Carbonisable substances Dissolve 0.5 g with shaking in 5 ml of sulfuric acid R. After 5 min, the solution is not more intensely coloured than reference solution Y5 (Appendix 9.3, method 1). Oxidisable substances Dissolve 0.2 g in 10 ml of boiling water. Cool, shake and filter. To the filtrate add 1 ml of dilute sulfuric acid R and 0.2 ml of 0.1 N potassium permanganate VS. After 5 min, the solution is still coloured pink. Chlorinated compounds All glassware used must be chloride-free and may be prepared by soaking overnight in a 35% solution of nitric acid R, rinsed with water and stored full of water. It is recommended that glassware be reserved for this test. Solution (1): Dissolve 6.7 g of the substance to be examined in a mixture of 40 ml of 1 M sodium hydroxide R and 50 ml of ethanol (96%) R and dilute to 100 ml with water. To 10.0 ml of this solution add 7.5 ml of dilute sodium hydroxide solution R and 0.125 g of nickel-aluminium alloy R and heat on a water-bath for 10 min. Allow to cool to 122

room temperature, filter into a 25 ml volumetric flask and wash with three quantities, each of 2 ml, of ethanol (96%) R. Dilute the filtrate and washings to 25 ml with water. Solution (2): In the same manner, prepare a similar solution (1) without the substance to be examined. In four 25 ml volumetric flasks, place separately 10 ml of solution (1), 10 ml of solution (2), 10 ml of chloride standard solution (8 ppm Cl) R [solution 3] and 10 ml of water [solution 4]. To each flask add 5 ml of ferric ammonium sulfate solution R, mix and add dropwise and with swirling 2 ml of nitric acid R and 5 ml of mercuric thiocyanate solution R. Shake. Dilute the contents of each flask to 25.0 ml with water and allow the solutions to stand in a water-bath at 20 °C for 15 min. Measure at 460 nm (Appendix 4.1) the absorbance of solution (1) using solution (2) as the compensation liquid, and the absorbance of solution (3) using the solution (4) as the compensation liquid. The absorbance of solution (1) is not greater than that of solution (3) (300 ppm).

Heavy metals Not more than 10 ppm (Appendix 9.4.8). 12 ml of solution S complies with limit test (2) for heavy metals. Prepare the standard using a mixture of 5 ml of lead standard solution (1 ppm Pb) R and 5 ml of ethanol (96%) R and 2 ml of solution S. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Weight accurately about 0.200 g the substance to be examined, dissolve in 20 ml of ethanol (96%) R and titrate with 0.1 N sodium hydroxide VS, using 0.1 ml of phenol red solution R as indicator, until the colour changes from yellow to violet-red. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 12.21 mg of C7H6O2. Storage Store in a well-closed container. Action and use Antifungal, antimicrobial preservative. BENZOSALI OINTMENT Unguentum Benzosalici Benzosali ointment for skin contains benzoic acid and salicylic acid in a suitable emulsifying basis.

Ingredient Benzoic acid (finely powder) Salicylic acid (finely powder) Emulsible excipient sufficient to produce

60 g 30 g 1000 g

VP V

The ointment complies with the requirements stated under “Topical semi-solid preparation” (Appendix 11.2) and with the following requirements.

Content of benzoic acid, C7H6O2, 5.7% to 6.3% (m/m). Content of salicylic acid, C7H6O3, 2.7% to 3.3% (m/m). Characters Opalescent ointment. Identification Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Toluene - glacial acetic acid (8 : 2). Test solution: To 1.0 g of the ointment add 10 ml of chloroform R, heat gently, cool and filter. Reference solution: Acid a 0.6% solution of benzoic acid and 0.3% of salicylic in chloroform R. Procedure: Apply separately to the place 2 µl of each of the above solutions. After removal of the plate, allow the plate to dry in air. Examine under ultraviolet light at 254 nm. The colour and Rf value of the principal spots in the chromatogram obtained with the test solution correspond to those of the principal spots in the chromatogram obtained with the reference solution. The test is not valid unless the chromatogram obtained with the reference solution shows two clearly separated spots. Examine under ultraviolet light at 365 nm. In the chromatogram obtained with the test solution, one blue fluorescent spot is observed, having colour and Rf value similar to that of the corresponding spot in the chromatogram obtained with the reference solution. Spray with a 5.0% solution of ferric chloride R. One purple spot in the chromatogram obtained with the test solution (at position of the above blue fluorescent spot under ultraviolet light at 365 nm) corresponds in colour and Rf value to that in the chromatogram obtained with the reference solution. Assay For benzoic acid: To 2 g of the preparation in a 300 ml conical flask, add 150 ml of water, heat gently until melted and titrate with 0.1 N sodium hydroxide VS using phenolphthalein solution R as indicator. Reserve the solution for Assay for salicylic acid. After the subtraction of 1 ml for each 13.81 mg of C7H6O3 found in Assay for salicylic acid, each ml of 0.1 N sodium hydroxide VS is equivalent to 12.21 mg of C7H6O2. For salicylic acid: Cool the titrated solution obtained in Assay for benzoic acid. Filter into a 250 ml volumetric flask, through a filter paper previously moistened with water. Then add 20 ml of water to the above conical flask, stir well and filter into the above volumetric flask. Continue

BENZYLPENICILLIN POTASSIUM

the washing and filtration two more times. Finally add sufficient water to volume. Mix and filter. Transfer 5 ml, accurately measured, of the filtrate into a 50 ml volumetric flask, add a 0.1% solution of ferric chloride in 0.1% (v/v) solution of nitric acid R to volume. Filter, if necessary, to remove haze and measure the absorbance of the resulting solution (test solution) at the maximum at 530 nm (Appendix 4.1), using a 0.1% solution of ferric chloride in 0.1% (v/v) solution of nitric acid R in the reference cell. In the same manner, measure the absorbance of the reference solution prepared as follows: To 5 ml of a 0.024% solution of salicylic acid in a 50 ml volumetric flask, add a 0.1% solution of ferric chloride in 0.1% (v/v) solution of nitric acid R to volume, shake well. Filter, if necessary, to remove haze. Calculate the content of salicylic acid, C7H6O3 in the ointment using the measured absorbances of the test solution, the reference solution and the declared content of C7H6O3 in the reference solution.

Storage Store in a well-closed container, at a cool place. BENZYLPENICILLIN POTASSIUM Benzylpenicillinum kalicum

C16H17KN2O4S  

M. 372.5

Benzylpenicillin potassium is potassium (2S,5R,6R)3,3-dimethyl-7-oxo-6-[(phenylacetyl)amino]-4-thia-1azabicyclo[3.2.0]heptane-2-carboxylate, produced by the growth of certain strains of Penicillium notatum or related organisms, or obtained by any other means. It contains not less than 96.0% and not more than 102.0% of C16H17KN2O4S, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder. Very soluble in water, practically insoluble in fatty oils and in liquid paraffin. Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of benzylpenicillin potassium RS. 123

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BENZYLPENICILLIN POTASSIUM

B. It complies with the test for Identification of penicillins (Appendix 8.2). C. It complies with the test B for Colour reactions of penicillins and cephalosporins (Appendix 8.3). D. It gives reaction (A) of potassium (Appendix 8.1).

pH 5.5 to 7.5 (Appendix 6.2). Dissolve 2.0 g in carbon dioxide-free water and dilute to 20 ml with the same solvent. Specific optical rotation +270° to +300°, calculated with reference to the dried substance (Appendix 6.4). Dissolve 0.500 g in carbon dioxide-free water R and dilute to 25.0 ml with the same solvent. Absorbance Dissolve 94.0 mg in water and dilute to 50.0 ml with the same solvent. Measure the absorbance (Appendix 4.1) of the solution at 325 nm, 280 nm and at the absorption maximum at 264 nm, diluting the solution, if necessary, for the measurement at 264 nm. The absorbances at 325  nm and 280  nm do not exceed 0.10 and that at the absorption maximum at 264 nm is 0.80 to 0.88, calculated on the basis of the undiluted (1.88 g/l) solution. Verify the resolution of the apparatus (Appendix 4.1); the ratio of the absorbances is at least 1.7. Related substances Examine by liquid chromatography (Appendix 5.3).  Prepare the solutions immediately before use. Buffer phophate solution pH 3.5: A 6.8% solution of potassium dihydrogen phosphate R adjusted to pH 3.5 with a 500 g/l solution of dilute phosphoric acid R. Mobile phase A: Buffer phophate solution pH 3.5 methanol - water (10 : 30 : 60). Mobile phase B: Buffer phophate solution pH 3.5 - water methanol (10 : 40 : 50). Test solution (1): Dissolve 50.0  mg of the substance to be examined in water and dilute to 50.0 ml with the same solvent. Test solution (2): Dissolve 80.0  mg of the substance to be examined in water and dilute to 20.0 ml with the same solvent. Reference solution (1): Dissolve 50.0  mg of  benzylpenicillin sodium RS in water and dilute to 50.0 ml with the same solvent. Reference solution (2): Dissolve 10.0  mg of  benzylpenicillin sodium RS and 10 mg of phenylacetic acid  R (impurity B) in water, then dilute to 50.0 ml with the same solvent. Reference solution (3): Dilute 4.0 ml of reference solution (1) to 100.0 ml with water. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 225 nm. 124

Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time

Mobile phase A

Mobile phase B

(min)

(% v/v)

(% v/v)

0 - tR

70

30

tR - (tR + 20)

70 → 0

30 → 100

(tR + 20) - (tR + 35)

0

100

(tR + 35) -(tR + 50)

70

30

tR = retention time of benzylpenicillin in the chromatogram obtained with reference solution (3)

Inject reference solutions (2) and (3) with isocratic elution at the initial mobile phase composition. Inject test solution (2) according to the elution gradient described above gradient. Inject  water as a blank according to the elution gradient described above gradient. If the mobile phase composition has been adjusted to achieve the required resolution, the adjusted composition will apply at time zero in the gradient and in the Assay. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurity B and benzylpenicillin is at least 6.0. If necessary, adjust the ratio A:B of the mobile phase. Limit: Any impurity: for each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (3) (1%). Note: Impurity A: (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia1-azabicyclo[3.2.0]heptane-2- carboxylic acid (6-aminopenicillanic acid). Impurity B: Phenylacetic acid. Impurity C: (2S,5R,6R)-6-[[(4-hydroxyphenyl)acetyl]amino]-3,3dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid. Impurity D: (3S,7R,7aR)-5-benzyl-2,2-dimethyl-2,3,7,7a-tetrahydro imidazo[5,1-b]thiazole-3,7-dicarboxylic acid (penillic acid of benzylpenicillin). Impurity E: (4S)-2-[carboxy[(phenylacetyl)amino]methyl]-5,5dime thylthiazolidine-4-carboxylic acid (penicilloic acids of benzylpenicillin). Impurity F: (2RS,4S)-2-[[(phenylacetyl)amino]methyl]-5,5dime thylthiazolidine-4-carboxylic acid (penilloic acids of benzylpenicillin).

Loss on drying Not more than 1.0% (Appendix 9.6). (1.000 g; 105 °C). Bacterial endotoxins Less than 0.16 EU/mg (Appendix 13.2, the end-point chromegenic test).

VP V

BENZYLPENICILLIN SODIUM

If intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins.

Assay Examine by liquid chromatography  (Appendix 5.3). Chromatographic system as described in the test for Related substances. Mobile phase: Initial composition of the mixture of mobile phases A and B, adjusted where applicable. Procedure: Inject test solution (1) and reference solution (1). Calculate the content of benzylpenicillin potassium, C16H17KN2O4S, using the areas of the principal peak in the chromatogram obtained with test solution (1), reference solution (1) and the declared content of benzylpenicillin sodium RS. Calculate the percentage content of C16H17KN2O4S by multiplying the content of benzylpenicillin sodium by 1.045. Storage Store in an airtight container. If the substance is sterile, store in a sterile, airtight container. Action and use Penicillin antibacterial. Preparation Injection. BENZYLPENICILLIN SODIUM Benzylpenicillinum natricum

C16H17N2NaO4S

M. 356.4

Benzylpenicillin sodium is sodium (2S,5R,6R)-3,3-dimethyl7-oxo-6-[(phenylacetyl)amino]-4-thia-1-azabicyclo[3.2.0] heptane-2-carboxylate, a substance produced by the growth of certain strains of Penicillium notatum or related organisms, or obtained by any other means. It contains not less than 96.0% and not more than 102.0% of C16H17N2NaO4S, calculated with reference to the dried substance.

Characters A white or almost white, crystalline powder, very soluble in water, practically insoluble in fatty oils and in liquid paraffin.

Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D. A.The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of benzylpenicillin sodium RS. B. Examine by thin-layer chromatography described under Identification of penicillins (Appendix 8.2). C. It gives reaction B described under “Colour reactions of penicillins and cephalosphorins” (Appendix 8.3). D. It gives the reaction of sodium (Appendix 8.1). pH 5.5 to 7.5 (Appendix 6.2). Dissolve 2.0 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent. Specific optical rotation +285° to +310°, calculated with reference to the dried substance (Appendix 6.4). Dissolve 0.500 g in carbon dioxide-free water R and dilute to 25.0 ml with the same solvent. Absorbance Dissolve 90.0 mg in water and dilute to 50.0 ml with the same solvent. Measure the absorbance of the solution (Appendix 4.1) at 325 nm, 280 nm and at the maximum at 264 nm, diluting the solution, if necessary, for the measurement at 264 nm. The absorbances at 325 nm and 280 nm do not exceed 0.10 and the absorbance at the absorption maximum at 264 nm is 0.80 to 0.88, calculated on the basis of the undiluted (1.80 g/l) solution. Verify the resolution of the apparatus (Appendix 4.1); the ratio of the absorbances is at least 1.7. Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use. Mobile phase A: Buffer solution pH 3.5 - methanol - water (10 : 30 : 60). Mobile phase B: Buffer solution pH 3.5 - water - methanol (10 : 40 : 50). Buffer solution pH 3.5: A 6.8% solution of  potassium dihydrogen phosphate R adjusted to pH 3.5 with a solution containing 500 g/l of dilute phosphoric acid R. Test solution (1): Dissolve 50.0 mg of the substance to be examined in water and dilute to 50.0 ml with the same solvent. Test solution (2): Dissolve 80.0 mg of the substance to be examined in water and dilute to 20.0 ml with the same solvent. Reference solution (1): Dissolve 50.0 mg of benzylpenicillin sodium RS in water and dilute to 50.0 ml with the same solvent. Reference solution (2): Dissolve 10.0 mg of benzylpenicillin sodium RS and 10.0 mg of phenylacetic acid R (impurity 125

VP V

BENZYLPENICILLIN FOR INJECTION

B) in water, then dilute to 50.0 ml with the same solvent. Reference solution (3): Dilute 4.0 ml of reference solution (1) to 100.0 ml with water.  Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 225 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - tR

70

30

tR - (tR + 20)

70 → 0

30 → 100

(tR + 20) - (tR + 35)

0

(tR + 35) - (tR + 50)

70

100 30

tR = Retention time of benzylpenicillin determined with chromatogram of reference solution (3).

Inject reference solutions (2) and (3) and elute isocratically with the mobile phase composition at the time zero in the gradient. Inject test solution (2) according to the elution gradient described above gradient; inject water as a blank according to the elution gradient described above gradient. If the mobile phase composition has been adjusted to achieve the required resolution, the adjusted composition will apply at time zero in the gradient and in the Assay. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurity B and benzylpenicillin is at least 6.0; if necessary, adjust the ratio A : B of the mobile phase. Limit: Any impurity: For each impurity, the peak area is not more than the area of the principal peak in the chromatogram obtained with reference solution (3) (1%).

Note: Impurity A: (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia-1azabicyclo[3.2.0]heptane-2-carboxylic acid (6-aminopenicillanic acid). Impuriry B: Phenylacetic acid. Impurity C: (2S,5R,6R)-6-[[(4-hydroxyphenyl)acetyl]amino]-3,3dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid. Impurity D: (3S,7R,7aR)-5-benzyl-2,2-dimethyl-2,3,7,7a-tetrahydroimidazo [5,1-b]thiazole-3,7-dicarboxylic acid (penillic acid of benzylpenicillin). Impurity E: (4S)-2-[carboxy[(phenylacetyl)amino]methyl]-5,5dimethylthiazolidine-4-carboxylic acid (penicilloic acids of benzylpenicillin). Impurity F: (2RS,4S)-2-[[(phenylacetyl)amino]methyl]-5,5dimethylthiazolidine-4-carboxylic acid (penilloic acids of benzylpenicillin).

126

2-Ethylhexanoic acid Not more than 0.5% (w/w) (Appendix 10.17). Loss on drying  Not more than 1.0% (Appendix 9.6). (1.000 g; 105 °C). Bacterial endotoxins Less than 0.16 EU/mg (Appendix 13.2, the end-pointchromogenic test). If intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins. Assay Examine by liquid chromatography (Appendix 5.3). Chromatographic system as described in the test for Related substances. Mobile phase: Isocratic elute with composition of the mixture of mobile phases A and B at zerotime in the gradient, adjusted where applicable. Inject test solution (1) and reference solution (1). Calculate the content of C16H17N2NaO4S in the substance to be examined, using the areas of the principal peaks in the chromatograms obtained with test solution (1), reference solution (1) and the declared content of C16H17N2NaO4S in benzylpenicillin sodium RS. Storage In an airtight container. If the substance is sterile, store in a sterile, airtight, tamper-proof container. Action and use Antibacterial. Preparation Injection. BENZYLPENICILLIN FOR INJECTION Benzylpenicillini pro injectione Benzylpenicillin for injection is a sterile crystalline powder of benzylpenicillin potassium or benzylpenicillin sodium. It is supplied in a sterile, airtight glass ampoule or vial. It is prepared before use by dissolving benzylpenicillin for injection in the requisite amount of water for injections. The preparation complies with the general requirements prescribed under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Content of benzylpenicillin potassium, C16H17N2KO4S or benzylpenicillin sodium, C16H17N2NaO4S, 95.0% to 105.0% of the stated amount. Characters A white, crystalline powder with a slightly bitter taste and a special odour.

VP V

Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of benzylpenicillin potassium RS or benzylpenicillin sodium RS. B. It yields reaction characteristic of potassium salts or sodium salts (Appendix 8.1), and appropriate reaction of benzylpenicillin (Appendix 8.2). Acidity or alkalinity The pH of a solution containing the equivalent of 10% of benzylpenicillin in carbon dioxide-free water R is 5.5 to 7.5 (Appendix 6.2). Appearance of solution Dissolve the contents of 5 containers of the preparation to be examined in water to produce a solution containing 60 mg/ml (calculated on the basis of the stated amount). The solution is clear (Appendix 9.2) and colourless (Appendix 9.3). Loss on drying Not more than 1.0% (Appendix 9.6). Use 1 g of the preparation to be examined, dry to constant mass at 100 °C. Pyrogens The preparation to be examined complies with the test for pyrogens (Appendix 13.4). Use per kg of the rabbit’s weight 1 ml of a solution in water for injections containing the equivalent of 1.5 mg of benzylpenicillin. Sterility The preparation to be examined complies with the test for sterility (Appendix 13.7). Take 120 mg of the preparation to be examined, inactivate by penicillinase and carry out the method of membrane filtration. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase A: A Mixture of 10 volumes of a 6.8% solution of potassium dihydrogen phosphate adjusted to pH 3.5 with a 50% solution of phosphoric acid R, 30 volumes of methanol R and 60 volumes of water. Mobile phase B: A Mixture of 10 volumes of a 6.8% solution of potassium dihydrogen phosphate, adjusted to pH 3.5 with a 50% solution of phosphoric acid R, 50 volumes of methanol R and 40 volumes of water. Mobile phase: Mobile phase A - mobile phase B (70 : 30). Reference solution: A 0.10% solution of benzylpenicillin potassium RS or benzylpenicillin sodium RS in water. Resolution solution: A solution containing 0.020% of benzylpenicillin potassium RS or benzylpenicillin sodium RS and 0.020% of phenylacetic acid RS in water. Test solution: Determine quickly the weights of the contents of 10 containers and mix. Dissolve an accurately weighed quantity containing about 80 mg of benzylpenicillin in

BERBERINE CHLORIDE

20.0 ml of water, filter. Dilute 1 volume of the filtrate to 4 volumes with water. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm) (Hypersil ODS is suitable). Detector: A spectrophotometer set at 225 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Inject resolution solution, the resolution factor between the two principal peaks is not less than 6.0 (if necessary, adjust the ratio of mobile phase A and mobile phase B). Inject separately the test solution and the reference solution. Calculate the content of benzylpenicillin, in one container using the peak areas in the chromatograms obtained with the test solution and the reference solution, and the declared content of benzylpenicillin potassium or benzylpenicillin sodium in the reference standard. The theoretical potency of benzylpenicillin potassium is 1600 Units per mg. The theoretical potency of benzylpenicillin sodium is 1670 Units per mg.

Storage Store in a cool, dry place, at a temperature not exceeding 30 °C. Action and use Antibiotic. Usual strength 0.125 g; 0.312 g; 0.625 g or 200 000 UI; 500 000 UI and 1 000 000 UI. BERBERINE CHLORIDE Berberini chloridum

C20H18NO4Cl,2H2O

M. 407.9

Berberine chloride is 9,10-dimethoxy-5,6-dihydro[1,3] dioxolo[4,5-g]-isoquino[3,2-a]isoquinolin-7-ium chloride hydrate. It contains not less than 95.0% and not more than 102.0% of C20H18NO4Cl, calculated with reference to the anhydrous substance.

Characters Yellow crystals or crystalline powder, odourless, very bitter. Soluble in hot water, slightly soluble in ethanol and water, very slightly soluble in chloroform, insoluble in ether. 127

VP V

BERBERINE CHLORIDE

Identification A.The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of berberine chloride RS. B. Dissolve 0.1 g of the substance to be examined in 20 ml of water by warming. Add 0.5 ml of concentrated nitric acid R, cool, allow standing for 10 min and filter. To 3 ml of the filtrate add 1 ml of a 2% solution of silver nitrate R, white precipitate is produced which does not dissolve in dilute nitric acid R, but dissolves in an excess amount of ammonia R. C. Dissolve 5 mg of the substance to be examined in 10 ml of a 10% solution of hydrochloric acid R. Shake well, add a small quantity of chloramine B powder R, a cherry-red colour is produced. Acidity Shake thoroughly 0.10 g of the substance to be examined with 30 ml of carbon dioxide-free water R, filter. To the filtrate add 2 drops of phenolphthalein solution R and 0.10 ml of 0.1 N sodium hydroxide VS, the yellow colour changes to orange and to red colour. Related substances Examine by liquid chromatography (Appendix 5.3) as described under Assay. Test solution: Dissolve accurately 0.010 g of the substance to be examined in 100.0 ml of the mobile phase. Reference solution: Pipet accurately 4 ml of the test solution, dilute with the mobile phase to 100.0 ml. Procedure: Inject the reference solution, adjust detection sensitivity so that the peak height of berberine in the chromatogram obtained with the reference solution is about 10% of the full scale. Inject the test solution. The run time is 2 times for twice the retention time of berberine. Limit: In the chromatogram obtained with the test solution, the sum of the areas of peaks other than berberine is not greater than the area of the principal peak in the chromatogram obtained with the reference solution. Sulfates Not more than 0.048%. Test solution: Shake 1.0 g of the substance to be examined with 48 ml of water and 2 ml of a 10% solution of hydrochloric acid R for 1 min, filter. Discard the first 5 ml of the filtrate, to the subsequent 25 ml of the filtrate, add water to produce 50 ml in a Nessler cylinder. Reference solution: To 0.50 ml of 0.01 N sulfuric acid R add 1 ml of a 10% solution of hydrochloric acid R, add 5 drops to 10 drops of a 0.1% solution of bromophenol blue R in ethanol (50%) and add water to produce 50 ml in a Nessler cylinder. Add 2 ml of 0.5 M barium chloride R to each cylinder, shake well, allow to stand for 10 min, any opalescence produced in the test solution is not more intense than that in the reference solution. 128

Heavy metals  Not more than 30 ppm (Appendix 9.4.8). 1.0 g complies with the limit test for Heavy metals, method 3. Prepare the standard using 3 ml of lead standard solution (10 ppm Pb) R. Water  8% to 12% (Appendix 10.3). Determined on 0.1 g. Sulphated ash Not more than 0.10% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 3.4 g of potassium dihydrogen phosphate R and 1.7 g of sodium lauryl sulphate R in a mixture of water - acetonitrile (1 : 1) and dilute to 1000 ml with the same solvent. Test solution: Weigh accurately about 0.010 g of the substance to be examined, dissolve in the mobile phase and dilute to 100.0 ml with the same solvent. Reference solution: Weigh accurately about 0.010 g of berberine chloride RS, dissolve in the mobile phase and dilute to 100.0 ml with the same solvent. Resolution solution: Dissolve 1 mg of  berberine chloride RS and 1 mg of palmatin chloride RS in 10 ml of the mobile phase. Chromatographic system: A column (25 cm × 4 mm) packed with stationary phase C (5 µm). Column temperature: 40 °C. Detector: A spectrophotometer set at 345 nm. Flow rate: Adjust the flow rate to obtain the retention time of berberine is about 10 min. Volume of injection: 10 µl. Procedure: System suitability: Inject the resolution solution. Elution order: palmatin, berberine. The resolution between two peaks is not less than 1.5. Inject separately five times the reference solution, the relative standard deviation of the peak areas of berberine is not more than 1.5%. Inject separately the test solution and the reference solution. Calculate the content of berberine chloride, C20H18NO4Cl in the substance to be examined, using the areas of the principal peaks in the chromatograms obtained with the test solution, the reference solution, and the declared content of C20H18NO4Cl in berberine chloride RS. Storage Store in an airtight container, at a cool place, protected from light. Action and use Antibacterial. Preparation Tablets.

VP V

BERBERINE CHLORIDE TABLETS Tabellae Berberini chloridi Berberine chloride tablets contain berberine chloride. They may be film-coated. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements:

Content of berberine chloride, C20H18ClNO4,2H2O, 90.0% to 110.0% of the stated amount. Identification A. Weigh a quantity of the powdered tablets containing about 100 mg of berberine chloride, add 10 ml of water, heat gently, shake well and filter. To 5 ml of the filtrate, add 2 drops of 1 M sodium hydroxide R, an orange-red colour is produced. Cool, add 4 drops of acetone R, a turbidity is produced immediately, allow it to stand, a yellow precipitate is produced. To the supernatant liquid, add acetone R dropwise until the alkaloid is completely precipitated. The filtrate gives reaction A of chlorides (Appendix 8.1). To 0.5 ml of the filtrate, add 2 ml of 10% solution of hydrochloric acid R, shake well, add a small quantity of chloramine B R, a cherry-red colour is produced. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of berberine peak in the chromatogram obtained with the reference solution. Dissolution Apparatus: Basket. Medium: 1000 ml of water. Rotation speed: 120 rpm. Time: 45 min. Procedure: After the specified time, withdraw a sample of the medium and filter. Dilute the filtrate with water if necessary. Measure the absorbance of the resulting solution at the maximum at 263 nm (Appendix 4.1), in a 1-cm cell and using water as the blank. Calculate the content of berberine chloride, C20H18ClNO4, 2H2O, dissolved taking 724 as the value of A (1%, 1 cm) at the maximum at 263 nm. Tolerance: Not less than 70% (Q) the content of berberine chloride, C20H18ClNO4,2H2O, of the stated amount is dissolved in 45 min. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 3.4 g of potassium dihydrogen phosphate R and 1.7 g of sodium laurylsulfate R in a mixture of water and acetonitrile R (1 : 1) and dilute to 1000 ml with the same solvent. Test solution: Weigh 20 tablets, calculate the average mass and finely powder. Weigh accurately a quantity equivalent to 10 mg of berberine chloride, add 70 ml of the mobile

BETAMETHASONE

phase, sonicate for 15 min, allow to cool and add sufficient the mobile phase to produce 100.0 ml. Filter. Reference solution: Weigh accurately about 0.010 g of berberine chloride RS, dissolve with the mobile phase, and add sufficient the mobile phase to produce 100.0 ml. Filter. Resolution solution: Dissolve 1 mg of berberine chloride RS and 1 mg of palmatine RS in 10 ml of the mobile phase. Filter. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 345 nm. Column temperature: 40 ºC. Flow rate: Adjust to obtain a retention time of about 10 min for berberine. Volume of injection: 10 µl. Procedure: System suitability: Inject the resolution solution, the elution order is berberine and palmatine, respectively. The resolution factor between the two peaks is not less than 1.5. Inject separately 5 times the reference solution, the relative standard deviation of the berberine peak areas for replicate injections is not more than 1.5%. Inject the test solution and the reference solution. Calculate the content of berberine chloride, C20H18ClNO4, 2H2O, in the tablets using the areas of the principal peaks in the chromatograms obtained with the test solution, the reference solution and the declared content of C20H18ClNO4,2H2O in berberine chloride RS.

Storage Store at a dry and cool place, protected from light. Action and use Antibacterial. Usual strength 10 mg; 50 mg and 100 mg. BETAMETHASONE Betamethasonium

C22H29FO5 M. 392.5 Betamethasone is 9-fluoro-11β,17,21-trihydroxy-16βmethylpregna-1,4-diene-3,20-dione. It contains not less than 97.0% and not more than 103.0% of C22H29FO5, calculated with reference to the dried substance. 129

VP V

BETAMETHASONE

Characters A white or almost white, crystalline powder. Practically insoluble in water, sparingly soluble in ethanol, very slightly soluble in methylene chloride. Identification Apply one of the two following identifications: First identification: A, C. Second identification: B, C, D, E. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of betamethasone RS. If the spectra obtained in the solid state with the substance to be examined and the reference substance show differences, dissolve the substance to be examined and the reference substance separately in the smallest necessary quantity of methylene chloride R and evaporate to dryness on a water-bath. Using the residues, record the spectra again. B. Dissolve 10.0 mg in ethanol R and dilute to 100.0 ml with the same solvent. Place 2.0 ml of the solution in a stoppered tube, add 10.0 ml of phenylhydrazine-sulfuric acid solution R, mix and heat in a water-bath at 60 °C for 20 min. Cool immediately. The absorbance (Appendix 4.1) of the solution measured at 419 nm is not greater than 0.10. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Butanol saturated with water - toluene ether (5 : 10 : 85) Test solution: Dissolve 10 mg of the substance to be examined in a mixture of methanol-methylene chloride (1 : 9) and dilute to 10 ml with the same mixture of solvents. Reference solution (1): Dissolve 20 mg of betamethasone RS in a mixture of methanol - methylene chloride (1 : 9) and dilute to 20 ml with the same mixture of solvents. Reference solution (2): Dissolve 10 mg of dexamethasone RS in reference solution (1) and dilute to 10 ml with the same solution. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (1). Spray with ethanolic sulfuric acid solution R. Heat at 120 °C for 10 min or until the spots appear. Allow to cool. Examine the chromatograms in daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) shows two spots which may however not be completely separated. D. Mix about 5 mg of substance to be examined with 45 mg of heavy magnesium oxide R and ignite in a crucible until an almost white residue is obtained (usually less than 5 min). Allow to cool, add 1 ml of water, 0.05 ml 130

of phenolphthalein solution R1 and about 1 ml of dilute hydrochloric acid R to render the solution colourless. Filter. Add 1.0 ml of the filtrate to a freshly prepared mixture of 0.1 ml of alizarin S solution R and 0.1 ml of zirconyl nitrate solution R. Mix, allow to stand for 5 min and compare the colour of the solution with that of a blank prepared in the same manner. The test solution is yellow and the blank is red. E. Add about 2 mg to 2 ml of sulfuric acid R and shake to dissolve. Within 5 min, a deep reddish-brown colour develops. Add the solution to 10 ml of water and mix. The colour is discharged and a clear solution remains.

Specific optical rotation Dissolve 0.125 g in methanol R and dilute to 25.0 ml with the same solvent. The specific optical rotation is + 118° to + 126°, calculated with reference to the dried substance (Appendix 6.4). Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: In a 1000 ml volumetric flask mix 250 ml of acetonitrile R with 700  ml of water and allow to equilibrate; adjust the volume to 1000 ml with water and mix again. Mobile phase B: Acetonitrile R. Test solution: Dissolve 25.0 mg of the substance to be examined in a mixture of equal volumes of acetonitrile R and methanol R and dilute to 10.0 ml with the same solvent. Reference solution (1): Dissolve 2 mg of betamethasone RS and 2 mg of methylprednisolone RS in mobile phase A and dilute to 100.0 ml with the same mobile phase. Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with mobile phase A. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Column temperature: 45 °C. Detector: A spectrophotometer set at 254 nm. Flow rate: 2.5 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

Comments

0

100

0

Isocratic

15

100

0

Linear gradient

40

0

100

End the gradient, back to 100% of mobile phase A

41

100

0

Equilibrate with mobile phase A

46 = 0

100

0

End equilibration. Start new injection

VP V

Equilibrate the column with mobile phase B for at least 30 min and then with mobile phase A for 5 min. For subsequent chromatograms, use the conditions described from 40 min to 46 min. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with reference solution (2) is not less than 50% of the full scale of the recorder. Inject reference solution (1). The retention times: methylprednisolone about 11.5 min and betamethasone about 12.5 min. The test is not valid unless the resolution between the peaks corresponding to methylprednisolone and betamethasone is at least 1.5; if necessary, adjust the concentration of acetonitrile in mobile phase A. Inject separately the mixture of equal volumes of acetonitrile R and methanol R as a blank, the test solution and reference solution (2). Limits: In the chromatogram obtained with the test solution: The area of any peak, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (2) (1.0% ) and not more than one such peak having area greater than half the area of the principal peak in the chromatogram obtained with reference solution (2) (0.5% ). The sum of the areas of all the peaks, apart from the principal peak, is not greater than twice the area of the principal peak in the chromatogram obtained with reference solution (2) (2.0%). Disregard any peak due to the blank and any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (2).

Loss on drying Not more than 0.5% (Appendix 9.6). (0.500 g; 100 °C to 105 °C). Assay Dissolve 0.100 g in ethanol (96%) R and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml of the solution to 100.0 ml with ethanol (96%) R. Measure the absorbance (Appendix 4.1) of this solution at the maximum at 238.5 nm. Calculate the content of C22H29FO5 taking the value of A(1%, 1 cm) at 238.5 nm is 395. Storage Store in an airtight container, protected from light. Action and use Corticosteroid. Preparation Tablets.

BETAMETHASONE TABLETS

BETAMETHASONE TABLETS Tabellae Betamethasoni Betamethasone tablets contain betamethasone. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements:

Content of betamethasone, C22H29FO5, 90.0% to 110.0% of the stated amount. Identification A. Shake a quantity of the powdered tablets containing the equivalent of about 25 mg of betamethasone with 150 ml of dichloromethane R for 30 min and filter, wash the filtrate with 20 ml of water, filter through a filter paper containing anhydrous sodium sulfate R. Evaporate the filtrate to dryness at 105 °C for 2 h. The infrared absorption spectrum of the residue (Appendix 4.2), is concordant with the spectrum of betamethasone RS. B. In the Assay, the principal peak obtained from the chromatogram of test solution (1) has the same retention time as the peak due to betamethasone in the chromatogram obtained with the reference solution. Uniformity of content (Appendix 11.2) Examine by liquid chromatography (Appendix 5.3). Carry out the test protected from light. Mobile phase, chromatographic system and procedure: Proceed as described under Assay. Reference solution: A solution containing 0.0025% of betamethasone RS and 0.002% of hydrocortisone (internal standard) in methanol (50%) R. Test solution: Finely powder one tablet, add 20.0 ml of a 0.002% solution of hydrocortisone in methanol (50%) R, shake well for 10 min and filter. Assay Examine by liquid chromatography (Appendix 5.3). Carry out the test protected from light. Mobile phase: Water - methanol (53 : 47). Reference solution: A solution containing 0.0125% of betamethasone RS and 0.010% of hydrocortisone (internal standard) in methanol (50%) R. Internal reference solution: A solution containing 0.010% of hydrocortisone in methanol (50%) R. Test solution (1): Weigh 20 tablets, calculate the average mass, and powder finely. Transfer an accurately weighed quantity of the powdered tablets containing the equivalent of about 2.5 mg of betamethasone to a 20 ml volumetric flask, add methanol (50%) R and shake well for 10 min to dissolve and dilute to volume with the same solvent, filter. Test solution (2): Prepare test solution (2) in the same manner as test solution (1) but use the internal reference solution in place of methanol (50%) R. 131

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BETAMETHASONE DIPROPIONATE

Chromatographic system: A column (20 cm × 5 mm) packed with stationary phase C (5 - 10 µm) (Spherisorb ODS is suitable). Detector: A spectrophotometer set at 238 nm. Flow rate: 1.4 ml/min. Volume of injection: 20 µl. Procedure: Inject the reference solution and the test solution. Calculate the content of betamethasone, C22H29FO5, in the tablets using the response ratios (area or height) of betamethasone peak and hydrocortisone peak in the chromatograms obtained with test solution (2), the reference solution and the declared content of C22H29FO5 in betamethasone RS.

Storage Store in a well-closed container, protected from light, at a temperature not exceeding 30 °C. Action and use Corticosteroid. Usual strength 0.5 mg and 0.6 mg. BETAMETHASONE DIPROPIONATE Betamethasoni dipropionas

C28H37FO7

M. 504.6

Betamethasone dipropionate is 9-fluoro-11β-hydroxy16β-methyl-3,20-dioxopregna-1,4-diene-17,21-diyl dipropionate. It contains not less than 97.0% and not more than 102.0% of C28H37 FO7, calculated with reference to the anhydrous substance.

Characters A white or almost white crystalline powder, practically insoluble in water, freely soluble in acetone and in methylene chloride, sparingly soluble in ethanol (96%). Identification Apply one of the two following identifications: First identification: A. Second identification: B, C, D, E. 132

A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of betamethasone dipropionate RS. B. Dissolve 10.0 mg in ethanol R and dilute to 100.0 ml with the same solvent. Place 2.0 ml of the solution in a groundglass-stoppered tube, add 10.0 ml of phenylhydrazinesulfuric acid solution  R. Mix and heat in a water-bath at 60  °C for 20 min. Cool immediately. The absorbance (Appendix 4.1) of the solution measured at 419 nm is not more than 0.10. C. Examine by thin- layer chromatography (Appendix 5.4) Coating substance: Silica gel F254. Mobile phase: Add a mixture of 1.2 volumes of water and 8 volumes of methanol R to a mixture of 15 volumes of ether R and 77 volumes of methylene chloride R. Test solution (1): Dissolve 25 mg of the substance to be examined in methanol R with gentle heating and dilute to 5  ml with the same solvent (solution A). Dilute 2  ml of solution A to 10 ml with methylene chloride R. Test solution (2): Transfer 2 ml of solution A to a 15 ml glass tube with a ground-glass stopper or a polytetrafluoroethylene cap. Add 10 ml of saturated methanolic potassium hydrogen carbonate solution  R and immediately pass a current of nitrogen R briskly through the solution for 5 min. Stopper the tube. Heat in a water-bath at 45 °C for 2 h, protected from light. Allow to cool. Reference solution (1): Dissolve 25 mg of betamethasone dipropionate RS in methanol R with gentle heating and dilute to 5  ml with the same solvent (solution B). Dilute 2 ml of solution B to 10 ml with methylene chloride R. Reference solution (2): Transfer 2 ml of solution B to a 15  ml glass tube with a ground-glass stopper or a polytetrafluoroethylene cap. Add 10  ml of  saturated methanolic potassium hydrogen carbonate solution R and immediately pass a current of nitrogen R briskly through the solution for 5 min. Stopper the tube. Heat in a waterbath at 45 °C, protected from light, for 2 h. Allow to cool. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in each of the chromatograms obtained with the test solutions is similar in position and size to the principal spot in the chromatogram obtained with the corresponding reference solution. Spray the plate with ethanolic sulfuric acid  solution R. Heat at 120 °C for 10 min or until the spots appear. Allow to cool. Examine in daylight and in ultraviolet light at 365 nm. The principal spot in each of the chromatograms obtained with the test solutions is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with the corresponding reference solution. The principal spot in each of the chromatograms obtained with test solution (2) and reference solution (2) has an Rf value distinctly lower than that of the principal spot in each of the chromatograms obtained with test solution (1) and reference solution (1).

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D. Add 2  mg to 2  ml of  sulfuric acid  R  and shake to dissolve. Within 5 min, a deep reddish-brown colour develops. Add this solution to 10 ml of water and mix. The colour is discharged and a clear solution remains. E. Mix 5 mg with 45 mg of heavy magnesium oxide R and ignite in a crucible until an almost white residue is obtained (usually less than 5 min). Allow to cool, add 1 ml of water, 0.05 ml of phenolphthalein solution R1 and about 1 ml of dilute hydrochloric acid R to render the solution colourless. Filter. Add 1.0  ml of the filtrate to a freshly prepared mixture of 0.1 ml of alizarin S solution R and 0.1 ml of zirconyl nitrate solution R. Mix, allow to stand for 5 min. Compare the colour of the solution with that of a blank prepared in the same manner. The test solution is yellow and the blank is red.

Specific optical rotation +84 ° to +88 °, calculated with reference to dried substance (Appendix 6.4). Dissolve 0.250 g in ethanol R and dilute to 25.0 ml with the same solvent. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Mix carefully 35  ml of  water  and 56  ml of  acetonitrile R. Allow to equilibrate, dilute to 100 ml with water and mix. Test solution (1): Dissolve 60.0 mg of the substance to be examined in the mobile phase and dilute to 25.0 ml with the same solvent. Test solution (2):  Dilute 1.0  ml of test solution (1) to 10.0 ml with the mobile phase. Reference solution (1):  Dissolve 5  mg of  betamethasone dipropionate for system suitability RS (containing impurities B, C, D, E and G) in the mobile phase and dilute to 2.0 ml with the same solvent. Reference solution (2): Dilute 1.0 ml of test solution (1) to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (3): Dissolve 60 mg of betamethasone dipropionate RS in the mobile phase and dilute to 25.0 ml with the mobile phase. Dilute 1.0  ml of this solution to 10.0 ml with the mobile phase. Reference solution (4):  Dissolve 5  mg of  betamethasone dipropionate for peak identification RS (containing impurity H) in the mobile phase and dilute to 2.0 ml with the same solvent. Chromatographic system: A column (10 cm × 2.0 mm) packed with stationary phase C (2.5 µm). Column temperature: 20 °C ± 2 °C. Flow rate: 0.2 ml/min. Detector: A spectrophotometer set at 254 nm. Volume of injection: 5 µl. Procedure: Carry out the chromatography for 3 times the retention time of betamethasone dipropionate.

BETAMETHASONE DIPROPIONATE

Identification of impurities:  Use the chromatogram supplied with betamethasone dipropionate for system suitability RS and the chromatogram obtained with reference solution (1) to identify the peaks due to impurities B, C, D, E and G. Use the chromatogram supplied with  betamethasone  dipropionate for peak identification RS and the chromatogram obtained with reference solution (4) to identify the peak due to impurity H. Relative retention with reference to betamethasone dipropionate (retention time  =  about 10 min): Impurity B = about 0.4; impurity C = about 0.5; impurity D = about 0.7; impurity E  =  about 1.2; impurity H  =  about 1.7; impurity G = about 2.1. System suitability: In the chromatogram obtained with reference solution (1), the peak-to-valley ratio (Hp/Hv) is at least 4.0, where Hp is the height above the baseline of the peak due to impurity E and Hv is the height above the baseline of the lowest point of the curve separating this peak from the peak due to betamethasone dipropionate. Limits: The correction factors: For the calculation of content, multiply the peak areas of the following impurities by the corresponding correction factor: Impurity G  =  1.3; impurity H = 1.4. Impurity C: The area of peak due to impurity C is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.5%). Impurities B, H: For each impurity, the corrected peak area, if necessary, is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.3%). Impurities D, E, G: For each impurity, the corrected peak area, if necessary, is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (2) (0.2%). Unspecified impurities: For each impurity, the peak area if necessary, is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.10%). Sum of the areas of all the impurity peaks is not more than 10 times the area of the principal peak in the chromatogram obtained with reference solution (2) (1.0%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Notes: Impurity A: 9-Fluoro-11β,17,21-trihydroxy-16β-methylpregna1,4-diene-3,20-dione (betamethasone). Impurity B: 9-Fluoro-11β,21-dihydroxy-16β-methyl-3,20dioxopregna-1,4-dien-17-yl propanoate (betamethasone 17-propionate). Imputity C: 9-Fluoro-11β,17-dihydroxy-16β-methyl-3,20dioxopregna-1,4-dien-21-yl propanoate (betamethasone 21-propionate). Impurity D: 21-(Acetyloxy)-9-fluoro-11β-hydroxy-16β-methyl3,20-dioxopregna-1,4-dien-17-yl propanoate (betamethasone 21-acetate 17-propionate).

133

BETAMETHASONE SODIUM PHOSPHATE

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Identification Apply one of the two following identifications: First identification: B, C. Second identification: A, C, D, E, F. A. Dissolve 10.0 mg of the substance to be examined in 5 ml of water and dilute to 100.0 ml with ethanol R. Place 2.0 ml of this solution in a ground-glass-stoppered tube, add 10.0 ml of phenylhydrazine-sulfuric acid solution R, mix and heat in a water-bath at 60 °C for 20 min. Cool immediately. The absorbance (Appendix 4.1) of the solution measured at the Loss on drying  maximum at 450 nm is not more than 0.10. Not more than 1.0% (Appendix 9.6). B. The infrared absorption spectrophotometry (Appendix (0.500 g; 105 °C). 4.2) of the substance to be examined is concordant with the spectrum of betamethasone sodium phosphate RS. If Assay Examine by liquid chromatography (Appendix 5.3) as the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference described in the test for Related substances. substance separately in the minimum volume of ethanol Inject test solution (2) and reference solution (3). Calculate the percentage content of C28H37FO7 in the (96%) R, evaporate to dryness on a water-bath. Record substance to be examined, using the areas of the principal new spectra of the residues. peaks in the chromatograms obtained with test solution C. Examine by thin-layer chromatography (Appendix 5.4). (2), reference solution (3), and the declared content Coating substance: Silica gel F254. Mobile phase: Glacial acetic acid - water - butanol (20 : 20 : 60). of C28H37FO7 in betamethasone dipropionate RS. Test solution: Dissolve 10 mg of the substance to be examined Storage in methanol R and dilute to 10 ml with the same solvent. Store protected from light. Reference solution (1): Dissolve 10 mg of betamethasone sodium phosphate RS in methanol R and dilute to 10 ml Action and use with the same solvent. Corticosteroid. Reference solution (2): Dissolve 10 mg of prednisolone Preparations sodium phosphate RS in methanol R and dilute to 10 ml Tablets, cream. with the same solvent. Dilute 5 ml of this solution to 10 ml with reference solution (1). Procedure: Apply separately to the plate 5 µl of each BETAMETHASONE SODIUM PHOSPHATE solution. Develop over a path of 15 cm. Allow the plate Betamethasoni natrii phosphas to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (1). Spray the plate with ethanolic sulfuric acid solution R. Heat at 120 °C for 10 min or until the spots appear. Allow to cool. Examine in daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, colour in C22H28FNa2O8P  M.516.4   daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained Betamethasone sodium phosphate is 9-fluoro-11β,17- with reference solution (1). The test is not valid unless the dihydroxy-16β-methyl-3, 20-dioxopregna-1,4-diene-21- chromatogram obtained with reference solution (2) shows yl disodium phosphate, it contains not less than 96.0% and two spots which may however not be completely separated. not more than 103.0% of C22H28FNa2O8P, calculated with D. Add about 2 mg of the substance to be examined to 2 ml reference to the anhydrous substance. of sulfuric acid R and shake to dissolve. Within 5 min, an intense reddish-brown colour develops. Add the solution Characters to 10 ml of water and mix. The colour is discharged and a A white or almost white powder, very hygroscopic. clear solution remains. Freely soluble in water, slightly soluble in ethanol (96%), E. Mix about 5 mg of the substance to be examined with practically insoluble in methylene chloride. 45 mg of heavy magnesium oxide R and ignite in a crucible Impurity E: 9-Chloro-11β-hydroxy-16β-methyl-3,20-dioxopregna1,4-diene-17,21-diyl dipropanoate (beclometasone dipropionate). Impurity F: 9,11β-Epoxy-16β-methyl-3,20-dioxo-9β-pregna1,4-diene-17,21-diyl dipropanoate (9β,11β-epoxybetamethasone dipropionate). Impurity G: 9-Fluoro-16β-methyl-3,20-dioxopregna-1,4-diene11β,17,21-triyl tripropanoate (betamethasone tripropionate). Impurity H: 6α-Bromo-9-fluoro-11β-hydroxy-16β-methyl-3, 20-dioxopregna-1,4-diene-17,21-diyl dipropanoate (6α-bromobetamethasone dipropionate).

134

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until an almost white residue is obtained (usually less than 5 min). Allow to cool, add 1 ml of water, 0.05 ml of phenolphthalein solution R1 and about 1 ml of dilute hydrochloric acid R to render the solution colourless. Filter. Add 1.0 ml of the filtrate to a freshly prepared mixture of 0.1 ml of alizarin S solution R and 0.1 ml of zirconyl nitrate solution R. Mix, allow to stand for 5 min and compare the colour of the solution with that of a blank prepared in the same manner. The test solution is yellow and the blank is red. F. To about 40 mg of the substance to be examined add 2 ml of sulfuric acid R and heat carefully until white fumes are evolved. Add nitric acid R dropwise, continue the heating until the solution is almost colourless and cool. Add 2 ml of water, heat until white fumes are again evolved, cool, add 10 ml of water and neutralise with dilute ammonia solution R, using red litmus paper as indicator. The solution gives reaction of sodium and reaction (A) of phosphates (Appendix 8.1).

Appearance of solution Solution S: Dissolve 1.0 g of the substance to be examined in carbon dioxide-free water R and dilute to 20 ml with the same solvent. Solution S is clear (Appedix 9.2) and not more intensely coloured than reference solution B7 (Appendix 9.3, method 2). pH 7.5 to 9.0 (Appendix 6.2). Dilute 1 ml of solution S to 5 ml with carbon dioxide-free water R. Specific optical rotation +98° to +104°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.250 g of the substance to be examined in water and dilute to 25.0 ml with the same solvent. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Weigh 1.360 g of potassium dihydrogen phosphate R and 0.600 g of hexylamine R in a 250 ml conical flask, mix and allow to stand for 10 min, then dissolve in 185 ml of water. Add 65 ml of acetonitrile R, mix and filter through a 0.45 µm membrane filter. Test solution: Dissolve 62.5 mg of the substance to be examined in the mobile phase and dilute to 25.0 ml with the mobile phase. Reference solution (1): Dissolve 25 mg of betamethasone sodium phosphate RS and 25 mg of dexamethasone sodium phosphate RS in the mobile phase and dilute to 25.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 25.0 ml with the mobile phase. Reference solution (2): Dilute 1.0 ml of the test solution to 50.0 ml with the mobile phase.

BETAMETHASONE SODIUM PHOSPHATE

Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Equilibrate the column with the mobile phase for about 45 min. The run time is twice the retention time of betamethasone sodium phosphate. In chromatographic conditions described above, the retetion time of betamethasone sodium phosphate is about 14 min; of dexamethasone sodium phosphate about 15.5 min. System suitability: Inject reference solution (1), the resolution between the peaks corresponding to betamethasone sodium phosphate and dexamethasone sodium phosphate is at least 2.0. If necessary, increase the concentration of acetonitrile or increase the concentration of water in the mobile phase. In the chromatogram obtained with the test solution, the area of any peak, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (2) (2%) and not more than one such peak has an area greater than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (1%). The sum of the areas of all the peaks, apart from the principal peak, is not greater than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (3%). Disregard any peak with an area less than 0.025 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%).

Inorganic phosphate Not more than 1%. Dissolve 50 mg of the substance to be examined in water and dilute to 100 ml with the same solvent. To 10 ml of this solution add 5 ml of molybdovanadic reagent R, mix and allow to stand for 5 min. Any yellow colour in the solution is not more intense than that in a standard prepared at the same time and in the same manner. Using 10 ml of phosphate standard solution (5 ppm PO4) R to prepare the standard solution. Water Not more than 8.0% (Appendix 10.3). Determined on 0.200 g. Assay Dissolve 0.100 g of the substance to be examined in water and dilute to 100.0 ml with the same solvent. Dilute 5.0 ml of this solution to 250.0 ml with water. Measure the absorbance (Appendix 4.1) at the maximum at 241 nm. Calculate the content of C22H28FNa2O8P, taking 297 as the value of A (1%, 1 cm) at the maximum at 241 nm. 135

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BETAMETHASONE EYE DROPS

Storage Store in an airtight container, protected from light.

pH 7.0 to 8.5 (Appendix 6.2).

Action and use Glucocorticoid.

Related substances Examine by liquid chromatography (Appendix 5.3). Carry out the test protected from light. Mobile phase: Citro-phosphate buffer pH 5.0 - methanol (60 : 40). Test solution: Dilute a volume of the eye drops with water, if necessary, to obtain a solution containing about 0.10% of betamethasone sodium phosphate. Reference solution (1): Dilute 1.0 ml of test solution to 50.0 ml with water. Reference solution (2): A solution containing 0.0060% of betamethasone sodium phosphate RS and 0.0060% of betamethasone RS in water. Chromatographic system: A column (20 cm × 4.6 mm) packed with stationary phase C (10 µm, Spherisorb ODS is suitable). Column temperature: 60 °C. Detector: A spectrophotometer set at 241 nm. Flow rate: 2 ml/min. Volume of injection: 20 µl. Procedure: Inject reference solution (2), the test is not valid unless the resolution factor between the peaks due to betamethasone sodium phosphate and betamethasone is at least 3.5. Inject alternately the test solution and reference solution (1), record the chromatogram for three times the retention time of the principal peak. In the chromatogram obtained with test solution, the area of any peak corresponding to betamethasone is not greater than 1.3 times the area of the principal peak in the chromatogram obtained with reference solution (1); the area of any other secondary peak is not greater than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (1). The sum of the areas of all the secondary peaks is not greater than 2.5 times the area of the principal peak in the chromatogram obtained with reference solution (1). Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (1).

Preparations Tablets, injection, eye drops. BETAMETHASONE EYE DROPS Collyrium Betamethasoni Betamethasone eye drops are a sterile solution of betamethasone sodium phosphate in water. The eye drops comply with the requirements stated under “Eye drops” (Appendix 1.14) and with the following requirements.

Content of betamethasone sodium phosphate, C22H28FNa2O8P, 90.0% to 110.0% of the stated amount. Characters A clear, colourless solution. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Butan-1-ol - anhydride acetic - water (60 : 20 : 20), prepared immediately before use. Test solution: Diluted the eye drops, if necessary, with water to obtain a 0.1% solution of betamethasone sodium phosphate. Reference solution (1): A solution containing 0.1% of betamethasone sodium phosphate RS in water. Reference solution (2): A mixture of equal volumes of the test solution and reference solution (1). Reference solution (3): A mixture of equal volumes of the reference solution (1) and a 0.1% solution of prednisolone sodium phosphate RS in water. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm. After removal of the plate, allow to dry in the air, heat at 110 °C for 10 minutes and examine under ultraviolet light (254 nm). The chromatograms obtained with the test solution, reference solution (1) and reference solution (2) show single principal spots with similar Rf values. The test is not valid unless the chromatogram obtained with reference solution (3) shows two principal spots with almost identical Rf values. B. In the Assay, the chromatogram obtained with test solution (1) shows a peak with the same retention time as the peak due to betamethasone sodium phosphate in the chromatogram obtained with reference solution (2). C. To a volume of the eye drops containing 0.2 mg of betamethasone sodium phosphate, add slowly 1 ml of sulfuric acid R and allow to stand for 2 min. A brownish yellow colour but no red colour or yellowish green fluorescence is produced. 136

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Citro-phosphate buffer pH 5.0 - methanol (55 : 45). Internal standard solution: A 0.06% solution of hydrocortisone RS in methanol R. Reference solution (1): A 0.1% solution of betamethasone sodium phosphate RS with water. Reference solution (2): Transfer 5.0 ml reference solution (1) to a 25 ml volumetric flask, add 10.0 ml of internal standard solution, mix, dilute to volume with water.

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BETAMETHASONE VALERATE

Test solution (1): Mix a quantity of the eye drops containing 5 mg of betamethasone sodium phosphate with 10.0 ml of methanol R and dilute to 25.0 ml with water. Test solution (2): Prepare in the same manner as test solution (1) but using 10.0 ml of the internal standard solution in place of the methanol. Chromatographic system: A column (20 cm × 5 mm) packed with stationary phase C (10 µm, Spherisorb ODS1 is suitable). Column temperature: 60 °C. Detector: A spectrophotometer set at 241 nm. Flow rate: 2 ml/min. Volume of injection: 20 µl. Procedure: Calculate the content of betamethasone sodium phosphate, C22H28FNa2O8P, in reference solution (1) by measuring the absorbance (Appendix 4.1) of an (betamethasone) aliquot diluted to 0.002% with water at the maximum at 241 nm and take 297 as the value of A(1%, 1 cm) at the maximum at 241 nm. Inject alternately reference solution (2), test solution (1) and test solution (2). Calculate the content of C22H28FNa2O8P in the eye drops using the content of betamethasone sodium phosphate in reference solution (1) and the peak areas in the chromatogram obtained with reference solution (2), test solution (2).

Storage Store in a well-closed container, at a cool and dry place, and protected from light. Action and use Glucocorticoid. Usual strength 0.1%. BETAMETHASONE VALERATE Betamethasoni valeras

C27H37FO6

M. 476.6

Betamethasone valerate is 9-fluoro-11β,21-dihydroxy16β-methyl-3,20-dioxopregna-1,4-dien-17-yl pentanoate. It contains not less than 97.0% and not more than 103.0% of C27H37 FO6, calculated with reference to the anhydrous substance.

Characters A white or almost white crystalline powder. Practically insoluble in water, freely soluble in acetone and in methylene chloride, soluble in ethanol (96%). It melts at about 192 °C, with decomposition. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of betamethasone 17-valerate RS. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in the minimum volume of methylene chloride R, evaporate to dryness on a water-bath and record new spectra using the residues. B. In the test for related substances, the principal peak in the chromatogram obtained with the test solution is similar in retention time and size to the principal peak in the chromatogram obtained with reference solution (2). Specific optical rotation +77° to +83°, calculated with reference to dried substance (Appendix 6.4). Dissolve 0.250 g in  ethanol R and dilute to 25.0 ml with the same solvent. Related substances Examine by liquid chromatography (Appendix 5.3). Carry out the test protected from light. Prepare the solutions immediately before use. Mobile phase: Acetonitrile - water (50 : 50). Solvent mixture: Glacial acetic acid - mobile phase (1 : 1000). Test solution:  Dissolve 50 mg of the substance to be examined in the solvent mixture and dilute to 20.0 ml with the same solvent. Reference solution (1):  Dilute 1.0  ml of test solution to 100.0  ml with the solvent mixture. Dilute 1.0  ml of this solution to 10.0 ml with the solvent mixture. Reference solution (2): Dissolve 12.5  mg of  betamethasone valerate for system suitability RS (containing impurities D and G) in 5.0 ml of the solvent mixture. Use 1.0 ml of this solution to dissolve the content of a vial of betamethasone valerate impurity mixture RS (containing impurities C, H and I). Reference solution (3):  Dissolve 6  mg of  betamethasone  RS (impurity A) and 3 mg of betamethasone 21-valerate RS (impurity E) in 30.0 ml of the solvent mixture. Dilute 1.0 ml of this solution to 10.0 ml with the solvent mixture. Chromatographic system: A column (25 cm × 4.6 mm) packed with end-capped octadecylsilyl silica gel for chromatography (5 µm). Column temperature: 20 ºC. Detector: A spectrophotometer set at 239 nm. Flow rate: 1.0  ml/min. Volume of injection: 20 µl. 137

VP V

BIOTIN

Procedure: Carry out the chromatography for 2.5 times the retention time of betamethasone valerate. Identification of impurities: Use the chromatogram supplied with betamethasone valerate for system suitability RS and the chromatogram obtained with reference solution (2) to identify the peaks due to impurities C, D, G, H and I. Use the chromatogram obtained with reference solution (3) to identify the peaks due to impurities A and E. The relative retention with reference to betamethasone valerate (retention time  =  about 20 min): Impurity A = about 0.3; impurity I = about 0.6; impurity C = about 0.8; impurity H  =  about 1.3; impurity D  =  about 1.4; impurity E = about 1.6; impurity G = about 2.0. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurities H and D is at least 1.7. Limits: Impurity A: The area of peak due to impurity A is not more than 7 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.7%). Impurities E and G: For each impurity, the peak area is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.3%). Impurities C, H and I: For each impurity, the peak area is not more than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.15%). Unspecified impurities: For each impurity, the peak area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). Sum of the areas of all the impurity peaks is not more than 15 times the area of the principal peak in the chromatogram obtained with reference solution (1) (1.5%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Notes: Impurity A: 9-fluoro-11β,17,21-trihydroxy-16β-methylpregna1,4-dien-3,20-dione (betamethasone). Impurity B: 9-fluoro-11β,17-dihydroxy-16β-methylpregna-1,4dien-3,20-dione (21-deoxy-betamethasone). Imputity C: 9-fluoro-11β,21-dihydroxy-16α-methyl-3,20dioxopregna1,4-dien-17-yl pentanoate (dexamethasone 17-valerate). Impurity D: 9-bromo-11β,21dihydroxy-16β-methyl-3,20dioxopregna-1,4-dien-17-yl pentanoate (9-bromo-betamethasone valerate). Impurity E: 9-fluoro-11β,17-dihydroxy-16β-methyl-3,20dioxopregna1,4-dien-21-yl pentanoate (betamethasone 21 -valerate). Impurity F: 21-hydroxy-16β-methyl-3,20-dioxopregna-1,4,9(11)trien-17-yl pentanoate (betamethasone valerate δ-9(11)). Impurity G: : 6α-bromo-9-fluoro-11β,21-dihydroxy-16βmethyl3,20-dioxopregna-1,4-dien -17-yl pentanoate (6α-bromo-betamethasone valerate). Impurity H: 9-chloro-11β,21-dihydroxy-16β-methyl-3,20dioxopregna1,4-dien -17-yl pentanoate (beclomethasone 17-valerate). Impurity I: 9-fluoro-11β,21-dihydroxy-3,20-dioxopregna-1,4dien -17-yl pentanoate (9-fluoro-prednisolone 17-valerate).

138

Loss on drying  Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Assay Dissolve 50.0 mg of the substance to be examined in ethanol (96%) R and dilute to 100.0 ml with the same solvent. Dilute 2.0  ml of this solution to 50.0  ml with  ethanol (96%)  R. Measure the absorbance (Appendix 4.1) at the absorption maximum at 240 nm. Calculate the content of C27H37FO6 taking 325 is the value of the specific absorbance at 240 nm. Storage Store in a well-closed container, protected from light. Action and use Corticosteroid. Preparations Tablets, cream. BIOTIN Biotinum

C10H16N2O3S

M. 244.3

Biotin is 5-[(3aS,4S,6aR)-2-oxohexahydrothieno[3,4-d] imidazol-4-yl]pentanoic acid, it contains not less than 98.5% and not more than 101.0% of C10H16N2O3S, calculated with reference to the dried substance.

Characters A white, crystalline powder or colourless crystals, very slightly soluble in water and in ethanol (96%), practically insoluble in acetone, soluble in dilute solutions of alkali hydroxides. Identification Apply one of the two following identifications: First identification: A. Second identification: B, C. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of biotin RS. B. Examine the chromatograms obtained in the test for related substances (see Related substances). The principal spot in the chromatogram obtained with test solution (2) is similar in position and size to the principal spot in the chromatogram obtained with reference solution (1).

VP V

C. Dissolve about 10 mg in 20 ml of water with heating. Allow to cool. Add 0.1 ml of bromine water R. The bromine water is decolourised.

Appearance of solution Solution S: Dissolve 0.250 g in a 0.4% solution of sodium hydroxide R and dilute to 25.0 ml with the same solvent. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). Specific optical rotation +89° to +93°, calculated with reference to the dried substance (Appendix 6.4). Determined on solution S. Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance : Silica gel (5 µm). Mobile phase: Methanol - glacial acetic acid - toluene (5 : 25 : 75) Prepare the solutions immediately before use and keep protected from bright light. Test solution (1): Dissolve 50 mg of the substance to be examined in glacial acetic acid R and dilute to 10 ml with the same solvent. Test solution (2): Dilute 1 ml of test solution (1) to 10 ml with glacial acetic acid R. Reference solution (1): Dissolve 5 mg of biotin RS in glacial acetic acid R and dilute to 10 ml with the same solvent. Reference solution (2): Dilute 1 ml of test solution (2) to 20 ml with glacial acetic acid R. Reference solution (3): Dilute 1 ml of test solution (2) to 40 ml with glacial acetic acid R. Procedure: Apply to the plate 10 µl of each solution. Develop over a path of 15 cm. Dry the plate in a current of warm air. Allow to cool and spray with 4-dimethylaminocinnamaldehyde solution R. Examine immediately in daylight. Any spot in the chromatogram obtained with test solution (1), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (2) (0.5% ) and at most one such spot is more intense than the spot in the chromatogram obtained with reference solution (3) (0.25%). Heavy metals Not more than 10 ppm (Appendix 9.4.8). 1.0 g complies with limit test for Heavy metals, method 3. Prepare the reference solution using 10 ml of lead standard solution (1 ppm Pb) R. Loss on drying Not more than 1.0% (Appendix 9.6). (1.000 g, 100 °C to 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g.

BIOTIN TABLETS

Assay Suspend 0.200 g in 5 ml of dimethylformamide R. Heat until the substance has dissolved completely. Add 50 ml of ethanol R and titrate with 0.1 M tetrabutylammonium hydroxide VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 M tetrabutylammonium hydroxide VS is equivalent to 24.43 mg of C10H16N2O3S. Storage Protected from light. Action and use Vitamin. BIOTIN TABLETS Tabellae Biotini Biotin tablets contain biotin. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of biotin, C10H16N2O3S, 90.0% to 110.0% of the stated amount. Identification A. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the principal peak in the chromatogram obtained with the reference solution. B. To a quantity of powdered tablets containing about 10 mg of biotin, add 20 ml of water, heat to dissolve and allow to cool. Add 0.1 ml of bromine water R. The bromine water is decolourised. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Transfer 85 ml of acetonitrile R, 1 g of sodium perchlorate R and 1 ml of phosphoric acid R to a 1000 ml volumetric flask, dilute with water to volume, mix, filter and degas. Adjust if necessary. Reference solution: Transfer about 67 mg of biotin RS, accurately weighed, to a 200 ml volumetric flask, dissolve with dimethyl sulfoxide R. Dilute to volume with dimethyl sulfoxide R. Mix well. Transfer 3.0 ml of the resulting solution to a 200.0 ml volumetric flask, dilute with water to volume and mix well. Test solution: Weigh 20 tablets, calculate the average mass, and powder finely. Transfer an accurately weighed quantity of the powdered tablets containing about 1 mg of biotin to a 200 ml volumetric flask. Add 3 ml of dimethyl sulfoxide R and shake on a wrist-action shaker to moisten. Heat the flask above in a water bath maintained at 60 °C to 70 °C for 5 min and then sonicate for 5 min. Dilute to volume with the water, mix and filter. 139

VP V

BISACODYL

Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase B (3 µm). Detector: A spectrophotometer set at 200 nm. Flow rate: 1.2 ml/min. Volume of injection: 100 µl. Procedure: System suitability: Inject the reference solution and record the chromatogram. The relative standard deviation of the peak areas for 6 replicate injections is not more than 3%. Inject separately the reference solution the test solution. Calculate the content of biotin, C10H16N2O3S, in the tablets using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C10H16N2O3S in biotin RS.

Storage Store in a well-closed container, at a temperature not exceeding 30 °C. Action and use Vitamin. Usual strength 0.3 mg; 0.6 mg and 5 mg. BISACODYL Bisacodylum

C22H19NO4

Acidity or alkalinity To 1.0 g add 20 ml of carbon dioxide-free water  R, shake, heat to boiling, cool and filter. Add 0.2  ml of 0.01  N  sodium hydroxide  VS and 0.1 ml of methyl red solution R. The solution is yellow. Not more than 0.4 ml of 0.01 N hydrochloric acid VS is required to change the colour of the indicator to red. M. 361.4

Bisacodyl is 4,4′-(pyridin-2-ylmethylene) diphenyl diacetate. It contains not less than 98.0% and not more than 101.0% of C22H19 NO4, calculated with reference to the dried substance.

Characters A white or almost white, crystalline powder, practically insoluble in water, soluble in acetone, sparingly soluble in ethanol (96%). It dissolves in dilute mineral acids. Identification Apply one of the two following identifications. First identification: A. Second identification: B,C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of bisacodyl RS. If the spectra obtained in the solid state show 140

differences, dissolve the substance to be examined and the reference substance separately in chloroform R, evaporate to dryness and record new spectra using the residues. B. Dissolve 10.0 mg in a 0.6% solution of  potassium hydroxide R in methanol R and dilute to 100.0 ml with the same solvent. Dilute 10.0 ml of this solution to 100.0 ml with a 0.6% solution of potassium hydroxide R in methanol R. The ultraviolet absorption spectrum (Appendix 4.1) of the solution in the range 220 nm to 350 nm, shows an absorption maximum at 248 nm and a shouder at 290 nm. The specific absorbance at the absorption maximum is 632 to 672. C. Melting point: 131 °C to 135 °C (Appendix 6.7). D. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Butan-2-one - xylene (50 : 50). Test solution: Dissolve 20 mg of the substance to be examined in acetone R and dilute to 10 ml with the same solvent. Reference solution: Dissolve 20 mg of bisacodyl RS in acetone R and dilute to 10 ml with the same solvent. Procedure: Apply separately to the plate 10 μl of each solution. Develop over a path of 10 cm. Allow the plate to dry in air or if necessary heating at 100 - 105 °C. Spray with a mixture of equal volumes of 0.05  M  iodine  and  dilute sulfuric acid  R. Examine in daylight. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution.

Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use. Mobile phase: Acetonitrile - buffer solution pH 5.0 (45 : 55). Buffer solution pH 5.0: A 0.158% solution of ammonium formate R adjusted to pH 5.0 with anhydrous formic acid R. Solvent mixture: Glacial acetic acid  -  acetonitrile  water  (4 : 30 : 66). Test solution:  Dissolve 50 mg of the substance to be examined in 25 ml of acetonitrile R and dilute to 50.0 ml with the solvent mixture. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 10.0 ml with the solvent mixture. Reference solution (2): Dissolve 2.0 mg of bisacodyl for system suitability RS (containing impurities A, B, C, D and E) in 1.0 ml of acetonitrile R and dilute to 2.0 ml with the solvent mixture.

VP V

Reference solution (3):  Dissolve 5.0  mg of  bisacodyl for peak identification RS (containing impurity F) in 2.5  ml of acetonitrile R and dilute to 5.0 ml with the solvent mixture. Chromatographic system: A column (25 cm × 4.6 mm) packed with end-capped octadecylsilyl silica gel for chromatography (5 µm). Detector: A spectrophotometer set at 265 nm. Flow rate: 1.5  ml/min. Volume of injection: 20 µl. Procedure: Carry out the chromatography for 3.5 times the retention time of bisacodyl. Identification of impurities:  Use the chromatogram supplied with bisacodyl for system suitability RS and the chromatogram obtained with reference solution (2) to identify the peaks due to impurities A, B, C, D and E. Relative retention with reference to bisacodyl (retention time  =  about 13 min): Impurity A  =  about 0.2; impurity B = about 0.4; impurity C = about 0.45; impurity D = about 0.8; impurity E = about 0.9; impurity F = about 2.6. System suitability: In the chromatogram obtained with reference solution (2), the peak-to-valley ratio (Hp/Hv) is at least 1.5, where Hp is the height above the baseline of the peak due to impurity E and Hv is the height above the baseline of the lowest point of the curve separating this peak from the peak due to bisacodyl. Limits: The correction factor: For the calculation of content, multiply the peak area of impurity A by 0.7. Impurities A, B: For each impurity, the peak area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%). Impurities C, E: For each impurity, the peak area is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). Impurity D: The area of peak due to impurity D is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%). Impurity F: The area of peak due to impurity F is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.3%). Unspecified impurities: For each impurity, the peak area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). Sum of the areas of all the impurity peaks is not more than 10 times the area of the principal peak in the chromatogram obtained with reference solution (1) (1.0%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Notes Impurity A: 4,4′-(pyridin-2-ylmethylene)diphenol. Impurity B: 2-[(RS)-(4-hydroxyphenyl)(pyridin-2-yl)methyl] phenol. Imputity C: 4-[(RS)-(4-hydroxyphenyl)(pyridin-2-yl)methyl]phenyl acetate. Impurity E: 2-[(RS)-[4-(acetyloxy)phenyl](pyridin-2-yl)methyl] phenyl acetate. Impurities D and F: Unknown structure.

GASTRO-RESISTANT BISACODYL TABLETS

Loss on drying Not more than 0.5% (Appendix 9.6). (0.500 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.300  g in 60  ml of  anhydrous acetic acid  R. Titrate with 0.1 N perchloric acid VS determining the endpoint potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 36.14 mg of C22H19NO4. Storage Store in a well-closed container, protected from light. Action and use Stimulant laxative. Preparations Gastro - resistant tablets, suppository. GASTRO-RESISTANT BISACODYL TABLETS Tabellae Bisacodyli Gastro-resistant bisacodyl tablets contain bisacodyl. They are covered with a gastro-resistant coating. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements:

Content of bisacodyl, C22H19NO4, 90.0% to 110.0% of the stated amount. Identification B. Shake a quantity of the powdered tablets containing about 50 mg of bisacodyl with chloroform R, filter, evaporate the filtrate to dryness. Dissolve the residue in 10 ml of a 0.5% solution of sulfuric acid R (solution A). To 2 ml of solution A, add 0.05 ml of potassium tetraiodomercurate solution R. A white precipitate is produced. B. To 2 ml of solution A, add sulfuric acid R. A reddish violet colour is produced. C. Boil 2 ml of solution A with a little of nitric acid R, a yellow colour is produced. Cool and add 5 M sodium hydroxide R. The colour becomes yellowish brown. D. In the test for Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of the bisacodyl peak in the chromatogram obtained with the referene solution. Dissolution (Appendix 11.4) Acid stage Apparatus: Paddle. Medium: 500 ml of 0.1 M hydrochlorid acid R. Rotation speed: 100 rpm. Time: 2 h. 141

VP V

BISOPROLOL FUMARATE

Procedure: Determine the content of bisacodyl dissolved by liquid chromatography, the mobile phase and chromatographic system as described under the test for Assay. Test solution: After 2 h, withdraw 10 ml of the medium and filter. Reference solution: Weigh accurately 25 mg of bisacodyl RS, dissolve in sufficient 0.1 M hydrochloric acid R to produce 100.0 ml. Dilute 1.0 ml of the resulting solution to 100.0 ml with 0.1 M hydrochloric acid R. Dilute 5.0 ml of the resulting solution to 20.0 ml with 0.1 M hydrochloric acid R. Tolerance: Not more than 5% of the stated amount of bisacodyl is dissolved in 2 h. Buffer stage Apparatus: Paddle. Medium: Replace the acid medium in the vessels with 900 ml of phosphate buffer pH 7.4, previously heated at 37 ± 0.5 °C. Phosphate buffer pH 7.4: Dissolve 1.56 g of sodium hydroxide R and 7.80 g of sodium dihydrogen phosphate R in water and dilute to 1000 ml with water. Add 5.00 g of sodium dodecylsulfate R and heat to dissolve, adjust the pH to 7.4 if necessary. Rotation speed: 100 rpm. Time: 45 min. Procedure: Determine the content of bisacodyl dissolved by liquid chromatography with the mobile phase and chromatographic system as described under the test for Assay. Test solution: After 45 min, withdraw a sample of the medium and filter. Reference solution: Weigh accurately about 28 mg of bisacodyl RS, put into a 100-ml volumetric flask, add 3 ml of acetonitrile R, shake to dissolve completely, dilute to volume with phosphate buffer pH 7.4, mix. Dilute 1.0 ml of the resulting solution to 50.0 ml with phosphate buffer pH 7.4. Tolerance: Not less than 75% of bisacodyl of the stated amount is dissolved in 45 min.

Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Butan-2-one - xylene (1 : 1). Test solution: Shake a quantity of powdered tablets containing 50 mg of bisacodyl with 5 ml of acetone R for 10 min, centrifuge and use the supernatant liquid. Reference solution: Dilute 3 volumes of the test solution to 100 volumes with acetone R. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm. Remove the plate, allow it to dry in air and examine under ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. 142

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - 0.025 M ammonium format adjusted to pH 5.0 with anhydrous formic acid solution R (45 : 55). Solvent mixture: Anhydrous acetic acid - acetonitrile water (4 : 30 : 66). Reference solution: A 0.005% solution of bisacodyl RS in the solvent mixture. Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Weigh accurately a quantity of the powdered tablets containing 10 mg of bisacodyl, transfer to a 50-ml volumetric flask. Add about 40 ml of the solvent mixture, shake well, dilute to 50 ml with the solvent mixture, mix. Filter, discard the first portion of the filtrate. Dilute 25.0 ml of the filtrate to 100.0 ml with the solvent mixture. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 265 nm. Flow rate: 1.5 ml/min. Volume of injection: 50 µl. Procedure: Inject the reference solution. The test is not valid unless the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of bisacodyl, C22H19NO4, in the tablets using the peak areas obtained with the test solution, reference solution and the declared content of C22H19NO4 in bisacodyl RS. Storage Store at a cool and dry place, protected from light. Action and use Stimulant laxative. Usual strength 5 mg. BISOPROLOL FUMARATE Bisoprololi fumaras

C40H66N2O12

M. 767

Bisoprolol fumarate is (2RS)-1-[4-[[2-(1-methylethoxy) ethoxy]methyl]phenoxy]-3-[(1-methylethyl)amino] propan-2-ol fumarate. It contains not less than 99.0% and

VP V

BISOPROLOL FUMARATE

not more than 101.0% of C40H66N2O12, calculated with reference to the anhydrous substance.

Characters White or almost white, slightly hygroscopic powder, it polymorphism. Very soluble in water, freely soluble in methanol. Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of bisoprolol fumarate RS. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in methanol R, evaporate and dry the residues at 60 °C at a pressure not exceeding 0.7 kPa and record new spectra using the residues. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: A 10 g/l solution of phosphoric acid R. Mobile phase B: A 10 g/l solution of phosphoric acid R in acetonitrile R1. Solvent mixture: Acetonitrile R1 - water for chromatography (20 : 80). Test solution: Dissolve 25 mg of the substance to be examined in the solvent mixture and dilute to 25.0 ml with the solvent mixture. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with the solvent mixture. Dilute 2.0 ml of this solution to 10.0 ml with the solvent mixture. Reference solution (2): Dissolve the contents of a vial of bisoprolol for peak identification RS (containing impurities A and E) in 1.0 ml of the solvent mixture. Reference solution (3): Dissolve the contents of a vial of bisoprolol for system suitability RS (containing impurity G) in 1.0 ml of the solvent mixture. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Column temperature: 20 °C ± 2 °C. Detector: A spectrophotometer set at 225 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0-4

95

5

4-8

95 → 80

5 → 20

8 - 15

80

20

15 - 34

80 → 20

20 → 80

34 - 36

20

80

Identification of impurities: Use the chromatogram supplied with bisoprolol for peak identification RS and the chromatogram obtained with reference solution (2) to identify the peaks due to fumaric acid and impurities A, E; use the chromatogram supplied with bisoprolol for system suitability RS and the chromatogram obtained with reference solution (3) to identify the peak due to impurity G. Relative retention with reference to bisoprolol (retention time = about 18 min): Impurity A = about 0.5; impurity G = about 1.1; impurity E = about 1.2. System suitability: In the chromatogramobtained with reference solution (3), peak-to-valley ratio (Hp/Hv) is at least 2.5, where Hp = height above the baseline of the peak due to impurity G and Hv = height above the baseline of the lowest point of the curve separating this peak from the peak due to bisoprolol. Limits: Impurity G: The area of the peak due to impurity G is not more than 2.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). Impurity A: The area of the peak due to impurity A is not more than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.3%). Impurity E: The area of the peak due to impurity E is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%). Any other impurity: For each impurity, the area is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). The sum of the peak areas of all impurities is not more than 2.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). Disregard any peak with an area less than 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%) and disregard the peak due to fumaric acid. Note: Impurity A: (2RS)-1-(4-hydroxymethyl-phenoxy)-3-isopropylaminopropan-2-ol. Impurity B: (2RS)-1-isopropylamino-3-[4-(2-propoxyethoxymethyl)phenoxy]propan-2-ol. Impurity C: 1-[4-[4-(2-hydroxy-3-isopropylamino-propoxy) benzyl]phenoxy]-3- isopropylaminopropan-2-ol. Impurity D: 1-[4-[4-(2-hydroxy-3-isopropylaminopropoxy) benzyloxylmethyl]phenoxy]-3- isopropylaminopropan-2-ol. Impurity E: (EZ)-[3-[4-(2-isopropoxy-ethoxymethyl)phenoxy] allyl]isopropylamine. Impurity F: (2RS)-2-[4-(2-isopropoxy-ethoxymethyl)phenoxy]3-isopropylaminopropan-2-ol. Impurity G: (2RS)-1-[4-[[(2-isopropoxyethoxy)methoxy] methyl]phenoxy]-3- isopropylaminopropan-2-ol. Impurity K: 2-isopropoxyethyl 4-[[(2RS)-2-hydroxy-3(isopropylamino)propyl]oxy]benzoate. Impurity L: 4-[[(2RS)-2-hydroxy-3-(isopropylamino)propyl] oxy]benzaldehyde.

143

VP V

BORIC ACID Impurity N: (2RS)-1-[4-[(2-ethoxyethoxy)methyl]phenoxy]-3isopropylaminopropan-2-ol. Impurity Q: (2RS)-1-(isopropylamino)-3-[4-(2-methoxyethoxy) methyl]phenoxypropan-2-ol. Impurity R: (2RS)-1-(isopropylamino)-3-(4-methylphenoxy) propan-2-ol. Impurity S: 4-hydroxybenzaldehyde. Impurity T: 4-[(3-isopropyl-2-oxo-1,3-oxazolidin-5-yl)methoxy] benzaldehyde. Impurity U: 5-[[4-(hydroxymethyl)phenoxy]methyl]-3-isopropyl -1,3-oxazolidin-2-one.

Water Not more than 0.5% (Appendix 10.3). Determined on 1.000 g.

Assay Dissolve 0.300 g of the substance to be examined in 50 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 38.35 mg of C40H66N2O12. Storage Store in an airtight container, protected from light. Action and use Beta-adrenoceptor antagonist. Preparations Tablets. BORIC ACID Acidum boricum

Solubility in ethanol (96%) Dissolve 1.0 g in 10 ml of boiling ethanol (96%) R. The solution is not more opalescent than reference suspension II (Appendix 9.2) and is colourless (Appendix 9.3, method 2).

Sulfates Not more than 0.045% (Appendix 9.4.14). 10 ml of solution S diluted to 15 ml with water complies with the limit test for sulfates. Heavy metals Not more than 15 ppm (Appendix 9.4.8) 12 ml of solution S complies with limit test for heavy metals, method 1. Prepare the standard using a mixture of 2.5 ml of lead standard solution (2 ppm Pb) R and 7.5 ml of water. Assay Dissolve 1.000 g with heating in 100 ml of water containing 15 g of mannitol R. Titrate with 1 N sodium hydroxide VS, using 0.5 ml of phenolphthalein solution R as indicator, until a pink colour is obtained. 1 ml of 1 N sodium hydroxide VS is equivalent 61,80 mg H3BO3. BORIC ACID OINTMENT (10%) Unguentum Acidi borici 10%

M. 61.8  

Boric acid contains not less than 99.0% and not more than 100.5% of H3BO3.

Characters A white, crystalline powder, colourless, shiny plates greasy to the touch, or white crystals. Soluble in water and in ethanol (96%), freely soluble in boiling water and in glycerol (85%). Identification A. Dissolve 0.1 g by gently heating in 5 ml of methanol R, add 0.1 ml of sulfuric acid R. Ignite the solution, the flame has a green border. B. Solution S (see Appearance of solution) is acid. 144

pH The pH of solution S is 3.8 to 4.8 (Appendix 6.2).

Organic matter It does not darken on progressive heating to dull redness.

Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g.

H3BO3

Appearance of solution Solution S: Dissolve 3.3 g in 80 ml of boiling distilled water, cool and dilute to 100 ml with carbon dioxide-free water R. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2).

Boric acid ointment intended for external use contains boric acid which is previously very finely powdered and passed through a sieve No. 125.

Composition Boric acid (very finely powdered) Excipients emulsified



qs

10 g 100 g

The ointment complies with the requirements stated under "Topical semi-solid preparations" (Appendix 1.12) and with the following requirements.

Content of boric acid, H3BO3, 9.0% to 11.0%. Characters A white or pale yellow ointment.

VP V

Identification Place 1 g of the ointment in a porcelain dish, add 4 ml of ethanol R and one drop of sulfuric acid R. Ignite the mixture while stirring with a glass rod. The flame has a green border. Assay Weigh accurately about 1.0 g of the ointment in a beaker, add 20 ml of water and 20 ml of glycerol R previously neutralised to phenolphthalein solution R with 0.1 N sodium hydroxide VS and mix. Heat on a water bath to dissolve, and mix. Titrate with 0.1 N sodium hydroxide VS, using phenolphthalein solution R as indicator, until a permanent pink color is produced. Each ml of 0.1 N sodium hydroxide VS is equivalent to 6.18 mg of H3BO3. Storage Store in an airtight glass or porcelain container.

BROMHEXINE HYDROCHLORIDE

1 ml of 0.1 N sodium hydroxide VS is equivalent to 6.18 mg of H3BO3.

Microbial contamination The solution complies with the requirements under Microbial limit test (Appendix 13.6). If the solution is used for eye washing it complies with the requirements under Test for sterility (Appendix 13.7).

Storage

Store in an airtight container.

Action and use Antiseptic. BROMHEXINE HYDROCHLORIDE Bromhexini hydrochloridum

BORIC ACID SOLUTION (3%) Solutio Acidi borici 3% Boric acid solution (3%) is an aqueous solution for external use containing boric acid.

Composition Boric acid 3 g Purified water sq 100 ml Dissolve boric acid in boiling water. Allow to cool, add sufficient water to produce 100 ml. Filter. The solution complies with the requirements stated under “Solutions” (Appendix 1.3) and the following requirements. Content of boric acid, H3BO3, 95.0% to 105.0% of the stated amount. Characteristics A clear, colourles and odourless solution, slightly acidic. Identification A. Put 3 ml of the solution into a porcelain dish, heat in a water-bath to dryness. Add 5 ml of methanol R, swirl to dissolve (heat on a water-bath if necessary). Add 0.1 ml of sulfuric acid R and ignite. The flame has a green border. B. To 5 ml add 1 drop of methyl orange solution R, a yellow colour is produced. Add 5 ml of glycerol R, the colour changes to orange-red. Assay Measure accurately 5 ml, add 20 ml of glycerol R previously neutralised (use the phenolphthalein solution R as indicator) and titrate with 0.1 N sodium hydroxide VS until a pink colour is obtained. Then add 5 ml of glycerol R previously neutralised to phenolphthalein solution R, if the pink colour disappears continue to titrate with 0.1 N sodium hydroxide VS until a pink colour persists.

C14H21Br2ClN2

M. 412.6 

Bromhexine hydrochloride is N-(2-amino-3,5-dibromobenzyl)-N-methylcyclohexanamine hydrochloride. It contains not less than 98.5% and not more than 101.5% of C14H21Br2ClN2, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder, polymorphism. Very slightly soluble in water, slightly soluble in ethanol (96%) and in dichloromethane. Identification Apply one of the two following identifications: First identification: A, E. Second identification: B, C, D, E. A. Infrared absorption spectrophotometry (Appendix 4.2) of the substance to be examined is concordant with the spectrum obtained with bromhexine hydrochloride RS. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in methanol R, evaporate to dryness and record new spectra using the residues. B. Examin by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel F254. Mobile phase: Glacial acetic acid - water - butanol (17 : 17 : 66) Test solution: Dissolve 20 mg of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. 145

VP V

BROMHEXINE HYDROCHLORIDE TABLETS

Reference solution: Dissolve 20 mg of bromhexine hydrochloride RS in methanol R and dilute to 10 ml with methanol R. Procedure: Apply separately to the plate 20 µl of each solution. Develop over 3/4 of the plate. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. C. Dissolve about 25 mg in a mixture of 1 ml of a 10% solution of sulfuric acid R and 50 ml of water. Add 2 ml of dichloromethane R and 5 ml of a 2% solution of chloramine T R and shake. A brownish-yellow colour develops in the lower layer. D. Dissolve about 1 mg in 3 ml of 0.1 M hydrochloric acid. The solution gives the reaction of primary aromatic amine (Appendix 8.1). E. Dissolve about 20 mg in 1 ml of methanol R and add 1 ml of water. The solution gives reaction (A) of chloride (Appendix 8.1).

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Mix 0.5 ml of phosphoric acid R in 950 ml of water, adjust to pH 7.0 with triethylamine R (about 1.5 ml) and dilute to 1000 ml with water. Mix 20 volumes of this solution with 80 volumes of acetonitrile R. Test solution: Dissolve 50 mg of the substance to be examined in methanol R and dilute to 10.0 ml with the same solvent. Reference solution (1): Dissolve 5 mg of bromhexine impurity C RS in methanol R, add 1.0 ml of the test solution and dilute to 10.0 ml with the same solvent. Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with methanol R. Dilute 1.0 ml of this solution to 10.0 ml with methanol R. Chromatographic system: A column (12 cm × 4.6 mm) packed with end-capped octadecylsilyl silica gel for chromatography (3 µm). Detector: A spectrophotometer set at 248 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: The run time is 2.5 times the retention time of bromhexine. Relative retention with reference to bromhexine (retention time = about 11 min), impurity A = about 0.1; impurity B = about 0.2; impurity C = about 0.4; impurity D = about 0.5. System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to impurity C and bromhexine is not less than 12.0. Limits: In the chromatogram obtained with the test solution, the area of any secondary peak is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (2) (0.2%), and not more than one such peak has an area greater than the area of the principal 146

peak in the chromatogram obtained with reference solution (2) (0.1%); the sum of areas of all impurities is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.3%). Disregard any peak with an area not greater than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Note: Impurity A: (2-amino-3,5-dibromophenyl)methanol. Impurity B: 2-amino-3,5-dibromobenzaldehyde. Impurity C: N-(2-aminobenzyl)-N-methylcyclo hexanamine. Impurity D: N-(2-amino-5-bromobenzyl)-N-methylcyclohexanamine.

Loss on drying Not more than 1.0% (Appendix 9.6). (1.000 g; 100 °C to 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.300 g in 70 ml of ethanol (96%) and add 1 ml of 0.1 M hydrochloric acid R. Carry out a potentiometric titration (Appendix 10.2), using 0.1 N sodium hydroxide VS. Read the volume between the 2 points of inflexion. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 41.26 mg of C14H21Br2ClN2. Storage Protected from light. Action and use Mucolytic. Preparation Tablets. BROMHEXINE HYDROCHLORIDE TABLETS Tabellae Bromhexini hydrochloridi Bromhexine hydrochloride tablets contain bromhexine hydrochloride. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of bromhexine hydrochloride, C14H20Br2N2, HCl, 93.0% to 107.0% of the stated amount. Identification A. In the test for Uniformity of content, the ultraviolet absorption spectrum (Appendix 4.1) of the test solution in the range 230 nm to 350 nm exhibits absorption maxima at 249 nm and 310 nm. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is similar

VP V

to that of the principal peak in the chromatogram obtained with the reference solution.

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase and chromatographic system are described under the Assay. Test solution: Weigh accurately a quantity of powdered tablets containing about 50 mg of bromhexine hydrochloride, transfer into a 20-ml volumetric flask, add 15 ml of methanol R, sonicate to dissolve. Dilute to volume with methanol R, mix and filter. Reference solution: Dilute 1.0 ml of the test solution to 100.0 ml with methanol R, mix. Procedure: Inject the reference solution. Adjust the sensitivity of the system so that the height of the principal peak corresponds to 20% of the full scale of the recorder. Inject the test solution into the chromatographic system, record the chromatogram for about 3 times the retention time of bromhexine hydrochloride. The sum of any secondary peak areas in the chromatogram obtained with the test solution is not more than the area of the principal peak in the chromatogram obtained with the reference solution (1.0%). Uniformity of content (Appendix 11.2) Triturate one tablet in a mortar with ethanol (96%) R and transfer with the same solvent to a 50-ml volumetric flask. Warm on a water bath and shake occasionally. Allow to cool, dilute with ethanol (96%) R to volume and mix well. Filter, discard the first portion of the filtrate. Dilute 5.0 ml of the filtrate to 50.0 ml with ethanol (96%) R. Measure the absorbance of the resulting solution at the maximum at 249 nm (Appendix 4.1), in a 1-cm cell and using ethanol (96%) R as the blank. Calculate the content of bromhexine hydrochloride, C14H20Br2N2,HCl, in the tablets taking 270 as the value of A (1%, 1 cm) at the maximum at 249 nm. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of water. Rotation speed: 75 rpm. Time: 45 min. Procedure: Examine by liquid chromatography (Appendix 5.3). Mobile phase and chromatographic system are described under the test for Assay with volume of injection being 50 µl. Test solution: After the specified time, withdraw a sample of the medium, filter, discarding the first 20 ml of the filtrate. Reference solution: Weigh accurately about 16 mg of bromhexine hydrochloride, transfer into a 100-ml volumetric flask, add 4 ml of ethanol R, shake to dissolve, dilute to volume with water, mix well. Dilute the resulting solution with water to obtain a solution having the same concentration as the test solution.

BROMHEXINE HYDROCHLORIDE TABLETS

Calculate the content of bromhexine hydrochloride, C14H20Br2N2,HCl, dissolved using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C14H20Br2N2,HCl in bromhexine hydrochloride RS. Tolerance: Not less than 70% (Q) of the stated amount of bromhexine hydrochloride, C14H20Br2N2,HCl, is dissolved in 45 min.

Assay Examine by liquid chromatography (Appendix 5.3). Phosphate buffer solution: Dissolve 1.0 g of potassium dihydrogen phosphate R in 900 ml of water, adjust the pH to 7.0 with 0.5 M sodium hydroxide R, dilute to 1000 ml with water. Mobile phase: Phosphate buffer solution - acetonitrile (20 : 80). Adjust if necessary. Test solution: Weigh 20 tablets, calculate the average mass and finely powder. Weigh accurately a quantity containing 12.5 mg of bromhexine hydrochloride, transfer into a 25-ml volumetric flask, add 20 ml of methanol R, shake to dissolve completely. Dilute to volume with methanol R, mix well and filter. Reference solution: Dissolve a quantity of bromhexine hydrochloride RS in methanol R to obtain a solution having a known concentration of about 0.5 mg of bromhexine hydrochloride per ml. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Column temperature: 40 °C. Detector: A spectrophotometer set at 245 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject the reference solution. The resolution factor between bromhexine hydrochloride peak and adjacent impurity peak (if any) is not less than 1.5. The relative standard deviation of the peak areas for replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of bromhexine hydrochloride, C14H20Br2N2,HCl, in the tablets using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C14H20Br2N2, HCl in bromhexine hydrochloride RS. Storage Store in a cool and dry place, protected from light. Action and use Expectorant, mucolytic agent. Usual strength 8 mg. 147

VP V

BUPIVACAINE HYDROCHLORIDE

BUPIVACAINE HYDROCHLORIDE Bupivacaini hydrochloridum

Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2).

Acidity or alkalinity To 10 ml of solution S add 0.2 ml of 0.01 M sodium hydroxide VS, the pH is not less than 4.7. Add 0.4 ml of 0.01 M hydrochloric acid VS, the pH is not greater than 4.7. C18H28N2O,HCl,H2O

M. 342.9

Bupivacaine hydrochloride is (2RS)-1-butyl-N-(2,6dimethylphenyl) piperidine-2-carboxamide hydrochloride monohydrate. It contains not less than 98.5% and not more than 101.0% of C18H28N2O,HCl, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder or colourless crystals. Soluble in water, freely soluble in ethanol (96%). Melting point: about 254 °C, with decomposition. Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum obtained with bupivacaine hydrochloride RS. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Ammonia - methanol (0.1 : 100). Test solution: Dissolve 25 mg of the substance to be examined in methanol R and dilute to 5 ml with the same solvent. Reference solution: Dissolve 25 mg of bupivacaine hydrochloride RS in methanol R and dilute to 5 ml with the same solvent. Procedure: Apply to the plate 5 µl of each solution. Develop over a path of 10 cm. Allow the plate to dry in air. Spray with dilute potassium iodobismuthate solution R. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. C. Dissolve 0.1 g in 10 ml of water, add 2 ml of dilute sodium hydroxide solution R and shake with 2 quantities, each of 15 ml, of ether R. Dry the combined ether layers over anhydrous sodium sulfate R and filter. Evaporate the ether, recrystallise the residue from ethanol (90%) R and dry under reduced pressure. The crystals melt (Appendix 6.7) at 105 °C to 108 °C. D. It gives reaction (A) of chlorides (Appendix 8.1). Appearance of solution Solution S: Dissolve 1.0 g in carbon dioxide-free water R and dilute to 50 ml with the same solvent. 148

Related substances Examine by gas chromatography (Appendix 5.2). Internal standard solution: Dissolve 25 mg of methyl behenate R in methylene chloride R and dilute to 500 ml with the same solvent. Test solution: Dissolve 50.0 mg of the substance to be examined in 2.5 ml of water, add 2.5 ml of dilute sodium hydroxide solution R and extract with 2 quantities, each of 5 ml, of the internal standard solution. Filter the lower layer. Reference solution (1): Dissolve 10 mg of the substance to be examined, 10 mg of bupivacaine impurity B RS and 10 mg of bupivacaine impurity E RS in 2.5 ml of water, add 2.5 ml of dilute sodium hydroxide solution R and extract with 2 quantities, each of 5 ml, of the internal standard solution. Filter the lower layer and dilute to 20 ml with the internal standard solution. Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with the internal standard solution. Reference solution (3): Dilute 5.0 ml of reference solution (2) to 10.0 ml with the internal standard solution. Reference solution (4): Dilute 1.0 ml of reference solution (2) to 10.0 ml with the internal standard solution. Chromatographic system: A fused silica column (30 m × 0.32 mm) coated with poly (dimethyl)(diphenyl)siloxane (film thickness 0.25 µm). Split ratio: 1 : 12. Detection: Flame ionisation. Temperature:

Column

Time (min)

Temperature (°C)

0 0 - 10 10 - 15

180 180 → 230 230

Injection port

250

Detector

250

Volume of injection: 1 µl. Proceduce: Under the conditions described above, relative retention with reference to bupivacaine (retention time = about 10 min): impurity C = about 0.5; impurity A = about 0.6; impurity B = about 0.7; impurity D = about 0.8; impurity E = about 1.1; internal standard = about 1.4. System suitability: Inject reference solution (1), resolution between the peaks due to bupivacaine and impurity E is minimum 3.0.

VP V

Limits: Impurity B: Calculate the ratio (R1) of the area of the principal peak to the area of the peak due to the internal standard from the chromatogram obtained with reference solution (3); from the chromatogram obtained with the test solution, calculate the ratio of the area of the peak due to impurity B to the area of the peak due to the internal standard: this ratio is not greater than R1 (0.5%), Any other impurity: Calculate the ratio (R2) of the area of the principal peak to the area of the peak due to the internal standard from the chromatogram obtained with reference solution (4); from the chromatogram obtained with the test solution, calculate the ratio of the area of any peak, apart from the principal peak, the peak due to impurity B and the peak due to the internal standard, to the area of the peak due to the internal standard: this ratio is not greater than R2 (0.1%), Total: Calculate the ratio (R3) of the area of the principal peak to the area of the peak due to the internal standard from the chromatogram obtained with reference solution (2); from the chromatogram obtained with the test solution, calculate the ratio of the sum of the areas of any peaks, apart from the principal peak and the peak due to the internal standard, to the area of the peak due to the internal standard: this ratio is not greater than R3 (1.0%). Disregard limit: Ratio less than 0.01 times R3 (0.01%). Note: Impurity A: N-(2,6-dimethylphenyl)pyridine-2-carboxamide. Impurity B: (2RS)-N-(2,6-dimethylphenyl)piperidine-2carboxamide. Impurity C: 1-(2,6-dimethylphenyl)-1,5,6,7-tetrahydro-2Hazepin- 2-one. Impurity D: (2RS)-2,6-dichloro-N-(2,6-dimethylphenyl) hexanamide. Impurity E: 6-(butylamino)-N-(2,6-dimethylphenyl)hexanamide. Impurity F: 2,6-dimethylaniline.

2,6-Dimethylaniline Not more than 0.01%. Dissolve 0.50 g in methanol R and dilute to 10 ml with the same solvent. To 2 ml of the solution add 1 ml of a freshly prepared 1.0% solution of dimethylaminobenzaldehyde R in methanol R and 2 ml of glacial acetic acid R and allow to stand for 10 min. Any yellow colour in the solution is not more intense than that in a standard prepared at the same time and in the same manner using 2 ml of a 0.0005% solution of 2,6-dimethylaniline R in methanol R. Heavy metals Not more than 10 ppm (Appendix 9.4.8). Dissolve 2.0 g in a mixture of 15 volumes of water R and 85 volumes of methanol R and dilute to 20 ml with the same mixture of solvents. 12 ml of the solution complies with limit test 2. Prepare the standard using lead standard solution (1 ppm Pb) obtained by diluting lead standard solution (100 ppm Pb) R with a mixture of 15 volumes of water R and 85 volumes of methanol R.

CAFFEINE

Loss on drying 4.5% to 6.0% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.250 g in a mixture of 20 ml of water and 25 ml of ethanol (96%) R. Add 5.0 ml of 0.01 M hydrochloric acid VS. Carry out a potentiometric titration (Appendix 10.2), using 0.1 M ethanolic sodium hydroxide VS. Read the volume added between the 2 points of inflexion. 1 ml of 0.1 M ethanolic sodium hydroxide VS is equivalent to 32.49 mg of C18H28N2O,HCl. Storage Protected from light. Action and use Local anaesthetic. Preparation Injection. CAFFEINE Caffeinum

C8H10N4O2

M. 194.2

Caffeine is 1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6dione. It contains not less than 98.5% and not more than 101.5% of C8H10N4O2, calculated with reference to the dried substance.

Characters A white or almost white, crystalline powder or silky, white or almost white, crystals. Sublimes readily. Sparingly soluble in water, freely soluble in boiling water. Slightly soluble in ethanol (96%). It dissolves in concentrated solutions of alkali benzoates or salicylates. Identification Apply one of the two following identifications. First identification: A, B, F. Second identification: B,C, D, E, F. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of caffeine RS. 149

VP V

CAFFEINE

B. Melting point: 234 °C to 239 °C (Appendix 6.7). C. To 2 ml of a saturated solution add 0.05 ml of iodine iodide solution R. The solution remains clear. Add 0.1 ml of dilute hydrochloric acid R, a brown precipitate is formed. Neutralise with dilute sodium hydroxide solution  R, the precipitate dissolves. D. In a ground-glass-stoppered tube, dissolve about 10.0 mg of the substance to be examined in 0.25 ml of a mixture of 0.5 ml of acetylacetone R and 5 ml of dilute sodium hydroxide solution R. Heat in a water-bath at 80 °C for 7 min. Cool and add 0.5  ml of  dimethylaminobenzaldehyde solution  R2. Heat again in a water-bath at 80 °C for 7 min. Allow to cool and add 10 ml of water, an intense blue colour develops. E. It gives the reaction of xanthines (Appendix 8.1). F. It complies with the test for Loss on drying. Appearance of solution Solution S: Dissolve 0.5 g with heating in 50 ml of carbon dioxide-free water  R prepared from distilled water, cool and dilute to 50 ml with the same solvent. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2).

Acidity To 10 ml of solution S add 0.05 ml of bromothymol blue solution R1; the solution is green or yellow. Not more than 0.2 ml of 0.01 N sodium hydroxide VS is required to change the colour of the indicator to blue. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Tetrahydrofuran - acetonitrile - buffer solution pH 4.5 (20 : 25 : 955). Buffer solution pH 4.5: A 0.082% solution of  sodium acetate R adjusted to pH 4.5 with glacial acetic acid R. Test solution:  Dissolve 0.100  g of the substance to be examined in the mobile phase and dilute to 50.0 ml with the same solvent. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (1): Dilute 2.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (2): Dissolve 5 mg of caffeine for system suitability RS  (containing impurities A, C, D and F) in the mobile phase and dilute to 5.0  ml with the same solvent. Dilute 2.0 ml of this solution to 10.0 ml with the mobile phase. Chromatographic system: A column (15 cm × 4.6 mm) packed with base-deactivated end-capped octadecylsilyl silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 275 nm. Flow rate: 1.0  ml/min. Volume of injection: 10 µl. Procedure: The run time is 1.5 times the retention time of caffeine. 150

Identification of impurities:  Use the chromatogram supplied with caffeine for system suitability RS and the chromatogram obtained with reference solution (2) to identify the peaks due to impurities A, C, D and F. Retention time of caffeine = about 8 min. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurities C and D is at least 2.5 and that is at least 2.5 between the peaks due to impurities F and A. Limits: Unspecified impurities: For each impurity, the peak area is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). Sum of the peak areas of all impurities is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%). Disregard any peak with an area less than 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Notes: Impurity A: 1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione (theophylline). Impurity B: N-(6-amino-1,3-dimethyl-2,4-dioxo-1,2,3,4tetrahydropyrimidin-5-yl)formamide. Imputity C: 1,3,9-trimethyl-3,9-dihydro-1H-purine-2,6-dione (isocaffeine). Impurity D: 3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione (theobromine). Impurity E: N,1-dimethyl-4-(methylamino)-1H-imidazole-5carboxamide (caffeidine). Impurities F: 1,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione.

Sulfates  Not more than 0.05% (Appendix 9.4.14). Determined on 15 ml of solution S. Prepare the standard using a mixture of 7.5 ml of sulfate standard solution (10 ppm SO4) R and 7.5 ml of distilled water.  Heavy metals  Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with the limit test for heavy metals, method 3. Prepare the reference solution using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying  Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C; 1 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 170  mg of the substance to be examined with heating in 5 ml of anhydrous acetic acid R. Allow to cool, add 10 ml of acetic anhydride R and 20 ml of toluene R. Titrate with 0.1 N perchloric acid VS, determining the endpoint potentiometrically (Appendix 10.2).

VP V

1 ml of 0.1 N perchloric acid VS is equivalent to 19.42 mg of C8H10N4O2.

Storage Store in a well-closed container. Action and use Central nervous system stimulant, diuretic. Preparations Aspirin and Caffeine tablets. CAFFEINE AND SODIUM BENZOATE INJECTION Injectio Coffeini et Natrii benzoas It is a sterile solution of caffeine and sodium benzoate in water for injection. The injection complies with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements:

Content of caffeine, C8H10N4O2, 90.0% to 110.0% of the stated amount. Content of sodium benzoate, C7H5NaO2, 90.0% to 110.0% of the stated amount. Characters Clear and colourless solution. Identification A. The infrared absorption spectrum (Appendix 4.2) of the residue obtained in the Assay is concordant with the spectrum of caffeine RS. B. Dip the end of a platinum wire or a glass rod in a portion of the injection. Introduce it into a colourless flame. The flame is colour intensely yellow. C. To 0.5 ml of the injection, add a few drops of a 10.5% solution of ferric chloride R, a pink precipitate is produced. D. To 5 ml of the injection, add 0.3 ml of hydrochloride acid R, a white precipitate is produced.

CALCITRIOL

chloroform extract through a filter previously moistened with chloroform R into a tared dish (retain the water layer for the Assay for sodium benzoate). Wash the separating funnel and the filter with 10 ml of hot chloroform R, add the washings to the dish above. Evaporate the combined chloroform on a water bath, add 2 ml of ethanol R just before the last trace of chloroform is expelled. Complete the evaporation of the solvent, dry the residue, consisting of C8H10N4O2, at 80 °C for 4 h, allow to cool and weigh. For sodium benzoate: To the water layer obtained in the Assay for caffeine, add 75 ml of ether R and 5 drops of methyl orange solution R. Titrate with 0.1 N hydrochloric acid VS, mixing the liquids by vigorous shaking, until a permanent pink color is produced in the water layer. Each ml of 0.1 N hydrochloric acid VS is equivalent to 14.41 mg of C7H5NaO2.

Storage Protected from light. Action and use Central nervous system stimulant and diuretic agent. Usual strength 250 mg of caffeine and 350 mg of sodium benzoate in 1 ml of the injection. CALCITRIOL Calcitriolum

pH 6.5 to 8.5 (Appendix 6.2). Endotoxine Not more than 0.7 EU/mg of caffeine and sodium benzoate, based on the total, in mg, of the stated amounts (Appendix 13.2). Assay For caffeine: Measure accurately a volume of the injection, equivalent to about 0.21 g of caffeine, transfer to a small separating funnel, add 5 ml of water, 1 drop of phenolphthalein solution R as indicator, and add 0.1 N sodium hydroxide R, dropwise, until a permanent pink colour is just produced. Extract the mixture with at least three quantities, each of 20 ml, of chloroform R. Filter each

C27H44O3  

M. 416.6

Calcitriol is (5Z,7E)-9,10-secocholesta-5,7,10(19)-triene1α,3β,25-triol. It contains not less than 97.0% and not more than 103.0% of C27H44O3.

Characters White or almost white crystals. It is sensitive to air, heat and light. In solution, depending on temperature and time, a reversible isomerisation to pre-calcitriol takes place. Freely soluble in ethanol (96%), soluble in fatty oils, practically insoluble in water. 151

VP V

CALCITRIOL SOFT CAPSULES

Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of calcitriol RS. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of the principal peak in the chromatogram obtained with reference solution (1). Related substances Examine by liquid chromatography (Appendix 5.3). Carry out the test as rapidly as possible, avoiding exposure to actinic light and air. Mobile phase: A 0.1% solution of tris (hydroxymethyl) aminomethane R adjusted to pH 7.0 to 7.5 with a mixture of phosphoric acid - acetonitrile (45 : 55). Test solution: Dissolve 1.00 mg of the substance to be examined without heating in 10.0 ml of the mobile phase. Reference solution (1): Dissolve 1.00 mg of calcitriol RS without heating in 10.0 ml of the mobile phase. Reference solution (2): Dilute 1.0 ml of reference solution (1) to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (3): Heat 2 ml of reference solution (1) at 80 °C for 30 min. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase B (5 µm). Column temperature: 40 °C. Detector: A spectrophotometer set at 230 nm. Flow rate: 1 ml/min. Volume of injection: 50 µl. Procedure: The run time is twice the retention time of calcitriol. Relative retention with reference to calcitriol (retention time = about 14 min): Impurity C = about 0.4; pre-calcitriol = about 0.88; impurity A = about 0.95; impurity B = about 1.1. System suitability: In the chromatogram obtained with reference solution (1), the number of theoretical plates calculated for the peak due to calcitriol is not less than 10 000. In the chromatogram obtained with reference solution (3), the resolution between the peaks due to precalcitriol and calcitriol is at least 3.5. Limits: Use the normalisation procedure to calculate percentage of impurities. Impurities A, B, C: For each impurity, not more than 0.5%. Unspecified impurities: For each impurity, not more than 0.10%. Total: Not more than 1.0%. Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%) and disregard the peak due to pre-calcitriol. 152

Note: Impurity A: (5E,7E)-9,10-secocholesta-5,7,10(19)-triene-1α,3β,25 -triol (trans-calcitriol). Impurity B: (5Z,7E)-9,10-secocholesta-5,7,10(19)-triene-1β,3β, 25-triol (1β-calcitriol). Impurity C: (6aR,7R,9aR)-11-[(3S,5R)-3,5-dihydroxy-2-methylcyclohex-1-enyl]-7-[(1R)-5-hydroxy-1,5-dimethylhexyl]6a-methyl-2-phenyl-5,6,6a,7,8,9,9a,11-octahydro-1H,4aHcyclopenta[f][1,2,4]triazolo[1,2-a]cinnoline-1,3(2H)-dione (triazoline adduct of pre-calcitriol).

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase, chromatographic system, test solution, reference solution (1) as described in the test for Related substances. Inject the test solution and reference solution (1). System suitability: In the chromatogram obtained with reference solution (1), the relative standard deviation of the peak areas for 6 replicate injections is not more than 1.0%. Calculate the content of C27H44O3, using the chromatograms obtained with the test solution, reference solution (1) and the declared content of C27H44O3 in calcitriol RS. Storage Under nitrogen, protected from light, at a temperature of 2 °C to 8 °C. The contents of an opened container are to be used immediately. Action and use Vitamin D analogue. Preparation Capsules. CALCITRIOL SOFT CAPSULES Molles capsulae calcitrioli Calcitriol soft capsules contain a solution of calcitriol in a suitable fixed-oil. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of calcitriol, C27H44O3, 90.0% to 110.0% of the stated amount. Identification In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that of the calcitriol peak in the chromatogram obtained with the reference solution.

VP V

CALCIUM CARBONATE

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Hexane - tetrahydrofuran - ethanol (59 : 40 : 1). Test solution: Mix the contents of at least 20 capsules. Transfer an accurately weighed quantity of the resulting mixture containing the equivalent of 5 µg of calcitriol to a 10 ml volumetric brown-glass flask, dilute to volume with the mobile phase, mix well. Reference solution: A 0.5 µg/ml solution of calcitriol RS in the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase A (5 μm) (A Lichrosorb Si60 column is suitable). Detector: A spectrophotometer set at 265 nm. Flow rate: 1.0 ml/min. Volume of injection: 100 μl. Procedure: Inject alternately the reference solution and the test solution. Calculate the content of calcitriol, C27H44O3, in the capsules using the peak areas due to calcitriol in the chromatograms obtained with the test solution, the reference solution and the declared content of C27H44O3 in calcitriol RS. Storage Store in an airtight container, protected from light. Action and use Vitamin D analogue.

Chlorides Not more than 0.033% (Appendix 9.4.5). 3 ml of solution S diluted to 15 ml with water complies with the limit test for chlorides.  Sulfates Not more than 0.25% (Appendix 9.4.14). 1.2 ml of solution S diluted to 15 ml with water complies with the limit test for sulfates. Arsenic Not more than 4 ppm (Appendix 9.4.2). 5.0 ml of solution S complies with the limit test for arsenic, method A. Barium To 10 ml of solution S add 10 ml of calcium sulfate solution R. After at least 15 min, any opalescence in the solution is not more intense than that in a mixture of 10 ml of solution S and 10 ml of distilled water. Iron Not more than 0.02% (Appendix 9.4.13). Dissolve 50 mg in 5 ml of dilute hydrochloric acid R and dilute to 10 ml with water. The solution complies with the limit test for iron.

Usual strength 0.25 µg; 0.5 µg. CALCIUM CARBONATE Calcii carbonas CaCO3

Substances insoluble in acetic acid Not more than 0.2%. Wash any residue obtained during the preparation of solution S with 4 quantities, each of 5 ml, of hot water and dry at 100 °C to 105 °C for 1 h. The residue weighs not more than 10 mg.

M. 100.1

Calcium carbonate contains not less than 98.5% and not more than 100.5% of CaCO3, calculated with reference to the dried substance.

Characters A white or almost white powder. Practically insoluble in water. Identification A. It gives the reaction of carbonates (Appendix 8.1). B. Solution S: Dissolve 5.0  g in 80  ml of  dilute acetic acid  R. When the effervescence ceases, boil for 2 min. Allow to cool and dilute to 100 ml with dilute acetic acid  R, filter through a sintered-glass filter. The filtrate is solution S. 0.2 ml of solution S gives the reactions of calcium (Appendix 8.1). The residue kept for the test for Substances insoluble in acetic acid.

Magnesium and alkali metals Not more than 1.5%. Dissolve 1.0  g in 12  ml of  dilute hydrochloric acid  R. Boil the solution for about 2 min and add 20 ml of water, 1 g of ammonium chloride  R and 0.1 ml of methyl red solution R. Add dropwise of a 10% solution of ammonia R until the colour of the indicator changes and then add 2.0 ml of a 10% solution of ammonia R. Heat to boiling and add 50 ml of a hot 4% solution of ammonium oxalate R. Allow to stand for 4 h, dilute to 100 ml with water and filter through a suitable filter. To 50 ml of the filtrate add 0.25 ml of sulfuric acid R and evaporate to dryness on a water-bath. Ignite the residue to constant mass at (600 ± 50 °C). The residue weighs not more than 7.5 mg. Heavy metals Not more than 20 ppm (Appendix 9.4.8). 12 ml of solution S complies with the limit test for heavy metals, method 1. Prepare the reference solution using lead standard solution (1 ppm Pb) R. Loss on drying Not more than 2.0% (Appendix 9.6). (1.000 g; 200 °C ± 10 °C). 153

VP V

CALCIUM AND VITAMIN D TABLETS

Assay Dissolve 0.150 g in a mixture of 3 ml of dilute hydrochloric acid R and 20 ml of water. Boil for 2 min, allow to cool and dilute to 50 ml with water. Carry out the complexometric titration of calcium (Appendix 10.5). 1 ml of 0.1 M sodium edetate VS is equivalent to 10.01 mg of CaCO3.

50 ml of water. Titrate the excess of disodium edetate with 0.05 M zinc chloride solution VS using eriochrome black T solution R as indicator. Each ml of 0.05 M disodium edetate solution VS is equivalent to 2.004 mg of Ca.

Content of calcium, Ca, 85.0% to 115.0% of the stated amount.

For colecalciferol Examine by liquid chromatography (Appendix 5.3). Carry out the test protected from light. Mobile phase: Methanol - water (97 : 3). Reference solution: A solution in methanol contains accurately about 20 IU of colecalciferol RS per ml. Test solution: Weigh a quantity of powdered tablets containing the equivalent of about 2000 IU of colecalciferol, accurately weighed, put into a 100 ml volumetric flask, add about 80 ml of methanol (90%), shake for 5 min and sonicate for 5 min. Add methanol (90%) to volume, mix well. Centrifuge and filter. Use the filtrate. Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 264 nm. Flow rate: 1.0 ml/min. Volume of injection: 50 µl. Procedure: Inject alternately the test solution and the reference solution. Calculate the content of colecalciferol, C27H44O, in the tablets using the peak areas (or heights) in the chromatograms obtained with the test solution, the reference solution and the declared content of C27H44O in the colecalciferol RS. Note: This method does not apply to the tablets containing microcapsule material.

Content of colecalciferol, C27H44O, 90.0% to 120.0% of the stated amount.

Storage Store at a cool and dry place, protected from light.

Identification A. Weigh a quantity of the powdered tablets containing the equivalent of about 40 mg of calcium add 10 ml of 2 M acetic acid R. The effervescence is produced. Add 10 ml of water, shake well and filter. The filtrate yields reactions characteristic of calcium (Appendix 8.1). B. In the Assay for colecalciferol, the principal peak in the chromatogram obtained with the test solution has the same retention time as the peak due to colecalciferol in the chromatogram obtained with the reference solution.

Action and use Calcium and vitamin D supplement.

FUNCTIONALITY-RELATED CHARACTERISTICS The characteristics of Particle-size distribution and Powder flow may be relevant for calcium carbonate used as filler in tablets and capsules. Storage Store in a well-closed container and in dry place. Action and use Antacid. Preparations Tablets; calcium and colecalciferol tablets. CALCIUM AND VITAMIN D3 TABLETS Tabellae Calcii carbonatis et Vitamini D3 Calcium and vitamin D3 tablets contain calcium carbonate and colecalciferol. They may be coated. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Assay For calcium: Weigh 20 tablets (remove the coating in case of coating tablets), calculate the average mass, and powder finely. To a quantity of the powder containing the equivalent of 50 mg of calcium, accurately weighed, add 50 ml of water and 5 ml of hydrochloric acid R. Heat gently to boiling and continue to boil for about 2 min. Allow to cool and add 50.0 ml of 0.05 M disodium edetate solution VS. Neutralise the solution with 2 M sodium hydroxide R, add 10 ml of ammonia buffer pH 10.0 R and 154

Usual strength 500 mg of calcium and 200 IU of vitamin D. CALCIUM CHLORIDE DIHYDRATE Calcii chloridum dihydricum CaCl2,2H2O

M. 147.0

Calcium chloride dihydrate contains not less than 97.0% and not more than 103.0% of CaCl2,2H2O.

Characters White, crystalline powder, hygroscopic. Freely soluble in water, soluble in ethanol (96%). Identification A. Solution S (see Appearance of solution) gives reaction (A) of chlorides (Appendix 8.1).

VP V

B. It gives the reactions of calcium (Appendix 8.1). C. It complies with the limits of the Assay.

Appearance of solution Solution S: Dissolve 10.0 g in carbon dioxide-free water R and dilute to 100 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution Y6 (Appendix 9.3, method 2). Acidity or alkalinity To 10 ml of freshly prepared solution S add 0.1 ml of phenolphthalein solution R. If the solution is red, not more than 0.2 ml of 0.01 M hydrochloric acid VS is required to discharge the colour. If the solution is colourless, not more than 0.2 ml of 0.01 M sodium hydroxide VS is required to turn it red. Sulfates Not more than 0.03% (Appendix 9.4.14). Dilute 5 ml of solution S to 15 ml with water. Aluminium To 10 ml of solution S add 2 ml of a 10% solution of ammonium chloride R and 1 ml of dilute ammonia R and boil the solution. No turbidity or precipitate is formed. If intended for use in the manufacture of dialysis solutions, it complies with the following limit test for aluminium (Appendix 9.4.9) which replaces the test prescribed above. Maximum 1 ppm. Test solution: Dissolve 4 g in 100 ml of water and add 10 ml of acetate buffer solution pH 6.0 R. Reference solution: Mix 2 ml of aluminium standard solution (2 ppm Al), 10 ml of acetate buffer solution pH 6.0 and 98 ml of water. Blank solution: Mix 10 ml of acetate buffer solution pH 6.0 and 100 ml of water. Barium To 10 ml of solution S add 1 ml of calcium sulfate solution R. After at least 15 min, any opalescence in the solution is not more intense than that in a mixture of 10 ml of solution S and 1 ml of water. Heavy metals Not more than 20 ppm (Appendix 9.4.8). 12 ml of solution S complies with the limit test for heavy metals, method 1. Prepare the reference solution using lead standard solution (2 ppm Pb) R. Iron Not more than 10 ppm (Appendix 9.4.13). 10 ml of solution S complies with the limit test for iron. Magnesium and alkali metals Not more than 0.5%. To a mixture of 20 ml of solution S and 80 ml of water add 2 g of ammonium chloride R and 2 ml of a 10% solution of

CALCIUM CHLORIDE INJECTION (10%)

ammonia R, heat to boiling and pour the boiling solution into a hot solution of 5 g of ammonium oxalate R in 75 ml of water. Allow to stand for 4 h, dilute to 200 ml with water and filter. To 100  ml of the filtrate add 0.5 ml of sulphuric acid R. Evaporate to dryness on a water-bath and ignite to constant mass at 600 °C. The residue weighs a maximum of 5 mg.

Assay Dissolve 0.280 g in 100 ml of water and carry out the complexometric titration of calcium (Appendix 10.5). 1 ml of 0.1M Trilon B solution VS is equivalent to 14.70 mg of CaCl2,2H2O. Srorage In an airtight container. Action and use Mineral. Preparation Injection. CALCIUM CHLORIDE INJECTION (10%) Injectio Calcii chloridi 10% Calcium chloride injection is a sterile solution of calcium chloride in water for injections. The injection complies with the general requirements prescribed under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Content of calcium chloride, CaCl2,2H2O, 95.0% to 105.0% of the stated amount. Characters A clear, colourless solution or it is not more intensely coloured than reference solution BY6 (Appendix 9.3, method 2). Identification A. To 1 ml of the injection, add a few drops of a 4% solution of ammonium oxalate R, a white precipitate is produced which is sparingly soluble in 6 M acetic acid R, but is soluble in hydrochloric acid R. B. It gives the reactions of chlorides (Appendix 8.1). pH 5.0 to 8.0 (Appendix 6.2). Bacterial endotoxins Not more than 0.2 EU/mg calcium chloride (Appendix 13.2). Assay Transfer an accurately measured volume of the injection containing the equivalent of about 0.3 g of calcium chloride to a 500 ml conical flask, dilute with water to 300 ml. Add 155

VP V

CALCIUM GLUCONATE

6 ml of 10 M sodium hydroxide R and 15 mg of calcon mixture R. Titrate with 0.1 M Trilon B solution VS until the colour changes from violet to full blue. 1 ml of 0.1 M Trilon B solution VS is equivalent to 14.7 mg of CaCl2,2H2O.

Storage Store at a cool and dry place.

Organic impurities and boric acid Introduce 0.5  g of the substance to be examind into a porcelain dish previously rinsed with sulfuric acid R and placed in a bath of iced water. Add 2 ml of cooled sulfuric acid  R and mix. No yellow or brown colour develops. Add 1 ml of chromotrope II B solution R. A violet colour develops and does not become dark blue. The solution is not more intensely coloured than that of a mixture of 1 ml of chromotrope II B solution R and 2 ml of cooled sulfuric acid R.

Action and use Treatment of blood calcium deficiency. Usual strength 10%. CALCIUM GLUCONATE Calcii gluconas

C12H22CaO14, H2O

M. 448.4

Calcium gluconate is calcium D-gluconate monohydrate. It contains not less than 98.5% and not more than 102.0% of C12H22CaO14.H2O.

Characters A white or almost white, crystalline or granular powder, sparingly soluble in water, freely soluble in boiling water. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Ethyl acetate - concentrated ammonia water - ethanol (96%) (10 : 10 : 30 : 50). Test solution: Dissolve 20  mg of the substance to be examined in 1 ml of water, heating if necessary in a waterbath at 60 °C. Reference solution: Dissolve 20 mg of calcium gluconate RS in 1 ml of water, heating if necessary in a water-bath at 60 °C. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 10 cm. Remove the plate, dry at 100 °C for 20 min. Allow to cool and spray with a 5% solution of potassium dichromate R in a 40% (m/m) solution of sulfuric acid R. After 5 min, examine the chromatograms in daylight. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. B. Solution S: Dissolve 1.0  g of the substance to be examind in water heated to 60 °C and dilute to 50 ml with the same solvent. Solution S gives the reactions of calcium (Appendix 8.1). 156

Appearance of solution At 60 °C, solution S is not more intensely coloured than reference solution Y6 (Appendix 9.3, method 2). After cooling, it is not more opalescent than reference suspension II (Appendix 9.2)

Sucrose and reducing sugars Dissolve 0.5 g of the substance to be examined in a mixture of 2 ml of a 25% solution of hydrochloric acid R and 10 ml of water. Boil for 5 min. Allow to cool. Add 10 ml of a 10% solution of sodium carbonate R and allow to stand for 10 min. Dilute to 25 ml with water and filter. To 5 ml of the filtrate add 2 ml of Fehling reagent R and boil for 1 min. Allow to stand for 2 min. No red precipitate is formed. Chlorides Not more than 0.02% (Appendix 9.4.5). Dilute 12.5 ml of solution S to 15 ml with water. Sulfates Not more than 0.01% (Appendix 9.4.14). Dissolve 10.0  g of the substance to be examined with heating in a mixture of 10 ml of acetic acid R and 90 ml of distilled water. Magnesium and alkaline-earth metals Not more than 0.4% (Appendix 9.4.16). Dissolve 1.00 g of the substance to be examined in 100 ml of boiling water add 10 ml of a 10% solution of ammonium chloride R, 1 ml of ammonia R and, dropwise 50 ml of a hot 4% solution of ammonium oxalate R. Allow to stand for 4 h, dilute to 200 ml with water and filter. Evaporate 100  ml of the filtrate to dryness and ignite. The residue weighs not more than 2 mg. Heavy metals Not more than 10 ppm (Appendix 9.4.8). 2.0 g complies with the limit test for heavy metals, method 4. Heat the substance to be examined gradually and with care until it is almost completely transformed into a white mass and then ignite. Prepare the reference solution using 2 ml of lead standard solution (10 ppm Pb) R.

VP V

Microbial contamination (Appendix 13.6). (The total viable aerobic microbial count) TAMC: Not more than 103 CFU/g. The total viable yeast and mould count (TYMC): Not more than 102 CFU/g. Assay Dissolve 0.8000  g of the substance to be examined in 20 ml of hot water. Allow to cool and dilute to 300 ml with  water. Carry out the complexometric titration of calcium (Appendix 10.5). 1 ml of 0.1 M sodium edetate VS is equivalent to 44.84 mg of C12H22CaO14,H2O. Storage Store in a well-closed container. Action and use Treatment of calcium deficiency. Preparations Tablets, effervescent tablets. EFFERVESCENT CALCIUM GLUCONATE TABLETS Tabellae effervescenti Calcii gluconatis Effervescent calcium gluconate tablets contain calcium gluconate in a suitable effervescent basis. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of calcium gluconate, C12H22CaO14,H2O, 95.0% to 105.0% of the stated amount. Identification A. Dissolve a quantity of the powdered tablets containing about 1 g of calcium gluconate in 20 ml of hot water, cool and filter. To 0.5 ml of the filtrate add 0.05 ml of 10.5% solution of iron (III) chloride R. An intense yellow colour is produced. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Ethyl acetate - concentrated ammonia water - ethanol (96%) (10 : 10 : 30 : 50). Test solution: Dissolve a quantity of the powdered tablets containing about 0.4 g of calcium gluconate in 20 ml water, heat if necessary in a water-bath at 60 °C, cool and filter. Reference solution: Dissolve 20 mg of calcium gluconate RS in 1 ml of water, heating if necessary in a water-bath at 60 °C Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm. After removal of the plate, dry the plate at 100 °C for 20 minutes.

CALCIUM GLUCONATE FOR INJECTION

Allow to cool and spray with a 5% solution of potassium dichromate R in a 40% (m/m) solution of sulfuric acid R. After 5 min, examine in daylight, the principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. C. Dissolve a quantity of the powdered tablets containing about 1 g of calcium gluconate in 20 ml of hot water, cool and filter. The filtrate gives reaction of calcium (Appendix 8.1). D. The tablets effervesce on the addition of water.

Assay Weigh and powder 20 tablets. Ignite a quantity of the powder containing about 500 mg of calcium gluconate, cool and dissolve the residue with gentle heating 5 ml of 2 M hydrochloric acid R. Filter, wash the residue on the filter with water and dilute the combined filtrate and washings to 50 ml with water. Neutralise with 5 M ammonia R, using methyl orange solution R as indicator, add 5 ml of 8 M sodium hydroxide R and titrate with 0.05 M disodium edetate VS using 300 mg of hydroxy naphthol blue R as indicator and continue the titration to a blue end point. Each ml of 0.05 M disodium edetate VS is equivalent to 22.42 mg of C12H22CaO14,H2O. Storage Store in a well-closed container, at a dry place, and protected from light. Action and use Calcium supplement. Usual strength 600 mg. Labelling The label states that the tablets should be dissolved in water immediately before use. CALCIUM GLUCONATE FOR INJECTION Calcii gluconas pro injectione

C12H22CaO14,H2O 

M. 448.4  

Calcium gluconate for injection contains not less than 99.0% and not more than 101.0% of C12H22CaO14,H2O.

Characters A white, crystalline or granular powder, sparingly soluble in water, freely soluble in boiling water. 157

VP V

CALCIUM GLUCONATE FOR INJECTION

Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G Mobile phase: Ethyl acetate - concentrated ammonia water - ethanol (96%) (10 : 10 : 30 : 50). Test solution: Dissolve 20 mg of the substance to be examined in 1 ml of water, heating if necessary in a waterbath at 60 °C. Reference solution: Dissolve 20 mg of calcium gluconate RS in 1 ml of water, heating if necessary in a water-bath at 60 °C. Procedure: Apply to the plate 5 µl of each solution. Develop over a path of 10 cm. Dry the plate at 100 °C for 20 min. Allow to cool and spray with a 5% solution of potassium dichromate R in a 40% (m/m) solution of sulfuric acid R. After 5 min examine the plate, the principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. B. Solution S (see Appearance of solution) gives reaction of calcium (Appendix 8.1). Appearance of solution Solution S: To 10.0 g add 90 ml of boiling water and boil with stirring, for 10 s to completely dissolve, dilute to 100.0 ml with the same solvent. Solution S at 60 °C is not more intensely coloured than reference solution B7 (Appendix 9.3, method 2). After cooling to 20 °C, it is not more opalescent than reference suspension II (Appendix 9.2). pH Dissolve 1.0 g in 20.0 ml of carbon dioxide-free water R, by heating on a water-bath. The pH of the solution is 6.4 to 8.3 (Appendix 6.2). Organic impurities and boric acid Introduce 0.5 g into a porcelain dish previously rinsed with sulfuric acid R and placed in a bath of iced water. Add 2 ml of cooled sulfuric acid R and mix. No yellow or brown colour develops. Add 1 ml of chromotrope II B solution R. A violet colour develops and does not become dark blue. The solution is not more intensely coloured than that of a mixture of 1 ml of chromotrope II B solution R and 2 ml of cooled sulfuric acid R. Oxalate Not more than 100 ppm. Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 0.212 g of anhydrous sodium carbonate R and 63 mg of sodium hydrogen carbonate R in water for chromatography R and dilute to 1000.0 ml with the same solvent. Test solution: Dissolve 1.00 g of the substance to be examined in water for chromatography R and dilute to 100.0 ml with the same solvent. 158

Reference solution: Dissolve 1.00 g of the substance to be examined in water for chromatography R, add 0.5 ml of a 0.0152% solution of sodium oxalate R in water for chromatography R and dilute to 100.0 ml with the same solvent. Chromatographic system: A guard column (30 mm × 4 mm) packed with a suitable strong anion exchange resin (30 µm to 50 µm). Two columns each (25 cm × 4 mm) packed with a suitable strong anion exchange resin (30 µm to 50 µm). A micromembrane anion-suppressor column, connected in series with the guard and analytical columns; the anionsuppressor column is equipped with a micromembrane that separates the mobile phase from the suppressor regeneration solution, flowing countercurrent to the mobile phase at a rate of 4 ml/min. Suppressor regeneration solution is a 0,123% solution of sulfuric acid R in water for chromatography R. Detector: Conductance detector. Flow rate: 2 ml/min. Volume of injection: 50 µl. Procedure: Inject the reference solution five times. The test is not valid unless the relative standard deviation of the peak areas for oxalate in the chromatograms obtained with the reference solution is not more than 2.0%. Inject reference solution, test solution, each solution three times. Calculate the content of oxalate in parts per million using the following expression:

S t × 50 S r − St

St: Average area of the oxalate peaks in the chromatograms obtained with the test solution, Sr: Average area of the oxalate peaks in the chromatograms obtained with the reference solution.

Sacharose and reducing sugars Dissolve 0.5 g in a mixture of 2 ml of a 25% solution of hydrochloric acid R and 10 ml of water. Boil for 5 min, allow to cool, add 10 ml of a 10% solution of sodium carbonate R and allow to stand for 10 min. Dilute to 25 ml with water and filter. To 5 ml of the filtrate add 2  ml of Fehling reagent R and boil for 1 min. Allow to stand for 2 min. No red precipitate is formed. Chlorides Not more than 50 ppm (Appendix 9.4.5). To 10 ml of previously filtered solution S add 5 ml of water. The solution complies with the limit test for chlorides. Phosphates Not more than 100 ppm (Appendix 9.4.12). Dilute 1 ml of solution S to 100 ml with water. The solution complies with the limit test for phosphates. Sulfates Not more than 50 ppm (Appendix 9.4.14).

VP V

15 ml of previously filtered solution S complies with the limit test for sulfates. Prepare the reference solution using a mixture of 7.5 ml of sulfate standard solution (10 ppm SO4) R and 7.5 ml of water.

Iron Not more than 5 ppm of Fe. Determined by atomic absorption spectrometry (Appendix 4.4, method 1). Test solution: Introduce 2.0 g into a 100 ml polytetrafluoroethylene beaker and add 5 ml of nitric acid R. Boil, evaporating almost to dryness. Add 1 ml of strong hydrogen peroxide solution R and evaporate again almost to dryness. Repeat the hydrogen peroxide treatment until a clear solution is obtained. Using 2 ml of nitric acid R, transfer the solution into a 25 ml volumetric flask. Dilute to 25.0 ml with dilute hydrochloric acid R. Blank solution: In the same manner, prepare a compensation solution using 0.65 g of calcium chloride tetrahydrate R instead of the substance to be examined. Reference solutions: Prepare the reference solutions from iron standard solution (20 ppm Fe)R diluted with dilute hydrochloric acid R. Measure the absorbance at 248.3 nm, using an iron hollowcathode lamp as source of radiation and an air-acetylene flame. Carry out a basic correction using a deuterium lamp. Heavy metals Not more than 10 ppm (Appendix 9.4.8). 12 ml of solution S complies with the limit test for heavy metals, method 1. Prepare the standard using lead standard solution (1 ppm Pb) R. Magnesium and alkali metals Not more than 0.4%. To 0.50 g add a mixture of 1.0 ml of dilute acetic acid R and 10.0 ml of water and rapidly boil, whilst shaking, until completely dissolved. To the boiling solution add 5.0 ml of a 4% solution of ammonium oxalate R and allow to stand for at least 6 h. Filter through a sintered-glass filter (Porosity number 1.6) into a porcelain crucible. Carefully evaporate the filtrate to dryness and ignite. The residue weighs not more than 2 mg. Microbial contamination (Appendix13.6) Total aerobic microbial count: Not more than 102 CFU/g, determined by plate count. Absence of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus. Bacterial endotoxins Not more than 167 EU/g (Appendix 13.2). Assay Dissolve 0.350 g in 20 ml of hot water, allow to cool and dilute to 300 ml with water. Carry out the complexometric titration of calcium (Appendix 10.5). Use 50 mg of calcon mixture R.

CALCIUM GLUCONATE INJECTION

1 ml of 0.1 M Trilon VS is equivalent to 44.84 mg of C12H22CaO14,H2O.

Storage Store in a well-closed container. Action and use Treatment of calcium deficiency. Preparation Calcium gluconate injection. CALCIUM GLUCONATE INJECTION Injectio Calcii gluconatis Calcium gluconate injection is a sterile solution of calcium gluconate in water for injections. Not more than 5.0% of the calcium gluconate can be replaced with suitable calcium salts for the purpose of stabilization. The preparation complies with the general requirements prescribed under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Content of calcium, Ca, 8.5% to 9.4% of the stated amount of calcium gluconate, C12H22O14Ca,H2O. Characters A clear, colourless solution. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Ethyl acetate - concentrated ammonia water - ethanol (96%) (10 : 10 : 30 : 50). Test solution: Dilute the injection with water to obtain a solution containing about 20 mg of calcium gluconate per ml. Reference solution: Dissolve 20 mg of calcium gluconate RS in 1 ml of water, heating if necessary in a water-bath at 60 °C. Procedure: Apply to the plate 5 µl of each solution. Develop over a path of 10 cm. Dry the plate at 100 °C for 20 min. Allow to cool and spray with a 5% solution of potassium dichromate R in a 40% (m/m) solution of sulfuric acid R. After 5 min, the principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. B. To 1 ml of the injection add 0.05 ml of a 10.5% solution of iron (III) chloride solution R. An intense yellow colour is produced. C. The injection gives the reactions characteristic of calcium (Appendix 8.1). pH 6.0 to 8.2 (Appendix 6.2). 159

VP V

CALCIUM GLYCEROPHOSPHATE

Bacterial endotoxins Comply with the test for bacterial endotoxins (Appendix 13.2). Dilute the injection in water for BET if necessary to obtain a solution containing 100 mg of calcium gluconate per ml (solution A). The endotoxin limit concentration of solution A is 16.7 EU per ml. Assay Take an accurately measured volume of the injection containing the equivalent of about 0.5 g of calcium gluconate and carry out the complexometric titration of calcium (Appendix 10.5). Storage Store at a cool and dry place, protected from light. Action and use Calcium supplement. Usual strength 10%.

M. 210.1  

Calcium glycerophosphate is a mixture in variable proportions of calcium (RS)-2,3- dihydroxypropyl phosphate, and of calcium 2-hydroxy-1-(hydroxymethyl)ethyl phosphate, which may be hydrated. Calcium glycerophosphate contains not less than 18.6% and not more than 19.4% of Ca, calculated with reference to the dried substance.

Characters A white powder, hygroscopic, sparingly soluble in water, practically insoluble in ethanol (96%). Identification A. Mix 1 g with 1 g of potassium hydrogen sulphate R in a test tube fitted with a glass tube. Heat strongly and direct the white vapour towards a piece of filter paper impregnated with a freshly prepared 1% solution of sodium nitroprusside R. The filter paper develops a blue colour in contact with piperidine R. B. Ignite 0.1 g in a crucible. Moisten the residue with 5 ml of nitric acid R and heat on a water-bath for 1 min. Filter. The filtrate gives reaction of phosphates (Appendix 8.1). C. It gives reaction (B) of calcium (Appendix 8.1). Clarity of solution Solution S: Dissolve 1.5 g in carbon dioxide-free water R and dilute to 150 ml with the same solvent. Solution S is not more opalescent than reference suspension III (Appendix 9.2). 160

Citric acid Shake 5.0 g with 20 ml of carbon dioxide-free water R and filter. To the filtrate add 0.15 ml of sulfuric acid R and filter again. To the filtrate add 5 ml of mercuric sulfate solution R and heat to boiling. Add 0.5 ml of a 0.32% solution of potassium permanganate R and again heat to boiling. No precipitate is formed. Glycerol and ethanol (96%)-soluble substances Not more than 0.5%. Shake 1.000 g with 25 ml of ethanol (96%) R for 1 min. Filter. Evaporate the filtrate to dryness on a water-bath and dry the residue at 70 °C for 1 h. The residue weighs not more than 5 mg. Chlorides Not more than 0.05% (Appendix 9.4.5). Dissolve 0.1 g in a mixture of 2 ml of acetic acid R and 8 ml of water and dilute to 15 ml with water. The solution complies with the limit test for chlorides.

CALCIUM GLYCEROPHOSPHATE Calcii glycerophosphas C3H7CaO6P 

Acidity or alkalinity To 100 ml of solution S add 0.1 ml of phenolphthalein solution R. Not more than 1.5  ml of 0.1 N hydrochloric acid VS or 0.5 ml of 0.1 N sodium hydroxide VS is required to change the colour of the indicator.

Phosphates Not more than 0.04% (Appendix 9.4.12). Dilute 2.5 ml of solution S to 100 ml with water. The solution complies with the limit test for phosphates. Sulfates Not more than 0.1% (Appendix 9.4.14). 15 ml of solution S complies with the limit test for sulphates. Arsenic Not more than 3 ppm (Appendix 9.4.2). Dissolve 0.33 g in water and dilute to 25 ml with the same solvent. The solution complies with limit test for arsenic, method A. Heavy metals Not more than 20 ppm (Appendix 9.4.8). Dissolve 2.0 g in 10 ml of acetate buffer solution pH 3.5 R and dilute to 20 ml with water. 12 ml of the solution complies with limit test for heavy metals, method 1. Prepare the reference solution using lead standard solution (2 ppm Pb) R. Iron Not more than 50 ppm (Appendix 9.4.13). Determined on 0.2 g. Loss on drying Not more than 12.0% (Appendix 9.6). (1.000 g; 150 °C; 4 h).

VP V

CALCIUM LACTATE PENTAHYDRATE

Assay Dissolve 0.200 g in water. Carry out the complexometric titration of calcium (Appendix 10.5). 1 ml of 0.1 M sodium edetate VS is equivalent to 4.008 mg of Ca.

Sulfates Not more than 0.4% (Appendix 9.4.14). Dissolve 0.15 g in a mixture of 5 ml of dilute hydrochloric acid R and 10 ml of water and dilute to 60 ml with water. 15 ml of the solution complies with the limit test for sulfates.

Srorage In an airtight container.

Arsenic Not more than 4 ppm (Appendix 9.4.2). Dissolve 0.50 g in 5 ml of brominated hydrochloric acid R and dilute to 50 ml with water. 25 ml of the solution complies with the limit test for arsenic, method A.

Action and use Calcium supplement. CALCIUM HYDROXIDE Calcii hydroxydum Ca(OH)2  

M. 74.09  

Calcium hydroxide contains not less than 95.0% and not more than 100.5% of Ca(OH)2.

Characters A white, fine powder, practically insoluble in water. Identification A. To 0.80 g in a mortar, add 10 ml of water and 0.5 ml of phenolphthalein solution R and mix well. The suspension turns red. On addition of 17.5 ml of 1 M hydrochloric acid, the suspension becomes colourless without effervescing. The red colour occurs again when the mixture is triturated for 1 min. On addition of a further 6 ml of 1 M hydrochloric acid R and triturating, the solution becomes colourless. B. Dissolve about 0.1 g in dilute hydrochloric acid R and dilute to 10 ml with water. 5 ml of the solution give reaction of calcium (Appendix 8.1). Matter insoluble in hydrochloric acid Not more than 0.5%. Dissolve 2.0 g in 30 ml of hydrochloric acid R. Boil the solution and filter. Wash the residue with hot water and ignite to constant mass. The residue weighs not more than 10 mg. Carbonates Not more than 5.0% of CaCO3. Add 5.0 ml of 1 N hydrochloric acid VS to the titrated solution obtained under Assay and titrate with 1 N sodium hydroxide VS using 0.5 ml of methyl orange solution R as indicator. 1 ml of 1 N hydrochloric acid VS is equivalent to 50.05 mg of CaCO3. Chlorides Not more than 0.033% (Appendix 9.4.5). Dissolve 0.30 g in a mixture of 2 ml of nitric acid R and 10 ml of water and dilute to 30 ml with water. 15 ml of the solution complies with the limit test for chlorides.

Magnesium and alkali metals Not more than 4.0%, calculated as sulfates. Dissolve 1.0 g in a mixture of 10 ml of hydrochloric acid R and 40 ml of water. Boil and add 50 ml of a 6.3% solution of oxalic acid R. Neutralise with ammonia R and dilute to 200 ml with water. Allow to stand for 1 h and filter through a suitable filter. To 100 ml of the filtrate, add 0.5 ml of sulfuric acid R. Cautiously evaporate to dryness and ignite. The residue weighs not more than 20 mg. Heavy metals Not more than 20 ppm (Appendix 9.4.8). Dissolve 1.0 g in 10 ml of a 25% solution of hydrochloric acid R and evaporate to dryness on a water- bath. Dissolve the residue in 20 ml of water and filter. 12 ml of the filtrate complies with limit test for heavy metals, method 1. Prepare the reference solution using lead standard solution (1 ppm Pb) R. Assay To 1.500 g in a mortar, add 25 ml of water and 0.5 ml of phenolphthalein solution R. Titrate with 1 N hydrochloric acid VS by triturating the substance until the red colour disappears. The final solution is used in the test for carbonates. 1 ml of 1 N hydrochloric acid VS is equivalent to 37.05 mg of Ca(OH)2. Srorage In an airtight container. Acetion and use Antacid. CALCIUM LACTATE PENTAHYDRATE Calcii lactas pentahydricus

C6H10CaO6,5H2O 

M. (anhydrous) 218.2  

Calcium lactate pentahydrate is calcium bis-2hydroxypropanoate or mixture of calcium (2R)-, (2S)- and (2RS)-2-hydroxypropanoates pentahydrates. It contains not 161

VP V

CALCIUM LACTATE PENTAHYDRATE

less than 98.0% and not more than 102.0% of C6H10CaO6, calculated with reference to the dried substance.

Characters White or almost white, crystalline or granular powder, slightly efflorescent. Soluble in water, freely soluble in boiling water, very slightly soluble in ethanol (96%). Identification A. It complies with the test for Loss on drying. B. It gives the reaction of lactates and reaction (B) of calcium (Appendix 8.1). Appearance of solution Solution S: Dissolve 7.1 g (equivalent to 5.0 g of the dried substance) with heating in carbon dioxide-free water R, allow to cool and dilute to 100  ml with the same solvent. Solution S is not more opalescent than reference suspention II (Appendix 9.2) and not more intensely coloured than reference solution BY6 (Appendix 9.3, method 2). Acidity or alkalinity To 10 ml of solution S add 0.1 ml of phenolphthalein solution R and 0.5 ml of 0.01 N hydrochloric acid VS. The solution is colourless. Not more than 2.0 ml of 0.01 N sodium hydroxide VS is required to change the colour of the indicator to pink. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 0.75 g of calcium phosphate monohydrate R in 20 ml of water. Add 1.0 ml of phosphoric acid R and dilute to 1000 ml with water. The pH is 2.2. Adjust if necessary with phosphoric acid R. Test solution: Dissolve 0.250 g of the substance to be examined in water and dilute to 10.0 ml with the same solvent. Reference solution: Dilute 1.0 ml of the test solution to 200.0 ml with water. Resolution solution: Dissolve 25 mg of calcium lactate R and 25 mg of sodium 2- hydroxybutyrate R in water and dilute to 50.0 ml with the same solvent. Chromatographic system: A column (30 cm × 7.8 mm) packed with strong cation exchange resin (calcium form) R (8 µm). Column temperature: 85 °C. Detector: A spectrophotometer set at 215 nm. Flow rate: 0.4 ml/min. Volume of injection: 20 µl. Procedure: The run time is twice the retention time of lactic acid. System suitability: In the chromatogram obtained with the resolution solution, the resolution between the peaks due to lactic acid (retention time = about 24.3 min) and (2RS)2-hydroxybutanoic acid (relative retention times with reference to lactic acid is 1.16) is at least 4. 162

Limits: In the chromatogram obtained with the test solution. The area of (2RS)-2-hydroxybutanoic acid multiply by 0.6, is not more than 0.4 times the area of the principal peak in the chromatogram obtained with the reference solution (0.2%). The area of any secondary peak is not more than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (0.1%). The total of area of the secondary peaks (apart area of lactic acid peak) is not more than the area of the principal peak in the chromatogram obtained with reference solution (0.5%). Disregard any peak with an area not greater than 0.1 times the area of the principal peak in the chromatogram obtained with the reference solution (0.05%) and disregard any peak with a retention time less than 14 min.

Chlorides Not more than 0.02% (Appendix 9.4.5). Dilute 5 ml of solution S to 15 ml with water. Sulfates Maximum 0.04% (Appendix 9.4.14). Dilute 7.5 ml of solution S to 15 ml with water. Barium To 10 ml of solution S add 1 ml of calcium sulfate solution R. Allow to stand for 15 min. Any opalescence in the solution is not more intense than that in a mixture of 1 ml of water and 10 ml of solution S. Heavy metals Not more than 10 ppm (Appendix 9.4.8). Dissolve a quantity equivalent to 2.0 g of the dried substance in water and dilute to 20 ml with the same solvent. 12 ml of the solution complies with limit test for heavy metal, method 1. Prepare the reference solution using lead standard solution (1 ppm Pb) R. Iron Not more than 50 ppm (Appendix 9.4.13). Dilute 4.0 ml of solution S to 10 ml with water. Magnesium and alkali salts Maximum 1%. To 20 ml of solution S add 20 ml of water, 2 g of ammonium chloride R and 2 ml of dilute ammonia R. Heat to boiling and rapidly add 40 ml of a hot 4% solution of ammonium oxalate R. Allow to stand for 4 h, dilute to 100 ml with water and filter. To 50.0 ml of the filtrate add 0.5 ml of sulfuric acid R. Evaporate to dryness and ignite the residue to constant mass at 600 °C. The residue weighs not more than of 5 mg. Loss on drying 22.0% to 27.0% (Appendix 9.6). (0.500 g; 125 °C).

VP V

CALCIUM LACTATE TRIHYDRATE

Assay Dissolve a quantity equivalent to 0.200 g of the dried substance in water and dilute to 300 ml with the same solvent. Carry out the complexometric titration of calcium (Appendix 10.5). 1 ml of 0.1 M sodium edetate VS is equivalent to 21.82 mg of C6H10CaO6. Storage Store in a well-closed container. Action and use Treatment of calcium deficiency. CALCIUM LACTATE TRIHYDRATE Calcii lactas trihydricus

C6H10CaO6,3H2O

M. (anhydrous) 218.2  

Calcium lactate trihydrate is calcium bis-2-hydroxypropanoate or mixture of calcium (2R)-, (2S)- and (2RS)2- hydroxypropanoates trihydrates. It contains not less than 98.0% and not more than 102.0% of C6H10CaO6 Calculated with reference to the dried substance.

Characters White or almost white, crystalline or granular powder. Soluble in water, freely soluble in boiling water, very slightly soluble in ethanol (96%). Identification A. It complies with the test for Loss on drying. B. It gives the reaction of lactates and reaction B of calcium (Appendix 8.1). Appearance of solution Solution S: Dissolve 6.2 g (equivalent to 5.0 g of the dried substance) with heating in carbon dioxide-free water R, allow to cool and dilute to 100  ml with the same solvent. Solution S is not more opalescent than reference suspention II (Appendix 9.2) and not more intensely coloured than reference solution BY6 (Appendix 9.3, method 2). Acidity or alkalinity To 10 ml of solution S add 0.1 ml of phenolphthalein solution R and 0.5 ml of 0.01 N hydrochloric acid. The solution is colourless. Not more than 2.0 ml of 0.01 N sodium hydroxide VS is required to change the colour of the indicator to pink. Related substances Examine by liquid chromatography (Appendix 5.3).

Mobile phase: Dissolve 0.75 g of calcium phosphate monohydrate R in 20 ml of water. Add 1.0 ml of phosphoric acid R and dilute to 1000 ml with water. The pH is 2.2. Adjust pH if necessary with phosphoric acid R. Test solution: Dissolve 0.250 g of the substance to be examined in water and dilute to 10.0 ml with the same solvent. Reference solution: Dilute 1.0 ml of the test solution to 200.0 ml with water. Resolution solution: Dissolve 25 mg of calcium lactate R and 25 mg of sodium 2- hydroxybutyrate R in water and dilute to 50.0 ml with the same solvent. Chromatographic system: A column (30 cm × 7.8 mm) packed with strong cation exchange resin (calcium form) R (8 µm). Column temperature: 85 °C. Detector: Spectrophotometer at 215 nm. Flow rate: 0.4 ml/min. Volume of injection: 20 µl. Procedure: The run time is twice the retention time of lactic acid. System suitability: In the chromatogram obtained with the resolution solution, the resolution is minimum 4 between the peaks due to lactic acid (retention time = about 24.3 min) and (2RS)-2-hydroxybutanoic acid (relative retention times with reference to lactic acid is 1.16). Inject the test solution and reference solution. Limits: In the chromatogram obtained with the test solution: The area of (2RS)-2-hydroxybutanoic acid multiply by 0.6, is not more than 0.4 times the area of the principal peak in the chromatogram obtained with the reference solution (0.2%). The area of any secondary peak is not more than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (0.1%). The total of area of the secondary peaks (apart from lactic acid peak) is not more than the area of the principal peak in the chromatogram obtained with reference solution (0.5%). Disregard any peak with an area not greater than 0.1 times the area of the principal peak in the chromatogram obtained with the reference solution (0.05%) and disregard any peak with a retention time less than 14 min.

Chlorides Not more than 0.02% (Appendix 9.4.5). Dilute 5 ml of solution S to 15 ml with water. Sulfates Not more than 0.04% (Appendix 9.4.14). Dilute 7.5 ml of solution S to 15 ml with water. Barium To 10 ml of solution S add 1 ml of calcium sulfate solution R. Allow to stand for 15 min. Any opalescence in the solution is not more intense than that in a mixture of 1 ml of water and 10 ml of solution S. Heavy metals Not more than 10 ppm (Appendix 9.4.8). 163

VP V

CALCIUM PANTOTHENATE

Dissolve a quantity equivalent to 2.0 g of the dried substance in water and dilute to 20 ml with the same solvent. 12 ml of the solution complies with limit test for heavy metal, method 1. Prepare the reference solution using lead standard solution (1 ppm Pb) R.

Iron Not more than 50 ppm (Appendix 9.4.13). Dilute 4.0 ml of solution S to 10 ml with water. Magnesium and alkali salts Not more than 1%. To 20 ml of solution S add 20 ml of water, 2 g of ammonium chloride R and 2 ml of dilute ammonia R. Heat to boiling and rapidly add 40 ml of a hot 4% solution of ammonium oxalate R. Allow to stand for 4 h, dilute to 100.0 ml with water and filter. To 50.0  ml of the filtrate add 0.5 ml of sulfuric acid R. Evaporate to dryness and ignite the residue to constant mass at 600 °C. The residue weighs not more than 5 mg.

Identification A. It complies with the test for Specific optical rotation. B. In the test for 3-aminopropionic acid, the principal spot in the chromatogram obtained with test solution (2) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). C. To 1 ml of solution S add 1 ml of dilute sodium hydroxide solution R and 0.1 ml of a 12.5% solution of copper sulfate R. A blue colour develops. D. It gives reaction of calcium (Appendix 8.1). Appearance of solution Solution S: Dissolve 5.0 g in carbon dioxide-free water R and dilute to 100 ml with the same solvent. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). pH The pH of solution S is 6.8 to 8.0 (Appendix 6.2).

Loss on drying 15.0% to 20.0% (Appendix 9.6). (0.500 g; 125 °C).

Specific optical rotation +25.5° to +27.5°, determined on solution S and calculated with reference to the dried substance (Appendix 6.4).

Assay Dissolve a quantity equivalent to 0.200 g of the dried substance in water and dilute to 300 ml with the same solvent. Carry out the complexometric titration of calcium (Appendix 10.5). 1 ml of 0.1 M sodium edetate VS is equivalent to 21.82 mg of C6H10CaO6.

3-Aminopropionic acid Not more than 0.5%. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Water - ethanol (96%) (35 : 65). Test solution (1): Dissolve 0.2 g of the substance to be examined in water and dilute to 5 ml with the same solvent. Test solution (2): Dilute 1 ml of test solution (1) to 10 ml with water. Reference solution (1): Dissolve 20 mg of calcium pantothenate RS in water and dilute to 5 ml with the same solvent. Reference solution (2): Dissolve 10 mg of 3-aminopropionic acid R in water and dilute to 50 ml with the same solvent. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 12 cm. Dry the plate in a current of air and spray with ninhydrin solution R. Heat at 110 °C for 10 min. Any spot corresponding to 3-aminopropionic acid in the chromatogram obtained with test solution (1) is not more intense than the spot in the chromatogram obtained with reference solution (2).

Storage Store in a well-closed container. Action and use Used in treatment of calcium deficiency. CALCIUM PANTOTHENATE Calcii pantothenas H

OH H N

HO H3C CH O 3

C18H32CaN2O10 

COO . Ca2+ 2

M. 476.5

Calcium pantothenate is calcium bis[(R)-3-(2,4-dihydroxy -3,3 dimethylbutyramido)propanoate]. It contains not less than 98.0% and not more than 101.0% of C18H32CaN2O10, calculated with reference to the dried substance.

Characters A white powder, slightly hygroscopic, freely soluble in water, slightly soluble in ethanol (96%), practically insolube in ether. 164

Chlorides Not more than 0.02% (Appendix 9.4.5). 5 ml of solution S diluted to 15 ml with water complies with the limit test for chlorides. Heavy metals Not more than 20 ppm (Appndix 9.4.8). 12 ml of solution S complies with limit test for heavy metals, method 1. Prepare the reference solution using lead standard solution (1 ppm Pb) R.

VP V

Loss on drying Not more than 3.0% (Appendix 9.6). (1.000 g; 100 °C to 105 °C). Assay Dissolve 0.180 g in 50 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS determining the endpoint potentiometrically (Appendix 6.12). 1 ml of 0.1 N perchloric acid VS is equivalent to 23.83 mg of C18H32CaN2O10. Storage Store in an airtight container. CALCIUM PHOSPHATE Calcii phosphas Calcium phosphate consists of a mixture of calcium phosphates. It contains not less than 35.0% and not more than 40.0% of Ca.

Characters A white or almost white powder, practically insoluble in water. It dissolves in dilute hydrochloric acid and in dilute nitric acid. Identification A. Dissolve 0.1 g in 5 ml of a 25% (V/V) solution of nitric acid R. To 2 ml of the solution, add 2 ml of ammonium molypdate solution R. A yellow precipitate is formed. B. Place 0.2 g in a porcelain crucible and ignite. Allow to cool. Add 0.5 ml of a 4.25% solution of silver nitrate R, a yellow precipitate is produced. C. It gives reaction A of calcium (Appendix 8.1). Filter before adding potassium ferrocyanide solution R. Chlorides Not more than 0.15% (Appendix 9.4.5). Dissolve 0.22 g in a mixture of 1 ml of nitric acid R and 10 ml of water and dilute to 100 ml with water. 15 ml of the solution complies with the limit test for chlorides. Fluorides Not more than 75 ppm, determined potentiometrically (Appendix 10.2) using a fluoride-selective indicator electrode and a silver-silver chloride reference electrode. Test solution: In a 50 ml volumetric flask, dissolve 0.250 g in 0.1 M hydrochloric acid, add 5.0 ml of fluoride standard solution (1 ppm F) R and dilute to 50.0 ml with 0.1 M hydrochloric acid. To 20.0 ml of the solution add 20.0 ml of total ionic strength adjustment buffer R and 3 ml of a 8.2% solution of anhydrous sodium acetate R. Adjust to pH 5.2 with ammonia R and dilute to 50.0 ml with water. Reference solutions: To 5.0 ml, 2.0 ml, 1.0 ml, 0.5 ml and 0.25 ml of fluoride standard solution (10 ppm F) R add 20.0 ml of total ionic strength adjustment buffer R and dilute to 50.0 ml with water.

CALCIUM PHOSPHATE

Carry out the measurements on 20.0 ml of each solution. Calculate the concentration of fluorides using the calibration curve, taking into account the addition of fluoride to the test solution.

Sulfates Not more than 0.5% (Appendix 9.4.14). Solution S: Dissolve 2.50 g in 20 ml of a 2 M solution of hydrochloric acid R. If the solution is not clear, filter. Add a 10% solution of ammonia R dropwise until a precipitate is formed. Dissolve the precipitate by adding 2 M hydrochloric acid R and dilute to 50 ml with water. Dilute 1 ml of solution S to 25 ml with water. 15 ml of the solution complies with the limit test for sulfates. Arsenic Not more than 4 ppm (Appendix 9.4.2). 5 ml of solution S complies with limit test for arsenic, method A. Heavy metals Not more than 30 ppm (Appendix 9.4.8). Dilute 13 ml of solution S to 20 ml with water. 12 ml of the solution complies with limit test for heavy metals, method 1. Prepare the reference solution using lead standard solution (1 ppm Pb) R. Iron Not more than 0.04% (Appendix 9.4.13). Dilute 0.5 ml of solution S to 10 ml with water. The solution complies with the limit test for iron. Acid-insoluble matter Not more than 0.2%. Dissolve 5.0 g in a mixture of 10 ml of hydrochloric acid R and 30 ml of water. Filter, wash the residue with water and dry to constant mass at 100 °C to 105 °C. The residue weighs not more than 10 mg. Loss on ignition Not more than 8.0%. (1.000 g; 800 °C ± 50 °C; 30 min). Assay Dissolve 0.200 g in a mixture of 1 ml of a 25% solution of hydrochloric acid R and 5 ml of water. Add 25.0 ml of 0.1 M sodium edetate VS and dilute to 200 ml with water. Adjust pH to 10.0 with concentrated ammonia R. Add 10 ml of ammonium chloride buffer solution pH 10.0 R and a few milligrams of Eriochrome black T mixture R. Titrate the excess sodium edetate with 0.1 M zinc sulfate VS until the colour changes from blue to violet. 1 ml of 0.1 M sodium edetate VS is equivalent to 4.008 mg of Ca. Storage Store in a well-closed container. 165

VP V

DRIED CALCIUM SULFATE

DRIED CALCIUM SULFATE Calci sulfat ustus CaSO4,½H2O



50 °C - 70 °C), freely soluble in fatty oils, very slightly soluble in glycerol. M. 145.1  

Dried calcium sulfate is prepared by heating powdered gypsum, CaSO4,2H2O, at about 150 °C in a controlled manner such that it is substantially converted into the hemihydrate, CaSO4,½H2O, with minimum production of the anhydrous phases of calcium sulfate. It may contain suitable setting accelerators or decelerators.

Characters A white or almost white powder; odourless or almost odourless, hygroscopic. Slightly soluble in water; more soluble in dilute mineral acids, practically insoluble in ethanol (96%). Identification It gives the reactions of calcium and of sulfates (Appendix 8.1). Setting properties 20 g mixed with 10 ml of water at 15 °C to 20 °C in a cylindrical mould about 2.4 cm in diameter sets in 4 min to 11 min. The mass thus produced, after standing for 3 h, possesses sufficient hardness to resist pressure of the fingers at the edges, which retain their sharpness of outline and do not crumble. Loss on ignition 4.5% to 8.0%. (1.00 g, 600 ºC).

NATURAL CAMPHOR Camphora

M. 152.2

Camphor is (1R,4R)-1,7,7-trimethylbicyclo[2.2.1]heptan2-one.

Characters A white or almost white, crystalline powder or friable, colourless crystalline masses, highly volatile even at room temperature. Slightly soluble in water, very soluble in ethanol (96%) and in petroleum ether (boiling range 166

Identification Apply one of the two following identifications: First identification: A, D. Second identification: B,C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of racemic camphor RS. B. Melting point: 175 °C to 179 °C (Appendix 6.7). C. Dissolve 1.0  g in 30  ml of  methanol R. Add 1.0 g of hydroxylamine hydrochloride R and 1.0 g of anhydrous sodium acetate R. Boil under a reflux condenser for 2 h. Allow to cool and add 100 ml of water. A precipitate is formed. Filter, wash with 10 ml of water and recrystallise from 10 ml of a mixture of ethanol (96%) - water (4 : 6). The crystals, dried in vacuo, melt at 118 °C to 121 °C. D. It complies with the test for specific optical rotation. Appearance of solution Solution S: Dissolve 2.50 g in 10 ml of ethanol (96%) R and dilute to 25.0 ml with the same solvent. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). Acidity or alkalinity To 10 ml of solution S add 0.1 ml of phenolphthalein solution  R1, the solution is colourless. Not more than 0.2 ml of 0.1 N sodium hydroxide VS is required to change the colour of the indicator to pink. Optical rotation +41° to +44°(Appendix 6.4). Determined on solution S.

Storage Store in an airtight container.

C10H16O

Carry out the weighings rapidly for the following tests:

Related substances Examine by gas chromatography (Appendix 5.2). Test solution: Dissolve 2.50 g in heptane R and dilute to 25.0 ml with the same solvent. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with heptane R.  Reference solution (2): Dilute 10.0 ml of reference solution (1) to 20.0 ml with heptane R. Reference solution (3): Dissolve 0.50 g of borneol R in heptane R and dilute to 25.0 ml with the same solvent. Dilute 5.0 ml of the solution to 50.0 ml with heptane R. Reference solution (4): Dissolve 50  mg of  linalol R and 50  mg of  bornyl acetate  R in heptane R and dilute to 100.0 ml with the same solvent. Chromatographic system: A column (30 m × 0.25 mm) coated with macrogol 20 000 R (0.25 µm). Carrier gas: Helium for chromatography R. Split ratio: 1 : 70. Flow rate: 45 cm/s.

VP V

RACEMIC CAMPHOR

Temperature:

Column

Time (min)

Temperature (°C)

0 - 10 10 - 35 35 - 45 45 - 55

50 50 → 100 100 → 200 200

Injection port

220

Detector

250

Detector: Flame - ionization detector. Volume of injection: 1 µl. Procedure: System suitability: Inject reference solution (4), the resolution between the peaks due to bornyl acetate and linalol is at least 3.0. Inject the test solution and reference solutions (1), (2), and (3). Limits: In the chromatogram obtained with the test solution: Borneol: The area of the peak due to borneol is not more than the area of the principal peak in the chromatogram obtained with reference solution (3) (2.0%). Any other impurity: For each impurity, the peak area is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). Sum of the peak areas of all the other impurities is not more than 4 times the area of the principal peak in the chromatogram obtained with reference solution (1) (4.0%). Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Note: Impurity A: 2,6,6-trimethylbicyclo[3.1.1]hept-2-ene (α-pinene). Impurity B: 2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane (camphene). Impurity C: 6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane (β-pinene). Impurity D: 1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane (cineole). Impurity E: 1,3,3-trimethylbicyclo[2.2.1]heptan-2-one (fenchone). Impurity F: exo-1,3,3-trimethylbicyclo[2.2.1]heptan-2-ol (fenchol). Impurity G: exo-2,3,3-trimethylbicyclo[2.2.1]heptan-2-ol (camphene hydrate). Impurity H: endo-2,3,3-trimethylbicyclo[2.2.1]heptan-2-ol (methylcamphenilol). Impurity I: exo-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ol (exoborneol). Impurity J: endo-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ol (endoborneol).

Halogens Not more than 0.01% (Appendix 9.4.5). Dissolve 1.0 g in 10 ml of 2-propanol R in a distillation flask. Add 1.5  ml of  dilute sodium hydroxide solution  R and 50 mg of nickel-aluminium alloy R. Heat on a waterbath until the 2-propanol R has evaporated. Allow to cool and add 5 ml of water. Mix and filter through a wet filter previously washed with water until free from chlorides.

Dilute the filtrate to 10.0 ml with water. To 5.0 ml of this solution, add nitric acid  R dropwise until the precipitate which forms is redissolved and dilute to 15 ml with water. The solution complies with the limit test for chlorides.

Water Dissolve 1 g in 10 ml of  petroleum ether (boiling range 50 °C - 70 °C) R. The solution is clear (Appendix 9.2). Residue on evaporation Not more than 0.05%. Evaporate 2.0  g on a water-bath and dry at 100 °C to 105 °C for 1 h. The residue weighs not more than 1 mg. Storage Store in a well-closed container, at a dry and cool place.

Note Incompatibilities: Produceable deliquescent mixtures (transparent, consistent liquid mixtures) with phenol, menthol, thymol, salol, naphthol, resorcinol, pyrocatechol, pyrogallol, salicylic acid, phenylsalicylate, chloral hydrate, antipyrine,...

Action and use Counter-irritant, analgesic, itch reliever. RACEMIC CAMPHOR Camphora racemica CH3 O

H3C

and enantiomer

H3C H

C10H16O

M. 152.2

Camphor is (1RS,4RS)-1,7,7-trimethylbicyclo[2.2.1]heptan2-one.

Characters A white or almost white, crystalline powder or friable, colourless crystalline masses, highly volatile even at room temperature. Slightly soluble in water, very soluble in ethanol (96%) and in petroleum ether (boiling range 50 °C - 70 °C), freely soluble in fatty oils, very slightly soluble in glycerol. Carry out the weighings rapidly for the following tests: Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of racemic camphor RS. Examine the substance as mulls in liquid paraffin R. B. Melting point : 172 °C to 180 °C (Appendix 6.7). 167

VP V

CAPTOPRIL

C. Dissolve 1.0  g in 30  ml of  methanol R. Add 1.0 g of hydroxylamine hydrochloride R and 1.0 g of anhydrous sodium acetate R. Boil under a reflux condenser for 2 h. Allow to cool and add 100 ml of water. A precipitate is formed. Filter, wash with 10 ml of water and recrystallise from 10 ml of a mixture of ethanol (96%)- water (4 : 6). The crystals, dried in vacuo, melt at 118 °C to 121 °C. D. It complies with the test for Optical rotation.

Appearance of solution Solution S: Dissolve 2.50 g in 10 ml of ethanol (96%) R and dilute to 25.0 ml with the same solvent. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). Acidity or alkalinity To 10 ml of solution S add 0.1 ml of phenolphthalein solution  R1, the solution is colourless. Not more than 0.2 ml of 0.1 N sodium hydroxide VS is required to change the colour of the indicator to pink. Optical rotation -0.15° to +0.15° (Appendix 6.4). Determined on solution S. Related substances Examine by gas chromatography (Appendix 5.2). Test solution: Dissolve 50 mg in of hexane R and dilute to 50.0 ml with the same solvent. Reference solution (1): Dissolve 50 mg of the substance to be examined and 50 mg of bornyl acetate R in hexane R and dilute to 50.0 ml with the same solvent. Reference solution (2): Dilute 1.0 ml of the test solution to 200.0 ml with hexane R. Chromatographic system: A column (2 m × 2 mm) packed with diatomaceous earth for gas chromatography R impregnated with 10% (m/m) of macrogol 20 000 R. Carrier gas: Nitrogen for chromatography R. Flow rate: 30  ml/min. Temperature: Column: 130 °C; injection port and detector: 200 °C. Detector: Flame - ionization detector. Volume of injection: 1 µl. Procedure: The run time is 3 times the retention time of camphor. System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to camphor and bornyl acetate is at least 1.5. In the chromatogram obtained with reference solution (2), the signal-to-noise ratio is at leat 5 for the principal peak. Limits: Any impurity: For each impurity, the peak area is not more than 2% of the area of the principal peak. Sum of the peak areas of all impurities is not more than 4% of the area of the principal peak. 168

Disregard any peak with an area equal to the area of the principal peak in the chromatogram obtained with reference solution (2).

Halogens Not more than 0.01% (Appendix 9.4.5). Dissolve 1.0 g in 10 ml of 2-propanol R in a distillation flask. Add 1.5  ml of  dilute sodium hydroxide solution  R and 50 mg of nickel-aluminium alloy R. Heat on a waterbath until the 2-propanol has evaporated. Allow to cool and add 5 ml of water. Mix and filter through a wet filter previously washed with water until free from chlorides. Dilute the filtrate to 10.0 ml with water. To 5.0 ml of this solution, add nitric acid  R dropwise until the precipitate which forms is redissolved and dilute to 15 ml with water. The solution complies with the limit test for chlorides. Water Dissolve 1  g in 10  ml of  petroleum ether boiling range (50 °C - 70 °C) R. The solution is clear (Appendix 9.2). Residue on evaporation Not more than 0.05%. Evaporate 2.0 g on a water-bath and dry at 100 °C to 105 °C for 1 h. The residue weighs not more than 1 mg. Storage Store in a well-closed container, at a dry and cool place. Action and use Counter-irritant, analgesic, itch reliever. CAPTOPRIL Captoprilum

C9H15NO3S  

M. 217.3

Captopril is (2S)-1-[(2S)-2-methyl-3-sulfanylpropanoyl] pyrrolidine-2-carboxylic acid. It contains not less than 98.0% and not more than 101.5% of C9H15NO3S, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder. Soluble in water, freely soluble in dichloromethane and in methanol. It dissolves in dilute solutions of alkali hydroxides. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of captopril RS. B. It complies with the test for Specific optical rotation.

VP V

CAPTOPRIL

Appearance of solution Solution S: Dissolve 0.5  g in  carbon dioxide-free water R and dilute to 25.0 ml with the same solvent. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). pH 2.0 to 2.6 (Appendix 6.2) Determined on solution S. Specific optical rotation -132° to -127°, calculated with reference to the dried substance (Appendix 6.4). Dissolve 0.250 g in ethanol R and dilute to 25.0 ml with the same solvent. Impurity F Not more than 0.2%. Examine by gas chromatography (Appendix 5.2). Reagent solution: Add 2.8 ml of acetyl chloride R dropwise to 17.2 ml of anhydrous methanol R at 0 °C and mix. Allow to stand for 20 min at room temperature before use. Test solution: Introduce 20.0 mg of the substance to be examined into a vial and add 1.0 ml of the reagent solution. Mix and heat at 60 °C for 30 min. Evaporate to dryness under a stream of nitrogen R. Dissolve the residue in 0.5 ml of  ethyl acetate  R, add 0.5  ml of  pentafluoropropionic anhydride R, mix and heat at 60 °C for 30 min. Evaporate to dryness under a stream of nitrogen R. Dissolve the residue in 1.0 ml of butyl acetate R. Reference solution (1): Dissolve the contents of a vial of captopril for system suitability RS (containing impurity F) in 1.0 ml of the reagent solution. Prepare as described for the test solution. Reference solution (2): Mix 0.25 ml of reference solution (1) and 0.75 ml of butyl acetate R. Chromatographic system: A fused-silica capillary column (25 m × 0.32 mm) coated with poly(dimethyl)(diphenyl)siloxane R (film thickness 1 µm). Carrier gas: Helium for chromatography. Flow rate: 1.2 ml/min. Split ratio: 1 : 20. Temperature programme:

Column

Time (min)

Temperature (°C)

0 - 10 10 - 14 14 - 34

200 200 → 240 240

Injection port

270

Detector

300

Detector: A flame-ionisation detector. Volume of injection: 1 µl. Procedure: Relative retention with reference to captopril (retention time = about 6 min): impurity F = about 0.96.

System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to impurity F and  captopril is at least 1.5. In the chromatogram obtained with reference solution (2), the signal-to-noise ratio of the peak due to impurity F is not less than 10. Calculate the percentage content of impurity F using the following expression: A × 100 A + B Where: A is the area of the peak due to impurity F in the chromatogram obtained with the test solution. B is the area of the peak due to captopril in the chromatogram obtained with the test solution.

Related substances Examine by liquid chromatography (Appendix 5.3). Solvent mixture: Phosphoric acid -  acetonitrile  R1 - water (0.08 : 10 : 90). Mobile phase A: Phosphoric acid - water (0.08 : 100). Mobile phase B:  Phosphoric acid  -  acetonitrile  R1 water (0.08 : 50 : 50). Test solution: Dissolve 0.125  g of the substance to be examined in the solvent mixture and dilute to 25.0 ml with the solvent mixture. Reference solution (1): Dissolve 4.0  mg of  captopril  impurity J RS, 5.0 mg of captopril impurity B RS, 5.0 mg of captopril impurity C RS and 5.0 mg of captopril impurity D RS in the solvent mixture and dilute to 50.0 ml with the solvent mixture. Dilute 1.0 ml of the solution to 20.0 ml with the solvent mixture. Prepare immediately before use. Reference solution (2): Dissolve 5  mg of the substance to be examined and 5  mg of  captopril  impurity E RS in  acetonitrile  R  and dilute to 25.0  ml with the same solvent. Dilute 4  ml of the solution to 50.0  ml with the solvent mixture. Reference solution (3): In order to prepare impurity A in situ, introduce 1.0 ml of the test solution into a volumetric flask and add 230 µl of 0.1 N iodine R. If the solution is not colourless, add 0.1 M sodium thiosulfate R dropwise until it becomes colourless, and dilute to 50.0 ml with the solvent mixture. Dilute 5.0 ml of this solution to 20.0 ml with the solvent mixture. Reference solution (4): Dilute 1.0 ml of the test solution to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 10.0 ml with the solvent mixture. Chromatographic system: A column (30 cm × 3.9 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (10 µm). Column temperature: 50 °C. Detector: A spectrophotometer set at 210 nm. Flow rate: 1.5 ml/min. Volume of injection: 25 µl. Procedure: Carry out a linear gradient elution using the following conditions: 169

VP V

CAPTOPRIL TABLETS

Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0-5 5 - 20 20 - 45

90 90 → 50 50

10 10 → 50 50

Identification of impurities: Use the chromatogram obtained with reference solution (1) to identify the peaks due to impurities B, C, D and J; use the chromatogram obtained with reference solution (2) to identify the peak due to impurity E; use the chromatogram obtained with reference solution (3) to identify the peak due to impurity A. Relative retention with reference to captopril (retention time  =  about 15 min): impurity C  =  about 0.6; impurity D = about 0.8; impurity E = about 0.9; impurity B = about 1.17; impurity J = about 1.22; impurity A = about 1.7. System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to impurities B and J is at least 1.5. In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurity E and captopril is at least 2.0. Limits: Impurity A: The area of the peak due to impurity A is not more than 10 times the area of the principal peak in the chromatogram obtained with reference solution (4) (1.0%). Impurity J: The area of the peak due to impurity J is not more than 2.5 times the area of the corresponding peak in the chromatogram obtained with reference solution (1) (0.2%). Impurities B, C, D: For each impurity, the area is not more than 1.5 times the area of the corresponding peak in the chromatogram obtained with reference solution (1) (0.15%); Impurity E: The area of the peak due to impurity E is not more than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (4) (0.15%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (4) (0.10%). Total peak area of all impurities is not more than 1.2%. Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (4) (0.05%). Note: ImpurityA: 1,1′-[disulfanediylbis[(2S)-2-methyl-1-oxopropane -3,1diyl]]bis[(2S)-pyrrolidine-2-carboxylic] acid (captopril disulfide). Impurity B: (2S)-1-[(2S)-3-bromo-2-methylpropanoyl]pyrrolidine -2-carboxylic acid. Impurity C: (2RS)-2-methyl-3-sulfanylpropanoic acid. Impurity D: (2RS)-3-bromo-2-methylpropanoic acid. Impurity E: (2S)-1-(2-methylpropanoyl)pyrrolidine-2-carboxylic acid. Impurity F: (2S)-1-[(2R)-2-methyl-3-sulfanylpropanoyl]pyrrolidine -2-carboxylic acid (epi-captopril). Impurity G: (2RS)-3-(acetylsulfanyl)-2-methylpropanoic acid.

170

Impurity H: (2S)-1-[(2S)-3-[[(2R)-3-(acetylsulfanyl)-2-methylpropanoyl]sulfanyl]-2-methylpropanoyl] pyrrolidine -2-carboxylic acid. Impurity I: (2S)-1-[(2S)-3-[[[(2S)-1-[(2S)-2-methyl-3-sulfanyl propanoyl] pyrrolidin-2-yl]carbonyl]sulfanyl]-2-methyl propanoyl] pyrrolidine-2-carboxylic acid. Impurity J: (2S)-1-[(2S)-3-(acetylsulfanyl)-2-methylpropanoyl] pyrrolidine-2-carboxylic acid (acetylcaptopril), Impurity L: 1,1′-[methylenebis[sulfanediyl[(2S)-2-methyl-1oxopropane-3,1-diyl]]]bis[(2S)-pyrrolidine-2-carboxylic] acid. Impurity M: (2S)-1-[(2S)-3-[[(2S)-2-carboxypropyl]disulfanyl]2-methylpropanoyl]pyrrolidine-2-carboxylic acid. Impurity N: 3,3′-disulfanediylbis[(2S)-2-methylpropanoic] acid. Impurity O: 1,1′-[propane-2,2-diylbis[sulfanediyl[(2S)-2-methyl -1-oxopropane-3,1-diyl]]]bis[(2S)-pyrrolidine-2-carboxylic] acid.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). Slovent: Water. 0.5 g complies with limit test for heavy metals, method 8. Prepare the standard using 1 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 1.0% (Appendix 9.6, method 2). (1.000 g; in vacuo; 60 °C; 3 h). Sulfated ash Not more than 0.2% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.150 g of the substance to be examined in 30 ml of water. Titrate with 0.1 N iodine VS, determining the endpoint potentiometrically (Appendix 10.2). Use a combined platinum electrode. 1 ml of 0.1 N  iodine VS is equivalent to 21.73 mg of C9H15NO3S. Storage Store in an airtight container. Action and use Angiotensin converting enzyme inhibitor. Preparation Tablets. CAPTOPRIL TABLETS Tabellae Captoprili Captopril tablets or coated tablets contain captopril. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of captopril, C9H15NO3S, 90.0% to 110.0% of the stated amount.

VP V

Identification A. To a quantity of the powdered tablets containing about 50 mg of captopril, add 5 ml of ethanol (96%) R, shake well for 5 min and filter. To 2 ml of the filtrate, add a few crystals of sodium nitrate R and 10 ml of 10% solution of sulfuric acid R. Shake vigorously, a red colour is produced. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is similar with that of captopril peak in the chromatogram obtained with the reference solution. Dissolution (Appendix 11.4) Note: Degas the medium to minimize the exposure of captopril to air and analyze the sample immediately. Apparatus: Basket. Medium: 900 ml of 0.01 M hydrochloric acid R. Rotation speed: 50 rpm. Time: 20 min. Procedure: After the specified time, withdraw a sample of the medium and filter. Dilute the filtrate with the medium to obtain a solution having a suitable concentration. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum at 205 nm, using 0.01 M hydrochloric acid R as the blank. Compare to a reference solution having the same concentration of captopril in the medium as the test solution. Calculate the content of captopril, C9H15NO3S, using the absorbances of the test solution, the reference solution and the declared content of C9H15NO3S in captopril RS. Tolerance: Not less than 80% (Q) of the stated amount of captopril, C9H15NO3S, is dissolved in 20 min. Captopril disulfide Examine by liquid chromatography (Appendix 5.3). Mobile phase and chromatographic system are described in the Assay. Test solution: Weigh accurately a quantity of the powdered tablets containing about 25 mg of captopril, transfer into a centrifuge tube, add 25.0 ml of methanol R and centrifuge for 15 min. Use the supernatant liquid. Reference solution (1): A solution containing 0.0030% of captopril disulfide RS in methanol R. Reference solution (2): Dilute 1 volume of the test solution to 100 volumes with reference solution (1). The test is not valid unless the resolution factor between captopril peak and captopril disulfide peak in the chromatogram obtained with reference solution (2) is at least 2.0. In the chromatogram obtained with the test solution the area of any peak corresponding to captopril disulfide is not greater than the area of the peak in the chromatogram obtained with reference solution (1) (3%).

CARBAMAZEPINE

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol - water - phosphoric acid (550 : 450 : 0.5). Reference solution: A solution containing 0.01% of captopril RS and 0.0005% of captopril disulfide RS in the mobile phase. Test solution: Weigh 20 tablets (remove the coating in case of coated tablets), calculate the average mass and finely powder. Weigh accurately a quantity of the powdered tablets containing about 25 mg of captopril, transfer into a centrifuge tube, add 25.0 ml of the mobile phase, sonicate for 15 min and centrifuge. Dilute 5.0 ml of the supernatant liquid to 50.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (10 µm). Detector: A spectrophotometer set at 220 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Inject the reference solution. The test is not valid unless the resolution factor between the peaks due to captopril and captopril disulfide is at least 2.0. Inject alternately the test solution and the reference solution. Calculate the content of captopril, C9H15NO3S, in the tablets using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C9H15NO3S in captopril RS. Storage Store in a cool and dry place, protected from light. Action and use Antihypertensive. Usual strength 12.5 mg; 25 mg. CARBAMAZEPINE Carbamazepinum

C15H22N2O

M. 236.3

Carbamazepine is 5H-Dibenzo[b,f]azepine-5-carboxamide. It contains not less than 98.0% and not more than 102.0% of C15H22N2O, calculated with reference to the anhydrous substance. 171

VP V

CARBAMAZEPINE

Characters A white or almost white, crystalline powder, polymorphism. The acceptable crystalline form corresponds to carbamazepine CRS. Very slightly soluble in water, freely soluble in methylene chloride, sparingly soluble in acetone and in ethanol (96%).

Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of carbamazepine RS. Examine the substances as discs without prior treatment. B. Melting point: 189 °C to 193 °C (Appendix 6.7). Acidity or alkalimity To 1.0 g add 20 ml of carbon dioxide-free water  R, shake for 15 min and filter. To 10  ml of the filtrate add 0.05  ml of  phenolphthalein solution R1  and 0.5  ml of 0.01 N sodium hydroxide VS; the solution is red. Add 1.0 ml of 0.01 N hydrochloric acid VS; the solution is colourless. Add 0.15 ml of methyl red solution R; the solution is red. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Tetrahydrofuran - methanol R2 - water (3 : 12 : 85). To 1000 ml of the mixture add 0.2 ml of anhydrous formic acid R and 0.5 ml of triethylamine R. Test solution (1):  Dissolve 60.0  mg in  methanol R2 and dilute to 20.0 ml with the same solvent. Sonicate. Dilute 10.0 ml of this solution to 20.0 ml with water. Test solution (2): Dilute 10.0  ml of test solution (1) to 50.0 ml with a mixture of methanol R2 - water (50 : 50). Reference solution (1): Dissolve 7.5 mg of carbamazepine RS, 7.5 mg of carbamazepine impurity A RS and 7.5 mg of iminodibenzyl R (impurity E) in methanol R2 and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of this solution to 50.0 ml with a mixture of methanol R2 - water (50 : 50). Reference solution (2): Dissolve 60.0 mg of carbamazepne RS in methanol R2 and dilute to 20.0 ml with the same solvent. Sonicate. Dilute 5.0 ml of this solution to 50.0 ml with a mixture of methanol R2 - water (50 : 50). Chromatographic system: A column (25 cm × 4.6 mm) packed with nitrile silica gel for chromatography R1 (10 µm). Detector: A spectrophotometer set at 230 nm. Flow rate: 2.0  ml/min. Volume of injection: 20 µl. Procedure: Inject separately test solution (1) and reference solution (1). Carry out the chromatography for 8 times the retention time of carbamazepine. Relative retention with reference to carbamazepine (retention time = about 10 min): Impurity A = about 0.9; impurity E = about 3.5. 172

System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to impurity A and carbamazepine is at least 1.7. Limits: Impurities A, E: For each impurity, the peak area is not more than 1.5 times the area of the corresponding peak in the chromatogram obtained with reference solution (1) (0.15%). Unspecified impurities: For each impurity, the peak area is not more than the area of the peak due to carbamazepin in the chromatogram obtained with reference solution (1) (0.10%). Sum of the areas of all the impurity peaks is not more than 5 times the area of the peak due to carbamazepine in the chromatogram obtained with reference solution (1) (0.5%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Notes: Impurity A: 10,11-dihydro-5H-dibenzo[b,f]azepine-5-carboxamide (10,11-dihydrocarbamazepine). Impurity B: 9-methylacridine. Imputity C: (5H-dibenzo[b,f]azepin-5-ylcarbonyl)urea (N-carbamoylcarbamazepine). Impurity D: 5H-dibenzo[b,f]azepine (iminostilbene). Impurity E: 10,11-dihydro-5H-dibenzo[b,f]azepine (iminodibenzyl). Impurity F: 5H-dibenzo[b,f]azepine-5-carbonyl chloride (5-chlorocarbonyliminostilbene). Impurity G: 10-bromo-5H-dibenzo[b,f]azepine-5-carboxamide (10-bromocarbamazepine).

Chlorides Not more than 140 ppm (Appendix 9.4.5). Suspend 0.715 g in 20 ml of water and boil for 10 min. Cool and dilute to 20 ml with water. Filter through a membrane filter (nominal pore size 0.8 µm). Dilute 10 ml of the filtrate to 15 ml with water. This solution complies with the limit test for chlorides. Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with the limit test for heavy metals, method 3. Prepare the reference solution using 2 ml of lead standard solution (10 ppm Pb) R.  Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C; 2 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase and chromatographic system are described in the test for Related substances.

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CARBAMAZEPINE TABLETS

Inject test solution (2) and reference solution (2). System suitability: Calculate the relative standard deviation of peak areas of carbamazepine in the chromatogram obtained with reference solution (2). Calculate the percentage content of carbamazepine in the substance to be examined, using the areas of the principal peaks in the chromatograms obtained with test solution (2), reference solution (2) and the declared content of C15H12N2O in carbamazepine RS.

Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm. After removal of the plate, dry in air for 15 min. Spray with a 0.5% solution of potassium dichromate in 20% (v/v) solution of sulphuric acid. Allow the plate to dry in air. Examine under day light or ultraviolet light at 365 nm. Any secondary spot in the chromatogram obtained with test solution (1) is not more intense than the spot in the chromatogram obtained with reference solution (1).

Storage Store in a well-closed container.

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: Dilute 24 ml of a 10% solution of hydrochloric acid R to 1000 ml with water. Rotation speed: 150 rpm. Time: 60 min. Procedure: After the specified time, withdraw a sample of the medium, filter and discard the first portion of the filtrate. Dilute a portion of the filtrate with the medium to obtain a solution containing about 6 to 15 µg of carbamazepine per ml. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum at 285 nm, in a 1 cm cell, using medium as a blank. Calculate the content of carbamazepine, C15H12N2O, dissolved taking 518 as the value of A (1%, 1 cm) at the maximum at 285 nm. Tolerance: Not less than 65% (Q) of the stated amount of carbamazepine, C15H12N2O, is dissolved in 60 min.

Action and use Antiepileptic. Preparation Tablets. CARBAMAZEPINE TABLETS Tabellae Carbamazepini Carbamazepine tablets contain carbamazepine. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of carbamazepine, C15H12N2O, 90.0% to 110.0% of the stated amount. Identification A. In the Assay, the ultraviolet absorption spectrum (Appendix 4.1) of the test solution in the range 220 nm to 350 nm exhibits absorption maxima at 238 nm and 285 nm. B. In the Related substances, the principal spot in the chromatogram obtained with test solution (2) corresponds in position, colour and size to that in the chromatogram obtained with reference solution (2). Related substances A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Toluene - methanol (95 : 5). Test solution (1): Extract a quantity of the powdered tablets containing the equivalent of 0.2 g of carbamazepine with three 10-ml quantities of chloroform R. Combine the extracts and filter. Evaporate the filtrate to dryness. Dissolve the residue in 10.0 ml of chloroform R. Test solution (2): Dilute 1 volume of test solution (1) to 10 volumes with chloroform R. Reference solution (1): A 0.006% solution of iminodibenzyl RS in methanol R. Reference solution (2): A 0.2% solution of carbamazepine RS in chloroform R.

Assay Weigh 20 tablets, calculate the average mass and powder finely. Transfer a quantity of the powdered tablets containing the equivalent of about 50 mg of carbamazepine, accurately weighed, to a 100 ml volumetric flask, add 60 ml of ethanol (96%) R, warm on a water bath for 15 minutes, shake continuously and allow to cool. Dilute to volume with ethanol (96%) R and mix well. Filter, discard the initial filtrate. Dilute 5.0 ml of the filtrate to 250.0 ml with ethanol (96%) R and mix. Measure the absorbance of the resulting solution at the maximum at about 285 nm (Appendix 4.1), in a 1 cm cell, using ethanol (96%) R as a blank. Calculate the content of carbamazepine, C15H12N2O, in the tablets taking 490 as the value of A (1%, 1 cm) at the maximum at 285 nm. Storage Store in a cool and dry place, protected from light. Action and use Treatment of epilepsy. Usual strength 200 mg.

173

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CARBIDOPA

CARBIDOPA Carbidopum

C10H14N2O4,H2O



M. 244.2

Carbidopa is (2S)-3-(3,4-dihydroxyphenyl)-2-hydrazino2-methylpropanoic acid monohydrate. It contains not less than 98.5% and not more than 101.0% of C10H14N2O4, calculated with reference to the dried substance.

Characters White or yellowish-white powder. Sparingly soluble in methanol, slightly soluble in water, very slightly soluble in ethanol (96%), practically insoluble in dichloromethane and diethyl ether. It dissolves in dilute solutions of mineral acids. Identification Apply one of the two following identifications: First identification: A, C. Second identification: B, C, D, E. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum obtained with carbidopa RS. B. Dissolve 50.0 mg in a 8.5 g/L solution of hydrochloric acid R in methanol R and dilute to 100.0 ml with the same solution. Dilute 10.0 ml of this solution to 100.0 ml with a 8.5 g/L solution of hydrochloric acid R in methanol R. The ultraviolet absorption spectrum (Appendix 4.1) of this solution in the range 230 nm to 350 nm exhibits an absorption maximum at 283 nm. The A (1%, 1 cm) at 283 nm is 135 to 150, calculated with reference to the dried substance. C. It complies with the test for Specific optical rotation. D. Shake vigorously about 5 mg with 10 ml of water for 1 min and add 0.3 ml of 1.3% solution of iron (III) chloride R. An intense green colour is produced, which quickly turns to reddish-brown. E. Suspend about 20 mg in 5 ml of water and add 5 ml of cupri-tartaric solution R. On heating, the colour of the solution changes to dark brown and a red precipitate is formed. Appearance of solution Dissolve 0.25 g in 25 ml of 1 M hydrochloric acid R. The solution is clear (Appendix 9.2) and not more intensely coloured than reference solution BY6 or B6 (Appendix 9.3, method 2). Specific optical rotation -22.5° to -26.5°, calculated with reference to the dried substance (Appendix 6.4). Dissolve completely 0.250 g in aluminium chloride solution R with the aid of an ultrasonic bath, dilute to 25.0 ml with the same solution. 174

Methyldopa and methylcarbidopa Examine by liquid chromatography (Appendix 5.3). Mobile phase: 1.4% solution of potassium dihydrogen phosphate - methanol (2 : 98). Test solution: Dissolve 0.100 g of the substance to be examined in 0.1 M hydrochloric acid R and dilute to 10.0 ml with the same acid. Reference solution: Dissolve 1 mg methylcarbidopa RS and 1 mg of methyldopa RS in 0.1 M hydrochloric acid R and dilute to 20.0 ml with the same acid. Resolution solution: Dissolve 5 mg of carbidopa RS and 5 mg of methyldopa RS in 0.1 M hydrochloric acid R and dilute to 10.0 ml with the same acid. Chromatographic system:   A column (25 cm × 4.6 mm) packed with stationary phase B (5 µm). Detector: A spectrophotometer set at 282 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Procedure Inject the resolution solution. The test is not valid unless the resolution between the peaks corresponding to methyldopa and carbidopa is at least 4.0. Limits: Methyldopa and methylcarbidopa: the area of each peak in the chromatogram obtained with the test solution is not more than the area of the corresponding peak in the chromatogram obtained with the reference solution (0.5%). Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying 6.9% to 7.9% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.150 g with gentle heating in 75 ml of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 22.62 mg of C10H14N2O4. Storage Protected from light. Action and use Treatment of Parkinson's disease. Preparation Tablets (combination with levodopa).

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CARBOMERS Carbomera Carbomer is high-molecular-mass polymers of acrylic acid cross-linked with alkenyl ethers of sugars or polyalcohols. It contains not less than 56.0% and not more than 68.0% of carboxylic acid (-COOH) groups, calculated with reference to the dried substance.

Characters White or almost white, fluffy, hygroscopic powder. Swells in water and in other polar solvents after dispersion and neutralisation with sodium hydroxide solution. Identification Apply one of the two following identifications: First identification: A. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined has main bands at 1710 ± 5 cm-1, 1454 ± 5 cm-1, 1414 ± 5 cm-1, 1245 ± 5 cm-1, 1172 ± 5 cm-1, 1115 ± 5 cm-1 and 801 ± 5 cm-1, with the strongest band at 1710 ± 5 cm-1. B. Disperse 10 g of the substance to be examined in 1 L water, adjust to about pH 7.5 with 1 M sodium hydroxide. A highly viscous gel is formed. C. Add 2 ml of a 10% solution of calcium chloride R, with continuous stirring, to 10 ml of the gel from identification test B. A white precipitate is immediately produced. D. Add 0.5 ml of thymol blue solution R to 10 ml of a 1% dispersion in water. An orange colour is produced. Add 0.5 ml of cresol red solution R to 10 ml of a 1% dispersion in water. A yellow colour is produced. Free acrylic acid Examine by liquid chromatography (Appendix 5.3). Mobile phase A: A 1.361 g/L solution of potassium dihydrogen phosphate R adjusted to pH 2.5 using 10% solution of phosphoric acid R. Mobile phase B: A 1.361 g/L solution of potassium dihydrogen phosphate R - acetonitrile for chromatography (50 : 50). Test solution: Mix 0.125 g of the substance to be examined with a 2.5% solution of aluminium potassium sulfate R and dilute to 25.0 ml with the same solution. Heat the suspension at 50 °C for 20 min with shaking, then shake the suspension at room temperature for 60 min. Centrifuge and use the clear supernatant solution as the test solution. Reference solution: Dissolve 62.5 mg of acrylic acid RS in a 2.5% solution of aluminium potassium sulfate R and dilute to 100.0 ml with the same solution. Dilute 1.0 ml of this solution to 50.0 ml with a 2.5% solution of aluminium potassium sulfate R. Chromatographic system: A column (12 cm × 4.6 mm) packed with stationary phase C (5 µm).

CARBOMERS

Detector: A spectrophotometer set at 205 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0-8

100

0

8-9

100 → 0

0 → 100

9 - 20

0

100

Retention time of acrylic acid is about 6.0 min. Limits: In the chromatogram obtained with the test solution, the area of the acrylic acid peak is not more than the area of the corresponding peak in the chromatogram obtained with the reference solution (0.25%).

Benzene Residual slovent (Appendix 10.14, system A). Solution A: Dissolve 0.100 g of benzene R in dimethyl sulfoxide R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of the solution to 100.0 ml with water. Dilute 1.0 ml of this solution to 100.0 ml with water. Test solution: Weigh 50.0 mg of the substance to be examined into an injection vial and add 5.0 ml of water and 1.0 ml of dimethyl sulfoxide R. Reference solution: Weigh 50.0 mg of the substance to be examined into an injection vial and add 4.0 ml of water, 1.0 ml of dimethyl sulfoxide R and 1.0 ml of solution A. Close the vials with a tight rubber membrane stopper coated with polytetrafluoroethylene and secure with an aluminium crimped cap. Shake to obtain a homogeneous dispersion. Procedure: Examine by gas chromatography (Appendix 5.2), static head-space conditions that may be used: Equilibration temperature: 80 °C. Equilibration time: 60 min. Transfer-line temperature: 90 °C. Injection 1 ml of the gaseous phase of the test solution and 1 ml of the gaseous phase of the reference solution; repeat these injections twice more. System suitability: The relative standard deviation of the differences in area between the analyte peaks obtained from the 3 replicate pair injections of the reference solution and the test solution is not more than 15%. Limit: The mean area of the peak due to benzene in the chromatograms obtained with the test solution is not greater than 0.5 times the mean area of the peak due to benzene in the chromatograms obtained with the reference solution (2 ppm). Heavy metals Not more than 20 ppm (Appendix 9.4.8). 175

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CARMELLOSE CALCIUM

1.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R.

Loss on drying Not more than 3.0% (Appendix 9.6). (1.000 g; in vacuo at 80 °C; 60 min). Sulfated ash Not more than 4.0% (Appendix 9.9, method 2). Determined on 1.000 g. Assay Slowly add 50 ml of water to 0.120 g of the substance to be examined whilst stirring and heating at 60 °C for 15 min. Stop heating, add 150 ml of water and continue stirring for 30 min. Add 2 g of potassium chloride R and titrate with 0.2 N sodium hydroxide VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.2 N sodium hydroxide VS is equivalent to 9.0 mg of carboxylic acid (-COOH) groups. Storage In an airtight container. Action and use Excipients. FUNCTIONALITY-RELATED CHARACTERISTICS The following characteristics may be relevant for carbomers used as viscosity-increasing agents and gelling agents. Apparent viscosity Appendix 6.3, method III. The nominal apparent viscosity is typically between 300 mPa.s and 115 000 mPa·s. For a product with a nominal apparent viscosity of 20 000 mPa·s or greater, the apparent viscosity is typically 70.0% to 130.0% of the nominal value; for a product with a nominal apparent viscosity of less than 20 000 mPa·s, the apparent viscosity is typically 50.0% to 150.0% of the nominal value. Dry the substance to be examined in vacuo at 80 °C for 1 h. Carefully add 2.50 g of the previously dried substance to be examined to 500 ml of water in a 1000 ml beaker while stirring continuously at (1000 ± 50) r/min, with the stirrer shaft set at an angle of 60° to one side of the beaker. Add the previously dried substance over a period of 45 s to 90 s, at a uniform rate, ensuring that loose agglomerates of powder are broken up, and continue stirring at (1000 ± 50) r/min for 15 min. Remove the stirrer and place the beaker containing the dispersion in a water-bath at (25 ± 1) °C for 30 min. Insert the stirrer to a depth necessary to ensure that air is not drawn into the dispersion and, while stirring at (300 ± 25) r/min, titrate with a glass-calomel electrode system to pH 7.3 to 7.8 by adding a 18% solution of sodium hydroxide R below the surface, determining the end-point potentiometrically (Appendix 10.2). The total volume of 176

the 18% solution of sodium hydroxide R used is about 6.2 ml. Allow to stand 2 min to 3 min before the final pH determination. If the final pH exceeds 7.8, discard the preparation and prepare another using a smaller amount of sodium hydroxide for titration. Return the neutralised preparation to the water-bath at (25 ± 1) °C for 1 h, then perform the viscosity determination without delay to avoid slight viscosity changes that occur 75 min after neutralisation. Determine the viscosity using a rotating viscometer with a spindle rotating at 20 r/min, using a spindle suitable for the expected apparent viscosity.

Carboxylic acid groups See Assay. CARMELLOSE CALCIUM Carmelosum calcicum Carmellose calcium is calcium salt of a partly O-carboxymethylated cellulose.

Characters White or yellowish-white powder, hygroscopic after drying. Practically insoluble in acetone, in alcohol and in toluene. It swells with water to form a suspension. Identification A. Shake 0.1 g of the substance to be examined thoroughly with 10 ml of water. Add 2 ml of dilute sodium hydroxide solution R and allow to stand for 10 min (solution A). Dilute 1 ml of solution A to 5 ml with water. To 0.05 ml add 0.5 ml of a 0.05% solution of chromotropic acid sodium salt R in a 75% solution of sulfuric acid R and heat on a water-bath for 10 min. A reddish-violet colour develops. B. Shake 5 ml of solution A obtained in identification test A with 10 ml of acetone R. A white, flocculent precipitate is produced. C. Shake 5 ml of solution A obtained in identification test A with 1 ml of a 10,5% solution of ferric chloride R. A brown, flocculent precipitate is formed. D. Ignite 1 g of the substance to be examined and dissolve the residue in a mixture of 5 ml of acetic acid R and 10 ml of water. Filter if necessary and boil the filtrate for a few minutes. Cool and neutralise with dilute ammonia R. The solution gives reaction (A) of calcium (Appendix 8.1). Alkalinity Shake 1.0 g of the substance to be examined thoroughly with 50 ml of carbon dioxide-free water and add 0.05 ml of phenolphthalein solution R. No red colour develops. Chlorides Not more than 0.36% (Appendix 9.4.5). Solution S: Shake 1.0 g of the substance to be examined with 50 ml of distilled water, add 5 ml of dilute sodium

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hydroxide solution R and dilute to 100 ml with distilled water. Heat 28 ml of solution S with 10 ml of dilute nitric acid R on a water-bath until a flocculent precipitate is produced. Cool, centrifuge and separate the supernatant liquid. Wash the precipitate with 3 quantities, each of 10 ml, of water, centrifuging each time. Combine the supernatant liquid and the washings and dilute to 100 ml with water. To 25 ml add 6 ml of dilute nitric acid R and dilute to 50 ml with water. Dilute 10 ml of the solution to 15 ml with water.

Sulfates Not more than 1% (Appendix 9.4.14). Heat 20 ml of solution S with 1 ml of hydrochloric acid R on a water-bath until a flocculent precipitate is produced. Cool, centrifuge and separate the supernatant liquid. Wash the precipitate with 3 quantities, each of 10 ml, of distilled water, centrifuging each time. Combine the supernatant liquid and the washings and dilute to 100 ml with distilled water. To 25 ml add 1 ml of dilute hydrochloric acid R and dilute to 50 ml with distilled water. Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 4. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 10% (Appendix 9.6). (1.000 g; 105 °C; 4 h). Sulfated ash 10.0% to 20.0% (Appendix 9.9, method 2). Determined on 1.0 g, in a platinum crucible. Storage In an airtight container. Action and use Excipients, bulk laxative. CARMELLOSE SODIUM Carmellosum natricum Carmellose sodium (carboxymethylcellulose sodium) is the sodium salt of a partly O-carboxymethylated cellulose. It contains not less than 6.5% and not more than 10.8% of sodium (Na), calculated with reference to the dried substance.

Characters A white or almost white, granular powder, hygroscopic after drying. Practically insoluble in acetone, in ethanol and in toluene. It is easily dispersed in water giving colloidal solutions.

CARMELLOSE SODIUM

Identification Solution S: Sprinkle a quantity of the substance to be examined equivalent to 1.0 g of the dried substance onto 90 ml of carbon dioxide-free water at 40 °C to 50 °C stirring vigorously. Continue stirring until a colloidal solution is obtained, cool and dilute to 100 ml with carbon dioxide-free water. A. To 10 ml of solution S add 1 ml of copper sulfate solution R. A blue, cotton-like precipitate is formed. B. Boil 5 ml of solution S for a few minutes. No precipitate is formed. C. The solution prepared in the test for Heavy metals gives the reactions of sodium (Appendix 8.1). Appearance of solution Solution S is not more opalescent than reference suspension III (Appendix 9.2). and not more intensely coloured than reference solution Y6 (Appendix 9.3, method 2). pH The pH of solution S is from 6.0 to 8.0 (Appendix 6.2). Apparent viscosity 75% to 140% of the value stated on the label (Appendix 6.3). While stirring, introduce a quantity of the substance to be examined equivalent to 2.00 g of the dried substance into 50 ml of water heated to 90 °C. Allow to cool, dilute to 100.0 ml with water and stir until dissolution is complete. Determine the viscosity using a rotating viscometer at 20 °C and a shear rate of 10 s-1. If it is impossible to obtain a shear rate of exactly 10 s-1, use a shear rate slightly higher and a rate slightly lower and interpolate. For a product of low viscosity, use if necessary, the quantity required to give the concentration indicated on the label. Sodium glycollate Not more than 0.4%. Test solution: Place a quantity of the substance to be examined equivalent to 0.500 g of dried substance in a beaker. Add 5 ml of acetic acid R and 5 ml of water. Stir until dissolution is complete (about 30 min). Add 80 ml of acetone R and 2 g of sodium chloride R. Filter through a fast filter paper impregnated with acetone R into a volumetric flask, rinse the beaker and filter with acetone R and dilute the filtrate to 100.0 ml with the same solvent. Allow to stand for 24 h without shaking. Use the clear supernatant liquid to prepare the test solution. Reference solution: Dissolve 0.310 g of glycollic acid R, previously dried in vacuo over diphosphorus pentoxide R, in water in a volumetric flask and dilute to 1000.0 ml with the same solvent. Place 5.0 ml of this solution in a volumetric flask, add 5 ml of acetic acid R and allow to stand for about 30 min. Add 80 ml of acetone R and 2 g of sodium chloride R and dilute to 100.0 ml with acetone R. Use this solution to prepare the reference solution. Place 2.0 ml of each solution in a separate 25 ml volumetric flask. Heat on a water-bath to eliminate acetone. Cool to room temperature and add 5.0 ml of 2,7- dihydroxynaphthalene 177

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CEFACLOR

solution R to each flask. Shake and add 15.0 ml of 2,7dihydroxynaphthalene solution R. Close the flasks with aluminium foil and heat on a water-bath for 20 min. Cool under running water and dilute to 25.0 ml with sulfuric acid R. Within 10 min, transfer 10.0 ml of each solution to a flat-bottomed tube. Examine the solutions viewing vertically. The test solution is not more intensely coloured than the reference solution.

Chlorides Not more than 0.25% (Appendix 9.4.5). Dilute 2 ml of solution S to 15 ml with water. Heavy metals Not more than 20 ppm (Appendix 9.4.8). To the residue obtained in the determination of the Sulfated ash, add 1 ml of hydrochloric acid R and evaporate on a water-bath. Take up the residue in 20 ml of water. 12 ml of solution complies with limit test for heavy metals, method 1. Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on drying Not more than 10.0% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash 20.0% to 33,3%, calculated with reference to the dried substance (Appendix 9.9, method 2). These limits correspond to a content of 6.5% to 10.8% of sodium (Na). Determined on 1.0 g of the substance to be examined, calculated with reference to the dried substance, using a mixture of equal volumes of sulfuric acid R and water. Storage Store in an airtight container. Labelling The label states the apparent viscosity in millipascal seconds for a 20 g/L solution. For a product of low viscosity, the label states the concentration of the solution to be used and the apparent viscosity in millipascal seconds. Action and use Excipients, bulk laxative. CEFACLOR Cefaclorum

C15H14ClN3O4S,H2O 178



M. 385.8

Cefaclor is (6R,7R)-7-[[(2R)-2-amino-2-phenylacetyl] amino]-3-chloro-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2ene-2-carboxylic acid monohydrate, semi-synthetic product derived from a fermentation product. It contains not less than 96.0% and not more than 102.0% of C15H14ClN3O4S, calculated with reference to the anhydrous substance.

Characters A white or slightly yellow powder. Slightly soluble in water, practically insoluble in methanol and in methylene chloride. Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of cefaclor RS. pH Shake 0.250  g of the substance to be examined in  carbon dioxide-free water R and dilute to 10 ml with the same solvent. The pH of the suspension is 3.0 to 4.5 (Appendix 6.2). Specific optical rotation + 101° to + 111°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.250 g of the substance to be examined in a 1% solution of hydrochloric acid R and dilute to 25.0 ml with the same solvent. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: A 0.78% solution of sodium dihydrogen phosphate R adjusted to pH 4.0 with phosphoric acid R. Mobile phase B: Mix 450 ml of acetonitrile R with 550 ml of mobile phase A. Diluent: A 0.27% solution of sodium dihydrogen phosphate R adjusted to pH 2.5 with phosphoric acid R. Test solution:  Dissolve 50.0  mg of the substance to be examined in 10.0 ml of the diluent. Reference solution (1): Dissolve 2.5 mg of cefaclor RS and 5.0 mg of delta-3-cefaclor RS (impurity D) in 100.0 ml of the diluent. Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with the diluent. Chromatographic system: A column (25 cm × 4.6 mm) packed with end-capped octadecylsilyl silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 220 nm. Flow rate: 1.0  ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions:

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CEFACLOR CAPSULES

Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 30

95 → 75

5 → 25

30 - 45

75 → 0

25 → 100

45 - 55

0

100

System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to cefaclor and impurity D is at least 2.0. The symmetry factor of the cefaclor peak is at most 1.2. If necessary, adjust the acetonitrile content of the mobile phase. Limits: Any impurity: The area of each impurity peak is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.5%). Sum of the areas of all the impurity peaks is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (2) (2%). Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.1%).

Notes: Impurity A: (2R)-2-amino-2-phenylacetic acid (phenylglycine). Impurity B: (6R,7R)-7-amino-3-chloro-8-oxo-5-thia-1-azabicyclo [4.2.0]oct-2-ene-2-carboxylic acid. Imputity C: (6R,7R)-7-[[(2S)-2-amino-2-phenylacetyl]amino] -3chloro-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid. Impurity D: (2R,6R,7R)- and (2S,6R,7R)-7-[[(2R)-2-amino-2phenylacetyl]amino]-3-chloro-8-oxo-5-thia- 1-azabicyclo[4.2.0] oct-3-ene-2-carboxylic acid (delta-3-cefaclor). Impurity E: 2-[[(2R)-2-amino-2-phenylacetyl]amino]-2-(5-chloro -4-oxo-3,4-dihydro-2H-1,3-thiazin-2-yl)acetic acid. Impurity F: 3-phenylpyrazin-2-ol. Impurity G: (2R,6R,7R)- and (2S,6R,7R)-7-[[(2R)-2-amino-2 -phenylacetyl]amino]-3-methylene-8-oxo-5-thia-1-azabicyclo [4.2.0]octane-2-carboxylic acid (isocefalexine). Impurity H: (6R,7R)-7-[[(2R)-2-[[(2R)-2-amino-2-phenylacetyl] amino]-2-phenylacetyl]amino]-3-chloro-8-oxo-5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (N-phenylglycyl cefaclor).

Heavy metals Not more than 30 ppm (Appendix 9.4.8). 1.0 g complies with the limit test for heavy metals, method 3. Prepare the standard using 3 ml of lead standard solution (10 ppm Pb) R. Water 3.0% to 6.5% (Appendix 10.3). Determined on 0.200 g. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 1 g of sodium pentanesulfonate R in a mixture of 780 ml of water and 10 ml of triethylamine R. Add 220 ml of methanol R to the mixture and adjust to pH 2.5 with phosphoric acid R.

Test solution: Dissolve 15.0  mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the same solvent. Reference solution (1): Dissolve 15.0 mg of cefaclor RS in the mobile phase and dilute to 50.0 ml with the same solvent. Reference solution (2): Dissolve 3.0 mg of cefaclor RS and 3.0 mg of delta-3-cefaclor RS (impurity D) in the mobile phase and dilute to 10.0 ml with the same solvent. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 265 nm. Flow rate: 1.5  ml/min. Volume of injection: 20 µl. Procedure: System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to cefaclor and impurity D is at least 2.5. If necessary, adjust the concentration of methanol in the mobile phase. The symmetry factor of the cefaclor peak is at most 1.5. In the chromatogram obtained with reference solution (1), the relative standard deviation of the cefaclor peak areas for 6 replicate injections is not more than 1.0%. Calculate the percentage content of cefaclor in the substance to be examined using the areas of the principal peaks in the chromatograms obtained with the test solution, reference solution (1) and the declared content of cefaclor RS.

Storage Store in an airtight container. Action and use Cephalosporin antibacterial. Preparations Tablets, capsules, oral suspensions. CEFACLOR CAPSULES Capsulae Cefaclori Cefaclor capsules contain cefaclor. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of anhydrous cefaclor, C15H14ClN3O4S, 90.0% to 110.0% of the stated amount. Identification A. Shake a quantity of the capsule contents containing the equivalent of 0.3 g of anhydrous cefaclor with 100 ml of water, filter and dilute 1 ml of the filtrate to 100 ml with water. The ultraviolet absorption spectrum (Appendix 4.1) of the resulting solution in the range 190 nm to 310 nm exhibits absorption maximum only at 264 nm. 179

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CEFACLOR POWDER FOR ORAL SUSPENSION

B. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the principal peak in the chromatogram obtained with the reference solution.

Water Not more than 8.0% (Appendix 10.3). Use 0.5 g of the capsule contents. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of water. Rotation speed: 50 rpm. Time: 30 min. Procedure: After the specified time, withdraw a sample of the medium and filter, discard the first 20 ml of the filtrate. Dilute the filtrate with water (if necessary) to obtain a solution containing about 0.025% of anhydrous cefaclor. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum at about 264 nm, in a 1 cm cell, using water as a blank. Calculate the content of anhydrous cefaclor, C15H14ClN3O4S, is dissolved in the each capsule using the absorbance of a reference solution of cefaclor RS having the same concentration in the same medium. Tolerance: Not less than 80% (Q) of the stated amount of anhydrous cefaclor, C15H14ClN3O4S, is dissolved in 30 min. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 1 g of sodium pentane sulphonate R in a mixture of 780 ml of water and 10 ml of triethylamine R, adjust the pH to 2.5 ± 0.1 using phosphoric acid R, add 220 ml of methanol R and mix well. Reference solution: Dissolve an accurately weighed quantity of cefaclor RS in the mobile phase to obtain a solution containing about 0.3 mg per ml. Sonicate, if necessary, to dissolve and avoid heating. Test solution: Weigh 20 capsules, calculate the average weight of the capsule contents and powder finely. Transfer an accurately weighed quantity of the contents of the capsules containing the equivalent of about 15 mg of anhydrous cefaclor to a 50 ml volumetric flask, add about 35 ml of the mobile phase and sonicate for 15 min, avoid heating. Dilute to volume with the mobile phase and mix, filter. Resolution solution: Dissolve a quantity of cefaclor RS and delta-3-cefaclor RS in the mobile phase to obtain a solution containing about 0.3 mg of each substance per ml. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 265 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the resolution solution. The relative retention times for cefaclor and delta-3-cefaclor 180

are 0.8 and 1.0, respectively; the resolution between cefaclor peak and delta-3-cefaclor peak is not less than 2.5; the symmetry factor of cefaclor peak is not more than 1.5. Inject the reference solution. The relative standard deviation of the cefaclor peak areas for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of cefaclor, C15H14ClN3O4S, in the capsules using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C15H14ClN3O4S in cefaclor RS.

Storage Pack in aluminum blister or closed bottle. Store in a cool and dry place at a temperature not exceeding 30 °C, protected from light. Action and use Cephalosporin antibacterial. Usual strength 250 mg; 500 mg. CEFACLOR POWDER FOR ORAL SUSPENSION Pulveres Cefaclori ad suspensionum peroralum Cefaclor powder for oral suspension contains cefaclor. Suitable excipients such as flavors, colours, preservatives, stabilizing agents… may be added. The constituted suspension prepared as directed on the label complies with the requirements stated under “Suspension” (Appendix 1.5). The powder complies with the requirements stated under “Powder” (Appendix 1.7) and with the following requirements:

Content of anhydrous cefaclor, C15H14ClN3O4S, 90.0% to 120.0% of the stated amount. Characters The powder should be loose and dry, with homogeneous colour. Identification A. Shake a quantity of the powder containing the equivalent of 0.3 g of anhydrous cefaclor with 100 ml of water, filter and dilute 1 ml of the filtrate to 100 ml with water. The ultraviolet absorption spectrum (Appendix 4.1) of the resulting solution in the range 190 nm to 310 nm exhibits a maximum only at 264 nm. B. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the principal peak in the chromatogram obtained with the reference solution.

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CEFACLOR POWDER FOR ORAL SUSPENSION

Water Not more than 2.0% (Appendix 10.3). Determined on 0.5 g. pH 2.5 to 5.0 (Appendix 6.2). Use the suspension constituted as directed on the label. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: A 0.78% solution of sodium dihydrogen phosphate R adjusted to pH 4.0 with phosphoric acid R. Mobile phase B: Acetonitrile - mobile phase A (45 : 55). Diluent: A 0.27% solution of sodium dihydrogen phosphate R adjusted to pH 2.5 with phosphoric acid R. Prepare the following solutions immediately before use. Test solution: Transfer an accurately weighed quantity of the powder or the suspension constituted as directed on the label containing the equivalent to about 0.25 g of anhydrous cefaclor to a 250 ml volumetric flask, add 200 ml of diluent and shake to dissolve. Dilute to volume with the same solvent and mix well. Filter. Reference solution: A 0.001% solution of cefaclor RS. Resolution solution: A solution containing 0.0025% of cefaclor RS and 0.005% of delta-3-cefaclor RS. Placebo solution (where applicable): Transfer an accurately weighed quantity of the mixed excipients (with the same ratio in the preparation) equivalent to the powder contaning 0.25 g of anhydrous cefaclor to a 250 ml volumetric flask, add 200 ml of diluent and shake to dissolve. Dilute to volume with the same solvent and mix well. Filter. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 μm). Detector: A spectrophotometer set at 220 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 μl. Procedure: Equilibrate the column with the mixture of mobile phase A and mobile phase B (95 : 5) for at least 15 min. Inject the test solution, the reference solution and the placebo solution (where applicable). Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

Comment

0 - 30

95 → 75

5 → 25

Linear gradient

30 - 45

75 → 0

25 → 100

Linear gradient

45 - 55

0

100

Isocratic

55 - 70

0 → 95

100 → 5

Re-equilibration

System suitability: In the chromatogram obtained with the resolution solution, the resolution factor between the

peaks due to cefaclor and delta-3-cefaclor is at least 2.0; adjust the proportion of acetonitrile in the mobile phase, if neccessary. Limits: In the chromatogram obtained with the test solution: The area of any secondary peak is not more than the area of the principal peak in the chromatogram obtained with the reference solution (1.0%) and the sum of the areas of all the secondary peaks is not greater 3 times the area of the principal peak in the chromatogram obtained with the reference solution (3.0%). Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with the reference solution (0.1%). Disregard any peak corresponding to those obtained with the placebo solution.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 1.0 g of sodium pentanesulfonate R in a mixture of 780 ml of water and 10 ml of triethylamine R, adjust to pH 2.5 with phosphoric acid R, add 220 ml of methanol R and mix. Reference solution: Dissolve an accurately weighed quantity of cefaclor RS in the mobile phase to obtain a solution having a known concentration of 0.3 mg per ml. Sonicate to dissolve, if necessary, avoid heating the solution. Test solution: For single-dose preparations, transfer an accurately weighed quantity of the mixed powder obtained from the test for Uniformity of mass; for multidose preparations, transfer an accurately weighed quantity of the suspension constituted as directed on the label containing the equivalent to 60 mg of anhydrous cefaclor to a 200 ml volumetric flask, add 150 ml of the mobile phase and sonicate for 15 min, avoid heating. Dilute to volume with the same solvent, mix and filter. Resolution solution: Dissolve a quantity of cefaclor RS and delta-3-cefaclor RS in the mobile phase to obtain a solution containing about 0.3 mg of each substance per ml. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 μm). Detector: A spectrophotometer set at 265 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 μl. Procedure: System suitability: Inject the resolution solution. The relative retention times for cefaclor and delta-3-cefaclor are 0.8 and 1.0, respectively; the resolution factor between the peaks due to cefaclor and delta-3-cefaclor is at least 2.5; the tailing factor of cefaclor peak is not more than 1.5. Inject the reference solution, the relative standard deviation of the peak areas due to cefaclor for 6 replicate injections is not more than 2.0%. Inject separately the reference solution and the test solution. Calculate the contents of cefaclor, C15H14ClN3O4S, in the preparation from the peak areas in the chromatograms 181

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CEFADROXIL MONOHYDRATE

obtained with the test solution and the reference solution, the declared content of C15H14ClN3O4S in cefaclor RS and the density of the constituted suspension (for multi-dose preparations, Appendix 6.5).

Storage Store the powder in an airtight container, in a cool and dry place, protected from light. The suspension after constitution is kept at the temperature and used within the period as stated on the label. Labelling The lable states the appropriate direction for the oral suspension preparation and the equivalent amount of cefaclor (C15H14ClN3O4S) per given volume of the constituted suspesion (for multi-dose preparations). Action and use Cephalosporin antibacterial. Usual strength Single-dose preparations: 125 mg; 250 mg. Multi-dose preparations: 125 mg/5 ml; 250 mg/5 ml. CEFADROXIL MONOHYDRATE Cefadroxilum monohydricum

C16H17N3O5S,H2O

M. 381.4

Cefadroxil is (6R,7R)-7-[[(2R)-2-amino-2-(4-hydroxyphenyl) acetyl]amino]-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0] oct-2-ene-2-carboxylic acid monohydrate, semi-synthetic product derived from a fermentation product. It contains not less than 95.0% and not more than 102.0% of C16H17N3O5S, calculated with reference to the anhydrous substance.

Characters A white or almost white powder. Slightly soluble in water, very slightly soluble in ethanol (96%). Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of cefadroxil RS. pH 4.0 to 6.0 (Appendix 6.2). Suspend 1.0 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent. 182

Specific optical rotation +165° to +178°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.500  g of the substance to be examined in water and dilute to 50.0 ml with the same solution. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Phosphate buffer solution pH 5.0 R. Mobile phase B: Methanol R2. Test solution:  Dissolve 50.0  mg of the substance to be examined in mobile phase A and dilute to 50.0 ml with the same solution. Reference solution (1):  Dissolve 10.0  mg of  D-α-(4hydroxyphenyl)glycine RS (impurity A) in mobile phase A and dilute to 10.0 ml with the same solution. Reference solution (2): Dissolve 10.0  mg of  7-aminodes acetoxycephalosporanic acid RS (impurity B) in phosphate buffer solution pH 7.0  R5 and dilute to 10.0 ml with the same solution. Reference solution (3): Dilute 1.0 ml of reference solution (1) and 1.0 ml of reference solution (2) to 100.0 ml with mobile phase A. Reference solution (4): Dissolve 10  mg of dimethylformamide R and 10 mg of dimethylacetamide R in mobile phase A and dilute to 10.0 ml with the same solution. Dilute 1.0 ml of this solution to 100.0 ml with mobile phase A. Reference solution (5): Dilute 1.0 ml of reference solution (3) to 25.0 ml with mobile phase A. Chromatographic system: A column (10 cm × 4.6 mm) packed with spherical octadecylsilyl silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 220 nm. Flow rate: 1.5  ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0-1

98

2

1 - 20

98 → 70

2 → 30

The relative retention with reference to cefadroxil (retention time = about 6 min): Dimethylformamide  =  about 0.4; dimethylacetamide = about 0.75. System suitability: In the chromatogram obtained with reference solution (3), the resolution between the peaks due to impurities A and B is at least 5.0. In the chromatogram obtained with reference solution (5),  the signal-to-noise ratio is at least 10 for the second peak. Limits: Impurity A: The area of peak due to impurity A is not more than the area of the first peak in the chromatogram obtained with reference solution (3) (1.0%),

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Any other impurity: The area of each impurity peak is not more than the area of the second peak in the chromatogram obtained with reference solution (3) (1.0%), Sum of the areas of all the impurity peaks is not more than 3 times the area of the second  peak in the chromatogram obtained with reference solution (3) (3.0%), Disregard any peak with an area less than 0.05 times the area of the second  peak in the chromatogram obtained with reference solution (3) (0.05%). Disregard the peaks due to dimethylformamide and dimethylacetamide. Notes: Impurity A: (2R)-2-amino-2-(4-hydroxyphenyl)acetic acid. Impurity B: (6R,7R)-7-amino-3-methyl-8-oxo-5-thia-1-azabicyclo [4.2.0]oct-2-ene-2-carboxylic acid (7-ADCA). Imputity C: (2R,5RS)-2-[(R)-[[(2R)-2-amino-2-(4-hydroxyphenyl) acetyl]amino]carboxymethyl]-5-methyl-5,6-dihydro-2H-1,3thiazine-4-carboxylic acid. Impurity D: 6R,7R)-7-[[(2S)-2-amino-2-(4-hydroxyphenyl) acetyl]amino]-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2ene-2-carboxylic acid (L-cefadroxil). Impurity E: (6RS)-3-(aminomethylene)-6-(4-hydroxyphenyl) piperazine-2,5-dione. Impurity F: (6R,7R)-7-[[(2R)-2-[[(2RS)-2-amino-2-(4-hydroxyphenyl) acetyl]amino]-2-(4-hydroxyphenyl)acetyl]amino]-3-methyl-8oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid. Impurity G: 3-hydroxy-4-methylthiophen-2(5H)-one. Impurity H: (6R,7R)-7-[(2,2-dimethylpropanoyl)amino]-3methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (7-ADCA pivalamide).

N,N-Dimethylaniline Not more than 20 ppm (Appendix 10.16, method 2). Water 4.0% to 6.0% (Appendix 10.3). Determined on 0.200 g. Sulfated ash Not more than 0.5% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile  - 0.272% solution of potassium dihydrogen phosphate (4 : 96 ). Test solution: Dissolve 50.0  mg of the substance to be examined in the mobile phase and dilute to 100.0 ml with the same solvent. Reference solution (1):  Dissolve 50.0  mg of  cefadroxil RS in the mobile phase and dilute to 100.0 ml with the same solvent. Reference solution (2): Dissolve 5 mg of cefadroxil RS and 50  mg of  amoxicillin trihydrate RS in the mobile phase and dilute to 100 ml with the same solvent. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm).

CEFADROXIL CAPSULES

Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0  ml/min. Volume of injection: 20 µl. Procedure: System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to cefadroxil and amoxicillin is at least 5.0. Inject the test solution and reference solution (1). Calculate the content of cefadroxil in the substance to be examined, using the areas of peaks due to cefadroxil in the chromatograms obtained with the test solution, reference solution (1), and the declared content of C16H17N3O5S in cefadroxil RS.

Storage Store in an airtight container, protected from light. Action and use Cephalosporin antibacterial. Preparations Capsules, oral suspensions. CEFADROXIL CAPSULES Capsulae Cefadroxili Cefadroxil capsules contain cefadroxil monohydrate. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of anhydrous cefadroxil, C16H17N3O5S, 90.0% to 110.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4). TLC plate coated with 0.25 mm layer of binder-free silica gel. In a chamber containing a mixture of n-hexane and tetradecane (95 : 5) to a depth of about 1 cm, allow the solvent front to move the length of the plate, remove the plate from the chamber and allow the solvent to evaporate. Mobile phase: 0.1 M citric acid - 0.1 M disodium hydrogen phosphate - solution of ninhydrin in acetone containing 1 g per 15 ml (60 : 40 : 1.5) Test solution: Dissolve a quantity of the contents of the capsules containing the equivalent of 20 mg of cefadroxil in 10 ml of water, filter. Reference solution: A 0.2% solution of cefadroxil RS in water. Procedure: Apply separately to the plate 20 µl of each solution. Develop over three-fourths of the length of the plate. After removal of the plate, allow it to dry in air. Spray with a 0.2% solution of ninhydrin in ethanol R (protect this solution from light), heat the plate at 110 °C for 10 min. Examine in day light. 183

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The principal spot in the chromatogram obtained with the test solution corresponds in position, colour and size to that in the chromatogram obtained with the reference solution. B. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the principal peak in the chromatogram obtained with the reference solution.

Water Not more than 7.0% (Appendix 10.3). Use 0.5 g of the capsule contents. Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 900 ml of water. Rotation speed: 100 rpm. Time: 30 min. Procedure: After the specified time, withdraw a sample of the medium and filter, discard the first 20 ml of the filtrate. Dilute the filtrate with water (if necessary) to obtain a suitable concentration. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum at about 263 nm, in a 1 cm cell, using water as a blank. Calculate the content of cefadroxil, C16H17N3O5S, is dissolved in comparison with a reference solution of cefadroxil RS having the same concentration in the same medium. Tolerance: Not less than 80% (Q) of the stated amount of cefadroxil, C16H17N3O5S, is dissolved in 30 minutes. Assay Examine by liquid chromatography (Appendix 5.3). Phosphate buffer pH 5.0: Dissolve 13.6 g of potassium dihydrogen phosphate R in sufficient water to produce 2000 ml and adjust the pH to 5.0 with 10 M potassium hydroxide solution R. Mobile phase: Acetonitril - phosphate buffer pH 5.0 (4 : 96). Reference solution: Dissolve an accurately weighed quantity of cefadroxil RS in phosphate buffer pH 5.0 to obtain a solution containing about 1.0 mg per ml. Test solution: Weigh 20 capsules, calculate the average weight of the capsule contents and powder finely. Transfer a quantity of the contents of the capsules equivalent to about 100 mg of cefadroxil, accurately weighed, to a 100 ml volumetric flask, add about 75 ml of phosphat buffer pH 5.0 and sonicate for 5 minutes. Dilute to volume with phosphat buffer pH 5.0, mix well and filter. Note: Use the reference solution and the test solution on the day prepared. Chromatographic system: A column (25 cm × 4 mm) packed with stationary phase C (5 µm or 10 µm). Detector: A spectrophotometer set at 230 nm. Flow rate: 1.5 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject the reference solution. The capacity factor, k’, is between 2.0 to 3.5; the column efficiency is not 184

less than 1800 theoretical plates; the symmetry factor is not more than 2.2; the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of cefadroxil, C16H17N3O5S, in the capsules using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C16H17N3O5S in cefadroxil RS.

Storage Store in a well-closed container, in a cool and dry place, protected from light. Action and use Cephalosporin antibacterial. Usual strength 500 mg. CEFADROXIL POWDER FOR ORAL SUSPENSION Pulveres Cefadroxili ad suspensionum peroralum Cefadroxil powder for oral suspension contains cefadroxil. It may contain suitable flavors, colors, preservatives, stabilizers… The oral suspension prepared as directed on the label complies with the requirements stated under Suspension (Appendix 1.5). The powder complies with the requirements stated under “Powders” (Appendix 1.7) and with the following requirements.

Content of anhydrous cefadroxil, C16H17N3O5S, 90.0% to 110.0% of the stated amount. Characters Powder should be loose and dry, not be moist and lumpy, with homogeneous colour. Identification A. Examine by thin-layer chromatography (Appendix 5.4). TLC plate coated with 0.25 mm layer of binder-free silica gel. In a chamber containing a mixture of n-hexane and tetradecane (95 : 5) to a depth of about 1 cm, allow the solvent front to move the length of the plate, remove the plate from the chamber and allow the solvent to evaporate. Mobile phase: 0.1 M citric acid - 0.1 M disodium hydrogen phosphate - solution of ninhydrin in acetone containing 1 g per 15 ml (60 : 40 : 1.5) Test solution: Dissolve a quantity of the contents of the powder containing the equivalent of 20 mg of cefadroxil in 10 ml of water, filter. Reference solution: A 0.2% solution of cefadroxil RS in water. Procedure: Apply separately to the plate 20 µl of each solution. Develop over three-fourths of the length of the

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plate. After removal of the plate, allow it to dry in air. Spray with a 0.2% solution of ninhydrin in ethanol R (protect this solution from light), heat the plate at 110 °C for 10 min. Examine in day light. The principal spot in the chromatogram obtained with the test solution corresponds in position, colour and size to that in the chromatogram obtained with the reference solution. B. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the principal peak in the chromatogram obtained with the reference solution.

Water Not more than 2.0% (Appendix 10.3). Use 1.0 g of the powder. Assay Examine by liquid chromatography (Appendix 5.3). Phosphate buffer pH 5.0: Dissolve 13.6 g of potassium dihydrogen phosphate R in sufficient water to produce 2000 ml and adjust the pH to 5.0 with 10 M potassium hydroxide solution R. Mobile phase: Acetonitrile - phosphate buffer pH 5.0 (4 : 96). Reference solution: Dissolve an accurately weighed quantity of cefadroxil RS in phosphate buffer pH 5.0 to obtain a solution containing about 1.0 mg per ml. Test solution: Transfer a quantity of the powder received from the test for Uniformity of weight equivalent to about 100 mg of cefadroxil, accurately weighed, to a 100 ml volumetric flask, add about 75 ml of phosphat buffer pH 5.0 and sonicate for 5 min. Dilute to volume with phosphat buffer pH 5.0, mix well and filter. Note: Use the reference solution and the test solution on the day prepared.

Chromatographic system: A column (25 cm × 4 mm) packed with stationary phase C (5 µm or 10 µm). Detector: A spectrophotometer set at 230 nm. Flow rate: 1.5 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject the reference solution. The capacity factor, k’, is between 2.0 to 3.5; the column efficiency is not less than 1800 theoretical plates; the symmetry factor is not more than 2.2; the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of cefadroxil, C16H17N3O5S, in the preparation using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C16H17N3O5S in cefadroxil RS.

CEFADROXIL TABLETS

Storage Store in a well-closed container, in a cool and dry place, protected from light. Action and use Cephalosporin antibacterial. CEFADROXIL TABLETS Tabellae Cefadroxili Cefadroxil tablets contain cefadroxil. They are tablets or film-coated tablets. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of anhydrous cefadroxil, C16H17N3O5S, 90.0 to 110.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4). TLC plate coated with 0.25 mm layer of binder-free silica gel. In a chamber containing a mixture of n-hexane and tetradecane (95 : 5) to a depth of about 1 cm, allow the solvent front to move the length of the plate, remove the plate from the chamber and allow the solvent to evaporate. Mobile phase: 0.1 M citric acid - 0.1 M disodium hydrogen phosphate - solution of ninhydrin in acetone containing 1 g per 15 ml (60 : 40 : 1.5) Test solution: Dissolve a quantity of the powdered tablets containing the equivalent of 20 mg of cefadroxil in 10 ml of water, filter. Reference solution: A 0.2% solution of cefadroxil RS in water. Procedure: Apply separately to the plate 20 µl of each solution. Develop over three-fourths of the length of the plate. After removal of the plate, allow it to dry in air. Spray with a 0.2% solution of ninhydrin in ethanol R (protect this solution from light), heat the plate at 110 °C for 10 minutes. Examine in day light. The principal spot in the chromatogram obtained with the test solution corresponds in position, colour and size to that in the chromatogram obtained with the reference solution. B. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the principal peak in the chromatogram obtained with the reference solution. Water Not more than 8.0% (Appendix 10.3). Use 0.5 g of the powdered tablets. Dissolution (Appendix 11.4) Apparatus: Paddle. 185

VP V

CEFALOTIN SODIUM

Medium: 900 ml of water. Rotation speed: 50 rpm. Time: 30 min. Procedure: After the specified time, withdraw a sample of the medium and filter (discard the first 20 ml of the filtrate). Dilute the filtrate with water (if necessary) to obtain a suitable concentration. Measure the absorbance at the maximum at about 263 nm (Appendix 4.1), in a 1 cm cell, using water as a blank. Calculate the content of cefadroxil, C16H17N3O5S, dissolved in comparison with a reference solution of cefadroxil RS having the same concentration in the same medium. Tolerance: Not less than 75% (Q) of the stated amount of cefadroxil, C16H17N3O5S, is dissolved in 30 min.

Assay Examine by liquid chromatography (Appendix 5.3). Phosphate buffer pH 5.0: Dissolve 13.6 g of potassium dihydrogen phosphate R in sufficient water to produce 2000 ml and adjust the pH to 5.0 with 10 M potassium hydroxide solution R. Mobile phase: Acetonitrile - phosphate buffer pH 5.0 (4 : 96). Reference solution: Dissolve an accurately weighed quantity of cefadroxil RS in phosphate buffer pH 5.0 to obtain a solution containing about 1.0 mg per ml. Test solution: Weigh 20 tablets (remove the coating in case of coated tablets), calculate the average mass, and powder finely. Transfer a quantity of the powdered tablets equivalent to about 100 mg of cefadroxil, accurately weighed, to a 100 ml volumetric flask, add about 75 ml of phosphat buffer pH 5.0 and sonicate for 5 minutes. Dilute to volume with phosphat buffer pH 5.0, mix well and filter. Note: Use the reference solution and the test solution on the day prepared. Chromatographic system: A column (25 cm × 4 mm) packed with stationary phase C (5 µm or 10 µm). Detector: A spectrophotometer set at 230 nm. Flow rate: 1.5 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject the reference solution. The capacity factor, k’, is between 2.0 to 3.5; the column efficiency is not less than 1800 theoretical plates; the symmetry factor is not more than 2.2; the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of cefadroxil, C16H17N3O5S, in the tablets using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C16H17N3O5S in cefadroxil RS. 186

Storage Pack in aluminum blister or closed bottle, in a cool and dry place, protected from light. Action and use Cephalosporin antibacterial. Usual strength 500 mg; 1 g. CEFALOTIN SODIUM Cefalotinum natricum

C16H15N2NaO6S2

M. 418.4

Cefalotin sodium is sodium (6R,7R)-3-[(acetyloxy) methyl]-8-oxo-7-[(thiophen-2-ylacetyl)amino]-5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxylate. It contains not less than 96.0% and not more than 102.0% of C16H15N2NaO6S2, calculated with reference to the anhydrous substance. Semi-synthetic product derived from a fermentation product.

Characters White or almost white powder. Freely soluble in water, slightly soluble in anhydrous ethanol. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of cefalotin sodium RS. B. It gives reaction of sodium (Appendix 8.1). Appearance of solution Solution S: Dissolve 2.50 g of the substance to be examined in carbon dioxide-free water and dilute to 25.0 ml with the same solvent. Solution S is clear (Appendix 9.2) and its absorbance (Appendix 4.1) at 450 nm is not greater than 0.20. pH 4.5 to 7.0 (Appendix 6.2) Determined on solution S. Specific optical rotation +124° to +134°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 1.25 g of the substance to be examined in water and dilute to 25.0 ml with the same solvent.

VP V

CEFALOTIN SODIUM

Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use. Solution A: Dissolve 1.742 g of dipotassium hydrogen phosphate R in 1000 ml water, adjusted to pH 2.5 with phosphoric acid R. Mobile phase A: Acetonitrile - solution A (3 : 97). Mobile phase B: Acetonitrile - solution A (40 : 60). Test solution (1): Dissolve 75.0 mg of the substance to be examined in water and dilute to 25.0 ml with the same solvent. Test solution (2): Dilute 5.0 ml of test solution (1) to 50.0 ml with water. Reference solution (1): Dilute 1.0 ml of test solution (1) to 100.0 ml with water. Reference solution (2): Dissolve 75.0 mg of cefalotin sodium RS in water and dilute to 25.0 ml with the same solvent. Dilute 5.0 ml of the solution to 50.0 ml with water. Reference solution (3): Dissolve 5 mg of cefalotin for impurity B identification RS in water and dilute to 5 ml with the same solvent. Resolution solution: Mix 1 ml of test solution (1), 1 ml of a 25% solution of hydrochloric acid R and 8 ml of water. Heat at 60 °C for 12 min and cool to room temperature in iced water. Inject immediately. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase end-capped octadecylsilyl silicagel for chromatography R (5 µm). Column temperature: 40 °C. Detector: A spectrophotometer set at 220 nm. Flow rate: 1,0 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 30

100 → 0

0 → 100

30 - 35

0

100

Inject the blank solution, test solution (1), reference solutions (1), (3) and the resolution solution. Relative retention with reference to cefalotin (retention time about 26 min): Impurity C = about 0.2; impurity B = about 0.7; impurity D = about 0.88; impurity A = about 0.96. System suitability: In the chromatogram obtained with resolution solution, the resolution between the peaks due to impurity D and cefalotin is at least 7.0. Limits: In the chromatogram obtained with test solution (1) Impurity B: The area of the peak due to impurity B is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (1.0%). Impurity D: The area of the peak due to impurity D is not more than 0.5 times the area of the principal peak in the

chromatogram obtained with reference solution (1) (0.5%). Any other impurity: For each impurity, the area is not more than 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.25%). The sum of the areas of all impurity peaks is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (1) (3.0%). Disregard limit: Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A: (6R,7R)-3-methyl-8-oxo-7-[(thiophen-2-ylacetyl) amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (deacetoxycefalotin). Impurity B: (6R,7R)-3-(hydroxymethyl)-8-oxo-7-[(thiophen2-ylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2carboxylic acid (deacetylcefalotin). Impurity C: (6R,7R)-3-[(acetyloxy)methyl]-7-amino-8-oxo-5thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (7-ACA). Impurity D: (5aR,6R)-6-[(thiophen-2-ylacetyl)amino]-5a,6dihydro-3H,7H-azeto[2,1-b]furo[3,4-d][1,3]thiazine-1,7(4H)dione (cefalotin lactone).

N,N-Dimethylaniline Not more than 20 ppm (Appendix 10.16, method 2). 2-Ethylhexanoic acid Not more than 0.5% (m/m) (Appendix 10.17). Water Not more than 1.5% (Appendix 10.3). Determined on 0.500 g. Bacterial endotoxins Not more than 0.13 EU/mg (Appendix 13.2). If intended for use in the manufacture of parenteral preparations forms without a further appropriate procedure for the removal of bacterial endotoxins. Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances with the following modifications. Solution B: Dissolve 6.967 g dipotassium hydrogen phosphate R in 1000 ml water, adjust to pH 6.0 with phosphoric acid R. Mobile phase: Acetonitrile - solution B (14 : 86). Detection: Spectrophotometer set at 260 nm. Volume of injection: 5 µl. Inject test solution (2) and reference solution (2). Run time is 1.5 times the retention time of cefalotin (retention time = about 10 min). Calculate the content of cefalotin sodium, C16H15N2NaO6S2, using the chromatogram obtained with test solution (2), reference solution (2) and using the declared content of C16H15N2NaO6S2 in cefalotin sodium RS. 187

VP V

CEFAMANDOLE NAFATE

Storage Protected from light. If the substance is sterile, store in a sterile, airtight container. Action and use Cephalosporin antibacterial.

Appearance of solution Solution S: Dissolve 2.5 g of the substance to be examined in carbon dioxide-free water and dilute to 25 ml with the same solvent. Solution S is clear (Appendix 9.2) and its absorbance (Appendix 4.1) at 475 nm is not greater than 0.03.

Preparations Tablets, capsules, injection powder.

pH 6.0 to 8.0 (Appendix 6.2). Determine on solution S, measured after 30 min.

CEFAMANDOLE NAFATE Cefamandoli nafas

Specific optical rotation -35.0° to -45.0° (Appendix 6.4) (calculate with reference to the anhydrous and sodium carbonate-free substance). Dissolve 1.00 g of the substance to be examined in acetate buffer solution pH 4.7 R1 and dilute to 10.0 ml with the same solvent.

R H O O Compound

CO2Na

O H N

S

N H H

R

S

N CH3 N N N

Molecular Formula

Mr

Cefamandole nafate

CHO

C19H17N6NaO6S2

512.5

Cefamandole sodium

H

C18H17N6NaO5S2

484.5

Cefamandole nafate is mixture of sodium (6R,7R)-7[[(2R)-2-(formyloxy)-2-phenylacetyl]amino]-3-[[(1methyl-1H-tetrazol-5-yl)sulfanyl]methyl]-8-oxo-5-thia1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate and sodium (6R,7R)-7-[[(2R)-2-hydroxy-2-phenylacetyl]amino]-3-[[(1methyl-1H-tetrazol-5-yl)sulfanyl]methyl]-8-oxo-5-thia -1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate(cefamandole sodium), with sodium carbonate. Semi-synthetic product derived from a fermentation product.

Content Cefamandole nafate: Not less than 93.0% and not more than 102.0% of C19H17N6NaO6S2 (calculated with reference to the anhydrous and sodium carbonate-free substance), for the sum of the content of cefamandole nafate and cefamandole sodium expressed as cefamandole nafate. Cefamandole sodium: Not more than 10.0% of C18H17N6NaO5S2 (calculated with reference to the anhydrous and sodium carbonate-free substance). Sodium carbonate: Not less than 4.8% and not more than 6.4% of Na2CO3. Characters White or almost white powder. Freely soluble in water, sparingly soluble in methanol. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of cefamandole nafate RS. B. It gives reaction of sodium (Appendix 8.1). 188

Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use. Triethylamine phosphate solution: Dissolve 2.0 g of sodium pentanesulfonate R in 350 ml of water, add 40 ml of triethylamine R, adjust to pH 2.5 with phosphoric acid R and dilute to 700 ml with water. Mobile phase A: Triethylamine phosphate solution - water (1 : 2). Mobile phase B: Triethylamine phosphate solution methanol - acetonitrile (1 : 1 : 1). Solvent mixture: Mix 18 volumes of acetonitrile R and 75 volumes of a 10% (v/v) solution of triethylamine R previously adjusted to pH 2.5 with phosphoric acid R. Test solution: Dissolve 0.100 g of the substance to be examined in the solvent mixture and dilute to 10.0 ml with the solvent mixture. Reference solution: Dilute 1.0 ml of the test solution to 100.0 ml with the solvent mixture. Resolution solution: Dilute 1 ml of the test solution to 10 ml with the solvent mixture, then heat at 60 °C for 30 min. Chromatographic system: A column (250 mm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0-1

100

0

1 - 35

100 → 0

0 → 100

Inject the blank solution, the test solution, the reference solution and the resolution solution. Relative retention with reference to cefamandole nafate (retention time = about 24 min): Cefamandole = about 0.8.

VP V

System suitability: In the chromatogram obtained with the resolution solution, the resolution between the peaks due to cefamandole and cefamandole nafate is at least 5.0. Limits: In the chromatogram obtained with the test solution: Any impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with the reference solution (1.0%). The sum of the areas of all impurity peaks is not more than 5 times the area of the principal peak in the chromatogram obtained with the reference solution (5.0%). Disregard limit: Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with the reference solution (0.1%).

Note: Impurity A: (6R,7R)-7-[[(2R)-2-(formyloxy)-2-phenylacetyl] amino]-3-methyl-8-oxo-5-thia-1-azabicyclo [4.2.0]oct-2-ene-2-carboxylic acid (formylmandeloyl-7-aminodesacetoxy-cephalosporanic acid). Impurity C: (6R,7R)-7-[[(2R)-2-(acetyloxy)-2-phenylacetyl] amino]-3-[[(1-methyl-1H-tetrazol-5-yl)sulfanyl] methyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2carboxylic acid (O-acetylcefamandole). Impurity D: 1-methyl-1H-tetrazole-5-thiol. Impurity E: (6R,7R)-7-[[(2R)-2-(formyloxy)-2-phenylacetyl] amino]-3-[(acetyloxy)methyl]-8-oxo-5-thia-1-azabicyclo[4.2.0] oct-2-ene-2-carboxylic acid (formylmandeloyl-7- ACA).

2-Ethylhexanoic acid Not more than 0.3% (m/m) (Appendix 10.17). Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 3. Prepare the standard solution using 2 ml of lead standard solution (10 ppm Pb) R. Water Not more than 2.0% (Appendix 10.3). Determined on 0.500 g. Bacterial endotoxins Less than 0.15 EU/mg (Appendix 13.2). If intended for use in the manufacture of parenteral preparations without a further appropriate procedure for the removal of bacterial endotoxins. Assay Cefamandole nafate Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use. Mobile phase: Mix 25 volumes of acetonitrile R and 75 volumes of a 10% v/v solution of triethylamine R previously adjusted to pH 2.5 with phosphoric acid R. Test solution: Dissolve 50.0 mg of the substance to be examined in the mobile phase and dilute to 100.0 ml with the mobile phase. Standard solution: Dissolve 50.0 mg of cefamandole nafate RS in the mobile phase and dilute to 100.0 ml with the mobile phase.

CEFAZOLIN SODIUM

Resolution solution: Dilute 1 ml of the test solution to 10 ml with the mobile phase, then heat at 60 °C for 30 min. Chromatographic system: A column (250 mm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl Procedure: System suitability: In the chromatogram obtained with resolution solution, the resolution between the 2 principal peaks is at least 7.0. Repeatability: The relative standard deviation is not more than 0.8% after a series of single injections of at least 3 freshly prepared standard solutions. Inject the standard solution and the test solution. Using the chromatogram obtained with the test solution, the standard solution and the declared content of cefamandole nafate RS, calculate the percentage content of cefamandole nafate (C16H15N2NaO6S2) from the sum of the contents of cefamandole nafate and cefamandole sodium (expressed as cefamandole nafate). 1 mg of cefamandole sodium is equivalent to 1.0578 mg of cefamandole nafate. Sodium carbonate Dissolve 0.500 g of the substance to be examined in 50 ml of water. Titrate with 0.1 N hydrochloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 mL of 0.1 N hydrochloric acid VS is equivalent to 5.3 mg of Na2CO3.

Storage In an airtight container, protected from light. If the substance is sterile, store in a sterile, airtight container. Action and use Cephalosporin antibacterial. Preparations Tablets, capsules, injection powder. CEFAZOLIN SODIUM Cefazolinum natricum

C14H13N8NaO4S3

M. 476.5

Cefazolin sodium is sodium (6R,7R)-3-[[(5-methyl-1,3,4thiadiazol-2-yl)sulfanyl]methyl]-8-oxo-7-[(1H-tetrazol189

VP V

CEFAZOLIN SODIUM

1-ylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene2-carboxylate, semi-synthetic product derived from a fermentation product. It contains not less than 95.0% and not more than 102.0% of C14H13N8NaO4S3, calculated with reference to the anhydrous substance.

Characters A white or almost white powder, very hygroscopic. It shows  polymorphism. Freely soluble in water, very slightly soluble in ethanol (96%). Identification A. Dissolve 0.150  g of the substance to be examined in 5  ml of water, add 0.5  ml of  dilute acetic acid  R, swirl and allow to stand for 10 min in iced water. Filter the precipitate and rinse with 1 ml to 2 ml of water. Dissolve the precipitate in a mixture of water – acetone R (1 : 9). Evaporate the solvent almost to dryness, then dry in an oven at 60 °C for 30 min. The infrared absorption spectrum (Appendix 4.2) of the residue is concordant with the spectrum of cefazolin RS. B. It gives reaction (A) of sodium (Appendix 8.1). Appearance of solution Solution S: Dissolve 2.50 g of the substance to be examined in carbon dioxide-free water R and dilute to 25.0 ml with the same solvent. Solution S is clear (Appendix 9.2) and its absorbance (Appendix 4.1) at 430 nm is not greater than 0.15. pH 4.0 to 6.0 (Appendix 6.2). Determined on the solution S. Specific optical rotation -15° to -24°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 1.25  g in water  and dilute to 25.0  ml with the same solvent. Absorbance Dissolve 0.100 g in water and dilute to 100.0 ml with the same solvent. Dilute 2.0  ml of the solution to 100.0  ml with a 4.2% solution of sodium hydrogen carbonate R. Examined between 220 nm and 350 nm (Appendix 4.1), the solution shows an absorption maximum at 272 nm. The specific absorbance at the maximum is 260 to 300 (calculated with reference to the anhydrous substance). Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Dissolve 14.54 g of disodium hydrogen phosphate R and 3.53 g of potassium dihydrogen phosphate R in water­and dilute to 1000 ml with the same solvent. Mobile phase B: Acetonitrile for chromatography R. Test solution:  Dissolve 50.0  mg of the substance to be examined in mobile phase A and dilute to 20.0 ml with the same solvent. 190

Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with mobile phase A. Reference solution (2): Dissolve 20 mg of the substance to be examined in 10 ml of a 0.2% solution of sodium hydroxide R. Allow to stand for 15 to 30 min. Dilute 1.0 ml of the solution to 20 ml with mobile phase A. Chromatographic system: A column (12.5 cm × 4.0 mm) packed with stationary phase C (3 µm). Column temperature: 45 °C. Detector: A spectrophotometer set at 254 nm. Flow rate: 1.2  ml/min. Volume of injection: 5 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0-2

98

2

2-4

98 → 85

2 → 15

4 - 10

85 → 60

15 → 40

10 - 11.5

60 → 35

40 → 65

11.5 - 12

35

65

12 - 15

35 → 98

65 → 2

15 - 21

98

2

System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to cefazolin and impurity L is at least 2.0 (Figure 1). Limits: Any impurity: The area of each impurity peak is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (1.0%). Sum of the areas of all the impurity peaks is not more than 3.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (3.5%). Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%).

Notes: Impurity A: (6R,7R)-7-amino-3-[[(5-methyl-1,3,4-thiadiazol-2yl)sulfanyl]methyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene2-carboxylic acid. Impurity B: (6R,7R)-7-[(2,2-dimethylpropanoyl)amino]-3-[[(5methyl-1,3,4-thiadiazol-2-yl)sulfanyl]methyl]-8-oxo-5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid. Imputity C: (6R,7R)-3-methyl-8-oxo-7-[(1H-tetrazol-1-ylacetyl) amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid. Impurity D: (6R,7R)-3-[(acetyloxy)methyl]-8-oxo-7-[(1H-tetrazol1-ylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid. Impurity E: 5-methyl-1,3,4-thiadiazol-2-thiol (MMTD). Impurity G: (5aR,6R)-6-[(1H-tetrazol-1-ylacetyl)amino]-5a,6dihydro-3H,7H-azeto[2,1-b]furo[3,4-d][1,3]thiazine-1,7(4H)-dione. Impurity H: (6R,7R)-3-[(acetyloxy)methyl]-7-amino-8-oxo-5thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (7-ACA).

VP V

CEFAZOLIN SODIUM

Impurity I: 2-[carboxy[(1H-tetrazol-1-ylacetyl)amino]methyl]5-[[(5-methyl-1,3,4-thiadiazol-2-yl)sulfanyl]methyl]-5,6dihydro-2H-1,3-thiazine-4-carboxylic acid (cefazoloic acid). Impurity J: 2-[carboxy[(1H-tetrazol-1-ylacetyl)amino]methyl]5-methylidene-5,6-dihydro-2H-1,3-thiazine-4-carboxylic acid. Impurity K: (6R,7R)-3-[[(5-methyl-1,3,4-thiadiazol-2-yl)sulfanyl] methyl]-8-oxo-7-[(1H-tetrazol-1-ylacetyl)amino]-5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxamide (cefazolinamide). Impurity L: (6R,7S)-3-[[(5-methyl-1,3,4-thiadiazol-2-yl)sulfanyl] methyl]-8-oxo-7-[(1H-tetrazol-1-ylacetyl)amino]-5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid.

N,N-Dimethylaniline Not more than 20 ppm (Appendix 10.16, method 2). Water Not more than 6.0% (Appendix 10.3). Determined on 0.300 g. Bacterial endotoxins Less than 0.15 IU/mg (Appendix 13.2), if intended for use in the manufacture of parenteral preparations without a further appropriate procedure for the removal of bacterial endotoxins. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - phosphate buffer solution (10 : 90). Phosphate buffer solution: Dissolve 2.77 g of disodium hydrogen phosphate R and 1.86 g of citric acid R in water and dilute to 1000 ml with the same solvent.

1. unknown structure

2. impurity E

Test solution: Dissolve 50.0  mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the same solvent. Reference solution (1):  Dissolve 50.0  mg of  cefazolin RS in the mobile phase and dilute to 50.0 ml with the same solvent. Reference solution (2):  Dissolve 5.0  mg of cefuroxime sodium RS in 10.0 ml of reference solution (1) and dilute to 100.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 270 nm. Flow rate: 1.0  ml/min. Volume of injection: 20 µl. Procedure: System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to cefazolin and cefuroxime is at least 2.0. Inject alternately the test solution and reference solution (1). Calculate the percentage content of cefazolin in the substance to be examined, using the areas of the principal peaks in the chromatograms obtained with the test solution, reference solution (1), and the declared content of cefazolin in cefazolin RS. Calculate the percentage content of cefazolin sodium by multiplying the percentage content of cefazolin by 1.048.

Storage Store in an airtight container, protected from light. If the substance is sterile, store in a sterile, airtight, tamper-proof container.

3. unknown structure

4. cefazolin

5. impurity L

Figure 1 - Chromatogram of reference solution (2) in the test for Related substances.

191

CEFAZOLIN FOR INJECTION

VP V

Action and use Cephalosporin antibacterial.

Procedure: Apply separately to the plate 50 µl of each solution under a stream of nitrogen. Develop the chromatogram. After removal of the plate, allow it to dry at room temperature. Examine under ultraviolet light at 254 nm. Expose the plate to iodine vapour in an airtight tank until the spots appear clearly and examine again. By each method of visualisation any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (1) (4%) and not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (2) (1.5%).

Preparations Injections, eye drops. CEFAZOLIN FOR INJECTION Cefazolini pulvis injectionem Cefazolin for injection is a sterile crystalline powder of cefazolin sodium. It is supplied in a sealed glass vial. Dissolve with sterile water for injections immediately before use. The preparation complies with the general requirements prescribed under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Content of cefazolin, C14H14N8O4S3, 90.0% to 105.0% of the stated amount. Characters Almost white crystals or crystalline powder. Identification A. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the peak due to cefazolin sodium in the chromatogram obtained with the reference solution. B. It yields reaction characteristic of sodium (Appendix 8.1). Acidity or alkalinity The pH of a solution containing the equivalent of 10% of cefazolin in carbon dioxide-free water R is 4.0 to 6.0 (Appendix 6.2). Clarity of solution A solution containing the equivalent of 10.0% of cefazolin in carbon dioxide-free water R is clear (Appendix 9.2). The absorbance of the above solution measured at 430 nm is not greater than 0.15 (Appendix 4.1). Water Not more than 6.0% (Appendix 10.3). Use 0.3 g of the preparation. Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Glacial acetic acid - water - acetone - ethyl acetate (10 : 10 : 20 : 50). Test solution: Dissolve a portion of the preparation in sufficient water to produce a solution containing the equivalent of 5.0% of cefazolin. Reference solution (1): Dilute 1 volume of the test solution to 25 volumes with water. Reference solution (2): Dilute 3 volumes of the test solution to 200 volumes with water. 192

Bacterial endotoxins Carry out the test for bacterial endotoxins (Appendix 13.2). Dissolve a portion of the preparation in water BET R to give a solution containing 10 mg of cefazolin per ml (solution A). The endotoxin limit concentration of solution A is 1.5 EU per ml. The maximum valid dilution of solution A is calculated from the declared sensitivity of the lysate used in the test. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - solution containing 0.277% of anhydrous disodium hydrogen phosphate and 0.186% of citric acid (10 : 90). Test solution: Dissolve an accurately weighed quantity of the preparation in water to produce a solution containing the equivalent of 0.1% of cefazolin. Reference solution: A solution containing 0.10% of cefazolin sodium RS in water. Resolution solution: A solution containing 0.01% of cefazolin sodium RS and 0.005% of cefuroxime sodium RS in water. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm to 10 µm) (Sperisorb ODS is suitable). Detector: A spectrophotometer set at 270 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the resolution solution. Adjust the sensitivity of the detector so that the heights of the peaks are at least 50% of the full scale of the recorder. The test is not valid unless the resolution factor between the peaks corresponding to cefazolin and cefuroxime is not less than 2.0. If necessary, adjust the concentration of acetonitrile in the mobile phase. Inject alternately the test solution and the reference solution. Calculate the content of cefazolin, C14H14N8O4S3, in one unit of the preparation using the areas of the principal peaks in the chromatograms obtained with the test solution, the reference solution and the declared content of C14H14N8O4S3 in cefazolin sodium RS.

VP V

CEFDINIR

Storage Store in a cool, dry place, at a temperature not exceeding 30 °C. Action and use Cephalosporin antibacterial. Usual strength 250 mg; 500 mg; 1000 mg. CEFDINIR Cefdinirum

C14H13N5O5S2

M. 395.41

Cefdinir is (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2(hydroxyimino)acetylamino]-8-oxo-3-vinyl-5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid. It contains not less than 940 µg/mg and not more than 1030 µg/mg of C14H13N5O5S2, calculated with reference to the anhydrous substance.

Characters A white to yellowish, crystalline powder. Practically insoluble in water, ethanol (96%) and diethylether, sparingly soluble in 0.1 M mixed phosphate buffer pH 7.0. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of cefdinir RS. B. In the test for Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of the principal peak in the chromatogram obtained with standard solution. Specific optical rotation -61° to -67°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.25 g in buffer solution in the test for Assay and dilute to 25.0 ml with the same solvent. Related substances Examine by liquid chromatography (Appendix 5.3). Solution A, solution B, solution C, solution D and buffer solution as described in the test for Assay Mobile phase E: Add 0.4 ml of solution D to 1000 ml of solution C. Mobile phase F:  Acetonitrile - methanol - solution C solution D (300 : 200 : 500 : 0.4)

Test stock solution: Dissolve 200.0 mg of the substance to be examined in buffer solution and dilute to 20.0 ml with the same solvent. Test solution: Dilute test stock solution with solution C to obtain 1.5 mg/ml of cefdinir. Prepare fresh immediately before use. Reference solution (1): Dilute the test solution with solution C to obtain 15 µg/ml of cefdinir. Reference solution (2): Dilute reference solution (1) with solution C to obtain 1.5 µg/ml of cefdinir. Reference solution (3): A solution contains 1.5 mg/ml of cefdinir RS and 0.1 mg/ml of cefdinir related compound A RS in solution C (dissolved initially in buffer solution corresponding to 15% of the final volume). Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 µm). Column temperature: 40 °C. Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase E (% v/v)

Mobile phase F (% v/v)

0

95

5

2

95

5

22

75

25

32

50

50

37

50

50

38

95

5

58

95

5

The run time is 1.8 times the retention time of cefdinir. System suitability: In the chromatogram obtained with reference solution (3), cefdinir impurity A should produce four peaks: First peak and second peak elute before cefdinir peak, third peak and fourth peak elute after cefdinir peak; the resolution between the peaks due to cefdinir and the third peak of cefdinir related compound A is at least 1.5; the relative standard deviation of the cefdinir peak areas a for 6 replicate injections of the reference solution (3) is not more than 2.0%. Response ratio: The response of the area of the peak due to cefdinir from reference solution (2) is 7% to 13% of that from reference solution (1). Limits: Calculate the percentage of each impurity base on peak area of each impurity and sum of all the peak areas in the chromatogram obtained with the test solution. Relative retention time and acceptance criteria of each impurity: 193

VP V

CEFDINIR

i(Z)-2-(2-Aminothiazol-4-yl)-2-(hydroxyimino)-N-{[(2RS,5RS)

Name

Relative retention time

Acceptance criteria

Thiazolylacetyl glycine oximea

0.10

0.5

Thiazolylacetyl glycine oxime acetalb

0.12

0.5

3-Methyl cefdinirc

0.74

0.7

Cefdinir related compound A (cefdinir open ring lactone a)d,e

0.85

Cefdinir related compound A (cefdinir open ring lactone b)d,e

Sulfated ash Not more than 0.20% (Appendix 9.9, method 2). Determined on 1.0 g.

0.93

Cefdinir related compound A (cefdinir open ring lactone c)d,e

1.11

Cefdinir related compound A (cefdinir open ring lactone d)d,e

1.14

Cefdinir lactonef

1.22

0.5

Assay Examine by liquid chromatography (Appendix 5.3). Solution A: Dissolve 14.2 g of anhydrous disodium hydrogen phosphate R in 1000 ml of water. Solution B:  Dissolve 13.6 g of potassium dihydrogen phosphate R in 1000 ml of water. Solution C: Dilute tetramethylammonium hydroxide R with water to obtain a 0.1% solution. Adjust to a pH of 5.5 with a 10% solution of phosphoric acid R. Solution D: Dissolve 37.2 g of edetate disodium R in 1000 ml of water. Buffer solution: Combine appropriate amounts of solution A and solution B (about 2 : 1) to obtain a solution with a pH of 7.0. Mobile phase: Acetonitrile - methanol - solution C solution D (300 : 200 : 4500 : 2). Test solution: Dissolve 20.0 mg of the substance to be examined in buffer solution and dilute to 100.0 ml with the same solvent. Standard solution: Dissolve 20.0 mg of cefdinir RS in buffer solution and dilute to 100.0 ml with the same solvent. Resolution solution: A solution contains 0.2 mg/ml of cefdinir RS and 0.5 mg/ml of cefdinir related compound A RS in buffer solution. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 µm). Column temperature: 40 °C. Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 5 µl. Procedure: System suitability: In the chromatogram obtained with resolution solution, cefdinir impurity A should produce four peaks; the resolution between the peaks due to the second peak of cefdinir related compound A and cefdinir is at least 1.2; the symmetry factor of the peak due to cefdinir is not more than 1.5. The relative standard deviation of the cefdinir peak areas for 6 replicate injections of the standard solution is not more than 1.0%.

Cefdinir isoxazole

analogg

0.7

1.36

0.5

E-Cefdinirh

1.51

0.7

Cefdinir decarboxy open ring lactone ai,j

1.61

Cefdinir decarboxy open ring lactone bi,j

1.64

Any other individual, unidentified impurity

—

0.2

Total impurities

—

3.0

0.5

Note: a 1N-[(Z)-2-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acetyl]glycine. b (Z)-2-(2-Aminothiazol-4-yl)-N-(2,2-dihydroxyethyl)-2(hydroxyimino) acetamide. c (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-(hydroxyimino) acetamido]-3-methyl-8-oxo-5-thia-1-azabicyclo [4.2.0]oct-2-ene2-carboxylic acid. d 2(R)-2-[(Z)-2-(2-Aminothiazol-4-yl)-2-(hydroxyimino)acetamido)] -2-[(2RS,5RS)-5-methyl-7-oxo-2,4,5,7tetrahydro-1H-furo[3,4-d][1,3]thiazin-2-yl]acetic acid. e Cefdinir related compound A is a mixture of 4 isomers labeled cefdinir open ring lactones a, b, c, and d. The sum of the values is reported. The limit for the sum of the 4 isomers is 0.7%. f (Z)-2-(2-Aminothiazol-4-yl)-2-(hydroxyimino)-N-{(3RS,5aR,6R) -3-methyl-1,7-dioxo-1,3,4,5a,6,7-hexahydroazeto [2,1-b]furo[3,4-d][1,3]thiazin-6-yl}acetamide. g (6R,7R)-7-(4-Hydroxyisoxazole-3-carboxamido)-8-oxo-3-vinyl5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid. h(6R,7R)-7-[(E)-2-(2-Aminothiazol-4-yl-)-2-(hydroxyimino) acetamido] -8-oxo-3-vinyl-5-thia-1-azabicyclo[4.2.0]oct-2-ene2-carboxylic acid.

194

-5-methyl-7-oxo-2,4,5,7-tetrahydro-1H-furo[3,4-d][1,3]thiazin2-yl]methyl}acetamide. j Cefdinir decarboxy open ring lactone is a mixture of 2 isomers labeled cefdinir decarboxy open ring lactones a and b. The sum of the values is reported. The limit for sum of the 2 isomers is 0.5%.

Water Not more than 2.0%. Use formamide - methanol (2 : 1) as solvent.

VP V

Calculate the content of cefdinir, C14H13N5O5S2, using the areas for the cefdinir peaks in the chromatogram obtained with the test solution, the standard solution and the declared content of C14H13N5O5S2 in cefdinir RS.

Storage Store in an airtight container, in a cool place, protected from light. Action and use Cephalosporin antibacterial. Preparations Tablets, capsules, powder. CEFDINIR CAPSULES Capsulae Cefdiniri Cefdinir capsules contain cefdinir. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of cefdinir, C14H13N5O5S2, 90.0% to 110.0% of the stated amount. Identification A. Examine by ultraviolet and visible absorption spectrophotometry (Appendix 4.1). Buffer solution: Prepare as directed in the Assay. Reference solution: Dissolve a quantity of cefdinir RS in the buffer solution to obtain a solution containing about 10 µg of cefdinir per ml. Test solution: Dissolve a quantity of the content in capsules in the buffer solution to obtain a solution containing about 10 µg of cefdinir per ml, filter. Procedure: Measure the ultraviolet absorption spectrum of the test solution and the reference solution, in the range 220 nm to 350 nm, in a 1 cm cell, using the buffer solution as a blank. The ultraviolet absorption spectrum of the test solution exhibits the same maxima and minima as those of the reference solution. B. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the principal peak in the chromatogram obtained with the reference solution. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of phosphate buffer pH 6.8 (Mix 250 ml of 0.2 M potassium dihydrogen phosphate R and 112.0 ml of 0.2 M sodium hydroxyde R and add water to 1000 ml). Rotation speed: 50 rpm. Time: 30 min.

CEFDINIR CAPSULES

Procedure: Test solution: After the specified time, withdraw a sample of the medium, filter. Dilute the filtrate with the medium, if necessary, to obtain a solution having a known concentration of about 10 µg per ml. Reference solution: Dissolve an accurately weighed quantity of cefdinir RS in the medium to obtain a solution having the same concentration as the test solution. Measure the absorbance (Appendix 4.1) of the test solution and the reference solution at 290 nm, in a 1 cm cell, using the medium as a blank. Calculate the content of cefdinir, C14H13N5O5S2, dissolved using the absorbance of the test solution and the reference solution and the declared content of C14H13N5O5S2 in cefdinir RS. Tolerance: Not less than 80% (Q) of the stated amount of cefdinir, C14H13N5O5S2, is dissolved in 30 minutes.

Assay Examine by liquid chromatography (Appendix 5.3). Solution A: Dissolve 14.2 g of anhydrous disodium hydrogen phosphate R in water to produce 1000 ml. Solution B: Dissolve 13.6 g of potassium dihydrogen phosphate R in water to produce 1000 ml. Solution C: Dilute tetramethylammonium hydroxide R quantitatively with water to obtain a 0.1% solution. Adjust to pH 5.5 ± 0.1 with a 10% solution of phosphoric acid R. Solution D: Dissolve 3.72 g of sodium edetate R in water to produce 100 ml. Buffer solution: Mix a suitable ratio of solution A and solution B (about 2 : 1) to obtain a solution having a pH of 7.0. Mobile phase: Acetonitril - methanol - solution C - solution D (300 : 200 : 4500 : 2), make adjustments if necessary. Resolution solution: Dissolve a quantity of cefdinir RS and cefdinir related compound A RS in the buffer solution to obtain a solution contaning 0.2 mg of cefdinir and 0.5 mg of cefdinir related compound A per ml. Reference solution: Dissolve an accurately weighed quantity of cefdinir RS in the buffer solution to obtain a solution containing 0.2 mg of cefdinir per ml. Test solution: Weigh 20 capsules and calculate the average mass of the capsule contents and finely powder. Transfer an accurately weighed quantity of the powder containing the equivalent of about 100 mg of cefdinir, to a 100 ml volumetric flask, add 70 ml of the buffer solution and sonicate for 30 min. Dilute to volume with the same solvent and mix well, filter. Dilute 10.0 ml of the filtrate to 50.0 ml with the buffer solution, mix well. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 μm), maintained at 40 °C. Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min, the flow rate may be adjusted so that the retention time of cefdinir is about 8 min. Volume of injection: 5 μl. 195

CEFDINIR POWDER FOR ORAL SUSPENSION

Procedure: System suitability: Inject the reference solution and the resolution solution. In the chromatogram obtained, cefdinir related compound A consists of four peaks: two peaks eluted before cefdinir peak and two other peaks eluted after cefdinir peak. The test is not valid unless the resolution factor between the second peak of cefdinir related compound A and cefdinir is at least 1.2; the tailing factor for cefdinir peak is not more than 1.5; the relative standard deviation of the cefdinir peak areas for replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of cefdinir, C14H13N5O5S2, in the capsules using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C14H13N5O5S2 in cefdinir RS. Note: Cefdinir related compound A: A mixture of cefdinir open ring lactones.

Storage Store in an airtight container, in a cool and dry place, protected from light. Action and use Cephalosporin antibacterial. Usual strength 300 mg. CEFDINIR POWDER FOR ORAL SUSPENSION Pulveres Cefdiniri ad suspensionum peroralum Cefdinir powder for oral suspension contains cefdinir. Suitable excipients such as flavours, colours, preservatives, stabilizing agents … may be added. The constituted suspension prepared as directed on the label complies with the requirements stated under “Suspension” (Appendix 1.5). The powder complies with the requirements stated under “Powder” (Appendix 1.7) and with the following requirements.

Content of cefdinir, C14H13N5O5S2, 90.0% to 110.0% of the stated amount. Characters The powder should be loose and dry, with homogeneous colour. Identification In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the cefdinir peak in the chromatogram obtained with the reference solution. 196

VP V

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of phosphate buffer pH 6.8 (Mix 250 ml of 0.2 M potassium dihydrogen phosphate R and 112.0 ml of 0.2 M sodium hydroxyde R and add water to 1000 ml). Rotation speed: 50 rpm. Time: 30 min. Procedure: For single-dose preparations, disperse the content of a container in 5 ml of water. For multi-dose preparations, constitute the content of a container as directed on the label, weigh accurately 5 ml of the constituted suspension. Test solution: After the specified time, withdraw a sample of the medium, filter, dilute the filtrate (if necessary) with the medium to obtain a solution containing 10 µg of cefdinir per ml. Reference solution: Dissolve an accurately weighed quantity of cefdinir RS in the medium to obtain a solution having the same concentration of cefdinir as the test solution. Measure the absorbance (Appendix 4.1) of the test solution and the reference solution at 290 nm, in a 1 cm cell, using the medium as the blank. Calculate the content of cefdinir, C14H13N5O5S2, dissolved using the absorbances of the test solution, reference solution and the declared content of C14H13N5O5S2, in cefdinir RS. Tolerance: Not less than 80% (Q) of the stated amount of cefdinir, C14H13N5O5S2, is dissolved in 30 minutes. pH 3.2 to 4.8 (Appendix 6.2). Use a suspension constituted as directed on the label. Assay Examine by liquid chromatography (Appendix 5.3). Solution A: Dissolve 14.2 g of anhydrous disodium hydrogen phosphate R in water to produce 1000 ml. Solution B: Dissolve 13.6 g of potassium dihydrogen phosphate R in water to produce 1000 ml. Solution C: Dilute tetramethylammonium hydroxyde R with water to obtain a 0.1% solution. Adjust to pH 5,5 ± 0,1 with a 10% solution of phosphoric acid R. Solution D: Dissolve 3.72 g of sodium edetate R in water to produce 100 ml. Buffer solution: Mix a suitable ratio of solution A and solution B (about 2 : 1) to obtain a solution having a pH of 7.0. Mobile phase: Acetonitrile - methanol - solution C - solution D (300 : 200 : 4500 : 2), make adjustments if necessary. Resolution solution: Dissolve a quantity of cefdinir RS and cefdinir related compound A RS in the buffer solution to obtain a solution contaning 0.2 mg of cefdinir and 0.5 mg of cefdinir related compound A per ml. Reference solution: Dissolve an accurately weighed quantity of cefdinir RS in the buffer solution to obtain a solution containing 0.2 mg of cefdinir per ml.

VP V

Test solution: For single-dose preparations, transfer an accurately weighed quantity of the mixed powder received from the test for Uniformity of mass; for multi-dose preparations, transfer an accurately weighed quantity of the suspension prepared as directed on the label containing the equivalent to 100 mg of cefdinir to a 100 ml volumetric flask, add 70 ml of the buffer solution and sonicate for 30 min. Dilute to volume with the same solvent, mix and centrifuge to obtain a clear supernatant liquid. Dilute 10.0 ml of the supernatant to 50.0 ml with the buffer solution, mix. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 μm), maintained at 40 °C. Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min; the flow rate may be adjusted so that the retention time of cefdinir is about 8 minutes. Volume of injection: 5 μl. Procedure: System suitability: Inject the reference solution, resolution solution. In the chromatograms obtained, cefdinir related compound A consists of four peaks: two peaks eluted before cefdinir peak and two additional peaks eluted after cefdinir peak. The test is not valid unless the resolution factor between the peak corresponding to the second peak of cefdinir related compound A and cefdinir is at least 1.2; the tailing factor for cefdinir peak is not more than 1.5; the relative standard deviation of the cefdinir peak areas for replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of cefdinir, C14H13N5O5S2, in the oral suspension using the peak areas in the chromatograms obtained with the test solution and the reference solution, the declared content of C14H13N5O5S2 in cefdinir RS and the density of the constituted suspension (for multi-dose preparations, Appendix 6.5). Note: Cefdinir related compound A: A mixture of cefdinir open ring lactones.

Storage Store in airtight containers, in a cool and dry place, protected from light. The multi-dose suspension after constitution is kept at the temperature and used within the period as stated on the label. Labelling The label states the direction for the constitution of the powder and the equivelent amount of cefdinir (C14H13N5O5S2) in a given volume of the constituted suspension (for multi-dose preparations). Action and use Cephalosporin antibacterial. Usual strength Single-dose preparations: 125 mg; 250 mg and 300 mg. Multi-dose preparations: 125 mg/5 ml; 250 mg/5 ml.

CEFEPIME HYDROCHLORIDE MONOHYDRATE

CEFEPIME HYDROCHLORIDE MONOHYDRATE Cefepimi hydrochloridum monohydricum

C19H24N6O5S2,2HCl,H2O 

M. 571.5

Cefepim hydrochloride monohydrate is (6R,7R)-7[[(2Z)-(2-aminothiazol-4-yl)(methoxyimino)acetyl] amino]-3-[(1-methylpyrrolidinio)methyl]-8-oxo-5-thia-1azabicyclo [4.2.0]oct-2-ene-2-carboxylate dihydrochloride monohydrate. It contains not less than 97.0% and not more than 102.0% of C19H24N6O5S2,2HCl, calculated with reference to the anhydrous substance. Semi-synthetic product derived from a fermentation product.

Characters White or almost white, crystalline powder. Freely soluble in water and in methanol, practically insoluble in methylene chloride. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of cefepime dihydrochloride monohydrate RS. B. It gives reaction (A) of chlorides (Appendix 8.1). Appearance of solution Dissolve 2.0 g in water and dilute to 20 ml with the same solvent. The solution is clear (Appendix 9.2) and not more intensely coloured than reference solution Y3 (Appendix 9.3, method 2). Specific optical rotation +40° to +45° calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.250 g in water and dilute to 25.0 ml with the same solvent. Impurity G Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use. Mobile phase: Acetonitrile - 0.01 M nitric acid (1 : 100), filter through a 0.2 µm filter. Test solution: Dissolve 0.100 g of the substance to be examined in 0.01 M nitric acid R and dilute to 10.0 ml with the same solvent. Reference solution (1): Dilute 0.250 g of N-methylpyrrolidine R (impurity G) to 100.0 ml with water. Dilute 2.0 ml of this solution to 100.0 ml with 0.01 M nitric acid R. 197

VP V

CEFEPIME HYDROCHLORIDE MONOHYDRATE

Reference solution (2): Dilute 0.250 g of pyrrolidine R in 0.01 M nitric acid R and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml of the solution to 100.0 ml with 0.01 M nitric acid R. Mix 5 ml of this solution with 5 ml of reference solution (1). Chromatographic system: A column (5 cm × 4.6 mm) packed with stationary phase strong cation-exchange resin R (5 µm). Detector: Conductivity detector. Flow rate: 1.0 ml/min. Volume of injection: 100 µl. Procedure: The run time is 1.1 times the retention time of cefepime. With described chromatographic system, the retention time of cefepime is about 50 min, eluting as a broadened peak. System suitability: In the chromatogram obtained with reference solution (1), the symmetry factor is not more than 2.5 for impurity G and the relative standard deviation of the peak areas due to impurity G for 6 replicate injections of reference solution (1) is not more than 5.0%. In the chromatogram obtained with reference solution (2), peak-to-valley ratio (Hp/Hv) between the peaks due to pyrrolidine and impurity G is at least 3. Calculate the percentage content of impurity G in the test solution using reference solution (1). Limit: Impurity G: Not more than 0.5%.

Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use or keep refrigerated at 4 °C to 8 °C for not more than 12 h. Mobile phase A: Acetonitrile - solution A (10 : 90). Mobile phase B: Acetonitrile - solution A (50 : 50). Solution A: Dissolve 0.68 g of potassium dihydrogen phosphate R in 1000 ml water, adjust to pH 5.0 with 0.5 M potassium hydroxide R. Test solution: Dissolve 70.0 mg of the substance to be examined in mobile phase A and dilute to 50.0 ml with mobile phase A. Sonicate for 30 s and stir for about 5 min. Reference solution (1): Dissolve 70.0 mg of cefepime dihydrochloride monohydrate RS in mobile phase A and dilute to 50.0 ml with mobile phase A. Sonicate for 30 s and stir for about 5 min. Reference solution (2): Dilute 1.0 ml of the test solution to 10.0 ml with mobile phase A. Dilute 2.0 ml of this solution to 100.0 ml with mobile phase A. Reference solution (3): Dissolve 7 mg of cefepime dihydrochloride monohydrate for system suitability RS (containing impurities A, B and F) in mobile phase A and dilute to 5 ml with mobile phase A. Reference solution (4): Dissolve 2 mg of cefepime impurity E RS in mobile phase A and dilute to 25.0 ml with mobile phase A. Dilute 1.0 ml of the solution to 10.0 ml with mobile phase A. 198

Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 10

100

0

10 - 30

100 → 50

0 → 50

30 - 35

50

50

35 - 36

50 → 100

50 → 0

36 - 45

100

0

Inject the blank, the test solution and reference solutions (2), (3) and (4). Identification of impurities: Use the chromatogram supplied with cefepime dihydrochloride monohydrate for system suitability RS and the chromatogram obtained with reference solution (3) to identify the peaks due to impurities A, B and F; use the chromatogram obtained with reference solution (4) to identify the peak due to impurity E. Relative retention with reference to cefepime (retention time = about 7 min): Impurity E = about 0.4; impurity F = about 0.8; impurity A = about 2.5; impurity B = about 4.1. System suitability: In the chromatogram obtained with reference solution (3), the resolution between the peaks due to impurity F and cefepime is at least 1.5. Limits: Correction factors: For the calculation of content, multiply the peak areas of the following impurities by the corresponding correction factor: Impurity A = 1.4; impurity B = 1.4; impurity E = 1.8; In the chromatogram obtained with the test solution: Impurity A: The corrected area of the peak due to impurity A is not more than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.3%). Impurities B, F: For each impurity, the area, corrected if necessary, is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.2%); Impurity E: The corrected area of the peak due to impurity E is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.1%). Any other impurity: For each impurity, the area is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.10%).

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The sum of the peak areas of all impurities is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (1.0%). Disregard any peak with an area less than 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Note: Impurity A: (6R,7R)-7-[[(2E)-(2-aminothiazol-4-yl)(methoxyimino) acetyl]amino]-3-[(1- methylpyrrolidinio)methyl]-8-oxo-5-thia1-azabicyclo[4.2.0]oct-2-ene-2- carboxylate (anti-cefepime). Impurity B: (6R,7R)-7-[[(2Z)-[2-[[(2Z)-(2-aminothiazol-4-yl) (methoxyimino)acetyl]amino]thiazol-4-yl](methoxyimino) acetyl]amino]-3-[(1-methylpyrrolidinio)methyl]-8-oxo-5-thia-1azabicyclo[4.2.0]oct-2-ene-2- carboxylate. Impurity C: (2Z)-2-(2-aminothiazol-4-yl)-N-(formylmethyl)-2(methoxyimino)acetamide. Impurity D: (2Z)-(2-aminothiazol-4-yl)(methoxyimino)acetic acid. Impurity E: (6R,7R)-7-amino-3-[(1-methylpyrrolidinio)methyl]8-oxo-5-thia-1-azabicyclo [4.2.0]oct-2-ene-2-carboxylate. Impurity F: (6R,7R)-7-[[[(6R,7R)-7-[[(2Z)-(2-aminothiazol-4-yl) (methoxyimino)acetyl]amino]-3-[(1-methylpyrrolidinio)methyl]8-oxo-5-thia-1- azabicyclo[4.2.0]oct-2-en-2-yl]carbonyl]amino]-3[(1-methylpyrrolidinio)methyl]-8- oxo-5-thia-1-azabicyclo[4.2.0] oct-2-ene-2-carboxylate. Impurity G: 1-methylpyrrolidine (N-methylpyrrolidine).

Water 3.0% to 4.5% (Appendix 10.3). Determined on 0.400 g. Bacterial endotoxins Less than 0.04 EU/mg (Appendix 13.2). If intended for use in the manufacture of parenteral preparations without a further appropriate procedure for the removal of bacterial endotoxins. Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. Mobile phase: Mobile phase A. Inject the test solution and reference solution (1). The run time is 1.4 times the retention time of cefepime. Calculate the content of C19H26Cl2N6O5S2, using the areas for the peaks in the chromatogram obtained with the test solution, reference solution (1) and the content of C19H26Cl2N6O5S2 in cefepime dihydrochloride monohydrate RS. Storage Protected from light. If the substance is sterile, store in a airtight, sterile container. Action and use Cephalosporin antibacterial. Preparations Tablets, capsules, injection powder.

CEFIXIME

CEFIXIME Cefiximum

C16H15N5O7S2,3H2O

M. 507.5

Cefixime is (6R,7R)-7-[[(Z)-2-(2-aminothiazol-4-yl)-2[(carboxymethoxy)imino]acetyl]amino]-3-ethenyl-8oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid trihydrate, semi-synthetic product derived from a fermentation product. It contains not less than 95.0% and not more than 102.0% of C16H15N5O7S2, calculated with reference to the anhydrous substance. Characters A white or almost white powder, slightly hygroscopic. Slightly soluble in water, soluble in methanol, sparingly soluble in anhydrous ethanol, practically insoluble in ethyl acetate. Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of cefixime RS. If the spectra obtained show differences, dissolve the substance to be examined and the reference substance separately in methanol R, evaporate to dryness and record new spectra using the residues. pH Suspend 0.5  g of the substance to be examined in  carbon dioxide-free water R and dilute to 10 ml with the same solvent. The pH of the suspension is 2.6 to 4.1 (Appendix 6.2). Ethanol (Appendix 10.14) Not more than 1.0% (w/w), determined by head-space gas chromatography  (Appendix 5.2), using the standard additions method. Test solution: Dissolve 0.250  g of the substance to be examined in a mixture of dimethylacetamide R - water (1 : 4) and dilute to 25.0 ml with the same mixture of solvents. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Acetonitrile - tetrabutylammonium hydroxide solution (250 : 750). Tetrabutylammonium hydroxide solution: Dissolve 8.2 g of tetrabutylammonium hydroxide R in water and dilute to 800 ml with the same solvent, adjust to pH 6.5 with  2 M phosphoric acid solution R and dilute to 1000 ml with water. Test solution:  Dissolve 25.0  mg of the substance to be examined in the mobile phase and dilute to 25.0 ml with the same solvent. 199

VP V

CEFIXIME CAPSULES

Reference solution (1): Dissolve 25.0 mg of cefixime RS in the mobile phase and dilute to 25.0 ml with the same solvent. Reference solution (2): Dilute 1.0 ml of reference solution (1) to 100.0 ml with the mobile phase. Reference solution (3): Dissolve 10 mg of cefixime RS in 10 ml of water. Heat on a water-bath for 45 min and cool (in situ preparation of impurity D). Inject immediately. Chromatographic system: A column (12.5 cm × 4.0 mm) packed with stationary phase C (5 µm). Column temperature: 40 °C. Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0  ml/min.Volume of injection: 10 µl. Procedure: Inject the test solution and reference solutions (2) and (3). Carry out the chromatography for 3 times the retention time of cefixime. System suitability: In the chromatogram obtained with reference solution (3), the resolution between the peaks due to cefixime and impurity D is at least 2.0. if necessary, adjust the concentration of acetonitrile in the mobile phase. Limits: Any impurity: The area of each impurity peak is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.5%). Sum of the areas of all the impurity peaks is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (2) (3%). Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.1%).

Notes: Impurity A: 2-[[(Z)-2-(2-aminothiazol-4-yl)-2-[(carboxymethoxy) imino]acetyl]amino]-2-[(2R)-5-methyl-7-oxo-1,2,5,7-tetrahydro4H-furo[3,4-d][1,3]thiazin-2-yl]acetic acid, Impurity B: 2-[[[(Z)-1-(2-aminothiazol-4-yl)-2-[[[(2R,5RS)-5methyl-7-oxo-1,2,5,7-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2yl]methyl]amino]-2-oxoethylidene]amino]oxy]acetic acid, Imputity C: (6R,7S)-7-[[(Z)-2-(2-aminothiazol-4-yl)-2-[(carboxymethoxy)imino]acetyl]amino]-3-ethenyl-8-oxo5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (cefixime 7-epimer), Impurity D: (6R,7R)-7-[[(E)-2-(2-aminothiazol-4-yl)-2-[(carboxymethoxy)imino]acetyl]amino]-3-ethenyl-8-oxo5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (cefixime (E)-isomer), Impurity E: (6R,7R)-7-[[(Z)-2-(2-aminothiazol-4-yl)-2-[(carboxymethoxy)imino]acetyl]amino]-3-methyl-8-oxo-5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, Impurity F: (6R,7R)-7-[[(Z)-2-(2-aminothiazol-4-yl)-2-[(2-ethoxy2-oxoethoxy)imino]acetyl]amino]-3-ethenyl-8-oxo-5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid.

Water 9.0% to 12.0% (Appendix 10.3). Determined on 0.200 g. Sulfated ash Not more than 0.2% (Appendix 9.9, method 2). Determined on 1.0 g. 200

Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. Inject the test solution and reference solution (1). System suitability: In the chromatogram obtained with reference solution (1), the relative standard deviation of the areas of peak due to cefixime for 6 replicate injections is not more than 1.0%. Calculate the content of C16H15N5O7S2 in the substance to be examined, using the areas of peak due to cefixime in the chromatograms obtained with the test solution and reference solution (1), and the declared content of C16H15N5O7S2, in cefixime RS. Storage Store in an airtight container, protected from light. Action and use Cephalosporin antibacterial Preparations Coated tablets, capsules, powder. CEFIXIME CAPSULES Capsulae Cefiximi Cefixime capsules contain cefixime. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of anhydrous cefixime, C16H15N5O7S2, 90.0% to 110.0% of the stated amount. Identification A. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the principal peak in the chromatogram obtained with the reference solution. B. Dissolve a quantity of the powder in phosphate buffer pH 7.0 (as directed in the Assay) to obtain a solution having a known concentration of about 10 µg/ml of cefixime, filter. The ultraviolet absorption spectrum (Appendix 4.1) of the filtrate exhibits an absorption maximum at 288 nm. Water Not more than 12.0% (Appendix 10.3). Determine on 0.3 g. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase, phosphate buffer pH 7.0 and Chromatographic system: Prepare as directed in the Assay. Test solution: Transfer an accurately weighed quantity of the contents of the capsules containing the equivalent of about 0.1 g of cefixime to a 100 ml volumetric flask, add 75 ml of phosphate buffer pH 7.0 and sonicate for 15 min.

VP V

Dilute with phosphate buffer pH 7.0 to volume and mix well, filter. Reference solution: Dilute 1.0 ml of the test solution to 100.0 ml with phosphate buffer pH 7.0. Procedure: Inject the reference solution. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained is not less than 50% of the full scale of the recorder. Inject the test solution and continue the chromatography for 3 times the retention time of the principal peak. In the chromatogram obtained with the test solution, the area of any peak, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with the reference solution (1.0%); the sum of areas of all secondary peaks, is not more than 6 times the area of the principal peak in the chromatogram obtained with the reference solution (6.0%).

Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 900 ml of 0.05 M phosphate buffer pH 7.2 prepared as follows: Dissolve 6.8 g of potassium dihydrogen phosphate R in 1000 ml of water and adjust to pH 7.2 with 1 M sodium hydroxide R. Rotation speed: 100 rpm. Time: 45 min. Procedure: Test solution: After the specified time (45 min) withdraw a sample of the medium, filter. Dilute the filtrate with the medium to obtain a solution containing 10 µg/ml of cefixime. Reference solution: Dissolve an accurately weighed quantity of cefixime RS in the medium to obtain a solution having the same concentration of cefixim as in the test solution. An amount of methanol R not to exceed 0.1% of the total volume of the reference solution may be used to dissolve the reference substance prior to dilution with the medium, and the solution may be sonicated. Measure the absorbance of the resulting solutions at the maximum at about 288 nm (Appendix 4.1), in a 1 cm cell, using the medium as the blank. Calculate the content of cefixime, C16H15N5O7S2, dissolved using the absorbance of the test solution and the reference solution and the declared content of C16H15N5O7S2 in cefixime RS. Tolerance: Not less than 75% (Q) of the stated amount of cefixime, C16H15N5O7S2, is dissolved in 45 min. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: A mixture of 250 volumes of acetonitrile R and 750 volumes of tetrabutylammonium hydroxide solution. Tetrabutylammonium hydroxide solution: Dissolve 8.2 g of tetrabutylammonium hydroxide R in 800 ml of water, adjust to pH 6.5 with dilute phosphoric acid solution R, dilute to 1000 ml with water. Potassium dihydrogen phosphate solution: Dissolve 6.8 g of potassium dihydrogen phosphate R in 500 ml of water.

CEFIXIME CAPSULES

Phosphate buffer pH 7.0: Dissolve 7.1 g of anhydrous disodium hydrogen phosphate R in 500 ml of water. Adjust to pH 7.0 with potassium dihydrogen phosphate solution. Resolution solution: Dissolve cefixime RS in water to obtain a solution containing about 0.5 mg per ml. Heat in a water bath at 95 °C for 45 min. Allow to cool, filter and use the filtrate immediately. Reference solution: Dissolve an accurately weighed quantity of cefixime RS in phosphate buffer pH 7.0 to obtain a solution containing about 0.1 mg per ml. Test solution: Weigh 20 capsules and calculate the average mass of the contents, and powder finely. Transfer an accurately weighed quantity of the powder containing the equivalent of about 0.2 g of anhydrous cefixime to a 100 ml volumetric flask, add 75 ml of phosphate buffer pH 7.0 and sonicate for 15 min, dilute with phosphate buffer pH 7.0 to volume and mix well, filter. Transfer 5.0 ml of the filtrate to a 100 ml volumetric flask and dilute with phosphate buffer pH 7.0 to volume. Mix well. Chromatographic system: A column (12.5 cm × 4.0 mm) packed with stationary phase C (5 μm), maintained at 40 °C. Detector: A spectrophotometer set at 254 nm. The flow rate is adjusted so that the retention time of cefixime is about 10 min. Volume of injection: 20 μl. Procedure: System suitability: Inject the resolution solution, the relative retention times for cefixime and cefixime E-isomer are about 1.0 and 0.9, respectively; the resolution factor between cefixime and cefixime E-isomer is at least 2.0. The theoretical plates of cefixime peak is not less than 4000. Inject the reference solution, the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of anhydrous cefixime, C16H15N5O7S2, in the capsules using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C16H15N5O7S2 in cefixime RS.

Storage Pack in aluminum blister or airtight bottle. Store in a cool and dry place, at a temperature not exceeding 30 °C, protected from light. Action and use Cephalosporin antibacterial. Usual strength 100 mg, 200 mg.

201

CEFIXIME POWDER FOR ORAL SUSPENSION

CEFIXIME POWDER FOR ORAL SUSPENSION  Pulveres Cefiximi ad suspensionum peroralum Cefixime powder for  oral  suspension  contains cefixime. Suitable excipients such as  flavors and preservatives… may be added. The powder complies with the requirements stated under “Powder” (Appendix 1.7) and with the following requirements.

Content of cefixime, C16H15N5O7S2, 90.0% to 110.0% of the stated amount. Characters A dry powder with a homogeneous colour. Identification A. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the principal peak in the chromatogram obtained with the reference solution. B. Dissolve a quantity of the powder in phosphate buffer pH 7.0 (as directed in the Assay) to obtain a solution having a known concentration of about 10 µg/ml of cefixime, filter. The ultraviolet absorption spectrum (Appendix 4.1) of the filtrate exhibits an absorption maximum at 288 nm. Water Not more than 9.0% (Appendix 10.3). Determinde on 1.0 g. pH pH of the suspension constituted as directed in the label is 2.5 to 4.5 (Appendix 6.2). Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase, phosphate buffer pH 7.0 and Chromatographic system: Prepare as directed in the Assay. Test solution: Transfer an accurately weighed quantity of the powder containing the equivalent of about 0.1 g of cefixime to a 100 ml volumetric flask, add 75 ml of phosphate buffer pH 7.0 and sonicate for 15 min. Dilute with phosphate buffer pH 7.0 to volume and mix well, filter. Reference solution: Dilute 1.0 ml of the test solution to 100.0 ml with phosphate buffer pH 7.0, mix. Procedure: Inject the reference solution. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained is not less than 50% of the full scale of the recorder. Inject the test solution and continue the chromatography for 3 times the retention time of the principal peak. In the chromatogram obtained with the test solution, the area of any peak, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with the reference solution (1.0%); the sum of areas of all secondary peaks, is not more than 6 times the 202

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area of the principal peak in the chromatogram obtained with the reference solution (6.0%).

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - tetrabutylammonium hydroxide solution (250 : 750). Tetrabutylammonium hydroxide solution: Dissolve 8.2 g of tetrabutylammonium hydroxide R in 800 ml of water, adjust to pH 6.5 with dilute phosphoric acid solution R, dilute to 1000 ml with water. Potassium dihydrogen phosphate solution: Dissolve 6.8 g of potassium dihydrogen phosphate R in 500 ml of water. Phosphate buffer pH 7.0: Dissolve 7.1 g of anhydrous disodium hydrogen phosphate R in 500 ml of water. Adjust to pH 7.0 with potassium dihydrogen phosphate solution. Resolution solution: Dissolve a quantity of cefixime RS in water to obtain a solution containing about 0.5 mg per ml. Heat in a water-bath at 95 °C for 45 min. Allow to cool, filter and use the filtrate immediately. Reference solution: Dissolve an accurately weighed quantity of cefixime RS in phosphate buffer pH 7.0 to obtain a solution containing about 0.1 mg per ml. Test solution: Use the contents in the Uniformity of mass, mix. Transfer an accurately weighed quantity of the powder containing the equivalent to about 0.2 g of anhydrous cefixime to a 100 ml volumetric flask, add 75 ml of phosphate buffer pH 7.0 and sonicate for 15 min, dilute with phosphate buffer pH 7.0 to volume and mix well, filter. Transfer 5.0 ml of the filtrate to a 100 ml volumetric flask and dilute with phosphate buffer pH 7.0 to volume. mix well. Chromatographic system: A column (12.5 cm × 4.0 mm) packed with stationary phase C (5 μm), maintained at 40 °C. Detector: A spectrophotometer set at 254 nm. The flow rate is adjusted so that the retention time of cefixime is about 10 min. Volume of injection: 20 μl. Procedure: System suitability: Inject the resolution solution, the relative retention times for cefixime and cefixime E-isomer are about 1.0 and 0.9, respectively; the resolution factor between cefixime and cefixime E-isomer is at least 2.0. The theoretical plates of cefixime peak is not less than 4000. Inject the reference solution, the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of anhydrous cefixime, C16H15N5O7S2, in the powder using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C16H15N5O7S2 in cefixime RS. Storage Store in an airtight container, in a cool and dry place, protected from light, at a temperature not exceeding 30 °C.

VP V

Action and use Cephalosporin antibacterial. Usual strength 100 mg, 200 mg. CEFIXIME TABLETS Tabellae Cefiximi Cefixime tablets are film-coated tablets containing cefixime. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of anhydrous cefixime, C16H15N5O7S2, 90.0% to 110.0% of the stated amount. Identification In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of the principal peak in the chromatogram obtained with the reference solution. Water Not more than 10.0% (Appendix 10.3). Use 0.3 g of the powdered tablets. Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 900 ml of 0.05 M phosphate buffer pH 7.2 prepared as follows: Dissolve 6.8 g of potassium dihydrogen phosphate R in 1000 ml of water and adjust to pH 7.2 with 1 M sodium hydroxide R. Rotation speed: 100 rpm. Time: 45 min. Procedure: Withdraw a sample of the medium, filter. Dilute the filtrate with the medium to obtain a solution containing a suitable concentration (if necessary). Measure the absorbance of the resulting solution at the maximum at about 288 nm (Appendix 4.1), in a 1-cm cell, using the medium as the blank. Calculate the content of cefixime, C16H15N5O7S2, dissolved using the absorbance of a reference solution of cefixime RS having the same concentration in the same medium. [Note: An amount of methanol not exceeding 0.1% of the total volume of the reference solution may be used to dissolve the reference substance prior to dilution with the medium, and the solution may be sonicated.] Tolerance: Not less than 75% (Q) of the stated amount of cefixime, C16H15N5O7S2, is dissolved in 45 min. Assay Examine by liquid chromatography (Appendix 5.3). Tetrabutylammonium hydroxide solution: Dilute 25 ml of 0.4 M tetrabutylammonium hydroxide solution with

CEFIXIME TABLETS

sufficient water to produce 1000 ml, adjust the pH to 6.5 with 1.5 M phosphoric acid solution R. Mobile phase: A mixture of 250 volumes of acetonitrile R and 750 volumes of tetrabutylammonium hydroxide solution. Potassium dihydrogen phosphate solution: Dissolve 6.8 g of potassium dihydrogen phosphate R in sufficient water to produce 500 ml. Phosphate buffer pH 7.0: Dissolve 7.1 g of anhydrous disodium hydrogen phosphate R in sufficient water to produce 500 ml. Adjust the pH to 7.0 with potassium dihydrogen phosphate solution. Resolution solution: Dissolve cefixime RS in water to obtain a solution containing about 0.5 mg per ml. Heat in a water bath at 95 °C for 45 min. Allow to cool, filter and use the filtrate immediately. Reference solution: Dissolve cefixime RS in phosphate buffer pH 7.0 to obtain a solution containing about 0.1 mg per ml. Test solution: Weigh 20 tablets, calculate the average weight and powder finely. Weigh accurately a quantity of the powdered tablets containing about 0.2 g of anhydrous cefixime to a 100- ml volumetric flask, add 75 ml of phosphate buffer pH 7.0, sonicate for 15 min, dilute with phosphate buffer pH 7.0 to volume and mix well, filter. Transfer 5.0 ml of the filtrate to a 100-ml volumetric flask and dilute with phosphate buffer pH 7.0 to volume, mix well. Chromatographic system: A column (12.5 cm × 4.0 mm) packed with stationary phase C (5 µm), maintained at 40 °C. Detector: A spectrophotometer set at 254 nm. Flow rate: Adjust to obtain a retention time of about 10 min for cefixime. Volume of injection: 20 µl. Procedure: System suitability: Inject the resolution solution. The resolution factor between two peaks due to cefixime and cefixime E-isomer is not less than 2.0. Inject the reference solution, the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of anhydrous cefixime, C16H15N5O7S2, in the tablets using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C16H15N5O7S2 in cefixime RS.

Storage Pack in aluminum blister or closed bottle. Store in a cool and dry place, protected from light and at a temperature not exceeding 30 °C. Action and use Cephalosporin antibacterial. Usual strength 100 mg.

203

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CEFOPERAZONE SODIUM

CEFOPERAZONE SODIUM Cefoperazonum natricum

C25H26N9NaO8S2  

M. 668.0

Cefoperazone sodium is sodium (6R,7R)-7-[[(2R)-2-[[(4ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]-2-(4hydroxyphenyl)acetyl]amino]-3-[[(1-methyl-1H-tetrazol5-yl)sulphanyl]methyl]-8-oxo-5-thia-1-azabicyclo[4.2.0] oct-2-ene-2-carboxylate. It contains not less than 95.0% and not more than 102.0% of C25H26N9NaO8S2, calculated with reference to the anhydrous substance.

Characters A white or slightly yellow powder, hygroscopic. Freely soluble in water, soluble in methanol, slightly soluble in ethanol (96%). If crystalline, it shows polymorphism. Identification A. Dissolve the substance to be examined in methanol R and evaporate to dryness. The infrared absorption spectrum (Appendix 4.2) of the residue is concordant with the reference spectrum of cefoperazone sodium. B. In the chromatograms obtained in the Assay, the retention time and size of the principal peak in the chromatogram obtained with test solution (1) are approximately the same as those of the principal peak in the chromatogram obtained with reference solution (1). C. It gives reaction (A) of sodium (Appendix 8.1). Appearance of solution Dissolve 2.5 g in water and dilute to 25.0 ml with the same solvent. The solution is clear (Appendix 9.2). The absorbance of the solution measured at 430 nm (Appendix 4.1) is not greater than 0.15. pH Dissolve 2.5 g in carbon dioxide-free water R and dilute to 10 ml with the same solvent. The pH of the solution is 4.5 to 6.5 (Appendix 6.2). Related substances Examine by liquid chromatography (Appendix 5.3) as prescribed under Assay. Prepare the solutions immediately before use. 204

Inject reference solution (2) and adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained is at least 50% of the full scale of the recorder. Inject test solution (2). Continue the chromatography for at least 2.5 times the retention time of the principal peak. In the chromatogram obtained with test solution (2): the area of any peak, apart from the principal peak, is not greater than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (1.5%). The sum of the areas of all such peaks is not greater than 4.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (4.5%). Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.1%).

Acetone Not more than 2.0%. Determined by head-space gas chromatography (Appendix 5.2), using the standard additions method. Test solution: Dissolve 0.500 g of the substance to be examined in water and dilute to 10.0 ml with the same solvent. Acetone solution: Dissolve 0.350 g of acetone R in water and dilute to 100.0 ml with the same solvent. Dilute 10.0 ml of the solution to 100.0 ml with water. Prepare each of 4 injection vials as shown in the table below: Vial No.

Sample solution (ml)

Solvent solution (ml)

Water (ml)

1 2 3 4

1.0 1.0 1.0 1.0

0 1.0 2.0 3.0

4.0 3.0 2.0 1.0

The chromatographic procedure may be carried out using system B of the test for Residual solvents (Appendix 10.14.1) with the following static head-space injection conditions:   - Equilibration time: 15 min,   - Transfer-line temperature: 110 °C, Maintaining the temperature of the column at 40 °C for 10 min.

Heavy metals Not more than 5 ppm (Appendix 9.4.8). 2.0 g complies with limit test 3. Prepare the standard solution using 1 ml of lead standard solution (10 ppm Pb) R. Water Not more than 5.0% (Appendix 10.3). Determined on 0.200 g. Bacterial endotoxins Less than 0.20 IU/mg (Appendix 13.2).

VP V

If intended for use in the manufacture of parenteral preparations forms without a further appropriate procedure for the removal of bacterial endotoxins.

Assay Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use. Mobile phase: Water - acetonitrile - 1 M acetic acid triethylamine acetate solution (884 : 110 : 3.5 : 2.5). Triethylamine acetate solution: Dilute 14 ml of triethylamine R and 5.7 ml of glacial acetic acid R to 100 ml with water. Test solution (1): Dissolve 25.0 mg of the substance to be examined in the mobile phase and dilute to 250.0 ml with the mobile phase. Test solution (2): Dissolve 25.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Reference solution (1): Dissolve 25.0 mg of cefoperazone dihydrate RS in the mobile phase and dilute to 250.0 ml with the mobile phase. Reference solution (2): Dilute 5.0 ml of reference solution (1) to 100.0 ml with the mobile phase. Chromatographic system: A column (15 cm × 4.6 mm) packed with end-capped octadecylsilyl silica gel for chromatography (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Procedure: Inject reference solution (1). When the chromatogram is recorded in the prescribed conditions, the retention time is about 15 min for cefoperazone. The test is not valid unless in the chromatogram obtained with reference solution (1), the number of theoretical plates calculated for the principal peak is at least 5000 and its symmetry factor is at most 1.6. If necessary adjust the content of acetonitrile in the mobile phase. Inject reference solution (1) 6 times. The test is not valid unless the relative standard deviation of the peak area is at most 1.0%. Inject alternately test solution (1) and reference solution (1). Calculate the percentage content of cefoperazone sodium by multiplying the percentage content of cefoperazone by 1.034. Storage In an airtight container, protected from light, at a temperature of 2 °C to 8 °C. If the substance is sterile, store in a sterile, airtight, tamperproof container. Action and use Cephalosporin antibacterial. Preparation Injection.

CEFOPERAZONE FOR INJECTION

CEFOPERAZONE FOR INJECTION Cefoperazoni pulvis ad injectionem Cefoperazone is a sterile powder of cefoperazone sodium with or without excipients. It is supplied in a sealed glass container. The powder complies with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements:

Content of cefoperazone, C25H27N9O8S2, 90.0% to 110.0% of the stated amount. Characters A white or almost white powder. Identification A. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that of cefoperazone peak in the chromatogram obtained with the reference solution. B. It gives the reactions of sodium (Appendix 8.1). pH 4.5 to 6.5 (Appendix 6.2). Dissolve 2.5 g of the substance to be examined in carbon dioxide-free water R and dilute to 10 ml with the same solvent. Clarity of solution Dissolve 2.5 g of the substance to be examined in water and dilute to 25 ml with the same solvent. The solution is clear (Appendix 9.2) and free from visible particles (Appendix 11.8). Absorbance The absorbance (Appendix 4.1) of the obtained solution in the Clarity of solution measured at 430 nm is not greater than 0.15. Water Not more than 5.0%, except that where it is in the freezedried form, the limit is not more than 2.0% (Appendix 10.3). Bacterial endotoxins Not more than 0.2 EU per mg of cefoperazone (Appendix 13.2). Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Water - acetonirile - 1 M acetic acid triethylamine acetate solution (884 : 110 : 3.5 : 2.5) Triethylamine acetate solution: Dilute 14 ml of triethylamine R and 5.7 ml of glacial acetic acid R to 100 ml with water. Test solution: Determine the average mass of the contents of 10 containers, and mix. Transfer an accurately weighed 205

VP V

CEFOPERAZONE AND SULBACTAM FOR INJECTION

quantity of the mixed contents containing the equivalent of about 25 mg of cefoperazone to a 250 ml volumetric flask, dissolve and dilute to volume with the mobile phase. Reference solution: Dissolve an accurately weighed quantity of cefoperazone sodium RS in the mobile phase to obtain a solution containing the equivalent of 0.01% of cefoperazone. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 μm). Detector: A spectrophotometer set at 254 nm. Flow rate: 2.0 ml/min. Volume of injection: 20 μl. Procedure: System suitability: Inject the reference solution. The theoretical plates for the principal peak are not less than 5000, the symmetry factor is not more than 1.6 and the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject separately the reference solution and the test solution. Calculate the content of cefoperazone, C25H27N9O8S2, in the injections using the peak areas of cefoperazone in the chromatograms obtained with the test solution and the reference solution, and the declared content of C25H27N9O8S2 in cefoperazone sodium RS.

Storage Store in a cool place, protected from light. Action and use Cephalosporin antibacterial. Usual strength 1 g, 2 g of cefoperazone. CEFOPERAZONE AND SULBACTAM FOR INJECTION Cefoperazoni et sulbactami pulvis injectionem Cefoperazone and sulbactam for injection is a sterile powder containing cefoperazone sodium and sulbactam sodium for injections. The ratio of the stated amount of cefoparezone and sulbactam is 1 : 1. The powder complies with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Content of cefoperazone, C25H27N9O8S2, 90.0% to 110.0% of the stated amount. Content of sulbactam, C8H11NO5S, 90.0% to 110.0% of the stated amount. Characters A white or almost white powder. 206

Identification A. In the Assay, the retention times of the two principal peaks in the chromatogram obtained with the test solution correspond to those in the chromatogram obtained with the reference solution. B. It gives the reactions of sodium (Appendix 8.1). pH Dissolve a quantity of the substance to be examined in carbon dioxide-free water R to obtain a solution containing 125 mg/ml of cefoperazone, pH of this solution is 3.5 to 6.5 (Appendix 6.2). Clarity of solution A 10% solution of the substance to be examined in water is clear (Appendix 9.2) and free from visible particles (Appendix 11.8). Water Not more than 4.0% (Appendix 10.3). Bacterial endotoxins Not more than 0.2 EU per mg of the substance to be examined (Appendix 13.2). Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: 0.005 M tetrabutylammonium hydroxide acetonitrie (75 : 25), make adjustments if necessary. 0.005 M tetrabutylammonium hydroxide:  Dilute 6.6 ml of a 40% solution of tetrabutylammonium hydroxide to 1800 ml with water. Adjust to pH 5.0 ± 0.1with 1 M phosphoric acid R, dilute to 2000 ml with water. Reference solution: Dissolve an accurately weighed quantity of cefoperazone sodium RS and sulbactam sodium RS equivalent to about 100 mg of cefoperazone and 100 mg of sulbactam in the mobile phase and dilute to 100.0 ml with the same solvent, mix. Resolution solution: Dissolve a quantity of about 50 mg of cefoperazone sodium RS in 50 ml water. Heat this solution at 60 °C in a water-bath for 30 min and allow to cool. Dissolve a quantity of about 50 mg of sulbactam sodium RS in this solution and mix. Test solution: Determine the average weight of the contents of 10 containers, and mix. Transfer an accurately weighed quantity of the mixed content containing the equivalent of about 100 mg of cefoperazone to a 100 ml volumetric flask. Add 70 ml of the mobile phase, shake to dissolve and dilute to volume with the mobile phase, mix. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 μm). Detector: A spectrophotometer set at 220 nm Flow rate: 1.5 ml/min. Volume of injection: 10 μl.

VP V

CEFOTAXIME SODIUM

Procedure: System suitability: Inject the resolution solution. The relative retention times for degradation product of cefoperazon and sulbactam are about 0.9 and 1.0, respectively. The assay is not valid unless, the resolution factor between the degradation product and sulbactam peaks is not less than 1.5. Inject the reference solution, the theoretical plates of the peak due to cefoperazone is not less than 3000, the symmetry factor of the peak due to cefoperazone is not more than 2 and the relative standard deviation for replicate injections is not more than 2.0%. Inject separately the reference solution and the test solution. Calculate the contents of cefoperazone, C25H27N9O8S2, and sulbactam, C8H11NO5S, in the injection from the peak areas of cefoperazone and sulbactam in the chromatograms obtained with the test solution and the reference solution, and the declared content of C25H27N9O8S2 and C8H11NO5S in cefoperazone sodium RS and sulbactam sodium RS, respectively.

Storage Store in an airtight container, in a cool place, protected from light. Action and use Antibacterial. Usual strength 0.5 g of cefoperazone and 0.5 g of sulbactam. 1.0 g of cefoperazone and 1.0 g of sulbactam.

Appearance of solution Solution S: Dissolve 2.5 g in carbon dioxide-free water R and dilute to 25.0 ml with the same solvent. Solution S is clear (Appendix 9.2). Add 1 ml of glacial acetic acid R to 10 ml of solution S. The solution, examined immediately, is clear. pH 4.5 to 6.5 (Appendix 6.2). Determined on solution S. Specific optical rotation +58° to + 64°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.100 g in water and dilute to 10.0 ml with the same solvent. Absorbance Not more than 0.40 (Appendix 4.1). Determined on solution S at 430 nm. Specific absorbance Dissolve 20.0  mg of the substance to be examined in  water and dilute to 100.0 ml with the same solvent. Dilute 10.0 ml of the solution to 100.0 ml with water. The specific absorbance determined at the maximum at 235 nm is 360 to 390, calculated with reference to the anhydrous substance.

CEFOTAXIME SODIUM Cefotaximum natricum

C16H16N5NaO7S2

Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of cefotaxime sodium RS. B. It gives reaction (A) of sodium (Appendix 8.1).

M. 477.4

Cefotaxime sodium is sodium (6R,7R)-3-[(acetyloxy) methyl]-7-[[(2Z)-2-(2-aminothiazol-4-yl)-2(methoxyimino)acetyl]amino]-8-oxo-5-thia-1-azabicyclo -[4.2.0]oct-2-ene-2-carboxylate. It contains not less than 96.0% and not more than 102.0% of C16H16N5NaO7S2, calculated with reference to the anhydrous substance. Semi-synthetic product derived from a fermentation product. Characters A white or slightly yellow powder, hygroscopic. Freely soluble in water, sparingly soluble in methanol.

Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use. Mobile phase A: A 0.71% solution of  disodium hydrogen phosphate R adjusted to pH 6.25 with phosphoric acid R. Mobile phase B: Methanol R. Solvent mixture: Mobile phase B - mobile phase A (14 : 86). Test solution: Dissolve 40.0  mg of the substance to be examined in solvent mixture and dilute to 50.0 ml with the same solvent. Reference solution (1):  Dissolve 8.0  mg of  cefotaxime acid RS in solvent mixture and dilute to 10.0 ml with the same solvent. Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with solvent mixture. Reference solution (3): Add 1.0 ml of dilute hydrochloric acid R to 4.0 ml of the test solution. Heat the solution at 40 °C for 2 h. Add 5.0 ml of buffer solution pH 6.6 R and 1.0 ml of 1 M sodium hydroxide solution R. 207

VP V

CEFOTAXIME SODIUM

Reference solution (4): Dissolve 4 mg of cefotaxime for peak identification RS (containing impurities A, B, C, E and F) in 5 ml of solvent mixture. Chromatographic system: A column (15 cm × 3.9 mm) packed with stationary phase C (5 µm). Column temperature: 30 °C. Detector: A spectrophotometer set at 235 nm. Flow rate: 1.0  ml/min. Volume of injection: 10 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0-7

86

14

7-9

86 → 82

14 → 18

9 - 16

82

18

16 - 45

82 → 60

18 → 40

45 - 50

60

40

50 - 55

60 → 86

40 → 14

55 - 60

86

14

Inject the test solution and reference solutions (2), (3) and (4). Identification of impurities:  Use the chromatogram supplied with cefotaxime for peak identification RS and the chromatogram obtained with reference solution (4) to identify the peaks due to impurities A, B, C, E and F. Relative retention with reference to cefotaxime (retention time  =  about 13 min): Impurity B  =  about 0.3; impurity A = about 0.5; impurity E = about 0.6; impurity C = about 1.9; impurity D  =  about 2.3; impurity F  =  about 2.4; impurity G = about 3.1. System suitability: In the chromatogram obtained with reference solution (3), the resolution between the peaks due to impurity E and cefotaxime is at least 3.5; the symmetry factor of the peak due to cefotaxime is not more than 2.0. Limits: Impurities A, B, C, D, E, F: For each impurity peak, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (1.0%). Any other impurity: The area of each impurity peak is not more than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.2%). Sum of the areas of all the impurity peaks is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (2) (3.0%). Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Notes: Impurity A: (6R,7R)-7-[[(2Z)-2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetyl]amino]-3-methyl-8-oxo-5-thia-1-azabicyclo [4.2.0]oct -2-ene-2-carboxylic acid (deacetoxycefotaxime).

208

Impurity B: (6R,7R)-7-[[(2Z)-2-(2-aminothiazol-4-yl)-2(methoxyimino) acetyl]amino]-3-(hydroxymethyl)-8-oxo-5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (deacetylcefotaxime). Imputity C: (6R,7R)-3-[(acetyloxy)methyl]-7-[[(2Z)-2-[2(formylamino)thiazol-4-yl]-2-(methoxyimino)acetyl]amino]8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (N-formylcefotaxime). Impurity D: (6R,7R)-3-[(acetyloxy)methyl]-7-[[(2E)-2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetyl]amino]-8-oxo-5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (E-cefotaxime). Impurity E: (5aR,6R)-6-[[(2Z)-2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetyl]amino]-5a,6-dihydro-3H,7H-azeto[2,1-b]furo[3,4-d] [1,3]thiazine-1,7(4H)-dione (deacetylcefotaxime lactone). Impurity F: (6R,7R)-3-[(acetyloxy)methyl]-7-[[(2Z)-2-[2[[[(6R,7R)-7-[[(2Z)-2-(2-aminothiazol-4-yl)-2-(methoxyimino) acetyl]amino]-2-carboxy-8-oxo-5-thia-1-azabicyclo[4.2.0]oct2-en-2-yl]methyl]amino]thiazol-4-yl]-2-(methoxyimino)acetyl] amino]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (cefotaxime dimer). Impurity G: (6R,7R)-3-[(acetyloxy)methyl]-7-[[(2Z)-2-[2-[[(2Z) -2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetyl]amino] thiazol-4-yl]-2-(methoxyimino)acetyl]amino]-8-oxo-5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (ATA cefotaxime).

Ethanol Not more than 1.0% (Appendix 10.14, System A). N,N-Dimethylaniline Not more than 20 ppm (Appendix 10.16, method 2). 2-Ethylhexanoic acid Not more than 0.5% (w/w) (Appendix 10.17). Water Not more than 3.0% (Appendix 10.3). Determined on 0.300 g. Bacterial endotoxins Less than 0.05 IU/mg (Appendix 13.2), if intended for use in the manufacture of parenteral preparations without a further appropriate procedure for the removal of bacterial endotoxins. Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. Inject the test solution and reference solution (1). Calculate the content of cefotaxime in the substance to be examined, using the areas of peaks due to cefotaxime in the chromatograms obtained with the test solution and reference solution (1), and the declared content of cefotaxime RS. Calculate the content of C16H16N5NaO7S2 by multiplying the percentage content of cefotaxime by 1.048.

VP V

CEFOTAXIME FOR INJECTION

Storage Store in an airtight container, protected from light. If the substance is sterile, store in a sterile, airtight, tamper-proof container. Action and use Cephalosporin antibacterial. Preparation Injections.

Procedure: Inject the test solution and the reference solution. Allow the chromatography to proceed for at least 8 times the retention time (about 6 minutes) of cefotaxime and record the chromatogram. In the chromatogram obtained with the test solution the area of any secondary peak is not greater than the area of the principal peak in the chromatogram obtained with the reference solution (1.0%) and the sum of the areas of all the secondary peaks is not greater than 4 times the area of the principal peak in the chromatogram obtained with the reference solution (4.0%).

CEFOTAXIME FOR INJECTION Cefotaximi pulvis ad injectionem

Loss on drying Not more than 3.0% (Appendix 9.6). Dry 1.0 g of the preparation at 100 °C to 105 °C.

Cefotaxime for injection is a sterile powder of cefotaxime sodium. It may contain excipients. It is supplied in an airtight container. The preparation complies with the general requirements prescribed under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Bacterial endotoxins Dissolve the preparation in water BET R to give a solution containing 10 mg of cefotaxime per ml (solution A). The endotoxin limit concentration of solution A is 0.5 EU per ml (Appendix 13.2).

Content of cefotaxime, C16H17N5O7S2, 90.0% to 110.0% of the stated amount. Characters White or slightly yellow powder. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined, is concordant with the spectrum of cefotaxime sodium RS. B. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the principal peak in the chromatogram obtained with the reference solution. C. It gives reaction of sodium (Appendix 8.1). Acidity Solution S: Solution contains the equivalent of 10.0% of cefotaxime in carbon dioxide-free water R. pH of solution S is from 4.5 to 6.5 (Appendix 6.2). Appearance of solution Solution S is clear (Appendix 9.2). The absorbance of solution S at 430 nm is not greater than 0.60 (Appendix 4.1). Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase and chromatographic system: Proceed described under the Assay. Test solution: Dissolve a quantity of the preparation the mobile phase to obtain a solution containing 0.1% cefotaxime. Reference solution: The solution contains 0.0011% cefotaxime sodium RS in the mobile phase. Note: Use freshly prepared solutions.

as in of of

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 3.5 g of potassium dihydrogen phosphate R and 11.6 g of disodium hydrogen phosphate R in 1000 ml of water at pH 7.0 and add 375 ml of methanol R. Reference solution: Dissolve an accurately weighed quantity of cefotaxime sodium RS in the mobile phase to obtain a solution containing 0.01% of cefotaxime. Test solution: Dissolve an accurately weighed quantity of the preparation in the mobile phase to obtain a solution containing 0.01% of cefotaxime. Resolution solution: Add 1 ml of 2 M hydrochloric acid solution R to 4 ml of the test solution, heat the solution at 40 °C for 2 hours, add 5 ml of phosphate buffer pH 6.6 R and 1 ml of 2 M sodium hydroxide solution R and mix well. Note: Use freshly prepared solutions. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 235 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject the reference solution and the resolution solution. Adjust the sensitivity of the detector so that the heights of the peaks in the chromatogram obtained with the resolution solution are at least 50% of the full scale of the recorder. The test is not valid unless cefotaxime is eluted as the second of the principal peaks and the resolution factor between the two principal peaks is at least 3.5; the symmetry factor of the cefotaxime peak in the chromatogram obtained with the reference solution is less than 2.0. 209

VP V

CEFPODOXIME PROXETIL

When the chromatograms are recorded under the prescribed conditions the retention time of cefotaxime peak is about 6 minutes. If necessary use another stationary phase or adjust the concentration of methanol in the mobile phase. Inject alternately the test solution and the reference solution. Calculate the content of cefotaxime, C16H17N5O7S2, in one unit of the preparation using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C16H17N5O7S2 in cefotaxime sodium RS. Each mg of cefotaxime sodium, C16H16N5NaO7S2, is equivalent to 0.9539 mg of cefotaxime, C16H17N5O7S2.

Storage Store in a well-closed container, in a cool and dry place, protected from light. Action and use Cephalosporin antibacterial. Usual strength 1 g. CEFPODOXIME PROXETIL Cefpodoximum proxetili

C21H27N5O9S2

M: 557.6

Cefpodoxime proxetil is (1RS)-1-[[(1-methylethoxy) carbonyl]oxy]ethyl(6R,7R)-7-[[(2Z)-2-(2-aminothiazol-4yl)-2-(methoxyimino)acetyl]amino]-3-(methoxymethyl)-8oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate. It contains not less than 94.0% and not more than 102.0% of C21H27N5O9S2, calculated with reference to the anhydrous substance.

Characters White or pale yellow or light brown, amorphous powder. Very slightly soluble or practically insoluble in water, very soluble in acetonitrile and in methanol, freely soluble in anhydrous ethanol. Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of cefpodoxime proxetil RS. 210

Diastereoisomer ratio Examine by liquid chromatography (Appendix 5.3) as described under Assay. Use the normalisation procedure. In the chromatogram obtained with the test solution, the ratio of the area of the peak due to cefpodoxime proxetil II to the sum of the areas of the peaks due to cefpodoxime proxetil I and II is between 0.5 and 0.6. Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use or store them at 2 °C - 8 °C. Mobile phase A: 0.02 M ammonium acetate R. Mobile phase B: Acetonitrile R. Solvent mixture: Water - acetonitrile (2 : 1). Test solution: Transfer about 50 mg of of the substance to be examined to a 50 ml volumetric flask, add 5 ml of methanol R, sonicate to dissolve, and dilute to volume with solvent mixture, and mix. This solution should be injected promptly or may be analyzed within 24 hours when stored at 8 °C. System suitability solution: Dissolve a quantity of cefpodoxime proxetil RS in solvent mixture to obtain a solution containing about 10 µg per ml (Note: A volume of methanol not exceeding 10% of the total volume in the final solution may be used to facilate dissolution). Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Column temperature: 30 °C. Detector: A spectrophotometer set at 260 nm. Flow rate: 2 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0

90

10

0 - 10

90 → 68

10 → 32

10 - 40

68

32

40 - 80

68 → 50

32 → 50

80 - 85

50

50

85 - 90

50 → 25

50 → 75

90 - 95

25

75

95 - 100

25 → 90

75 → 10

Inject the system suitability solution: The retention time of the peak due to cefpodoxime proxetil II is 37 min to 42 min; the relative retention times of the peak due to cefpodoxime proxetil I is about 0.9 and cefpodoxime proxetil II is 1.0 The resolution between the peaks due to cefpodoxime proxetil I and cefpodoxime proxetil II is at least 4.0; the column efficiency determined from the peak due to

VP V

cefpodoxime proxetil II is not less than 19000 theoretical plates; the relative standard deviation for replicate injections determined from the sum of the areas of the peaks due to cefpodoxime proxetil I and cefpodoxime proxetil II is not more than 2.0%. Limit: Use the normalisation procedure. Any peak at a relative retention time of about 0.86: Not more than 3.0%. Any peak at relative retention times of about 1.27, 1.36 or higher than 2.0: Not more than 1.0%. Any other individual impurity: Not more than 0.5%. Total impurities: Not more than 6.0%. Disregard any impurity peaks of less than 0.05% .

Water Not more than 2.5% (Appendix 10.3). Determined on 0.500 g.

CEFPODOXIME CAPSULES

Action and use Cephalosporin antibacterial. Preparations Tablets, capsules, powder for suspension. CEFPODOXIME CAPSULES Capsulae Cefpodoximi Cefpodoxime capsules contain cefpodoxime proxetil. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of cefpodoxime, C15H17N5O6S2, 90.0% to 110.0% of the stated amount.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol - water (9 : 11). Solvent mixture: A 20 mg/L solution of anhydrous citric acid R in acetonitrile R. Test solution: Dissolve 30.0 mg of the substance to be examined in solvent mixture and dilute to 50.0 ml with the same solution. Reference solution: Dissolve 30.0 mg of cefpodoxime proxetil RS in solvent mixture and dilute to 50.0 ml with the same solution. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (5 µm). Column temperature: 40 °C. Detector: A spectrophotometer set at 240 nm. Flow rate: 0.8 ml/min. Volume of injection: 10 µl. Procedure: Inject the test solution and the reference solution. The run time is 1.2 times the retention time of cefpodoxime proxetil II. Retention time of the peak due to cefpodoxime proxetil II is about 30 min. System suitability: In the chromatogram obtained with reference solution, the resolution between the peaks due to cefpodoxime proxetil I and II is at least 4.0. Calculate the percentage content of cefpodoxime proxetil, C21H27N5O9S2, using the sum of the areas of the 2 peaks due to cefpodoxime proxetil I and cefpodoxime proxetil II in the chromatogram obtained with the test solution, the reference solution and using the declared content of C21H27N5O9S2 in cefpodoxime proxetil RS.

Identification In the Assay, the retention times of two principal peaks in the chromatogram obtained with the test solution correspond to those of the peaks due to cefpodoxime proxetil S-epimer và cefpodoxime proxetil R-epimer in the chromatogram obtained with the reference solution.

Storage Store in an airtight container, protected from light.

Water Not more than 5.0% (Appendix 10.3).

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: Transfer 54.5 g of glycine R and 42.6 g of sodium chloride R to a 1000 volumetric flask. Add 500 ml of water, shake to dissolve. Cautiously add, with swirling, 14.2 ml of hydrochloric acid R, and allow to cool. Dilute with water to volume, and mix. Dilute 50 ml of this solution to 900 ml with water to obtain a solution having a pH of 3.0 ± 0.1 (Adjust the pH of the medium with 1 M sodium hydroxide R, if necessary). Rotation speed: 75 rpm. Time: 30 min. Procedure: Test solution: After the specified time, withdraw a sample of the medium and filter. Dilute the filtrate with the medium, if necessary. Reference solution: Transfer an accurately weighed quantity of cefpodoxime proxetil RS equivalent to 25 mg of cefpodoxime to a 50 ml volumetric flask, add 30 ml of methanol R and shake to dissolve. Dilute to volume with methanol R. Dilue this solution quantitatively with the medium to obtain a solution having the same concentration of cefpodoxime as the test solution. Measure the absorbances (Appendix 4.1) of the test solution and the reference solution at the maximum at about 259 nm, in a 1 cm cell, using the medium as a blank. Tolerance: Not less than 70% (Q) of the stated amount of cefpodoxime, C15H17N5O6S2, is dissolved in 30 min.

211

VP V

CEFPODOXIME POWDER FOR ORAL SUSPENSION

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: 0.02 M ammonium acetate - acetonitrile (6 : 4). Make adjustments if necessary. Diluent: Water - acetonitrile (6 : 4). Reference solution: Transfer an accurately weighed quantity of cefpodoxime proxetil RS, equivalent to 50 mg of cefpodoxime, to a 100 ml volumetric flask, add 10 ml of methanol R, dissolve by sonicating for 5 min. Dilute to volume with the diluent and mix. Dilute 5.0 ml of this solution to 100.0 ml with the diluent. Test solution: Weigh 20 capsules, calculate the average weight of the capsule contents and finely powder. Transfer an accurately weighed quantity of the powder containing the equivalent of 50 mg of cefpodoxime to a 100 ml volumetric flask, add 70 ml of the diluent, dissolve by sonicating for 10 min. Dilute to volume with the same solvent, mix well and filter. Dilute 5.0 ml of the filtrate to 100.0 ml with the same solvent. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 μm). Column temperature: 30 °C. Detector: A spectrophotometer set at 235 nm. Flow rate: 2.0 ml/min. Volume of injection: 20 μl. Procedure: System suitability: Inject the reference solution. In the obtained chromatogram, the relative retention times for cefpodoxime proxetil S-epimer peak and cefpodoxime proxetil R-epimer peak are about 0.9 and 1.0, respectively and the resolution factor between those peaks is at least 2.5. The symmetry factor of the peak due to cefpodoxime proxetil R-epimer is not more than 1.5. The relative standard deviation of the sum of the peak areas due to cefpodoxime proxetil S-epimer and cefpodoxime proxetil R-epimer for six replicate injections is not more than 2.0%. Inject separately the reference solution and the test solution. Calculate the contents of cefpodoxime, C15H17N5O6S2, in the capsules using the sums of the peak responses for cefpodoxime proxetil S-epimer and cefpodoxime proxetil R-epimer in the chromatograms obtained with the test solution and the reference solution and the declared content of C15H17N5O6S2 in cefpodoxime proxetil RS. Storage Store in an airtight container, in a cool and dry place, protected from light. Action and use Cephalosporin antibacterial. Usual strength 100 mg; 200 mg of cefpodoxime.

212

CEFPODOXIME POWDER FOR ORALSUSPENSION Pulveres Cefpodoximi ad suspensionum peroralum Cefpodoxime powder for oral suspension contains cefpodoxime proxetil. Suitable excipients such as flavors, colors, preservatives, stabilizing agents … may be added. The constituted suspension prepared as directed on the label complies with the requirements stated under “Suspension” (Appendix 1.5). The powder complies with the requirements stated under “Powder” (Appendix 1.7) and with the following requirements:

Content of cefpodoxime, C15H17N5O6S2, 90.0% to 110.0% of the stated amount. Characters Powder should be loose and dry, with homogeneous colour. Identification In the Assay, the retention times of two principal peaks in the chromatogram obtained with the test solution correspond to those of the peaks due to cefpodoxime proxetil S-epimer và cefpodoxime proxetil R-epimer in the chromatogram obtained with the reference solution. Water Not more than 1.5% (Appendix 10.3). Determined on 1.0 g. pH 4.0 to 5.5 (Appendix 6.2). Use a suspension constituted as directed on the label. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: 0.02 M ammonium acetate - acetonitrile (6 : 4). Make adjustments if necessary. Diluent: Water - acetonitrile (6 : 4). Reference solution: Transfer an accurately weighed quantity of cefpodoxime proxetil RS containing the equivalent of about 50 mg of cefpodoxime to a 100 ml volumetric flask, add 10 ml of methanol R, dissolve by sonicating for 5 min. Dilute to volume with the diluent and mix. Dilute 5.0 ml of this solution to 100.0 ml with the diluent. Test solution: For single-dose preparations: Use the powder obtained from the test for Uniformity of mass and finely powder. Transfer an accurately weighed quantity of the powder containing the equivalent of about 100 mg of cefpodoxime to a 200 ml volumetric flask, add 20 ml of water, shake to disperse, add 40 ml of acetonitrile R, sonicate for 15 min, allow to cool. Dilute to volume with the diluent and filter. Dilute 5.0 ml of the filtrate to 100.0 ml with the diluent. For multi-dose preparations: Constitute the content of a container directed on the label, shake thoroughly. Determine the density of the resulting suspension (Appendix 6.5).

VP V

Transfer an accurately weighed quantity of the suspension containing the equivalent of about 100 mg of cefpodoxime, to a 200 ml volumetric flask. Add 20 ml of water and 20 ml of acetonitrile R, sonicate for 15 min, allow to cool and dilute to volume with the diluent, mix well and filter. Dilute 5.0 ml of the filtrate to 100.0 ml with the diluent. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 μm). Column temperature: 30 °C. Detector: A spectrophotometer set at 235 nm. Flow rate: 2.0 ml/min. Volume of injection: 20 μl. Procedure: System suitability: Inject the reference solution. In the obtained chromatogram, the relative retention times for cefpodoxime proxetil S-epimer peak and cefpodoxime proxetil R-epimer peak are about 0.9 and 1.0, respectively and the resolution factor between those peaks is not less than 2.5. The symmetry factor of the peak due to cefpodoxime proxetil R-epimer is not more than 1.5. The relative standard deviation of the sum of the peak areas due to cefpodoxime proxetil S-epimer and cefpodoxime proxetil  R-epimer for 6 replicate injections is not more than 2.0%. Inject separately the reference solution and the test solution. Calculate the contents of cefpodoxime, C15H17N5O6S2, in the tablets using the sums of the peak areas due to cefpodoxime proxetil  S-epimer and cefpodoxime proxetil R-epimer in the chromatograms obtained with the test solution and the reference solution, the declared content of C15H17N5O6S2 in cefpodoxime proxetil RS and the density of the suspension (for multi-dose preparations).

Storage Store the powderin an airtight container, in a cool and dry place, protected from light. Store the constituted suspension at a temperature and within the period as stated on the label. Labelling The label states the direction for the constitution of the powder and the equivelent amount of cefpodoxim (C15H17N5O6S2) in a given volume of the constituted suspension (for multidose preparations). Action and use Cephalosporin antibacterial. Usual strength Single-dose preparations: 20 mg; 100 mg of cefpodoxime. Multi-dose preparations: 50 mg/5 ml of cefpodoxime.

CEFPODOXIME TABLETS

CEFPODOXIME TABLETS Tabellae cefpodoximi Cefpodoxime tablets contain cefpodoxime proxetil. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of cefpodoxime, C15H17N5O6S2, 90.0% to 110.0% of the stated amount. Identification In the Assay, the retention times of the two principal peaks in the chromatogram obtained with the test solution correspond to those of the cefpodoxime proxetil R-epimer peak and cefpodoxime proxetil  S-epimer peak in the chromatogram obtained with the reference solution. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: Transfer 54.5 g of glycine R and 42.6 g of sodium chloride R in a 1000 ml volumetric flask. Add about 500 ml of water, shake to dissolve. Cautiously add, with swirling, 14.2 ml of hydrochloric acid R, and allow to cool. Dilute with water to volume, and mix. Dilute 50 ml of this solution to 900 ml with water to obtain a solution having a pH of 3.0 ± 0.1 [Adjust the pH of the medium with 1 M sodium hydroxide R, if necessary]. Rotation speed: 75 rpm. Time: 30 min. Procedure: Test solution: After the specified time (30 min), withdraw a sample of the medium, filter. Dilute the filtrate with the medium, if necessary. Reference solution: Transfer an accurately weighed quantity of cefpodoxime proxetil RS, equivalent to 25 mg of cefpodoxime to a 50 ml volumetric flask, add 30 ml of methanol R and shake to dissolve. Dilute to volume with methanol R. Dilue this solution quantitatively with the medium to obtain a solution having the same concentration of cefpodoxime to that in the test solution. Measure the absorbance (Appendix 4.1) of the test solution and the reference solution at the maximum at about 259 nm, in a 1 cm cell, using the medium as a blank. Calculate the content of cefpodoxime, C15H17N5O6S2, dissolved using the absorbance of the test solution, the reference solution and the declared content of C15H17N5O6S2 in cefpodoxime proxetil RS. Tolerance: Not less than 70% (Q) of the stated amount of cefpodoxime, C15H17N5O6S2, is dissolved in 30 minutes. Water Not more than 5.0% (Appendix 10.3). Assay Examine by liquid chromatography (Appendix 5.3). 213

VP V

CEFRADINE

Mobile phase: 0.02 M ammonium acetate - acetonitrile (6 : 4). Make adjustments if necessary. Diluent: Water - acetonitrile (6 : 4). Reference solution: Transfer an accurately weighed quantity of about 60 mg of cefpodoxime proxetil RS to a 100 ml volumetric flask, add 10 ml of methanol R, dissolve by sonicating for 5 min. Dilute to volume with the diluent and mix. Dilute 5.0 ml of this solution to 100.0 ml with the diluent. Pass through a 0.45 µm filter. Test solution: Weigh 20 tablets (with the coating layer removed, if necessary), calculate the average mass and powder finely. Transfer an accurately weighed quantity of the powdered tablets containing the equivalent of about 50 mg of cefpodoxime to a 100 ml volumetric flask, add 70 ml of the diluent, dissolve by sonicating for 10 min. Dilute to volume with the same solvent and filter. Dilute 5.0 ml of the filtrate to 100.0 ml with the same solvent. Pass through a 0.45 µm filter. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 μm), maintained at 30 °C. Detector: A spectrophotometer set at 235 nm. Flow rate: 2.0 ml/min. Volume of injection: 20 μl. Procedure: System suitability: Inject the reference solution. In the obtained chromatogram, the relative retention times for cefpodoxime proxetil S-epimer peak and cefpodoxime proxetil R-epimer peak are about 0.9 and 1.0, respectively. The test is not valid unless the resolution factor between those peaks is at least 2.5. The symmetry factor of the peak due to cefpodoxime proxetil  R-epimer is not more than 2.0. The relative standard deviation of the sum of the peak areas due to cefpodoxime proxetil S-epimer and cefpodoxime proxetil R-epimer for replicate injections is not more than 2.0%. Inject separately the reference solution and the test solution. Calculate the content of cefpodoxime, C15H17N5O6S2, in the tablets using the sum of the peak areas due to cefpodoxime proxetil  S-epimer and cefpodoxime proxetil R-epimer in the chromatograms obtained with the test solution and the reference solution and the declared content of C15H17N5O6S2 in cefpodoxime proxetil RS.

Storage Store in an airtight container, in a cool and dry place, protected from light. Action and use Cephalosporin antibacterial. Usual strength 100 mg; 200 mg of cefpodoxime 214

CEFRADINE Cefradinum

Cefradine with main component is (6R,7R)-7-[[(2R)amino(cyclohexa-1,4-dienyl)acetyl]amino]-3-methyl8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (cefradine), semi-synthetic product derived from a fermentation product. It contains not less than 90.0% of cefradine and not more than 5.0% of cefalexin, not more than 2.0% of 4′,5′-dihydrocefradine, calculated with reference to the anhydrous substance; The sum of the percentage contents of cefradine, cefalexin and 4′,5′-dihydrocefradine is not less than 95.0% and not more than 102.0%, calculated with reference to the anhydrous substance.

Characters A white or slightly yellow, hygroscopic powder. Sparingly soluble in water, practically insoluble in ethanol (96%) and in hexane. Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of cefadine RS. If the spectra obtained in the solid state show differences, dissolve 30 mg of the substance to be examined and 30 mg of the reference substance separately in 10 ml of methanol R, evaporate to dryness at 40 °C at a pressure less than 2 kPa and record new spectra using the residues. Appearance of solution Solution S: Dissolve 2.5 g in sodium carbonate solution R and dilute to 25.0 ml with the same solvent. Solution S is not more opalescent than reference suspension II  (Appendix 9.2). Allow solution S to stand for 5 min. The absorbance (Appendix 4.1) of solution S measured at 450 nm is not greater than 0.60. pH Dissolve 0.100 g in carbon dioxide-free water R and dilute to 10 ml with the same solvent. The pH (Appendix 6.2) of the solution is 3.5 to 6.0.

VP V

CEFRADINE

Specific optical rotation +80° to +90°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.250  g of the substance to be examined in acetate buffer solution pH 4.6 R and dilute to 25.0 ml with the same solvent. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: A 0.272  % solution of  potassium dihydrogen phosphate  R adjusted to pH 3.0 with 2 M phosphoric acid R; Mobile phase B: Methanol R2. Test solution:  Dissolve 0.300 g of the substance to be examined in mobile phase A and dilute to 50.0 ml with the same solvent. Reference solution (1):  Dissolve 3.0  mg of  cyclohexa1,4-dienylglycine RS (impurity B) in mobile phase A and dilute to 100.0 ml with the same solvent. Reference solution (2): Dissolve 3 mg of the substance to be examined and 3 mg of cefalexin RS in mobile phase A and dilute to 25 ml with the same solvent. Reference solution (3): Dilute 1.0 ml of the test solution to 100.0 ml with mobile phase A. Reference solution (4): Dissolve 6  mg of  cefradine for peak identification RS (containing impurities C, D and E) in 1.0 ml of mobile phase A. Reference solution (5): Dissolve the contents of a vial of cefradine impurity mixture RS (impurities A and G) in 1.0 ml of mobile phase A. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 µm). Column temperature: 30 oC. Detector: A spectrophotometer set at 220 nm. Flow rate: 1.0  ml/min. Volume of injection: 25 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 2.5

99.5 → 97

0.5 → 3

2.5 - 11

97 → 75

3 → 25

11 - 13

75 → 60

25 → 40

13 - 16

60

40

16 - 19

60 → 20

40 → 80

19 - 19.1

20 → 99.5

80 → 0.5

19.1 - 25

99.5

0.5

Identification of impurities:  Use the chromatogram supplied with cefradine for peak identification RS and the chromatogram obtained with reference solution (4) to identify the peaks due to impurities C, D and E. Use

the chromatogram obtained with reference solution (1) to identify the peak due to impurity B; use the chromatogram supplied with cefradine impurity mixture RS and the chromatogram obtained with reference solution (5) to identify the peaks due to impurities A and G. Relative retention with reference to cefradine (retention time = about 15 min): Impurity A = about 0.27; impurity B = about 0.32; impurity C = about 0.53; impurity D = about 0.63; impurity E = about 0.80; impurity F = about 0.92; cefalexin  =  about 0.95; 4′,5′-dihydrocefradine  =  about 1.06; impurity G = about 1.32. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to cefalexin and cefradine is at least 4.0. Limits: Correction factor: For the calculation of content, multiply the peak area of impurity B by 3.4. Impurities A, B, C, D, E, F, G: For each impurity, the peak area, corrected if necessary, is not more than 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (3) (0.25%). Any other impurity: The area of each impurity peak is not more than 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (3) (0.25%). Sum of the areas of all the impurity peaks is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (3) (2.0%). Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (3) (0.05%), disregard the peaks due to cefalexin and 4′,5′-dihydrocefradine. Notes: Impurity A: (6R,7R)-7-amino-3-methyl-8-oxo-5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (7-aminodeacetoxycephalosporanic acid, 7-ADCA). Impurity B: (2R)-amino(cyclohexa-1,4-dienyl)acetic acid (D-dihydrophenylglycine, cyclohexa-1,4-dienylglycine), Imputity C: (6R,7R)-7-[[(2R)-amino(cyclohexa-1,4-dienyl) acetyl]amino]-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2ene-2-carboxylic acid 5-oxide (isomer 1). Impurity D: (6R,7R)-7-[[(2R)-amino(cyclohexa-1,4-dienyl) acetyl]amino]-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2ene-2-carboxylic acid 5-oxide (isomer 2). Impurity E: (6R,7R)-7-[[(2R)-amino(2-hydroxyphenyl)acetyl] amino]-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2carboxylic acid. Impurity F: 3-hydroxy-4-methylthiophen-2(5H)-one. Impurity G: (6R,7R)-7-[(2,2-dimethylpropanoyl)amino]-3-methyl8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (7ADCA pivalamide).

N,N-Dimethylaniline Not more than 20 ppm (Appendix 10.16, method 2). Water Not more than 6.0% (Appendix 10.3). Determined on 0.300 g. 215

VP V

CEFRADINE CAPSULES

Sulfated ash Not more than 0.2% (Appendix 9.9, method 2). Determined on 1.0 g.

Preparations Capsules, oral suspensions.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol -  phosphate buffer solution pH 5.0  (25 : 75 ). Test solution:  Dissolve 50.0 mg of the substance to be examined in phosphate buffer solution pH 5.0 R and dilute to 100.0 ml with the same solvent. Reference solution (1):  Dissolve 50.0  mg of  cefradine RS  (containing 4′,5′-dihydrocefradine) in  phosphate buffer solution pH 5.0 R and dilute to 100.0 ml with the same solvent. Reference solution (2): Dissolve 5.0  mg of  cefalexin RS in phosphate buffer solution pH 5.0  R and dilute to 100.0 ml with the same solvent. Reference solution (3): Dilute 1 ml of reference solution (1) to 10 ml with phosphate buffer solution pH 5.0 R. Mix 5 ml of this solution and 5 ml of reference solution (2). Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.5  ml per minute. Volume of injection: 5 µl. Procedure: The run time is twice the retention time of cefradine. Relative retention with reference to cefradine (retention time  =  about 3 min): Cefalexin  =  about 0.7; 4′,5′-dihydrocefradine = about 1.5. System suitability: In the chromatogram obtained with reference solution (3), the resolution between the peaks due to cefalexin and cefradine is at least 4.0. Calculate the percentage content of cefradine in the substance to be examined, using the areas of peak due to cefradine in the chromatograms obtained with the test solution and reference solution (1), and the declared content of cefradine in cefradine RS. Calculate the percentage content of cefalexin in the substance to be examined, using the areas of peak due to cefalexin in the chromatograms obtained with the test solution, reference solution (2) and the declared content of cefalexin in cefalexin RS. Calculate the percentage content of 4′,5′-dihydrocefradine in the substance to be examined, using the area of 4′,5′-dihydrocefradine peak in the chromatograms obtained with the test solution and cefalexin peak obtained from reference solution (2), and multiplying the area of the peak due to 4′,5′-dihydrocefradine by a correction factor of 1.6.

CEFRADINE CAPSULES Capsulae Cefradini

Storage Store in an airtight container, protected from light, at a temperature of 2 °C to 8 °C. Action and use Cephalosporin antibacterial 216

Cefradine capsules contain cefradine. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of cephalosporins, calculated as the sum of cefradine, C16H19N3O4S, and cefalexin, C16H17N3O4S, 90.0% to 110.0% of the stated amount of cefradine. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: 0.25 mm layer of binder-free silica gel. Place the plate in a chamber containing a mixture of n-hexane and tetradecane (95 : 5) to a depth of about 1 cm, allow the solvent front to move the length of the plate, remove the plate from the chamber and allow the solvent to evaporate. Mobile phase: 0.1 M citric acid solution - 0.1 M disodium hydrogen phosphate solution - a solution of ninhydrin in acetone R containing 1 g per 15 ml (60 : 40 : 1.5). Test solution: Dissolve a quantity of the contents of the capsules containing the equivalent of 30 mg of cefradine with 10 ml of water, filter. Reference solution: A 0.3% solution of cefradine RS in water. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of about 10 cm. After removal of the plate, allow it to dry in air and heat it at 110 °C for 10 min. Examine in day light. The principal spot in the chromatogram obtained with the test solution corresponds in position, colour and size to that in the chromatogram obtained with the reference solution. B. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the principal peak in the chromatogram obtained with the reference solution. Loss on drying Not more than 7.0% (Appendix 9.6). Weigh accurately about 100 mg of the powdered capsules, dry in vacuo (5 mm Hg) at 60 °C for 3 hours. Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 900 ml of 0.12 M hydrochloric acid R. Rotation speed: 100 rpm. Time: 45 min. Procedure: After the specified time, withdraw a sample of the medium, filter, discard the first 20 ml of the filtrate.

VP V

Dilute the filtrate with medium to contain a suitable concentration (if necessary). Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum at 255 nm, in a 1 cm cell, using the medium as a blank. Calculate the content of cephalosporins (sum of cefradine, C16H19N3O4S, and cefalexin, C16H17N3O4S), dissolved in each capsules in comparison with a reference solution of cefradine RS having the same concentration in the same medium. Tolerance: Not less than 75% (Q) of the stated amount of cefradine, calculated as the sum of cefradine and cephalexin, is dissolved in 45 min.

Cephalexin Not more than 10.0% of the sum of cefradine, C16H19N3O4S, and cefalexin, C16H17N3O4S. Calculate the content of cephalexin, C16H17N3O4S, in the capsules using cefalexin and cefradine peak area obtained with the test solution in the assay. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Water - methanol - 0.5 M sodium acetate 0.7 M acetic acid (782 : 200 : 15 : 3). Reference solution: A 0.05% solution of cefradine RS in the mobile phase. Test solution: Weigh 20 capsules, calculate the average weight of the capsule contents, powder finely and mix well. Transfer an accurately weighed quantity of the contents of the capsules containing the equivalent of about 50 mg of cefradine to a 100 ml volumetric flask, add about 70 ml of the mobile phase and sonicate for 15 minutes. Dilute to volume with the mobile phase, mix well and filter. Resolution solution: Dissolve two quantities of cefradine RS and cefalexin RS in the mobile phase to obtain a solution containing about 0.5 mg of each substance/ml. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm to 10 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject the resolution solution. The relative retention times are 0.8 for cefalexin and 1.0 for cefradine; the resolution between cefradine peak and cefalexin peak is not less than 2.0. Inject the reference solution: the relative standard deviation of cefradin peak areas for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of cephalosporins using the sum of the cefradine and cefalexin peak areas in the chromatograms obtained with the test solution, the reference solution and the declared total contents of cefradine, C16H19N3O4S, and cefalexin, C16H17N3O4S, in cefradine RS.

CEFTAZIDIME PENTAHYDRATE

Storage Store in a well-closed container, in a cool and dry place, protected from light, at a temperature not exceeding 30 °C. Action and use Cephalosporin antibacterial. Usual strength 250 mg; 500 mg. CEFTAZIDIME PENTAHYDRATE Ceftazidium pentahydricum

C22H22N6O7S2,5H2O

M. 637

Ceftazidime pentahydrate is (6R,7R)-7-[[(2Z)-2-(2aminothiazol-4-yl)-2-[(1-carboxy-1- methylethoxy)imino] acetyl]amino]-8-oxo-3-[(pyridin-1-ium-1-yl)methyl]5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate pentahydrate. It contains not less than 95.0% and not more than 102.0% of C22H22N6O7S2, calculated with reference to the anhydrous substance.

Characters A white or almost white, crystalline powder. Slightly soluble in water and in methanol, practically insoluble in acetone and in ethanol (96%). It dissolves in acid and alkali solutions. Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum obtained with ceftazidime RS. Appearance of solution Solution S: Dissolve 0.25 g in carbon dioxide-free water R and dilute to 50.0 ml with the same solvent. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). pH The pH of solution S is 3.0 to 4.0 (Appendix 6.1). Related substances A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel F254. Mobile phase: Butanol - sodium acetate buffer solution pH 4.5 - butyl acetate - glacial acetic acid (6 : 26 : 32 : 32). 217

VP V

CEFTAZIDIME PENTAHYDRATE

Test solution: Dissolve 0.100 g of the substance to be examined in a 3.6% solution of disodium hydrogen phosphate R and dilute to 2.0 ml with the same solution. Reference solution: Dilute 1.0 ml of the test solution to 200 ml with a 3.6% solution of disodium hydrogen phosphate R. Procedure: Apply separately to the plate 2 µl of each solution. Develop over a path of 15 cm. Dry the plate in a current of warm air and examine in ultraviolet light at 254 nm. Any spot with an Rf value greater than that of the principal spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution (0.5%). B. Examine by liquid chromatography (Appendix 5.3) Mobile phase: Mixture of acetonitrile - 2.26% solution of ammonium dihydrogen phosphate R (7 : 93), adjusted to pH 3.9 with a 10% solution of phosphoric acid R. Test solution: Dissolve 0.100 g of the substance to be examined in the mobile phase and dilute to 20.0 ml with the mobile phase. Dilute 5.0 ml of the solution to 20.0 ml with the mobile phase. Reference solution: Dissolve 5.0 mg of ceftazidime impurity A RS in the mobile phase and dilute to 20.0 ml with the mobile phase. Dilute 1.0 ml of the solution to 20.0 ml with the mobile phase. Resolution solution: Dissolve 5.0 mg of ceftazidime impurity A RS and 5.0 mg of ceftazidime RS in the mobile phase and dilute to 20.0 ml with the mobile phase. Dilute 1.0 ml of the solution to 20.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Column temperature: 35 °C. Detector: A spectrophotometer set at 255 nm. Flow rate: 1.3 ml/min. Volume of injection: 20 µl. Procedure: Inject the resolution solution, adjust the sensitivity of the system so that the heights of the 2 peaks in the chromatogram obtained are at least 50% of the full scale of the recorder. The test is not valid unless the resolution between the peaks corresponding to ceftazidime and impurity A is at least 5.9. Inject the test solution and reference solution. Allow the chromatography of the test solution to procees for 3 times the retention time of ceftazidime. The area of any secondary peak in the chromatogram obtained with the test solution is not greater than half the area of the principal peak in the chromatogram obtained with the reference solution (0.5%). The sum of the areas of all the peaks, apart from the principal peak is not greater than 2 times the area of the principal peak in the chromatogram obtained with the reference solution (2.0%). Disregard any peak with an area less than 10% the area of the principal peak in the chromatogram obtained with the reference solution. Note: Impurity A: (2RS,6R,7R)-7-[[(2Z)-2-(2-aminothiazol-4-yl)-2-[(1carboxy-1-methylethoxy)imino]acetyl]amino]-8-oxo-3-[(pyridin1-ium-1-yl)methyl]-5-thia-1-azabicyclo[4.2.0]oct-3-ene-2carboxylate (Δ-2-ceftazidime).

218

Pyridine Not more than 0.05%. Examine by liquid chromatography (Appendix 5.3) Mobile phase: 2.88% solution of ammonium dihydrogen phosphate previously adjusted to pH 7.0 with amonia acetonitrile - water (8 : 24 : 68). Prepare the following solutions immediately before use. Test solution: Dissolve 0.500 g of the substance to be examined in the mixture of 10 volumes of phosphate buffer solution pH 7.0 R4 and 90 volumes of water (mixture A). Dilute to 100.0 ml with the same mixture. Reference solution: Dissolve 1.0 g pyridine R in water and dilute to 100.0 ml with the same solvent. Dilute 5.0 ml of the solution to 200.0 ml with water. To 1.0 ml of the solution, add 10 ml of phosphate buffer solution pH 7.0 R4 and dilute to 100.0 ml with water. Resolution solution: Dilute 1.0 ml of the test solution to 200.0 ml with mixture A. To 1.0 ml of the solution, add 20 ml of the reference solution and dilute to 200.0 ml with mixture A. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 255 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Inject the resolution solution, the test is not valid unless, in the chromatogram obtained, the resolution between the peaks due to ceftazidime and pyridine is at least 7.0. Inject the reference solution, adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained is at least 50% of the full scale of the recorder. Inject alternately the test solution and reference solution, the area of any peak corresponding to pyridine in the chromatogram obtained with the test solution is not greater than the area of the principal peak in the chromatogram obtained with the reference solution. Water 13.0% to 15.0% (Appendix 10.3). Determined on 0.200 g. Bacterial endotoxins Less than 0.10 EU/mg, if intended for use in the manufacture of parenteral preparations without a further appropriate procedure for the removal of bacterial endotoxins (Appendix 13.2). Assay Examine by liquid chromatography (Appendix 5.3) Mobile phase: Dissolve 4.26 g of disodium hydrogen phosphate R and 2.73 g of potassium dihydrogen phosphate R in 980 ml of water, then add 20 ml of acetonitrile R.

VP V

Test solution: Dissolve 25.0 mg of the substance to be examined in the mobile phase and dilute to 25.0 ml with the mobile phase. Reference solution: Dissolve 25.0 mg of ceftazidime RS in the mobile phase and dilute to 25.0 ml with the mobile phase. Resolution solution: Dissolve 5.0 mg of ceftazidime impurity A RS in 5.0 ml with the reference solution. Chromatographic system: A column (15 cm × 4.6 mm) packed with hexylsilyl silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 245 nm. Flow rate: 2.0 ml/min. Volume of injection: 20 µl. Procedure: Inject the resolution solution, adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained is at least 50% of the full scale of the recorder. The test is not valid unless in the chromatogram obtained, the resolution between the peaks corresponding to ceftazidime and impurity A is at least 1.5. Inject alternately the test solution and reference solution. Calculate the content of ceftazidime (C22H22N6O7S2) using the area of the peaks in the chromatograms obtained with the test solution, the reference solution and the declared content of C22H22N6O7S2 in ceftazidime RS.

Storage Storage in an airtight container. If the substance is sterile, store in a sterile, airtight, tamper-proof container. Action and use Cephalosporin antibacterial. Preparation Injection. CEFTAZIDIME FOR INJECTION Ceftazidimi pulvis ad injectionem Ceftazidime for injection is a sterile mixture of ceftazidime pentahydrate and anhydrous sodium carbonate. It is supplied in a sealed glass container. The powder complies with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Content of ceftazidime, C22H22N6O7S2, 90.0% to 105.0% of the stated amount, calculated with reference to the dried and sodium carbonate-free basis. Content of sodium carbonate, Na2CO3, 8.0% to 10.0%. Characters A white or yellowish powder.

CEFTAZIDIME FOR INJECTION

Identification A. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that of the ceftazidime peak in the chromatogram obtained with the reference solution. B. It gives the reaction (A) of carbonate (Appendix 8.1). pH 5.0 to 7.5 (Appendix 6.2), determined on a solution of the powder in carbon dioxide-free water R containing 10% of ceftazidime. Clarity of solution A solution of the powder in carbon dioxide-free water R containing 10% of ceftazidime is clear (Appendix 9.2). Loss on drying Not more than 13.5% (Appendix 9.6). Use 0.3 g of the powder, dry in vacuum at a pressure not exceeding 0.67 kPa at 25 °C for 4 h; then heat the residue in vacuum at a pressure not exceeding 0.67 kPa at 100 °C for 3 h. Bacterial endotoxins Not more than 0.10 EU per mg of ceftazidime (Appendix 13.2). Pyridin Examine by liquid chromatography (Appendix 5.3). Mobile phase: A 2.88% solution of ammonium dihydrogen phosphate adjusted to pH 7.0 with ammonia - acetonitrile - water (8 : 24 : 68). Prepare following solutions immediately before use. Diluent: A mixture of 10 volumes of phosphate buffer pH 7.0 R4 and 90 volumes of water. Test solution: Disperse a quantity of the powder containing the equivalent to 0.5 g of ceftazidime, with 10 ml of the diluent and dilute to 100.0 ml with the same solvent. Reference solution: Transfer 1.0 ml of a solution containing 0.025% of pyridin to a 100 ml volumetric flask, add 10.0 ml of phosphate buffer pH 7.0 R4 and dilute to volume with water. Resolution solution: Dilute 1.0 ml of the test solution to 200.0 ml with the diluent. To 1.0 ml of the resulting solution, add 20.0 ml of the reference solution and dilute to 200 ml with the diluent. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 μm). Detector: A spectrophotometer set at 255 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 μl. Procedure: Inject the resolution solution, the test is not valid unless the resolution factor between the peaks due to ceftazidime and pyridin is at least 7.0. Inject the reference solution. Adjust the sensitivity of the system so that the height of the principal peak in the 219

VP V

CEFTRIAXONE SODIUM

chromatogram obtained is not less than 50% of the full scale of the recorder. Inject alternately the reference solution and the test solution. The area of the peak corresponding to pyridin in the chromatogram obtained with the test solution is not greater than 10 times the area of the pyridin peak in the chromatogram obtained with the reference solution (0.5%).

Sodium carbonate Examine by atomic absorption spectrophotometry (Appendix 4.4, method 1). Sodium stock solution: A solution contains 1000 μg of sodium per ml. Potassium chloride solution (4%): Dissolve 4 g of potassium chloride R in water to produce 100.0 ml. Reference solutions of sodium: Dilute 10.0 ml of sodium stock solution (1000 μg/ml) to 100.0 ml with water to obtain a solution having a known concentration of 100 μg of sodium per ml. Prepare the sodium reference solutions with concentrations 2.0, 4.0, 6.0 and 8.0 μg of sodium per ml as directed in the table below: Concentration of sodium (µg/ml)

Volume of sodium reference solution (100 μg/ml) (ml)

Volume of potassium chloride solution (4%) (ml)

Sufficient water to produce (ml)

0 (blank)

0

10

100

2.0

2.0

10

100

4.0

4.0

10

100

6.0

6.0

10

100

8.0

8.0

10

100

Test solution: Transfer an accurately weighed quantity of the powder containing the equivalent to 100 mg of ceftazidime, to a 100 ml volumetric flask, add 30 ml of water, shake to dissolve and dilute to volume with water, mix well (solution A). Dilute solution A with water to obtain a solution contaning about 4 µg/ml of sodium. Add 4% potassium chloride solution to the final solution with the ratio of 1 : 10. Procedure: Use atomic absorption spectrophotometer with a sodium hollow-cathode lamp and an air-acetylene flame. Measure the absorbances of the reference solutions and the test solution at 589.0 nm. From the absorbances of the reference solutions and the test solution, plot a calibration curve representing the correlation between the absorbance and the concentration of sodium. Calculate the concentration of sodium in the test solution from the calibration curve. 1 mg of sodium equivalent to 2.305 mg of sodium carbonate, Na2CO 3.

Assay Examine by liquid chromatography (Appendix 5.3). 220

Mobile phase: Dissolve 4.26 g of disodium hydrogen phosphate R and 2.73 g of potassium dihydrogen phosphate R in 980 ml of water, add 20 ml of acetonitrile R. Test solution: Disperse an accurately weighed quantity of the powder containing the equivalent to 100 mg of ceftazidime with 10 ml of the mobile phase, dilute to 100.0 ml with the same solvent. Reference solution: A 0.1% solution of ceftazidime RS in the mobile phase. Resolution solution: Dissolve 5.0 mg of ceftazidime related compound A in 5.0 ml of the reference solution. Chromatographic system: A column (15 cm × 4.6 mm) packed with hexylsilyl silica gel for chromatography (5 μm). Detector: A spectrophotometer set at 245 nm. Flow rate: 2.0 ml/min. Volume of injection: 20 μl. Procedure: System suitability: Inject the resolution solution, adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained is not less than 50% of the full scale of the recorder. The test is not valid unless the resolution factor between the peaks due to ceftazidime and ceftazidime related compound A is at least 1.5. Inject the reference solution, the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of ceftazidime, C22H22N6O7S2, in the injection using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C22H22N6O7S2 in ceftazidime RS.

Storage Store in a dry place, protected from light and at a temparature not exeeding 30 °C. Action and use Cephalosporin antibacterial. Usual strength 500 mg; 1 g of ceftazidime. CEFTRIAXONE SODIUM Ceftriaxonum natricum

, C18H16N8Na2O7S3,3½H2O

M. 662

VP V

Ceftriaxone sodium is disodium (6R,7R)-7-[[(2Z)-(2aminothiazol-4-yl)(methoxyimino)acetyl]amino]-3-[[(2methyl-6-oxido-5-oxo-2,5-dihydro-1,2,4-triazin-3-yl) sulfanyl]methyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct2-ene-2-carboxylate 3.5 hydrate, semi-synthetic product derived from a fermentation product. It contains not less than 96.0% and not more than 102.0% of C18H16N8Na2O7S3, calculated with reference to the anhydrous substance.

Characters A almost white or yellowish crystalline powder, slightly hygroscopic. Freely soluble in water, sparingly soluble in methanol, very slightly soluble in anhydrous ethanol. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of ceftriaxone sodium RS. B. It gives reaction (A) of sodium (Appendix 8.1). Appearance of solution Solution S: Dissolve 2.40 g of the substance to be examined in carbon dioxide-free water R and dilute to 20.0 ml with the same solvent. Dilute 2 ml of solution S to 20 ml with water. The solution is clear (Appendix 9.2) and not more intensely coloured than reference solution Y5 or BY5 (Appendix 9.3). pH 6.0 to 8.0 (Appendix 6.2). Determined on the solution S. Specific optical rotation -155° to -170°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.250 g in water and dilute to 25.0 ml with the same solvent. Related substances Examine by liquid chromatography (Appendix 5.3). Citrate buffer solution pH 5.0: Dissolve 20.17 g of citric acid R in 800 ml of water, adjust to pH 5.0 with a 42% solution of sodium hydroxide  R and dilute to 1000.0 ml with water. Mobile phase: Dissolve 2.0  g of  tetradecylammonium bromide R and 2.0 g of tetraheptylammonium bromide R in a mixture of 440 ml of water, 55 ml of 0.067 M phosphate buffer solution pH 7.00 R, 5.0 ml of citrate buffer solution pH 5.0 and 500 ml of acetonitrile R. Test solution:  Dissolve 30.0  mg of the substance to be examined in the mobile phase and dilute to 100.0 ml with the same solvent. Reference solution (1):  Dissolve 30.0  mg of ceftriaxone sodium RS in the mobile phase and dilute to 100.0 ml with the same solvent. Reference solution (2): Dissolve 5.0  mg of  ceftriaxone sodium RS and 5.0 mg of ceftriaxone impurity A RS in the mobile phase and dilute to 100.0 ml with the same solvent.

CEFTRIAXONE SODIUM

Reference solution (3): Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.5  ml/min. Volume of injection: 20 µl. Procedure: Inject the test solution and reference solutions (2), (3). The run time is twice the retention time of ceftriaxone. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to ceftriaxone and impurity A is at least 3.0. Limits: Any impurity: The area of each impurity peak is not more than the area of the principal peak in the chromatogram obtained with reference solution (3) (1.0%). Sum of the areas of all the impurity peaks is not more than 4 times the area of the principal peak in the chromatogram obtained with reference solution (3) (4.0%). Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (3) (0.1%).

Notes: Impurity A: (6R,7R)-7-[[(2E)-(2-aminothiazol-4-yl)(methoxyimino) acetyl]amino]-3-[[(2-methyl-5,6-dioxo-1,2,5,6-tetrahydro-1,2,4triazin-3-yl)sulfanyl]methyl]-8-oxo-5-thia-1-azabicyclo[4.2.0] oct-2-ene-2-carboxylic acid ((E)-isomer). Impurity B: (5aR,6R)-6-[[(2Z)-(2-aminothiazol-4-yl)(methoxyimino) acetyl]amino]-5a,6-dihydro-3H,7H-azeto[2,1-b]furo[3,4-d][1,3] thiazine-1,7(4H)-dione. Imputity C: 2-methyl-3-sulfanyl-1,2-dihydro-1,2,4-triazine-5,6dione. Impurity D: S-benzothiazol-2-yl (2Z)-(2-aminothiazol-4-yl) (methoxyimino)thioacetate. Impurity E: (6R,7R)-7-amino-3-[[(2-methyl-5,6-dioxo-1,2,5,6tetrahydro-1,2,4-triazin-3-yl)sulfanyl]methyl]-8-oxo-5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid.

N,N-Dimethylaniline Not more than 20 ppm (Appendix 10.16, method 2). 2-Ethylhexanoic acid Not more than 0.8% w/w (Appendix 10.17). Water 8.0% - 11.0% (Appendix 10.3). Determined on 0.100 g. Bacterial endotoxins Less than 0.08 IU/mg (Appendix 13.2), if intended for use in the manufacture of parenteral preparations without a further appropriate procedure for the removal of bacterial endotoxins. Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. 221

VP V

CEFTRIAXONE FOR INJECTION

Inject the test solution and reference solution (1). Calculate the percentage content of C18H16N8Na2O7S3 in the substance to be examined, using the areas of the principal peaks in the chromatograms obtained with the test solution, reference solution (1) and the declared content of ceftriaxone sodium RS.

Storage Store in an airtight container, protected from light. If the substance is sterile, store in a sterile, airtight, tamper-proof container. Action and use Cephalosporin antibacterial. Preparation Injections. CEFTRIAXONE FOR INJECTION Ceftriaxoni pulvis ad injectionem Ceftriaxone for injection is a sterile crystalline powder of ceftriaxone sodium. It is supplied in an airtight glass vial. It is dissolved immediately before use in requisite solvent. The preparation complies with the general requirements prescribed under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Content of ceftriaxone, C18H18N8O7S3, 92.0% to 108.0% of the stated amount. Characters Almost white crystals or crystalline powder. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined, is concordant with the spectrum of ceftriaxone sodium RS. B. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the peak due to ceftriaxone in the chromatogram obtained with the reference solution. C. It gives reaction of sodium (Appendix 8.1). Acidity or alkalinity The pH of a solution containing 10% of the preparation in carbon dioxide-free water R is 6.0 to 8.0 (Appendix 6.2). Clarity of solution A solution containing 1.2% of the preparation in carbon dioxide-free water R is clear (Appendix 9.2). Related substances Examine by liquid chromatography as described under the Assay. 222

Allow the chromatography to proceed for at least twice the retention time of the principal peak. In the chromatogram obtained with the test solution the area of any secondary peak is not greater than the area of the principal peak in the chromatogram obtained with the diluted test solution (1%) and the sum of the areas of all the secondary peaks is not greater than 5 times the area of the principal peak in the chromatogram obtained with the diluted test solution (5%). Disregard any peak with an area less than 10% of the area of the principal peak in the chromatogram obtained with the diluted test solution.

Water Not more than 11.0% (Appendix 10.3). Use 0.2 g of the preparation. Bacterial endotoxins (Appendix 13.2) Dissolve the preparation in water BET R to give a solution containing 10 mg of ceftriaxone per ml (solution A). The endotoxin limit concentration of solution A is 2.0 EU per ml. Use the maximum valid dilution of solution A calculated from the declared sensitivity of the lysate used in the test. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 2 g of tetradecylammonium bromide R and 2 g of tetraheptylammonium bromide R in a mixture of 440 ml of water, 55 ml of phosphate buffer pH 7.0 R, 5 ml of citrate buffer solution pH 5.0 (prepared by dissolving 20.17 g of citric acid R in 800 ml of water, adjusting to pH 5.0 with 10 M sodium hydroxide solution R and diluting to 1000 ml with water), add 500 ml of acetonitrile R and mix. Test solution: Weigh the contents of 10 containers, calculate the average weight and mix well. Dissolve an accurately weighed quantity of the preparation in the mobile phase to obtain a solution containing the equivalent of 0.030% of ceftriaxone. Reference solution: A solution contains 0.030% of ceftriaxone sodium RS in the mobile phase. Resolution solution: A solution contains 0.0050% of ceftriaxone sodium RS and 0.0050% of ceftriaxone sodium E-isomer RS in the mobile phase. Diluted test solution: Dilute 1 volume of the test solution to 100 volumes with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm) (Lichrosphere RP-18 is suitable). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: Inject the resolution solution. Adjust the sensitivity of the detector so that the heights of the peaks are at least 50% of the full scale of the recorder. The test is not valid unless the resolution factor between the two principal peaks in

VP V

CEFUROXIME AXETIL

the chromatogram obtained with the resolution solution is at least 3.0. Inject alternately the test solution and the reference solution. Calculate the content of ceftriaxone, C18H18N8O7S3, in one container of the preparation using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C18H18N8O7S3 in ceftriaxone sodium RS. Each mg of ceftriaxone sodium (C18H16N8Na2O7S3,3½H2O) is equivalent to 0.8383 mg of ceftriaxone (C18H18N8O7S3).

Storage Store in a well-closed container, at a temperature not exceeding 30 °C. Action and use Cephalosporin antibacterial. Usual strength 250 mg; 500 mg and 1000 mg, calculated as ceftriaxone. CEFUROXIME AXETIL Cefuroximum axetili

C20H22N4O10S

M. 510.5

Cefuroxime axetil is a mixture of the 2 diastereoisomers of (1RS)-1-(acetyloxy)ethyl (6R,7R)-3-[(carbamoyloxy) methyl]-7-[[(Z)-2-(furan-2-yl)-2(methoxyimino)acetyl] amino]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2carboxylate, semi-synthetic product derived from a fermentation product. It contains not less than 96.0% and not more than 102.0% of C20H22N4O10S, calculated with reference to the anhydrous substance.

Characters A white or almost white powder. Slightly soluble in water and in ethanol (96%), soluble in acetone, in ethyl acetate and in methanol. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of cefuroxime acetil RS. B. Examine the chromatograms obtained in the Assay, the retention time and size of the principal peaks in the chromatogram obtained with the test solution correspond to

those of the peaks due to cefuroxime axetil diastereoisomers A and B in the chromatogram obtained with reference solution (4).

Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the test solution and reference solution (4) immediately before use. Mobile phase: Methanol - a 0.23% solution of ammonium dihydrogen phosphate (38 : 62). Test solution:  Dissolve 10.0  mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the same solvent. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Reference solution (2): In order to prepare in situ impurity A, heat 5 ml of the test solution at 60 °C for 1 h. Reference solution (3): In order to prepare in situ impurity B, expose 5 ml of the test solution to ultraviolet light at 254 nm for 24 h. Reference solution (4): Dissolve 10.0  mg of  cefuroxime axetil RS in the mobile phase and dilute to 50.0 ml with the same solvent. Chromatographic system: A column (25 cm × 4.6 mm) packed with trimethylsilyl silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 278 nm. Flow rate: 1.0  ml/min. Volume of injection: 20 µl. Procedure: Inject the test solution and reference solutions (1), (2) and (3). Identification of impurities: Use the chromatogram obtained with reference solution (2) to identify the pair of peaks due to impurity A and use the chromatogram obtained with reference solution (3) to identify the pair of peaks due to impurity B. The relative retention with reference to cefuroxime axetil diastereoisomer A: Cefuroxime axetil diastereoisomer B = about 0.9; impurity A = about 1.2; impurity B = 1.7 and 2.1. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to cefuroxime axetil diastereoisomer A and impurity A is at least 1.5. Limits: Impurity A: Not more than 1.5% for the sum of the pair of peaks. Impurity B: Not more than 1.0% for the sum of the pair of peaks. Impurity E: Not more than 0.5%. Any other impurity: Not more than 0.5% for each impurity. Sum of the impurities: Not more than 3.0%. Disregard any peak with an area less than 0.05 times the area of the 2 principal peaks in the chromatogram obtained with reference solution (1) (0.05%). 223

VP V

CEFUROXIME POWDER FOR ORAL SUSPENSION  Notes: ImpurityA: 1-(acetyloxy)ethyl (6R,7R)-3-[(carbamoyloxy) methyl]-7-[[(Z)-2-(furan-2-yl)-2-(methoxyimino)acetyl]amino]8-oxo-5-thia-1-azabicyclo[4.2.0]oct-3-ene-2-carboxylate (Δ3isomers). Impurity B: (1RS)-1-(acetyloxy)ethyl (6R,7R)-3-[(carbamoyloxy) methyl]-7-[[(E)-2-(furan-2-yl)-2-(methoxyimino)acetyl]amino]8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2carboxylate((E)isomers). Imputity C: (6R,7R)-7-[[(Z)-2-(furan-2-yl)-2-(methoxyimino) acetyl]amino]-8-oxo-3-[[[(trichloroacetyl)carbamoyl]oxy] methyl]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid. Impurity D: Cefuroxime. Impurity E: (5aR,6R)-6-[[(2Z)-2-(furan-2-yl)-2-(methoxyimino) acetyl]amino]-5a,6-dihydro-3H,7H-azeto[2,1-b]furo[3,4-d] [1,3]thiazine-1,7(4H)-dione (descarbamoylcefuroxime lactone).

Diastereoisomer ratio Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. In the chromatogram obtained with the test solution, the ratio of the area of the peak due to cefuroxime axetil diastereoisomer A to the sum of the areas of the peaks due to cefuroxime axetil diastereoisomers A and B is between 0.48 and 0.55. Acetone Not more than 1.1% (Appendix 10.14). Water Not more than 1.5% (Appendix 10.3). Determined on 0.400 g. Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. Inject the test solution and reference solution (4). System suitability: In the chromatogram obtained with reference solution (4), the resolution between the peaks due to cefuroxime axetil diastereoisomer A and B is at least 1.5; the relative standard deviation of the sum of the areas of peaks due to cefuroxime axetil diastereoisomers A and B after 6 injections is not more than 2.0%. Calculate the percentage content of C20H22N4O10S from the sum of the areas of the 2 diastereoisomer peaks in the chromatograms obtained with the test solution, reference solution (4) and the declared content of C20H22N4O10S in cefuroxime axetil RS. Storage Store in an airtight container, protected from light. Action and use Cephalosporin antibacterial. Preparations Tablets, powder for suspension.

224

CEFUROXIME POWDER FOR ORAL SUSPENSION  Pulveres Cefuroximi ad suspensionum peoralum Cefuroxime powder for oral suspension contains cefuroxime acetil. The powder complies with the requirements stated under “Powder” (Appendix 1.7) and with the following requirements.

Content of cefuroxime, C16H16N4O8S, 90.0% to 110.0% of the stated amount. Characters A dry powder with a homogeneous colour. Identification In the Assay, the retention times of the two principal peaks (cefuroxime acetil A and cefuroxime acetil B) in the chromatogram obtained with the test solution correspond to those in the chromatogram obtained with the reference solution. Water Not more than 6.0% (Appendix 10.3). Use 0.5 g. pH 3.5 to 7.0 (Appendix 6.2) Determine on a suspension constituted as directed in the labeling. Related substances In the chromatogram obtained with the test solution in the Assay, the sum of the areas of the pair of peaks corresponding to the E-isomers in the chromatogram obtained with resolution solution (2) is not greater than 1.5% of the total areas of all the peaks. The sum of the areas of any peaks corresponding to the Δ3-isomers in the chromatogram obtained with resolution solution (1) is not greater than 2.0% of the total areas of all the peaks. The area of any other secondary peak is not greater than 1.0% of the total areas of all the peaks. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of phosphate buffer pH 7.0 Phosphate buffer pH 7.0: Dissolve 3.7 g of sodium dihydrogen phosphate R and 5.7 g of disodium hydrogen phosphate R in 1000 ml of water. Rotation speed: 50 rpm. Time: 30 min. Procedure: Constitute the content of a container as directed in the labeling. For single-dose preparations, test the total amount of the obtained suspension. For multi-dose preparations test 5.0 ml of constituted suspension equivalent to 125 or 250 mg of cefuroxime.

VP V

After the specified time (30 min) withdraw a sample of the medium, filter, dilute the filtrate with the medium to obtain a solution containing a suitable concentration (if necessary). Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum at about 280 nm, in a 1 cm cell, using the medium as the blank and in comparison with a reference solution of cefuroxime axetil RS having the same concentration in the same medium. Calculate the content of cefuroxime dissolved, using the absorbances of the test solution, reference solution and the declared content cefuroxime, C16H16N4O8S, in cefuroxime axetil RS. Tolerance: Not less than 60% (Q) of the stated amount of cefuroxime, C16H16N4O8S, is dissolved in 30 minutes.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: A mixture of 38 volumes of methanol R and 62 volumes of 0.2 M ammonium dihydrogen phosphate. Reference solution: Dissolve an accurately weighed quantity of cefuroxime axetil RS in the mobile phase to obtain a solution containing about 0.25 mg of cefuroxime per ml. Test solution: Use the contents in the Uniformity of mass, mix. Transfer an accurately weighed quantity of the powder containing the equivalent of about 500 mg of cefuroxime to a 100 ml volumetric flask, add 5 ml of 0.2 M ammonium dihydrogen phosphate, previously adjusted to pH 2.4 with phosphoric acid R, shake and immediately add methanol R to volume, mix well and filter. Dilute 5.0 ml of the filtrate to 100.0 ml with the mobile phase. Resolution solution (1): Heat a portion of the test solution at 60 °C for 60 minutes or until impurities Δ3 - isomers could be detected, filter. Resolution solution (2): Expose a portion of the test solution to ultraviolet light (254 nm) for 24 hours or until impurities E-isomers could be detected, filter. Note: The test solution and the reference solution should be used immediately or stored in the dark at a temperature between 2 °C and 8 °C before analysis.

Chromatographic system: A column (25 cm × 4.6 mm) packed with particles of trimethylsilyl silica gel for chromatography (5 μm) (Hypersil SAS is suitable). Detector: A spectrophotometer set at 278 nm. Flow rate: 1.2 ml/min. Volume of injection: 20 μl. Procedure: System suitability: Inject the reference solution, resolution solution (1) and resolution solution (2). The relative retention times are approximately 0.9 for cefuroxime axetil diastereoisomer B, 1.0 for cefuroxime axetil diastereoisomer A, 1.2 for Δ3-isomers and 1.7 and 2.1 for the E-isomers. The test is not valid unless the resolution factors between the peaks corresponding to the cefuroxime

CEFUROXIME TABLETS

axetil diastereoisomers A and B in the reference solution and between the peaks corresponding to cefuroxime axetil diastereoisomer A and the cefuroxime axetil Δ3-isomer in resolution solution (1) are each not less than 1.5. Adjust the concentration of methanol R in the mobile phase, if necessary. Inject the reference solution 6 times. The relative standard deviation of the sums of cefuroxime axetil diastereoisomer A and cefuroxime axetil diastereoisomer B peak areas is not more than 2.0%. Inject alternately the test solution and the reference solution. Calculate the content of cefuroxime, C16H16N4O8S, using the sums of cefuroxime axetil diastereoisomer A and cefuroxime axetil diastereoisomer B peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C16H16N4O8S in cefuroxime axetil RS. Each mg of cefuroxime axetil, C20H22N4O10S, is equivalent to 0.8313 mg of cefuroxime, C16H16N4O8S.

Storage Store in an airtight container, in a cool and dry place, protected from light. Action and use Cephalosporin antibacterial. Usual strength 125 mg; 250 mg. CEFUROXIME TABLETS Tabellae Cefuroximi Cefuroxime tablets contain cefuroxime axetil. They are film-coated tablets. The tablets comply with the requirements stated under “Tablets”, “Coated tablets” item (Appendix 1.20) and with the following requirements.

Content of cefuroxime, C16H16N4O8S, 90.0% to 110.0% of the stated amount. Identification A. Extract a quantity of the powdered tablets containing the equivalent of 0.1 g of cefuroxime with 5 ml of dichloromethane R, filter and evaporate the filtrate to dryness. The infrared absorption spectrum of the residue (Appendix 4.2) is concordant with the reference spectrum of cefuroxime axetil or the spectrum of cefuroxime axetil RS. B. In the Assay, the retention times of two principal peaks in the chromatogram obtained with the test solution correspond to those of the peaks due to diastereoisomers A and B of cefuroxime axetil in the chromatogram obtained with the reference solution. 225

VP V

CEFUROXIME TABLETS

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of 0.1 M hydrochloric acid R. Rotation speed: 50 rpm. Time: 45 min. Procedure: After the specified time, withdraw a sample of the medium, filter and discard the first 20 ml of the filtrate. Dilute the filtrate with the medium to obtain a solution containing a suitable concentration (if necessary). Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum at 278 nm, in a 1 cm cell, using the medium as a blank and in comparison with a reference solution of cefuroxime axetil RS having the same concentration in the same medium. Calculate the content of cefuroxime dissolved, using the declared content cefuroxime, C16H16N4O8S, in cefuroxime axetil RS. Tolerance: Not less than 70% (Q) of the stated amount of cefuroxime is dissolved in 45 minutes. Related substances In the chromatogram obtained with the test solution in the Assay, the sum of the areas of the pair of peaks corresponding to the E-isomers in the chromatogram obtained with resolution solution (2) is not greater than 1.5% of the total areas of all the peaks. The sum of the areas of any peaks corresponding to the ∆3-isomers in the chromatogram obtained with resolution solution (1) is not greater than 2.0% of the total areas of all the peaks. The area of any other secondary peak is not greater than 1.0% of the total areas of all the peaks. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: A mixture of 38 volumes of methanol R and 62 volumes of 0.2 M ammonium dihydrogen phosphate solution. Reference solution: Dissolve an accurately weighed quantity of cefuroxime axetil RS in the mobile phase to obtain a solution containing about 0.25 mg of cefuroxime per ml. Test solution: Weigh 20 tablets (with the coating layer removed), calculate the average mass, and powder finely. Transfer an accurately weighed quantity of the powdered tablet containing the equivalent of about 500 mg of cefuroxime to a 100 ml volumetric flask, add 5 ml of 0.2 M ammonium dihydrogen phosphate solution, previously adjusted to pH 2.4 with phosphoric acid R, immediately add methanol R to volume, mix well and filter. Dilute 5.0 ml of the filtrate to 100.0 ml with the mobile phase. Resolution solution (1): Heat a potion of the test solution at 60 °C for 60 min or until impurities (∆3-isomers) could be detected, filter. Resolution solution (2): Expose a potion of the test solution to ultraviolet light (254 nm) for 24 h or until impurities (E-isomers) could be detected, filter. 226

Note: The test solution and the reference solution, should be used immediately, or stored in the dark at a temperature between 2 °C and 8 °C before analysis.

Chromatographic system: A column (25 cm × 4.6 mm) packed with particles trimethylsilyl silica gel for chromatography (5 µm) (Hypersil SAS is suitable). Detector: A spectrophotometer set at 278 nm. Flow rate: 1.2 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution, resolution solution (1) and resolution solution (2). The relative retention times are approximately 0.9 for cefuroxime axetil diastereoisomer B, 1.0 for cefuroxime axetil diastereoisomer A, 1.2 for the ∆3-isomers and 1.7 and 2.1 for the E-isomers. The test is not valid unless the resolution factors between the peaks corresponding to the cefuroxime axetil diastereoisomers A and the cefuroxime axetil diastereoisomers B in the reference solution and between the peaks corresponding to cefuroxime axetil diastereoisomer A and the cefuroxime axetil ∆3-isomer in resolution solution (1) are each not less than 1.5. Adjust the concentration of methanol R in the mobile phase, if necessary. Inject the reference solution 6 times. The relative standard deviation of the sums of cefuroxime axetil diastereoisomer A and cefuroxime axetil diastereoisomer B peak areas is not more than 2.0%. Inject alternately the test solution and the reference solution. Calculate the content of cefuroxime, C16H16N4O8S, using the sums of cefuroxime axetil diastereoisomer A and cefuroxime axetil diastereoisomer B peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C16H16N4O8S in cefuroxime axetil RS. Each mg of cefuroxime axetil, C20H22N4O10S, is equivalent to 0.8313 mg of cefuroxime, C16H16N4O8S.

Storage Store in aluminum blister or airtight container, in a cool and dry place, protected from light, at a temperature not exceeding 30 °C. Action and use Antibacterial. Usual strength 100 mg; 250 mg; 500 mg.

VP V

CEFUROXIME SODIUM

CEFUROXIME SODIUM Cefuroximum natricum

C16H15N4NaO8S



M. 446.4

Cefuroxime sodium is sodium (6R,7R)-3- [(carbamoyloxy) methyl]-7-[[(Z)-(furan-2-yl)(methoxyimino)acetyl] amino]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2carboxylate. It contains not less than 96.0% and not more than 102.0% of C16H15N4NaO8S, calculated with reference to the anhydrous substance.

Characters White or almost white powder, slightly hygroscopic. Freely soluble in water, very slightly soluble in ethanol. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of cefuroxime sodium RS. B. It gives reaction (A) of sodium (Appendix 8.1). Appearance of solution Solution S: Dissolve 2.0 g of the substance to be examined in carbon dioxide-free water R and dilute to 20.0 ml with the same solvent, and mix. Solution S is not more opalescent than reference suspension II (Appendix 9.2). The absorbance (Appendix 4.1) of solution S measured at 450 nm is not greater than 0.25. pH 5.5 to 8.0 (Appendix 6.2). Dilute 2 ml of solution S to 20 ml with carbon dioxide-free water R, and mix. Specific optical rotation +59° to +66°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.500 g of the substance to be examined in acetate buffer solution pH 4.6 R and dilute to 25.0 ml with the same buffer solution. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Mix 1 volume of acetonitrile R and 99 volumes of acetate buffer solution pH 3.4, prepared by dissolving 6.01 g of glacial acetic acid R and 0.68 g of sodium acetate R in water and diluting to 1000 ml with the same solvent.

Test solution (1): Dissolve 25.0 mg of the substance to be examined in water and dilute to 25.0 ml with the same solvent, and mix. Test solution (2): Dilute 5.0 ml of test solution (1) to 50.0 ml with water, and mix. Reference solution (1): Dissolve 25.0 mg of cefuroxime sodium RS in water and dilute to 25.0 ml with the same solvent, and mix. Dilute 5.0 ml of this solution to 50.0 ml with water, and mix. Reference solution (2): Place 20 ml of reference solution (1) in a water-bath at 80°C for 15 min. Cool and inject immediately. Reference solution (3): Dilute 1.0 ml of test solution (1) to 100.0 ml with water, and mix. Chromatographic system: A column (25 cm × 4.6 mm) packed with hexylsilyl silica gel for chromatography (5 µm). Detector: A spectrophotometer set at 273 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: The run time is 3 times the retention time of the principal peak. System suitability: Inject reference solution (2). The test is not valid unless the resolution between the peaks due to cefuroxime and impurity A (descarbamoylcefuroxime) is at least 2.0. Limits: In the chromatogram obtained with test solution (1): The area of any secondary peak is not greater than the area of the principal peak in the chromatogram obtained with reference solution (3) (1.0%) The sum of the areas of all the secondary peaks is not greater than 3 times the area of the principal peak in the chromatogram obtained with reference solution (3) (3.0%). Disregard any peak with an area less than 0.05 times that of the principal peak in the chromatogram obtained with reference solution (3) (0.05%).

N,N-Dimethylaniline Not more than 20 ppm (Appendix 10.16, method 2). 2-Ethylhexanoic acid Not more than 0.5% w/w (Appendix 10.17). Water Not more than 3.5% (Appendix 10.3). Determined on 0.400 g. Sterility If intended for use in the manufacture of parenteral preparations forms without a further appropriate sterilisation procedure, it complies with the test for sterility (Appendix 13.7). Bacterial endotoxins Less than 0.10 EU/mg (Appendix 13.2), if intended for use in the manufacture of parenteral preparations without a 227

VP V

CEFUROXIME FOR INJECTION

further appropriate procedure for the removal of bacterial endotoxins.

Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. Inject alternately test solution (2) and reference solution (1). Calculate the content of C16H15N4NaO8S in the substance to be examined, using the areas of the principal peaks obtained from the test solution, the reference solution and the declared content of C16H15N4NaO8S in cefuroxime sodium RS. Storage Store in an airtight container, protected from light. If the substance is sterile, store in a sterile, airtight container. Action and use Cephalosporin antibacterial. Preparation Injection. CEFUROXIME FOR INJECTION Cefuroximi pro injectione Cefuroxime for injection is a sterile material consisting of cefuroxime sodium with or without excipients. It is supplied in a sealed container. The preparation complies with the general requirements prescribed under “Injections, infusions ” (Appendix 1.19) and with the following requirements.

Content of cefuroxime, C16H16N4O8S, 90.0% to 110.0% of the stated amount. Characters White or almost white powder. Identification A. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the principal peak in the chromatogram obtained with reference solution (1). B. It gives reaction of sodium (Appendix 8.1). pH pH of a solution containing the equivalent of 10.0% of cefuroxime in carbon dioxide-free water R, 5.5 to 8.5, (Appendix 6.2). Clarity of solution A solution containing the equivalent of 10.0% of cefuroxime in carbon dioxide-free water is not more opalescent than reference suspension II (Appendix 9.2). 228

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - acetate buffer pH 3.4 (1 : 10). Test solution: Dissolve a quantity of the contents of the injection containing about 25.0 mg of cefuroxime sodium in water, and dilute to 50.0 ml with water. Reference solution (1): Dissolve 25.0 mg cefuroxime sodium RS in water and dilute to 50.0 ml with water. Reference solution (2): Dilute 5.0 ml of reference solution (1) to 25.0 ml with water. Heat in a water-bath at 60 °C in 10 min, cool and inject immediately. Reference solution (3): Dilute 1.0 ml the test solution to 100.0 ml with water. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase hexylsilyl sillicagel for chromatography R (5 µm). Detector: A spectrophotometer set at 273 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: Inject reference solution (2): The chromatogram obtained shows two principal peaks corresponding to cefuroxime and descarbamoyl cefuroxime. The test is not valid unless the resolution factor between the two principal peaks is at least 2.0. If necessary, adjust the concentration of acetonitrile in the mobile phase. Inject reference solution (3): Adjust the sensitivity of the system so that the height of the principal peak is not less than 25% of the full scale of the recorder. The test is not valid unless the symmetry factor of the cefuroxime peak is 1.5 or less. Inject the test solution and allow the chromatography to proceed for at least 3 times the retention time of the principal peak. In the chromatogram obtained, the area of any secondary peak is not greater than the area of the principal peak in the chromatogram obtained with reference solution (3) (1%) and the sum of the areas of all the secondary peaks is not greater than 3 times the area of the principal peak in the chromatogram obtained with reference solution (3) (3%). Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (3) (0.1%). Water Not more than 3.5% (Appendix 10.3). Determined on 0.4 g. Bacterial endotoxins Carry out the test for bacterial endotoxins. Dissolve a quantity of the content of the injection in water BET to give a solution containing the equivalent of 10 mg of cefuroxime per ml (solution A). The endotoxin limit concentration of solution A is 1.0 IU per ml. Carry out the test using the maximum valid dilution of solution A

VP V

CELECOXIB

calculated from the declared sensitivity of the lysate used in the test (Appendix 13.2).

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase, chromatographic system and procedure as described in the test for Related substances. Inject the test solution and reference solution (1). Calculate the content of C16H16N4O8S, using the peak areas in the chromatograms obtained with the test solution, reference solution (1) and the declared content of C16H16N4O8S in cefuroxime sodium RS. Storage Store in a cool and dry place, and protected from light. Action and use Cephalosporin antibacterial. Usual strength 0.5 g; 0.75g; 1 g or 1.5 g of cefuroxime. CELECOXIB Celecoxibum

C17H14F3N3O2S  

M. 381.4

Celecoxib is 4-[5-(4-methylphenyl)-3-(trifluoromethyl)1H-pyrazol-1-yl]benzenesulfonamide. It contains not less than 98.0% and not more than 102.0% of C17H14F3N3O2S, calculated with reference to the anhydrous substance.

Characters White or almost white, crystalline or amorphous powder, it shows polymorphism. Practically insoluble in water, freely soluble to soluble in anhydrous ethanol, soluble in methylene chloride. Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of celecoxib RS. If the spectra obtained with the substance to be examined and the reference substance show differences, dissolve separately the substance to be examined and celecoxib RS in 2-propanol R, evaporate to dryness and record new spectra using the residues.

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Mix 10 volumes of acetonitrile R1, 30 volumes of methanol R2 and 60 volumes of a 0.27% solution of potassium dihydrogen phosphate R previously adjusted to pH 3.0 with phosphoric acid R. Solvent mixture: Water - methanol R2 (25 : 75). Test solution: Dissolve 50.0 mg of the substance to be examined in the solvent mixture and dilute to 100.0 ml with the solvent mixture. Reference solution (1): Dissolve 50.0 mg of celecoxib RS in the solvent mixture and dilute to 100.0 ml with the solvent mixture. Reference solution (2): Dissolve 3 mg of celecoxib impurity A RS and 3 mg of celecoxib impurity B RS in the solvent mixture and dilute to 50.0 ml with the solvent mixture. Dilute 1.0 ml of the solution to 25.0 ml with reference solution (1). Reference solution (3): Dilute 1.0 ml of the test solution to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 10.0 ml with the solvent mixture. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase end-capped phenylsilyl silica gel for chromatography R (5 µm). Column temperature: 60 °C. Detector: A spectrophotometer set at 215 nm. Flow rate: 1.5 ml/min. Volume of injection: 25 µl. Procedure: Inject the test solution and reference solutions (2), (3). The run time is 1.5 times the retention time of celecoxib. Identification of impurities: Use the chromatogram obtained with reference solution (2) to identify the peaks due to impurities A and B. Relative retention with reference to celecoxib (retention time = about 27 min): Impurity A = about 0.9; impurity B = about 1.1. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurity A and celecoxib is at least 1.8; the resolution between the peaks due to celecoxib and impurity B is at least 1.8. Calculation of percentage contents for all impurities, use the concentration of celecoxib in reference solution (3). Limits: Impurity A: Not more than 0.4%. Unspecified impurities: For each impurity, not more than 0.10%. Total: Not more than 0.5%. Reporting threshold: 0.05%. Note: Impurity A: 4-[5-(3-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol -1-yl]benzenesulfonamide. Impurity B: 4-[3-(4-methylphenyl)-5-(trifluoromethyl)-1H-pyrazol -1-yl]benzenesulfonamide.

229

VP V

CELLULOSE ACETATE

Heavy metals Not more than 20 ppm (Appendix 9.4.8). Solvent mixture: Water - acetone (15 : 85). 0.5 g complies with limit test for heavy metals, method 8. Prepare the standard using 1 ml of lead standard solution (10 ppm Pb) R. Water Not more than 0.5% (Appendix 10.3). Determined on 0.400 g. Sulfated ash Not more than 0.2% (Appendix 9.9, method 2). Determined on 1.0 g in a platinum crucible. Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. Inject the test solution and reference solution (1). Calculate the content of C17H14F3N3O2S using the areas for the celecoxib peaks in the chromatogram obtained with the test solution, reference solution (1) and the content of C17H14F3N3O2S in celecoxib RS.

Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of cellulose acetate RS. Preparation: Dissolve 1 g of cellulose acetate, previously dried, in 10 ml dioxane R, and spread 1 drop of the solution between 2 sodium chloride plates; separate the plates, heat them both at 105 °C for 1 h, and reassemble the dried plates. Free acid Not more than 0.1%, calculated as acetic acid (dried substance). To 5.00 g of the substance to be examined in a 250 ml conical flask, add 150 ml of carbon dioxide-free water, insert the stopper, swirl the suspension gently and allow to stand for 3 h. Filter, then wash the flask and the filter with carbon dioxide-free water. Combine these washings to the filtrate. Add 0.1 ml of phenolphthalein solution R1 and titrate with 0.01 N sodium hydroxide VS until a pale pink colour is obtained. 1 ml of 0.01 N sodium hydroxide VS is equivalent to 0.6005 mg of free acid, calculated as acetic acid.

Storage Store in an airtight container, protected from light and moisture at room temperture.

Heavy metals Not more than 10 ppm (Appendix 9.4.8). 2.0 g complies with limit test for heavy metals, method 4. Prepare the standard solution using 2 ml of lead standard solution (10 ppm Pb) R.

Action and use Cyclo-oxygenase (COX-2) inhibitor; analgesic; antiinflammatory.

Loss on drying Not more than 5.0% (Appendix 9.6). (1.00 g; 105 °C; 3 h).

Preparations Tablets, capsules, injection.

Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g.

CELLULOSE ACETATE Cellulosi acetas

Microbial contamination Total aerobic microbial count (TAMC): Not more than 103 CFU/1 g. Total combined yeasts/moulds count (TYMC): Not more than 102 CFU/1 g. Determined by plate count (Appendix 13.6). Absence of Escherichia coli and Salmonella (Appendix13.6).

Cellulose acetat is partly or completely O-acetylated cellulose. It contains not less than 29.0% and not more than 44.8% of acetyl groups (C2H3O), calculated with reference to the dried substance. It contains not less than 90.0% and not more than 110.0% of the labelled amount of the nominal acetyl content, calculated with reference to the dried substance.

Characters White, yellowish-white or greyish-white, hygroscopic powder or granules. Practically insoluble in ethanol (96%) and in water. Soluble in acetone, in formic acid and in a mixture of equal volumes of methanol and methylene chloride.

230

Acetyl groups (C2H3O) Cellulose acetate containing not more than 42.0% of acetyl groups. To 2.000 g in a 500 ml conical flask, add 100 ml of acetone R and 10 ml of water. Close the flask and stir with a magnetic stirrer until dissolution is complete. Add 30.0 ml of 1 N sodium hydroxide VS with constant stirring. A finely divided precipitate of regenerated cellulose is obtained. Close the flask and stir with a magnetic stirrer for 30 min. Add 100 ml of water at 80 °C, washing down the sides of the flask, stir for 2 min and cool to room temperature. Titrate

VP V

MICROCRYSTALLINE CELLULOSE

with 1 N sulfuric acid VS, using 0.1 ml of phenolphthalein solution R as indicator. Carry out a blank titration. Calculate the percentage content of acetyl groups using the following expression:

4.305 (n2 − n1 ) (100 − d ) x m

x

MICROCRYSTALLINE CELLULOSE Cellulosum microcrystallinum

100

Where: d: loss on drying as a percentage (%). m: mass of the substance to be examined (g). n1: number of millilitres of 1 N sulfuric acid VS used in the tes solution. n2: number of millilitres of 1 N sulfuric acid VS used in the blank titration. Cellulose acetate containing more than 42.0 per cent of acetyl groups. To 2.000 g in a 500 ml conical flask, add 30 ml of dimethyl sulfoxide R and 100 ml of acetone R. Close the flask and stir with a magnetic stirrer for 16 h. Add 30.0 ml of 1 N sodium hydroxide VS with constant stirring. Close the flask and stir with a magnetic stirrer for 6 min. Allow to stand without stirring for 60 min. Resume stirring and add 100 ml of water at 80 °C, washing down the sides of the flask, stir for 2 min and cool to room temperature. Titrate with 0.5 N hydrochloric acid VS, using 0.1 ml of phenolphthalein solution R as indicator. Add 0.5 ml of 0.5 N hydrochloric acid VS in excess, stir for 5 min and allow to stand for 30 min. Titrate with 0.5 N sodium hydroxide VS, until a persistent pink colour is obtained, stirring with a magnetic stirrer. Calculate the net number of millimoles of 0.5 N sodium hydroxide VS consumed, taking the mean of 2 blank titrations into consideration. Calculate the percentage content of acetyl groups using the following expression:

4.305 n × 100 (100 − d ) × m Where: d: loss on drying as a percentage (%). m: mass of the substance to be examined (g). n: net number of millimoles of 0.5 N sodium hydroxide VS consumed.

Storage In an airtight container. Action and use Excipients. Labelling The label states the percentage content of acetyl groups.

C6nH10n+2O5n+1 Purified cellulose, partly depolymerised cellulose prepared by treating alpha-cellulose, obtained as a pulp from fibrous plant material, with mineral acids.

Characters White or almost white, fine or granular powder. Practically insoluble in water, in acetone, in anhydrous ethanol, in toluene, in dilute acids and in a 5% solution of sodium hydroxide. Identification A. Place about 10 mg of the substance to be examined on a watch-glass and disperse in 2 ml of iodinated zinc chloride solution R. The substance becomes violet-blue. B. The degree of polymerisation is not more than 350. Transfer 1.300 g of the substance to be examined to a 125 ml conical flask. Add 25.0 ml of water and 25.0 ml of cupriethylenediamine hydroxide solution R. Immediately purge the solution with nitrogen R, insert the stopper and shake until completely dissolved. Transfer an appropriate volume of the solution to a suitable capillary viscometer (Appendix 6.3). Equilibrate the solution at 25 °C ± 0.1 °C for at least 5 min. Record the flow time (t1) in seconds between the 2 marks on the viscometer. Calculate the kinematic viscosity (v1) of the solution using the following expression: t1 × k1 where k1 is the viscometer constant. Dilute a suitable volume of cupriethylenediamine hydroxide solution R with an equal volume of water and measure the flow time (t2) using a suitable capillary viscometer. Calculate the kinematic viscosity (v2) of the solvent using the following expression: t2 × k2 where k2 is the viscometer constant. Determine the relative viscosity (ηrel) of the substance to be examined using the following expression: v1/v2 Determine the intrinsic viscosity [n]c by interpolation, using the intrinsic viscosity table (Table 1 - The intrinsic viscosity table). Calculate the degree of polymerisation (P) using the following expression: 231

MICROCRYSTALLINE CELLULOSE

VP V

95[η ]c 100 − b  m  100 

Shake 5.0 g with 80 ml of water for 10 min. Filter through a filter paper with the aid of vacuum into a tared flask. Evaporate to dryness on a water-bath avoiding charring. Dry at 105 °C for 1 h, allow to stand in a desiccator and weigh. Carry out a blank determination. The difference between the mass of the residue and the mass obtained from a blank determination not more than 12,5 mg.

Where: m is the mass in grams of the substance to be examined (g). b is the loss on drying as a percentage (%).

Solubility Dissolve 50 mg of the substance to be examined in 10 ml of ammoniacal solution of copper tetrammine R. It dissolves completely, leaving no residue. pH 5.0 to 7.5 for the supernatant liquid (Appendix 6.2). Shake 5 g of the substance to be examined with 40 ml of carbon dioxide-free water for 20 min and centrifuge. Conductivity The conductivity of the test solution does not exceed the conductivity of the water by more than 75 µS·cm-1. (Appendix 6.10). Use as test solution the supernatant liquid obtained in the test for pH. Measure the conductivity of the supernatant liquid after a stable reading has been obtained and measure the conductivity of the water used to prepare the test solution. Ether-soluble substances Not more than 0.05%. Place 10.0 g of the substance to be examined in a chromatography column about 20 mm in internal diameter and pass 50 ml of peroxide-free ether R through the column. Evaporate the eluate to dryness. Dry the residue at 105 °C for 30 min, allow to cool in a desiccator and weigh. Carry out a blank determination. The difference between the weight of the residue and the weight obtained from a blank determination not more than 5 mg.

Loss on drying Not more than 7.0% (Appendix 9.6). (1.000 g; 105 °C; 3 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Microbial contamination Total aerobic microbial count (TAMC): Not more than 103 CFU/1 g. Total combined yeasts/moulds count (TYMC): Not more than 102 CFU/1 g. Determined by plate count (Appendix 13.6). Absence of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella (Appendix13.6). Storage In an airtight container. Action and use Excipients (Avicel). Labelling The label states the degree of polymerisation.

Water-soluble substances Not more than 0.25%.

232

Heavy metals Not more than 10 ppm (Appendix 9.4.8). 2.0 g complies with limit test for heavy metals, method 3. Prepare the standard solution using 2 ml of lead standard solution (10 ppm Pb) R.

Table 1 - Intrinsic viscosity table Intrinsic viscosity [η]  c at different values of  relative viscosity ηrel       

 

 

 

 

ηrel

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

1.1

0.098

0.106

0.115

0.125

0.134

0.143

0.152

0.161

0.170

0.180

1.2

0.189

0.198

0.207

0.216

0.225

0.233

0.242

0.250

0.259

0.268

1.3

0.276

0.285

0.293

0.302

0.310

0.318

0.326

0.334

0.342

0.350

1.4

0.358

0.367

0.375

0.383

0.391

0.399

0.407

0.414

0.422

0.430

1.5

0.437

0.445

0.453

0.460

0.468

0.476

0.484

0.491

0.499

0.507

1.6

0.515

0.522

0.529

0.536

0.544

0.551

0.558

0.566

0.573

0.580

1.7

0.587

0.595

0.602

0.608

0.615

0.622

0.629

0.636

0.642

0.649

1.8

0.656

0.663

0.670

0.677

0.683

0.690

0.697

0.704

0.710

0.717

1.9

0.723

0.730

0.736

0.743

0.749

0.756

0.762

0.769

0.775

0.782

[η]c

VP V

MICROCRYSTALLINE CELLULOSE  

 

 

 

 

 

 

 

 

 

Intrinsic viscosity [η]c at different values of relative viscosity ηrel [η]c

ηrel

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

2.0

0.788

0.795

0.802

0.809

0.815

0.821

0.827

0.833

0.840

0.846

2.1

0.852

0.858

0.864

0.870

0.876

0.882

0.888

0.894

0.900

0.906

2.2

0.912

0.918

0.924

0.929

0.935

0.941

0.948

0.953

0.959

0.965

2.3

0.971

0.976

0.983

0.988

0.994

1.000

1.006

1.011

1.017

1.022

2.4

1.028

1.033

1.039

1.044

1.050

1.056

1.061

1.067

1.072

1.078

2.5

1.083

1.089

1.094

1.100

1.105

1.111

1.116

1.121

1.126

1.131

2.6

1.137

1.142

1.147

1.153

1.158

1.163

1.169

1.174

1.179

1.184

2.7

1.190

1.195

1.200

1.205

1.210

1.215

1.220

1.225

1.230

1.235

2.8

1.240

1.245

1.250

1.255

1.260

1.265

1.270

1.275

1.280

1.285

2.9

1.290

1.295

1.300

1.305

1.310

1.314

1.319

1.324

1.329

1.333

3.0

1.338

1.343

1.348

1.352

1.357

1.362

1.367

1.371

1.376

1.381

3.1

1.386

1.390

1.395

1.400

1.405

1.409

1.414

1.418

1.423

1.427

3.2

1.432

1.436

1.441

1.446

1.450

1.455

1.459

1.464

1.468

1.473

3.3

1.477

1.482

1.486

1.491

1.496

1.500

1.504

1.508

1.513

1.517

3.4

1.521

1.525

1.529

1.533

1.537

1.542

1.546

1.550

1.554

1.558

3.5

1.562

1.566

1.570

1.575

1.579

1.583

1.587

1.591

1.595

1.600

3.6

1.604

1.608

1.612

1.617

1.621

1.625

1.629

1.633

1.637

1.642

3.7

1.646

1.650

1.654

1.658

1.662

1.666

1.671

1.675

1.679

1.683

3.8

1.687

1.691

1.695

1.700

1.704

1.708

1.712

1.715

1.719

1.723

3.9

1.727

1.731

1.735

1.739

1.742

1.746

1.750

1.754

1.758

1.762

4.0

1.765

1.769

1.773

1.777

1.781

1.785

1.789

1.792

1.796

1.800

4.1

1.804

1.808

1.811

1.815

1.819

1.822

1.826

1.830

1.833

1.837

4.2

1.841

1.845

1.848

1.852

1.856

1.859

1.863

1.867

1.870

1.874

4.3

1.878

1.882

1.885

1.889

1.893

1.896

1.900

1.904

1.907

1.911

4.4

1.914

1.918

1.921

1.925

1.929

1.932

1.936

1.939

1.943

1.946

4.5

1.950

1.954

1.957

1.961

1.964

1.968

1.971

1.975

1.979

1.982

4.6

1.986

1.989

1.993

1.996

2.000

2.003

2.007

2.010

2.013

2.017

4.7

2.020

2.023

2.027

2.030

2.033

2.037

2.040

2.043

2.047

2.050

4.8

2.053

2.057

2.060

2.063

2.067

2.070

2.073

2.077

2.080

2.083

4.9

2.087

2.090

2.093

2.097

2.100

2.103

2.107

2.110

2.113

2.116

233

VP V

MICROCRYSTALLINE CELLULOSE

234

[η]  c at different values of  relative viscosity ηrel    Intrinsic viscosity     [η]c

 

 

 

 

ηrel

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

5.0

2.119

2.122

2.125

2.129

2.132

2.135

2.139

2.142

2.145

2.148

5.1

2.151

2.154

2.158

2.160

2.164

2.167

2.170

2.173

2.176

2.180

5.2

2.183

2.186

2.190

2.192

2.195

2.197

2.200

2.203

2.206

2.209

5.3

2.212

2.215

2.218

2.221

2.224

2.227

2.230

2.233

2.236

2.240

5.4

2.243

2.246

2.249

2.252

2.255

2.258

2.261

2.264

2.267

2.270

5.5

2.273

2.276

2.279

2.282

2.285

2.288

2.291

2.294

2.297

2.300

5.6

2.303

2.306

2.309

2.312

2.315

2.318

2.320

2.324

2.326

2.329

5.7

2.332

2.335

2.338

2.341

2.344

2.347

2.350

2.353

2.355

2.358

5.8

2.361

2.364

2.367

2.370

2.373

2.376

2.379

2.382

2.384

2.387

5.9

2.390

2.393

2.396

2.400

2.403

2.405

2.408

2.411

2.414

2.417

6.0

2.419

2.422

2.425

2.428

2.431

2.433

2.436

2.439

2.442

2.444

6.1

2.447

2.450

2.453

2.456

2.458

2.461

2.464

2.467

2.470

2.472

6.2

2.475

2.478

2.481

2.483

2.486

2.489

2.492

2.494

2.497

2.500

6.3

2.503

2.505

2.508

2.511

2.513

2.516

2.518

2.521

2.524

2.526

6.4

2.529

2.532

2.534

2.537

2.540

2.542

2.545

2.547

2.550

2.553

6.5

2.555

2.558

2.561

2.563

2.566

2.568

2.571

2.574

2.576

2.579

6.6

2.581

2.584

2.587

2.590

2.592

2.595

2.597

2.600

2.603

2.605

6.7

2.608

2.610

2.613

2.615

2.618

2.620

2.623

2.625

2.627

2.630

6.8

2.633

2.635

2.637

2.640

2.643

2.645

2.648

2.650

2.653

2.655

6.9

2.658

2.660

2.663

2.665

2.668

2.670

2.673

2.675

2.678

2.680

7.0

2.683

2.685

2.687

2.690

2.693

2.695

2.698

2.700

2.702

2.705

7.1

2.707

2.710

2.712

2.714

2.717

2.719

2.721

2.724

2.726

2.729

7.2

2.731

2.733

2.736

2.738

2.740

2.743

2.745

2.748

2.750

2.752

7.3

2.755

2.757

2.760

2.762

2.764

2.767

2.769

2.771

2.774

2.776

7.4

2.779

2.781

2.783

2.786

2.788

2.790

2.793

2.795

2.798

2.800

7.5

2.802

2.805

2.807

2.809

2.812

2.814

2.816

2.819

2.821

2.823

7.6

2.826

2.828

2.830

2.833

2.835

2.837

2.840

2.842

2.844

2.847

7.7

2.849

2.851

2.854

2.856

2.858

2.860

2.863

2.865

2.868

2.870

7.8

2.873

2.875

2.877

2.879

2.881

2.884

2.887

2.889

2.891

2.893

7.9

2.895

2.898

2.900

2.902

2.905

2.907

2.909

2.911

2.913

2.915

VP V

MICROCRYSTALLINE CELLULOSE

[η]  c at different values of  relative viscosity ηrel   Intrinsic viscosity     [η]c

 

 

 

 

ηrel

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

8.0

2.918

2.920

2.922

2.924

2.926

2.928

2.931

2.933

2.935

2.937

8.1

2.939

2.942

2.944

2.946

2.948

2.950

2.952

2.955

2.957

2.959

8.2

2.961

2.963

2.966

2.968

2.970

2.972

2.974

2.976

2.979

2.981

8.3

2.983

2.985

2.987

2.990

2.992

2.994

2.996

2.998

3.000

3.002

8.4

3.004

3.006

3.008

3.010

3.012

3.015

3.017

3.019

3.021

3.023

8.5

3.025

3.027

3.029

3.031

3.033

3.035

3.037

3.040

3.042

3.044

8.6

3.046

3.048

3.050

3.052

3.054

3.056

3.058

3.060

3.062

3.064

8.7

3.067

3.069

3.071

3.073

3.075

3.077

3.079

3.081

3.083

3.085

8.8

3.087

3.089

3.092

3.094

3.096

3.098

3.100

3.102

3.104

3.106

8.9

3.108

3.110

3.112

3.114

3.116

3.118

3.120

3.122

3.124

3.126

9.0

3.128

3.130

3.132

3.134

3.136

3.138

3.140

3.142

3.144

3.146

9.1

3.148

3.150

3.152

3.154

3.156

3.158

3.160

3.162

3.164

3.166

9.2

3.168

3.170

3.172

3.174

3.176

3.178

3.180

3.182

3.184

3.186

9.3

3.188

3.190

3.192

3.194

3.196

3.198

3.200

3.202

3.204

3.206

9.4

3.208

3.210

3.212

3.214

3.215

3.217

3.219

3.221

3.223

3.225

9.5

3.227

3.229

3.231

3.233

3.235

3.237

3.239

3.241

3.242

3.244

9.6

3.246

3.248

3.250

3.252

3.254

3.256

3.258

3.260

3.262

3.264

9.7

3.266

3.268

3.269

3.271

3.273

3.275

3.277

3.279

3.281

3.283

9.8

3.285

3.287

3.289

3.291

3.293

3.295

3.297

3.298

3.300

3.302

9.9  

3.304  

3.305 3.307 3.309 3.311 3.313 3.316 3.318  Intrinsic viscosity         [η]c at different values of relative viscosity ηrel  [η]c

3.320  

3.321  

ηrel

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

10

3.32

3.34

3.36

3.37

3.39

3.41

3.43

3.45

3.46

3.48

11

3.50

3.52

3.53

3.55

3.56

3.58

3.60

3.61

3.63

3.64

12

3.66

3.68

3.69

3.71

3.72

3.74

3.76

3.77

3.79

3.80

13

3.80

3.83

3.85

3.86

3.88

3.89

3.90

3.92

3.93

3.95

14

3.96

3.97

3.99

4.00

4.02

4.03

4.04

4.06

4.07

4.09

15

4.10

4.11

4.13

4.14

4.15

4.17

4.18

4.19

4.20

4.22

16

4.23

4.24

4.25

4.27

4.28

4.29

4.30

4.31

4.33

4.34

17

4.35

4.36

4.37

4.38

4.39

4.41

4.42

4.43

4.44

4.45

18

4.46

4.47

4.48

4.49

4.50

4.52

4.53

4.54

4.55

4.56

19

4.57

4.58

4.59

4.60

4.61

4.62

4.63

4.64

4.65

4.66

235

VP V

CEPHALEXIN

CEPHALEXIN Cephalexinum

C16H17N3O4S,H2O

M. 365.4

Cephalexin is (6R,7R)-7-[[(2R)-2-amino-2-phenylacetyl] amino]-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2ene-2-carboxylic acid monohydrate. It contains not less than 95.0% and not more than 102.0% of C16H17N3O4S, calculated with reference to the anhydrous substance.

Characters White or almost white, crystalline powder. Sparingly soluble in water, practically insoluble in ethanol (96%). Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum obtained with cephalexin RS. pH Dissolve 50 mg in carbon dioxide-free water R and dilute to 10 ml with the same solvent. The pH of this solution is 4.0 to 5.5 (Appendix 6.2). Specific optical rotation +149° to +158°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.125 g in phthalate buffer solution pH 4.4 R and dilute to 25.0 ml with the same solvent. Absorbance Dissolve 50 mg in water and dilute to 100.0 ml with the same solvent. The absorbance of the solution determined at 330 nm (Appendix 4.1) is not greater than 0.05. Dilute 2.0 ml of the solution to 50.0 ml with water. The ultraviolet absorption spectrum of the resulting solution in the range 220 nm to 300 nm exhibits an absorption maximum at 262 nm. The specific absorbance at this maximum is 220 to 245, calculated with reference to the anhydrous substance. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Phosphate buffer solution pH 5.0 R. Mobile phase B: Methanol R. Test solution: Dissolve 50.0 mg of the substance to be examined in mobile phase A and dilute to 50.0 ml with the same solvent. Reference solution (1): Dissolve 10.0 mg of D-phenylglycine RS in mobile phase A and dilute to 10.0 ml with the same solvent. 236

Reference solution (2): Dissolve 10.0 mg of 7-aminodesacetoxycephalosporanic acid RS in 2 ml of phosphate buffer solution pH 7.0 R and dilute to 10.0 ml with mobile phase A. Reference solution (3): Pipet 1.0 ml of reference solution (1) and 1.0 ml of reference solution (2) into 100 ml volumetric flask, make up to volume with mobile phase A, mix well. Reference solution (4): Dissolve 10 mg of dimethylformamide R and 10 mg of dimethylacetamide R in mobile phase A and dilute to 10.0 ml with the same solvent. Dilute 1.0 ml of this solution to 100.0 ml with mobile phase A. Reference solution (5): Dilute 1.0 ml of reference solution (3) to 20.0 ml with mobile phase A. Reference solution (6): Dissolve 10 mg of cefotaxime sodium RS in mobile phase A and dilute to 10.0 ml with the same solvent. To 1.0 ml of the solution add 1.0 ml of the test solution and dilute to 100 ml with mobile phase A. Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 220 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min) 0-1 1 - 20 20 - 23 23 - 30

Mobile phase A (% v/v) 98 98 → 70 70 → 98 98

Mobile phase B (% v/v) 2 2 → 30 30 → 2 2

Inject the test solution and reference solutions (3), (4), (5) and (6). System suitability: The resolution between the peaks corresponding to impurity A (D-phenylglycine) and to impurity B (7-aminodesacetoxy cephalosporanic acid) in the chromatogram obtained with reference solution (3) is at least 2.0. The resolution between the peaks corresponding to cephalexin and to cefotaxime in the chromatogram obtained with reference solution (6) is at least 1.5. Limits: In the chromatogram obtained with the test solution: The area of the peak corresponding to the second peak in the chromatogram obtained with reference solution (3) (impurity B) is not greater than the area of the second peak in the chromatogram obtained with reference solution (3) (1.0%). The area of any other impurity (disregard the peaks due to dimethylformamide and dimethylacetamide) is not greater than the area of the first peak in the chromatogram obtained with reference solution (3) (1.0%). The sum of the areas of all peaks, apart from the principal peak, is not greater than three times the area of the the first peak in the chromatogram obtained with reference solution (3) (3.0%).

VP V

Disregard any peak with an area less than the area of the second peak in the chromatogram obtained with reference solution (5) (0.05%).

N,N-Dimethylaniline Not more than 20 ppm (Appendix 10.16, method 2). Water 4.0% to 8.0% (Appendix 10.3). Determined on 0.300 g. Sulfated ash Not more than 0.2% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol - acetonitrile - a 1.36% solution of potassium dihydrogen phosphate - water (2 : 5 : 10 : 83). Test solution: Dissolve 50.0 mg of the substance to be examined in water and dilute to 100.0 ml with the same solvent. Reference solution: Dissolve 50.0 mg of cephalexin monohydrate RS in water and dilute to 100.0 ml with the same solvent. Resolution solution: Dissolve 10 mg of cefradine RS in 20 ml of the reference solution and dilute to 100 ml with water. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: Inject the resolution solution, the test solution and the reference solution. System suitability: The resolution between the peaks corresponding to cephalexin and cefradine is at least 4.0. Calculate the content of cephalexin using the peak areas in the chromatograms obtained with the test solution and the reference solution. Storage Store protected from light. Action and use Cephalosporin antibacterial. Preparations Tablets; capsules; powder for oral suspension. CEPHALEXIN CAPSULES Capsulae Cephalexini Cephalexin capsules contain cephalexin. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

CEPHALEXIN CAPSULES

Content of anhydrous cephalexin, C16H17N3O4S, 90.0% to 110.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Binder-free silica gel. Place the plate in a chamber containing a mixture of n-hexane and tetradecane (95 : 5) to a depth of about 1 cm, allow the solvent front to move the length of the plate, remove the plate from the chamber and allow the solvent to evaporate. Mobile phase: 0.1 M citric acid solution - 0.1 M disodium hydrogen phosphate solution - a solution of ninhydrin in acetone containing 1 g per 15 ml (60 : 40 : 1.5) Test solution: Dissolve a quantity of the contents of the capsules containing about 30 mg of cephalexin in 10 ml of water, filter. Reference solution: Solution contains 0.3% of cephalexin RS in water. Procedure: Apply separately to the plate 10 µl of each solution. Develop the chromatogram over a path of about three fourths of the length of the plate. After removal of the plate, allow it to dry in air. Heat the plate at 110 °C for 10 min. Examine in day light. The principal spot in the chromatogram obtained with the test solution corresponds in position, colour and size to that in the chromatogram obtained with the reference solution. B. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the peak due to cephalexin in the chromatogram obtained with the reference solution. Water Not more than 10.0% (Appendix 10.3). Use 0.3 g of the contents of the capsules. Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 900 ml of water. Rotation speed: 100 rpm. Time: 30 min. Procedure: After the specified time, withdraw w a sample of the medium, filter, discard the first portion of the filtrate. Dilute the filtrate with water to obtain a solution containing about 20 µg of cephalexin per ml. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum at 262 nm, in a 1 cm cell, using water as a blank, in comparison with the reference solution of cephalexin RS having the same concentration in the same medium. Calculate the content of cephalexin, C16H17N3O4S, dissolved in each capsule using the absorbances obtained and the declared content of C16H17N3O4S in cephalexin RS. Tolerance: Not less than 80% (Q) of the stated amount of cephalexin is dissolved in 30 minutes. Assay Examine by liquid chromatography (Appendix 5.3). 237

VP V

CEPHALEXIN POWDER FOR ORAL SUSPENSION

Mobile phase: Dissolve 1.0 g of sodium pentanesulfonate R in 1015 ml of a mixture of water - acetonitrile - methanol - triethylamine (850 : 100 : 50 : 15), adjust to pH 3.0 ± 0.1 with phosphoric acid R. Internal standard solution: Transfer 300 mg of 1-hydroxy benzotriazole, accurately weighed, to a 1000 ml volumetric flask, dissolve in 10 ml of methanol R, dilute with the mobile phase to volume and mix. Reference solution: Dissolve an accurately weighed quantity of cephalexin RS in water to obtain a stock solution having a concentration of about 1.0 mg per ml. Transfer 10.0 ml of the stock solution to a ground-glassstoppered conical flask, add 15.0 ml of the internal standard solution and mix well. Test solution: Weigh 20 capsules, calculate the average weight of the capsule contents and powder finely. Transfer an accurately weighed quantity of powdered capsules containing the equivalent of about 100 mg of cephalexin, to a 100 ml volumetric flask, add about 75 ml of water and sonicate for 15 min, dilute to volume with water, mix and filter. Transfer 10.0 ml of the filtrate to a groundglass-stoppered conical flask, add 15.0 ml of the internal standard solution and mix. Chromatographic system: A column (25 cm × 4 mm) packed with stationary phase C (5 µm or 10 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution. The resolution factor between the internal standard and cephalexin peak is not less than 5, the relative standard deviation of the area ratios of cephalexin peak relative to the internal standard peak for replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of cephalexin, C16H17N3O4S, in the capsules using the area ratios of cephalexin peak relative to the internal standard peak in the chromatograms obtained with the test solution, the reference solution and the declared content of C16H17N3O4S in cephalexin RS.

CEPHALEXIN POWDER FOR ORAL SUSPENSION Pulveres Cephalexini ad suspensionum peroralum

Storage Packed in aluminum blister or airtight container. Store in a cool and dry place, protected from light, at a temperature not exceeding 30 °C.

Water Not more than 2.0% (Appendix 10.3). Use 0.5 g of the powder.

Action and use Cephalosporin antibacterial. Usual strength 250 mg; 500 mg. 238

Cephalexin powder for oral suspension contains cephalexin. Suitable excipients such as  buffers, preservatives, colors, diluents, and flavors… may be added. The constituted suspension prepared as directed in the label complies with the requirements stated under “Suspension” ” (Appendix 1.5).   The powder complies with the requirements stated under “Powder” (Appendix 1.7) and with the following requirements.

Content of anhydrous cephalexin, C16H17N3O4S, 90.0% to 110.0% of the stated amount. Characters Powder should be loose and dry, with homogeneous colour. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Binder-free silica gel. Place the plate in a chamber containing a mixture of n-hexane and tetradecane (95 : 5) to a depth of about 1 cm, allow the solvent front to move the length of the plate, remove the plate from the chamber and allow the solvent to evaporate. Mobile phase: 0.1 M citric acid solution - 0.1 M disodium hydrogen phosphate solution - a solution of ninhydrin in acetone containing 1 g per 15 ml (60 : 40 : 1.5) Test solution: Dissolve a quantity of the powders containing about 30 mg of cephalexin in 10 ml of water, filter. Reference solution: A 0.3% solution of cephalexin RS in water. Procedure: Apply separately to the plate 10 µl of each solution. Develop the chromatogram over a path of about three fourths of the length of the plate. After removal of the plate, allow it to dry in air. Heat the plate at 110 °C for 10 min. Examine in day light. The principal spot in the chromatogram obtained with the test solution corresponds in position, colour and size to that in the chromatogram obtained with the reference solution. B. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the peak due to cephalexin in the chromatogram obtained with the reference solution.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 1.0 g of sodium pentanesulfonate R in 1015 ml of a mixture of water - acetonitrile - methanol - triethylamine (850 : 100 : 50 : 15), adjust to pH 3.0 ± 0.1 with phosphoric acid R.

VP V

Internal standard solution: Transfer 300 mg of 1-hydroxy benzotriazole, accurately weighed, to a 1000 ml volumetric flask, dissolve in 10 ml of methanol R, dilute with the mobile phase to volume and mix. Reference solution: Dissolve an accurately weighed quantity of cephalexin RS in water to obtain a stock solution having a concentration of about 1.0 mg per ml. Transfer 10.0 ml of the stock solution to a ground-glassstoppered conical flask, add 15.0 ml of the internal standard solution and mix well. Test solution: Transfer an accurately weighed quantity of powders containing the equivalent of about 100 mg of cephalexin, to a 100 ml volumetric flask, add about 75 ml of water and sonicate for 15 minutes, dilute to volume with water, mix and filter. Transfer 10.0 ml of the filtrate to a ground-glass-stoppered conical flask, add 15.0 ml of the internal standard solution and mix. Chromatographic system: A column (25 cm × 4 mm) packed with stationary phase C (5 µm or 10 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution. The resolution factor between the internal standard and cephalexin peak is not less than 5, the relative standard deviation of the area ratios of cephalexin peak relative to the internal standard peak for replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of cephalexin, C16H17N3O4S, using the area ratios of cephalexin peak relative to the internal standard peak in the chromatograms obtained with the test solution, the reference solution and the declared content of C16H17N3O4S in cephalexin RS.

Storage Packed in air-tight aluminum or polyethylene bag. Store in a cool and dry place, protected from light, at a temperature not exceeding 30 °C. Action and use Cephalosporin antibacterial. Usual strength 125 mg; 250 mg; 500 mg. CEPHALEXIN TABLETS Tabellae Cephalexini Cephalexin tablets contain cephalexin. They are tablets or film-coated tablets. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

CEPHALEXIN TABLETS

Content of anhydrous cephalexin, C16H17N3O4S, 90.0% to 110.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Binder-free silica gel. Place the plate in a chamber containing a mixture of n-hexane and tetradecane (95 : 5) to a depth of about 1 cm, allow the solvent front to move the length of the plate, remove the plate from the chamber and allow the solvent to evaporate. Mobile phase: 0.1 M citric acid solution - 0.1 M disodium hydrogen phosphate solution - a solution of ninhydrin in acetone containing 1 g per 15 ml (60 : 40 : 1.5) Test solution: Dissolve a quantity of the powdered tablets containing about 30 mg of cephalexin in 10 ml of water, filter. Reference solution: The solution contains 0.3% of cephalexin RS in water. Procedure: Apply separately to the plate 10 µl of each solution. Develop the chromatogram over a path of about three fourths of the length of the plate. After removal of the plate, allow it to dry in air. Heat the plate at 110 °C for 10 min. Examine in day light. The principal spot in the chromatogram obtained with the test solution corresponds in position, colour and size to that in the chromatogram obtained with the reference solution. B. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the peak due to cephalexin in the chromatogram obtained with the reference solution. Water Not more than 9.0% (Appendix 10.3). Use 0.3 g of the powdered tablets. Dissolution Apparatus: Basket. Medium: 900 ml of water. Rotation speed: 100 rpm. Time: 30 min. Procedure: After the specified time, withdraw a sample of the medium, filter, discard the first portion of the filtrate. Dilute the filtrate with water to obtain a solution containing about 20 µg of cephalexin per ml. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum at 262 nm, in a 1 cm cell, using water as a blank, in comparison with the reference solution of cephalexin RS having the same concentration in the same medium. Calculate the content of cephalexin, C16H17N3O4S, dissolved in each tablet using the absorbances obtained and the declared content of C16H17N3O4S in cephalexin RS. Tolerance: Not less than 80% (Q) of the stated amount of cephalexin is dissolved in 30 min. 239

VP V

CETIRIZINE DIHYDROCHLORIDE

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 1.0 g of sodium pentanesulfonate R in 1015 ml of a mixture of water - acetonitrile - methanol - triethylamine (850 : 100 : 50 : 15), adjust to pH 3.0 ± 0.1 with phosphoric acid R. Internal standard solution: Transfer 300 mg of 1-hydroxy benzotriazole, accurately weighed, to a 1000 ml volumetric flask, dissolve in 10 ml of methanol R, dilute with the mobile phase to volume and mix. Reference solution: Dissolve an accurately weighed quantity of cephalexin RS in water to obtain a stock solution having a concentration of about 1.0 mg per ml. Transfer 10.0 ml of the stock solution to a ground-glassstoppered conical flask, add 15.0 ml of the internal standard solution and mix well. Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Transfer an accurately weighed quantity of powdered tablets containing the equivalent of about 100 mg of cephalexin, to a 100 ml volumetric flask, add about 75 ml of water and sonicate for 15 minutes, dilute to volume with water, mix and filter. Transfer 10.0 ml of the filtrate to a ground-glass-stoppered conical flask, add 15.0 ml of the internal standard solution and mix. Chromatographic system: A column (25 cm × 4 mm) packed with stationary phase C (5 µm or 10 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution. The resolution factor between the internal standard and cephalexin peak is not less than 5, the relative standard deviation of the area ratios of cephalexin peak relative to the internal standard peak for the replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of cephalexin, C16H17N3O4S, in the tablets using the area ratios of cephalexin peak relative to the internal standard peak in the chromatograms obtained with the test solution, the reference solution and the declared content of C16H17N3O4S in cephalexin RS. Storage Packed in aluminum blister or airtight container. Store in a cool and dry place, protected from light, at a temperature not exceeding 30 °C. Action and use Cephalosporin antibacterial. Usual strength 250 mg; 500 mg. 240

CETIRIZINE DIHYDROCHLORIDE Cetirizini hydrochloridum

acid enantiomer

C21H25ClN2O3,2HCl  

M. 461.8

Cetirizine dihydrochloride is (RS)-2-[2-[4-[(4chlorophenyl)phenylmethyl]piperazin-1-yl]ethoxy]acetic acid dihydrochloride. It contains not less than 99.0% and not more than 101.0% of C21H25ClN2O3,2HCl, calculated with reference to the dried substance.

Characters White or almost white powder. Freely soluble in water, practically insoluble in acetone and in methylene chloride. Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of cetirizine dihydrochloride RS. B. Dissolve 20.0 mg in 50 ml of 0.1 M hydrochloric acid R and dilute to 100.0 ml with the same solvent. Dilute 10.0 ml of this solution to 100.0 ml with 0.1 M hydrochloric acid R. The ultraviolet absorption spectrum (Appendix 4.1) of the the solution in the range 210 nm to 350 nm, exhibits an absorption maximum at 231 nm. The specific absorbance at this maximum is 359 to 381. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Ammonia - methanol - methylene chloride (1 : 10 : 90). Test solution: Dissolve 10 mg of the substance to be examined in water and dilute to 5 ml with the same solvent. Reference solution (1): Dissolve 10 mg of cetirizine dihydrochloride RS in water and dilute to 5 ml with the same solvent. Reference solution (2): Dissolve 10 mg of chlorphenamine maleate RS in water and dilute to 5 ml with the same solvent. Mix 1 ml of the solution and 1 ml of reference solution (1). Procedure: Apply separately to the plate 5 µl of each solution. Develop over 2/3 of the plate. Allow the plate to dry in a current of cold air. Examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with the reference solution (2) shows two clearly separated spots. D. It gives reaction (A) of chlorides (Appendix 8.1).

VP V

Appearance of solution Solution S: Dissolve 1.0 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution BY7 (Appendix 9.3, method 2). pH 1.2 to 1.8 (Appendix 6.2). Determined on solution S. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: A 10% solution of sulfuric acid - water acetonitrile (0.4 : 6.6 : 93). Test solution: Dissolve 20 mg of the substance to be examined in the mobile phase and dilute to 100.0 ml with the mobile phase. Reference solution (1): Dissolve 2 mg of cetirizine dihydrochloride RS and 2 mg of cetirizine impurity A RS in the mobile phase and dilute to 50.0 ml with the mobile phase. Dilute 1.0 ml of the solution to 100.0 ml with the mobile phase. Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (3): Dissolve the contents of a vial of cetirizine for peak identification RS (containing impurities B, C, D, E and F) in 5.0 ml of the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 230 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: The run time is 3 times the retention time of cetirizine. Identification of impurities: Use the chromatogram supplied with cetirizine for peak identification RS and the chromatogram obtained with reference solution (3) to identify the peaks due to impurities B, C, D, E and F; use the chromatogram obtained with reference solution (1) to identify the peak due to impurity A. Relative retention with reference to cetirizine (retention time = about 10 min): impurity D = about 0.6; impurity B = about 0.8; impurity C = about 0.9; impurity E = about 1.2; impurity F = about 1.37; impurity A = about 1.42. System suitability: In the chromatogram obtained with reference solution (3), peak-to-valley ratio (Hp/Hv) is at least 5, where Hp = heigh above the baseline of the peak due to impurity C and Hv = height above the baseline of the lowest point of the curve separating this peak from the peak due to cetirizine.

CETIRIZINE DIHYDROCHLORIDE

Limits: Correction factors: for the calculation of content, multiply the peak areas of the following impurities by the corresponding correction factor: impurity A = 0.7; impurity C = 1.9; impurity D = 0.6; impurity E = 1.3; impurity F = 1.9; Impurities A, B, C, D, E, F: For each impurity, the area is not more than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.15%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.10%). Total peak area of all impurities is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.3%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Note: Impurity A: (RS)-1-[(4-chlorophenyl)phenylmethyl]piperazine. Impurity B: (RS)-2-[4-[(4-chlorophenyl)phenylmethyl]piperazin1-yl]acetic acid. Impurity C: (RS)-2-[2-[4-[(2-chlorophenyl)phenylmethyl]piperazin -1-yl]ethoxy]acetic acid. Impurity D: 1,4-bis[(4-chlorophenyl)phenylmethyl]piperazine. Impurity E: (RS)-2-[2-[2-[4-[(4-chlorophenyl)phenylmethyl] piperazin-1-yl]ethoxy]ethoxy]acetic acid (ethoxycetirizine). Impurity F: 2-[2-[4-(diphenylmethyl)piperazin-1-yl]ethoxy]acetic acid. Impurity G: (RS)-2-[4-[(4-chlorophenyl)phenylmethyl]piperazin1-yl]ethan-1-ol.

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.2% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.100 g of the substance to be examined in 70 ml of a mixture of water - acetone R (30 : 70). Titrate with 0.1 N sodium hydroxide VS to the 2nd point of inflexion. Determine the end-point potentiometrically (Appendix 10.2). Carry out a blank titration. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 15.39 mg of C21H27Cl3N2O3. Storage Protected from light. Action and use Histamine H1 receptor antagonist. Preparation Tablets. 241

VP V

CETIRIZINE TABLETS

CETIRIZINE TABLETS Tabellae Cetirizini Cetirizine tablets contain cetirizine hydrochloride. They are film-coated tablets. The tablets comply with the requirements stated under “Tablets”, “Coated tablets” item (Appendix 1.20), and with the following requirements.

Content of cetirizine hydrochloride, C21H27Cl3N2O3, 90.0% to 110.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Ammonia - methanol - methylene chloride (1 : 10 : 90). Test solution: Dissolve a quantity of the powdered tablets containing about 10 mg of cetirizine hydrochloride with 5 ml of water, filter. Reference solution (1): Dissolve 10 mg of cetirizine hydrochloride RS in water and dilute to 5 ml with the same solvent. Reference solution (2): Dissolve 10 mg of chlorphenamine maleate RS in water and dilute to 5 ml with the same solvent. To 1 ml of the resulting solution add 1 ml of reference solution (1) and mix well. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of about three fourths of the length of the plate. After removal of the plate, allow it to dry in air. Examine under ultraviolet light at 254 nm. The test is not valid unless the chromatogram obtained with reference solution (2) shows two clearly separated spots. The principal spot in the chromatogram obtained with the test solution corresponds in position, colour and size to that in the chromatogram obtained with reference solution (1). B. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the principal peak in the chromatogram obtained with the reference solution. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 1000 ml of water. Rotation speed: 50 rpm. Time: 30 min. Procedure: Examine by liquid chromatography (Appendix 5.3). Mobile phase and chromatographic system: Proceed as directed in the Assay. Test solution: After the specified time, withdraw a sample of the medium and filter. Reference solution: Dissolve cetirizine hydrochloride RS in water to obtain a solution having the same concentration as the test solution. 242

Tolerance: Not less than 75% (Q) of the stated amount of cetirizine hydrochloride, C21H27Cl3N2O3, is dissolved in 30 min.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - 0.01 M potassium dihydrogen phosphate (150 : 850). Test solution: Weigh 20 tablets (with the coating removed), calculate the average mass, and powder finely. Transfer an accurately weighed quantity of the powdered tablet containing the equivalent of about 20 mg of cetirizine hydrochloride to a 100 ml volumetric flask, add 50 ml of the mobile phase, mix well and sonicate for 5 minutes. Add the mobile phase to volume and mix. Filter though a 0.45 µm filter. Reference solution: The solution contains 0.02% of cetirizine hydrochloride RS in the mobile phase. Filter though a 0.45 µm filter. Chromatographic system: A column (25 cm × 4 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 230 nm. Flow rate: 1.5 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject the reference solution, the relative standard deviation of the peak areas for 6 replicate injections is not more than 2%. Inject alternately the reference solution and the test solution. Calculate the content of cetirizine hydrochloride, C21H27Cl3N2O3, in the tablets using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C21H27Cl3N2O3 in cetirizine hydrochloride RS. Storage Store in a cool and dry place, protected from light. Action and use Histamine H1-receptor antagonist. Usual strength 5 mg; 10 mg. CETOSTEARYL ALCOHOL Alcohol cetylicus et stearylicus Cetostearyl alcohol is a mixture of solid aliphatic alcohols. It contains not less than 40.0% of stearyl alcohol (C18H38O; Mr. 270.5) and the sum of the contents of cetyl alcohol (C16H34O; M. 242.4) and of stearyl alcohol is not less than 90.0%.

VP V

Characters White or pale yellow, wax-like mass, granules. Practically insoluble in water, ether, soluble in ethanol (90%) and in When melted, it is miscible with fatty paraffin and with melted wool fat.

CETYL ALCOHOL

plates, flakes or freely soluble in light petroleum. oils, with liquid

Identification Examine the chromatograms obtained in the assay. The two principal peaks in the chromatogram obtained with the test solution have similar retention times to the principal peaks in the chromatograms obtained with reference solution (1) and reference solution (2), respectively. Appearance of solution Dissolve 0.5 g in 20 ml of boiling ethanol (96%) R. The solution is clear (Appendix 9.2) and not more intensely coloured than reference solution B6 (Appendix 9.3, method 2). Melting point 49 °C to 56 °C (Appendix 6.7). Acid value Not more than 1.0 (Appendix 7.2). Hydroxyl value 208 to 228 (Appendix 7.4, method A). Iodine value Not more than 2.0 (Appendix 7.5). Determine on 2.0 g, dissolved in 25 ml of chloroform R. Saponification value Not more than 2.0 (Appendix 7.7). Determine on 2.0 g. Assay Examine by gas chromatography (Appendix 5.2). Test solution: Dissolve 0.100 g of the substance to be examined in ethanol R and dilute to 10.0 ml with the same solvent. Reference solution (1): Dissolve 60.0 mg of cetyl alcohol RS in ethanol R and dilute to 10.0 ml with the same solvent. Reference solution (2): Dissolve 40.0 mg of stearyl alcohol RS in ethanol R and dilute to 10.0 ml with the same solvent. Reference solution (3): Mix 1 ml of reference solution (1) and 1 ml of reference solution (2) and dilute to 10.0 ml with ethanol R. Chromatographic system: A column (3 m × 4 mm) packed with diatomaceous earth for gas chromatography R impregnated with 10% (m/m) of poly(dimethyl)siloxane R. Carrier gas: Nitrogen for chromatography R. Flow rate: 30 ml/min. Detector: A flame-ionisation detector. Maintaining the temperature of the column at 200 °C and that of the injection port and of the detector at 250 °C.

Volume of injection: 2 µl. Procedure: Inject separately each solution. Adjust the flow rate so that the resolution between the two principal peaks in the chromatogram obtained with the test solution is not less than 1.25. System suitability: The chromatogram obtained with reference solution (3) shows two principal peaks with a signal-to-noise ratio of at least 5. Determine the content of cetyl alcohol and of stearyl alcohol from the chromatogram obtained with the test solution, using the normalisation procedure. Identify the peaks by comparison with the chromatograms obtained with reference solution (1) and reference solution (2), respectively.

Storage Store in a well-closed container, protected from light. Action and use Excipients. CETYL ALCOHOL Alcohol cetylicus Cetyl alcohol is a mixture of solid alcohols, mainly hexadecan-1-ol (C16H34O; Mr 242.4), of animal or vegetable origin. It contains not less than 95.0% of C16H34O.

Characters White or almost white, unctuous mass, powder, flakes or granules. Practically insoluble in water, freely soluble or sparingly soluble in ethanol (96%). When melted, it is miscible with vegetable and animal oils, with liquid paraffin and with melted wool fat. Identification In the test for Assay, the principal peak in the chromatogram obtained with the test solution is similar in retention time to the principal peak in the chromatogram obtained with reference solution (1). Appearance of solution Dissolve 0.50 g of the substance to be examined in 20 ml of boiling ethanol (96%) R. Allow to cool. The solution is clear (Appendix 9.2) and not more intensely coloured than reference solution B6 (Appendix 9.3, method 2). Melting point 46 °C to 52 °C (Appendix 6.7). Acid value Not more than 1.0 (Appendix 7.2). Hydroxyl value 218 to 238 (Appendix 7.4, method A). 243

VP V

ACTIVATED CHARCOAL

Iodine value Not more than 2.0 (Appendix 7.5, method A). Dissolve 2.00 g in methylene chloride R and dilute to 25 ml with the same solvent. Saponification value Not more than 2.0 (Appendix 7.7). Assay Examine by gas chromatography (Appendix 5.2), use the normalisation procedure. Test solution: Dissolve 0.100 g of the substance to be examined in ethanol (96%) R and dilute to 10.0 ml with the same solvent. Reference solution (1): Dissolve 50 mg of cetyl alcohol RS in ethanol (96%) R and dilute to 5 ml with the same solvent. Reference solution (2): Dissolve 50 mg of stearyl alcohol R in ethanol (96%) R and dilute to 10 ml with the same solvent. Reference solution (3): Mix 1 ml of reference solution (1) and 1 ml of reference solution (2) and dilute to 10 ml with ethanol (96%) R. Chromatographic system: A column (30 m × 0.32 mm) coated with a film of poly (dimethyl) siloxane R (1 µm). Carrier gas: Helium for chromatography. Flow rate: 1 ml/min. Split ratio: 1 : 100. Temperature programme:

Column

Time (min)

Temperature (°C)

0 - 20 20 - 40

150 → 250 250

Injection port

250

Detector

250

Detector: A flame-ionisation detector. Volume of injection: 1 µl. Procedure: Inject the test solution and the reference solutions (1) and (3). System suitability: In the chromatogram obtained with reference solution (3), the resolution between the peaks due to cetyl alcohol and stearyl alcohol is at least 5.0. Calculate the content of C16H34O using the areas for the peak due to cetyl alcohol in chromatograms obtained with the test solution and reference solution (1).

Storage Store in an airtight container, protected from light. Action and use Excipient.

244

ACTIVATED CHARCOAL Carbo activatus Activated charcoal is obtained from vegetable matter by suitable carbonisation processes intended to confer a high adsorption power.

Characters A black, light powder free from grittiness. Practically insoluble in all usual solvents. Identification A. When heated to redness it burns slowly without a flame. B. It complies with the test for Adsorption power. Solution S: To 2.0 g of the substance to be examined in a conical flask with a ground-glass neck add 50 ml of dilute hydrochloric acid R. Boil gently under a reflux condenser for 1 h, filter and wash the filter with dilute hydrochloric acid R. Evaporate the combined filtrate and washings to dryness on a water-bath, dissolve the residue in 0.1 M hydrochloric acid R and dilute to 50.0 ml with the same acid, and mix. Acidity or alkalinity To 2.0 g of the substance to be examined add 40 ml of water and boil for 5 min. Cool, restore to the original volume with carbon dioxide-free water R and filter. Reject the first 20 ml of the filtrate. To 10  ml of the filtrate add 0.25 ml of bromothymol blue solution R and 0.25 ml of 0.02 M sodium hydroxide VS. The solution is blue. Not more than 0.75 ml of 0.02 M hydrochloric acid VS is required to change the colour of the indicator to yellow. Acid-soluble substances Not more than 3%. To 1.0 g of the substance to be examined add 25 ml of dilute nitric acid R and boil for 5 min. Filter whilst hot through a sintered-glass filter No.10 and wash with 10 ml of hot water. Evaporate the combined filtrate and washings to dryness on a water-bath, add to the residue 1 ml of hydrochloric acid R, evaporate to dryness again and dry the residue to constant mass at 100 °C to 105 °C. The residue weighs not more than 30 mg. Alkali-soluble coloured substances To 0.25 g of the substance to be examined add 10 ml of dilute sodium hydroxide solution R and boil for 1 min. Cool, filter and dilute the filtrate to 10 ml with water, and mix. The solution is not more intensely coloured than reference solution GY4 (Appendix 9.3, method 2). Alcohol-soluble substances Not more than 0.5%. To 2.0 g of the substance to be examined add 50 ml of ethanol (96%) R and boil under a reflux condenser for 10 min. Filter immediately, cool, and dilute to 50 ml with

VP V

ethanol (96%) R, and mix. The filtrate is not more intensely coloured than reference solution Y6 or BY6 (Appendix 9.3, method 2). Evaporate 40  ml of the filtrate to dryness and dry to constant mass at 100 °C to 105 °C. The residue weighs not more than 8 mg.

Fluorescent substances In an intermittent-extraction apparatus, treat 10.0 g of the substance to be examined with 100 ml of cyclohexane R1 for 2 h. Collect the liquid and dilute to 100 ml with cyclohexane R1, and mix. Examine in ultraviolet light at 365 nm. The fluorescence of the solution is not more intense than that of a solution of 83 µg of quinine R in 1000 ml of 0.005 M sulfuric acid R examined in the same manner. Sulfides To 1.0 g of the substance to be examined in a conical flask add 5 ml of 25% solution of hydrochloric acid R and 20 ml of water. Heat to boiling. The fumes released do not turn lead acetate paper R brown. Copper Not more than 25 ppm. Examine by atomic absorption spectrometry (Appendix 4.4, method 1). Test solution: Use solution S. Reference solutions: Prepare the reference solutions using copper standard solution (0.1% Cu) R and diluting with 0.1 M hydrochloric acid R. Measure the absorbance at 325.0 nm using a copper hollow-cathode lamp as source of radiation and an airacetylene flame. Lead Not more than 10 ppm. Examine by atomic absorption spectrometry (Appendix 4.4, method 1). Test solution: Use solution S. Reference solutions: Prepare the reference solutions using lead standard solution (100 ppm Pb) R and diluting with 0.1 M hydrochloric acid R. Measure the absorbance at 283.3 nm using a lead hollowcathode lamp as source of radiation and an air-acetylene flame. Depending on the apparatus the line at 217.0 nm may be used. Zinc Not more than 25 ppm. Examine by atomic absorption spectrometry (Appendix 4.4, method 1). Test solution: Use solution S. Reference solutions: Prepare the reference solutions using zinc standard solution (100 ppm Zn) R and diluting with 0.1 M hydrochloric acid R. Measure the absorbance at 214.0 nm using a zinc hollowcathode lamp as source of radiation and an air-acetylene flame.

CHLORAL HYDRATE

Loss on drying Not more than 15% (Appendix 9.6). (1.00 g; 120 °C; 4 h). Sulfated ash Not more than 5.0% (Appendix 9.9, method 2). Determined on 1.0 g. Adsorption power To 0.300 g of the substance to be examined in a 100 ml ground-glass-stoppered conical flask add 25.0 ml of a freshly prepared solution of 0.5 g of phenazone R in 50 ml of water. Shake thoroughly for 15 min. Filter and reject the first 5 ml of filtrate. To 10.0 ml of the filtrate add 1.0 g of potassium bromide R, 20 ml of dilute hydrochloric acid R and 0.1 ml of methyl red solution R. Titrate with 0.1 N potassium bromate VS until the red colour is discharged. Titrate slowly (1 drop every 15 seconds) towards the end of the titration. Carry out a blank titration using 10.0 ml of the phenazone solution. Calculate the quantity of phenazone adsorbed per 100 g of activated charcoal from the expression:

2.353 × (a − b) m

Where: a = number of millilitres of 0.1 N potassium bromate VS used for the blank. b = number of millilitres of 0.1 N potassium bromate VS used for the test. m = mass in grams of the substance to be examined. Not less than 40 g of phenazone is adsorbed per 100 g of activated charcoal, calculated with reference to the dried substance.

Microbial contamination Total aerobic microbial count: Not more than 103 CFU/g. Determined by plate count (Appendix 13.6). Storage Store in an airtight container. Action and use Absorbing material. CHLORAL HYDRATE Chlorali hydras

C2H3Cl3O2

M. 165.4

Chloral hydrate is 2,2,2-trichloroethane-1,1-diol. It contains not less than 98.5% and not more than 101.0% of C2H3Cl3O2. 245

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CHLORAMPHENICOL

Characters Colourless, transparent crystals; with a characteristic odour and a pungent taste. Very soluble in water, freely soluble in ethanol (96%). Identification Solution S: Dissolve 2.5 g of the substance to be examined in carbon dioxide-free water R and dilute to 25 ml with the same solvent. A. To 10 ml of solution S add 2 ml of 2 M sodium hydroxide R. The mixture becomes cloudy and, when heated, gives off an odour of chloroform. B. To 1 ml of solution S add 2 ml of sodium sulphide solution R. A yellow colour develops which quickly becomes reddishbrown. On standing for a short time, a red precipitate may be formed.

volume of 1 N sulfuric acid VS used in the first titration and two-fifteenths of the volume of 0.1 N silver nitrate VS used in the second titration. Each ml of 1 N sodium hydroxide VS is equivalent to 165.4 mg of C2H3Cl3O2.

Storage Store in an airtight container. Action and use Hypnotics. CHLORAMPHENICOL Chloramphenicolum

Appearance of solution Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). pH The pH of solution S is 3.5 to 5.5 (Appendix 6.2).

C11H12Cl2N2O5

Heavy metals Not more than 20 ppm (Appendix 9.4.8). Dilute 10 ml of solution S to 20 ml with water. 12 ml of the resulting solution complies with limit test for heavy metals, method 1. Prepare the reference using lead standard solution (1 ppm Pb) R.

Chloramphenicol is 2,2-dichloro-N-[(1R,2R)-2-hydroxy -1-(hydroxymethyl)-2-(4-nitrophenyl)ethyl] acetamide, produced by the growth of certain strains of Streptomyces venezuelae in a suitable medium or normally prepared by synthesis. It contains not less than 98.0% and not more than 102.0% of C11H12Cl2N2O5, calculated with reference to the dried substance.

Chlorides Not more than 0.01% (Appendix 9.4.5). 5 ml of solution S diluted to 15 ml with water complies with the limit test for chlorides.

Characters A white, greyish-white or yellowish-white, crystalline powder or crystals, needles or elongated plates. Slightly soluble in water, freely soluble in ethanol (96%) and in propylene glycol. A solution in ethanol is dextrorotatory and a solution in ethyl acetate is laevorotatory.

Chloral alcoholate Warm 1.0 g with 10 ml of 2 M sodium hydroxide R, filter and add 0.1 N iodine VS dropwise until a yellow colour is obtained. Allow to stand for 1 hour. No precipitate is formed. Non-volatile residue Not more than 0.1%. Evaporate 2.000 g on a water-bath. The residue weighs not more than 2 mg. Assay Dissolve 4.000 g in 10 ml of water and add 40.0 ml of 1 N sodium hydroxide VS. Allow to stand for exactly 2 minutes and titrate with 1 N sulfuric acid VS, using 0.1 ml of phenolphthalein solution R as indicator. Titrate the neutralised solution with 0.1 N silver nitrate VS, using 0.2 ml of potassium chromate solution R as indicator. Calculate the number of millilitres of 1 N sodium hydroxide VS used by deducting from the volume of 1 N sodium hydroxide VS, added at the beginning of the titration, the 246

M. 323.1

Identification Apply one of the two following identifications: First identification: A, B. Second identification: B, C, D, E. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum obtained with chloramphenicol RS. B. Melting point: 149 °C to 153 °C (Appendix 6.7). C. In the test for Related substances, the principal spot in the chromatogram obtained with 1 µl of the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (1). D. Dissolve about 10 mg in 1 ml of ethanol (50%), add 3 ml of a 1% solution of calcium chloride R and 50 mg of zinc powder R and heat on a water-bath for 10 min. Filter the hot solution and allow to cool. Add 0.1 ml of benzoyl

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chloride R and shake for 1 min. Add 0.5 ml of a 10.5% solution of ferric chloride R and 2 ml of chloroform R and shake. The aqueous layer is coloured light violet-red to purple. E. To 50 mg in a porcelain crucible add 0.5 g of anhydrous sodium carbonate R. Heat over an open flame for 10 min, allow to cool. Dissolve the residue in 5 ml of 2 M nitric acid R and filter. To 1 ml of the filtrate add 1 ml of water. The solution gives reaction A of chlorides (Appendix 8.1).

Acidity or alkalinity To 0.1 g add 20 ml of carbon dioxide-free water R, shake and add 0.1 ml of bromothymol blue solution R. Not more than 0.1 ml of 0.02 N hydrochloric acid VS or 0.02 N sodium hydroxide VS is required to change the colour of the indicator. Specific optical rotation +18.5° to +20.5° (Appendix 6.4). Dissolve 1.50 g in ethanol R and dilute to 25.0 ml with the same solvent. Related substances Not more than 0.5%. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Chloroform - methanol - water (90 : 10 : 1). Test solution: Dissolve 0.10 g of the substance to be examined in acetone R and dilute to 10 ml with the same solvent. Reference solution (1): Dissolve 0.10 g of chloramphenicol RS in acetone R and dilute to 10 ml with the same solvent. Reference solution (2): Dilute 0.5 ml of reference solution (1) to 100 ml with acetone R. Procedure: Apply separately to the plate 1 µl and 20 µl of the test solution, 1 µl of the reference solution (1) and 20 µl of the reference solution (2). Develop over a path of 15 cm. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with 20 µl of the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with the reference solution (2) (0.5%). Chlorides Not more than 100 ppm (Appendix 9.4.5). To 1.00 g add 20 ml of water and 10 ml of nitric acid R and shake for 5 min. Filter through a filter paper previously washed by filtering 5 ml portions of water until 5 ml of filtrate no longer becomes opalescent on addition of 0.1 ml of nitric acid R and 0.1 ml of a 4.25% solution of silver nitrate R. 15 ml of the filtrate complies with the limit test for chlorides. Loss on drying Not more than 0.5% (Appendix 9.6). (1.0 g; 100 °C to 105 °C).

CHLORAMPHENICOL CAPSULES

Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determine on 2.0 g. Pyrogens If intended for use in the manufacture of a parenteral dosage form without a further appropriate procedure for the removal of pyrogens, it complies with the test for pyrogens (Appendix 13.4). Inject per kilogram of the rabbit’s weight 2.5 ml of a solution in water containing 2 mg of the substance to be examined per millilitre. Assay Dissolve 0.100 g in water and dilute to 500.0 ml with the same solvent. Dilute 10.0 ml of this solution to 100.0 ml with water. Measure the absorbance (Appendix 4.1) of the solution at the maximum at 278 nm. Calculate the content of C11H12Cl2N2O5 due to A (1%, 1 cm), taking 297 as the value of A (1%, 1 cm) at the maximum at 278 nm. Storage Store protected from light. If the substance is sterile, store in a sterile, airtight, tamper-proof container. Labelling The label states stored condition and states, where applicable, that the substance is apyrogenic. Action and use Antibacterial. Preparations Tablets; capsules; eye drops. CHLORAMPHENICOL CAPSULES Capsulae Chloramphenicoli Chloramphenicol capsules contain chloramphenicol. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of chloramphenicol, C11H12Cl2N2O5, 95.0% to 105.0% of the stated amount. Identification To a quantity of the contents of the capsules containing 0.1 g of chloramphenicol add 10 ml of ethanol R, shake and filter. Evaporate the filtrate to dryness. The residue complies with the following tests. A. Carry out the method for thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel GF254. Mobile phase: Chloroform - methanol - water (90 : 10 : 1). 247

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CHLORAMPHENICOL EAR DROPS

Test solution: A 1% solution of the residue in ethanol R. Reference solution: A 1% solution of chloramphenicol RS in ethanol R. Procedure: Apply separately to the plate 1 µl of each solution. After developing over a path of 15 cm, remove the plate, allow it to dry in air. Examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in Rf value to that in the chromatogram obtained with the reference solution. B. Dissolve 10 mg in 2 ml of ethanol (50%), add 4.5 ml of 1 M sulphuric acid R and 50 mg of zinc powder R and allow to stand for 10 min. Decant the supernatant liquid or filter if necessary. Cool the resulting solution in ice and add 0.5 ml of 10% sodium nitrite solution R and, after 2 min, 1 g of urea R followed by 1 ml of 2-naphthol alkaline solution R and 2 ml of 10 M sodium hydroxide R, a red colour is produced. Repeat the test omitting the zinc powder, no red colour is produced.

Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 900 ml of 0.1 M hydrochloric acid R. Rotation speed: 100 rpm. Time: 45 min. Procedure: After the specified time, withdraw a sample of the medium and filter, discarding the first portion of the filtrate. Measure the absorbance of the filtered sample, suitably diluted if necessary, at the maximum at 278 nm (Appendix 4.1) using an 1 cm cell and medium as the blank. Calculate the content of chloramphenicol, C11H12Cl2N2O5, in the medium taking 297 as the value of A(1%, 1 cm) at the maximum at 278 nm. Tolerance: Not less than 70% (Q) of the labelled amount of chloramphenicol, C11H12Cl2N2O5, is dissolved in 45 min. 2-Amino-1-(4-nitrophenyl)propane-1,3-diol Carry out the method for liquid chromatography (Appendix 5.3). Mobile phase: 0.21% solution of sodium pentanesulphonate - acetonitrile - glacial acetic acid (85 : 15 : 1). Test solution: Shake a quantity of the contents of the capsules containing 40 mg of chloramphenicol with 100 ml of the mobile phase for 10 min, add sufficient mobile phase to produce 200 ml, mix and filter. Reference solution: A 0.0002% solution of 2-amino-1-(4nitrophenyl) propane-1,3-diol RS in the mobile phase. Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase C (Nucleosil C18 is suitable) (5 µm). Detector: A spectrophotometer set at 272 nm. Flow rate: 2.0 ml/min. Volume of injection : 20 µl. 248

Procedure: Inject separately the reference solution and the test solution. In the chromatogram obtained with the test solution the area of any peak corresponding to 2-amino-1-(4-nitrophenyl) propane-1,3-diol is not greater than the area of the peak in the chromatogram obtained with the reference solution.

Assay Weigh 20 capsules, calculate the average weight of the capsule contents, and finely powder. Weigh accurately a quantity of the powder, equivalent to about 40 mg of chloramphenicol, add 4 ml of ethanol R, dilute to 200.0 ml with water. Filter, discard 20 ml of the first portion. Dilute 10.0 ml to 100.0 ml with water and measure the absorbance of the resulting solution at the maximum at 278 nm (Appendix 4.1) using an 1 cm cell and water as the blank. Calculate the content of C11H12Cl2N2O5 taking 297 as the value of A(1%, 1 cm) at the maximum at 278 nm. Storage Store in an airtight container, in a dry place, protected from light. Action and use Antibacterial. Usual strength 250 mg. CHLORAMPHENICOL EAR DROPS Auricularia Chloramphenicoli Chloramphenicol ear drops are a solution of chloramphenicol in a suitable vehicle. The ear drops comply with the requirements stated under “Ear drops and sprays” (Appendix 1.16) and with the following requirements.

Content of chloramphenicol, C11H12Cl2N2O5, 90.0% to 110.0% of the stated amount. Characters A clear liquid, colourless to slightly yellow. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Chloroform - methanol - water (90 : 10 : 1). Test solution: Dilute a volume of the ear drops containing 100 mg of chloramphenicol to 10 ml with ethanol (96%). Reference solution: Contains 1% of chloramphenicol RS in ethanol (96%). Procedure: Apply separately to the plate 1 µl of each solution. Develop over a path of 15 cm. After removal of the plate, allow to dry in the air. Examine under ultraviolet light (254 nm).

VP V

The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution . B. Dilute a volume of the ear drops containing 50 mg of chloramphenicol to 10 ml with ethanol (50%). To 2 ml add 4.5 ml of 1 M sulfuric acid R and 50 mg of zinc powder R and allow to stand for 10 minutes, decant the supernatant liquid or filter if necessary. Cool the resulting solution in ice and add 0.5 ml of 10% solution of sodium nitrite R and, after 2 minutes, 1 g of urea R followed by 1 ml of 2-naphthol solution R and 2 ml of 10 M sodium hydroxide R; a red colour is produced. Repeat the test omitting the zinc powder, no red colour is produced.

2-Amino-1-(4-nitrophenyl)propane-1,3-diol Examine by liquid chromatography (Appendix 5.3). Mobile phase: Sodium pentanesulfonate 0.21% - acetonitrile - glacial acetic acid (85 : 15 : 1). Test solution: Dilute a volume of the ear drops with the mobile phase to obtain a 0.050% solution of chloramphenicol. Reference solution: Contains 0.0025% of 2-amino-1(4-nitrophenyl) propane-1,3-diol RS in mobile phase. Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase C (5 µm, Nucleosil C18 is suitable). Detector: A spectrophotometer set at 272 nm. Flow rate: 2.0 ml/min. Volume of injection: 10 µl. Procedure: Inject separately the test solution and the reference solution. In the chromatograph obtained with the test solution, the area of any peak corresponding to 2-amino-1-(4-nitrophenyl) propane-1,3-diol is not greater than the area of the peak in the chromatogram obtained with the reference solution. Assay Dilute a volume of the ear drops containing 25 mg of chloramphenicol to 250.0 ml with water. Dilute 10.0 ml of the resulting solution to 100.0 ml with water and measure the absorbance of the obtained solution at the maximum at 278 nm, in a 1 cm cell, using water as a blank (Appendix 4.1). Calculate the content of chloramphenicol, C11H12Cl2N2O5 taking 297 as the value of A(1%, 1 cm) at the maximum at 278 nm. Storage Store in a airtight container, in a cool and dry place, and protected from light. Action and use Antibacterial. Usual strength 2.5%.

CHLORAMPHENICOL EYE DROPS

CHLORAMPHENICOL EYE DROPS Collyrium Chloramphenicoli Chloramphenicol eye drops are a sterile solution of chloramphenicol in purified water. The eye drops comply with the requirements stated under “Eye drops” (Appendix 1.14) and with the following requirements.

Content of chloramphenicol, C11H12Cl2N2O5, 90.0% to 110.0% of the stated amount. Characters Clear, colourless solution. Identification To a volume containing 50 mg of chloramphenicol add 15 ml of water and extract with four 25 ml quantities of ether R. Combine the extracts and evaporate to dryness. The dried residue complies with with the following tests. A. Carry out the method for thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel GF254. Mobile phase: Chloroform - methanol - water (90 : 10 : 1). Test solution: A 1% solution of the residue in ethanol R. Reference solution: A 1% solution of chloramphenicol RS in ethanol R Procedure: Apply separately to the plate 1 µl of each solution. After developing over a path of 15 cm, remove the plate, allow it to dry in air. Examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in Rf value to that in the chromatogram obtained with the reference solution. B. Dissolve 10 mg in 2 ml of ethanol (50%), add 4.5 ml of 1 M sulphuric acid R and 50 mg of zinc powder R and allow to stand for 10 min. Decant the supernatant liquid or filter if necessary. Cool the resulting solution in ice and add 0.5 ml of 10% sodium nitrite solution R and, after 2 min, 1 g of urea R followed by 1 ml of 2-naphthol alkaline solution R and 2 ml of 10 M sodium hydroxide R, a red colour is produced. Repeat the test omitting the zinc powder, no red colour is produced. pH 7.0 to 7.5 (Appendix 6.2). 2-Amino-1-(4-nitrophenyl)propane-1,3-diol Carry out the method for liquid chromatography (Appendix 5.3). Mobile phase:A0.21% solution of sodium pentanesulphonate - acetonitrile - glacial acetic acid (85 : 15 : 1). Test solution: Dilute the eye drops with the mobile phase to obtain a solution containing 0.050% of chloramphenicol. Reference solution: A 0.0040% solution of 2-amino-1-(4nitrophenyl)propane-1,3-diol RS in the mobile phase. 249

CHLORAMPHENICOL TABLETS

Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase C (Nucleosil C18 is suitable) (5 µm). Detector: A spectrophotometer set at 272 nm. Flow rate: 2.0 ml/min. Volume of injection : 10 µl. Procedure: Inject separately the reference solution and the test solution. In the chromatogram obtained with the test solution the area of any peak corresponding to 2-amino-1-(4-nitrophenyl) propane-1,3-diol is not greater than the area of the peak in the chromatogram obtained with the reference solution.

Assay Dilute a volume of the eye drops containing 20 mg of chloramphenicol to 200.0 ml with water. Dilute 10.0  ml to 100.0 ml with water and measure the absorbance of the resulting solution at the maximum at 278 nm (Appendix 4.1) using an 1 cm cell and water as the blank. Calculate the content of C11H12Cl2N2O5 taking 297 as the value of A (1%, 1 cm) at the maximum at 278 nm. Storage Store in an airtight container, protected from light, in a cool place. Action and use Antibacterial. Usual strength 0.4%, 0.5%. CHLORAMPHENICOL TABLETS Tabellae Chloramphenicoli Chloramphenicol tablets contain chloramphenicol. The tablets comply with the requirements stated under Tablets (Appendix 1.20) and with the following requirements.

Content of chloramphenicol, C11H12Cl2N2O5, 95.0% to 105.0% of the stated amount. Identification To a quantity of the powdered tablets containing 0.1 g of chloramphenicol add 10 ml of ethanol R, shake and filter. Evaporate the filtrate. The residue complies with the following tests. A. Carry out the method for thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel GF254. Mobile phase: Chloroform - methanol - water (90 : 10 : 1). Test solution: A 1% solution of the residue in ethanol R. Reference solution: A 1% solution of chloramphenicol RS in ethanol R Procedure: Apply separately to the plate 1 µl of each solution. After developing over a path of 15 cm, remove 250

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the plate, allow it to dry in air. Examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in Rf value to that in the chromatogram obtained with the reference solution. B. Dissolve 10 mg in 2 ml of ethanol (50%), add 4.5 ml of 1 M sulphuric acid R and 50 mg of zinc powder R and allow to stand for 10 min. Decant the supernatant liquid or filter if necessary. Cool the resulting solution in ice and add 0.5 ml of 10% sodium nitrite solution R and, after 2 min, 1 g of urea R followed by 1 ml of 2-naphthol alkaline solution R and 2 ml of 10 M sodium hydroxide R, a red colour is produced. Repeat the test omitting the zinc powder, no red colour is produced.

Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 900 ml of 0.1 M hydrochloric acid R. Rotation speed: 100 rpm. Time: 45 min. Procedure: After the specified time, withdraw a sample of the medium and filter, discarding the first portion of the filtrate. Measure the absorbance of the filtered sample, suitably diluted if necessary, at the maximum at 278 nm (Appendix 4.1) using an 1 cm cell and medium as the blank. Calculate the content of chloramphenicol, C11H12Cl2N2O5, in the medium taking 297 as the value of A (1%, 1 cm) at the maximum at 278 nm. Tolerance: Not less than 70% (Q) of the labelled amount of chloramphenicol, C11H12Cl2N2O5, is dissolved in 45 min. 2-Amino-1-(4-nitrophenyl)propane-1,3-diol Carry out the method for liquid chromatography (Appendix 5.3). Mobile phase: 0.21% solution of sodium pentanesulphonate - acetonitrile - glacial acetic acid (85 : 15 : 1). Test solution: Shake a quantity of the contents of the capsules containing 40 mg of chloramphenicol with 100 ml of the mobile phase for 10 min, add sufficient mobile phase to produce 200 ml, mix and filter. Reference solution: A 0.0002% solution of 2-amino-1(4-nitrophenyl)propane-1,3-diol RS in the mobile phase. Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase C (Nucleosil C18 is suitable) (5 µm). Detector: A spectrophotometer set at 272 nm. Flow rate: 2.0 ml/min. Volume of injection : 20 µl. Procedure: Inject separately the reference solution and the test solution. In the chromatogram obtained with the test solution the area of any peak corresponding to 2-amino-1-(4-nitrophenyl) propane-1,3-diol is not greater than the area of the peak in the chromatogram obtained with the reference solution.

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CHLORAMPHENICOL AND DEXAMETHASONE SODIUM PHOSPHATE CREAM

Assay Examine by ultraviolet and visible absorption spectrophotometry (Appendix 4.1). Weigh 20 tablets, calculate the average mass, powder finely. Weigh a quantity of the powdered tablets containing 40 mg of chloramphenicol, add 4 ml of ethanol R, dilute to 200.0 ml with water. Filter, discard the first 20 ml of the filtrate. Dilute 10.0 ml to 100.0 ml with water and measure the absorbance of the resulting solution at the maximum at 278 nm using an 1 cm cell and water as the blank. Calculate the content of C11H12Cl2N2O5 taking 297 as the value of A(1%, 1 cm) at the maximum at 278 nm. Storage Store in an airtight container, in a dry place, protected from light. Action and use Antibacterial. Usual strength 250 mg. CHLORAMPHENICOL AND DEXAMETHASONE SODIUM PHOSPHATE CREAM Cremoris Chloramphenicoli et Dexamethasoni natrii phosphas Chloramphenicol and dexamethasone sodium phosphate cream is a topical cream containing chloramphenicol and dexamethasone sodium phosphate. The cream complies to requirements stated under "Topical and mucosal semi-solid preparation" (Appendix 1.12) and with the following requirements.

Content of chloramphenicol, C11H12Cl2N2O5, 90.0% to 130.0% of the stated amount. Content of dexamethasone sodium phosphate, C22H28FNa2O8P, 90.0% to 115.0% of the stated amount. Characters A off white, smooth, soft and homogeneous cream. Identification Transfer a quantity of the substance to be examined containing about 50 mg of chloramphenicol into a separating funnel with 50 ml of ether R, add 10 ml of water, shake well and allow to separate absolutely. Repeat with three portions of 25 ml of ether R. Combine the ether layer and evaporate to dryness. Use the residue for test A and the water layer for test B as follows: A. Chloramphenicol identification Examine by thin layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254.

Mobile phase: Chloroform - methanol - water (90 : 10 : 1). Test solution: A 1% solution of the residue in ethanol R. Reference solution: A 1% solution of chloramphenicol RS in ethanol R. Procedure: Apply separately to the plate 1 µl of each solution. Develop over a path of about 15 cm. Remove the plate, allow it to dry in air. Examine under ultraviolet light at 254 nm. In the chromatogram obtained with the test solution, the Rf value of the principal spot is similar to that of the spot in the chromatogram obtained with the reference solution. B. Dexamethasone sodium phosphate identification Examine by thin layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Butanol - glacial acetic acid - water (60: 20 : 20). Test solution: Dilute the obtained solution above with methanol R to obtain a solution having a concentration of dexamethasone sodium phosphate of about 0.05% in methanol R. Reference solution (1): A 0.05% solution of dexamethasone sodium phosphate in methanol R. Reference solution (2): A solution containing 0.05% of each of dexamethasone sodium phosphate and prednisolone sodium phosphate in methanol R. Procedure: Apply separately to the plate 10 µl of each solution. After development, remove the plate, allow it to dry in air. Heat at 110 oC for 10 min, spray the hot plate with ethanolic sulfuric acid solution R and heat at 120 °C for 10 min. Allow to cool and examine in daylight and under ultraviolet light at 365 nm. In the chromatogram obtained with the test solution by each method of visualisation, the principal spot is similar in position, colour and size to that of the spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) exhibits two spots which may, however, not be completely separated. B. In the Assay, the retention times of two principal peaks in the chromatogram obtained with the test solution are similar to those of the chloramphenicol and dexamethasone sodium phosphate peaks in the chromatogram obtained with the reference solution.

Assay Examine by liquid chromatography (Appendix 5.3) Mobile phase: 0.01 M potassium dihydrogen phosphate methanol (55 : 45). Make adjustments if necessary. Reference solution (prepare immediately before use): Dissolve dexamethasone sodium phosphate RS and chloramphenicol RS in the mobile phase to obtain a solution having a known concentration of about 20 µg/ml of dexamethasone sodium phosphate and 20C µg/ml of chloramphenicol (C is the ratio of chloramphenicol and dexamethasone sodium phosphate according to the label), filter. 251

CHLORAMPHENICOL AND DEXAMETHASONE SODIUM PHOSPHATE EYE DROPS

Test solution: Weigh accurately a quantity of the cream containing about 10 mg of dexamethasone sodium phosphate, transfer into a 200-ml beaker, add 70 ml of methanol R, put in a boiled water-bath, occasionally shake to dissolve, transfer into a 100-ml volumetric flask containing 20 ml of methanol R. Allow to cool to room temperature, add methanol R to volume, mix well. Transfer 10.0 ml of this solution into a 50-ml volumetric flask, dilute to volume with the mobile phase, mix well and filter. Chromatographic system: A column (25 cm × 4.0 mm) packed with stationary C (5 µm). Nucleosil C18 column is suitable. Detector: A spectrophotometer set at 280 nm for chloramphenicol and 254 for dexamethasone sodium phosphate. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution, the relative standard deviations for six replicate injections of peak areas are not more than 2.0%, the resolution factor between the chloramphenicol and dexamethasone sodium phosphate peaks is not less than 2.5 Inject alternately the reference solution and the test solution. Calculate the content of chloramphenicol, C11H12Cl2N2O5 and of dexamethasone sodium phosphate, C22H28FNa2O8P, in 1 g of cream using the peak areas in the chromatogram obtained with the test solution, the reference solution, the declared content of C11H12Cl2N2O5 in chloramphenicol RS and the declared content of C22H28FNa2O8P in dexamethasone sodium phosphate RS.

Storage Store in a tight container, in a cool place and protect from light. Action and use Topical antibiotic and anti-inflammatory drug. Preparations 0.05% of dexamethasone sodium phosphate. 2% of chloramphenicol. CHLORAMPHENICOL AND DEXAMETHASONE SODIUM PHOSPHATE EYE DROPS Collyrium Chloramphenicoli et Dexamethasoni natrii phosphas Chloramphenicol and dexamethasone sodium phosphate eye drops is a sterile solution containing chloramphenicol and dexamethasone sodium phosphate in water for injection. The eye drops comply to requirements stated under “Eye drops” (Appendix 1.14) and with the following requirements. 252

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Content of chloramphenicol, C11H12Cl2N2O5, 90.0% to 130.0% of the stated amount. Content of dexamethasone sodium phosphate, C22H28FNa2O8P, 90.0% to 115.0% of the stated amount. Characters A clear and colourless solution. Identification Transfer a quantity of the substance to be examined containing about 50 mg of chloramphenicol into a separating funnel, extract with four portions of 25 ml of ether R. Combine the ether extract and evaporate to dryness. Use the residue for the test A and the water layer for the test B as follow: A. Chloramphenicol identification. Examine by thin layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Chloroform - methanol - water (90 : 10 : 1). Test solution: A 1% solution of the residue in ethanol R. Reference solution: A 1% solution of chloramphenicol RS in ethanol R. Procedure: Apply separately to the plate 1 µl of each solution. Develop over a path of about 15 cm. Remove the plate, allow it to dry in air. Examine under ultraviolet light at 254 nm. In the chromatogram obtained with the test solution, the Rf value of the principal spot is similar to that of the spot in the chromatogram obtained with the reference solution. B. Dexamethasone sodium phosphate identification Examine by thin layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Butanol - glacial acetic acid - water (60 : 20 : 20). Test solution: Dilute the water solution obtained above with methanol R to obtain a solution having a concentration of about 0.05% of dexamethasone sodium phosphate in methanol R. Reference solution (1): A 0.05% solution of dexamethasone sodium phosphate in methanol R. Reference solution (2): A solution containing 0.05% of each of dexamethasone sodium phosphate and 0.05% of prednisolone sodium phosphate in methanol R. Procedure: Apply separately to the plate 10 µl of each solution. After development, remove the plate, allow it to dry in air. Heat at 110oC for 10 min, spray the hot plate with ethanolic sulfuric acid solution R and heat at 120 °C for 10 min. Allow it to cool and examine in daylight and under ultraviolet light at 365 nm. In the chromatogram obtained with the test solution by each method of visualisation, the principal spot is similar in position, colour and size to that of the spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) exhibits two spots which may, however, not be completely separated.

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C. In the Assay, the retention times of two principal peaks in the chromatogram obtained with the test solution are similar to those of the chloramphenicol and dexamethasone sodium phosphate peaks in the chromatogram obtained with the reference solution.

CHLORAMPHENICOL PALMITATE

CHLORAMPHENICOL PALMITATE Chloramphenicoli palmitas

pH 6.5 to 8.0 (Appendix 6.2). Assay Examine by liquid chromatography (Appendix 5.3) Mobile phase: 0.01 M potassium dihydrogen phosphate methanol (55 : 45). Make adjustments if necessary. Reference solution (prepare immediately before use): Dissolve dexamethasone sodium phosphate RS and chloramphenicol RS in the mobile phase to obtain a solution having a known concentration of about 100 µg/ml of dexamethasone sodium phosphate and 400 µg/ml of chloramphenicol, filter. Test solution: To an accurate quantity of the eye drops containing about 10 mg of dexamethasone sodium phosphate, dilute to 100 ml with the mobile phase, mix well and filter. Chromatographic system: A column (25 cm × 4.0 mm) packed with stationary phase C (5 µm). Nucleosil C18 column is suitable. Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution, the relative standard deviation of the peak areas for six replicate injections is not more than 2.0%, the resolution factor between chloramphenicol and dexamethasone sodium phosphate peaks is not less than 2.5 (chloramphenicol is eluted before dexamethasone sodium phosphate). Inject alternately the reference solution, the test solution. Calculate the content of chloramphenicol, C11H12Cl2N2O5 and of dexamethasone sodium phosphate, C22H28FNa2O8P, in the eye drops using the peak areas in the chromatogram obtained with the test solution, the reference solution and the declared content of C11H12Cl2N2O5 in chloramphenicol RS and the declared content of C22H28FNa2O8P in dexamethasone sodium phosphate RS. Storage Store in a tight container, protected from light. Action and use Antibiotic and anti-inflammatory. Usual strength 0.4% of chloramphenicol and 0.1% of dexamethasone sodium phosphate.

C27H42Cl2N2O6

M. 561.6

Chloramphenicol palmitate is (2R,3R)-2-[(dichloroacetyl) amino]-3-hydroxy-3-(4-nitrophenyl)propyl hexadecanoate. It contains not less than 98.0% and not more than 102.0% of C27H42Cl2N2O6, calculated with reference to the dried substance.

Characters A white or almost white, fine powder. Practically insoluble in water, freely soluble in acetone, sparingly soluble in ethanol (96%), very slightly soluble in n-hexane. It melts at 87 °C to 95 °C. It shows polymorphism. The thermodynamically stable form has low bioavailability following oral administration. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silanised silica gel H. Mobile phase: 10.0% solution of ammonium acetate ethanol (96%) (30 : 70). Test solution: Dissolve 50 mg of the substance to be examined in a mixture of 1 ml of 1 M sodium hydroxide R and 5 ml of acetone R and allow to stand for 30 min. Add 1.1 ml of 1 M hydrochloric acid R and 3 ml of acetone R. Reference solution (1): Dissolve 10 mg of chloramphenicol RS in acetone R and dilute to 5 ml with the same solvent. Reference solution (2): Dissolve 10 mg of palmitic acid R in acetone R and dilute to 5 ml with the same solvent. Reference solution (3): Dissolve 10 mg of the substance to be examined in acetone R and dilute to 5 ml with the same solvent. Procedure: Apply separately to the plate 4 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in air and spray with a solution containing 0.02 % of dichlorofluorescein R and 0.01% of rhodamine B R in ethanol (96%) R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The chromatogram obtained with the test solution shows three spots corresponding in position to the principal spots in the chromatograms obtained with reference solutions (1), (2) and (3). B. Dissolve 0.2 g in 2 ml of pyridine R, add 2 ml of a 10% solution of potassium hydroxide R. Heat on a water-bath. A red colour is produced. C. Dissolve about 10 mg in 5 ml of ethanol (96%) R and add 4.5 ml of dilute sulphuric acid R and 50 mg of 253

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CHLORAMPHENICOL SODIUM SUCCINATE

zinc powder R. Allow to stand for 10 min, decant the supernatant liquid or filter if necessary. Cool the solution in iced water and add 0.5 ml of a 10% solution of sodium nitrite R. Allow to stand for 2 min, add 1 g of urea R, 2 ml of 10 M sodium hydroxide R and 1 ml of β-naphthol solution R. A red colour develops.

Acidity Dissolve 1.0 g in 5 ml of a mixture of equal volumes of ethanol (96%) R and ether R, warming to 35 °C. Add 0.2 ml of phenolphthalein solution R. Not more than 0.4 ml of 0.1 N sodium hydroxide VS is required to produce a pink colour persisting for 30 s. Specific optical rotation +22.5° to +25.5° (Appendix 6.4). Dissolve 1.25 g in ethanol (96%) R and dilute to 25.0 ml with the same solvent. Free chloramphenicol Not more than 450 ppm. Dissolve 1.0 g, with gentle heating, in 80 ml of xylene R. Cool and shake with 3 quantities, each of 15 ml, of water. Dilute the combined aqueous extracts to 50 ml with water and shake with 10 ml of toluene R. Allow to separate and discard the toluene layer. Centrifuge a portion of the aqueous layer and measure the absorbance (A) (Appendix 4.1) at the maximum at 278 nm. The blank solution is prepared by repeating the above procedure without the substance to be examined. The absorbance of the blank solution has to be not greater than 0.05. Calculate the content of free chloramphenicol in parts per million from the expression: A × 10 4 5.96

Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Cyclohexane - chloroform - methanol (50 : 40 : 10). Test solution: Dissolve 0.1 g of the substance to be examined in acetone R and dilute to 10 ml with the same solvent. Reference solution (1): Dissolve 20 mg of chloramphenicol palmitate isomer RS in acetone R and dilute to 10 ml with the same solvent. Dilute 1 ml of this solution to 10 ml with acetone R. Reference solution (2): Dissolve 20 mg of chloramphenicol dipalmitate RS in acetone R and dilute to 10 ml with the same solvent. Dilute 1 ml of this solution to 10 ml with acetone R. Reference solution (3): Dissolve 5 mg of chloramphenicol RS in acetone R and dilute to 10 ml with the same solvent. Dilute 1 ml of this solution to 10 ml with acetone R. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. 254

In the chromatogram obtained with the test solution, any spots due to chloramphenicol palmitate isomer and chloramphenicol dipalmitate are not more intense than the corresponding spots in the chromatograms obtained with reference solutions (1) and (2) respectively (2.0%); and any spot, apart from the principal spot and the spots due to chloramphenicol palmitate isomer and chloramphenicol dipalmitate, is not more intense than the principal spot in the chromatogram obtained with reference solution (3) (0.5%).

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 80 °C; diphosphorus pentoxide; pressure not exceeding 0.1 kPa; 3 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 90.0 mg in ethanol (96%) R and dilute to 100.0 ml with the same solvent. Dilute 10.0 ml of this solution to 250.0 ml with ethanol (96%) R. Measure the absorbance (Appendix 4.1) of the solution at the maximum at 271 nm. Calculate the content of C27H42Cl2N2O6 due to A (1%, 1 cm), taking 178 as the value of A (1%, 1 cm) at the maximum at 271 nm. Storage Store protected from light. Action and use Antibacterial. Preparations Tablets; capsules; powder for oral suspension. CHLORAMPHENICOL SODIUM SUCCINATE Chloramphenicoli natrii succinas

C15H15Cl2N2NaO8

M. 445.2

Chloramphenicol sodium succinate is a mixture in variable proportions of sodium (2R,3R)-2-[(dichloroacetyl) amino]-3-hydroxy-3-(4-nitrophenyl)propyl butanedioate (3 isomer) and of sodium (1R,2R)-2-[(dichloroacetyl) amino]-3-hydroxy-1-(4-nitrophenyl)propyl butanedioate (1 isomer). It contains not less than 98.0% and not

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more than 102.0% of C15H15Cl2N2NaO8, calculated with reference to the anhydrous substance.

Characters A white or yellowish-white powder, hygroscopic. Very soluble in water, freely soluble in ethanol (96%). Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: 2 M acetic acid - methanol - chloroform (1 : 14 : 85). Test solution: Dissolve 20 mg of the substance to be examined in 2 ml of acetone R. Reference solution (1): Dissolve 20 mg of chloramphenicol sodium succinate RS in 2 ml of acetone R. Reference solution (2): Dissolve 20 mg of chloramphenicol RS in 2 ml of acetone R. Procedure: Apply separately to the plate 2 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The two principal spots in the chromatogram obtained with the test solution are similar in position and size to those in the chromatogram obtained with reference solution (1). Their positions are different from that of the principal spot in the chromatogram obtained with reference solution (2). B. Dissolve about 10 mg in 1 ml of ethanol (50%) R, add 3 ml of a 1% solution of calcium chloride R and 50 mg of zinc powder R and heat on a water-bath for 10 min. Filter the hot solution and allow to cool. Add 0.1 ml of benzoyl chloride R and shake for 1 min. Add 0.5 ml of a 10.5% solution of ferric chloride R and 2 ml of chloroform R and shake. The upper layer is light violet-red to purple. C. Dissolve 50 mg in 1 ml of pyridine R. Add 0.5 ml of 2 M sodium hydroxide R and 1.5 ml of water. Heat in a waterbath for 3 min. A red colour develops. Add 2 ml of nitric acid R and cool under running water. Add 1 ml of 0.1 M silver nitrate R. A white precipitate is formed. D. It gives reaction A of sodium (Appendix 8.1). pH Dissolve 2.5 g in carbon dioxide-free water R and dilute to 10 ml with the same solvent. The pH of the solution is 6.4 to 7.0 (Appendix 6.2). Specific optical rotation +5.0° to +8.0°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.50 g in water and dilute to 10.0 ml with the same solvent. Chloramphenicol and chloramphenicol disodium disuccinate Examine by liquid chromatography (Appendix 5.3). Mobile phase: 2% solution of phosphoric acid - methanol - water (5 : 40 : 55).

CHLORAMPHENICOL SODIUM SUCCINATE

Test solution: Dissolve 25.0 mg of the substance to be examined in the mobile phase and dilute to 100.0 ml with the mobile phase. Reference solution (1): Dissolve 10.0 mg of chloramphenicol RS in the mobile phase and dilute to 100.0 ml with the mobile phase (solution A). Dilute 5.0 ml of this solution to 100.0 ml with the mobile phase. Reference solution (2): Dissolve 10.0 mg of chloramphenicol disodium disuccinate RS in the mobile phase and dilute to 100.0 ml with the mobile phase (solution B). Dilute 5.0 ml of this solution to 100.0 ml with the mobile phase. Resolution solution: Dissolve 25 mg of the substance to be examined in the mobile phase, add 5 ml of solution (A) and 5 ml of solution (B) and dilute to 100 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 275 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Inject the resolution solution, the test solution and each of the reference solutions. The test is not valid unless, in the chromatogram obtained with the resolution solution, the two peaks corresponding to those in the chromatograms obtained with reference solutions (1) and (2) are clearly separated from the peaks corresponding to the two principal peaks in the chromatogram obtained with the test solution. If necessary, adjust the methanol content of the mobile phase. Limits: In the chromatogram obtained with the test solution: The area of any peak corresponding to chloramphenicol is not greater than the area of the principal peak in the chromatogram obtained with reference solution (1) (2.0%). The area of any peak corresponding to chloramphenicol disodium disuccinate is not greater than the area of the principal peak in the chromatogram obtained with reference solution (2) (2.0%).

Water Not more than 2.0% (Appendix 10.3). Determined on 0.500 g. Pyrogens If intended for use in the manufacture of a parenteral dosage form without a further appropriate procedure for removal of pyrogens, it complies with the test for pyrogens (Appendix 13.4). Inject per kilogram of the rabbit’s weight 2.5 ml of a solution in water for injections R containing 2 mg of the substance to be examined per millilitre. Assay Dissolve 0.200 g in water and dilute to 500.0 ml with the same solvent. Dilute 5.0 ml of this solution to 100.0 ml 255

CHLORAMPHENICOL FOR INJECTION

with water. Measure the absorbance (Appendix 4.1) of the solution at the maximum at 276 nm. Calculate the content of C15H15Cl2N2NaO8 due to A (1%, 1 cm), taking 220 as the value of A (1%, 1 cm) at the maximum at 276 nm.

Storage Store in an airtight container, protected from light. If the substance is sterile, store in a sterile, airtight, tamper-proof container, protected from light. Labelling The label states, where applicable, that the substance is apyrogenic. Action and use Antibacterial. Preparation Injection. CHLORAMPHENICOL FOR INJECTION Chloramphenicoli pro injectione Chloramphenicol for injection is a sterile crystalline powder of chloramphenicol sodium succinate supplied in a sealed glass container. It is dissolved in water for injections just before use. The powder complies with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Content of chloramphenicol, C11H12Cl2N2O5, 95.0% to 105.0% of the stated amount. Characters A yellowish-white crystalline powder. Identification A. Carry out the method for thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel GF254. Mobile phase: 2 M acetic acid - methanol - chloroform (1 : 14 : 85). Test solution: Dissolve a quantity of the powder containing 50 mg of chloramphenicol sodium succinate in 5 ml of aceton R. Reference solution (1): Dissolve 50 mg of chloramphenicol sodium succinate RS in 5 ml of aceton R. Reference solution (2): Dissolve 50 mg of chloramphenicol RS in 5 ml of aceton R. Procedure: Apply separately to the plate 2 µl of each solution. After developing over a path of 15 cm, remove the plate, allow it to dry in air. Examine in ultraviolet light at 254 nm. The two principal spots in the chromatogram obtained with the test solution are similar in position and 256

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size to those in the chromatogram obtained with reference solution (1) and their positions are different from that of the principal spot in the chromatogram obtained with reference solution (2). B. Dissolve about 10 mg in 1 ml of ethanol (50%) R, add 3 ml of a 1% solution of calcium chloride R and 50 mg of zinc powder R and heat on a water bath for 10 min. Filter the hot solution, allow to cool, add 0.1 ml of benzoyl chloride R and shake for 1 min. Add 0.5 ml of a 10.5% iron(III) chloride solution R and 2 ml of chloroform R and shake. The aqueous layer is light violet-red to purple. C. Yields reaction of sodium salts (Appendix 8.1).

Acidity or alkalinity Dissolve a quantity of the powder equivalent to 2.0 g of chloramphenicol in 10 ml of carbon dioxide-free water R. pH of the solution 6.0 to 7.0 (Appendix 6.2). Chloramphenicol and chloramphenicol disodium disuccinate Examine by liquid chromatography (Appendix 5.3). Mobile phase: Water - methanol - 2% solution of phosphoric acid (55 : 40 : 5). Test solution: Dissolve a quantity of the powder in mobile phase to give a solution containing the equivalent of 0.018% of chloramphenicol. Reference solution (1):A0.0005% solution of chloramphenicol disodium disuccinate RS in the mobile phase. Reference solution (2):A0.0005% solution of chloramphenicol RS in the mobile phase. Resolution solution: A solution containing 0.0005% each of chloramphenicol disodium disuccinate RS and chloramphenicol RS and a sufficient quantity of the contents of the sealed container to give a solution containing the equivalent of 0.025% w/v of chloramphenicol in the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 275 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Inject separately the solutions above. The test is not valid unless the two peaks in the chromatogram obtained with the resolution solution corresponding to those in the chromatograms obtained with reference solutions (1) and (2) are clearly separated from the peaks corresponding to the two principal peaks in the chromatogram obtained with the test solution. If necessary, adjust the methanol content of the mobile phase. In the chromatogram obtained with the test solution the areas of any peaks corresponding to chloramphenicol and chloramphenicol disodium disuccinate are not greater than those of the principal peaks in the chromatograms

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CHLORHEXIDINE GLUCONATE SOLUTION

obtained with reference solutions (2) and (1) respectively (2% of each).

Water Not more than 5.0% (Appendix 10.3). Use 0.5 g. Bacterial endotoxins Carry out the test for bacterial endotoxins (Appendix 13.2). Dissolve the contents of the sealed container in water BET to give a solution containing 10 mg/ml (solution A). The endotoxin limit of solution A is 2.0 IU per mL. Carry out the test using the maximum allowable concentration of solution A calculated from the declared sensitivity of the lysate used in the test. Assay Weigh the contents of 10 containers, calculate the average mass. Dissolve a quantity of the mixed contents of the 10 containers containing the equivalent of 0.200 g of chloramphenicol in water and add sufficient water to produce 500.0 ml. Dilute 5.0 ml of this solution to 100.0 ml with water and measure the absorbance of the resulting solution at the maximum at 276 nm (Appendix 4.1), using water in the reference cell. Calculate the content of chloramphenicol, C11H12Cl2N2O5, in a container taking 297 as the value of A(1%, 1 cm) at the maximum at 276 nm. Storage Store in a cool and dry place, protected from light. Action and use Antibacterial. Usual strength 1000 mg, expressed as chloramphenicol. CHLORHEXIDINE GLUCONATE SOLUTION Chlorhexidini digluconatis solutio

Characters An almost colourless or pale-yellowish liquid. Miscible with water, soluble in acetone and in ethanol (96%). Identification Apply one of the two following identifications: First identification: A, B. Second identification: B, C, D. A. Examine by infrared absorption spectrophotometry (Appendix 4.2): To 1 ml add 40 ml of water, cool in iced water, make alkaline to titan yellow paper R by adding dropwise and with stirring 10 M sodium hydroxide R and add 1 ml in excess. Filter, wash the precipitate with water until the washings are free from alkali and recrystallise from ethanol (70%) (v/v) R. Dry at 100 °C to 105 °C. The infrared absorption spectrum of the result residue is concordant with the spectrum obtained with chlorhexidine RS. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Ethyl acetate - concentrated ammonia water - ethanol (96%) (10 : 10 : 30 : 50). Test solution: Dilute 10.0 ml of the solution to be examined to 50 ml with water. Reference solution: Dissolve 25 mg of calcium gluconate RS in 1 ml of water. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 10 cm. Dry the plate at 100 °C for 20 min, allow to cool and spray with a 5% solution of potassium dichromate in a 40% solution of sulfuric acid R. After 5 min, examine the chromatograms. The principal spot in the chromatogram obtained with the test solution is similar in colour, position and size to the principal spot in the chromatogram obtained with the reference solution. C. To 1 ml add 40 ml of water, cool in iced water, make alkaline to titan yellow paper R by adding dropwise and with stirring 10 M sodium hydroxide R and add 1 ml in excess. Filter, wash the precipitate with water until the washings are free from alkali and recrystallise from ethanol (70%) (v/v) R. Dry at 100 °C to 105 °C. The residue melts (Appendix 6.7) at 132 °C to 136 °C. D. To 0.05 ml add 5 ml of a 1% solution of cetrimide R, 1 ml of 10 M sodium hydroxide R and 1 ml of bromine water R, a deep red colour is produced. Relative density 1.06 to 1.07 (Appendix 6.5). Examine by pycnometer.

C22H30Cl2N10,2C6H12O7

M. 898.0

Chlorhexidine gluconate solution is an aqueous solution of 1,1’-(hexane-1,6-diyl)bis[5-(4-chlorophenyl)-biguanide] di-D-gluconate. It contains not less than 19.0% and not more than 21.0% of C22H30Cl2N10,2C6H12O7.

pH Dilute 5.0 ml to 100 ml with carbon dioxide-free water R. The pH of the solution is 5.5 to 7.0 (Appendix 6.2). Chloroaniline Not more than 0.25%. Dilute 2.0 ml to 100 ml with water. To 10 ml of the solution add 2.5 ml of 2 M hydrochloric acid R and dilute to 20 ml with water. Add rapidly and with thorough mixing 257

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CHLOROFORM

after each addition: 0.35 ml of a 10% solution of sodium nitrite R, 2 ml of a 5% solution of ammonium sulfamate R, 5 ml of a 0.1% solution of naphthyl ethylenediamine dihydrochloride R, 1 ml of ethanol (96%) R, dilute to 50.0 ml with water and allow to stand for 30 min. Any reddish-blue colour in the solution is not greater than that in a standard prepared at the same time in the same manner using a mixture of 10.0 ml of a 0.010 g/l solution of chloroaniline in 2 M hydrochloric acid R and 10 ml of water instead of the dilution of the solution to be examined.

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 2.0 g of sodium octanesulfonate R in a mixture of 120 ml of glacial acetic acid R, 270 ml of water and 730 ml of methanol R. Test solution: Dilute 5.0 ml of the substance to be examined to 50.0 ml with the mobile phase. Dilute 5.0 ml of this solution to 50.0 ml with the mobile phase. Reference solution (1): Dilute 3.0 ml of the test solution to 100 ml with the mobile phase. Reference solution (2): Dilute 1.0 ml of reference solution (1) to 50 ml with the mobile phase. Resolution solution: Dissolve 15 mg of chlorhexidine for performance test RS in the mobile phase and dilute to 10.0 ml with the mobile phase. Chromatographic system: A column (20 cm × 4.0 mm) packed with octadecylsilyl silica gel for chromatography (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: Equilibrate the column with mobile phase for at least 1 hour. Inject reference solution (1). Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with reference solution (1) is at least 50% of the full scale of the recorder. Inject the resolution solution. The test is not valid unless the resulting chromatogram is similar to the specimen chromatogram provided with chlorhexidine for performance test RS in that the peaks due to impurity A (1-(4-chlorophenyl)5-[6-(3-cyanoguanidino)-hexyl]biguanide) and impurity B([[[6-[5-(4-chlorophenyl)guanidino]hexyl]-amino] iminomethyl]urea) precede that due to chlorhexidine. If necessary, adjust the concentration of acetic acid in the mobile phase (increasing the concentration decreases the retention times). Inject the test solution, reference solutions (1) and reference solution (2). Record the chromatograms of reference solutions (1) and (2) until the peak due to chlorhexidine has been eluted. The run time of the test solution is six times the retention time of chlorhexidine. Limits: In the chromatogram obtained with the test solution: The sum of the areas of all peaks, apart from the principal peak is not greater than the area of the principal peak in the 258

chromatogram obtained with reference solution (1) (3.0%). Disregard any peak with a relative retention time of 0.25 or less with respect to the principal peak and any peak whose area is less than that of the principal peak in the chromatogram obtained with reference solution (2).

Assay Determine the density (Appendix 6.5) of the solution to be examined. Transfer 1.00 g to a 250 ml beaker and add 50 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS. Determine the end-point potentiometrically (Appendix 10.2). Each ml of 0.1 N perchloric acid VS is equivalent to 22.44 mg of C34H54Cl2N10O14. Storage Store protected from light. Action and use Disinfectant. Preparations Chlorhexidine irrigation solution, Chlorhexidine mouthwash, Lidocaine and chlorhexidine gel, Lignocaine and chlorhexidine gel. CHLOROFORM Chloroformium Trichloromethane CHCl3  

M. 119.4

Chloroform is trichloromethane to which either 1.0% to 2.0% of ethanol or 50 mg per litre of amylene has been added.

Characters A colourless, volatile liquid. Slightly soluble in water; miscible with absolute ethanol, with ether, with fixed and volatile oils and with most organic solvents. Identification The infrared absorption spectrum (Appendix 4.2) of the substance being examined after washing with water and drying with anhydrous sodium sulfate R is concordant with the reference spectrum of chloroform. Distillation range Not more than 5.0% v/v distils below 60° and the remainder distils at 60 °C to 62 °C (Appendix 6.8). Density 1.474 g/ml to 1.479 g/ml (Appendix 6.5). Acidity or alkalinity Solution S: Shake 10 ml with 20 ml of freshly boiled and cooled water for 3 min and allow to separate. Use the aqueous layer.

VP V

To 5 ml of the solution S, add 0.1 ml of neutral litmus solution. The colour produced is the same as that produced on adding 0.1 ml of the neutral litmus solution to 5.0 ml of freshly boiled and cooled water.

Chloride To 5 ml of the solution S, add 5 ml of water and 0.2 ml of a 5% solution of silver nitrate R (Appendix 9.2). The solution is clear. Free chlorine To 10 ml of the solution S, add 1 ml of a 5.0% w/v solution of zinc iodide R and 0.1 ml of starch mucilage R. No blue colour is produced. Aldehyde Shake 5 ml with 5 ml of water and 0.2 ml of alkaline potassium tetraiodomercurate solution R in a glassstoppered flask and allow to stand in the dark for 15 min. Not more than a pale yellow colour is produced. Foreign chlorine compounds Shake 20 ml for 5 min with 10 ml of sulfuric acid R in a glass-stoppered flask previously rinsed with sulfuric acid R, allow to stand in the dark for 30 min and discard the acid layer. Shake 15 ml of the chloroform layer with 30 ml of water in a glass-stoppered flask for 3 min and allow to separate. To the aqueous layer add 0.2 ml of 5% solution of silver nitrate R and allow to stand in the dark for 5 min. No opalescence is produced. Related substances Examine by gas chromatography (Appendix 5.2). Solution (1): 0.2% v/v of carbon tetrachloride, 0.2% v/v of 1,1,1-trichloroethane (internal standard), 0.2% v/v of dichloromethane, 0.2% v/v of ethanol, 0.5% v/v of bromochloromethane and 0.2% v/v of the substance being examined in propan-1- ol. Solution (2): The substance being examined. Solution (3): 0.2% v/v of the internal standard in the substance being examined. Solution (4): propan-1-ol. Chromatographic system: A glass column (4 m × 3.0 mm) packed with acid-washed kieselguhr (60 to 100 mesh) coated with 15% w/w of di-2cyanoethyl ether. Carrier gas: Nitrogen for chromatography. Flow rate: 30 ml/min. Temperature programme: Column 40 °C, injection port 100 °C, detector 100 °C. Detector: A flame-ionisation detector. Volume of injection: 0.1 µl. Procedure: Inject solution (1). System suitability: The test is not valid unless the column efficiency determined using the chloroform peak is greater

CHLOROFORM

than 700 plates per metre and the total number of plates is greater than 2,500. In the chromatogram obtained with solution (1), the peaks following: Carbon tetrachloride, 1,1,1- trichloroethane, dichloromethane, chloroform, ethanol, bromochloromethane and propan-1-ol (solvent). Using the chromatogram obtained with solution (4), make any corrections due to the contribution of secondary peaks from the solvent to the peaks in the chromatogram obtained with solution (1). Inject solution (3). the ratio of the areas of any peaks due to carbon tetrachloride, dichloromethane and bromochloromethane to the area of the peak due to the internal standard is not greater than the corresponding ratios in the chromatogram obtained with solution (1) and the ratio of the area of any other secondary peak that elutes prior to the solvent peak, except for the peak corresponding to ethanol, to the area of the peak due to the internal standard is not greater than the ratio of the area of the peak due to chloroform to the area of the peak due to the internal standard in the chromatogram obtained with solution (1). Calculate the percentage content of each of the specified impurities and also calculate the percentage content of each of any other impurities assuming the same response per unit volume as for chloroform. The total content of all impurities is not more than 1.0% v/v. Note: Impurity A: Carbon tetrachloride. Impurity B: Dichloromethane. Impurity C: Bromochloromethane.

Ethanol Carry out the following test for Chloroform that contains ethanol. Examine by gas chromatography (Appendix 5.2) as described in the test for related substances with the following modifications. Solution (1): 1.0% v/v of absolute ethanol R and 1.0% v/v of propan-1-ol (internal standard) in water. Solution (2): The substance being examined. Solution (3): 1.0% v/v of the internal standard in the substance being examined. System suitability: The test is not valid unless, in the chromatogram obtained with solution (2), the height of the trough separating the ethanol peak from the chloroform peak is less than 15% of the height of the ethanol peak. Calculate the percentage content of ethanol from the areas of the peaks due to ethanol and the internal standard in the chromatograms obtained with solutions (1) and (3). Non-volatile matter Not more than 0.004%. 25 ml of the substance to be examined, when evaporated to dryness and dried at 105 °C, leaves not more than 1 mg of residue. 259

VP V

CHLOROQUINE PHOSPHATE

Storage Chloroform should be kept in a well-closed container with a glass stopper or other suitable closure and protected from light. Labelling The label states whether it contains ethanol or amylene. Action and use General anaesthetic; antimicrobial preservative (for animals). CHLOROQUINE PHOSPHATE Cloroquini phosphas Cloroquine diphosphate, nivaquine phosphate, aralene

at 220 nm, 235 nm, 256 nm, 329 nm and 342 nm. The A (1%, 1 cm) at the maxima are respectively 600 to 660, 350 to 390, 300 to 330, 325 to 355 and 360 to 390. C. Dissolve 25 mg in 20 ml of water and add 5 ml of picric acid solution R, a yellow precipitate is produced. Filter and wash the precipitate, in turn, with water, with ethanol (96%) R and finally with methylene chloride R. This precipitate melts at 206 °C to 209 °C (Appendix 6.7). D. Dissolve 0.1 g in 10 ml of water, add 2 ml of 2 M sodium hydroxide R and shake with 2 quantities, each of 10 ml of methylene chloride R. The aqueous layer, acidified by the addition of nitric acid R, gives the reactions of phosphates (Appendix 8.1).

Appearance of solution Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution BY5 or GY5 (Appendix 9.3, method 2). pH Solution S: Dissolve 2.50 g in carbon dioxide-free water R and dilute to 25 ml with the same solvent. The pH of solution S is 3.8 to 4.3 (Appendix 6.2).

C18H26ClN3,2H3PO4

M. 515.9

Chloroquine phosphate is (RS)-4-(7-chloro-4-quinolylamino) pentyldiethylamine diphosphate. It contains not less than 98.5% and not more than 101.0% of C18H26ClN3,2H3PO4, calculated with reference to the dried substance.

Characters A white or almost white, crystalline powder; odourless; bitter; discoloured slowly on exposure to light; hygroscopic. Freely soluble in water; very slightly soluble in chloroform, in ethanol (96%), in ether and in methanol. It exists in 2 forms, one of which melts at about 195 °C and the other at about 218 °C. Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D. A. Dissolve 0.1 g of the substance to be examined in 10 ml of water, add 2 ml of 2 M sodium hydroxide R and shake with 2 quantities, each of 20 ml of methylene chloride R; combine the organic layers, wash with water, dry over anhydrous sodium sulphate R, evaporate to dryness and dissolve the residue in 2 ml of methylene chloride R. The infrared absorption spectrum (Appendix 4.2) of this solution is concordant with the spectrum of the solution obtained by treating 80 mg of chloroquine sulphate RS in the same manner. B. The ultraviolet absorption spectrum (Appendix 4.1) of a 0.001% solution of the substance to be examined in water, in the range 210 nm to 370 nm, shows absorption maxima 260

Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Chloroform - cyclohexane - diethylamine (50 : 40 : 10). Test solution: A 5% solution of the substance to be examined in water. Reference solution (1): A 0.050% solution of the substance to be examined in water. Reference solution (2): A 0.025% solution of the substance to be examined in water. Procedure: Apply separately to the plate 2 µl of each solution. Develop over a path of 12 cm. After removal of the plate, allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (1) and not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (2). Heavy metals Not more than 20 ppm (Appendix 9.4.8). Dissolve 2.0 g in 10 ml of water. Add 5 ml of 13.5 M ammonia R and shake with 40 ml of methylene chloride R. Filter the aqueous layer and neutralise the filtrate with glacial acetic acid R. Heat on a water-bath to eliminate methylene chloride, allow to cool and dilute to 20.0 ml with water. 12 ml of this solution complies with limit test for heavy metals, method 1. Prepare the standard using lead standard solution (2 ppm Pb) R.

VP V

Loss on drying Not more than 2.0% (Appendix 9.6). (1.000 g; 100 °C to 105 °C). Assay Dissolve 0.200 g in 50 ml of anhydrous acetic acid R. Add 2 drops of crystal violet solution R. Titrate with 0.1 N perchloric acid VS until the colour changes to green or determining the end-point potentiometrically (Appendix 10.2). Each ml of 0.1 N perchloric acid VS is equivalent to 25.79 mg of C18H26ClN3,2H3PO4. Storage Store in an airtight container, protected from light. Action and use Antimalarial. Preparations Injection; sugar-coated tablets; tablets. CHLOROQUINE PHOSPHATE TABLETS Tabellae Chloroquini phosphatis Chloroquine phosphate tablets contain chloroquine phosphate. The tablets comply with the requirements stated under Tablets (Appendix 1.20) and with the following requirements.

Content of chloroquine phosphate, C18H26ClN3,2H3PO4, 93.0% to 107.0% of the stated amount. Identification A. Dissolve a quantity of the powdered tablets containing 15 mg of chloroquine phosphate in 100 ml of water, mix well and filter. Dilute 5 ml of the filtrate to 100 ml with water. The ultra violet spectrum of the resulting solution (Appendix 4.1) is concordant with the reference spectrum of chloroquine phosphate RS at the same concentration in water and measured simultaneously. The absorbance ratio A343/A329 is 1.00 to 1.15. B. Shake a quantity of the powdered tablets containing 20 mg of chloroquine phosphate with 20 ml of water, filter and add 5 ml of 1% picric acid solution R to the filtrate, a yellow precipitate is formed. Filter through a 0.45 µm membrane filter by a vacuum filter and wash the precipitate with water until the washing is colorless. The melting point of the precipitate is about 205 °C to 210 °C (Appendix 6.7). C. Shake a quantity of the powdered tablets containing 0.5 g of chloroquine phosphate with 25 ml of water and filter. To the filtrate add 2.5 ml of 5 M sodium hydroxide R and extract with three 10 ml quantities of ether R. The aqueous layer, after neutralisation with dilute nitric acid solution R, yields the reactions of phosphates (Appendix 8.1).

CHLOROQUINE PHOSPHATE TABLETS

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of water. Rotation speed: 100 rpm. Time: 45 min. Procedure: After the specified time, withdraw a sample of the medium and filter, discard the first portion of the filtrate. Dilute the filtrate with water to obtain an appropriate concentration. Measure the absorbance of the resulting solution at the maximum at 343 nm (Appendix 4.1) in a 1-cm cell, using water as the blank. Compare with a reference solution having the same concentration of chloroquine phosphate. Tolerance: Not less than 75% (Q) of the stated amount of chloroquine phosphate, C18H26ClN3,2H3PO4, is dissolved in 45 min. Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Chloroform - cyclohexane - diethylamine (50 : 40 : 10). Test solution: Shake a quantity of the powdered tablets containing 1 g of chloroquine phosphate with 20 ml of water for 30 min, centrifuge and use the supernatant liquid. If necessary, filter through a sintered-glass filter. Reference solution (1): Dilute 1 ml of the test solution to 100 ml with water. Reference solution (2): Dilute 25 ml of the reference solution (1) to 50 ml with water. Procedure: Apply separately to the plate 2 µl of each solution. After developing over a path of 12 cm, remove the plate, allow it to dry in air. Examine under ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (1) and not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (2). Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Buffer solution - methanol (78 : 22). Buffer solution: Dissolve 13.6 g of potassium dihydrogen phosphate R in 2 L of water. Add 2.0 ml of perchloric acid R, mix and adjust the pH to 2.5 with phosphoric acid R. Reference solution: A solution containing 0.015% chloroquin phosphate RS in water. Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Weigh accurately a quantity of the powdered tablets containing 15 mg of chloroquine phosphate and transfer into a 100-ml volumetric flask, add 70 ml of water, sonicate for 20 min. Dilute to volume with water, mix and filter. 261

VP V

CHLORPHENIRAMINE MALEATE

Resolution solution: A solution containing 0.015% of chloroquine phosphate RS and 0.015% of amodiaquine hydrochloride RS in water. Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 224 nm. Flow rate: 1.2 ml/min. Volume of injection: 20 µl. Procedure: Inject the resolution solution, in the chromatogram obtained, the relative retention time is 1 for chloroquine phosphate and 1.3 for amodiaquine; the resolution factor between amodiaquine and chloroquine phosphate peaks is not less than 1.5; the symmetry factors of two peaks are not more than 1.5; and the relative standard deviation of the peak areas for the replicate injections is not more than 2.0%. Inject alternately the test solution and the reference solution. Calculate the content of chloroquine phosphate, C18H26ClN3,2H3PO4, in the tablets using the areas of the principal peaks in the chromatograms obtained with the reference solution, the test solution and the declared content of C18H26ClN3,2H3PO4 in chloroquine phosphate RS.

Storage Store in a well-closed container, protected from light. Action and use Antimalarial. Usual strength 50 mg; 500 mg. CHLORPHENIRAMINE MALEATE Chlorpheniramini maleas

C16H19ClN2,C4H4O4

M. 390.9

Chlorpheniramine maleate is (3RS)-3-(4-chlorophenyl)N,N-dimethyl-3-( pyridine-2-yl)propan-1-amin hydrogen (Z)-butenedioate. It contains not less than 98.0% and not more than 101.0% of C16H19ClN2.C4H4O4, calculated with reference to the dried substance.

Characters A white or almost white, crystalline powder. Freely soluble in water, soluble in ethanol (96%). 262

Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of chlorpheniramine maleate RS. B. Melting point: 130 °C to 135 °C (Appendix 6.7). C. It complies with the test for Specific optical rotation. Appearance of solution Solution S: Dissolve 2.0 g in water and dilute to 20.0 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution BY6 (Appendix 9.3, method 2). Specific optical rotation -0.10° to +0.10°, determined on solution S (Appendix 6.4). Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile R - a 8.57 g/L solution of ammonium dihydrogen phosphate R previously adjusted to pH 3.0 with phosphoric acid R. Test solution: Dissolve 0.100 g of the substance to be examined in mobile phase and dilute to 100.0 ml with the same solvent. Reference solution (1): Dilute 0.5 ml of test solution to 100.0 ml with mobile phase. Reference solution (2): Dilute 1.0 ml of reference solution (1) to 10.0 ml with mobile phase. Reference solution (3): Dissolve 5 mg of chlorpheniramine impurity C RS in 5 ml of test solution and dilute to 50.0 ml with mobile phase. Dilute 2 ml of the solution to 20.0 ml with mobile phase. Reference solution (4): Dissolve 5 mg of 2,2’-dipyridylamine R (impurity B) in mobile phase and dilute to 100.0 ml with the same solvent. Reference solution (5): Dissolve the contents of a vial of chlorpheniramine impurity A RS in 2 ml of the test solution. Sonicate for 5 min. Chromatographic system: A column (30 cm × 3.9 mm) packed with stationary phase C (10 µm). Detector: A spectrophotometer set at 225 nm. Flow rate: 1.2 ml/min. Volume of injection: 20 µl. Procedure: The run time is 3.5 times the retention time of chlorpheniramine. Relative retention with reference to chlorpheniramine (retention time = about 11 min): Maleic acid = about 0.2; impurity A = about 0.3; impurity B = about 0.4; impurity C = about 0.9; impurity D = about 3.0. System suitability: In the chromatogram obtained with reference solution (3), the resolution between the peaks due to impurity C and chlorpheniramine is at least 1.5.

VP V

Limits: Correction factors: For the calculation of contents, multiply the peak areas of impurity A with 1.5; impurity B with 1.4. Impurity A: The corrected area of the peak due to impurity A is not more than 0.4 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%). Impurities B, C, D: For each impurity, the corrected area, if necessary, is not more than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%); Any other impurity: For each impurity, the area is not more than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). Total peak area of all impurities is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). Disregard the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Disregard the peaks due to solvent and maleic acid. Note: Impurity A: 2-(4-chlorophenyl)-4-(dimethylamino)-2-[2(dimethylamino) ethyl]butanenitrile. ImpurityB:N-(pyridin-2-yl)pyridin-2-amine(2,2’-dipyridylamine). Impurity C: (3RS)-3-(4-chlorophenyl)-N-methyl-3-(pyridin-2yl)propan-1-amine. Impurity D: (2RS)-2-(4-chlorophenyl)-4-(dimethylamino)-2(pyridin-2-yl)butanenitrile.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C; 4 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.150 g of the substance to be examined in 25 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 19.54 mg of C16H19ClN2.C4H4O4. Storage Store in an airtight container, protected from light. Action and use Histamine H1 receptor antagonist. Preparation Tablets.

CHLORPHENIRAMINE TABLETS

CHLORPHENIRAMINE TABLETS Tabellae Chlorpheniramini Chlorpheniramine tablets contain chlorpheniramine maleate. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of chlorphenamine maleate, C16H19ClN2, C4H4O4, 90.0% to 110.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: 1 M acetic acid - methanol - ethyl acetate (20 : 30 : 50). Test solution: Shake a quantity of the powdered tablets containing 5 mg of chlorphenamine maleate with chloroform R, filter, evaporate the filtrate to dryness and dissolve the residue in 1 ml of chloroform R. Reference solution: A solution containing 0.5% of chlorpheniramine maleate RS in chloroform R. Procedure: Apply separately to the plate 2 µl of each solution. After development, remove the plate, allow it to dry in air and examine under ultraviolet light at 254 nm. The two principal spots in the chromatogram obtained with the test solution are similar in position and colour to those in the chromatogram obtained with the reference solution. Spray the plate with dilute potassium iodobismuthate solution R. The principal spot in the chromatogram obtained with the test solution is similar in position and colour to that in the chromatogram obtained with the reference solution. B. In the Assay, the retention time of two principal peaks in the chromatogram obtained with the test solution are similar to those of two principal peaks in the chromatogram obtained with the reference solution. Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Diethylamine - chloroform - cyclohexane (10 : 40 : 50). Test solution: Shake a quantity of the powdered tablets containing 50 mg of chlorpheniramine maleate with chloroform R, filter, evaporate to dryness and dissolve the residue in 1 ml of chloroform R. Reference solution: Dilute 1 volume of the test solution to 500 volumes with chloroform R. Procedure: Apply separately to the plate 10 µl of each solution. After developing over a path of 12 cm, remove the plate, allow it to dry in air and examine under ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than 263

VP V

CHLORPROMAZINE HYDROCHLORIDE

the spot in the chromatogram obtained with the reference solution (0.2%). Disregard any spot remaining on the line of application.

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: Dilute 2.5 ml of a solution of 10% hydrochloric acid solution R to 250 ml with water. Rotation speed: 50 rpm. Time: 45 min. Procedure: After the specified time, withdraw a sample of the medium and filter. Measure the absorbance of the test solution at 264 nm. Calculate the content of chlorpheniramine maleate dissolved taking 217 as the value of A (1%, 1 cm) at 264 nm. Tolerance: Not less than 75% (Q) of the stated amount of chlorpheniramine maleate, C16H19ClN2,C4H4O4, is dissolved in 45 min.

The test is not valid unless the theoretical plates determined on chlorpheniramine peak are not less than 4000. Inject alternately the reference solution and the test solution. Calculate the content of chlorpheniramine maleate, C16H19ClN2,C4H4O4, in tablets using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C16H19ClN2,C4H4O4 in chlorpheniramine maleate RS.

Storage Protected from light. Action and use Histamine antagonist. Usual strength 2 mg, 4 mg.

Uniformity of content (Appendix 11.2) Examined by liquid chromatography (Appendix 5.3). The reference solution and chromatographic system are described in the Assay. Test solution: Transfer one tablet into a 25-ml volumetric flask for 2 mg tablet and into a 50-ml volumetric flask for 4 mg tablet, add 20 ml of the mobile phase, shake to disintegrate completely, dilute to volume with the mobile phase, shake well and filter.

CHLORPROMAZINE HYDROCHLORIDE Chlorpromazini hydrochloridum

Assay Examine by liquid chromatography (Appendix 5.3). Buffer solution: Dissolve 11.5 g of ammonium dihydrophosphate R in water, add 1 ml of phosphoric acid R and dilute to 1000 ml with water. Mobile phase: Acetonitrile - buffer solution (20 : 80). Adjust if necessary. Reference solution: Dissolve a quantity of chlorpheniramine maleate RS in the mobile phase to obtain a solution having a known concentration of about 0.08 mg/ml. Test solution: Weigh 20 tablets, calculate the average mass and finely powder. Weigh accurately a quantity of the powdered tablets containing about 4 mg of chlorpheniramine maleate, transfer into a 50-ml volumetric flask, add 30 ml of the mobile phase and shake with the aid of ultrasound for 10 min. Dilute to volume with the mobile phase, mix and filter. Chromatographic system: A column (25 × 4.6 mm) packed with stationary phase C (10 µm). Column temperature: 30 °C. Detector: A spectrophotometer set at 262 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject the reference solution. The elution order is maleic acid and chlorpheniramine, respectively.

C17H19ClN2S,HCl  

264



M. 355.3

Chlorpromazine hydrochloride is 3-(2-chloro-10Hphenothiazin-10-yl)-N,N-dimethylpropan-1-amine hydrochloride. It contains not less than 99.0% and not more than 101.0% of C17H19ClN2S,HCl, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder, it shows polymorphism. It decomposes on exposure to air and light. Very soluble in water, freely soluble in ethanol (96%). Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of chlorpromazine hydrochloride RS. Determined on a 6.0% solution of the substance to be examined in methylene chloride R using a 0.1 mm cell. B. The ultraviolet absorption spectrum (Appendix 4.1): Dissolve 50.0 mg of the substance to be examined in 0.1 M hydrochloric acid R and dilute to 500.0 ml with the same solution. Dilute 5.0 ml of the solution to 100.0 ml with 0.1 M hydrochloric acid R.

VP V

The ultraviolet absorption spectrum of the solution in the range 230 nm to 340 nm exhibits two absorption maxima at 254 nm and 306 nm. A(1%, 1 cm) at 254 nm is 890 to 960. Prepare the solutions protected from bright light and measure the absorbances immediately. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Kieselguhr G. Impregnate the plate by placing it in a closed tank containing the necessary quantity of the impregnation mixture composed of a solution containing 10% v/v of phenoxyethanol R and 5% of macrogol 300 R in acetone R so that the plate dips about 5 mm beneath the surface of the liquid. When the impregnation mixture has risen at least 17 cm from the lower edge of the plate, remove the plate and use immediately for chromatography. Carry out the chromatography in the same direction as the impregnation. Mobile phase: A mixture of 50 ml of light petroleum R (50 °C to 70 °C) and 1 ml of diethylamine R saturated with phenoxyethanol R (add about 3 ml to 4 ml of phenoxyethanol R to the above mixture of solvents to give a persistent cloudiness on shaking, decant, and use the supernatant liquid, even if it is cloudy). Test solution: Dissolve 20 mg of the substance to be examined in chloroform R and dilute to 10 ml with the same solvent. Reference solution: Dissolve 20 mg of chlorpromazine hydrochloride RS in chloroform R and dilute to 10 ml with the same solvent. Procedure: Apply separately to the plate 2 µl of each solution. Develop in the dark over a path of 15 cm. After development place the plate under ultraviolet light at 365 nm and examine after a few minutes. The spot in the chromatogram obtained with the test solution is similar in position, fluorescence and size to the spot in the chromatogram obtained with the reference solution. Spray with a 10% solution of sulfuric acid R in alcohol R. The spot in the chromatogram obtained with the test solution is of the same colour as that in the chromatogram obtained with the reference solution and has similar stability over a period of at least 20 min. D. It gives reaction (B) of chlorides (Appendix 8.1).

pH 3.5 to 4.5 (Appendix 6.2). Dissolve 1.0 g in carbon dioxide-free water R and dilute to 10 ml with the same solvent. Carry out the test protected from light and use freshly prepared solutions. Impurity F Examine by thin-layer chromatography (Appendix 5.4). Prepare the solutions immediately before use and protect from light. Coating substance: Silica gel GF254.

CHLORPROMAZINE HYDROCHLORIDE

Mobile phase: Acetone - diethylamine - cyclohexane (10 : 10 : 80). Solvent mixture: Diethylamine - methanol (5 : 95). Test solution: Dissolve 0.100 g of the substance to be examined in the solvent mixture and dilute to 5.0 ml with the solvent mixture. Reference solution (1): Dissolve the contents of a vial of chlorpromazine impurity F RS in 2.0 ml of the solvent mixture. Reference solution (2): Dilute 300 µL of reference solution (1) to 10.0 ml with the solvent mixture. Reference solution (3): Dissolve 0.10 g of the substance to be examined in the solvent mixture, add 1.0 ml of reference solution (1) and dilute to 5.0 ml with the solvent mixture. Procedure: Apply separately to the plate 10 µl of the test solution and reference solutions (2) and (3). Develop over 3/4 of the plate. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Retardation factors: Impurity F = about 0.5; chlorpromazine = about 0.6. The test is not valid unless the chromatogram obtained with the reference solution (3) shows 2 clearly separated spots due to impurity F and chlorpromazine. In the chromatogram obtained with the test solution, any spot due to impurity F is not more intense than the spot in the chromatogram obtained with reference solution (2) (0.15%).

Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use and protect from light. Mobile phase: Mix 0.2 volumes of thiodiethylene glycol R with 50 volumes of acetonitrile R and 50 volumes of a 0.5% v/v solution of trifluoroacetic acid R previously adjusted to pH 5.3 with tetramethylethylenediamine R. Test solution: Dissolve 40.0 mg of the substance to be examined in the mobile phase and dilute to 100.0 ml with the mobile phase. Reference solution (1): Dissolve 4 mg of chlorpromazine impurity D RS in the mobile phase and dilute to 10.0 ml with the mobile phase. To 1 ml of the solution add 1 ml of the test solution and dilute to 100.0 ml with the mobile phase. Reference solution (2): Dilute 1.0 ml of the test solution to 20.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (3): Dissolve 4.0 mg of chlorpromazine impurity A RS in the mobile phase and dilute to 100.0 ml with the mobile phase. Dilute 1.0 ml of the solution to 100.0 ml with the mobile phase. Reference solution (4): Dissolve 4 mg of promazine hydrochloride RS (impurity C) and 4.0 mg of chlorpromazine impurity E RS in the mobile phase and dilute to 100.0 ml with the mobile phase. Dilute 1.0 ml of the solution to 100.0 ml with the mobile phase. 265

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CHLORPROMAZINE HYDROCHLORIDE INJECTION

Chromatographic system: A column (25 cm × 4.0 mm) packed with stationary phase base-deactivated octylsilyl silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: The run time of the test solution is 4 times the retention time of chlorpromazine. Identification of impurities: Use the chromatogram obtained with reference solution (3) to identify the peak due to impurity A; use the chromatogram obtained with reference solution (4) to identify the peaks due to impurities C and E; use the chromatogram obtained with reference solution (1) to identify the peak due to impurity D. Relative retention with reference to chlorpromazine (retention time = about 8 min): impurity A = about 0.4; impurity B = about 0.5; impurity C = about 0.7; impurity D = about 0.9; impurity E = about 3.4. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurity D and chlorpromazine is at least 2.0. Limits: Impurities B, C, D: For each impurity, the area is not more than 0.6 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.3%). Impurity A: The area of the peak due to impurity A is not more than 1.5 times the area of the corresponding peak in the chromatogram obtained with reference solution (3) (0.15%). Impurity E: The area of the peak due to impurity E is not more than 1.5 times the area of the corresponding peak in the chromatogram obtained with reference solution (4) (0.15%). Any other impurity: For each impurity, the area is not more than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.10%). Total: Not more than 1.0%. Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Note: Impurity A: 3-(2-chloro-10H-phenothiazin-10-yl)-N,Ndimethylpropan-1-amine S-oxide (chlorpromazine sulfoxide). Impurity B: N-[3-(2-chloro-10H-phenothiazin-10-yl)propyl]N,N',N'-trimethylpropane-1,3- diamine. Impurity C: 3-(10H-phenothiazin-10-yl)-N,N-dimethylpropan1-amine (promazine). Impurity D: 3-(2-chloro-10H-phenothiazin-10-yl)-N-methylpropan-1-amine (desmethylchlorpromazine). Impurity E: 2-chloro-10H-phenothiazine. Impurity F: 3-(4-chloro-10H-phenothiazin-10-yl)-N,Ndimethylpropan-1-amine.

Heavy metals Not more than 10 ppm (Appendix 9.4.8). Solvent: Water. 266

0.25 g complies with limit test for heavy metals, method 8. Prepare the standard using 0.25 ml of lead standard solution (10 ppm Pb) R.

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.250 g of the substance to be examined in a mixture of 5.0 ml of 0.1 N hydrochloric acid VS and 50 ml of ethanol (96%) R. Carry out a potentiometric titration (Appendix 10.2), using 0.1 N hydrochloric acid VS. Read the volume added between the 2 points of inflexion. 1 ml of 0.1 N hydrochloric acid VS is equivalent to 35.53 mg of C17H20Cl2N2S. Storage Store in an airtight container, protected from light. Action and use Dopamine receptor antagonist; neuroleptic. Preparations Oral solution, injection solution, tablets. CHLORPROMAZINE HYDROCHLORIDE INJECTION Injectio Chlorpromazini hydrochloridi Chlorpromazine hydrochloride injection is a sterile solution of chlorpromazine hydrochloride in water for injections. The injection complies with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Content of chlorpromazine hydrochloride, C17H19ClN2S, HCl, 95.0% to 105.0% of the stated amount. Characters A clear, colourless or almost colourless solution. Identification A. To a volume containing 0.1 g of chlorpromazine hydrochloride add 20 ml of water and 2 ml of 10 M sodium hydroxide R. Shake and extract with 25 ml of ether R. Rinse the ether layer with two portions of 5 ml of water, filter the ether layer through anhydrous sodium sulfate R, evaporate the ether layer and dissolve the residue in 1 ml of chloroform R. The infrared absorption spectrum of the resulting solution (Appendix 4.2), is concordant with the reference spectrum of chlorpromazine.

VP V

B. The absorbance (Appendix 4.1) of the test solution in the Assay in the range 220 nm to 340 nm exhibits two maxima at 254 nm and 306 nm. C. Yields reaction of chlorides (Appendix 8.1).

pH pH 4.0 to 6.5 (Appendix 6.2). Related substances Examine by thin-layer chromatography (Appendix 5.4). Carry out the procedure protected from light. Coating substance: Silica gel GF254. Mobile phase: Cyclohexane - acetone - diethylamine (80 : 10 : 10). Test solution: Dilute a volume of the injection, if necessary, with a sufficient volume of a mixture of methanol diethylamine (95 : 5) to obtain a solution containing 0.5% of chlorpromazine hydrochloride. Reference solution (1): Dilute 1 volume of the test solution to 20 volumes with a mixture of methanol - diethylamine (95 : 5). Reference solution (2): Dilute 1 volume of the test solution to 200 volumes with a mixture of methanol - diethylamine (95 : 5). Procedure: Apply separately to the plate 10 µl of each solution. After developing over a path of 15 cm, remove the plate, allow it to dry in air and examine under ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (1) and not more than one such secondary spot is more intense than the spot in the chromatogram obtained with reference solution (2). Bacterial endotoxins (Appendix 13.2) Not more than 6.9 EU per 1 mg of chlorpromazine hydrochloride. Assay Carry out the following procedure protected from light. Dilute a suitable volume of the injection in 0.1 M hydrochloric acid R to obtain a solution containing about 0.0005% of chlorpromazine hydrochloride. Measure the absorbance of the resulting solution at the maximum at 254 nm (Appendix 4.1) using 0.1 M hydrochloric acid R as the blank. Calculate the content of chlorpromazine hydrochloride, C17H19ClN2S,HCl, taking 915 as the value of A(1%, 1 cm) at the maximum at 254 nm. Storage Store in a cool place, protected from light. Action and use Antipsychotic, antiemetic, antidyskinetic. Usual strength 25 mg/ml.

CHLORPROMAZINE HYDROCHLORIDE TABLETS

CHLORPROMAZINE HYDROCHLORIDE TABLETS Tabellae Chlorpromazini hydrochloridi Chlorpromazine hydrochloride tablets contain chlorpromazine hydrochloride. They are coated. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of chlorpromazine hydrochloride, C17H19ClN2S, HCl, 92.5% to 107.5% of the stated amount. Identification A. To a quantity of the powdered tablets containing 40 mg of chlorpromazine hydrochloride add 10 ml of water and 2 ml of 10 M sodium hydroxide R. Shake and extract with 15 ml of ether R. Wash the ether layer with two 5 ml quantities of water, dry with anhydrous sodium sulphate R and evaporate the ether. Dissolve the residue in 0.4 ml of chloroform R. The infrared absorption spectrum of the resulting solution (Appendix 4.2) is concordant with the reference spectrum of chlorpromazine. B. The absorbance (Appendix 4.1) of the test solution in the Assay in the range 220 nm to 340 nm exhibits two maxima at 254 nm and 306 nm. C. Shake a quantity of the powdered tablets containing 25 mg of chlorpromazine hydrochloride with 25 ml of water. Filter, the filtrate yields reaction of chlorides (Appendix 8.1). Related substances Carry out the following procedure protected from light. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Cyclohexane - acetone - diethylamine (80 : 10 : 10). Test solution: Shake a quantity of the powdered tablets containing 0.1 g of chlorpromazine hydrochloride with 10 ml of a mixture of methanol - diethylamine (95 : 5), filter. Reference solution: Dilute 1 volume of test solution to 200 volumes with a mixture of methanol - diethylamine (95 : 5). Procedure: Apply separately to the plate 10 µl of each solution. After developing over a path of 15 cm, remove the plate, allow it to dry in air and examine under ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution (0.5%). Disregard any spot remaining on the line of application. Dissolution (Appendix 11.4) Carry out the following procedure protected from light. Apparatus: Paddle. Medium: 900 ml of 0.1 M hydrochloric acid R. Rotation speed: 50 rpm. Time: 45 min. 267

VP V

CHYMOTRYPSIN

Procedure: After the specified time, withdraw a sample of the medium and filter, and dilute a suitable volume of the filtrate with 0.1 M hydrochloric acid R to produce a solution containing 0.0005% of chlorpromazine hydrochloride. Measure the absorbance of this solution (Appendix 4.1) at 254 nm using 0.1 M hydrochloric acid R in the reference cell. Calculate the total content of C17H19ClN2S,HCl in the medium taking 915 as the value of A (1%, 1 cm) at the maximum at 254 nm. Tolerance: Not less than 70% (Q) of the stated amount of chlorpromazine hydrochloride, C17H19ClN2S, HCl, is dissolved in 45 min.

Assay Carry out the following procedure, protected from light. Weigh 20 tablets, calculate the average mass, finely powder, if necessary discard the coating. Weigh a quantity of the powdered tablets equivalent to 25 mg of chlorpromazine hydrochloride in a 250-ml volumetric flask. Add about 150 ml of 0.1 M hydrochloric acid R. Shake for 15 min, add sufficient 0.1 M hydrochloric acid R to produce 250 ml, mix. Filter, discarding the first 20 ml of the filtrate. Dilute 5.0 ml of the filtrate to 100.0 ml with 0.1 M hydrochloric acid R. Measure immediately the absorbance of the resulting solution at the maximum at 254 nm (Appendix 4.1), in a 1-cm cell, using 0.1 M hydrochloric acid R in the reference cell. Calculate the content of chlorpromazine hydrochloride, C17H19ClN2S,HCl, taking 915 as the value of A (1%, 1 cm) at the maximum at 254 nm. Storage Store in an airtight container, protected from light. Action and use Antipsychotic, antiemetic, antidyskinetic. Usual strength 10 mg and 25 mg. CHYMOTRYPSIN Chymotrypsinum Chymotrypsin is a proteolytic enzyme crystallized from an extract of the pancreas gland of ox, Bos taurus Linne’ (Fam. Bovidae). It contains not less than 1000 chymotrypsin units in each mg, calculated with reference to the dried substance, and not less than 90.0% and not more than 110.0% of the labelled potency.

Characters A white, crystalline or amorphous powder. Sparingly soluble in water. The amorphous form is hygroscopic. Identification A. Dissolve about 10 mg of the substance to be examined in 10 ml of carbon dioxide-free water R. To 0.05 ml of this 268

solution in a depression on a white spot plate, add 0.2 ml of substrate solution. A purple colour develops within 3 min. Substrate solution: Weigh accurately 237.0 mg of acetyltyrosine ethyl ester R into a 100 ml volumetric flask; add 2 ml of ethanol (96%) R, and swirl until dissolution is completely. Add 20 ml of phosphate buffer solution pH 7.0 (prepared in the Assay) and 1 ml of methyl red - methylene blue solution, and dilute to volume with water. Methyl red - methylene blue solution: Mix equal volumes of a 0.1% solution of methyl red in ethanol (96%) R and a 0.05% solution of methylene blue in ethanol (96%) R. B. Dissolve about 30 mg in 0.001 M hydrochloric acid R and dilute to 100 ml with the same solvent. The ultraviolet absorption spectrum (Appendix 4.1) of the solution in the range 220 nm to 320 nm shows an absorption maximum at about 281 nm and a minimum at about 250 nm.

Loss on drying Not more than 5.0% (Appendix 9.6). (1.00 g; 60 °C; in vacuo; 4 h). Sulfated ash Not more than 2.5% (Appendix 9.9, method 2). Determine on 0.100 g. Trypsin Not more than 1% (m/m). Test solution: Dissolve 100 mg of the substance to be examined in 10.0 ml of water. Reference solution: A 0.01% solution of trypsin RS in water. Tris(hydroxymethyl)aminomethane buffer solution pH 8.1 (0.08 M): Dissolve 294 mg of calcium chloride R in 40 ml of 0.20 M tris(hydroxymethyl)aminomethane, adjust to pH 8.1 with 1 N hydrochloric acid R, and dilute with water to 100 ml. Substrate solution: Transfer 98.5 mg of tosylarginine methyl ester hydrochloride R to a 25 ml volumetric flask. Add 5 ml of tris(hydroxymethyl)aminomethane buffer solution pH 8.1, and swirl until the substrate dissolves. Add 0.25 ml of methyl red - methylene blue solution (prepared in the Identification), and dilute with water to volume. Procedure: By means of a micropipet, transfer 50 µl of the test solution and the reference solution to depressions on two separate white spot plates. Add 0.2 ml of the substrate solution to each solution: within 3 min, no purple colour develops in the plate containing the test solution and a purple colour develops in the plate containing the reference solution. Assay Phosphate buffer solution pH 7.0 (0.067 M): Dissolve 4.54 g of potassium dihydrogen phosphate R in water and dilute to 500 ml with the same solvent (solution A). Dissolve 4.73 g of anhydrous dibasic sodium hydrogenphosphate

VP V

CHYMOTRYPSIN TABLETS

R in water and dilute to 500 ml with the same solvent (solution B). Mix 38.9 ml of solution A with 61.1 ml of solution B. If necessary, adjust to pH 7.0 by the dropwise addition of solution B. Substrate solution: Dissolve 23.7 mg of acetyltyrosine ethyl ester R (suitable for use in assay chymotrypsin) in about 50 ml of phosphate buffer solution pH 7.0, with warming. Allow the solution to cool, dilute with phosphate buffer solution pH 7.0 to 100 ml. Note: Substrate solution may be stored in the frozen state and used after thawing, but it is important to freeze it immediately after preparation. Test solution: Dissolve a sufficient quantity of the substance to be examined (W), accurately weighed, in 0.0012 M hydrochloric acid to yield a solution containing 12 to 16 chymotrypsin units per ml. The dilution is correct if, during the conduct of the assay, there is a change in absorbance of between 0.008 and 0.012 in each 30-second interval. Procedure: Conduct the assay in a suitable spectrophotometer equipped to maintain a temperature of 25 ± 0.1 °C in the cell compartment. Determine the temperature in the reaction cell before and after the measurement of absorbance in order to ensure that the temperature does not change by more than 0.5 °C. Pipet accurately 0.2 ml of 0.0012 M hydrochloric acid and 3.0 ml of substrate solution into a 1-cm cell. Place this cell in the spectrophotometer, and adjust the instrument so that the absorbance will read 0.200 at 237 nm. Pipet accurately 0.2 ml of the test solution into another 1-cm cell, add accurately 3.0 ml of substrate solution, mix well. Place the cell in the spectrophotometer and measure the absorbance immediately. Read the absorbance at 30-second intervals for not less than 5 minutes. Repeat the procedure on the same dilution at least once. Absolute absorbance values are less important than a constant rate of absorbance decrease. If the rate of absorbance decrease fails to remain constant for not less than 3 min, repeat the test and, if necessary, use a suitable concentration. Plot a curve of absorbance against time, take the absorbance value as the ordinate and time as the abscissa. Use the linear portion of the curve within 3 min to calculate the potency of the substance to be examined. One chymotrypsin unit is the activity causing a change in absorbance of 0.0075 per minute under the conditions specified in this assay. Calculate the potency (units) of chymotrypsin per mg taken by the following formula:

( A1 × A2 ) × D T × 0.0075 × W × 0.2 In which: A1: The absorbance straight-line initial reading. A2: The absorbance straight-line final reading.

T: The elapsed time between the initial and final reading (min). D: The dilution factor of the test solution. W: The weight of the substance to be examined to prepare the test solution (mg).

Microbial contamination Absence of Pseudomonas aeruginosa and Salmonella species and Staphylococcus aureus (Appendix 13.6). Storage In an airtight container, in a dry and cool place and protected from light. Action and use Proteolytic enzyme. CHYMOTRYPSIN TABLETS Tabellae Chymotrypsini Chymotrypsin tablets contain chymotrypsin. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of chymotrypsin, 90.0% to 120.0% of the stated amount. Identification A. Dissolve a quantity of the powdered tablets containing about 4.2 mg of chymotrypsin in 4 ml of water freshly boiled and cooled, filter. Transfer 0.05 ml of the filtrate to a white dish and add 0.2 ml of substrate solution. A purple color is produced within 3 min. Prepare substrate solution as follows: Substrate solution: Transfer 237.0 mg of N-acetyl-Ltyrosine ethyl ester to a 100 ml volumetric flask, add 2 ml of ethanol R, and swirl until dissolved completely. Add 20 ml of phosphate buffer pH 7.0 (prepared as directed in the Assay), add 10 ml of methyl red-methylene blue solution, and dilute with water to volume. Methyl red-methylene blue solution: Mix an equal volume of each of the solutions: 0.1% solution of methyl red in ethanol 96% and 0.05% solution of methylene blue in ethanol 96%. B. Dissolve a quantity of the powdered tablets containing about 30 mg of chymotrypsin in 0.001 M hydrochloric acid, dilute to 100 ml with the same solvent, filter. The ultraviolet absorption spectrum (Appendix 4.1) of the solution in the range 220 nm to 320 nm exhibits absorption maximum at 281 nm and minimum at 250 nm. Assay Phosphate buffer pH 7.0: Dissolve 4.54 g of potassium dihydrogen phosphate in water and dilute to 500 ml 269

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with the same solvent (solution A). Dissolve 4.73 g of anhydrous disodium hydrogen phosphate R in water and dilute to 500 ml with the same solvent (solution B). Mix 38.9 ml of the solution A with 61.1 ml of solution B. If necessary, adjust to pH 7.0 by the drop wise addition of solution B. Substrate solution: Dissolve 23.7 mg of acetyltyrosine ethyl ester R (suitable for use in assaying chymotrypsin) in about 50 ml of phosphate buffer pH 7.0 with warming. Allow to cool, dilute with phosphate buffer pH 7.0 to 100 ml. Note: Substrate solution may be stored in the frozen state and used after thawing, but it is important to freeze it immediately after preparation. Test solution: Weigh 20 tablets, calculate the average mass, and powder finely. Dissolve a suitable quantity of the powdered tablets, accurately weighed, in 0.0012 M hydrochloric acid to obtain a solution containing between 12 and 16 USP chymotrypsin units per ml. Use the solution having a lower or upper concentration of chymotrypsin (if necessary) so that during the conduct of the assay, there is a change in absorbance of between 0.008 and 0.012 in each 30-second interval. Procedure:

Note: Determine the suitability of the substrate and check the adjustment of the spectrophotometer by performing the Procedure using chymotrypsin RS in place of the assay specimen.

Conduct the assay in a suitable spectrophotometer equipped to maintain a temperature of (25 ± 0.1) °C in the cell compartment. Determine the temperature in the reaction cell before and after the measurement of absorbance in order to assure that the temperature does not change by more than 0.5 °C. Pipet 0.2 ml of 0.0012 M hydrochloric acid and 3.0 ml of substrate solution into a 1 cm cell. Place this cell in the spectrophotometer, and adjust the instrument so that the absorbance will read 0.200 at 237 nm. Pipet 0.2 ml of the test solution and 3.0 ml of substrate solution into a 1 cm cell. Place this cell in the spectrophotometer. (Note: Carefully follow this order of addition, and begin timing the reaction from the addition of the substrate solution). Read the absorbance at 30 second intervals for not less than 5 minutes. Repeat the procedure on the same dilution at least once. Absolute absorbance values are less important than a constant rate of absorbance change. If the rate of change fails to remain constant for not less than 3 minutes, repeat the test and, if necessary, use solution having suitable concentration of chymotrypsin. The duplicate determination at the same dilution matches the first determination in rate of absorbance change. Determine the average absorbance change per minute, using only the values within the 3 min portion of the curve where the rate of absorbance change is constant. Plot a curve of absorbance against time. 270

One USP chymotrypsin unit is the activity causing a change in absorbance of 0.0075 per minute under the conditions specified in this assay. Calculate the content (%), of labeled amount of chymotrypsin in the tablets by the formula:

( A1 − A2 ) × D × M × 100 T × 0.0075 × W × 0.2 × L

in which: A1 is the absorbance straight-line initial reading. A2 is the absorbance straight-line final reading. T is the elapsed time, in minutes, between the initial and final readings. D is the dilute factor of the test solution. W is the weight of sample taken, in mg. M is the average weight of tablets, in mg L is the content in the label (the USP chymotrypsin units).

Storage Store in well-closed container, in a cool and dry place, protected from light. Action and use Proteolytic enzyme. Usual strength 4.2 mg; 6.3 mg; 8.4 mg (equivalent to 4200, 6300, 8400 USP chymotrypsin units). CILASTATIN SODIUM Cilastatinum natricum

C16H25N2NaO5S

M. 380.4

Cilastatin sodium is sodium (Z)-7-[[(R)-2-amino-2carboxyethyl]sulfanyl]-2-[[[(1S)-2,2-dimethylcyclopropyl] carbonyl]amino]hept-2-enoate. It contains not less than 98.0% and not more than 101.5% of C16H25N2NaO5S, calculated with reference to the anhydrous substance.

Characters White or light yellow amorphous, hygroscopic powder. Very soluble in water and in methanol, slightly soluble in anhydrous ethanol, very slightly soluble in dimethyl sulfoxide, practically insoluble in acetone and in methylene chloride. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of cilastatin sodium RS.

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CILASTATIN SODIUM

B. It complies with the test Specific optical rotation. C. It gives reaction (A) of sodium (Appendix 8.1).

Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

Appearance of solution Solution S: Dissolve 1.0 g in carbon dioxide-free water and dilute to 100 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution Y6 (Appendix 9.3, method 2).

0 - 30

15 → 100

85 → 0

30 - 46

100

0

46 - 56

100 → 15

0 → 85

pH 6.5 to 7.5 (Appendix 6.2). Determined on solution S. Specific optical rotation +41.5° to +44.5°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.250 g in a mixture of 1 volume of hydrochloric acid R and 120 volumes of methanol R, then dilute to 25.0 ml with the same mixture of solvents. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Mobile phase A: Acetonitrile - a 0.1% v/v solution of phosphoric acid R in water (30 : 70). Mobile phase B: A 0.1% v/v solution of phosphoric acid R in water. Test solution: Dissolve 32.0 mg of the substance to be examined in water and dilute to 20.0 ml with the same solvent. Reference solution (1): Dilute 2.0 ml of the test solution to 100.0 ml with water. Dilute 5.0 ml of this solution to 100.0 ml with water. Reference solution (2): Dilute 5.0 ml of the test solution to 100.0 ml with water. Dilute 2.0 ml of this solution to 20.0 ml with water. Reference solution (3): Dissolve 16 mg of the substance to be examined in dilute hydrogen peroxide solution R and dilute to 10.0 ml with the same solution. Allow to stand for 30 min. Dilute 1 ml of this solution to 100 ml with water. Reference solution (4): Dissolve 32 mg of mesityl oxide R (impurity D) in 100 ml of water. Dilute 1 ml of this solution to 50 ml with water. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Column temperture: 50 °C. Detector: A spectrophotometer set at 210 nm. Flow rate: 2.0 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions:

System suitability: The chromatogram obtained with reference solution (3) shows 3 principal peaks: The first 2 peaks (impurity A) may elute without being completely resolved. Mass distribution ratio: At least 10 for the peak due to cilastatin (3rd peak) in the chromatogram obtained with reference solution (3). Signal-to-noise ratio: At least 5.0 for the principal peak in the chromatogram obtained with reference solution (1). Inject the reference solution (2), the reference solution (4) and the test solution. Limits: In the chromatogram obtained with the test solution: Any impurities: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.5%). Sum of the areas of all auxiliary peaks is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (2) (1.0%). Disregard any peak with an area less than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%) and disregard any peak corresponding to the peak due to impurity D in the chromatogram obtained with reference solution (4). Note: Impurity A: (Z)-7-[(RS)-[(R)-2-amino-2-carboxyethyl]sulfinyl]2-[[[(1S)-2,2-dimethylcyclopropyl]carbonyl] amino]hept-2-enoic acid. Impurity B: (Z)-7-[[(R)-2-[[(1RS)-1-methyl-3-oxobutyl]amino] 2-carboxyethyl]sulfanyl]-2-[[[(1S)-2,2-dimethyl cyclopropyl]carbonyl]amino]hept-2- enoic acid. Impurity C: (Z)-7-[[(R)-2-[(1,1-dimethyl-3-oxobutyl)amino]2carboxyethyl]sulfanyl]-2-[[[(1S)-2,2-dimethyl cyclopropyl]carbonyl]amino]hept-2- enoic acid. Impurity D: 4-methylpent-3-en-2-one (mesityl oxide).

Impurity D, acetone and methanol Examine by gas chromatography (Appendix 5.2). Internal standard solution: Dissolve 0.5 ml of propanol R in water and dilute to 1000 ml with the same solvent. Test solution: Dissolve 0.200 g of the substance to be examined in water, add 2.0 ml of the internal standard solution and dilute to 10.0 ml with water. Reference solution: Dissolve 2.0 ml of acetone R, 0.5 ml of methanol R and 0.5 ml of mesityl oxide R (impurity D) in water and dilute to 1000 ml with the same solvent. To 2.0 ml of this solution add 2.0 ml of the internal standard solution and dilute to 10.0 ml with water. This solution 271

VP V

CIMETIDINE

contains 316 µg of acetone, 79 µg of methanol and 86 µg of impurity D per millilitre. Chromatographic system: A fused-silica capillary column (30 m long and 0.53 mm in internal diameter) coated with a film of macrogol 20 000 R (film thickness 1.0 µm). Carrier gas: Helium for chromatography. Flow rate: 9 ml/min. Temperature programme:

Column

Time (min)

Temperature (°C)

0 - 2.5

50

2.5 - 5

50 → 70

5 - 5.5

70

Injection port

160

Detector

220

Detector: A flame-ionisation detector. Volume of injection: 1 µl. Procedure: Calculate the percentage contents of acetone, methanol and impurity D using the following expression: R C   ×  u W   Rs

Storage In an airtight container, at a temperature not exceeding 8 °C. If the substance is sterile, store in a sterile, airtight container. Action and use Dehydropeptidase-I inhibitor; inhibition of the renal metabolism of imipenem. Preparation Injection. CIMETIDINE Cimetidinum

  

C: Concentration of the solvent in the reference solution (µg/ml). W: Quantity of cilastatin sodium in the test solution (mg). Ru: Ratio of the area of the solvent peak to the area of the propanol peak in the chromatogram obtained with the test solution. Rs: Ratio of the area of the solvent peak to the area of the propanol peak in the chromatogram obtained with the reference solution. Limits: Acetone: Not more than 1.0% m/m. Methanol: Not more than 0.5% m/m. Impurity D: Not more than 0.4% m/m.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 3. Prepare the standard solution using 2.0 ml of lead standard solution (10 ppm Pb) R. Water Not more than 2.0% (Appendix 10.3). Determined on 0.50 g. Bacterial endotoxins Less than 0.17 EU/mg (Appendix 13.2). If intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins. 272

Assay Dissolve 0.300 g of the substance to be examined in 30 ml of methanol R and add 5 ml of water. Add 0.1 N hydrochloric acid (VS) to a pH of about 3.0. Carry out a potentiometric titration (Appendix 10.2), using 0.1 N sodium hydroxide VS. 3 jumps of potential are observed. Titrate to the 3rd equivalence point. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 19.02 mg of C16H25N2NaO5S.

C10H16N6S  

M. 252.3

Cimetidine is 2-cyano-1-methyl-3-[-2-[[(5-methyl-1Himidazol-4-yl)methyl]sulfanyl]ethyl]guanidine. It contains not less than 98.5% and not more than 101.5% of C10H16N6S, calculated with reference to the dried substance.

Characters White or almost white powder, it shows polymorphism. Slightly soluble in water, soluble in ethanol (96%), practically insoluble in methylene chloride. It dissolves in dilute mineral acids. Identification Apply one of the two following identifications: First identification: A. Second identification: B, C. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of cimetidine RS. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in 2-propanol R, evaporate to dryness and record new spectra using the residues.

VP V

B. Melting point: 139 °C to 144 °C (Appendix 6.7). If necessary, dissolve the substance to be examined in 2-propanol R, evaporate to dryness and determine the melting point again. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Ammonia - methanol - ethyl acetate (15 : 20 : 65). Test solution: Dissolve 10 mg of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Reference solution: Dissolve 10 mg of cimetidine RS in methanol R and dilute to 10 ml with the same solvent. Procedure: Apply separately to the plate 5 µl of each solution. Develop over 3/4 of the plate. Dry the plate in a current of cold air and expose to iodine vapour until maximum contrast has been obtained and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution.

Appearance of solution Dissolve 3.0 g in 12 ml of 1 M hydrochloric acid R and dilute to 20 ml with water. The solution is clear (Appendix 9.2) and not more intensely coloured than reference solution Y5 (Appendix 9.3, method 2). Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Mix 0.4 volumes of diethylamine R and 780 volumes of a 0.11% solution of sodium hexanesulfonate R; adjust to pH 2.8 with phosphoric acid R; add 250 volumes of methanol R2; Mobile phase B: Methanol R2. Test solution: Dissolve 20 mg of the substance to be examined in mobile phase A and dilute to 50.0 ml with mobile phase A. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with mobile phase A. Dilute 2.0 ml of this solution to 10.0 ml with mobile phase A. Reference solution (2): Dissolve the contents of a vial of cimetidine for system suitability RS (containing impurities B, C, D, E, G and H) in 1.0 ml of mobile phase A. Reference solution (3): Dissolve 4 mg of cimetidine for peak identification RS (containing impurity F) in mobile phase A and dilute to 10.0 ml with mobile phase A. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 220 nm. Flow rate: 1.1 ml/min. Volume of injection: 50 µl.

CIMETIDINE

Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 60

100

0

60 - 65

100 → 90

0 → 10

65 - 120

90

10

Identification of impurities: Use the chromatogram supplied with cimetidine for system suitability RS and the chromatogram obtained with reference solution (2) to identify the peaks due to impurities B, C, D, E, G and H; use the chromatogram supplied with cimetidine for peak identification RS and the chromatogram obtained with reference solution (3) to identify the peak due to impurity F. Relative retention with reference to cimetidine (retention time = about 18 min): impurity G = about 0.2; impurity E = about 0.4; impurity D = about 1.5; impurity C = about 1.6; impurity B = about 2.0; impurity H = about 2.3; impurity F = about 4.6. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurities D and C is at least 1.5. Limits: Correction factors: for the calculation of content, multiply the peak areas of the following impurities by the corresponding correction factor: impurity C = 2.5; impurity D = 3.3; impurity E = 0.7; impurity G = 0.6. Impurities B, C, D, E, F, G, H: For each impurity, the area, corrected if necessary is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%). Any other impurity: For each impurity, the area is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). Sum of the peak areas of all impurities is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (1.0%). Disregard any peak with an area less than 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A. methyl 3-cyano-1-[2-[[(5-methyl-1H-imidazol-4yl)methyl]sulfanyl]ethyl]carbamimidothioate. Impurity B: methyl 3-cyano-1-[2-[[(5-methyl-1H-imidazol-4yl)methyl]sulfanyl]ethyl]carbamimidate. Impurity C: 1-[(methylamino)[[2-[[(5-methyl-1H- imidazol-4yl)methyl]sulfanyl]ethyl]amino]methylidene]urea. Impurity D: 1-methyl-3-[2-[[(5-methyl-1H-imidazol-4-yl) methyl]sulfanyl]ethyl]guanidine. Impurity E: 2-cyano-1-methyl-3[2-[[(5-methyl-1H- imidazol-4yl)methyl]sulfinyl]ethyl]guanidine.

273

VP V

CIMETIDINE TABLETS Impurity F: 2-cyano-1,3-bis[2-[[(5-methyl-1H-imidazol-4-yl) methyl]sulfanyl]ethyl]guanidine. Impurity G: 2-cyano-1,3-dimethylguanidine. Impurity H: 1,1’-(disulfanediyldiethylene)bis(2-cyano-3methylguanidine). Impurity I: (5-ethyl-1H-imidazol-4-yl)methanol. Impurity J: 2-[[(5-methyl-1H-imidazol-4- yl)methyl]sulfanyl] ethanamine.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.2% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.200 g of the substance to be examined in 60 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS determining the end point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 25.23 mg of C10H16N6S Storage Store in an airtight container, protected from light. Action and use Histamine H2 receptor antagonist. Preparations Tablets, injection, siro, oral suspension. CIMETIDINE TABLETS Tabellae Cimetidini Tablets or film-coated tablets contain cimetidine. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of cimetidine, C10H16N6S, 95.0% to 105.0% of the stated amount. Identification A. Shake a quantity of the powdered tablets containing 0.1 g of cimetidine with 10 ml of methanol R for 10 min, filter and evaporate to dryness. Dissolve the residue in 5 ml of chloroform R and evaporate to dryness. Dry the residue at 274

60 °C in vacuo. The infrared absorption spectrum of the residue (Appendix 4.2) is concordant with the reference spectrum of cimetidine. B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (2) corresponds to that in the chromatogram obtained with reference solution (4).

Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase A: Ammonia - methanol - ethyl acetate (15 : 20 : 65). Mobile phase B: Ammonia - methanol - ethyl acetate (8 : 8 : 84). Test solution (1): Add 20 ml of methanol R to a quantity of the powdered tablets containing 1 g of cimetidine, mix with the aid of ultrasound for 2 minutes, shake for 3 min and filter. Test solution (2): Dilute 1 volume of test solution (1) to 10 volumes with methanol R. Reference solution (1): Dilute 1 volume of test solution (2) to 20 volumes with methanol R. Reference solution (2): Dilute 1 volume of test solution (1) to 100 volumes with methanol R and dilute 20 volumes of this solution to 100 volumes with methanol R. Reference solution (3): Dilute 5 volumes of reference solution (2) to 10 volumes with methanol R. Reference solution (4): A solution containing 0.5% of cimetidine RS in methanol R. Procedure: A. Apply separately to the plate 4 µl of each solution. Allow the plate to stand for 15 min in the tank saturated with vapour from the mobile phase A. After development in the same mobile phase and removal of the plate, allow to dry in the air, expose to iodine vapour until appearence of the spots and examine under ultraviolet light at 254 nm. B. Apply separately to the plate 4 µl of each solution. Develop using mobile phase B. After removal of the plate, allow to dry, expose to iodine vapour until appearence of the spots and examine under ultraviolet light at 254 nm. The following limits apply to both methods: Any secondary spot in the chromatogram obtained with test solution (1) is not more intense than the principal spot in the chromatogram obtained with reference solution (1) (0.5%) and not more than two such spots are more intense than the principal spot in the chromatogram obtained with reference solution (2) (0.2% of each). The tests are not valid unless the chromatograms obtained with reference solution (3) show clearly visible spots. Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 900 ml of 0.01 M hydrochloric acid R.

VP V

Rotation speed: 100 rpm. Time: 15 min for tablets and 45 min for film-coated tablets. Procedure: After the specified time, withdraw a sample of the medium and filter, discard the first 10 ml of the filtrate. Dilute the filtrate with 0.01 M hydrochlorid acid R to obtain a solution containing 5 - 10 µg of cimetidine per 1 ml. Measure the absorbance of the obtained solution at the maximum at 218 nm (Appendix 4.1), using 0.01 M hydrochlorid acid R in the reference cell. Calculate the dissolution of cimetidine, C10H16N6S, is dissolved in each tablet, taking 774 as the value of A (1%, 1 cm) of cimetidine at 218 nm. Tolerance: Not less than 75% (Q) of the stated amount of cimetidine is dissolved in the prescribed time.

Assay Weigh 20 tablets, calculate the average mass, and powder finely. Weigh a quantity of the powdered tablets containing 100 mg of cimetidine in a 500 ml volumetric flask, add 300 ml of 0.05 M sulfuric acid R, shake for 20 min, add sufficient 0.05 M sulfuric acid R to volume and filter. Dilute 5.0 ml of the filtrate to 100.0 ml with 0.05 M sulfuric acid R. Prepare a 0.001% solution of cimetidine RS in 0.05 M sulfuric acid R. Measure the absorbance of two solutions at the maximum at 218 nm and at 260 nm (Appendix 4.1), using an 1-cm cell and 0.05 M sulfuric acid R in the reference cell. Calculate the content of cimetidine, C10H16N6S using the ratio of the difference between the absorbances of two solutions at the two wavelengths and the declared content of C10H16N6S in cimetidine RS.

CINEOLE

Identification A. It complies with the test for Refractive index. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Ethyl acetate - toluene (1 : 9). Test solution: Dilute 1 ml of solution S (see Appearance of solution) to 25 ml with ethanol (96%) R. Reference solution: Dissolve 80 mg of cineole RS in ethanol (96%) R and dilute to 10 ml with the same solvent. Procedure: Apply separately to the plate 2 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in air. Spray with anisaldehyde solution R, heat at 100 - 105 °C for 5 minutes. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution C. To 0.1 ml add 4 ml of concentrated sulfuric acid R. An orange-red colour develops. Add 0.2 ml of formaldehyde solution R. The colour changes to deep brown. Appearance of solution Solution S: Dissolve 2.00 g of the substance to be examined in ethanol (96%) R and dilute to 10.0 ml with the same solvent. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 1). Optical rotation The optical rotation of solution S is -0.10° to +0.10° (Appendix 6.4).

Storage Store in a cool, dry place, protected from light.

Refractive index 1.456 to 1.460 (Appendix 6.1).

Action and use Histamine H2-receptor antagonist.

Related substances Examine by gas chromatography (Appendix 5.2). Internal standard solution: Dissolve 1.0 g of camphor R in heptane R and dilute to 200 ml with the same solvent. Test solution (1): Dissolve 2.5 g of the substance to be examined in heptane R and dilute to 25.0 ml with the same solvent. Test solution (2): Dissolve 2.5 g of the substance to be examined in heptane R, add 5.0 ml of the internal standard solution and dilute to 25.0 ml with heptane R. Reference solution (1): To 2.0 ml of test solution (1) add 20.0 ml of the internal standard solution and dilute to 100.0 ml with heptane R. Reference solution (2): Dissolve 50 mg of 1,4-cineole R and 50 mg of the substance to be examined in heptane R and dilute to 50.0 ml with the same solvent. Chromatographic system: A capillary column (50 m × 0.25 mm), stationary phase is macrogol 20 000 (film thickness 0.25 µm). Carrier gas: Helium for chromatography. Linear velocity: 45 cm/s. Split-ratio: 1 : 70. Detector: A flame-ionisation detector.

Usual strength 200 mg, 300 mg, 400 mg, 800 mg. CINEOLE Cineolum Eucalyptol

C10H18O M. 154.3 Cineole is 1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane.

Characters Clear colourless liquid with characteristic odour. Practically insoluble in water, miscible with ethanol (96%) and with dichloromethane.

275

VP V

CINNARIZINE

Temperature of the column: Maintain the temperature of the column at 50 °C for 10 minutes, then raise the temperature at a rate of 2 °C per minute to 100 °C, continue to raise the temperature at a rate of 10 °C per minute to 200 °C and maintain for 10 minutes. Temperature of the injection port: 220 °C. Temperature of the detector: 250 °C. Volume of injection: 1 µl. Procedure: Inject the above solutions in succession. The test is not valid unless, in the chromatogram obtained with reference solution (2), the resolution between the two peaks due to 1,4-cineole and cineole is at least 10. Calculate the ratio R of the area of the peak due to cineole to the area of the peak due to the internal standard from the chromatogram obtained with reference solution (1). Limits: In the chromatogram obtained with test solution (2), calculate the ratio of the sum of the areas of any peaks, apart from the principal peak and the peak due to the internal standard, to the area of the peak due to internal standard: this ratio is not greater than R (2%). Disregard any peak with an area less than 0.025 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%).

Residue on evaporation Not more than 0.1%. To 2.0 g add 5 ml of water, evaporate to dryness on a waterbath and dry at 100 - 105 °C for 1 h. The residue weighs not more than 2 mg. Storage In an airtight container, protected from light. CINNARIZINE Cinnarizinum

C26H28N2

Appearance of solution Dissolve 0.5 g in methylene chloride R and dilute to 20 ml with the same solvent. The solution is clear (Appendix 9.2) and not more intensely coloured than reference solution BY7 (Appendix 9.3, method 2).

M. 368.5

Cinnarizine is (E)-1-(diphenylmethyl)-4-(3-phenylprop-2enyl)piperazine. It contains not less than 99.0% and not more than 101.0% of C26H28N2, calculated with reference to the dried substance.

Characters A white or almost white powder. Practically insoluble in water, freely soluble in methylene chloride, soluble in acetone, slightly soluble in ethanol (96%) and in methanol. 276

Identification Apply one of the two following identifications: First identification: A, B. Second identification: A, C, D. A. Melting point (Appendix 6.7): 118 °C to 122 °C. B. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum obtained with cinnarizine RS. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Using suitable octadecylsilyl silica gel with a fluorescent indicator having an optimal intensity at 254 nm. Mobile phase: 1 M sodium chloride - methanol - acetone (20 : 30 : 50). Test solution: Dissolve 10 mg of the substance to be examined in methanol R and dilute to 20 ml with the same solvent. Reference solution (1): Dissolve 10 mg of cinnarizine RS in methanol R and dilute to 20 ml with the same solvent. Reference solution (2): Dissolve 10 mg of cinnarizine RS and 10 mg of flunarizine hydrochloride RS in methanol R and dilute to 20 ml with the same solvent. Procedure: Apply separately to the plate 5 µl of each solution. Develop in an unsaturated tank over a path of 15 cm. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) shows two clearly separated spots. D. Dissolve 0.2 g of anhydrous citric acid R in 10 ml of acetic anhydride R in a water-bath at 80 °C and maintain the temperature of the water-bath at 80 °C for 10 min. Add about 20 mg of the substance to be examined. A purple colour develops.

Acidity or alkalinity Solution S: Suspend 0.5 g in 15 ml of water, boil for 2 minutes. Cool and filter. Dilute the filtrate to 20 ml with carbon dioxide-free water R. To 10 ml of solution S, add 0.1 ml of phenolphthalein solution R and 0.25 ml of 0.01 M sodium hydroxide VS. The solution is pink. To 10 ml of solution S, add 0.1 ml of methyl red solution R and 0.25 ml of 0.01 N hydrochloric acid VS. The solution is red. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: A 1% solution of ammonium acetate R.

VP V

CINNARIZINE TABLETS

Mobile phase B: A 0.2% (v/v) solution of glacial acetic acid in acetonitrile. Test solution: Dissolve 25.0 mg of the substance to be examined in methanol R and dilute to 10.0 ml with the same solvent. Reference solution: Dilute 1.0 ml of the test solution to 100.0 ml with methanol R. Dilute 5.0 ml of this solution to 20.0 ml with methanol R. Resolution solution: Dissolve 12.5 mg of cinnarizine RS and 15.0 mg of flunarizine hydrochloride RS in methanol R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of this solution to 20.0 ml with methanol R. Chromatographic system: A stainless steel column (10 cm × 4.0 mm) packed with basedeactivated octadecylsilyl silica gel for chromatography R (3 µm). Flow rate: 1.5 ml/min. Detector: A spectrophotometer set at 230 nm. Volume of injection: 10 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

Comment

0 - 20

75 → 10

25 90

Linear gradient

20 - 25

10

90

Isocratic elution

25 - 30

75

25

Switch to initial eluent composition

30 - 0

75

25

Restart gradient

Equilibrate the column for a least 30 minutes at the mixture of mobile phase A - mobile phase B (75 : 25). Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with the reference solution is at least 50% of the full scale of the recorder. If necessary, adjust the concentration of glacial acetic acid in mobile phase B to obtain a horizontal baseline in the chromatogram. Inject resolution solution. When the chromatogram is recorded in the prescribed conditions, the retention times are: cinnarizine about 11 min and flunarizine about 11.5 min. The test is not valid unless the resolution between the peaks corresponding to cinnarizine and flunarizine is at least 5.0. If necessary, adjust the time programme for the gradient elution. Inject methanol R as a blank, the test solution and the reference solution. Limits: In the chromatogram obtained with the test solution: The area of any peak, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with the reference solution (0.25%). The sum of the areas of the peaks, apart from the principal

peak, is not greater than twice the area of the principal peak in the chromatogram obtained with the reference solution (0.5%). Disregard any peak due to the blank and any peak with an area less than 0.2 times the area of the principal peak in the chromatogram obtained with the reference solution.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). Dissolve 1.0 g in a mixture of 15 volumes of water and 85 volumes of acetone R. Add dilute hydrochloric acid solution R until dissolution is complete, dilute to 20 ml with the same mixture of water and acetone R. 12 ml of the solution complies with limit test for heavy metals, method 2. Prepare the reference solution using 10 ml of lead standard solution (1 ppm Pb) obtained by diluting lead standard solution (100 ppm Pb) R with the mixture of water and acetone R above. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; in vacuo; 60 °C; 4 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Non-aqueous titration (Appendix 10.6). Dissolve 0.150 g in 50 ml of a mixture of 1 volume of anhydrous acetic acid R and 7 volumes of ethyl methyl ketone R. Titrate with 0.1 N perchloric acid VS, using 0.2 ml of naphtholbenzein solution R as indicator. Each ml of 0.1 N perchloric acid VS is equivalent to 18.43 mg of C26H28N2. Storage Store protected from light. Action and use Histamine H1-receptor antagonist. CINNARIZINE TABLETS Tabellae Cinnarizini Cinnarizine tablets contain cinnarizine. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements. Content of cinnarizine, C26H28N2, 90.0% to 110.0% of the stated amount.

Identification Shake well a quantity of the powdered tablets equivalent of about 0.25 g of cinnarizine in 25 ml of ethanol R, 277

VP V

CIPROFLOXACIN HYDROCHLORIDE

heat gently to dissolve and filter. Evaporate the filtrate to dryness, the residue complies with the following tests: A. Dissolve about 20 mg in 5 ml of ethanol R by warming, add 2 drops of a 6.5% solution of potassium hydroxide R, mix well. Add 2 - 3 drops of 0.02 M potassium permanganate R, the violet colour is immediately discharged. B. To about 10 mg, add several drops of a 2% solution of formaldehyd R in sulfuric acid R, a red colour develops. C. In a test tube, add about 50 mg. Cover the orifice with a piece of filter paper moistened with a 2% solution of trichloacetic acid R and 1 drops of a 5% solution of p-dimethylaminobenzaldehyde R in hydrochloric acid R. Ignite the tube to obtain a black mass. Fumes are evolved which turn the filter paper to violet.

Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 1000 ml of 0.1 M hydrochloric acid R. Rotation speed: 100 rpm. Time: 45 min. Procedure: After the specified time, withdraw a sample of the medium and filter, discard the first 20 ml of the filtrat. Dilute 5 ml of the filtrate with medium to 20 ml. Measure the absorbance of the solution at 253 nm (Appendix 4.1), using 0.01 M hydrochloric acid in the reference cell. Calculate the content of cinnarizine, C26H28N2, dissolved in the medium, taking 575 as the value of A (1%, 1 cm) of cinnarizine at 253 nm. Tolerance: Not less than 70% (Q) of the stated amount of cinnarizine is dissolved in 45 min. Assay Weigh 20 tablets, calculate the average mass, and powder finely. Weigh accurately a quantity of the powdered tablets equivalent to about 30 mg of cinnarizine in a 200 ml volumetric flask. Add 150 ml of 0.1 M hydrochloric acid R, shake well to dissolve and add 0.1 M hydrochloric acid R to volume. Mix well and filter. Dilute 5.0 ml of the filtrate with 0.1 M hydrochloric acid R in a 100 ml volumetric flask to volume, mix well. Measure the absorbance of the solution at 253 nm (Appendix 4.1), using 0.1 M hydrochloric acid in the reference cell. Calculate the content of cinnarizine, C26H28N2, in the tablets, taking 575 as the value of A (1%, 1 cm) of cinnarizine at 253 nm. Storage Store in a airtight containers, protected from light. Action and use Histamine H1-receptor antagonist. Usual strength 15 mg, 25 mg. 278

CIPROFLOXACIN HYDROCHLORIDE Ciprofloxacini hydrochloridum

C17H18FN3O3,HCl

M. 367.8

Ciprofloxacin hydrochloride is 1-cyclopropyl-6-fluoro-4oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid hydrochloride. It contains not less than 98.0% and not more than 102.0% of C17H18FN3O3,HCl, calculated with reference to the anhydrous substance.

Characters A pale yellow, crystalline powder, slightly hygroscopic. Soluble in water, slightly soluble in methanol, very slightly soluble in ethanol, practically insoluble in acetone, in ethyl acetate and in methylene chloride. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum obtained with ciprofloxacin hydrochloride RS. B. It gives reaction B of chlorides (Appendix 8.1). Appearance of solution Solution S: Dissolve 0.5 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent. Dilute 10 ml of solution S to 20 ml with carbon dioxidefree water R. The solution is clear (Appendix 9.2) and not more intensely coloured than reference solution GY5 (Appendix 9.3, method 2). pH The pH of solution S is 3.5 to 4.5 (Appendix 6.2). Fluoroquinolonic acid Not more than 0.2%. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Acetonitrile - concentrated ammonia methanol - methylene chloride (10 : 20 : 40 : 40). Test solution: Dissolve 50 mg of the substance to be examined in water and dilute to 5 ml with the same solvent. Reference solution: Dissolve 10 mg of fluoroquinolonic acid RS in a mixture of 0.1 ml of a ammonia R and 90 ml of water and dilute to 100 ml with water. Dilute 2 ml of the solution to 10 ml with water. Procedure: Apply separately to the plate 5 µl of each solution. At the bottom of a chromatographic tank, place an evaporating dish containing 50 ml of concentrated ammonia R. Expose the plate to the ammonia vapour for 15 min in the closed tank. Withdraw the plate, transfer

VP V

to a second chromatographic tank and proceed with development. Develop over a path of 15 cm. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot corresponding to fluoroquinolonic acid in the chromatogram obtained with the test solution is not more intense than the principal spot in the chromatogram obtained with the reference solution.

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Mix 13 volumes of acetonitrile R and 87 volumes of a 0.245% solution of phosphoric acid R, previously adjusted to pH 3.0 with triethylamine R. Test solution: Dissolve 25.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Reference solution (1): Dissolve 5 mg of ciprofloxacin hydrochloride for peak identification RS in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (2): Dilute 1.0 ml of the test solution to 50.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm), maintaining the temperature of the column at 40 °C. Detector: A spectrophotometer set at 278 nm. Flow rate: 1.5 ml/min. Volume of injection: 50 µl. Procedure: The run time is twice the retention time of ciprofloxacin. Identify the impure peaks: Use the chromatogram obtained with reference solution (1) and parameters of the relative retention times with reference to ciprofloxacin to identify the corresponding impure peaks. Inject reference solution (1). Relative retention times with reference to ciprofloxacin (retention time = about 9 minutes) are: impurity E = about 0.4; impurity F = about 0.5; impurity B = about 0.6; impurity C = about 0.7; impurity D = about 1.2. System suitability: The chromatogram obtained with reference solution (1), the resolution between the peaks due to impurity B and impurity C is at least 1.3. Litmits: Correction factors: for the calculation of contents, multiply the peak areas of the following impurities by the corresponding correction factor: impurity B = 0.7; impurity C = 0.6; impurity D = 1.4; impurity E = 6.7. In the chromatogram obtained with the test solution: The corrected area of any peak due to each impurity B, C, D and E, is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.2%). The area of any other impurity peak is not more than half the area of the principal peak in the chromatogram obtained with reference solution (2) (0.1%).

CIPROFLOXACIN HYDROCHLORIDE

The sum of the areas of all peaks, apart from the principal peak, is not more than 2.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.5%). Disregard any peak with an area less than or equal to 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%).

Note: Impurity A: 7-chloro-1-cyclopropyl-6-fluoro-4-oxo-1,4dihydroquinoline-3-carboxylic acid (fluoroquinolonic acid). Impurity B: 1-cyclopropyl-4-oxo-7-(piperazin-1-yl)-1,4dihydroquinoline-3-carboxylic acid (desfluoro compound). Impurity C: 7-[(2-aminoethyl)amino]-1-cyclopropyl-6-fluoro-4oxo-1,4-dihydroquinoline-3-carboxylic acid (ethylenediamine compound). Impurity D: 7-chloro-1-cyclopropyl-4-oxo-6-(piperazin-1-yl)1,4-dihydroquinoline-3-carboxylic acid. Impurity E: 1-cyclopropyl-6-fluoro-7-(piperazin-1-yl)quinolin4(1H)-one. Impurity F: 1-cyclopropyl-6-hydroxy-4-oxo-7-(piperazin-1-yl)1,4-dihydroquinoline-3-carboxylic acid.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). Dissolve 0.25 g in water and dilute to 30 ml with the same solvent, filter. The filtrate complies with limit test for heavy metals, method 5. Prepare the reference solution using 5 ml of lead standard solution (1 ppm Pb) R. Water Not more than 6.7% (Appendix 10.3). Determined on 0.200 g. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g in a platinum crucible. Assay Examine by liquid chromatography (Appendix 5.3). Chromatographic system as described in the test for Related substances with the following modifications. Volume of injection: 10 µl. Test solution: Dissolve 25.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Reference solution: Dissolve 25.0 mg of ciprofloxacin hydrochloride RS in the mobile phase and dilute to 50.0 ml with the mobile phase. Calculate the percentage content of C17H18FN3O3,HCl using the areas for the principal peaks in the chromatogram obtained with the test solution, the reference solution and the declared content of C17H18FN3O3,HCl in ciprofloxacin hydrochloride RS. Storage Store in an airtight container, protected from light. 279

VP V

CIPROFLOXACIN EYE DROPS

Action and use Fluoroquinolone antibacterial. Preparations Tablets, eye drops. CIPROFLOXACIN EYE DROPS Collyrium Ciprofloxacini Ciprofloxacin eye drops are a sterile, aqueous solution of ciprofloxacin hydrochloride, they may have suitable excipients. The eye drops comply with the requirements stated under “Eye drops” (Appendix 1.14) and with the following requirements.

Content of ciprofloxacin, C17H18FN3O3, 90.0% to 110.0% of the stated amount. Characters Clear, colourless solution. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Dichloromethane - methanol - ammonia acetonitrile (40 : 40 : 20 : 10). Test solution: Dilute the eye drops with water to produce a solution containing 3 mg of ciprofloxacin hydrochloride per ml. Reference solution: Dissolve a quantity of ciprofloxacin hydrochloride RS in water to obtain a solution containing about 3 mg/ml. Procedure: Apply separately to the plate 3 µl of each solution. Allow the plate to expose to ammonia vapour for 15 minutes in a chromatographic tank. After developing over a path of three-fourths of the length of the plate, remove the plate, allow it to dry in air and examine in ultraviolet light at 254 nm and at 365 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is the same as that of the principal peak in the chromatogram obtained with the reference solution. pH 4.0 to 5.5 (Appendix 6.2). Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: 0.025 M phosphoric acid adjusted to pH 3.0 with triethylamine - acetonitrile (87 : 13). Test solution: Dilute a volume of the eye drops equivalent to 6 mg of ciprofloxacin with mobile phase to produce 50.0 ml. Mix well and filter. 280

Reference solution: Weigh accurately a quantity of ciprofloxacin hydrochloride RS and dissolve in mobile phase to obtain a solution containing about 0.14 mg/ml. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (10 µm). Detector: A spectrophotometer set at 280 nm. Flow rate: 1.0 ml/min. Column temperature: 30 °C ± 1 °C. Volume of injection : 20 µl. Procedure: System suitability: Inject the reference solution, the relative standard deviation of the peak areas in 6 replicate injections is at most 2.0%. Inject the test solution. Calculate the content of ciprofloxacin, C17H18FN3O3 in the eye drops using the peak areas of ciprofloxacin in the chromatograms obtained from the test solution, the reference solution and and the declared content of C17H18FN3O3 in ciprofloxacin hydrochloride RS. Each mg of ciprofloxacin hydrochloride, C17H18FN3O3,HCl is equivalent to 0.9010 mg of ciprofloxacin C17H18FN3O3.

Storage Store in a cool place, protected from light. Action and use Fluoroquinolone antibacterial. Usual strength 0.3% solution. CIPROFLOXACIN TABLETS Tabellae Ciprofloxacini Ciprofloxacin tablets contain ciprofloxacin hydrochloride. They may be film-coated. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of ciprofloxacin, C17H18FN3O3, 95.0% to 105.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Dichloromethane - methanol - concentrated ammonia - acetonitrile (40 : 40 : 20 : 10). Test solution: Shake a quantity of the powdered tablets equivalent to 100 mg of ciprofloxacin with 10 ml of water, filter. Reference solution: Dissolve a quantity of ciprofloxacin hydrochloride RS in water to obtain a solution containing about 10 mg/ml.

VP V

Procedure: Apply separately to the plate 5 µl of each solution. Allow the plate to expose to ammonia vapour for 15 minutes in a chromatographic tank. After developing over a path of 15 cm with the mobile phase, remove the plate, allow it to dry in air for 15 min and examine in ultraviolet light at 254 nm and at 365 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is the same as that of the principal peak in the chromatogram obtained with the reference solution.

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of water Rotation speed: 50 rpm. Time: 30 min. Procedure: After the specified time, withdraw a sample of the medium, filter, dilute with water to obtain an appropriate concentration. Measure the absorbance at the maximum at 276 nm (Appendix 4.1), using an 1-cm cell and water in the reference cell. At the same time, measure the absorbance of a solution of ciprofloxacin hydrochloride RS (C17H18FN3O3,HCl) in water having the same concentration as the test solution. Calculate the content of ciprofloxacin, C17H18FN3O3, dissolved in the medium from the declared content of ciprofloxacin in ciprofloxacin hydrochloride RS (C17H18FN3O3,HCl). Each mg of ciprofloxacin hydrochloride, C17H18FN3O3,HCl, is equivalent to 0.9010 mg of ciprofloxacin, C17H18FN3O3. Tolerance: Not less than 80% (Q) of the stated amount of ciprofloxacin is dissolved in 30 min. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: 0.025 M phosphoric acid adjusted to pH 3.0 with triethylamine - acetonitrile (87 : 13). Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Weigh accurately a quantity of the powdered tablets equivalent to about 40 mg of ciprofloxacin in a 200 ml volumetric flask, add 150 ml of mobile phase and sonicate for 20 min, dilute with mobile phase to volume, mix well, filter. Reference solution: Dissolve a quantity of ciprofloxacin hydrochloride RS in water to obtain a solution containing about 0.2 mg of ciprofloxacin/ml. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm), Nucleosil 120-C18 is suitable. Column temperature: 40 °C. Detector : A spectrophotometer set at 278 nm. Flow rate: 1.5 ml/min.

CITRIC ACID MONOHYDRATE

Volume of injection : 10 µl. Procedure: System suitability: Inject the reference solution, the column efficiency (determined from the principal peak) is not less than 2,500 theoretical plates. The symmetry factor is not more than 2 and the relative standard deviation for 6 replicate injections of the reference solution is at most 1.5%. Inject the test solution. Calculate the content of ciprofloxacin, C17H18FN3O3 in the tablets using the peak areas of ciprofloxacin in the chromatograms obtained from test solution, reference solution and the content of C17H18FN3O3 in ciprofloxacin hydrochloride RS. Each mg of ciprofloxacin hydrochloride, C17H18FN3O3,HCl is equivalent to 0.9010 mg of ciprofloxacin C17H18FN3O3.

Storage Store in a well-closed container and in a cool place, protected from light. Action and use Fluoroquinolone antibacterial. Usual strength 250 mg, 500 mg, 750 mg. CITRIC ACID MONOHYDRATE Acidum citricum monohydricum

,

C6H8O7,H2O 

M. 210.1  

Citric acid monohydrate is 2-hydroxy-propane-1,2,3tricarboxylic acid. It contains not less than 99.5% and not more than 101.0% of C6H8O7, calculated with reference to the anhydrous substance.

Characters A white, crystalline powder, colourless crystals or granules, efflorescent. Very soluble in water, freely soluble in ethanol (96%), sparingly soluble in ether. Identification Apply one of the two following identifications. First identification: A, E. Second identification: B, C, D, E. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of citric acid monohydrate RS after drying both the substance to be examined and the reference substance at 100 - 105 °C for 2 h. 281

VP V

CLARITHROMYCIN

B. Dissolve 1 g in 10 ml of water. pH of this solution is less than 4 (Appendix 6.2). C. Add about 5 mg to a mixture of 1 ml of acetic anhydride R and 3 ml of pyridine R. A red colour develops. D. Dissolve 0.5 g in 5 ml of water, neutralise using 1 M sodium hydroxide R (about 7 ml), add 10 ml of calcium chloride solution R and heat to boiling. A white precipitate is formed. E. It complies with the test for Water.

Heavy metals Not more than 10 ppm (Appendix 9.4.8). Dissolve 5.0 g in several portions in 39 ml of dilute sodium hydroxide solution R and dilute to 50 ml with water. 12 ml complies with limit test 1 for heavy metals. Prepare the standard using lead standard solution (1 ppm Pb) R.

Appearance of solution Dissolve 2.0 g in water and dilute to 10 ml with the same solvent. The solution is clear (Appendix 9.2) and not more intensely coloured than reference solution Y7, BY7 or GY7 (Appendix 9.3, method 2).

Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g.

Readily carbonisable substances To 1.0 g in a cleaned test tube add 10 ml of sulphuric acid R and immediately heat the mixture in a water-bath at 90 ± 1 °C for 1 h. Immediately cool rapidly. The solution is not more intensely coloured than a mixture of 1 ml of red primary solution and 9 ml of yellow primary solution (Appendix 9.3, method 1). Oxalic acid Not more than 0.036%, calculated as anhydrous oxalic acid. Dissolve 0.80 g in 4 ml of water. Add 3 ml of hydrochloric acid R and 1 g of zinc R in granules. Boil for 1 min. Allow to stand for 2 min. Transfer the supernatant liquid to a test-tube containing 0.25 ml of a 1% solution of phenylhydrazine hydrochloride R and heat to boiling. Cool rapidly, transfer to a graduated cylinder and add an equal volume of hydrochloric acid R and 0.25 ml of a 5% solution of potassium ferricyanide. Shake and allow to stand for 30 min. Any pink colour in the solution is not more intense than that in a standard prepared at the same time in the same manner using 4 ml of a 0.01% solution of oxalic acid.

Water 7.5% to 9.0% (Appendix 10.3). Determined on 0.500 g.

Bacterial endotoxins Not more than 0.5 EU/mg (Appendix 13.2). If intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins, its maximum allowable endotoxin concentration is 0.5 unit per milligram (Appendix 13.2). Assay Dissolve 0.550 g in 50 ml of water. Titrate with 1 N sodium hydroxide VS, using 0.5 ml of phenolphthalein solution R as indicator. 1 ml of 1 N sodium hydroxide VS is equivalent to 64.03 mg of C6H8O7. Storage In an airtight container. CLARITHROMYCIN Clarithromycinum

Sulfates Not more than 0.015% (Appendix 9.4.14). Dissolve 1.0 g in water and dilute to 15 ml with the same solvent. The solution complies with the limit test for sulphates. Aluminium Not more than 0.2 ppm (Appendix 9.4.9). If intended for use in the manufacture of dialysis solutions, it complies with the test for aluminium. Test solution: Dissolve 20 g in 100 ml of water and add 10 ml of acetate buffer solution pH 6.0 R. Reference solution: A mixture of 2 ml of aluminium standard solution (2 ppm Al) R, 10 ml of acetate buffer solution pH 6.0 R and 98 ml of water. Blank solution: A mixture of 10 ml of acetate buffer solution pH 6.0 R and 100  ml of water. 282

C38H69NO13

M. 748.0

Clarithromycin is (3R,4S,5S,6R,7R,9R,11R,12R,13S,14R)4-[(2,6-dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyl)oxy]-14-ethyl-12,13-dihydroxy-7-methoxy3,5,7,9,11,13-hexamethyl-6-[[3,4,6-trideoxy-3-(dimethylamino)-β-D-xylo-hexopyranosyl]oxy] oxacyclotetradecane -2,10-dione (6-O-methylerythromycin A). It contains not less than 96.0% and not more than 102.0% of C38H69NO13, calculated with reference to the anhydrous substance.

VP V

Characters White or almost white, crystalline powder. Practically insoluble in water, soluble in acetone and in methylene chloride, slightly soluble in methanol. Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum obtained with clarithromycin RS. Appearance of solution Solution S: Dissolve 0.500 g in methylene chloride R and dilute to 50.0 ml with the same solvent. Solution S is clear or not more opalescent than reference suspension II (Appendix 9.2) and not more intensely coloured than reference solution Y7 (Appendix 9.3). Specific optical rotation -94° to -102°, calculated with reference to the anhydrous substance (Appendix 6.4). Determine on solution S. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: A 0.476% solution of potassium dihydrogen phosphate R adjusted to pH 4.4 with dilute phosphoric acid R or a 4.5% solution of potassium hydroxide R, filtered through a C18 filtration kit. Mobile phase B: Acetonitrile R. Test solution: Dissolve 75.0 mg of the substance to be examined in 25 ml of acetonitrile R and dilute to 50.0 ml with water. Reference solution (1): Dissolve 75.0 mg of clarithromycin RS in 25 ml of acetonitrile R and dilute to 50.0 ml with water. Reference solution (2): Dilute 5.0 ml of reference solution (1) to 100.0 ml with a mixture of equal volumes of acetonitrile R and water. Reference solution (3): Dilute 1.0 ml of reference solution (2) to 10.0 ml with a mixture of equal volumes of acetonitrile R and water. Reference solution (4): Dissolve 15.0 mg of clarithromycin for peak identification RS in 5.0 ml of acetonitrile R and dilute to 10.0 ml with water. Blank solution: Dilute 25.0 ml of acetonitrile R to 50.0 ml with water and mix. Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase octadecylsilyl silica gel (3.5 µm). Column temperature: 40 °C. Detector: A spectrophotometer set at 205 nm. Volume of injection: 10 µl. Flow rate: 1.1 ml/min. Procedure: Carry out a linear gradient elution using the following conditions:

CLARITHROMYCIN

Time (min) 0 - 32 32 - 34 34 - 36 36 - 42

Mobile phase A (% v/v) 75 → 40 40 40 → 75 75

Mobile phase B (% v/v) 25 → 60 60 60 → 25 25

Inject the blank solution, the test solution and reference solutions (2), (3) and (4). Identify the impure peaks: Use the chromatogram obtained with reference solution (4) to identify the corresponding G and H impure peaks. Relative retention times with reference to clarithromycin (retention time = about 11 minutes) are: impurity I = about 0.38; impurity A = about 0.42; impurity J = about 0.63; impurity L = about 0.74; impurity B = about 0.79; impurity M = about 0.81; impurity C = about 0.89; impurity D = about 0.96; impurity N = about 1.15; impurity E = about 1.27; impurity F = about 1.33; impurity P = about 1.35; impurity O = about 1.41; impurity K = about 1.59; impurity G = about 1.72; impurity H = about 1.82. System suitability: In the chromatogram obtained with reference solution (2), symmetry factor not more than 1.7 for the peak due to clarithromycin. In the chromatogram obtained with reference solution (4), peak-to-valley ratio (Hp/Hv) not less than 3.0. Hp is height above the baseline of the peak due to impurity D and Hv is height above the baseline of the lowest point of the curve separating impurity D peak from the peak due to clarithromycin. Limits: Correction factors: For the calculation of contents, multiply the peak areas of the following impurities by the corresponding correction factor: impurity G = 0.27; impurity H = 0.15. In the chromatogram obtained with the test solution: The area of any impurity peak is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (3) (1.0%), and not more than 4 such peaks have an area greater than 0.8 times the area of the principal peak in the chromatogram obtained with reference solution (3) (0.4%). The sum of the areas of all peaks, apart from the principal peak is not more than 7 times the area of the principal peak in the chromatogram obtained with reference solution (3) (3.5%). Disregard any peak with an area less than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (3) (0.1%). Disregard the peaks eluting before impurity I and after impurity H. Note: Impurity A: Clarithromycin F. Impurity B: 6-O-methyl-15-norerythromycin A. Impurity C: 6-O-methylerythromycin A (E)-9-oxime. Impurity D: 3’’-N-demethyl-6-O-methylerythromycin A. Impurity E: 6,11-di-O-methylerythromycin A.

283

VP V

CLARITHROMYCIN CAPSULES Impurity F: 6,12-di-O-methylerythromycin A. Impurity G: 6-O-methylerythromycin A (E)-9-(O-methyloxime). Impurity H: 3’’-N-demethyl-3’-N-formyl-6-O-methylerythromycin A. Impurity I: 3-O-decladinosyl-6-O-methylerythromycin A. Impurity J: Erythromycin A (E)-9-oxime. Impurity K: (1S,2R,5R,6S,7S,8R,9R,11Z)-2-ethyl-6-hydroxy -9-methoxy-1,5,7,9,11,13-hexamethyl-8-[[3,4,6-trideoxy3-(dimethylamino)-β-D-xylo-hexopyranosyl]oxy]-3,15dioxabicyclo[10.2.1]pentadeca-11,13-dien-4-one (3-O-decladinosyl8,9:10,11-dianhydro-6-O-methylery-thromycin A-9,12-hemiketal. Impurity L: 6-O-methylerythromycin A (Z)-9-oxime. Impurity M: 3’’-N-demethyl-6-O-methylerythromycin A (E)-9oxime. Impurity N: (10E)-10,11-didehydro-11-deoxy-6-O-methylerythromycin A. Impurity O: 6-O-methylerythromycin A (Z)-9-(O-methyloxime). Impurity P: 4’,6-di-O-methylerythromycin A.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). Dissolve 1.0 g in a mixture of water - dioxan (15 : 85) and dilute to 20 ml with the same mixture of solvents. 12 ml of the solution complies with limit test for heavy metals, method 2. Prepare the reference solution using lead standard solution (1 ppm Pb) obtained by diluting lead standard solution (100 ppm Pb) R with a mixture of water - dioxan (15 : 85). Water Not more than 2.0% (Appendix 10.3). Determined on 0.500 g. Sulfated ash Not more than 0.2% (Appendix 9.9, method 2). Determined on 0.5 g. Assay Examine by liquid chromatography (Appendix 5.3). Chromatographic system as described in the test for Related substances. Inject the test solution and reference solution (1). Calculate the percentage content of C38H69NO13, using the areas of the peaks due to clarithromycin in the chromatograms obtained with the test solution and reference solution (1). Storage Store in an airtight container, protected from light. Action and use Macrolide antibacterial. Preparations Tablets; capsules; powder for oral suspension.

284

CLARITHROMYCIN CAPSULES Capsulae Clarithromycini Clarithromycin capsules contain clarithromycin. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of clarithromycin, C38H69NO13, 90.0% to 110.0% of the stated amount. Identification In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is the same as that of clarithromycin peak in the chromatogram obtained with the reference solution. Loss on drying Not more than 6.0%. Determined on 0.25 g of the content of the capsules drying in an oven in vacuo at a pressure of 5 mmHg at 110 °C for 3 h (Appendix 9.6). Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of 0.1 M sodium acetate buffer. 0.1 M sodium acetate buffer: Transfer 13.61 g of sodium acetate trihydrate R to a 1000 ml volumetric flask, add water to dissolve, dilute with water to volume, and mix. Adjust with 0.1 M acetic acid R to a pH of 5.0. Rotation speed: 50 r/min. Time: 30 min. Procedure: Carry out the method for liquid chromatography (Appendix 5.3). Mobile phase, reference solution and chromatographic system carry out as in Assay. Test solution: After the specified time, withdraw a portion of the medium and filter, discarding the first 20 ml of the filtrate. Dilute the filtrate with mobile phase to obtain a solution containing 125 µg of clarithromycin/ml. Tolerance: Not less than 80% (Q) of the stated amount of clarithromycin, C38H69NO13, is dissolved in 30 min. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Prepare a mixture of methanol R and 0.067 M solution of potassium dihydrogen phosphate (65 : 35), adjust with phosphoric acid R to a pH of 4.0. Make adjustments if necessary. Test solution: Weigh 20 capsules, calculate the average weight of the capsule contents and powder finely. Weigh a quantity of the powder containing 0.2 g of clarithromycin in 50 ml volumetric flask, add 35 ml of methanol R, shake and by mechanical means for 30 minutes. Dilute with methanol R to volume, mix, and allow any insoluble matter to settle.

VP V

Transfer 3.0 ml of the supernatant to a 100 ml volumetric flask, dilute with mobile phase to volume, and mix. Reference solution: Dissolve an accurately weighed quantity of clarithromycin RS in methanol R, shaking and sonicating if necessary to effect dissolution, to obtain a stock solution having a known concentration of about 625 µg of clarithromycin/ml. Dilute 10.0 ml of this stock solution to 50.0 ml with mobile phase, mix well. Resolution solution: Prepare a solution of E impurity of clarithromycin RS (6,11-di-O-methylerythromycin A, C39H71NO13) in methanol R containing about 625 µg/ml. Transfer 10.0 ml of this solution and 10.0 ml of the reference solution to a 50 ml volumetric flask, dilute with mobile phase to volume, and mix. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 µm). Column temperature: 50 °C. Detector: A spectrophotometer set at 210 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the resolution solution, the relative retention times are about 0.75 for clarithromycin and 1.0 for E impurity; and the resolution between clarithromycin and E impurity is not less than 2.0. Inject the reference solution, the column efficiency, determined from the clarithromycin peak, is not less than 750 theoretical plates, the tailing factor is not less than 0.9 and not more than 2.0; and the relative standard deviation for 6 replicate injections is not more than 2.0%. Inject separately the test solution and the resolution solution. Calculate the content of clarithromycin, C38H69NO13, in the capsules using the areas of the principal peaks in the chromatograms obtained with the test solution, the reference solution, and the declared content of C38H69NO13 in clarithromycin RS.

Storage Store in an airtight container, at a temperature not exceed 30 °C. Action and use Macrolide antibacterial. Usual strength 250 mg, 500 mg. CLARITHROMYCIN TABLETS Tabellae Clarithromycini Clarithromycin tablets contain clarithromycin. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

CLARITHROMYCIN TABLETS

Content of clarithromycin, C38H69NO13, 90.0% to 110.0% of the stated amount. Identification In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is the same as that of clarithromycin peak in the chromatogram obtained with the reference solution. Loss on drying Not more than 6.0%. Determined on 0.25 g of powdered tablets by drying in an oven in vacuo at a pressure of 5 mmHg at 110 °C for 3 h (Appendix 9.6). Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of 0.1 M sodium acetate buffer. 0.1 M sodium acetate buffer: Transfer 13.61 g of sodium acetate trihydrate R to a 1000 ml volumetric flask, add water to dissolve, dilute with water to volume, and mix. Adjust with 0.1 M acetic acid R to a pH of 5.0. Rotation speed: 50 rpm. Time: 30 min. Procedure: Carry out the method for liquid chromatography (Appendix 5.3). Mobile phase, reference solution and chromatographic system carry out as in Assay. Test solution: After the specified time, withdraw a sample of the medium and filter, discarding the first 20 ml of the filtrate. Dilute the filtrate with mobile phase to obtain a solution containing 125 µg of clarithromycin/ml. Tolerance: Not less than 80% (Q) of the stated amount of clarithromycin, C38H69NO13, is dissolved in 30 min. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Prepare a mixture of methanol R and 0.067 M monobasic potassium phosphate (65 : 35), adjust with phosphoric acid R to a pH of 4.0. Make adjustments the mobile phase if necessary. Test solution: Weigh 20 tablets and calculate the average mass, powder finely. Weigh a quantity of the powdered tablets containing 0.2 g of clarithromycin in 50 ml volumetric flask, add 35 ml of methanol R, shake and by mechanical means for 30 minutes. Dilute with methanol R to volume, mix, and allow any insoluble matter to settle. Transfer 3.0 ml of the supernatant to a 100 ml volumetric flask, dilute with mobile phase to volume, and mix. Reference solution: Dissolve an accurately weighed quantity of clarithromycin RS in methanol R, shaking and sonicating if necessary to effect dissolution, to obtain a stock solution having a known concentration of about 625 µg of clarithromycin/ml. Dilute 10.0 ml of this stock solution to 50.0 ml with mobile phase, mix well. 285

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CLINDAMYCIN HYDROCHLORIDE

Resolution solution: Prepare a solution of E impurity of clarithromycin RS (6,11-di-O-methylerythromycin A, C39H71NO13) in methanol R containing about 625 µg/ml. Transfer 10.0 ml of this solution and 10.0 ml of the reference solution to a 50 ml volumetric flask, dilute with mobile phase to volume, and mix. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 µm). Column temperature: 50 °C. Detector: A spectrophotometer set at 210 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the resolution solution, the relative retention times are about 0.75 for clarithromycin and 1.0 for E impurity; and the resolution between clarithromycin and E impurity is not less than 2.0. Inject the reference solution, the column efficiency, determined from the clarithromycin peak, is not less than 750 theoretical plates, the tailing factor is not less than 0.9 and not more than 2.0; and the relative standard deviation for 6 replicate injections is not more than 2.0%. Inject separately the test solution and the resolution solution. Calculate the content of clarithromycin, C38H69NO13, in the tablets using the areas of the principal peaks in the chromatograms obtained with the test solution and the reference solution, and the declared content of C38H69NO13 in clarithromycin RS.

Storage Store in an airtight container, at a temperature not exceed 30 °C. Action and use Macrolide antibacterial. Usual strength 250 mg, 500 mg. CLINDAMYCIN HYDROCHLORIDE Clindamycini hydrochloridum

hydrochloride. It contains not less than 84.0% and not more than 93.0% of clindamycin, C18H33ClN2O5S, calculated with reference to the anhydrous substance.

Characters White or almost white, crystalline powder. Very soluble in water, slightly soluble in ethanol (96%). Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum obtained with clindamycin hydrochloride RS. B. Dissolve about 10 mg in 2 ml of dilute hydrochloric acid R and heat on a water-bath for 3 minutes. Add 3 ml of a 10% solution of sodium carbonate R and 1 ml of a 2% solution of sodium nitroprusside R. A violet-red colour develops. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Isopropanol - a 15% solution of ammonium acetate adjusted to pH 9.6 with ammonia - ethyl acetate (20 : 40 : 45). Mix well, allow to stand for separating and use the upper layer of the mobile phase. Test solution: Dissolve 10 mg of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Reference solution (1): Dissolve 10 mg of clindamycin hydrochloride RS in methanol R and dilute to 10 ml with the same solvent. Reference solution (2): Dissolve 10 mg of clindamycin hydrochloride RS and 10 mg of lincomycin hydrochloride RS in methanol R and dilute to 10 ml with the same solvent. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm. After removal of the plate, allow the plate to dry in air. Spray with a 0.1% solution of potassium permanganate R. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) shows two clearly separated spots. D. Dissolve 0.1 g in water and dilute to 10 ml with the same solvent. The solution gives reaction A of chlorides (Appendix 8.1). pH Dissolve 1.0 g in carbon dioxide-free water R and dilute to 10 ml with the same solvent. The pH of this solution is 3.0 to 5.0 (Appendix 6.2).

C18H33ClN2O5S,HCl

M. 461.5

Clindamycin hydrochloride is methyl 7-chloro-6,7,8trideoxy-6-[[[(2S,4R)-1-methyl-4-propylpyrrolidin-2-yl] carbonyl]amino]-1-thio-L-threo-D-galacto- octopyranoside 286

Specific optical rotation +135° to +150°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 1.000 g in water and dilute to 25.0 ml with the same solvent.

VP V

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase and chromatographic system as described in the test for assay. Test solution: Dissolve 50.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Reference solution (1): Dissolve 50.0 mg of clindamycin hydrochloride RS in the mobile phase and dilute to 50.0 ml with the mobile phase. Reference solution (2): Dilute 2.0 ml of the test solution to 100.0 ml with the mobile phase. Procedure: Carry out the chromatography of above solutions twice the retention time of clindamycin peak. System suitability: In the chromatogram obtained with reference solution (1): Relative retention times with reference to clindamycin (retention time = about 10 minutes) are: impurity A (lincomycin) = about 0.4; impurity B (clindamycin B) = about 0.65; impurity C (7-epiclindamycin) = about 0.8. Limits: In the chromatogram obtained with the test solution: Impurity B: The area of the peak due to clindamycin B is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (2.0%). Impurity C: The area of the peak due to 7-epiclindamycin is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (2) (4.0%). The area of any other impurity peaks, apart from the above impurity peaks, is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (1.0%). The sum of the areas of all peaks, apart from the principal peak is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (2) (6.0%). Disregard any peak with an area less than or equal to 0.025 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Water 3.0% to 6.0% (Appendix 10.3). Determined on 0.500 g. Sulfated ash Not more than 0.5% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by liquid chromatography (Appendix 5.3). Phosphate buffer solution pH 7.5: Dissolve 6.8 g of potassium dihydrogen phosphate R in 1000 ml of water and adjusted to pH 7.5 with a 25% solution of potassium hydroxide R. Mobile phase: Phosphate buffer solution pH 7.5 acetonitrile (550 : 450). Make adjustments if necessary

CLINDAMYCIN CAPSULES (Note: Increasing the proportion of acetonitrile in the mobile phase decreases the retention time, and decreasing it increases the resolution between 7-epiclindamycin and clindamycin).

Reference solution: Use reference solution (1) in the test for Related substances. Test solution: Use the test solution in the test for Related substances. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 210 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Procedure: System suitability: The relative standard deviation of the clindamycin peak areas, for 6 injections of the reference solution, is not more than 0.85%. Inject the reference solution and the test solution twice the retention time of the clindamycin peak. Calculate the content of clindamycin using the clindamycin peak areas in the chromatograms obtained with the reference solution and the test solution, and the declared content of clindamycin (C18H33ClN2O5S) in clindamycin hydrochloride RS.

Storage Store in an airtight container, at a temperature not exceeding 30 °C. Action and use Lincosamide antibiotic. Preparation Capsules. CLINDAMYCIN CAPSULES Capsulae Clindamycini Clindamycin capsules contain clindamycin hydrochloride. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of clindamycin, C18H33ClN2O5S, 90.0% to 110.0% of the stated amount. Identification A. In the Assay, the chromatogram obtained with the test solution shows a principal peak with the same retention time as the principal peak in the chromatogram obtained with the reference solution. B. The contents of the capsules comply with the test for chlorides (Appendix 8.1). Water Not more than 7.0%. Use 1.00 g (Appendix 10.3). 287

VP V

CLOFAZIMINE

Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 900 ml of phosphate buffer pH 6.8 R. Rotation speed: 100 rpm. Time: 30 min. Procedure: Examine by liquid chromatography (Appendix 5.3). Mobile phase and chromatographic system carry out as in Assay. Test solution: After the specified time, withdraw a portion of the medium and filter, discarding the first portion of the filtrate. Reference solution: A solution of clindamycin hydrochloride RS in water having the same concentration as test solution. Tolerance: Not less than 80% (Q) of the labelled amount of clindamycin, C18H33ClN2O5S is dissolved in 30 min. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol - solution A (30 : 21). Solution A: Dissolve 2.88 g ammonium dihydrogen phosphate R in 1000 ml of water, adjust pH to 3.0 with a 80% solution of phosphoric acid R. Test solution: Weigh individually 20 capsules, calculate the average weight of the capsule contents and powder finely, mix well. Weigh accurately a quantity of the powder, equivalent to about 50 mg of clindamycin hydrochloride, transfer to a 25 ml volumetric flask, add 20 ml of mobile phase, dissolve with the aid of ultrasound. Dilute to volume with mobile phase. Shake well and filter. Reference solution: Weigh accurately about 50 mg of clindamycin hydrochloride RS, dissolve and dilute to 25.0 ml with mobile phase. Chromatographic system: A column (25 cm × 4 mm) packed with stationary phase C (10 µm). Detector: A spectrophotometer set at 215 nm. Flow rate: 1.5 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject the reference solution, the column efficiency is not less than 1,300 theoretical plates. Separately inject the reference solution and the test solution. Calculate the content of clindamycin, C18H33ClN2O5S, in the capsules using the areas of the principal peaks in the chromatograms obtained with the test solution and the reference solution, and the declared content of clindamycin, C18H33ClN2O5S in clindamycin hydrochloride RS. Storage Store in an airtight container, at a temperature not exceed 30 °C. Action and use Antibacterial. Usual strength 75 mg, 150 mg and 300 mg (calculated as clindamycin). 288

CLOFAZIMINE Clofaziminum

C27H22Cl2N4  

M. 473.4

Clofazimine is N,5-bis(4-chlorophenyl)-3-[(1-methylethyl) imino]-3,5-dihydrophenazin-2-amine. It contains not less than 99.0% and not more than 101.0% of C27H22Cl2N4, calculated with reference to the dried substance.

Characters Reddish-brown, fine powder, it shows polymorphism. Practically insoluble in water, soluble in methylene chloride, very slightly soluble in ethanol (96%). Identification Apply one of the two following identifications: First identification: A. Second identification: B, C. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of clofazimine RS. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in methylene chloride R, evaporate to dryness and record new spectra using the residues. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Propanol - methylene chloride (6 : 85). Test solution: Dissolve 10 mg of the substance to be examined in methylene chloride R and dilute to 10 ml with the same solvent. Reference solution: Dissolve 10 mg of clofazimine RS in methylene chloride R and dilute to 10 ml with the same solvent. Procedure: Apply separately to the plate 5 µl of each solution. Develop over 2/3 of the plate. Dry the plate horizontally in air for 5 min. continue second development over 2/3 of the plate, allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. C. Dissolve 2 mg of the substance to be examined in 3 ml of acetone R and add 0.1 ml of hydrochloric acid R. An

VP V

intense violet colour is produced. Add 0.5 ml of 5 M sodium hydroxide R; the colour changes to orange-red.

Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use. Mobile phase: Dissolve 2.25 g of sodium laurilsulfate R, 0.85 g of tetrabutylammonium hydrogen sulfate R and 0.885 g of disodium hydrogen phosphate R in water. Adjust to pH 3.0 with dilute phosphoric acid R and dilute to 500 ml with water. Mix 35 volumes of this solution and 65 volumes of acetonitrile R. Test solution: Dissolve 50 mg of the substance to be examined in the mobile phase and dilute to 100 ml with the mobile phase. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (2): Dissolve 5.0 mg of clofazimine for system suitability RS in the mobile phase and dilute to 10.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase B (5 µm). Detector: A spectrophotometer set at 280 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: The run time is 3 times the retention time of clofazimine. Identification of impurities: Use the chromatogram supplied with clofazimine for system suitability RS to identify the peak due to impurity B. Relative retention with reference to clofazimine (retention time = about 15 min): impurity A = about 0.7; impurity B = about 0.8. System suitability: In the chromatogram obtained with reference solution (2), baseline separation between the peaks due to impurity B and clofazimine. Limits: Impurity A: The area of the peak due to impurity A is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%). Impurity B: The area of the peak due to impurity B is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.3%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%). Sum of the peak areas of all impurities is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%).

CLOFAZIMINE CAPSULES Note: Impurity A: N,5-bis(4-chlorophenyl)-3-imino-3,5-dihydrophenazin -2- amine. Impurity B: 5-(4-chlorophenyl)-3-[(1-methylethyl)imino]-N-phenyl3,5-dihydrophenazin-2-amine.

Heavy metals Not more than 10 ppm (Appendix 9.4.8). 2.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.400 g of the substance to be examined in 5 ml of methylene chloride R and add 20 ml of acetone R and 5 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 47.34 mg of C27H22Cl2N4. Storage Store in an airtight container. Action and use Antileprosy drug. Preparation Capsules. CLOFAZIMINE CAPSULES Capsulae Clofazimini Clofazimine capsules contain clofazimine. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of clofazimine, C27H22Cl2N4, 90.0% to 110.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4). In the test for Related substances, the principal spot in the chromatogram obtained with the test solution has the same Rf value as the spot in the chromatogram obtained with reference solution (1). B. The ultraviolet absorptiom spectrum (Appendix 4.1) of the test solution and the standard solution in the Assay, 289

VP V

CLOPIDOGREL HYDROGEN SULFATE

exhibits maxima and minima at the same wavelengths, concomitantly measured.

Related substances A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Immediately before use, expose the plate to ammonia vapours for 30 minutes by suspending the plate in a tank containing a shallow layer of approximately 25 ml of ammonia solution (Note: prevent the plate from coming into contact with the liquid). Ammonia solution: Dilute 1 ml ammonia R to 100 ml with water and mix well (use within 24 h). Mobile phase: Methylene chloride - propan-1-ol (10 : 1). Test solution: Dissolve a quantity of the contents of the capsules containing about 500 mg of clofazimine, add 25 ml of methylen chloride R and 25 ml of 0.1 M sodium hydroxide and sonicate for 30 min. Withdraw the methylene chloride R layer and filter through anhydrous sodium sulfate. Reference solutions: Dissolve an accurately weighed quantity of clofazimine RS in methylene chloride R to obtain reference solution (1) having a concentration of about 0.2 mg per ml. Dilute reference solution (1) with methylene chloride R to obtain reference solution (2) and reference solution (3) having known concentrations of about 0.1 mg and 0.04 mg per ml, respectively. Procedure: Apply separately to the plate 5 µl of each solution. Allow to dry and develop over a path of 15 cm. After removal of the plate, allow to dry in the air and examine under ultraviolet light (254 nm). No secondary spot in the chromatogram obtained with the test solution is larger or more intense than the principal spot in the chromatogram obtained with reference solution (1) (1.0%), and the sum of the intensities of all the secondary spots in the chromatogram obtained with the test solution corresponds to not more than 2.0%. Assay Test solution: Weigh 20 capsules, calculate the average mass of the contents, and powder finely. Dissolve an accurately weighed quantity of the powder in methylene chloride R to obtain a solution having a concentration of about 0.075 mg per ml, filter. Dilute 5.0 ml of the filtrate to 50.0 ml with 0.1 M methanolic hydrochloric acid R, and mix. Reference solution: Dissolve an accurately weighed quantity of clofazimine RS in methylene chloride R to obtain a solution having a concentration of about 0.075 mg per ml. Dilute 5.0 ml of this solution to 50.0 ml with 0.1 M methanolic hydrochloric R, and mix. Blank solution: Transfer 5 ml methylene chloride R to a 50.0 ml volumetric flask, dilute with 0.1 M methanolic hydrochloric acid R to volume, and mix. Measure the absorbance of the test solution, reference solution at the maximum at about 491 nm (Appendix 4.1), in a 1 cm cell, using the blank solution as a blank. 290

Calculate the content of clofazimine, C27H22Cl2N4, in the capsules using the absorbances of test solution, reference solution and the declared content of C16H17N3O4S in clofazimine RS.

Storage Store in a airtight container, in a cool and dry place, and protected from light. Action and use Antileprosy drug. Usual strength 50 mg. CLOPIDOGREL HYDROGEN SULFATE Clopidogreli hydrogenosulfas Clopidogel bisulfate

C16H16ClNO2S,H2SO4 



M: 419.9

Clopidogrel hydrogen sulfate is methyl (2S)-(2chlorophenyl)[6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl] acetate sulfate. It contains not less than 99.0% and not more than 101.0% of C16H16ClNO2S,H2SO4, calculated with reference to the anhydrous substance.

Characters White or almost white powder, it shows polymorphism. Freely soluble in water and in methanol, practically insoluble in cyclohexane. Identification Apply one of the two following identifications: First identification: A, B, D. Second identification: A, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of clopidogrel hydrogen sulfate RS. If the spectra obtained show differences, dissolve the substance to be examined and the reference substance separately in anhydrous ethanol R, evaporate to dryness and record new spectra using the residues (the substance may stick to the surface of the recipient used). B. Specific optical rotation: +54.0° to + 58.0°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.250 g of the substance to be examined in methanol R and dilute to 25.0 ml with the same solvent.

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C. It complies with the test for Enantiomeric purity. D. It gives reaction (A) of sulfates (Appendix 8.1).

Appearance of solution Dissolve 1.0 g in methanol R and dilute to 20.0 ml with the same solvent. The solution is clear (Appendix 9.2). and not more intensely coloured than reference solution Y6 (Appendix 9.3, method 1). Enantiomeric purity Examine by liquid chromatography (Appendix 5.3). Mobile phase: Anhydrous ethanol - heptane (15 : 85). Test solution: Dissolve 0.1 g of the substance to be examined in 25.0 ml of anhydrous ethanol R and dilute to 50.0 ml with heptane R. Reference solution: Dissolve 10 mg of clopidogrel for system suitability RS (containing impurities B and C) in 2.5 ml of anhydrous ethanol R and dilute to 5.0 ml with heptane R. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase silica gel OJ for chiral separations R (10 µm). Detector: A spectrophotometer set at 220 nm. Flow rate: 0.8 ml/min. Volume of injection: 10 µl. Procedure: The run time is 1.25 times the retention time of clopidogrel. Identification of impurities: Use the chromatogram supplied with clopidogrel for system suitability RS and the chromatogram obtained with the reference solution to identify the peaks due to impurities B and C. Relative retention with reference to clopidogrel (retention time = about 18 min): Impurity C = about 0.6; impurity B = about 0.7. System suitability: In the chromatogram obtained with reference solution, the resolution between the peaks due to impurities C and B is at least 2.0 and the signal-to-noise ratio of the peak due to impurity C is not less than 20. Limit: Impurity C: Not more than 0.5%. Note: Impurity C: Methyl (2R)-(2-chlorophenyl)[6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl]acetate.

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Mix 5 volumes of methanol R2 and 95 volumes of a 0.096% solution of sodium pentanesulfonate monohydrate R adjusted to pH 2.5 with phosphoric acid R. Mobile phase B: Methanol R2 - acetonitrile R1 (5 : 95). Solvent mixture: Mobile phase A - acetonitrile R1 (40 : 60). Test solution: Dissolve 65 mg of the substance to be examined in the solvent mixture and dilute to 10.0 ml with the solvent mixture.

CLOPIDOGREL HYDROGEN SULFATE

Reference solution (1): Dissolve 5 mg of clopidogrel impurity A RS in the solvent mixture and dilute to 25.0 ml with the solvent mixture. Reference solution (2): Dissolve 32 mg of clopidogrel for system suitability RS (containing impurities B and C) in the solvent mixture, add 0.5 ml of reference solution (1) and dilute to 5.0 ml with the solvent mixture. Reference solution (3): Dilute 1.0 ml of the test solution to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 10.0 ml with the solvent mixture. Chromatographic system: A column (15 cm × 3.9 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (5 µm). Column temperature: 30 °C. Detector: A spectrophotometer set at 220 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0-3

89.5

10.5

3 - 48

89.5 → 31.5

10.5 → 68.5

48 - 68

31.5

68.5

Inject the test solution, reference solutions (2) and (3). Identification of impurities: Use the chromatogram supplied with clopidogrel for system suitability RS and the chromatogram obtained with reference solution (2) to identify the peaks due to impurities A and B. Relative retention with reference to clopidogrel (retention time = about 25 min): Impurity A = about 0.4; impurity B = about 1.1. System suitability: In the chromatogram obtained with reference solution (2), peak-to-valley ratio (Hp/Hv) is at least 10, where Hp =height above the baseline of the peak due to impurity B and Hv = height above the baseline of the lowest point of the curves eparating this peak from the peak due to clopidogrel. Limits: Impurity B: The area of the peak due to impurity B is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (3) (0.3%). Impurity A: The area of the peak due to impurity A is not more than 2 times the area of the principal peak in the chromatogram obtained with reference solution (3) (0.2%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (3) (0.10%). Sum of the peak areas of all impurities is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (3) (0.5%). 291

CLOPIDOGREL TABLETS

Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (3) (0.05%). Note: Impurity A: (2S)-(2-chlorophenyl)[6,7-dihydrothieno[3,2-c] pyridin-5 (4H)-yl]acetic acid. Impurity B: Methyl (2S)-(2-chlorophenyl)[4,7-dihydrothieno -[2,3-c]pyridin-6(5H)-yl]acetate.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Water Not more than 0.5% (Appendix 10.3). Determined on 1.00 g. Replace the solvent after each titration. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.160 g of the substance to be examined in a mixture of 10 ml of acetone R, 10 ml of methanol R and 30 ml of water. Titrate with 0.1 N sodium hydroxide VS, determining the end-point potentiometrically (Appendix 10.2). A precipitate may be formed during the titration. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 20.99 mg of C16H18ClNO6S2. Storage Store in an airtight container, protected from light. Action and use Inhibitor of platelet aggregation. Preparation Tablets. CLOPIDOGREL TABLETS Tabellae Clopidogreli Clopidogrel tablets contain clopidogrel bisulfate. They may be coated. The tablets comply with the requirements stated under “Tablets” item (Appendix 1.20) and with the following requirements.

Content of clopidogrel, C16H16ClNO2S, 90.0% to 110.0% of the stated amount. Identification A. Dissolve a quantity of the powdered tablets containing the equivalent of 75 mg of clopidogrel in 100 ml of 0.1 M 292

VP V

hydrochloric acid R. Mix and filter. Dilute 10 ml of the filtrate to 50 ml with 0.1 M hydrochloric acid R. The ultraviolet absorption spectrum (Appendix 4.1) of the resulting solution in the range 250 nm to 300 nm is concordant with the spectrum of a solution of clopidogrel bisulfate RS prepared in the same solvent having the same concentration of clopidogrel. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that of clopidogrel peak in the chromatogram obtained with the reference solution.

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of 0.1 M hydrochloric acid R. Rotation speed: 50 rpm. Time: 30 min. Procedure: After the specified time withdraw a sample of the medium, filter. Dilute the filtrate with the medium to obtain a solution having a suitable concentration. Measure the absorbance (Appendix 4.1) of the resulting solution at 240 nm, in a 1 cm cell, using the medium as a blank. Calculate the content of clopidogrel, C16H16ClNO2S, dissolved using the absorbance of a reference solution prepared by dissolving an accurately weighed quantity of clopidogrel bisulfate RS in 20.0 ml of methanol R and then diluting with the medium to obtain a solution having the same concentration of clopidogrel to that in the test solution. Tolerance: Not less than 80% (Q) of the stated amount of clopidogrel, C16H16ClNO2S, is dissolved in 30 min. Assay Examine by liquid chromatography (Appendix 5.3). Buffer solution pH 7.0: Dissolve 5.75 g of ammonium dihydrogen phosphate R in 1000 ml of water, adjust to pH 7.0 with triethylamine R. Mobile phase: A mixture of 65 volumes of acetonitrile R and 35 volumes of buffer solution pH 7.0. Reference solution: Dissolve an accurately weighed quantity of about 50 mg of clopidogrel bisulfate RS in methanol R and dilute to 50.0 ml with methanol R. Dilute 5.0 ml of this solution to 50.0 ml with the mobile phase. Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Transfer an accurately weighed quantity of the powdered tablets containing the equivalent of 75 mg of clopidogrel to a 100 ml volumetric flask, add 70 ml of methanol R, dissolve by sonicating for 10 minutes. Dilute to volume with methanol R, mix and filter. Dilute 5.0 ml of the filtrate to 50.0 ml with the mobile phase. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase B (5 μm). Detector: A spectrophotometer set at 210 nm.

VP V

CLOTRIMAZOLE

Flow rate: 1.0 ml/min. Volume of injection: 20 μl. Procedure: System suitability: Inject the reference solution, the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of clopidogrel, C16H16ClNO2S, in the tablets using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C16H16ClNO2S in clopidogrel bisulfate RS.

Mobile phase: 18 M Ammonia - propanol - toluene (0.5 : 10 : 90). Test solution: Dissolve 50 mg of the substance to be examined in ethanol (96%) R and dilute to 5 ml with the same solvent. Reference solution: Dissolve 50 mg of clotrimazole RS in ethanol (96%) R and dilute to 5 ml with the same solvent. Procedure: Apply separately to the plate 10 µl of each solution. Develop over 2/3 of the plate. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution.

Storage Store in an airtight container, in a cool and dry place, at a temperature not exceeding 30 °C, protected from moisture.

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Dissolve 1.0 g of potassium dihydrogen phosphate R and 0.5 g of tetrabutylammonium hydrogen sulfate R1 in water and dilute to 1000 ml with the same solvent. Mobile phase B: Acetonitrile R1. Test solution: Dissolve 50.0 mg of the substance to be examined in acetonitrile R1 and dilute to 50.0 ml with the same solvent. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with acetonitrile R1. Dilute 1.0 ml of this solution to 10.0 ml with acetonitrile R1. Reference solution (2): Dissolve the contents of a vial of clotrimazole for peak identification RS (containing impurities A, B and F) in 1.0 ml of acetonitrile R1. Reference solution (3): Dissolve 5.0 mg of imidazole RS (impurity D) and 5.0 mg of clotrimazole impurity E RS in acetonitrile R1 and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of this solution to 25.0 ml with acetonitrile R1. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase spherical end-capped octylsilyl silica gel for chromatography R (5 µm). Column temperature: 40 °C. Detector: A spectrophotometer set at 210 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: Carry out a linear gradient elution using the following conditions:

Action and use Inhibitor of aggregation. Usual strength 75 mg of clopidogrel. CLOTRIMAZOLE

Clotrimazolum

C22H17ClN2 

M. 344.8

Clotrimazole is 1-[(2-chlorophenyl)diphenylmethyl]-1Himidazole. It contains not less than 98.5% and not more than 100.5% of C22H17ClN2, calculated with reference to the dried substance.

Characters White or pale yellow, crystalline powder. Practically insoluble in water, soluble in ethanol (96%) and in methylene chloride. Identification Apply one of the two following identifications: First identification: A. Second identification: B, C. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of clotrimazole RS. B. Melting point: 141 °C to 145 °C (Appendix 6.7). C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254.

Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0-3

75

25

3 - 25

75 → 20

25 → 80

25 - 30

20

80

Relative retention with reference to clotrimazole (retention time = about 12 min): impurity D = about 0.1; impurity F = about 0.9; impurity B = about 1.1; impurity E = about 1.5; impurity A = about 1.8. 293

VP V

CLOTRIMAZOLE CREAM

System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurity F and clotrimazole is at least 1.5; the chromatogram obtained is similar to the chromatogram supplied with clotrimazole for peak identification RS. Limits: Impurities A, B: For each impurity, the area is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%). Impurities D, E: For each impurity, the area is not more than the area of the corresponding peak in the chromatogram obtained with reference solution (3) (0.2%). Impurity F: The area of the peak due to impurity F is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). Sum of the peak areas of all impurities is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A: (2-chlorophenyl)diphenylmethanol. Impurity B: 1-[(4-chlorophenyl)diphenylmethyl]-1H-imidazole. Impurity C: 1-chloro-2-(chlorodiphenylmethyl)benzene. Impurity D: imidazole. Impurity E: (2-chlorophenyl)phenylmethanone (2-chlorobenzophenone). Impurity F: 1-(triphenylmethyl)-1H-imidazole (deschloroclotrimazole).

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.300 g of the substance to be examined in 80 ml of anhydrous acetic acid R. Using 0.3 ml of naphtholbenzein solution R as indicator, titrate with 0.1 N perchloric acid VS until the colour changes from brownish-yellow to green. 1 ml of 0.1 N perchloric acid VS is equivalent to 34.48 mg of C22H17ClN2. Storage Protected from light. Action and use Antifungal. Preparations Cream, pessaries. 294

CLOTRIMAZOLE CREAM Cremoris Clotrimazoli Clotrimazole cream contains clotrimazole in a suitable basis. The cream complies with the requirements stated under “Topical semi-solid preparations” (Appendix 1.12) and with the following requirements.

Content of clotrimazole, C22H17ClN2, 90.0% to 110.0% of the stated amount. Characters A milky, homogeneous cream, odourless. Identification A. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is the same as that of clotrimazole peak in the chromatogram obtained with the reference solution. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Ether R as the mobile phase in a chromatography tank containing 25 ml of ammonia R in a beaker. Test solution: Shake a quantity of the cream containing 20 mg of clotrimazole with 20 ml of ethanol R, heat on a water-bath, mix well. Allow to cool in ice for about 20 minutes and filter. Reference solution: A 0.1% solution of clotrimazole RS in ethanol R. Procedure: Apply separately to the plate 20 µl of each solution. Develop over a path of 15 cm. After removal of the plate, allow it to dry in a current of air. Examine under ultraviolet light or spray with Dragendorff reagent R. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol - 0.44% solution of dipotassium hydrogen phosphate (70 : 30) (Adjust the ratio if necessary). Reference solution: Dissolve 30 mg of clotrimazole RS, accurately weighed, in ethanol R and dilute to 50.0 ml with the same solvent. Test solution: Weigh a quantity of the cream containing 30 mg of clotrimazole in a beaker, add 25 ml of ethanol R, heat on a water-bath, mix to dissolve. Allow to cool in ice for at least 30 min. Decant and filter through a filter paper moistened with ethanol R. Repeat the extraction with two 10-ml quantities of ethanol R. Rinse the beaker and the filter with ethanol R. Combine the extracts and washings, dilute to 50.0 ml with ethanol R. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 254 nm.

VP V

Flow rate: 1.0 ml/min to 1.5 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution, the column efficiency, determined from the clotrimazole peak, is not less than 2500 theoretical plates. The relative standard deviation of the areas of clotrimazole peaks obtained from 6 replicate injections of the reference solution is not more than 2.0%. Inject separately the test solution and the reference solution. Calculate the content of clotrimazole, C22H17ClN2, using the areas of the principal peaks in the chromatograms obtained with the test solution and the reference solution, and the declared content of C22H17ClN2 in clotrimazole RS.

Storage Store in an airtight container, in a cool place, protected from light. Action and use Antifungal. Usual strength 1%, 3%. CLOTRIMAZOLE PESSARIES Tabellae vaginalis Clotrimazoli Clotrimazole pessaries are vaginal tablets containing clotrimazole. The pessaries comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of clotrimazole, C22H17ClN2, 90.0% to 110.0% of the stated amount. Characters White tablets or tablets having homogeneous colour. Identification A. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is the same as that of clotrimazole peak in the chromatogram obtained with the reference solution. B. Examine by thin-layer chromatography (Appendix5.4). Coating substance: Silica gel GF254. Mobile phase:Ammonia - propanol - toluene (0.5 : 10 : 90). Test solution: Shake a quantity of the powdered tablets containing 0.1 g of clotrimazole with 10 ml of ethanol R, filter. Reference solution: A 1% solution of clotrimazole RS in ethanol R. Procedure: Apply separately to the plate 10 µl of each solution. After developing over a path of 15 cm, remove the plate, allow it to dry in air and examine under ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, colour

CLOXACILLIN SODIUM

and size to the principal spot in the chromatogram obtained with the reference solution.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: A mixture of 30 volumes of 0.02 M phosphoric acid and 70 volumes of methanol R, the pH of the mixture being adjusted to 7.5 with a 10% v/v solution of triethylamine R in methanol R. Reference solution: Dissolve 20 mg of clotrimazole RS in 70 ml of methanol R, add sufficient 0.02 M phosphoric acid to produce 100.0 ml and dilute 1 volume of the resulting solution to 5 volumes with a mixture of 70 volumes of methanol R and 30 volumes of 0.02 M phosphoric acid. Test solution: Weigh 20 tablets, calculate the average mass, finely powder. Weigh a quantity of the powdered tablets equivalent to 0.1 g of clotrimazole add 50 ml of methanol R and shake for 20 minutes, dilute to 250.0 ml with methanol R, filter and to 10.0 ml of the filtrate add 60 ml of methanol R and sufficient 0.02 M phosphoric acid to produce 100.0 ml. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 215 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution, the column efficiency, determined from the clotrimazole peak, is not less than 2500 theoretical plates. Inject separately the test solution and the reference solution. Calculate the content of clotrimazole, C22H17ClN2 using the areas of the principal peaks in the chromatograms obtained with the test solution and the reference solution, and the declared content of C22H17ClN2 in clotrimazole RS. Storage Store in an airtight container, protected from light. Action and use Antifungal. Usual strength 100 mg and 500 mg. CLOXACILLIN SODIUM Cloxacillin natricum

C19H17ClN3NaO5S,H2O  

M. 475.9 295

VP V

CLOXACILLIN SODIUM

Cloxacillin sodium is sodium (2S,5R,6R)-6-[[[3-(2chlorophenyl)-5-methylisoxazol-4-yl]carbonyl]amino]3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane2-carboxylate monohydrate. It contains not less than 95.0% and not more than 102.0% of C19H17ClN3NaO5S, calculated with reference to the anhydrous substance. Semi-synthetic product derived from a fermentation product.

Characters White or almost white, hygroscopic, crystalline powder. Freely soluble in water and in methanol, soluble in ethanol (96%). Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of cloxacillin sodium RS. Examine the substances prepared as discs. B. It complies with the test for Identification of penicillins (Appendix 8.2), use mobile phase B. C. It complies with the test B for Colour reactions of penicillins and cephalosporins (Appendix 8.3). D. It gives reaction (A) of sodium (Appendix 8.1). Appearance of solution Solution S: Dissolve 2.50 g in carbon dioxide-free water R and dilute to 25.0 ml with the same solvent. Solution S is clear (Appendix 9.2) and its absorbance (Appendix 4.1) at 430 nm is not greater than 0.04. pH 5.0 to 7.0 (Appendix 6.2). Determined on solution S. Specific optical rotation +160° to +169° calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.250 g of the substance to be examined in water and dilute to 25.0 ml with the same solvent. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Mix 25 volumes of acetonitrile R and 75 volumes of a 0.27% solution of potassium dihydrogen phosphate R adjusted to pH 5.0 with dilute sodium hydroxide solution R. Test solution (1): Dissolve 50.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Test solution (2): Dilute 5.0 ml of test solution (1) to 50.0 ml with the mobile phase. Reference solution (1): Dissolve 50.0 mg of cloxacillin sodium RS in the mobile phase and dilute to 50.0 ml with 296

the mobile phase. Dilute 5.0 ml of this solution to 50.0 ml with the mobile phase. Reference solution (2): Dilute 5.0 ml of test solution (2) to 50.0 ml with the mobile phase. Reference solution (3): Dissolve 5 mg of flucloxacillin sodium RS and 5 mg of cloxacillin sodium RS in the mobile phase and dilute to 50.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.0 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 225 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Inject the test solution (1) and reference solutions (2) and (3). The run time of the test solution is 5 times the retention time of cloxacillin. System suitability: In the chromatogram obtained with reference solution (3), the resolution between the peaks due to cloxacillin (1st peak) and flucloxacillin (2nd peak) is at least 2.5. Limits: In the chromatogram obtained with test solution (1): Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (1.0%). Sum of the peak areas of all impurities is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (5.0%). Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Note: ImpurityA: (4S)-2-[carboxy[[[3-(2-chlorophenyl)-5-methylisoxazol4-yl]carbonyl]amino]methyl]-5,5-dimethylthiazolidine-4carboxylic acid (penicilloic acid of cloxacillin). Impurity B: (2RS,4S)-2-[[[[3-(2-chlorophenyl)-5-methylisoxazol4-yl]carbonyl]amino]methyl]-5,5-dimethylthiazolidine-4carboxylic acid (penilloic acid of cloxacillin). Impurity C: (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia-1azabicyclo[3.2.0]heptane-2- carboxylic acid (6-aminopenicillanic acid). Impurity D: 3-(2-chlorophenyl)-5-methylisoxazole-4-carboxylic acid. Impurity E: (2S,5R,6R)-6-[[[(2S,5R,6R)-6-[[[3-(2-chlorophenyl)5-methylisoxazol-4- yl]carbonyl]amino]-3,3-dimethyl-7-oxo4-thia-1-azabicyclo[3.2.0]hept-2-yl]carbonyl]amino]-3,3dimethyl-7-oxo-4-thia-1-azabicyclo [3.2.0]heptane-2- carboxylic acid (6-APA cloxacillin amide).

N,N-Dimethylaniline Not more than 20 ppm (Appendix 10.16, method 2). 2-Ethylhexanoic acid Maximum 0.8% m/m (Appendix 10.17).

VP V

Water 3.0% to 4.5% (Appendix 10.3). Determined on 0.300 g. Bacterial endotoxins Less than 0.20 EU/mg (Appendix 13.2), if intended for use in the manufacture of parenteral preparations without a further appropriate procedure for the removal of bacterial endotoxins. Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. Inject the test solution (2) and reference solution (1). System suitability: In the chromatogram obtained with reference solution (1), the relative standard deviation of the cloxacillin sodium peak areas for 6 replicate injections is not more than 1.0%. Calculate the content of cloxacillin sodium, C19H17ClN3NaO5S, using the areas of the peaks in the chromatograms obtained with the test solution (2), the reference solution (1) and the declared content of C19H17ClN3NaO5S in cloxacillin sodium RS. Storage In an airtight container, at a temperature not exceeding 25 °C. If the substance is sterile, store in a sterile, airtight, tamperproof container. Labelling The label states, where applicable, that the substance is free of bacterial endotoxins. Action and use Antibacterial. Preparations Capsules, infusion. CLOXACILLIN CAPSULES Capsulae Cloxacillini Cloxacillin capsules contain cloxacillin sodium. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of cloxacillin, C19H18ClN3NO5S, 90.0% to 110.0% of the stated amount. Identification A. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is the same as that of cloxacillin peak in the chromatogram obtained with the reference solution.

CLOXACILLIN CAPSULES

B. Shake a quantity of the content of the capsules containing the equivalent of 50 mg of cloxacillin with 2 ml of water, and filter. Acidify the filtrate with dilute acetic acid R, add 1 ml of magnesium uranyl acetate solution R, scratch the inside of the tube with a glass rod, a yellow crystalline precipitate is produced.

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of phosphate buffer pH 6.8 R. Rotation speed:100 rpm. Time: 45 min. Procedure: Examine by liquid chromatography (Appendix 5.3). Mobile phase, Reference solution and Chromatographic system: Prepare as directed in the Assay. Test solution: After the specified time, withdraw a sample of the medium and filter, discarding the first 20 ml of filtrate. Dilute an accurately measured volume of the filtrate with mobile phase to obtain a solution containing about 0.01% of cloxacillin. Separately inject the reference solution and the test solution. Calculate the content of cloxacillin, C19H18ClN3O5S, dissolved using the areas for the principal peaks in the chromatograms obtained with the test solution and the reference solution, and the declared content of C19H18ClN3O5S in cloxacillin sodium RS. Tolerance: Not less than 80% (Q) of the labelled amount of cloxacillin, C19H18ClN3O5S, is dissolved in 45 min. Water Not more than 5.0% (Appendix 10.3). Use 0.300 g of the content of the capsules. Assay Examine by liquid chromatography (Appendix 5.3). Buffer solution: Prepare a 0.02 M solution of potassium dihydrogen phosphate R in water, and adjust with 2 M sodium hydroxide R to a pH of 6.8. Mobile phase: Buffer solution - acetonitril (70 : 30). Reference solution: A 0.01% solution of cloxacillin sodium RS in the mobile phase. Test solution: Weigh 20 capsules, calculate the average weight of the capsule contents. Weigh accurately a quantity of the powder containing the equivalent of 100 mg of cloxacillin in 200 ml volumetric flask, add 150 ml of the mobile phase, shake to dissolve and dilute to volume with the same solvent, mix. Filter, and discard the first 20 ml of the filtrate. Dilute 5.0 ml of the filtrate to 25.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C. Detector: A spectrophotometer set at 225 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. 297

VP V

COCAINE HYDROCHLORIDE

Procedure: System suitability: Inject the reference solution, the relative standard deviation for 6 replicate injections is not more than 2.0%. Inject separately the reference solution and the test solution. Calculate the content of cloxacillin, C19H18ClN3O5S, in the capsules using the areas of the principal peaks in the chromatograms obtained with the test solution and the reference solution, and the declared content of C19H18ClN3O5S in cloxacillin sodium RS.

Storage Store in an airtight container and cool place.

the solution in the range 220 nm to 350 nm exhibits two absorption maxima, at 233 nm and 273 nm. The specific absorbance at 233 nm is 378 to 402. C. Dissolve 0.1 g in 5 ml of water and add 1 ml of dilute ammonia solution R. A white precipitate is formed. Initiate crystallisation by scratching the wall of the tube with a glass rod. The crystals, washed with water and dried in vacuo, melt at 96 °C to 99 °C. D. It gives the reactions characteristic of chlorides (Appendix 8.1). E. It gives the reactions characteristic of alkaloids (Appendix 8.1).

Acidity To 10 ml of a 2% solution of the substance to be examined in carbon dioxide-free water R add 0.05 ml of methyl red solution R. Not more than 0.2 ml of 0.02 N sodium hydroxide VS is required to change the colour of the indicator to yellow.

Action and use Antibacterial. Usual strength 250 mg; 500 mg.

Appearance of solution A 2.0% solution of the substance to be examined in water is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2).

COCAINE HYDROCHLORIDE Cocaini hydrochloridum

Specific optical rotation -70° to -73° (calculated with reference to the dried substance) (Appendix 6.4). Determine on a 2.5% solution of the substance to be examined in water. C17H21NO4,HCl

M. 339.8

Cocaine hydrochloride is methyl (1R,2R,3S,5S)-3(benzoyloxy)-8-methyl-8-azabicyclo[3.2.1]octane-2carboxylate hydrochloride. It contains not less than 98.5% and not more than 101.0% of C17H21NO4,HCl, calculated with reference to the dried substance.

Characters A white, crystalline powder or colourless crystals. Very soluble in water, freely soluble in ethanol (96%), slightly soluble in methylene chloride. Melting point is about 197 °C, with decomposition. Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D, E. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum obtained with cocaine hydrochloride RS. B. Dissolve 20.0 mg in 0.01 M hydrochloric acid R and dilute to 100.0 ml with the same acid. Dilute 5.0 ml of the solution to 50.0 ml with 0.01 M hydrochloric acid R. The ultraviolet absorption spectrum (Appendix 4.1) of 298

Foreign organic matter To 0.2 g add 2 ml of sulfuric acid R and allow to stand for 15 min. The solution is not more intensely coloured than reference solution BY5 (Appendix 9.3, method 1). Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Triethylamine - tetrahydrofuran acetonitrile - water (0.5 : 100 : 430 : 479.5). Test solution: Dissolve 25.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Reference solution (1): Dilute 1.0 ml of the test solution to 50.0 ml with the mobile phase. Dilute 5.0 ml of this solution to 100.0 ml with the mobile phase. Reference solution (2): Dissolve 25.0 mg of the substance to be examined in 0.01 M sodium hydroxide R and dilute to 10.0 ml with the same solvent. Dilute 1.0 ml of the solution to 10.0 ml with 0.01 M sodium hydroxide R. Allow the solution to stand for 15 min. Chromatographic system: A column (15 cm × 4.6 mm) packed with end-capped octadecylsilyl silica gel for chromatography R (5 µm) with a specific surface area of 335 m2/g, a pore size of 10 nm and a carbon loading of 19.1%.

VP V

CODEINE

Temperature of column: 35 °C. Detector: A spectrophotometer set at 216 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: In the chromatogram obtained with reference solution (2), the retention time of cocaine is about 7.4 min. The relative retention time for degradation product of cocaine comparing with reference to cocaine is about 0.7. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks corresponding to cocaine and to the degradation product is at least 5.0. Limits: The area of any impurity peak, eluting after the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%); the sum of the areas of all peaks, eluting after the principal peak, is not greater than 5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%).

CODEINE Codeinum monohydricum Codeine monohydrate

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 100 °C to 105 °C).

Identification Apply one of the two following identifications: First identification: A, C. Second identification: B, C, D, E. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of codeine RS. Examine the substances prepared as discs of potassium bromide R. B. To 2.0 ml of solution S (see Appearance of solution) add 50 ml of water then 10 ml of 1 M sodium hydroxide R and dilute to 100.0 ml with water. The ultraviolet absorption spectrum (Appendix 4.1) of the solution in the range 250 nm to 350 nm, exhibits an absorption maximum at 284 nm. The specific absorbance at this maximum is about 50, calculated with reference to the dried substance C. Melting point: 155 °C to 159 °C (Appendix 6.7). D. Add 1 ml of sulfuric acid R and 0.05 ml of a 1.3% solution of ferric chloride R to about 10 mg of the substance to be examined and heat on a water-bath. A blue colour develops. Add 0.05 ml of nitric acid R. The colour changes to red. E. It gives the reaction of alkaloids (Appendix 8.1).

Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determine on the residue from the test for Loss on drying. Assay Dissolve 0.250 g in a mixture of 5.0 ml of 0.01 M hydrochloric acid R and 50 ml of ethanol (96%) R. Titrate with 0.1 N sodium hydroxide VS. Determine the end point potentiometrically (Appendix 10.2). Read the volume added between the 2 points of inflexion. Each ml of 0.1 N sodium hydroxide VS is equivalent to 33.98 mg of C17H22ClNO4. Storage Protected from light. Action and use Anaesthetic. Preparation Local solution.



C18H21NO3,H2O 

, H2O

M. 317.4

Codeine is 7,8-didehydro-4,5a-epoxy-3-methoxy-17methylmorphinan-6a-ol monohydrate. It contains not less than 99.0% and not more than 101.0% of C18H21NO3, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder or colourless crystals. Soluble in boiling water, freely soluble in ethanol (96%).

Appearance of solution Solution S: Dissolve 50 mg in carbon dioxide-free water R and dilute to 10.0 ml with the same solvent. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). Specific optical rotation -142° to -146°, calculated with reference to the dried substance (Appendix 6.4). Dissolve 0.50 g in ethanol (96%) R and dilute to 25.0 ml with the same solvent. 299

VP V

CODEINE PHOSPHATE

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 1.08 g of sodium octanesulfonate R in a mixture of 20 ml of glacial acetic acid R and 250 ml of acetonitrile R and dilute to 1000 ml with water. Test solution: Dissolve 0.100 g of the substance to be examined and 0.100 g of sodium octanesulfonate R in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (1): Dissolve 5.0 mg of codeine impurity A RS in the mobile phase and dilute to 5.0 ml with the mobile phase. Reference solution (2): Dilute 1.0 ml of reference solution (1) to 20.0 ml with the mobile phase. Reference solution (3): Dilute 1.0 ml of the test solution to 50.0 ml with the mobile phase. Dilute 5.0 ml of this solution to 100.0 ml with the mobile phase. Reference solution (4): To 0.25 ml of the test solution, add 2.5 ml of reference solution (1). Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase end-capped octylsilyl silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 245 nm. Flow rate: 2.0 ml/min. Volume of injection: 10 µl. Procedure: The run time of the test solution is 10 times the retention time of codeine. Relative retention with reference to codeine (retention time = about 6 min): impurity B = about 0.6; impurity E = about 0.7; impurity A = about 2.0; impurity C = about 2.3; impurity D = about 3.6. System suitability: In the chromatogram obtained with reference solution (4), the resolution between the peaks due to codeine and impurity A is at least 3. Limits: Correction factor: for the calculation of content, multiply the peak area of impurity C by 0.25; Impurity A: The area of the peak due to impurity A is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (2) (1.0%). Impurities B, C, D, E: For each impurity, the area, corrected if necessary, is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (3) (0.2%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (3) (0.10%). The sum of the peak areas of all impurities other than impurity A is not more than 10 times the area of the principal peak in the chromatogram obtained with reference solution (3) (1.0%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (3) (0.05%). Note: Impurity A: 7,8-didehydro-4,5a-epoxy-3,6a-dimethoxy-17methylmorphinan (methylcodeine).

300

Impurity B: 7,8-didehydro-4,5a-epoxy-17-methylmorphinan3,6a-diol (morphine). Impurity C: 7,7’,8,8’-tetradehydro-4,5a:4’,5’a-diepoxy-3,3’dimethoxy-17,17’-dimethyl-2,2’-bimorphinanyl-6a,6’a-diol (codeine dimer). Impurity D: 7,8-didehydro-2-[(7,8-didehydro-4,5a-epoxy-6ahydroxy-17-methylmorphinan- 3-yl)oxy]-4,5a-epoxy-3-methoxy17-methylmorphinan-6a-ol (3-O-(codein-2- yl)morphine). Impurity E: 7,8-didehydro-4,5a-epoxy-3-methoxy-17-methylmorphinan-6a,10-diol. Impurity F: 7,8-didehydro-4,5a-epoxy-3-methoxy-17-methylmorphinan-6a,14-diol. Impurity G: 6,7,8,14-tetradehydro-4,5a-epoxy-3,6-dimethoxy17-methylmorphinan (thebaine).

Loss on drying 4.0% to 6.0% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.250 g of the substance to be examined in 10 ml of anhydrous acetic acid R. Add 20 ml of dioxan R. Titrate with 0.1 N perchloric acid VS, using 0.05 ml of crystal violet solution R as indicator. Carry out a blank titration. 1 ml of 0.1 N perchloric acid VS is equivalent to 29.94 mg of C18H21NO3. Storage Store in an airtight container, protected from light. Action and use Opioid receptor agonist; analgesic. Preparations Tablets, combination tablets. CODEINE PHOSPHATE

Codeini phosphas

, H3PO4 , 1/2 H2O

C18H21NO3,H3PO4,1/2H2O  

M. 406.4

VP V

Codeine phosphate is 7,8-didehydro-4,5α-epoxy-3methoxy-17-methylmorphinan-6α-ol phosphate hemihydrate. It contains not less than 98.5% and not more than 101.0% of C18H21NO3,H3PO4, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder or small, colourless crystals. Freely soluble in water, slightly soluble or very slightly soluble in ethanol (96%). Identification Apply one of the two following identifications: First identification: A, E, F. Second identification: B, C, D, E, F, G. A. Dissolve 0.20 g in 4 ml of water. Add 1 ml of a mixture of equal volumes of 10 M sodium hydroxide R and water and initiate crystallisation, if necessary, by scratching the wall of the tube with a glass rod and cooling in iced water. Wash the precipitate with water and dry at 100 °C to 105 °C. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the reference spectrum of codeine RS. Examine the dried precipitate prepared as discs using potassium bromide R. B. Solution S: Dissolve 1.00 g in carbon dioxide-free water R prepared from distilled water R and dilute to 25.0 ml with the same solvent. Dilute 1.0 ml of solution S to 100.0 ml with water. To 25.0 ml of this solution add 25 ml of water then 10 ml of 1 M sodium hydroxide R and dilute to 100.0 ml with water. The ultraviolet absorption spectrum (Appendix 4.1) of the solution in the range 250 nm to 350 nm, exhibits an absorption maximum at 284 nm. The specific absorbance at this maximum is about 38, calculated with reference to the dried substance C. Melting point for the precipitate obtained in Test A: 155 °C to 159 °C (Appendix 6.7). D. Add 1 ml of sulfuric acid R and 0.05 ml of a 1.3% solution of ferric chloride R to about 10 mg of the substance to be examined and heat on a water-bath. A blue colour develops. Add 0.05 ml of nitric acid R. The colour changes to red. E. It complies with the test for Loss on drying. F. Solution S gives reaction (A) of phosphates (Appendix 8.1). G. It gives the reaction of alkaloids (Appendix 8.1). pH 4.0 to 5.0 (Appendix 6.2). Determined on solution S. Specific optical rotation -98° to -102°, calculated with reference to the dried substance (Appendix 6.4). Dilute 5.0 ml of solution S to 10.0 ml with water.

CODEINE PHOSPHATE

Related substances Liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 1.08 g of sodium octanesulfonate R in a mixture of 20 ml of glacial acetic acid R and 250 ml of acetonitrile R and dilute to 1000 ml with water. Test solution: Dissolve 0.100 g of the substance to be examined and 0.100 g of sodium octanesulfonate R in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (1): Dissolve 5.0 mg of codeine impurity A RS in the mobile phase and dilute to 5.0 ml with the mobile phase. Reference solution (2): Dilute 1.0 ml of reference solution (1) to 20.0 ml with the mobile phase. Reference solution (3): Dilute 1.0 ml of the test solution to 50.0 ml with the mobile phase. Dilute 5.0 ml of this solution to 100.0 ml with the mobile phase. Reference solution (4): To 0.25 ml of the test solution add 2.5 ml of reference solution (1). Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase end-capped octylsilyl silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 245 nm. Flow rate: 2.0 ml/min. Volume of injection: 10 µl. Procedure: The run time is 10 times the retention time of codeine. Relative retention with reference to codeine (retention time = about 6 min): impurities (impurities B and E) B = about 0.6; impurity E = about 0.7; impurity A = about 2.0; impurity C = about 2.3; impurity D = about 3.6. System suitability: In the chromatogram obtained with reference solution (4), the resolution between the peaks due to codeine and impurity A is at least 3.0. Limits: Correction factor: for the calculation of content, multiply the peak area of impurity C by 0.25; Impurity A: The area of the peak due to impurity A is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (2) (1.0%). Sum of impurities B and E: The sum of the peak areas is not more than 4 times the area of the principal peak in the chromatogram obtained with reference solution (3) (0.4%). Impurities C, D: For each impurity, the area, corrected if necessary, is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (3) (0.2%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (3) (0.10%). The sum of the peak areas of all impurities other than impurity A is not more than 10 times the area of the principal peak in the chromatogram obtained with reference solution (3) (1.0%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (3) (0.05%). 301

VP V

CODEINE PHOSPHATE TABLETS

Loss on drying 1.5% to 3.0% for hemihydrate (Appendix 9.6). (1.000 g, 105 °C). Sulfates Not more than 0.1% (Appendix 9.4.14). Dilute 5 ml of solution S to 20 ml with distilled water R. Assay Dissolve 0.350 g of the substance to be examined in a mixture of 10 ml of anhydrous acetic acid R and 20 ml of dioxan R. Titrate with 0.1 N perchloric acid VS using 0.05 ml of crystal violet solution R as indicator. Carry out a blank titration. 1 ml of 0.1 N perchloric acid VS is equivalent to 39.74 mg of C18H21NO3,H3PO4. Storage Store in an airtight container, protected from light. Action and use Opioid receptor agonist; analgesic. Preparations Tablets, oral solution. CODEINE PHOSPHATE TABLETS Tabellae Codeini phosphatis Codeine phosphate tablets contain codeine phosphate. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of codeine phosphate, C18H21NO3, H3PO4,1/2H2O, 93.0% to 107.0% of the stated amount. Identification A. Weight a quantity of the powdered tablets containing about 100 mg of codeine phosphate, add 15 ml of water and 5 ml of 1 M sulfuric acid R, allow to stand for 1 hour. Filter, wash any undissolved residue with a few milliliters of water, make the filtrate alkaline with 6 M ammonia R, extract with two 10 ml quantities of chloroform R. Evaporate the chloroform extracts on a water bath to dryness. Continue to dry the residue at 80 °C for 4 hours. The infrared absorption spectrum (Appendix 4.2) of the residue is concordant with the spectrum of codeine obtained by similarly treating 10 ml of a 1% solution of codeine phosphate RS. B. To a quantity of the powdered tablets containing about 100 mg of codeine phosphate, add 10 ml of water and 2 drops of 2 M sulfuric acid R, heat for 15 minutes with frequent shaking. Filter, neutralize 5 ml of the filtrate with 6 M ammonia R, and add a 5% solution of silver nitrate R, a yellow precipitate of silver phosphate is formed, which is soluble in dilute nitric acid R and 6 M ammonia R. 302

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 500 ml of water. Rotation speed: 50 rpm. Time: 45 min. Procedure: Test solution: After the specified time, withdraw a sample of the medium and filter, discard the first 20 ml of the filtrate. Dilute the filtrate if necessary. Reference solution: Prepare a reference solution of codeine phosphate RS having the same concentration in the same medium as the test solution. Measure the absorbances of the test solution and the reference solution at the maximum at 284 nm. Calculate the content of codeine phosphate, C18H21NO3,H3PO4,1/2H2O, dissolved from the obtained absorbances of the test solution, the reference solution and declared content of C18H21NO3,H3PO4,1/2H2O in codeine phosphate RS. Tolerance: Not less than 75% (Q) of the stated amount of codeine phosphate, C18H21NO3,H3PO4,1/2H2O, is dissolved in 45 min.

Limit of morphine Dissolve about 50 mg of potassium ferricyanide R in 10 ml of water, and add 1 drop of a 10.5% solution of ferric chloride R and 1 ml of the filtrate obtained in test B for Identification, no blue colour is produced immediately. Assay Weigh 20 tablets and powder finely. Transfer a quantity of powdered tablets containing the equivalent of about 0.15 g of codeine phosphate, accurately weighed, to a 100 ml volumetric flask. Add 20 ml of 0.25 M sulfuric acid R, shake for 30 min and dilute to volume with water. Filter, make 50 ml of the filtrate alkaline with 6 M ammonia R and extract with four portions of chloroform R (25 ml, 15 ml, 15 ml, 15 ml). Combine the chloroform extracts, evaporate on water bath to dryness, add to the residue 25.0 ml of 0.02 N sulfuric acid VS, heat to dissolve, cool and add 2 drops of methyl red R and titrate with 0.02 N sodium hydroxide VS. Each ml of 0.02 N sulfuric acid VS is equivalent to 8.128 mg of C18H21NO3,H3PO4,1/2H2O. Storage Store in closed container, protected from light. Action and use Analgesic, anti-diarrhoeal, cough suppressant. Usual strength 15 mg; 30 mg.

VP V

COLCHICINE

COLCHICINE Colchicinum

it becomes green. Not more than 0.1 ml of 0.01 N sodium hydroxide VS is required to change the colour of the indicator to blue.

Specific optical rotation -235º to -250º (anhydrous substance) (Appendix 6.4). Dissolve 50.0 mg in ethanol (96%) R and dilute to 10.0 ml with the same solvent. C22H25NO6 

 

M. 399.4

Colchicine is (-)-N-[(7S,12aS)-1,2,3,10-tetramethoxy-9oxo-5,6,7,9-tetrahydrobenzo[a] heptalen-7-yl]acetamide. It contains not less than 97.0% and not more than 102.0% of C22H25NO6, calculated with reference to the anhydrous substance.

Characters Yellowish-white, amorphous or crystalline powder. Very soluble in water, rapidly recrystallising from concentrated solutions as the sesquihydrate, freely soluble in ethanol (96%), practically insoluble in cyclohexane. Identification Apply one of the two following identifications: First identification: A. Second identification: B, C, D. A. Infrared absorption spectrophotometry (Appendix 4.2) of the substance to be examined is concordant with the spectrum obtained with colchicine RS. Using discs of potassium bromide. B. Dissolve 5 mg in ethanol (96%) R and dilute to 100.0 ml with the same solvent. Dilute 5.0 ml of the solution to 25.0 ml with ethanol (96%) R. Examined between 230 nm and 400 nm (Appendix 4.1), the solution shows 2 absorption maxima, at 243 nm and 350 nm. The ratio of the absorbance measured at 243 nm to that measured at 350 nm is 1.7 to 1.9. C. To 0.5 ml of solution S (see Appearance of solution) add 0.5 ml of dilute hydrochloric acid R and 0.15 ml of a 10.5% solution of ferric chloride R. The solution is yellow and becomes dark green on boiling for 30 s. Cool, add 2 ml of methylene chloride R and shake. The methylene chloride layer is greenish-yellow. D. Dissolve about 30 mg in 1 ml of ethanol (96%) R and add 0.15 ml of a 10.5% solution of ferric chloride R. A brownishred colour develops. Appearance of solution Solution S: Dissolve 0.10 g in water and dilute to 20 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution GY3 (Appendix 9.3, method 2). Acidity or alkalinity To 10 ml of solution S add 0.1 ml of bromothymol blue solution R. Either the solution does not change colour or

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Mix 450 ml of a 0.68% solution of potassium dihydrogen phosphate and 530 ml of methanol R. After cooling to room temperature, adjust the volume to 1000 ml with methanol R. Adjust the apparent pH to 5.5 with dilute phosphoric acid R. Test solution: Dissolve 20.0 mg of the substance to be examined in a mixture of equal volumes of methanol R and water and dilute to 20.0 ml with the same mixture of solvents. Reference solution (1): Dissolve 20.0 mg of colchicine for system suitability RS in a mixture of equal volumes of methanol R and water and dilute to 20.0 ml with the same mixture of solvents. Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with a mixture of equal volumes of methanol R and water. Reference solution (3): Dilute 1.0 ml of reference solution (2) to 20.0 ml with a mixture of equal volumes of methanol R and water. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Proceduce: The run time is 3 times the retention time of colchicine. When the chromatograms are recorded in the conditions described above, relative retention with reference to colchicine (retention time = about 7 min): impurity D = about 0.4; impurity E = about 0.7; impurity B = about 0.8; impurity A = about 0.94; impurity C = about 1.2. System suitability: Inject reference solution (1), peak-tovalley ratio: Minimum 2, where HP = height above the baseline of the peak due to impurity A and HV = height above the baseline of the lowest point of the curve separating this peak from the peak due to colchicine. Limits: In the chromatogram obtained with the test solution, the area of the peak due to impurity A is not more than 3.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (3.5%); the area of any peak, apart from the principal peak and the peak due to impurity A, is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) 303

COLCHICINE TABLETS

(1%), the sum of the areas of all the peaks, apart from the principal peak, is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (5%). Disregard any peak with an area less than area of the principal peak in the chromatogram obtained with reference solution (3) (0.05%).

Colchiceine Not more than 0.2%. Dissolve 50 mg in water and dilute to 5 ml with the same solvent. Add 0.1 ml of a 10.5% solution of ferric chloride R. The solution is not more intensely coloured than a mixture of 1 ml of red primary solution, 2 ml of yellow primary solution and 2 ml of blue primary solution (Appendix 9.3, method 2). Chloroform Not more than 0.05% (Appendix 10.14). Ethyl acetate Not more than 6.0% (m/m) (Appendix 10.14). Water Not more than 2.0% (Appendix 10.3). Determined on 0.500 g. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 0.5 g. Assay Dissolve 0.250 g with gentle heating in a mixture of 10 ml of acetic anhydride R and 20 ml of toluene R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 39.94 mg of C22H25NO6. Storage Protected from light. Action and use Used in treatment of gout. Preparation Tablets. COLCHICINE TABLETS Tabellae Colchicini Colchicine tablets contain colchicine. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of colchicine, C22H25NO6, 90.0% to 110.0% of the stated amount. 304

VP V

Identification Triturate a quantity of the powdered tablets containing about 20 mg of colchicine with 20 ml of water, allow the solids to settle, and filter the clear supernatant liquid into a separator. Extract with 30 ml of chloroform R. Evaporate the chloroform extract, using mild heat, to dryness. The infrared absorption spectrum (Appendix 4.2) of the residue is concordant with the spectrum of colchicine RS. Dissolution (Appendix 11.4) Carry out the following procedure protected from light. Apparatus: Basket. Medium: 500 ml of water. Rotation speed: 100 rpm. Time: 30 min. Procedure: Test solution: After the specified time, withdraw a sample of the medium and filter, discard the first 20 ml of the filtrate. Reference solution: Dissolve a quantity of colchicine RS, accurately weighed, in water and dilute with the same solvent to obtain a reference solution having the same concentration as the test solution. Determine the content of colchicine dissolved by liquid chromatography (Appendix 5.3). Inject separately 50 µl of the test solution and the reference solution into the chromatographic system. Employ the mobile phase and chromatographic system as directed in the Assay. Tolerance: Not less than 75% (Q) of the stated amount of colchicine, C22H25NO6, is dissolved in 30 minutes. Uniformity of content (Appendix 11.2) Test solution: Powder one tablet finely, add about 50 ml of a mixture of methanol R and water (1 : 1), shake for 15 min, transfer to a volumetric flask with suitable volume and dilute with the same solvent to obtain a solution with concentration of about 5 µg/ml, filter. Reference solution: Dissolve a quantity of colchicine RS, accurately weighed, in a mixture of methanol and water (1 : 1) and dilute with the same solvent to obtain a solution with concentration of about 5 µg/ml. Determine content of colchicine in each tablet by liquid chromatography (Appendix 5.3). Using the mobile phase and chromatographic system as directed in the Assay. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dilute 45 ml of 0.5M potassium dihydrogen phosphate with water to 450 ml, add about 530 ml of methanol R, cool to room temperature and add methanol R to bring the volume to 1000 ml. Adjust to a pH of 5.5 ± 0.05 with 0.5 M phosphoric acid R. Reference solution: Dissolve a quantity of colchicine RS, accurately weighed, in a mixture of methanol R and water (1 : 1) and dilute with the same solvent to obtain a solution with concentration of about 6 µg/ml. This solution is stable for 4 months when stored tightly stoppered and in the dark.

VP V

COLECALCIFEROL

Test solution (Prepare immediately before use): Powder 20 tablets finely. Transfer a quantity of powdered tablets containing the equivalent of about 0.6 mg of colchicine, accurately weighed, to a 100 ml volumetric flask, add about 50 ml of a mixture of methanol R and water (1 : 1), shake by mechanical means for 15 min, rinsing down the walls of the flask and dilute with the same solvent to 100 ml, filter. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase B (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Inject the reference solution and record the peak responses. The column efficiency is not less than 4500 theoretical plates, calculated on the peak due to colchicine. The retention time of colchicine is between 5.5 and 9.5 minutes. The relative standard deviation of the peak areas (or peak height) for replicate injections is not more than 2.0%. Inject alternately the test solution and the reference solution. Calculate the content of colchicine, C22H25NO6, in tablets using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C22H25NO6 in colchicine RS.

Storage Store in a cool and dry place, protected from light. Action and use For the treatment of gout. Usual strength 0.25 mg; 0.5 mg. COLECALCIFEROL Cholecalciferolum Vitamin D3

C27H44O  

M. 384.6

Colecalciferol is (5Z,7E)-9,10-secocholesta-5,7,10(19)trien-3β-ol. It contains not less than 97.0% and not more than 102.0% of C27H44O.

1 mg of colecalciferol is equivalent to 40 000 IU of antirachitic activity (vitamin D) in rats.

Characters White or almost white crystals. It is sensitive to air, heat and light. Freely soluble in ethanol (96%), soluble in trimethylpentane and in fatty oils, practically insoluble in water. A reversible isomerisation to pre-colecalciferol takes place in solution, depending on temperature and time.Solutions in solvents without an antioxidant are unstable and are to be used immediately. Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of colecalciferol RS. Specific optical rotation +105° to +112° (Appendix 6.4). Dissolve 0.200 g of the substance to be examined rapidly in aldehyde-free alcohol R without heating and dilute to 25.0 ml with the same solvent. Determined within 30 min of preparing the solution. Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use, avoiding exposure to actinic light and air. Mobile phase: Pentanol - hexane (3 : 997). Test solution: Dissolve 10.0 mg of the substance to be examined in trimethylpentane R without heating and dilute to 10.0 ml with the same solvent. Reference solution (1): Dissolve 10.0 mg of colecalciferol RS in trimethylpentane R without heating and dilute to 10.0 ml with the same solvent. Reference solution (2): Dilute 1.0 ml of colecalciferol for system suitability RS (containing impurity A) to 5.0 ml with the mobile phase. Heat in a water-bath at 90 °C under a reflux condenser for 45 min and cool (formation of precholecalciferol). Reference solution (3): Dilute 10.0 ml of reference of the solution (1) to 100.0 ml with the mobile phase. Dilute 1.0 ml of the solution to 100.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 265 nm. Flow rate: 2.0 ml/min. Volume of injection: 5 µl. Procedure: Inject the test solution and reference solutions (2) and (3). The run time is twice the retention time of colecalciferol. Relative retention with reference to colecalciferol (retention time = about 19 min): pre-colecalciferol = about 0.5; impurity A = about 0.6. 305

COLECALCIFEROL TABLETS

System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to pre-colecalciferol and impurity A is at least 1.5. Limits: Impurity A: The area of the peak due to impurity A is not more than the area of the principal peak in the chromatogram obtained with reference solution (3) (0.1%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (3) (0.10%). The sum of the peak areas of all impurities is not more than 10 times the area of the principal peak in the chromatogram obtained with reference solution (3) (1.0%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (3) (0.05%); disregard the peak due to pre-cholecalciferol.

Note: Impurity A: (5E,7E)-9,10-secocholesta-5,7,10(19)-trien-3β-ol (trans-cholecalciferol, trans-vitamin D3). Impurity B: Cholesta-5,7-dien-3β-ol (7,8-didehydrocholesterol, provitamin D3). Impurity C: 9β,10α-cholesta-5,7-dien-3β-ol (lumisterol3). Impurity D: (6E)-9,10-secocholesta-5(10),6,8(14)-trien-3β-ol (iso-tachysterol3). Impurity E: (6E)-9,10-secocholesta-5(10),6,8-trien-3β-ol (tachysterol3).

Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. Inject the test solution and reference solution (1). Calculate the content of C27H44O using the areas for the peaks in the chromatograms obtained with the test solution, the reference solution (1) and the declared content of C27H44O in colecalciferol RS. Storage Under nitrogen, in an airtight container, protected from light, at a temperature of 2 °C to 8 °C. The contents of an opened container are to be used immediately. Action and use Vitamin. Preparations Tablets, calcium and colecalciferol tablets, vitamins A, C and D oral drops. COLECALCIFEROL TABLETS Tabellae Colecalciferoli Colecalciferol tablets contain colecalciferol. They may be coated. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements. 306

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Content of colecalciferol, C27H44O, 90.0% to 125.0% of the stated amount. Identification A. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the peak due to colecalciferol in the chromatogram obtained with the reference solution. B. Extract a quantity of the powdered tablets equivalent to 400 IU of vitamin D with 5 ml of ethanol-free chloroform R, filter. To 1 ml of the filtrate add 9 ml of antimony trichloride solution R, a brownish red colour is produced. Uniformity of content (Appendix 11.2) Examine by liquid chromatography (Appendix 5.3). Carry out the following procedure in subdued light: Mobile phase, reference solution, chromatographic conditions and procedure: Proceed as directed in the Assay Test solution: Transfer the fine powder of one tablet to a 50-ml conical flask, add 20.0 ml of methanol (90%) R, stopper, shake well and sonicate for 5 min. Centrifuge and filter through a sintered-glass filter (Whatman GF/C is suitable). If necessary, dilute the filtrate with methanol (90%) R to obtain a solution with concentration of colecalciferol of about 0.00005%. Calculate the content of colecalciferol in each tablet using the peak areas (or heights) in the chromatograms obtained with the test solution, the reference solution and the declared content of C27H44O in colecalciferol RS. Assay Examine by liquid chromatography (Appendix 5.3) in subdued light. Mobile phase: Methanol - water (97 : 3). Reference solution: A 0.00005% solution of colecalciferol RS in methanol 90% R. Test solution: Weigh 20 tablets (remove the coating in case of coated tablets), powder finely. Transfer a quantity of powdered tablets containing the equivalent of about 50 µg of colecalciferol, accurately weighed, to a 100 ml volumetric flask, add 70 ml of methanol (90%) R, shake for 5 min and sonicate for 5 min. Dilute to volume with methanol (90%) R, mix well. Centrifuge and filter through a sintered-glass filter (Whatman GF/C is suitable). Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase C (5 µm) (Hypersil 5 ODS is suitable). Detector: A spectrophotometer set at 264 nm. Flow rate: 1.0 ml/min. Volume of injection: 50 µl. Procedure: Inject alternately the test solution and the reference solution. Calculate the content of colecalciferol, C27H44O, in tablets using the peak areas (or heights) in the chromatograms

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ANHYDROUS COPPER SULFATE

obtained with the test solution and the reference solution and the declared content of C27H44O in colecalciferol RS.

Note: Each µg of colecalciferol is equivalent to 40 IU of vitamin D.

Storage Store in a cool and dry place, protected from light. Action and use Vitamin D analogue. Usual strength 400 IU. COPPER SULFATE Cupri sulfas CuSO4,5H2O

M. 249.7

Copper sulfate pentahydrate contains not less than 99.0% and not more than 101.0% of CuSO4,5H2O.

Characters A blue, crystalline powder or transparent, blue crystals. Freely soluble in water, soluble in methanol, practically insoluble in ethanol (96%). Identification A. Add several drops of 2 M ammonia R to 1 ml of solution S (see Clarity of solution), a blue precipitate is formed. On further addition of 2 M ammonia R, the precipate dissolves and a dark blue colour is produced. B. It complies with the test for Loss on drying. C. Dilute 1 ml of solution S to 5 ml with water, add 1 ml of 2 M hydrochloric acid R and several drops of a 5% solution of barium chloride R. A white precipitate is produced. Clarity of solution Solution S: Dissolve 5 g in water and dilute to 100 ml with the same solvent. Solution S is clear (Appendix 9.2). Chlorides Not more than 0.01% (Appendix 9.4.5). Dilute 10 ml of solution S to 15 ml with water. The solution complies with the limit test for chlorides. Examine the tubes laterally against a black background. Iron Not more than 0.01%. Determine by atomic absorption spectrometry (Appendix 4.4, method 1). Test solution: Dissolve 0.5 g in 10 ml of water, add 2.5 ml of lead-free nitric acid R and dilute to 25.0 ml with water. Reference solutions: Prepare the reference solutions using iron standard solution (20 ppm Fe) R, adding 2.5 ml of lead-free nitric acid R and diluting to 25.0 ml with water.

Measure the absorbance at 248.3 nm using an iron hollowcathode lamp as a source of radiation and an air-butane flame.

Lead Not more than 50 ppm. Determine by atomic absorption spectrometry (Appendix 4.4, method 1). Test solution: Dissolve 2.5 g in 10 ml of water, add 2.5 ml of lead-free nitric acid R and dilute to 25.0 ml with water. Reference solutions: Prepare the reference solutions using lead standard solution (100 ppm Pb) R, adding 2.5 ml of lead-free nitric acid R and diluting to 25.0 ml with water. Measure the absorbance at 217.0 nm using a lead hollowcathode lamp as a source of radiation and an air-butane flame. Loss on drying 35.0% to 36.5% (Appendix 9.6). (0.500 g; 250 °C). Assay Dissolve 0.200 g in 50 ml of water. Add 2 ml of sulfuric acid R and 3 g of potassium iodide R. Titrate with 0.1 N sodium thiosulfate VS, adding 1 ml of starch solution R towards the end of the titration. Each ml of 0.1 N sodium thiosulfate VS is equivalent to 24.97 mg of CuSO4,5H2O. Storage Store in an airtight container. ANHYDROUS COPPER SULFATE Cupri sulfas anhydricus CuSO4

M. 159.6

Anhydrous copper sulfate contains not less than 99.0% and not more than 101.0% of CuSO4, calculated with reference to the dried substance.

Characters A greenish-grey powder, very hygroscopic. Freely soluble in water, slightly soluble in methanol, practically insoluble in ethanol (96%). Identification A. Add several drops of 2 M ammonia R to 1 ml of solution S (see Clarity of solution), a blue precipitate is formed. On further addition of 2 M ammonia R, the precipate dissolves and a dark blue colour is produced. B. It complies with the test for Loss on drying. C. Dilute 1 ml of solution S to 5 ml with water, add 1 ml of 2 M hydrochloric acid R and several drops of a 5% solution of barium chloride R. A white precipitate is produced. Clarity of solution Solution S: Dissolve 1.6 g in water and dilute to 50 ml with the same solvent. Solution S is clear (Appendix 9.2). 307

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CORTISONE ACETATE

Chlorides Not more than 0.015% (Appendix 9.4.5). Dilute 10 ml of solution S to 15 ml with water. The solution complies with the limit test for chlorides. Examine the tubes laterally against a black background. Iron Not more than 0.015%. Determine by atomic absorption spectrometry (Appendix 4.4, method 1). Test solution: Dissolve 0.32 g in 10 ml of water, add 2.5 ml of lead-free nitric acid R and dilute to 25.0 ml with water. Reference solutions: Prepare the reference solutions using iron standard solution (20 ppm Fe) R, adding 2.5 ml of lead-free nitric acid R and diluting to 25.0 ml with water. Measure the absorbance at 248.3 nm using an iron hollowcathode lamp as a source of radiation and an air-acetylene flame. Lead Not more than 80 ppm. Determine by atomic absorption spectrometry (Appendix 4.4, method 1). Carry out one of the two following method: Method 1 Test solution: Dissolve 1.6 g in 10 ml of water, add 2.5 ml of lead-free nitric acid R and dilute to 25.0 ml with water. Reference solutions: Prepare the reference solutions using lead standard solution (100 ppm Pb) R, adding 2.5 ml of lead-free nitric acid R and diluting to 25.0 ml with water. Measure the absorbance at 217.0 nm using a lead hollowcathode lamp as a source of radiation and an air- acetylene flame. Method 2 Test solution: Dissolve 0.5 g in 10 ml of a 1% solution of nitric acid R (prepared by diluting lead-free nitric acid R in deionised distilled water) and dilute to 25.0 ml with the same solvent. Reference solutions: Dilute lead standard solution (1000 ppm Pb) R with a 1% solution of nitric acid R to obtain lead reference solutions having concentrations respectively of 1, 2, 4 ppm. Use these solutions to establish a standard curve. Measure the absorbance at 217.0 nm using a lead hollowcathode lamp as a source of radiation and an air- acetylene flame. Calculate the content of Pb (ppm), using the established standard curve and the absorbance measured from the test solution. Loss on drying Not more than 1.0% (Appendix 9.6). (0.500 g; 250 °C). Assay Dissolve 0.125 g in 50 ml of water. Add 2 ml of sulfuric acid R and 3 g of potassium iodide R. Titrate with 0.1 N 308

sodium thiosulfate VS, adding 1 ml of starch solution R towards the end of the titration. Each ml 0.1 N sodium thiosulfate VS is equivalent to 15.96 mg of CuSO4.

Storage Store in an airtight container. Action and use Copper deficiency. CORTISONE ACETATE Cortisoni acetas

C23H30O6

M. 402.5

Cortisone acetate is 17,21-dihydroxypregn-4-ene-3,11,20trione 21-acetate. It contains not less than 97.0% and not more than 103.0% of C23H30O6, calculated with reference to the dried substance.

Characters A white or almost white, crystalline powder, it shows polymorphism. Practically insoluble in water, freely soluble in methylene chloride, soluble in dioxan, sparingly soluble in acetone, slightly soluble in ethanol (96%), in ether and in methanol. Identification Apply one of the two following identifications: First identification: A, B. Second identification: C, D, E. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum obtained with cortisone acetate RS. If the spectra obtained in the solid state show differences, record new spectra using 5% solutions of the reference substance and of the substance to be examined in methylene chloride R in a 0.2 mm cell. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel F254. Mobile phase: Mix a mixture of 1.2 volumes of water and 8 volumes of methanol R to a mixture of 15 volumes of ether R and 77 volumes of methylene chloride R. Test solution: Dissolve 10 mg of the substance to be examined in a mixture of methanol - methylene chloride (1 : 9) and dilute to 10 ml with the same mixture of solvents. Reference solution (1): Dissolve 20 mg of cortisone acetate RS in a mixture of methanol - methylene chloride (1 : 9) and dilute to 20 ml with the same mixture of solvents.

VP V

Reference solution (2): Dissolve 10 mg of hydrocortisone acetate in reference solution (1) and dilute to 10 ml with reference solution (1). Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (1). Spray with ethanolic solution of sulphuric acid R. Heat at 120 °C for 10 min or until the spots appear. Allow to cool. Examine the plate in daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) shows two clearly separated spots. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel F254. Mobile phase: Mix a mixture of 1.2 volumes of water and 8 volumes of methanol R to a mixture of 15 volumes of ether R and 77 volumes of methylene chloride R. Test solution (1): Dissolve 25 mg of the substance to be examined in methanol R with gentle heating and dilute to 5 ml with the same solvent. This solution is also used to prepare test solution (2). Dilute 2 ml of the solution to 10 ml with methylene chloride R. Test solution (2): Transfer 2 ml of the solution obtained during preparation of test solution (1) to a 15 ml glass tube with a ground-glass stopper or a polytetrafluoroethylene cap. Add 10 ml of saturated methanolic potassium hydrogen carbonate solution R and immediately pass a stream of nitrogen R briskly through the solution for 5 min. Stopper the tube. Heat in a water-bath at 45 °C protected from light for 2 h and 30 min. Allow to cool. Reference solution (1): Dissolve 25 mg of cortisone acetate RS in methanol R with gentle heating and dilute to 5 ml with the same solvent. This solution is also used to prepare reference solution (2). Dilute 2 ml of the solution to 10 ml with methylene chloride R. Reference solution (2): Transfer 2 ml of the solution obtained during preparation of reference solution (1) to a 15 ml glass tube with a ground-glass stopper or a polytetrafluoroethylene cap. Add 10 ml of saturated methanolic potassium hydrogen carbonate solution R and immediately pass a stream of nitrogen R briskly through the solution for 5 min. Stopper the tube. Heat in a waterbath at 45 °C protected from light for 2 h 30 min. Allow to cool. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in air and examine in ultraviolet light at 254 nm.

CORTISONE ACETATE

The principal spot in each of the chromatograms obtained with the test solutions is similar in position and size to the principal spot in the chromatogram obtained with the corresponding reference solution. Spray with ethanolic solution of sulphuric acid R and heat at 120 °C for 10 min or until the spots appear. Allow to cool. Examine the plate in daylight and in ultraviolet light at 365 nm. The principal spot in each of the chromatograms obtained with the test solutions is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with the corresponding reference solution. The principal spots in the chromatograms obtained with test solution (2) and reference solution (2) have an Rf value distinctly lower than that of the principal spots in the chromatograms obtained with test solution (1) and reference solution (1). D. Add about 2 mg to 2 ml of sulfuric acid R and shake to dissolve. Within 5 min, a faint yellow colour develops. Add the solution to 10 ml of water and mix. The colour is discharged and a clear solution remains. E. About 10 mg gives the reaction characteristic of acetyl (Appendix 8.1).

Specific optical rotation +211° to +220°, calculated with reference to the dried substance (Appendix 6.4). Dissolve 0.250 g in dioxan R and dilute to 25.0 ml with the same solvent. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Mix 400 ml of acetonitrile R with 550 ml of water and allow to equilibrate; adjust the volume to 1000 ml with water and mix again. Test solution: Dissolve 25.0 mg of the substance to be examined in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (1): Dissolve 2 mg of cortisone acetate RS and 2 mg of hydrocortisone acetate RS in the mobile phase and dilute to 100.0 ml with the mobile phase. Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with octadecylsilyl silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Procedure: Equilibrate the column with the mobile phase at a flow rate of 1 ml/min for about 30 min. Inject reference solution (2). Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with reference solution (2) is at least 50% of the full scale of the recorder. 309

CORTISONE TABLETS

Inject reference solution (1). When the chromatograms are recorded in the prescribed conditions the retention times are: hydrocortisone acetate about 10 min and cortisone acetate about 12 min. The test is not valid unless the resolution between the peaks due to hydrocortisone acetate and cortisone acetate is at least 4.2; if necessary, adjust the concentration of acetonitrile in the mobile phase. Inject separately the test solution and reference solution (2). Continue the chromatography for twice the retention time of the principal peak. Limits: In the chromatogram obtained with the test solution: The area of any peak apart from the principal peak, is not greater than half the area of the principal peak in the chromatogram obtained with reference solution (2) (0.5%). The sum of the areas of all the peaks apart from the principal peak, is not greater than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (1.5%). Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (2).

Loss on drying Not more than 0.5% (Appendix 9.6). (0.500 g; 100 °C to 105 °C). Assay Dissolve 0.100 g in ethanol (96%) R and dilute to 100.0 ml with the same solvent. Dilute 2.0  ml of the solution to 100.0 ml with ethanol (96%) R. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum of 237 nm. Calculate the content of C23H30O6 taking 395 as the value of A (1%, 1 cm) at the maximum of 237 nm. Storage Store in an airtight container, protected from light. Action and use Corticosteroid. Preparation Tablets. CORTISONE TABLETS Tabellae Cortisoni Cortisone tablets contain cortisone acetate. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of cortisone acetate, C23H30O6, 90.0% to 110.0% of the stated amount. Identification A. To a quantity of the powdered tablets containing about 30 mg of cortisone acetate, add 15 ml of chloroform R, stir 310

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thoroughly for 15 min and filter. Evaporate the filtrate to dryness on a water bath. The residue complies with the following tests: Dissolve 1 mg of the residue in 10 ml of methanol R. To 1 ml of the resulting solution, add 8 ml of a freshly prepared phenylhydrazine sulfate solution R, heat at 70 °C for 15 min. A yellow colour is produced. Dissolve about 2 mg of the residue in 2 ml of sulfuric acid R, allow to stand for 5 min. A yellow or light orange colour is formed. The colour is faded when diluting with 10 ml of water, the solution remains clear. B. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the principal peak in the chromatogram obtained with the reference solution.

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of a 0.3% solution of sodium lauryl sulfate. Rotation speed: 50 rpm. Time: 45 min. Procedure: Reference solution: A 0.0028% solution of cortisone acetate RS in the medium. Test solution: After the specified time, withdraw a sample of the medium and filter, discard the first portion of the filtrate. Measure the absorbance (Appendix 4.1) of the obtained solutions at the maximum at about 242 nm in a 1-cm cell, using the dissolution medium as a blank. Calculate the content of cortisone acetate, C23H30O6, dissolved from the absorbances of the reference solution, the test solution and the concentration of C23H30O6 in the reference solution. Tolerance: Not less than 70% (Q) of the stated amount of cortisone acetate, C23H30O6, is dissolved in 45 minutes. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Mix 400 ml of acetonitrile R with 550 ml of water, allow to equilibrate, add water to 1000 ml, mix well. Prepare the following solutions immediately before use. Test solution: Weigh accurately a quantity of powdered tablets containing about 25 mg of cortisone acetate, add 10 ml of the mobile phase, sonicate for 10 minutes, filter. Reference solution: Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase, mix well. Resolution solution: A solution contains 0.002% each of cortisone acetate RS and hydrocortisone acetate RS in the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm) (Hypersil ODS is suitable). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Equilibrate the column with the mobile phase for 30 min. Inject the reference solution. Adjust the sensitivity of

VP V

the system so that the height of the principal peak in the chromatogram obtained is at least 50% of the full scale of the recorder. Inject the resolution solution. In the chromatogram obtained with the prescribed conditions, the retention times are: hydrocortisone acetate, about 10 minutes and cortisone acetate, about 12 min. The test is not valid unless the resolution factor between the peaks due to hydrocortisone acetate and cortisone acetate is at least 4.2. If necessary, adjust the concentration of acetonitrile in the mobile phase. Inject separately the test solution and the reference solution. Allow the chromatography to proceed for twice the retention time of the principal peak. In the chromatogram obtained with the test solution, the area of any secondary peak is not greater than half the area of the principal peak in the chromatogram obtained with the reference solution (0.5%); the sum of the areas of all the secondary peaks is not greater than 1.5 times the area of the principal peak in the chromatogram obtained with the reference solution (1.5%). Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with the reference solution (0.05%).

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol 60%. Reference solution: Prepare a 0.02% solution of cortisone acetate RS in methanol R. Dilute further 50.0 ml of the obtained solution to 100.0 ml with water, mix well. Resolution solution: Prepare a solution containing 0.02% each of cortisone acetate RS and prednisolone RS in methanol R. Dilute further 50.0 ml of the obtained solution to 100.0 ml with water, shake well. Test solution: Weight 20 tablets, powder finely. Weight accurately a quantity of powdered tablets containing the equivalent of about 10 mg of cortisone acetate, add 50 ml of methanol R, shake thoroughly and sonicate for 2 min, dilute to 100.0 ml with water, mix well, centrifuge. Use the clear supernatant liquid. Chromatographic system: A column (25 cm × 4.6 mm) packed with end-capped octadecylsilyl silica gel for chromatography (5 µm) (Hypersil ODS is suiltable). Detector: A spectrophotometer set at 240 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: Inject the resolution solution and record the chromatogram. The assay is not valid unless the resolution factor between the peaks due to cortisone acetate and prednisolone is at least 5.0. Inject alternately the reference solution and the test solution. Calculate the content of cortisone acetate, C23H30O6, in tablets using the peak areas (or heights) in the chromatograms obtained with the test solution and the reference solution and the concentration of C23H30O6 in the reference solution.

ABSORBENT COTTON

Storage Store in a cool and dry place, protected from light. Action and use Corticosteroid. Usual strength 25 mg. ABSORBENT COTTON Lanugo gossypii absorbens Absorbent cotton is the hair of the seed of various species of the genus Gossypium L., (Fam. Malvaceae), deprived of fatty matter, bleached and carefully carded.

Characters Absorbent cotton occurs as white, soft, fine filament-like hairs. It is odourless, tasteless and does not contain traces of leaf residue or seed-coat. Identification A. Examined under a microscope, each fibre is seen to consist of a single cell, up to about 4 cm long and up to 40 µm wide, in the form of a flattened tube with thick and rounded walls and often twisted. B. Dip a portion of the preparation in iodinated zinc chloride solution R, the fibres become violet. C. To 1 g add 10 ml of zinc chloride-formic acid solution R. Heat to 40 °C and allow to stand for 2 h 30 min, shaking occasionally. It does not dissolve. Acidity or alkalinity Place 15.0 g of the preparation in a suitable vessel, add 150 ml of carbon dioxide-free water R, close the vessel and allow macerate for 2 h. Decant the solution, squeeze the residual liquid carefully from the sample with a glass rod and mix. Reserve 10 ml of the solution for the test for Surface-active substances and filter the remainder. To 25 ml of the filtrate add 0.1 ml of phenolphthalein solution R and to another 25 ml add 0.05 ml of methyl orange solution R. Neither solution is pink. Surface-active substances Introduce the 10 ml portion of the solution reserved in the Acidity or alkalinity into a 25 ml graduated ground-glassstoppered cylinder with an external diameter of 18 to 22 mm, previously rinsed with sulfuric acid R and then with water. Shake vigorously 30 times in 10 seconds, allow to stand for 1 min and repeat the shaking. After 5 min, the height of foam layer above the surface of the water is not more than 2 mm. Sinking time  Prepare a test basket from copper wire 0.44 mm in diameter with spaces of 20 mm between the wires. The basket, weighing about 3.0 g, is in the form of cylinder 311

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STERILISED ABSORBENT COTTON

50.0 mm in diameter and 80.0 mm deep. Take 5.0 g of the preparation, place in the basket. Hold the basket on its side 12 mm above the surface of the water with 200 mm deep and maintained at 24 – 26 °C. The time to sink completely is not more than 8 seconds.

Water-holding capacity In the Sinking time, about 3 minutes after sinking, remove carefully the basket from the water. Place in a horizontal position over a sieve with appropriate numbers (1400 - 2000). Allow it to drain for 1 minute, transfer it to a beaker and weigh. The weight of water absorbed is not less than 100.0 g. Foreign fibres Dip 1.0 g of the preparation in to 0.5 M iodine solution R for 0.5 minute and wash carefully with water R. It shows no coloured fibre. Fluorescence Examine a layer of the preparation about 5 mm in thickness under ultraviolet light at 365 nm. It displays only slight brownish-violet fluorescence and a few yellow particles. It shows no intense blue fluorescence, apart from that which may be shown by a few isolated fibres. Extractable colouring matter Moisten 10.0 g of the preparation with ethanol (96%) R, allow to macerate for 4 hours. Transfer it in to a percolator, open the tap, add ethanol (96%) R until some drops of extract pass though. Close the tap and add ethanol (96%) R to submerge the preparation in alcohol (96%) R. Allow to settle for 24 hours. Open the tap until 38 ml of extract is obtained. Squeeze the residual liquid carefully from the sample with a glass rod and mix. Dilute with alcohol (96%) R to 50.0 ml. The liquid obtained is not more intensely coloured (Appendix 9.3, Method 2) than reference solution Y5, GY6 or a reference solution prepared as follows: to 3.0 ml of blue primary solution, add 7.0 ml of a 1% solution of hydrochloric acid R. Dilute 0.5 ml of this solution to 10.0 ml with a 1% solution of hydrochloric acid R.

Loss on drying Not more than 8.0% (Appendix 9.6). (5.000 g; 105 °C). Sulfated ash Not more than 0.4% (Appendix 9.9). Introduce 5.00 g of the preparation into a previously heated and cooled, tared crucible. Heat cautiously over a naked flame and then ignite carefully at 600 °C. Allow to cool, add a few drops of dilute sulfuric acid R, then heat and incinerate until all the black particles have disappeared. Allow to cool. Add a few drops of a 20% solution of ammonium carbonate R. Evaporate and incinerate carefully, allow to cool and weigh again. Repeat the incineration for periods of 5 minutes to constant mass. Storage Store in a dust-proof package, in a dry place. STERILISED ABSORBENT COTTON Lanugo gossypii absorbens sterilis Sterilised absorbent cotton complies with the requirements stated under “Absorbent cotton”. It can be yellowish colour because of being sterilised by heating.

Sterility The preparation complies with the test for Sterility (Appendix 13.7). Storage Store in an airtight package that maintain sterility and in a dry place. CYANOCOBALAMIN Cyanocobalaminum

Ether-soluble substances Not more than 0.5%. In an extraction apparatus (Soxhlet type), extract 5.0 g of the preparation with ether R for 4 hours at a rate of at least 4 extractions per hour. Evaporate the ether extract and dry the residue to constant mass at 105 °C. Water-soluble substances Not more than 0.5%. Boil 5.0 g of the preparation in 500 ml of water for 30 minutes, stirring frequently. Replace the water lost by evaporation. Decant the liquid, squeeze the residual liquid carefully from the sample with a glass rod and mix. Filter the liquid while hot. Evaporate 400 ml of the filtrate and dry the residue to constant mass at 105 °C. 312

C63H88CoN14O14P

M. 1355.0

VP V

Cyanocobalamin is α-(5,6-dimethylbenzimidazol-1-yl) cobamide cyanide. It contains not less than 96.0% and not more than 102.0% of C63H88CoN14O14P, calculated with reference to the dried substance.

Characters A dark-red, crystalline powder or dark-red crystals. Sparingly soluble in water and in ethanol (96%), practically insoluble in acetone and in ether. The anhydrous substance is very hygroscopic. Identification A. Dissolve 2.5 mg in water and dilute to 100.0 ml with the same solvent. The ultraviolet and visible absorption spectrum (Appendix 4.1) of the solution in the range 260 nm to 610 nm exhibits three absorption maxima, at 278 nm, 361 nm and at 547 nm to 559 nm. The ratio of the absorbance at the maximum at 361 nm to that at the maximum at 547 nm to 559 nm is 3.15 to 3.45. The ratio of the absorbance at the maximum at 361 nm to that at the maximum at 278 nm is 1.70 to 1.90. B. Examine by thin-layer chromatography (Appendix 5.4). Carry out the test protected from light. Coating substance: Silica gel G. Mobile phase: 10% solution of ammonia - methanol dichloromethane (9 : 30 : 45). Test solution: Dissolve 2 mg of the substance to be examined in 1 ml of a mixture of equal volumes of ethanol (96%) R and water. Reference solution: Dissolve 2 mg of cyanocobalamin RS in 1 ml of a mixture of equal volumes of ethanol (96%) R and water. Procedure: Apply separately to the plate 10 µl of each solution. Develop in a unsaturated tank over a path of 12 cm. Allow the plate to dry in air. Examine in daylight. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Mix 26.5 volumes of methanol R and 73.5 volumes of a 1.0% solution of disodium hydrogen phosphate adjusted to pH 3.5 using phosphoric acid R and use within 2 days. Test solution: Dissolve 10.0 mg of the substance to be examined in the mobile phase and dilute to 10.0 ml with the same solvents. Use within 1 h. Reference solution (1): Dilute 3.0 ml of the test solution to 100.0 ml with the mobile phase. Use within 1 h. Reference solution (2): Dilute 5.0 ml of the test solution to 50.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 100.0 ml with the mobile phase. Use within 1 h.

CYANOCOBALAMIN

Resolution solution: Dissolve 25 mg of the substance to be examined in 10 ml of water, warming if necessary. Allow to cool and add 5 ml of a 0.1% solution of chloramine T R and 0.5 ml of 0.05 M hydrochloric acid. Dilute to 25 ml with water. Shake and allow to stand for 5 min. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase and inject immediately. Chromatographic system: A column (25 cm × 4 mm) packed with octylsilyl silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 361 nm. Flow rate: 0.8 ml/min. Volume of injection: 20 µl. Procedure: The run time is 3 times the retention time of cyanocobalamin. Inject separately each solution and continue the chromatography for 3 times the retention time of cyanocobalamin. The test is not valid unless the chromatogram obtained with the resolution solution shows 2 principal peaks, the resolution between these peaks is not less than 2.5 and the chromatogram obtained with reference solution (2) shows one principal peak with a signal-to-noise ratio of not less than 5. Limits: In the chromatogram obtained with the test solution: The sum of the areas of any peaks apart from the principal peak is not greater than the area of the principal peak in the chromatogram obtained with reference solution (1) (3.0%). Disregard any peak whose area is less than that of the principal peak in the chromatogram obtained with reference solution (2).

Loss on drying Not more than 12.0% (Appendix 9.6). (20.00 mg; in vacuo; over phosphorus pentoxide; 100 °C to 105 °C; 2 h). Assay Dissolve 25.00 mg in water and dilute to 1000.0 ml with the same solvent. Measure the absorbance (Appendix 4.1) of the solution at the maximum at 361 nm. Calculate the content of C63H88CoN14O14P, taking 207 as the value of A (1%, 1 cm) at the maximum at 361 nm. Storage Store in an airtight container, protected from light. Action and use Vitamin. Preparations Tablets; injection.

313

VP V

CYANOCOBALAMIN INJECTION

CYANOCOBALAMIN INJECTION Injectio Cyanocobalamini Vitamin B12 injection Cyanocobalamin injection is a sterile solution of cyanocobalamin in water for injections. It may contain stabiliters The injection complies with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Content of cyanocobalamin, C63H88CoN14O14P, 95.0% to 115.0% of the stated amount. Characters A clear, pink to red solution. Identification The light absorption spectrum of the test solution in the Assay shows 3 absorption maxima, at 278 nm, 361 nm and at 547 nm to 559 nm. The ratio of the absorbance at the maximum at 361 nm to that at the maximum at 547 nm to 559 nm is 3.15 to 3.45. The ratio of the absorbance at the maximum at 361 nm to that at the maximum at 278 nm is 1.70 to 1.90. pH 4.0 to 6.0 (Appendix 6.2). Bacterial endotoxins Less than 0.4 EU/mg (Appendix 13.2). Assay Pipet accurately a volume of the injection, dilute with water to produce a solution containing 25 µg of cyanocobalamin in 1 ml. Measure the absorbance at the maximum at 361 nm (Appendix 4.1) in a 1-cm cell, using water as a blank. Calculate the content of C63H88CoN14O14P taking 207 as the value of A(1%, 1 cm) at the maximum at 361 nm. Storage In a cool place, protected from light. Action and use Vitamin. Usual strength 200 µg/ml, 500 µg/ml. CYPROHEPTADINE HYDROCHLORIDE Cyproheptadini hydrochloridum

, HCl , 11/2 H2O

C21H21N,HCl,1½H2O   314

M. 350.9

Cyproheptadine hydrochloride is 4-(5H-dibenzo[a,d][7] annulen-5-ylidene)-1-methylpiperidine hydrochloride sesquihydrate. It contains not less than 98.5% and not more than 101.0% of C21H21N,HCl, calculated with reference to the anhydrous substance.

Characters White or slightly yellow, crystalline powder. Slightly soluble in water, freely soluble in methanol, sparingly soluble in ethanol (96%). Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of cyproheptadine hydrochloride RS. B. A saturated solution in water gives reaction (B) of chlorides (Appendix 8.1). Acidity Dissolve 0.10 g in water and dilute to 25 ml with the same solvent. Add 0.1 ml of methyl red solution R. Not more than 0.15 ml of 0.01 N sodium hydroxiders VS is required to change the colour of the indicator. Related substances Examine by liquid chromatography (Appendix 5.3) Buffer solution pH 4.5: Dissolve 6.12 g of potassium dihydrogen phosphate R in 900 ml of water, adjust to pH 4.5 with phosphoric acid R and dilute to 1000 ml with water . Mobile phase A: Buffer solution pH 4.5 - acetonitrile for chromatography (60 : 40). Mobile phase B: Buffer solution pH 4.5 - acetonitrile for chromatography R (40 : 60). Test solution: Dissolve 40.0 mg of the substance to be examined in mobile phase A and dilute to 20.0 ml with mobile phase A. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with mobile phase A. Dilute 1.0 ml of this solution to 10.0 ml with mobile phase A. Reference solution (2): Dissolve 2.0 mg of dibenzocycloheptene RS (impurity A), 2.0 mg of dibenzosuberone RS (impurity B) and 2.0 mg of cyproheptadine impurity C RS in mobile phase A, add 1.0 ml of the test solution and dilute to 100.0 ml with mobile phase A. Reference solution (3): Dilute 1.0 ml of reference solution (2) to 10.0 ml with mobile phase A. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase B (5 µm). Detector: A spectrophotometer set at 230 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: Carry out a linear gradient elution using the following conditions:

VP V

CYPROHEPTADINE HYDROCHLORIDE TABLETS

Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 10.0

100

0

10.0 - 10.1

100 → 0

0 → 100

10.1 - 35

0

100

Relative retention with reference to cyproheptadine (retention time = about 8 min): impurity C = about 0.7; impurity B = about 2.6; impurity A = about 3.9. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurity C and cyproheptadine is at least 7.0. Limits: Impurities A, B, C: For each impurity, the area is not more than 1.5 times the area of the corresponding peak in the chromatogram obtained with reference solution (3) (0.15%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). The sum of the peak areas of all impurities is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A: 5H-dibenzo[a,d][7]annulene (dibenzocycloheptene). Impurity B: 10,11-dihydro-5H-dibenzo[a,d][7]annulen-5-one (dibenzosuberone). Impurity C: 5-(1-methylpiperidin-4-yl)-5H-dibenzo[a,d][7]annulen5-ol.

Water 7.0% to 9.0% (Appendix 10.3). Determined on 0.200 g. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.250 g of the substance to be examined in a mixture of 5.0 ml of 0.01 N hydrochloric acid VS and 50 ml of ethanol (96%) R. Carry out a potentiometric titration (Appendix 10.2), using 0.1 N sodium hydroxide VS. Read the volume added between the 2 points of inflexion. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 32.39 mg of C21H22ClN. Storage Protected from light. Action and use Antihistamine. Preparation Tablets.

CYPROHEPTADINE HYDROCHLORIDE TABLETS Tabellae Cyproheptadini hydrochloridi Cyproheptadine tablets contain cyproheptadine hydrochloride. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of cyproheptadine hydrochloride, C21H21N,HCl, 90.0% to 110.0% of the stated amount. Identification A. To a quantity of the powdered tablets containing the equivalent of 20 mg of anhydrous cyproheptadine hydrochloride add 10 ml of water and 2.5 ml of 0.1 M sodium hydroxide, extract with 10 ml of dichloromethane R, filter through anhydrous sodium sulphate R moistened with dichloromethane R on absorbent cotton and evaporate the filtrate to dryness. The infrared absorption spectrum of the residue (Appendix 4.2), is concordant with the reference spectrum of cyproheptadine or the spectrum of cyproheptadine hydrochloride RS determined in the same condition. B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (2) corresponds to that in the chromatogram obtained with reference solution (3). C. Extract a quantity of the powdered tablets containing the equivalent of 20 mg of anhydrous cyproheptadine hydrochloride with 7 ml of water, filter, add 0.3 ml of 5 M ammonia R to the filtrate and filter again. The filtrate yields reaction A characteristic of chlorides (Appendix 8.1). Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 500 ml of 0.1 M hydrochloric acid R. Rotation speed: 50 rpm. Time: 30 min. Procedure: After the specified time, withdraw a sample of the medium and filter, discarding the first 20 ml of the filtrate, dilute the filtrate with 0.1 M hydrochloric acid R if necessary. Measure the absorbance at the maximum at 285 nm (Appendix 4.1), using 0.1 M hydrochloric acid R in the reference cell. At the same time, measure the absorbance of a solution of cyproheptadine hydrochloride RS in 0.1 M hydrochloric acid R having the same concentration as the test solution. Tolerance: Not less than 80% (Q) of the stated amount of cyproheptadine hydrochloride, C21H21N,HCl, is dissolved in 30 min. Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel 60. Mobile phase: Methanol - dichloromethane (10 : 90). 315

VP V

DAPSONE

Test solution (1): Shake a quantity of the powdered tablets equivalent to 50 mg of anhydrous cyproheptadine hydrochloride is 5 ml of the mobile phase, shake mechanically for 10 min, filter. Test solution (2): Dilute 1 volume of test solution (1) to 10 volumes with the mobile phase. Reference solution (1): A 0.002% w/v solution of dibenzocycloheptene RS in the mobile phase. Reference solution (2): Dilute 1 volume of test solution (1) to 100 volumes with the mobile phase and dilute 1 volume to 10 volumes with the mobile phase. Reference solution (3): A 0.1% w/v solution of cyproheptadine hydrochloride RS in the mobile phase. Procedure: Apply separately to the plate 10 µl of each solution. After removal of the plate, allow it to dry in air and spray with solution of sulfuric acid in ethanol R. Heat at 110 °C for 30 min and examine under ultraviolet light at 365 nm. In the chromatogram obtained with test solution (1) any spot corresponding to dibenzocycloheptene is not more intense than the spot in the chromatogram obtained with reference solution (1) (0.2%) and any other secondary spot is not more intense than the spot in the chromatogram obtained with reference solution (2) (0.1%).

Assay Weigh 20 tablets, calculate the average mass and powder finely. To a quantity of the powder containing the equivalent of 1.5 mg of anhydrous cyproheptadine hydrochloride add sufficient ethanol (96%) R to produce 100.0 ml and filter if necessary. Measure the absorbance of the resulting solution at the maximum at 286 nm (Appendix 4.1). Calculate the content of C21H21N,HCl taking 355 as the value of A (1%, 1 cm) at the maximum at 286 nm. Storage Store in a well-closed container. Action and use Histamine H1-receptor antagonist. Usual strength 4 mg. DAPSONE Dapsonum H 2N

NH2 S O

C12H12N2O2S

O

M. 248.3

Dapsone is 4,4’-sulphonyldianiline. It contains not less than 99.0% and not more than 101.0% of C12H12N2O2S, calculated with reference to the dried substance. 316

Characters A white or slightly yellowish-white, crystalline powder, odourless. Very slightly soluble in water, freely soluble in acetone, freely soluble in dilute mineral acids, sparingly soluble in ethanol (96%). Identification A. Dissolve 50.0 mg in methanol R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of this solution to 100.0 ml with methanol R. The ultraviolet absorption spectrum (Appendix 4.1) of the solution in the range 230 nm to 350 nm exhibits two absorption maxima, at 260 nm and 295 nm. The A (1%, 1 cm) at 260 nm is 700 to 760 and at 295 nm is 1150 to 1250. B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (2) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). C. Melting point: 175 °C to 181 °C (Appendix 6.7). Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Concentrated ammonia - methanol - ethyl acetate - heptane (1 : 6 : 20 : 20). Test solution (1): Dissolve 0.10 g of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Test solution (2): Dilute 1 ml of test solution (1) to 10 ml with methanol R. Reference solution (1): Dissolve 10 mg of dapsone RS in methanol R and dilute to 10 ml with the same solvent. Reference solution (2): Dilute 1 ml of test solution (2) to 10 ml with methanol R. Reference solution (3): Dilute 2 ml of reference solution (2) to 10 ml with methanol R. Procedure: Apply separately to the plate 1 µl of test solution (2), 1  µl of reference solution (1), 10  µl of test solution (1), 10  µl of reference solution (2) and 10  µl of reference solution (3). Develop in an unsaturated tank over a path of about 15 cm. After removal of the plate, allow the plate to dry in air. Spray the plate with a 0.5% solution of sodium nitrite in 0.1 N hydrochloric acid, after that spray a 0.1% solution of naphthylethylenediamine dihydrochloride on the plate while it is wet. Examine in daylight. Any spot in the chromatogram obtained with test solution (1), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (2) (1.0%) and not more than 2 such spots are more intense than the spot in the chromatogram obtained with reference solution (3) (0.2%). Loss on drying Not more than 1.5% (Appendix 9.6). (1.000 g; 100 °C to 105 °C).

VP V

Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determine on 1.0 g. Assay Dissolve 0.100 g in 50 ml of dilute hydrochloric acid R, add 3.0 of potassium bromide R. Cool in iced water and titrate slowly with 0.1 M sodium nitrite VS. Determine the end-point potentiometrically (Appendix 10.2). Each ml of 0.1 M sodium nitrite VS is equivalent to 12.42 mg of C12H12N2O2S. Storage Protected from light. Action and use Antileprotic. Preparation Tablets. DAPSONE TABLETS Tabellae Dapsoni Dapsone tablets contain dapsone. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of dapsone, C12H12N2 O2S, 92.5% to 107.5% of the stated amount. Identification A. Shake well a quantity of the powdered tablets containing the equivalent of about 50 mg of dapsone with 50 ml of methanol R and filter. Dilute 1.0 ml of the filtrate to 200.0 ml with methanol R, mix well. The ultraviolet absorption spectrum (Appendix 4.1) of the obtained solution in the range 230 nm to 350 nm exhibits absorption maxima at about 261 nm and 296 nm. B. In the test for Related substances, the principal spot in the chromatogram obtained with the test solution (2) is similar in position, size and colour to that in the chromatogram obtained with the reference solution (3). Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 1000 ml of 0.25M hydrochloric acid R. Rotation speed: 100 rpm. Time: 60 min. Procedure: Reference solution: Prepare a solution of dapsone RS in the medium with concentration of about 0.05 mg/ml. Transfer 2.0 ml of the resulting solution into a 25 ml volumetric flask, add 5 ml of 1M sodium hydroxide R, dilute with water to volume, mix well.

DAPSONE TABLETS

Test solution: After the specified time, withdraw a sample of the medium and filter, discard the first portion of the filtrate. Transfer an accurate volume of the filtrate, containing about 0.1 mg of dapsone, into a 25 ml volumetric flask, add 5 ml of 1M sodium hydroxide R, dilute with water to volume, mix well. Measure the absorbance (Appendix 4.1) of the obtained solutions at the maximum at about 290 nm in a 1-cm cell. Calculate the content of dapsone, C12H12N2O2S, dissolved from the absorbances of the reference solution, the test solution and the concentration of C12H12N2O2S in the reference solution. Tolerance: Not less than 75% (Q) of the stated amount of dapsone, C12H12N2O2S, is dissolved in 60 min.

Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: n-heptane - ethyl acetate - methanol 13.5M ammonia (20 : 20 : 6 : 1). Test solution (1): Weight a quantity of powdered tablets containing the equivalent of about 100 mg of dapsone, add 10 ml of methanol R, shake thoroughly for 10 min, filter. Test solution (2): Dilute 1.0 ml of test solution (1) to 10.0 ml with methanol R, mix well. Reference solution (1): Dilute 1.0 ml of test solution (2) to 10.0 ml with methanol R, mix well. Resolution solution (2): Dilute 1.0 ml of reference solution (1) to 5.0 ml with methanol R, mix well. Reference solution (3): A 0.1% solution of dapsone RS in methanol R. Procedure: Apply separately to the plate 10 µl of each of test solution (1), reference solution (1) and (2), 1 µl of each of test solution (2) and reference solution (3). Develop the chromatograph in an unsaturated tank until the solvent front has moved about 15 cm. After removal of the plate, allow it to dry in air. Spray with a 0.1% solution of 4-dimethylaminocinnamaldehyde R in a 1% v/v solution of hydrochloric acid in ethanol (96%) R and examine in daylight. Any secondary spot in the chromatogram obtained with test solution (1) is not more intense than the spot in the chromatogram obtained with reference solution (1) (1.0%) and not more than two such spots are more intense than the spot in the chromatogram obtained with reference solution (2) (0.2%). Assay Weight 20 tablets, powder finely. Weight accurately a quantity of powdered tablets containing the equivalent of about 0.25 g of dapsone, add 30 ml of 1M hydrochloric acid R, shake well to dissolve, add 3 g of potassium bromide R, cool in ice and titrate slowly with 0.1 M sodium nitrite 317

VP V

DEXAMETHASONE

VS, stirring constantly and determining the end-point electrometrically (Appendix 10.1 or 10.2). Each ml of 0.1 M sodium nitrite VS is equivalent to 12.42 mg of C12H12N2O2S.

Storage Store in a cool and dry place, protected from light. Action and use Treatment of leprosy. Usual strength 50 mg; 100 mg. DEXAMETHASONE Dexamethasonum

C22H29FO5 



M. 392.5

Dexamethasone is 9-fluoro-11β,17,21-trihydroxy-16αmethylpregna-1,4-diene-3,20-dione. It contains not less than 97.0% and not more than 103.0% of C22H29FO5, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder. Practically insoluble in water, sparingly soluble in anhydrous ethanol, slightly soluble in methylene chloride. Identification Apply one of the two following identifications: First identification: A, C. Second identification: B, C, D, E. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of dexamethasone RS. B. Dissolve 10.0 mg of the substance to be examined in ethanol R and dilute to 100.0 ml with the same solvent. Place 2.0 ml of this solution in a stoppered test tube, add 10.0 ml of phenylhydrazine-sulfuric acid solution R, mix and heat in a water-bath at 60 °C for 20 min. Cool immediately. The absorbance (Appendix 4.1) measured at the absorption maximum at 419 nm is not less than 0.4. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel F254. Mobile phase: Butanol saturated with water - toluene ether (5 : 10 : 85). Solvent mixture: Methanol - methylene chloride (1 : 9). 318

Test solution: Dissolve 10 mg of the substance to be examined in the solvent mixture and dilute to 10 ml with the solvent mixture. Reference solution (1): Dissolve 20 mg of dexamethasone RS in the solvent mixture and dilute to 20 ml with the solvent mixture. Reference solution (2): Dissolve 10 mg of betamethasone RS in reference solution (1) and dilute to 10 ml with reference solution (1). Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of two thirds of the plate. Allow the plate to dry in air and examine in ultraviolet light at 254 nm.The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (1). Spray with alcoholic solution of sulfuric acid R. Heat at 120 °C for 10 min or until the spots appear. Allow to cool. Examine in daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with the reference solution (2) shows 2 spots which may, however, not be completely separated. D. Add about 2 mg to 2 ml of sulfuric acid R and shake to dissolve. Within 5 min, a faint reddish-brown colour develops. Add this solution to 10 ml of water and mix; the colour is discharged. E. Mix about 5 mg with 45 mg of heavy magnesium oxide R and ignite in a crucible until an almost white residue is obtained (usually less than 5 min). Allow to cool, add 1 ml of water, 0.05 ml of phenolphthalein solution R1 and about 1 ml of dilute hydrochloric acid R to render the solution colourless. Filter. To a freshly prepared mixture of 0.1 ml of alizarin S solution R and 0.1 ml of zirconyl nitrate solution R, add 1.0 ml of the filtrate. Mix, allow to stand for 5 min and compare the colour of the solution with that of a blank prepared in the same manner. The test solution is yellow and the blank solution is red.

Specific optical rotation +86° to +92°, calculated with reference to the dried substance (Appendix 6.4). Dissolve 0.250 g in anhydrous ethanol R and dilute to 25.0 ml with the same solvent. Related substances Examine by liquid chromatography (Appendix 5.3). Carry out the test protected from light. Mobile phase A: Mix 250 ml of acetonitrile R with 700 ml of water and allow to equilibrate; dilute to 1000.0 ml with water and mix again. Mobile phase B: Acetonitrile.

VP V

DEXAMETHASONE TABLETS

Test solution: Dissolve 25.0 mg of the substance to be examined in 1.5 ml of acetonitrile R and add 5 ml of mobile phase A. Mix with the aid of an ultrasonic bath until complete dissolution, and dilute to 10.0 ml with mobile phase A. Reference solution (1): Dissolve 5 mg of dexamethasone for system suitability RS (containing impurities B, F and G) in 0.5 ml of acetonitrile R and add 1 ml of mobile phase A. Mix with the aid of an ultrasonic bath until complete dissolution and dilute to 2.0 ml with mobile phase A. Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with mobile phase A. Dilute 1.0 ml of this solution to 10.0 ml with mobile phase A. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 µm). Column temperature: 45 °C. Detector: A spectrophotometer set at 254 nm. Flow rate: 1.2 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 15

100

0

15 - 40

100 → 0

0 → 100

Identification of impurities: Use the chromatogram supplied with dexamethasone for system suitability RS and the chromatogram obtained with reference solution (1) to identify the peaks due to impurities B, F and G. Relative retention with reference to dexamethasone (retention time = about 15 min): impurity B = about 0.94; impurity F = about 1.5; impurity G = about 1.7. System suitability: In the chromatogramobtained with reference solution (1), peak-to-valley ratio (Hp/Hv) is at least 2.0, where Hp =height above the baseline of the peak due to impurity B and Hv = height above the baseline of the lowest point of the curve separating this peak from the peak due to dexamethasone. Limits: Impurity G: The area of the peak due to impurity G is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.3%). Impurities B, F: For each impurity, the area is not more than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.15%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.10%). The sum of the peak areas of all impurities is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.5%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%).

Note: Impurity A: 14-fluoro-11β,17,21-trihydroxy-16α-methylpregna1,4-diene-3,20-dione. Impurity B: 9-fluoro-11β,17,21-trihydroxy-16β-methylpregna1,4-diene-3,20-dione (betamethasone). Impurity C: 9-fluoro-11β,17,21-trihydroxy-16α-methylpregn-4ene-3,20-dione. Impurity D: 17,21-dihydroxy-16α-methyl-9β,11β-epoxypregna1,4-diene-3,20-dione. Impurity E: 17,21-dihydroxy-16α-methylpregna-1,4,9(11)triene-3,20-dione. Impurity F: 9-fluoro-11β,21-dihydroxy-16α-methylpregna-1,4diene-3,20-dione. Impurity G: 9-fluoro-11β,17-dihydroxy-16α-methyl-3,20dioxopregna-1,4-dien-21-yl acetate (dexamethasone acetate). Impurity H: 17-hydroxy-16α-methyl-3,20-dioxopregna-1,4,9 -(11)-trien-21-yl acetate.

Loss on drying Not more than 0.5% (Appendix 9.6). (0.500 g; 105 °C). Assay Dissolve 0.100 g of the substance to be examined in ethanol (96%) R and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml of this solution to 100.0 ml with ethanol (96%) R. Measure the absorbance (Appendix 4.1) at the absorption maximum at 238.5 nm. Calculate the content of C22H29FO5 taking the specific absorbance A (1%, 1 cm) to be 394. Storage Store in an airtight container, protected from light. Action and use Glucocorticoid. Preparations Tablets, suspension eye drops. DEXAMETHASONE TABLETS Tabellae Dexamethasoni Dexamethasone tablets contain dexamethasone. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of dexamethasone, C22H29FO5, 90.0% to 110.0% of the stated amount. Identification Mix a quantity of the powdered tablets containing 20 mg of dexamethasone with 5 ml of 0.1 M sodium hydroxide R, add 50 ml of dichloromethane R, sonicate for 20 min, filter and evaporate to dryness. Dry the residue at 105 °C for 2 h. 319

VP V

DEXAMETHASONE TABLETS

The infrared absorption spectrum of the obtained residue (Appendix 4.2) is concordant with the reference spectrum of dexamethasone.

Relative substances Examine by liquid chromatography (Appendix 5.3) Mobile phase A: A 15% (v/v) solution of acetonitrile R. Mobile phase B: Acetonitrile R. Test solution: Weigh accurately a quantity of the powdered tablets containing 2.5 mg of dexamethasone, add 10 ml of acetonitrile R, sonicate and filter. Dilute 4 ml of the filtrate to 10 ml with water. Reference solution (1): Dilute 1 ml of the test solution to 100 ml with mobile phase A. Reference solution (2): Dilute 1 ml of reference solution (1) to 20 ml with mobile phase A. Resolution solution: Dissolve 2 mg of dexamethasone RS and 2 mg of methylprednisolone RS in mobile phase A and dilute to 100 ml with the same solvent. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm), Hypersil ODS is suitable. Column temperature: 45 °C. Detector: A spectrophotometer set at 254 nm. Flow rate: 2.5 ml/min. Volume of injection: 20 µl. Procedure: Chromatograph the solutions with the gradient as follow:

15

Mobile phase A (% v/v) 100 100 → 0

Mobile phase B (% v/v) 0 0 → 100

40

0

100

End chromatogram and return to 100% of mobile phase A

41

100

0

Equilibration with mobile phase A

46 = 0

100

0

End equilibration and begin next chromatogram

Time (min) 0

Note Isocratic Linear gradient

Inject the resolution solution, the retention time of methylprednisolone is about 13 min, of dexamethasone is about 16 min, the test is not valid unless the resolution factor between the two peaks is at least 2.8. Adjust the ratio of acetonitrile in mobile phase A if necessary. Inject reference solution (1), reference solution (2), and the test solution. In the chromatogram obtained with the test solution, the area of any secondary peak is not greater than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%), the sum of 320

the areas of secondary peaks is not greater than that of the principal peak in the chromatogram obtained with reference solution (1) (1.0%). Disregard the peaks due to mobile phase A and any peak with an area less than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%).

Uniformity of content The tablets comply with the requirements stated under Uniformity of content (Appendix 11.2). The mobile phase and chromatographic system are described in the test for Assay. Test solution: Powder one tablet, add sufficient methanol (50%) v/v to obtain a solution containing 0.0025% of dexamethasone, shake for 10 min and filter. Reference solution: A solution containing 0.0025% of dexamethasone RS in methanol (50%) (v/v) R. Assay Examine by liquid chromatography (Appendix 5.3) Mobile phase: Methanol - water (47 : 53). Reference solution: A solution containing 0.0125% of dexamethasone RS in methanol (50%) (v/v) R. Test solution: Weigh 20 tablets, calculate the average mass and finely powder. Weigh accurately a quantity of the powdered tablets containing 2.5 mg of dexamethasone, add 20.0 ml of methanol (50%) (v/v) R, shake for 20 min and filter. Chromatographic system: A column (20 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 238 nm. Flow rate: 1.4 ml/min. Volume of injection: 20 µl. Procedure: Inject alternately the test solution and the reference solution. Calculate the content of dexamethasone, C22H29FO5, in tablets using the peak areas in the chromatograms obtained from the test solution, the reference solution and the declared content of C22H29FO5 in dexamethasone RS. Storage Store in a well-closed container in a cool, dry place, protected from light. Action and use Glucocorticoid. Usual strength 0.5 mg.

VP V

DEXAMETHASONE ACETATE

DEXAMETHASONE ACETATE Dexamethasoni acetas

C24H31FO6 

M. 434.5

Dexamethasone acetate is 9-fluoro-11β,17-dihydroxy16α-methyl-3,20-dioxopregna-1,4-dien-21-yl acetate. It contains not less than 97.0% and not more than 103.0% of C24H31FO6, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder, it shows polymorphism. Practically insoluble in water, freely soluble in ethanol (96%), slightly soluble in methylene chloride. Identification Apply one of the two following identifications: First identification: A, C. Second identification: B, C, D, E, F. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of dexamethasone acetate RS. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in methylene chloride R, evaporate to dryness and record new spectra using the residues. B. Dissolve 10.0 mg of the substance to be examined in ethanol R and dilute to 100.0 ml with the same solvent. Place 2.0 ml of this solution in a stoppered test tube, add 10.0 ml of phenylhydrazine-sulfuric acid solution R, mix and heat in a water-bath at 60 °C for 20 min. Cool immediately. The absorbance (Appendix 4.1) measured at the absorption maximum at 419 nm is not less than 0.35. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Add a mixture of 1.2 volumes of water and 8 volumes of methanol R to a mixture of 15 volumes of ether R and 77 volumes of methylene chloride R. Solvent mixture: Methanol - methylene chloride (1 : 9). Test solution: Dissolve 10 mg of the substance to be examined in the solvent mixture and dilute to 10 ml with the solvent mixture. Reference solution (1): Dissolve 20 mg of dexamethasone acetate RS in the solvent mixture and dilute to 20 ml with the solvent mixture. Reference solution (2): Dissolve 10 mg of cortisone acetate R in reference solution (1) and dilute to 10 ml with reference solution (1).

Procedure: Apply separately to the plate 5 µl of each solution. Develop over 3/4 of the plate. Allow the plate to dry in air and examine in ultraviolet light at 254 nm.The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (1). Spray with alcoholic solution of sulfuric acid R. Heat at 120 °C for 10 min or until the spots appear. Allow to cool. Examine in daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with the reference solution (2) shows 2 clearly separated spots. D. Add about 2 mg to 2 ml of sulfuric acid R and shake to dissolve. Within 5 min, a faint reddish-brown colour develops. Add this solution to 10 ml of water and mix; the colour is discharged and a clear solution remains. E. Mix about 5 mg with 45 mg of heavy magnesium oxide R and ignite in a crucible until an almost white residue is obtained (usually less than 5 min). Allow to cool, add 1 ml of water, 0.05 ml of phenolphthalein solution R1 and about 1 ml of dilute hydrochloric acid R to render the solution colourless. Filter. To a freshly prepared mixture of 0.1 ml of alizarin S solution R and 0.1 ml of zirconyl nitrate solution R, add 1.0 ml of the filtrate. Mix, allow to stand for 5 min and compare the colour of the solution with that of a blank prepared in the same manner. The test solution is yellow and the blank solution is red. F. About 10 mg gives the reaction of acetyl (Appendix 8.1).

Specific optical rotation +94° to +99°, calculated with reference to the dried substance (Appendix 6.4). Dissolve 0.250 g in ethanol R and dilute to 25.0 ml with the same solvent. Related substances Examine by liquid chromatography (Appendix 5.3). Carry out the test protected from light. Mobile phase: Mix 380 ml of acetonitrile R with 550 ml of water and allow to equilibrate; dilute to 1000.0 ml with water and mix again. Test solution: Dissolve 25 mg of the substance to be examined in about 4 ml of acetonitrile R and dilute to 10.0 ml with water R. Reference solution (1): Dissolve 2 mg of dexamethasone RS (impurity A) and 2 mg of betamethasone acetate RS (impurity D) in 100.0 ml of the mobile phase and sonicate for about 10 min (solution A). Mix 6.0 ml of the test solution and 1.0 ml of solution A and dilute to 10.0 ml with the mobile phase. 321

VP V

DEXAMETHASONE SODIUM PHOSPHATE

Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (3): Dissolve the contents of a vial of dexamethasone acetate impurity E RS in 1.0 ml of the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.2 ml/min. Volume of injection: 20 µl. Procedure: The run time is 2.5 times the retention time of dexamethasone acetate. Identification of impurities: Use the chromatogram obtained with reference solution (1) to identify the peaks due to impurities A and D; use the chromatogram obtained with reference solution (3) to identify the peak due to impurity E. Relative retention with reference to dexamethasone acetate (retention time = about 22 min): impurity A = about 0.4; impurity D = about 0.9; impurity E = about 1.2. System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to impurity D and dexamethasone acetate is at least 3.3. Limits: Impurity D: The area of the peak due to impurity D is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.3%). Impurities A, E: For each impurity, the area is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (2) (0.2%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.10%). The sum of the peak areas of all impurities is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.5%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Note: Impurity A: 9-fluoro-11β,17,21-trihydroxy-16α-methylpregna1,4-diene-3,20-dione (dexamethasone). Impurity B: 14-fluoro-11β,17-dihydroxy-16α-methyl-3,20dioxopregna-1,4-dien-21-yl acetate. Impurity C: 9-fluoro-11β,17β-dihydroxy-16α-methyl-3,20dioxopregna-1,4-dien-21-yl acetate. Impurity D: 9-fluoro-11β,17-dihydroxy-16β-methyl-3,20dioxopregna-1,4-dien-21-yl acetate (betamethasone acetate). Impurity E: 9-fluoro-11β,17-dihydroxy-16α-methyl-3,20dioxopregn-4-en-21-yl acetate. Impurity F: 17-hydroxy-16α-methyl-3,20-dioxo-9β,11β-epoxypregna-1,4-dien-21-yl acetate.

322

Impurity G: 9-fluoro-11β-hydroxy-16α-methyl-3,20-dioxopregna-1,4-dien-21-yl acetate. Impurity H: 17-hydroxy-16α-methyl-3,20-dioxopregna-1,4,9 (11)-trien-21-yl acetate.

Loss on drying Not more than 0.5% (Appendix 9.6). (0.500 g; in vacuo; 105 °C). Assay Dissolve 0.100 g of the substance to be examined in ethanol (96%) R and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml of this solution to 100.0 ml with ethanol (96%) R. Measure the absorbance (Appendix 4.1) at the absorption maximum at 238.5 nm. Calculate the content of C24H31FO6 taking the specific absorbance A (1%, 1 cm) to be 357. Storage Store in an airtight container, protected from light. Action and use Glucocorticoid. Preparation Suspension for injection. DEXAMETHASONE SODIUM PHOSPHATE Dexamethasoni natrii phosphas

C22H28FNa2O8P 

M. 516.4

Dexamethasone sodium phosphate is 9-fluoro-11β,17dihydroxy-16α-methyl-3,20-dioxopregna-1,4-dien-21-yl disodium phosphate. It contains not less than 97.0% and not more than 102.0% of C22H28FNa2O8P, calculated with reference to the anhydrous substance.

Characters White or almost white, very hygroscopic powder, it shows polymorphism. Freely soluble in water, slightly soluble in ethanol (96%), practically insoluble in methylene chloride. Identification Apply one of the two following identifications: First identification: A, G. Second identification: B, C, D, E, F. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of dexamethasone sodium phosphate RS.

VP V

If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in the minimum volume of ethanol (96%) R, evaporate to dryness on a water-bath and record new spectra using the residues. B. Dissolve 10.0 mg in 5ml of water and dilute to 100.0 ml with ethanol R. Place 2.0 ml of this solution in a stoppered test tube, add 10.0 ml of phenylhydrazine-sulfuric acid solution R, mix and heat in a water-bath at 60 °C for 20 min. Cool immediately. The absorbance (Appendix 4.1) measured at the absorption maximum at 419 nm is not less than 0.20. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Glacial acetic acid - water - butanol (2 : 2 : 6). Test solution: Dissolve 10 mg of the substance to be examined in ethanol R and dilute to 10 ml with the same solvent. Reference solution (1): Dissolve 20 mg of dexamethasone sodium phosphate RS in ethanol R and dilute to 20 ml with the same solvent. Reference solution (2): Dissolve 10 mg of prednisolone sodium phosphate RS in reference solution (1) and dilute to 10 ml with reference solution (1). Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of three fourths of the plate. Allow the plate to dry in air and examine in ultraviolet light at 254 nm.The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (1). Spray with alcoholic solution of sulfuric acid R. Heat at 120 °C for 10 min or until the spots appear. Allow to cool. Examine in daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with the reference solution (2) shows 2 spots which may, however, not be completely separated. D. Add about 2 mg of the substance to be examined to 2 ml of sulfuric acid R and shake to dissolve. Within 5 min, a faint yellowish-brown colour develops. Add this solution to 10 ml of water and mix; the colour fades and a clear solution renains. E. Mix about 5 mg of the substance to be examined with 45 mg of heavy magnesium oxide R and ignite in a crucible until an almost white residue is obtained (usually less than 5 min). Allow to cool, add 1 ml of water, 0.05 ml of phenolphthalein solution R1 and about 1 ml of dilute hydrochloric acid R to render the solution colourless. Filter. To a freshly prepared mixture of 0.1 ml of alizarin S solution R and 0.1 ml of zirconyl nitrate solution R, add 1.0 ml of the filtrate. Mix, allow to stand for 5 min and compare the colour of the solution with that of a blank

DEXAMETHASONE SODIUM PHOSPHATE

prepared in the same manner. The test solution is yellow and the blank solution is red. F. To 40 mg of the substance to be examined add 2 ml of sulfuric acid R and heat gently until white fumes are evolved, add nitric acid R dropwise, continue the heating until the solution is almost colourless and cool. Add 2 ml of water, heat until white fumes are again evolved, cool, add 10 ml of water and neutralise to red litmus paper R with dilute ammonia R. The solution gives reaction (A) of sodium and reaction (B) of phosphates (Appendix 8.1). G. In the test for Assay, the principal peak in the chromatogram obtained with the test solution is similar in retention time and size to the principal peak in the chromatogram obtained with reference solution (2).

Appearance of solution Solution S: Dissolve 1.0 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution B7 (Appendix 9.3, method 2). pH 7.5 to 9.5 (Appendix 6.2). Dilute 1 ml of solution S to 5 ml with carbon dioxide-free water R. Specific optical rotation +75° to +83°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.250 g of the substance to be examined in water and dilute to 25.0 ml with the same solvent. Related substances Examine by liquid chromatography (Appendix 5.3). Carry out the test protected from light. Solution A: Dissolve 7.0 g of ammonium acetate R in 1000 ml of water. Mobile phase A: Mix 300 ml of solution A and 350 ml of water, adjust to pH 3.8 with acetic acid R, then add 350 ml of methanol R. Mobile phase B: Adjust 300 ml of solution A to pH 4.0 with acetic acid R, then add 700 ml of methanol R. Test solution: Dissolve 10 mg of the substance to be examined in mobile phase A and dilute to 10.0 ml with mobile phase A. Reference solution (1): Dissolve 2 mg of betamethasone sodium phosphate RS (impurity B) and 2 mg of dexamethasone sodium phosphate RS in mobile phase A, then dilute to 100.0 ml with mobile phase A. Reference solution (2): Dissolve 2 mg of dexamethasone sodium phosphate for peak identification RS (containing impurities A, C, D, E, F and G) in mobile phase A and dilute to 2.0 ml with mobile phase A. Reference solution (3): Dilute 1.0 ml of the test solution to 100.0 ml with mobile phase A. Dilute 1.0 ml of this solution to 10.0 ml with mobile phase A. 323

VP V

DEXAMETHASONE SODIUM PHOSPHATE

Chromatographic system: A column (12.5 cm × 4.6 mm) packed with stationary phase end-capped octylsilyl silica gel for chromatography R (5 µm). Column temperature: 30 °C. Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 3.5

90

10

3.5 - 23.5

90 → 60

10 → 40

23.5 - 34.5

60 → 5

40 → 95

34.5 - 50

5

95

Identification of impurities: Use the chromatogram supplied with dexamethasone sodium phosphate for peak identification RS and the chromatogram obtained with reference solution (2) to identify the peaks due to impurities A, C, D, E, F and G; use the chromatogram obtained with reference solution (1) to identify the peak due to impurity B. Relative retention with reference to dexamethasone sodium phosphate (retention time = about 22 min): impurity C = about 0.5; impurity D = about 0.6; impurity E = about 0.8; impurity F = about 0.92; impurity B = about 0.95; impurity A = about 1.37; impurity G = about 1.41. System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to impurity B and dexamethasone sodium phosphate is at least 2.0. Limits: Correction factor: for the calculation of content, multiply the peak area of impurity A by 0.75. Impurity A: The corrected area of the peak due to impurity A is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (3) (0.5%). Impurity G: The area of the peak due to impurity G is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (3) (0.3%). Impurities B, C, D, E, F: For each impurity, the area is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (3) (0.2%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (3) (0.10%). The sum of the peak areas of all impurities is not more than 10 times the area of the principal peak in the chromatogram obtained with reference solution (3) (1.0%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (3) (0.05%). 324

Note: Impurity A: 9-fluoro-11β,17,21-trihydroxy-16α-methylpregna1,4-diene-3,20-dione (dexamethasone), Impurity B: 9-fluoro-11β,17-dihydroxy-16β -methyl-3,20-dioxopregna-1,4-dien-21-yl dihydrogen phosphate (betamethasone phosphate). Impurity C. D, E, F: For each impurity, one or more diastereoisomer(s) of (9-fluoro-11β, 17α-dihydroxy-16-methyl-3,17-dioxo-D-homoandrosta-1,4-dien-17α-yl)methyl dihydrogen phosphate (undefined stereochemistry at C-16 and C-17a), or (9- fluoro-11β,17-dihydroxy16a-methyl-3,17α-dioxo-D-homo-androsta-1,4-dien-17-yl)methyl dihydrogen phosphate (undefined stereochemistry at C-17). Impurity G:. 9-fluoro-11β,17-dihydroxy-16α-methyl-3-oxoandrosta1,4-diene-17β -carboxylic acid. Impurity H: 9-fluoro-11β,17-dihydroxy-16α-methyl-3,20-dioxopregn-4-en-21-yl dihydrogen phosphate.

Inorganic phosphates

Not more than 1%. Dissolve 50 mg of the substance to be examined in water and dilute to 100 ml with the same solvent. To 10 ml of this solution add 5 ml of molybdovanadic reagent R, mix and allow to stand for 5 min. Any yellow colour in the solution is not more intense than that in a standard prepared at the same time in the same manner using 10 ml of phosphate standard solution (5 ppm PO4) R.

Ethanol Not more than 3.0% m/m. Examine by gas chromatography (Appendix 5.2). Internal standard solution: Dilute 1.0 ml of propanol R to 100.0 ml with water. Test solution: Dissolve 0.50 g of the substance to be examined in 5.0 ml of the internal standard solution and dilute to 10.0 ml with water. Reference solution: Dilute 1.0 g of anhydrous ethanol R to 100.0 ml with water. To 2.0 ml of this solution add 5.0 ml of the internal standard solution and dilute to 10.0 ml with water. Chromatographic system: A column (1 m × 3.2 mm) packed with ethylvinylbenzenedivinylbenzene copolymer (150 µm to 180 µm). Carrier gas: Nitrogen for chromatography. Flow rate: 30 ml/min. Detector: A frame-ionisation detector. Temperature: column: 150 °C; injection port: 250 °C; detector: 280 °C. Volume of injection: 2 µl. Ethanol and water Water content (Appendix 10.3). Determined on 0.200 g. The percentage content of water and the percentage content of ethanol obtained in the test for ethanol is not more than 13.0% m/m for the sum of the percentage contents.

VP V

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Mix 520 ml of water with 2 ml of phosphoric acid R. Adjust the temperature to 20 °C, then adjust to pH 2.6 with sodium hydroxide R. Mix this solution with 36 ml of tetrahydrofuran R and 364 ml of methanol R. Test solution: Dissolve 30.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Dilute 5.0 ml of the solution to 50.0 ml with the mobile phase. Standard solution (1): Dissolve 2 mg of dexamethasone RS (impurity A) and 2 mg of dexamethasone sodium phosphate RS in 2 ml of tetrahydrofuran R, then dilute to 100.0 ml with the mobile phase. Dilute 5.0 ml of this solution to 50.0 ml with the mobile phase. Standard solution (2): Dissolve 30.0 mg of dexamethasone sodium phosphate RS in the mobile phase and dilute to 50.0 ml with the mobile phase. Dilute 5.0 ml of the solution to 50.0 ml with the mobile phase. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (7 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: The run time is 3 times the retention time of dexamethasone sodium phosphate. Identification of impurities: Use the chromatogram obtained with standard solution (1) to identify the peak due to impurity A. Relative retention with reference to dexamethasone sodium phosphate (retention time = about 8 min): impurity A = about 2.0. System suitability: In the chromatogram obtained with standard solution (1), the resolution between the peaks due to dexamethasone sodium phosphate and impurity A is at least 6.0. Calculate the content of C22H28FNa2O8P, using the areas of the peaks in the chromatograms obtained with the test solution, the standard solution (2) and the declared content of C22H28FNa2O8P in dexamethasone sodium phosphate RS. Storage In an airtight container, protected from light. Action and use Glucocorticoid. Preparations Injection, eye drops.

DEXAMETHASONE INJECTION

DEXAMETHASONE INJECTION Injectio Dexamethasoni Dexamethasone injection is a sterile solution of dexamethasone sodium phosphate in water for injections. It may contain stabilisers. It complies with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Content of dexamethasone phosphate, C22H30FO8P, 90.0% to 110.0% of the stated amount. Characters A clear, colourless solution. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel F254. Mobile phase: Butanol - glacial acetic acid - water (60 : 20 : 20) Test solution: Dilute, if necessary, a volume of the injection to obtain a 0.1% solution of dexamethasone phosphate in methanol R. Reference solution (1): A 0.1% solution of dexamethasone phosphate in methanol R. Reference solution (2): A solution contains 0.1% each of dexamethasone phosphate and prednisolone sodium phosphate in methanol R. Procedure: Apply separately to the plate 5 µl of each solution. After removal of the plate, allow it to dry in air, heat at 110 °C for 10 min, spray the hot plate with ethanolic sulphuric acid (20%) R and heat at 120 °C for 10 min. Cool and examine in daylight and under ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to that in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) shows two spots which may, however, not be completely separated. B. In the Assay, the chromatogram obtained with the test solution shows a peak corresponding in retention time to the principal peak in the chromatogram obtained with the reference solution. pH 7.0 to 8.5 (Appendix 6.2). Endotoxine Not more than 31.3 EU/mg of dexamethasone phosphate (Appendix 13.2). Assay Examine by liquid chromatography (Appendix 5.3). 325

VP V

DEXCHLORPHENIRAMINE MALEATE

Mobile phase: A 0.01 M solution of potassium dihydrogen phosphate R in a mixture of equal volumes of methanol R and water. Test solution: Dilute a volume of the injection containing the equivalent of 8 mg of dexamethasone phosphate to 100.0 ml with the mobile phase. Reference solution: Solution of dexamethasone sodium phosphate RS in the mobile phase containing the equivalent of 80 µg of dexamethasone phosphate per ml (prepared just before use). Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Lichrosorb RP 18 is suitable. Detector: A spectrophotometer set at 254 nm. Flow rate: 1.6 ml/min. Volume of injection: 20 µl. Procedure: System suitability: The relative standard deviation of the peak areas for 6 replicate injections of the reference solution is not greater than 2.0%. Inject separately the test solution and the reference solution. Calculate the concentration of dexamethasone phosphate (mg/ml) in the preparation using the peak areas of dexamethasone sodium phosphate in the chromatograms obtained with the test solution, reference solution and the content of dexamethasone phosphate, C22H30FO8P, in dexamethasone sodium phosphate RS. Taking 472.45/516.41 as the factor to convert dexamethasone sodium phosphate into dexamethasone phosphate.

Storage In a cool place, protected from light. Action and use Glucocorticoid. Usual strength 4mg/2ml and 4 mg/1ml (calculated as dexamethasone phosphate). DEXCHLORPHENIRAMINE MALEATE Dexchlorpheniramini maleas

Characters A white crystalline powder. Very soluble in water, freely soluble in ethanol (96%), in methanol and in methylene chloride. Identification Apply one of the two following identifications: First identification: A, C, E. Second identification: B, C, D, E. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum obtained with dexchlorpheniramine maleate RS. B. Melting point: 110 °C to 115 °C (Appendix 6.7). C. It complies with the test for Specific optical rotation. D. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Water - anhydrous formic acid - methanol diisopropyl ether (3 : 7 : 20 : 70). Test solution: Dissolve 0.10 g of the substance to be examined in methanol R and dilute to 5.0 ml with the same solvent. Reference solution: Dissolve 56 mg of maleic acid R in methanol R and dilute to 10.0 ml with the same solvent. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 12 cm. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The chromatogram obtained with the test solution shows two clearly separated spots. The upper spot is similar in position and size to the spot in the chromatogram obtained with the reference solution. E. To 0.15 g in a porcelain crucible add 0.5 g of anhydrous sodium carbonate R. Heat over an open flame for about 10 min. Allow to cool. Dissolve the residue in 10 ml of dilute nitric acid R, filter. To 1 ml of the filtrate add 1 ml of water. The solution gives reaction (A) of chlorides (Appendix 8.1). Appearance of solution Solution S: Dissolve 2.0 g in water and dilute to 20.0 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution BY6 (Appendix 9.3, method 2). pH Dissolve 0.20 g in 20 ml of water. The pH of the solution is 4.5 to 5.5 (Appendix 6.2).

C16H19ClN2,C4H4O4

M. 390.9

Dexchlorpheniramine maleate is (3S)-3-(4-chlorophenyl)N,N-dimethyl-3-(pyridin-2-yl)propan-1-amine(Z)butanedioate. It contains not less than 98.0% and not more than 100.5% of C16H19ClN2,C4H4O4, calculated with reference to the dried substance. 326

Specific optical rotation +22° to +23°, calculated with reference to the dried substance (Appendix 6.4). Determined on solution S. Related substances Examine by gas chromatography (Appendix 5.2). Test solution: Dissolve 10.0 mg in 1.0 ml of methylene chloride R.

VP V

Reference solution: Dissolve 5.0 mg of brompheniramine maleate RS in 0.5 ml of methylene chloride R and add 0.5 ml of the test solution. Dilute 0.5 ml of this solution to 50.0 ml with methylene chloride R. Chromatographic system: A glass column (2.3 m × 2 mm) packed with acid- and base-washed silanised diatomaceous earth for gas chromatography R (135 µm to 175 µm) impregnated with 3% (m/m) of a mixture of 50% of poly(dimethyl)siloxane and 50% of poly(diphenyl)siloxane R. Carrier gas: Nitrogen for chromatography R. Flow rate: 20 ml per min. Detector: A flame-ionisation detector. Maintaining the temperature of the column at 205 °C and that of the injection port and of the detector at 250 °C. Volume of injection: 1 µl. Procedure: Inject the reference solution. The test is not valid unless, in the chromatogram obtained with the reference solution, the resolution between the two peaks corresponding to dexchlorpheniramine maleate and brompheniramine maleate is at least 1.5. Inject the test solution. Continue the chromatography for at least 2.5 times the retention time of the principal peak. Limits: In the chromatogram obtained with the test solution: none of the peaks, apart from the principal peak, has an area greater than 0.8 times the area of the peak corresponding to dexchlorpheniramine maleate in the chromatogram obtained with the reference solution (0.4%); the sum of the areas of all the peaks, apart from the principal peak, is not greater than twice the area of the peak corresponding to dexchlorpheniramine maleate in the chromatogram obtained with the reference solution (1%).

Enantiomeric purity Examine by liquid chromatography (Appendix 5.3). Mobile phase: Diethylamine - isopropanol - hexane (3 : 20 : 980). Test solution: Dissolve 10.0 mg in 3 ml of water. Add a few drops of concentrated ammonia R until an alkaline reaction is produced. Shake with 5 ml of methylene chloride R. Separate the layers. Evaporate the lower, methylene chloride layer to an oily residue on a water bath. Dissolve the oily residue in isopropanol R and dilute to 10.0 ml with the same solvent. Reference solution (1): Dissolve 10.0 mg of dexchlorpheniramine maleate RS in 3 ml of water and continue the procedure in the same manner as the test solution. Reference solution (2): Dissolve 10.0 mg of chlorpheniramine maleate RS in 3 ml of water and continue the procedure in the same manner as the test solution. Reference solution (3): Dilute 1.0 ml of the test solution to 50 ml with isopropanol R. Chromatographic system: A column (0.25 m × 4.6 mm) packed with amylose derivative of silica gel for chromatography R.

DEXCHLORPHENIRAMINE TABLETS

Detector: A spectrophotometer set at 254 nm. Flow rate: 1 ml/min. Volume of injection: 10 µl. Procedure: Under these conditions, the peak of the (S)-isomer appears first. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks corresponding to the (R)-enantiomer and to the (S)-enantiomer is at least 1.5 The retention times of the principal peaks in the chromatograms obtained with the test solution and reference solution (1) are identical ((S)-enantiomer). Limits: In the chromatogram obtained with the test solution, the area of the peak corresponding to the (R)-enantiomer is not greater than the area of the principal peak in the chromatogram obtained with reference solution (3) (2%) and the area of any peak, apart from the principal peak and any peak corresponding to the (R)-enantiomer, is not greater than 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (3) (0.5%).

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 65 °C; 4h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.150 g in 25 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS. Determine the endpoint potentiometrically (Appendix 10.2). Each ml of 0.1 N perchloric acid VS is equivalent to 19.54 mg of C16H19ClN2,C4H4O4. Storage Store in an airtight container, protected from light. Action and use Anti-histamine. DEXCHLORPHENIRAMINE TABLETS Tabellae Dexchlorpheniramini Dexdexchlorpheniramine tablets contain dexchlorpheniramine maleate. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements. 327

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DEXCHLORPHENIRAMINE TABLETS

Content of dexchlorpheniramine maleate, C16H19ClN2, C4H4O4, 90.0% to 110.0% of the stated amount. Identification Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: 1 M acetic acid - methanol - ethyl acetate (20 : 30 : 50). Test solution: Shake a quantity of the powdered tablets containing 5 mg of dexchlorphenamine maleate with chloroform R, filter, evaporate to dryness and dissolve the residue in 1 ml of chloroform R. Reference solution: A0.5% solution of dexchlorpheniramine maleate RS in chloroform R. Procedure: Apply separately to the plate 2 µl of each solution. After removal of the plate, allow it to dry in air and examine under ultraviolet light at 254 nm. The two principal spots in the chromatogram obtained with the test solution are similar in position and colour to those in the chromatogram obtained with the reference solution. Spray the plate with potassium iodobismuthate solution R. The principal spot in the chromatogram obtained with the test solution is similar in position and colour to that in the chromatogram obtained with the reference solution. B. Shake a quantity of finely powdered tablets, equivalent to about 150 mg of dexchlorpheniramine maleate, with 100 ml of 1 M acetic acid R for 10 min, filter through a sintered-glass funnel into a suitable vessel, adjust the filtrate with 10% solution of sodium hydroxide R to pH 11, and extract the solution with six 100-ml portions of hexane R. Evaporate the combined extracts on a water-bath, transfer the residue, with the aid of dimethylformamide R, to a suitable glass-stoppered graduated cylinder, dilute with dimethylformamide R to 15.0 ml, mix, and centrifuge if necessary: the optical rotation of the solution (Appendix 6.4) obtained, in a 100-mm tube, using dimethylformamide R as the blank, is between +0.24° and +0.35° (distinction from chlorpheniramine maleate). Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Diethylamine - chloroform - cyclohexane (10 : 40 : 50). Test solution: Shake a quantity of the powdered tablets containing 50 mg of dexchlorpheniramine maleate with chloroform R, filter, evaporate to dryness and dissolve the residue in 1 ml of chloroform R. Reference solution: Dilute 1 volume of the test solution to 500 volumes with chloroform R. Procedure: Apply separately to the plate 10 µl of each solution. After developing over a path of 12 cm, remove the plate, allow it to dry in air and examine under ultraviolet 328

light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution (0.2%). Disregard any spot remaining on the line of application.

Assay Ultraviolet absorption spectrophotometry (Appendix 4.1) Reference solution: Transfer about 40 mg of dexchlorpheniramine maleate RS, accurately weighed, to a 100-ml volumetric flask, add water to volume, and mix. Transfer 10.0 ml of this solution to a separator, adjust with 1 M sodium hydroxide R to pH 11, and cool to room temperature. Extract with two 50-ml portions of hexane R, shaking each portion for 2 min before separating the phases, and combining the hexane extracts in a second separator. Extract the hexane solution with two 40-ml portions of 0.1 M hydrochloric acid R, combine the acid extracts in a 100-ml volumetric flask, add 0.1 M hydrochloric acid R to volume, and mix. Filter, discarding the first 10 ml of the filtrate. The concentration of dexchlorpheniramine maleate RS is about 40 µg/ml. Test solution: Weigh 20 tablets, calculate the average mass and finely powder. Weigh accurately a quantity of the powdered tablets equivalent to about 8 mg of dexchlorpheniramine maleate, to a 250-ml separator, mix with 50 ml of water for 10 min, adjust with 10% sodium hydroxide solution R to pH 11, and cool to room temperature. Extract the mixture with two 75-ml portions of hexane R, and combine the extracts in a second separator. Extract the solvent hexane solution with three 50-ml portions of 0.1 M hydrochloric acid R, combining the acid extracts in a 200-ml volumetric flask. Add 0.1 M hydrochloric acid R to volume, and mix. Filter the solution, discard the first 10 ml of the filtrate. Measure the absorbance of the reference solution and the test solution at the maximum at 264 nm (Appendix 4.1), using 0.1 M hydrochloric acid R as the blank. Calculate the content of dexchlorpheniramine maleate, C16H19ClN2,C4H4O4 using the absorbances of the reference solution and the test solution and the declared content of C16H19ClN2,C4H4O4 in dexchlorpheniramine maleate RS. Storage Protected from light. Action and use Histamine H1-receptor antagonist. Usual strength 2 mg.

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DEXPANTHENOL

DEXPANTHENOL Dexpanthenolum

C9H19NO4

M. 205.3

Dexpanthenol is (2R)-2,4-dihydroxy-N-(3-hydroxypropyl)-3,3-dimethylbutanamide. It contains not less than 98.0% and not more than 101.0% of C9H19NO4, calculated with reference to the anhydrous substance.

Characters A colourless or slightly yellowish, viscous hygroscopic liquid, or a white, crystalline powder. Very soluble in water, freely soluble in ethanol (96%). Identification Apply one of the two following identifications: First identification: B, C, D. Second identification: A, B. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum obtained with dexpanthenol RS. Examine the substances using discs prepared as follows: prepare separately a 0.5% solution of the substance to be examined and the reference substance in ethanol R. Place dropwise 0.5 ml of this solution on a disc of potassium bromide R. Dry the disc at 100 °C to 105 °C for 15 min. B. It complies with the test for Specific optical rotation. C. In the test for 3-Aminopropanol, the principal spot in the chromatogram obtained with test solution (2) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). D. To 1 ml of solution S add 1 ml of dilute sodium hydroxide solution R and 0.1 ml of 12.5% solution of copper sulphate R. A blue colour develops. Appearance of solution Solution S: Dissolve 2.500 g in carbon dioxide-free water R and dilute to 50.0 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution B6 (Appendix 9.3, method 2). pH The pH of solution S is not greater than 10.5 (Appendix 6.2). Specific optical rotation +29.0° to +32.0°, calculated with reference to the anhydrous substance (Appendix 6.4). Determined on solution S.

3-Aminopropanol Not more than 0.5%. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Concentrated ammonia - methanol - butanol (20 : 25 : 55). Test solution (1): Dissolve 0.25 g of the substance to be examined in ethanol R and dilute to 5 ml with the same solvent. Test solution (2): Dilute 1 ml of test solution (1) to 10 ml with ethanol R. Reference solution (1): Dissolve 50 mg of dexpanthenol RS in ethanol R and dilute to 10 ml with the same solvent. Reference solution (2): Dissolve 25 mg of 3-aminopropanol R in ethanol R and dilute to 100 ml with the same solvent. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in air, spray with a 10% solution of trichloroacetic acid in methanol R and heat at 150 °C for 10 min. Spray with a 0.1% solution of ninhydrin in methanol R and heat at 120 °C until a colour appears. Any spot corresponding to 3-aminopropanol in the chromatogram obtained with test solution (1) is not more intense than the spot in the chromatogram obtained with reference solution (2). Heavy metals Not more than 20 ppm (Appendix 9.4.8). 12 ml of solution S complies with limit test for heavy metals, method 1. Prepare the standard using lead standard solution (1 ppm Pb) R. Water Not more than 1.0% (Appendix 10.3). Determined on 1.000 g. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay To 0.400 g add 50.0 ml of 0.1 N perchloric acid VS. Boil under a reflux condenser for 5 h, protected from humidity. Allow to cool. Add 50 ml of dioxan R by rinsing the condenser, protected from humidity. Add 0.2 ml of naphtholbenzein solution R and titrate with 0.1 M potassium hydrogen phthalate VS until the colour changes from green to yellow. Carry out a blank titration. Each ml of 0.1 N perchloric acid VS is equivalent to 20.53 mg of C9H19NO4. Storage Store in an airtight container. Action and use Similar to vitamin B5. 329

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DEXPANTHENOL TABLETS

DEXPANTHENOL TABLETS Tabellae Dexpanthenoli Dexpanthenol tablets contain dexpanthenol. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of dexpanthenol, C9H19NO4, 95.0% to 105.0% of the stated amount. Identification A. Add 1 ml of ethanol R to a quantity of the powdered tablets containing 5 mg of dexpanthenol, filter and place dropwise 0.5 ml of this solution on a disc of potassium bromide used for IR R while blowing a current of warm air to evaporate ethanol to produce a thin layer. The infrared absorption spectrum of the layer (Appendix 4.2) is concordant with the reference spectrum of dexpanthenol. B. Dissolve quantity of the powdered tablets containing 2.5 g of dexpanthenol in 50 ml of carbon dioxide-free water R. To 1 ml filtrate, add 1 ml of dilute sodium hydroxide solution R, 0.1 ml of a 12.5% solution of copper sulfate R, a blue colour develops. C. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is the same as that of dexpanthenol peak in the chromatogram obtained with the reference solution. Assay Examine by liquid chromatography (Appendix 5.3) Mobile phase: Solution A - methanol (60 : 40). Solution A: Add 1 ml of phosphoric acid R to 1000 ml of water, mix. Test solution: Weigh 20 tablets, calculate the average mass and finely powder. Weigh accurately a quantity of the powder containing 0.1 g of dexpanthenol in a 100 ml volumetric flask, add 60 ml of the mobile phase, shake well, add the mobile phase to volume and mix. Filter, discarding the first 20 ml of the filtrate. Dilute 10.0 ml filtrate to 50.0 ml with the mobile phase. Reference solution: A solution contains 0.2 mg of dexpanthenol RS in 1 ml of the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 or 10 µm). Detector: A spectrophotometer set at 215 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: System suitability: The relative standard deviation of the peak areas for 6 replicate injections of the reference solution is not greater than 2%. Inject the test solution and the reference solution. 330

Calculate the content of dexpanthenol, C9H19NO4, in each tablet using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C9H19NO4 in dexpanthenol RS.

Storage Store in a well-closed container. Action and use Analogue of vitamin B5. Usual strength 100 mg. DEXTROMETHORPHAN HYDROBROMIDE Dextromethorphani hydrobromidum

, HBr , H2O

C18H25NO,HBr,H2O 

M. 370.3 

Dextromethorphan hydrobromide is ent-3-methoxy17-methylmorphinan hydrobromide monohydrate. It contains not less than 99.0% and not more than 101.0% of C18H25NO,HBr, calculated with reference to the anhydrous substance.

Characters Almost white, crystalline powder. Sparingly soluble in water, freely soluble in ethanol (96%). Melting point: About 125 °C, with decomposition. Identification Apply one of the two following identifications: First identification: A, C, D. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of dextromethorphan hydrobromide RS. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Ammonia - methylene chloride - methanol ethyl acetate - toluene (2 : 10 : 13 : 20 : 55). Test solution: Dissolve 25 mg of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Reference solution: Dissolve 25 mg of dextromethorphan hydrobromide RS in methanol R and dilute to 10 ml with the same solvent. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of two thirds of the plate.

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Allow the plate to dry in air and spray with potassium iodobismuthate solution R. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. D. It gives reaction (A) of bromides (Appendix 8.1).

Appearance of solution Solution S: Dissolve 1.0 g in ethanol (96%) R and dilute to 20 ml with the same solvent. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). Acidity or alkalinity Dissolve 0.4 g in carbon dioxide-free water R with gentle heating, cool and dilute to 20 ml with the same solvent. Add 0.1 ml of methyl red solution R and 0.2 ml of 0.01 N sodium hydroxide VS. The solution is yellow. Not more than 0.4 ml of 0.01 N hydrochloric acid VS is required to change the colour of the indicator to red. Specific optical rotation +28° to +30°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.200 g in 0.1 M hydrochloric acid R and dilute to 10.0 ml with the same solvent. Related substances Examine by liquid chromatography (Appendix 5.3) Mobile phase: Dissolve 3.11 g of docusate sodium R in a mixture of 400 ml of water and 600 ml of acetonitrile R, add 0.56 g of ammonium nitrate R and adjust to apparent pH 2.0 with glacial acetic acid R. Test solution: Dissolve 10.0 mg of the substance to be examined in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (1): Dissolve 2 mg of dextromethorphan impurity A RS in 2 ml of the test solution and dilute to 25.0 ml with the mobile phase. Reference solution (2): Dilute 1.0 ml of the test solution to 200.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 280 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: The run time is twice the retention time of dextromethorphan. Relative retention with reference to dextromethorphan (retention time = about 22 min): impurity B = about 0.4; impurity C = about 0.8; impurity D = about 0.9; impurity A = about 1.1. System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to dextromethorphan and impurity A is at least 1.5.

DEXTROMETHORPHAN HYDROBROMIDE

Limits: Correction factor: for the calculation of content, multiply the peak area of impurity C by 0.2; Impurities A, B, C, D: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.5%) and not more than 1 such peak has an area greater than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.25%). Any other impurity: For each impurity, the area is not more than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.10%). The sum of the peak areas of all impurities is not more than 2 times the area of the principal peak in the chromatogram obtained with reference solution (2) (1.0%). Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Note: Impurity A: ent-3-methoxymorphinan. Impurity B: ent-17-methylmorphinan-3-ol. Impurity C: ent-3-methoxy-17-methylmorphinan-10-one. Impurity D: ent-(14S)-3-methoxy-17-methylmorphinan.

N,N-Dimethylaniline Not more than 10 ppm. Dissolve 0.5 g with heating in 20 ml of water. Allow to cool, add 2 ml of dilute acetic acid R and 1 ml of a 1% solution of sodium nitrite R and dilute to 25 ml with water. The solution is not more intensely coloured than a reference solution prepared at the same time and in the same manner using 20 ml of a 0.25 mg/L solution of dimethylaniline R. Water 4.0% to 5.5% (Appendix 10.3). Determined on 0.200 g. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.300 g of the substance to be examined in a mixture of 5.0 ml of 0.01 N hydrochloric acid VS and 20 ml of ethanol (96%) R. Titrate with 0.1 N sodium hydroxide VS, determining the end- point potentiometrically (Appendix 10.2). Read the volume added between the 2 points of inflexion. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 35.23 mg of C18H25NO,HBr. Storage Store in an airtight container, protected from light. Action and use Cough suppressant. 331

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DEXTROMETHORPHAN HYDROBROMIDE TABLETS

Preparations Tablets, siro, oral solution. DEXTROMETHORPHAN HYDROBROMIDE TABLETS Tabellae Dextromethorphani hydrobromidi Dextromethorphan hydrobromide tablets contain dextromethorphan hydrobromide. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of dextromethorphan hydrobromide, C18H25NO,HBr, 90.0% to 110.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Ammonia - methylene chloride - methanol ethyl acetate - toluene (2 : 10 : 13 : 20 : 55). Test solution: Shake a quantity of the powdered tablets containing 15 mg of dextromethorphan hydrobromide with 5 ml of methanol R, centrifuge. Reference solution: A 0.3% solution of dextromethorphan hydrobromide RS in methanol R. Procedure: Apply separately to the plate 5 µl of each solution. After removal of the plate, allow it to dry in air. Spray the plate with potassium iodobismuthate solution R. The principal spot in the chromatogram obtained with the test solution is similar in position and colour to the principal spot in the chromatogram obtained with the reference solution. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is the same as that of the principal peak in the chromatogram obtained with the reference solution. C. Shake a quantity of the powdered tablets containing 20 mg of dextromethorphan hydrobromide with 5 ml of water, centrifuge. To the clear solution, add 5 drops of 10% nitric acid solution R and 1 ml of 2% solution of silver nitrate R, a pale yellow precipitate is formed. Filter, wash the precipitate with three quantities, each of 1 ml, of water. Suspend the precipitate obtained in 2 ml of water and add 1.5 ml of 10 M ammonia R. The precipitate dissolves with difficulty. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: A solution containing 0.007 M docusate sodium R and 0.007 M ammonium nitrate R in a mixture of acetonitrile - water (70 : 30). Adjust to apparent pH 3.4 with glacial acetic acid R. Test solution: Weigh 20 tablets, calculate the average mass and finely powder. Weigh accurately a quantity of the 332

powdered tablets containing 10 mg of dextromethorphan hydrobromide in a 100 ml volumetric flask, dissolve with the mobile phase and add the mobile phase to volume, mix and filter. Reference solution: A 0.01% solution of dextromethorphan hydrobromide RS in the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 280 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution, the relative standard deviation of the peak areas for 6 replicate injections is not greater than 2.0%. The symmetry factor for the dextromethorphan peak is not more than 2.5. Inject the test solution and the reference solution. Calculate the content of dextromethorphan hydrobromide, C18H25NO,HBr in each tablet using the peak areas obtained with the test solution and the reference solution and the declared content of C18H25NO,HBr in dextromethorphan hydrobromide RS.

Storage Store in a well-closed container, protected from light. Action and use Cough suppressant. Usual strength 5 mg, 10 mg, 15 mg. DIAZEPAM Diazepamum

C16H13ClN2O  

M. 284.7

Diazepam is 7-chloro-1-methyl-5-phenyl-1,3-dihydro-2H1,4-benzodiazepin-2-one. It contains not less than 99.0% and not more than 101.0% of C16H13ClN2O, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder. Very slightly soluble in water, soluble in ethanol (96%).

VP V

Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of diazepam RS. Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions protected from bright light. Mobile phase: Mix 22 volumes of acetonitrile R, 34 volumes of methanol R and 44 volumes of a 0.34% solution of potassium dihydrogen phosphate R previously adjusted to pH 5.0 with dilute sodium hydroxide solution R. Test solution: Dissolve 25.0 mg of the substance to be examined in 0.5 ml of acetonitrile R and dilute to 50.0 ml with the mobile phase. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (2): Dissolve the contents of a vial of diazepam for system suitability RS (containing impurities A, B and E) in 1.0 ml of the mobile phase. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase spherical end-capped octylsilyl silica gel for chromatography R (5 µm). Column temperature: 30 °C. Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: The run time is 4 times the retention time of diazepam. Identification of impurities: Use the chromatogram supplied with diazepam for system suitability RS and the chromatogram obtained with reference solution (2) to identify the peaks due to impurities A, B and E. Relative retention with reference to diazepam (retention time = about 9 min): impurity E = about 0.7; impurity A = about 0.8; impurity B = about 1.3. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurities E and A is at least 2.5; the resolution between the peaks due to impurity A and diazepam is at least 6.0. Limits: Correction factors: for the calculation of content, multiply the peak areas of the following impurities by the corresponding correction factor: impurity B = 1.3; impurity E = 1.3; Impurities A, B, E: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%).

DIAZEPAM INJECTION

The sum of the peak areas of all impurities is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%).

Note: Impurity A: 7-chloro-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin -2-one (nordazepam). Impurity B: 2-chloro-N-(4-chloro-2-benzoylphenyl)-Nmethylacetamide. Impurity D: [5-chloro-2-(methylamino)phenyl]phenylmethanone. Impurity C: 3-amino-6-chloro-1-methyl-4-phenylquinolin-2(1H)one. Impurity E: 6-chloro-1-methyl-4-phenylquinazolin-2(1H)-one. Impurity F: 7-chloro-2-methoxy-5-phenyl-3H-1,4-benzodiazepine.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 2.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 4.0 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 60 °C; in vacuo, 4 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.200 g of the substance to be examined in 50 ml of acetic anhydride R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). Carry out a blank titration. 1 ml of 0.1 N perchloric acid VS is equivalent to 28.47 mg of C16H13ClN2O. Storage Protected from light. Action and use Anxiolytic, hypnotic. Preparations Tablets, suppository, injection. DIAZEPAM INJECTION Injectio Diazepami Diazepam injection is a sterile solution of diazepam in water for injections or other suitable solvent. It may contain suitable stabilizers. The preparation complies with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements. 333

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DIAZEPAM TABLETS

Content of diazepam, C16H13ClN2O, 90.0% to 110.0% of the stated amount. Characters A clear and colourless solution. Identification A. In the Assay, the ultraviolet absorption spectrum (Appendix 4.1) of the test solution exhibits the maximum absorbance at 368 nm. B. Examine by thin layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Chloroform - methanol (100 : 10). Test solution: Dilute the substance to be examined in methanol R to obtain a solution having a concentration of 0.10% of diazepam. Reference solution: Dissolve a quantity of diazepam RS in methanol R to obtain a solution having a concentration of 0.10% of diazepam. Procedure: Apply separately to the plate 10 µl of each solution. After development, remove the plate and spray it with a 10% solution of sulfuric acid R in ethanol R. Heat the plate in an oven at 105 °C in 10 min. Examine the plate in the ultraviolet light (365 nm). The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to that in the chromatogram obtained with the reference solution. pH 6.2 to 7.0 (Appendix 6.2). Assay To an accurately measured volume of the injection containing 10 mg of diazepam, add 20 ml of phosphate buffer solution pH 7.0 R, shake well. Extract with four 20-ml portions of chloroform R, filter the extract through the same funnel containing 5 g of anhydrous sodium sulfate R. Combine the extracts into a 100-ml volumetric flask, dilute to volume with chloroform R, shake well. Evaporate 10.0 ml of this solution in a current of nitrogen to dryness. Dissolve the dryness in 25.0 ml of 0.05 M sulfuric acid R in methanol R. Measure the absorbance (Appendix 4.1) of the solution at the maximum at 368 nm. Calculate the content of diazepam, C16H13ClN2O, taking 151 as the value of A (1%, 1 cm) at the maximum at 368 nm. Storage Protect from light. Action and use Anxiolytic, hypnotic and anticonvulsant agent Usual strength 10 mg/2 ml; 50 mg/10 ml. 334

DIAZEPAM TABLETS Tabellae Diazepami Diazepam tablets contain diazepam. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of diazepam, C16H13ClN2O, 92.5% to 107.5% of the stated amount. Identification A. The ultraviolet absorption spectrum (Appendix 4.1) of the final solution obtained in the Assay in the range 230 nm to 350 nm exhibits two maxima, at 242 nm and 284 nm. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Chloroform - methanol (100 : 10). Test solution: Shake a quantity of the powdered tablets containing 50 mg of diazepam with 10 ml of methanol R, allow to settle and decant the supernatant liquid. Reference solution: A 5 mg/ml solution of diazepam RS in methanol R. Procedure: Apply separately to the plate 2 µl of each solution. After removal of the plate, spray it with ethanolic sulfuric acid R, heat at 105 °C for 10 min and examine under ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. Related substances and decomposition products Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Ethyl acetate - hexane (1: 1). Test solution: Shake a quantity of the powdered tablets containing 50 mg of diazepam with 5 ml of ethanol (96%) R, filter. Reference solution: Dilute 1 ml of the test solution to 50 ml with ethanol (96%) R. Procedure: Apply separately to the plate 20 µl of the test solution and 5 µl of the reference solution. Allow the solvent front to ascend 12 cm above the line of application. After removal of the plate, allow the solvent to evaporate and examine under ultraviolet light 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 900 ml of 0.1 M hydrochloric acid R. Rotation speed: 100 rpm. Time: 45 min.

VP V

Procedure: After the specified time, withdraw a sample of the medium and filter, discarding the first 20 ml of the filtrate. Dilute a suitable volume of the filtrate with 0.1 M hydrochloric acid R to produce a solution having an appropriate concentration of diazepam. Measure the absorbance of this solution (Appendix 4.1) at 286 nm using 0.1 M hydrochloric acid R in the reference cell. Calculate the content of diazepam, C16H13ClN2O, in the medium taking 488 as the value of A(1%, 1 cm) at the maximum at 286 nm. Tolerance: Not less than 75% (Q) of the stated amount of diazepam is dissolved in 45 min.

Uniformity of content Tablets containing less than 10 mg of diazepam comply with the requirement stated under Uniformity of content (Appendix 11.2) using the method of analysis described in the Assay. Test solution: To one tablet add 1 ml of water, allow the tablet to disintegrate and stand for 15 min. Add 80 ml of a 0.5% solution of sulfuric acid R in methanol R, shake for 15 min, add sufficient of the methanolic sulfuric acid to produce 100.0 ml and filter. Dilute, if necessary, with the same solvent to obtain an appropriate concentration. Assay Weigh 20 tablets, calculate the average mass, finely powder. Weigh accurately a quantity of the powdered tablets containing 10 mg of diazepam in a 100 ml volumetric flask, add 5 ml of water, mix and allow to stand for 15 min. Add 70 ml of a 0.5% w/v solution of sulfuric acid R in methanol R, shake for 15 min, add sufficient of the methanolic sulfuric acid to volume and filter. Dilute 10.0 ml of the filtrate to 100.0 ml with the same solvent and measure the absorbance (Appendix 4.1) of the resulting solution at the maximum at 284 nm, in a 1-cm cell, using a 0.5% solution of sulfuric acid R in methanol R in the reference cell. Calculate the content of C16H13ClN2O taking 450 as the value of A(1%, 1 cm) at the maximum at 284 nm. Storage Store protected from light. Action and use Anxiolytic, hypnotic and anticonvulsant agent. Usual strength 2 mg, 5 mg, 10 mg.

DICLOFENAC DIETHYLAMINE

DICLOFENAC DIETHYLAMINE Diclofenacum diethylaminum

C18H22Cl2N2O2  

M: 369.29

Diclofenac diethylamine is diethylammonium 2-[(2,6-dichloroanilino) phenyl]acetate. It contains not less than 99.0% and not more than 101.0% of C18H22Cl2N2O2, calculated with reference to the dried substance.

Characters A white to light beige, crystalline powder. Sparingly soluble in water and in acetone, freely soluble in ethanol (96%) and in methanol, practically insoluble in 1M sodium hydroxide. It melts at about 154°, with decomposition. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the reference spectrum of diclofenac diethylamine. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Hydrochloric acid - water - glacial acetic acid - ethyl acetate (1 : 1 : 6 : 11). Test solution: A 5.0% solution of the substance being examined in methanol R. Reference solution: A 5.0% solution of diclofenac diethylamine RS in methanol R. Procedure: Apply separately to the plate 2 µl of each solution. Develop over a path of 15 cm. Dry the plate in a current of warm air for 10 min. Spray with ninhydrin solution R and heat at 110 °C for 15 min. The test is not valid unless the chromatogram obtained with reference solution shows two clearly separated spots. The two principal spots in the chromatogram obtained with the test solution are similar in position, colour and size to the corresponding spots in the chromatogram obtained with the reference solution. Acidity or alkalinity pH of a 1% solution in ethanol (10%) is from 6.4 to 8.4 (Appendix 6.2). Appearance of solution A 5% solution of the substance being examined in methanol is clear (Appendix 9.2). The absorbance of the solution measured at 440 nm is not greater than 0.05 (Appendix 4.1). 335

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DICLOFENAC SODIUM

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Solution A - methanol (34 : 66). Solution A: A solution contains 0.5 g/L of phosphoric acid R and 0.8 g/L of sodium dihydrogen phosphate R adjusted to pH 2.5 with phosphoric acid R. Test solution: A 0.10% solution of the substance being examined in mobile phase. Reference solution (1): Dilute 2.0 ml of the test solution to 100.0 ml with mobile phase. Dilute 1.0 ml of the solution to 10.0 ml with mobile phase. Reference solution (2): Dissolve 1 mg of diclofenac impurity A RS in 1 ml of the test solution and dilute to 200 ml with mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase end-capped octylsilyl silica gel for chromatography (5 µm) (end-capped Zorbax C8 is suitable). Detector: A spectrophotometer set at 254 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Procedure: The run time is 1.5 times the retention time of diclofenac. In the chromatogram obtained with reference solution (2), the retention times are about 25 minutes for diclofenac and about 12 min for diclofenac impurity A. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to diclofenac and diclofenac impurity A is at least 6.5. Limits: In the chromatogram obtained with the test solution: The area of any secondary peak is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%). The sum of the areas of any secondary peaks is not more than 2.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). Disregard any peak with an area less than 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A: 1-(2,6-dichlorophenyl)-1,3-dihydro-2H-indol-2-one. Impurity B: 2-[(2,6-dichlorophenyl)amino]benzaldehyde. Impurity C: [2-[(2,6-dichlorophenyl)amino]phenyl]methanol. Impurity D: 2-[2-[(2-bromo-6-chlorophenyl)amino]phenyl]acetic acid. Impurity E: 1,3-dihydro-2H-indol-2-one.

Heavy metals Not more than 10 ppm (Appendix 9.4.8). 2 g complies with limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1 g; at a pressure not exceeding 1 kPa; 24 h). 336

Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1 g. Assay Dissolve 0.500 g of the substance being examined in 30 ml of anhydrous acetic acid R and titrate with 0.1 N perchloric acid VS. Determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 36.93 mg of C18H22Cl2N2O2. Storage In an airtight container and protected from light. Action and use Cyclo-oxygenase inhibitor; analgesic; anti-inflammatory. Preparation Skin gel. DICLOFENAC SODIUM

Diclofenacum natricum

C14H10Cl2NNaO2 

M. 318.1

Diclofenac sodium is sodium 2-[(2,6-dichlorophenyl) amino]phenyl]acetate. It contains not less than 99.0% and not more than 101.0% of C14H10Cl2NNaO2, calculated with reference to the dried substance.

Characters White or slightly yellowish, slightly hygroscopic, crystalline powder. Freely soluble in methanol, soluble in ethanol (96%), sparingly soluble in water, slightly soluble in acetone. Melting point: About 280 °C, with decomposition. Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of diclofenac sodium RS. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Concentrated ammonia - methanol - ethyl acetate (10 : 10 : 80). Test solution: Dissolve 25 mg of the substance to be examined in methanol R and dilute to 5 ml with the same solvent.

VP V

DICLOFENAC SODIUM INJECTION

Reference solution (1): Dissolve 25 mg of diclofenac sodium RS in methanol R and dilute to 5 ml with the same solvent. Reference solution (2): Dissolve 10 mg of indometacin RS in reference solution (1) and dilute to 2 ml with the same solution. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 10 cm. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with the reference solution (2) shows two clearly separated spots. C. Dissolve about 10 mg in 10 ml of ethanol (96%) R. To 1 ml of this solution add 0.2 ml of a mixture, prepared immediately before use, of equal volumes of a 0.6% solution of potassium ferricyanide R and a 0.9% solution of ferric chloride R. Allow to stand protected from light for 5 min. Add 3 ml of 0.1 M hydrochloric acid R. Allow to stand, protected from light, for 15 min. A blue colour develops and a precipitate is formed. D. Dissolve 60 mg in 0.5 ml of methanol R and add 0.5 ml of water. The solution gives reaction of sodium (Appendic 8.1).

System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurity A and diclofenac is at least 6.5. Limits: In the chromatogram obtained with the test solution. The area of any second peak is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%). The sum of the peak areas of all impurities is not more than 2.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). Disregard any peak with an area less than 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%).

Appearance of solution Dissolve 1.25 g of the substance to be examined in methanol R and dilute to 25.0 ml with the same solvent. The solution is clear (Appendix 9.2) and its absorbance (Appendix 4.1) at 440 nm is not greater than 0.05.

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 100 °C to 105 °C; 3 h).

Related substances Examine by liquid chromatography (Appendix 5.3) Mobile phase: Mixture A - methanol (34 : 66). Mixture A: A solution containing 0.5 g/L of phosphoric acid R and 0.8 g/L of sodium dihydrogen phosphate R, adjusted to pH 2.5 with phosphoric acid R. Test solution: Dissolve 50.0 mg of the substance to be examined in methanol R and dilute to 50.0 ml with the same solvent. Reference solution (1): Dilute 2.0 ml of the test solution to 100.0 ml with methanol R. Dilute 1.0 ml of this solution to 10.0 ml with methanol R. Reference solution (2): To 1.0 ml of the test solution in a 200 ml volumetric flask, add 5.0 ml of a 0.2 mg/ml solution of diclofenac impurity A RS in methanol R and dilute to volume with methanol R, mix well. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase end-capped octylsilyl silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Procedure: The run time of the test solution is 1.5 times the retention time of diclofenac. Retention times: impurity A = about 12 min; diclofenac = about 25 min.

Note: Impurity A: 1-(2,6-dichlorophenyl)-1,3-dihydro-2H-indol-2-one.

Heavy metals Not more than 10 ppm (Appendix 9.4.8). Dissolve 2.0 g in 20 ml of methanol R and the solution complies with limit test for heavy metals, method 8. Prepare the standard using lead standard solution (1 ppm Pb) obtained by diluting lead standard solution (100 ppm Pb) R with methanol R.

Assay Dissolve 0.250 g of the substance to be examined in 60 ml of glacial acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 31.81 mg of C14H10Cl2NNaO2. Storage In an airtight container, protected from light. Action and use Anti-inflammatory non-steroide. Preparations Tablets, injection. DICLOFENAC SODIUM INJECTION Injectio Diclofenaci natrii Diclofenac injection is a sterile solution of diclofenac sodium in water for injections. It complies with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Content of diclofenac sodium, C14H10Cl2NNaO2, 95.0% to 105.0% of the stated amount. 337

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DICLOFENAC GASTRO-RESISTANT TABLETS

Characters A clear, colourless solution or pale yellow solution. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Chloroform - acetone - formic acid (90 : 5 : 5). Test solution: Dilute, if necessary, a volume of the injection containing the equivalent of 25 mg of diclofenac sodium in 10 ml of methanol R. Reference solution: A 0.25% solution of diclofenac sodium in methanol R. Procedure: Apply separately to the plate 2 µl of each solution. Allow the solvent front to ascend 15 cm above the line of application. After removal of the plate, allow it to dry in a current of warm air. Examine under ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with test solution is similar in position, colour and size to that in the chromatogram obtained with reference solution. B. In the Assay, the chromatogram obtained with the test solution shows a principal peak corresponding in retention time to the principal peak in the chromatogram obtained with the reference solution. pH 8.0 to 9.0 (Appendix 6.2). Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: 0.01 M phosphoric acid - 0.01 M sodium dihydrogen phosphate - methanol (10 : 25 : 65). Test solution: Dilute a volume of the injection, accurately mesured, with a mixture of methanol - water (65 : 35) to obtain a 0.005% solution of diclofenac sodium. Reference solution: A 0.005% solution of diclofenac sodium RS in a mixture of methanol - water (65 : 35). Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase B (5 µm). Column temperature: 35 °C. Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: System suitability: The relative standard deviation of the peak areas for 6 replicate injections of the reference solution is not greater than 2.0%. Inject separately the reference solution and the test solution. Calculate the concentration of diclofenac sodium, C14H10Cl2NNaO, in the preparation using the peak areas in the chromatograms obtained with the test solution, reference solution and the content of diclofenac sodium, C14H10Cl2NNaO2, in diclofenac sodium RS. 338

Storage Store in sealed glass container, at a cool place, protected from light. Action and use Non steroidal anti-inflammatory. Usual strength 75 mg/2 ml, 75 mg/3 ml. GASTRO-RESISTANT DICLOFENAC TABLETS Tabellae Diclofenaci Gastro-resistant diclofenac tablets contain diclofenac sodium. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of diclofenac sodium, C14H10Cl2NNaO2, 90.0% to 110.0% of the stated amount. Identification Remove the coating and finely powder. To a quantity of the powdered tablets containing 150 mg of diclofenac sodium add 0.5 ml of glacial acetic acid R and 15 ml of methanol R and sonicate. Filter and collect the filtrate in a beaker containing 15 ml of water, a precipitate is formed, filter in a vacuum and collect the precipitate. Wash the precipitate with four portion of 5 ml of water. Dry at 105 °C for 2 to 3 h. The infrared absorption spectrum of the obtained precipitate (Appendix 4.2) is concordant with the reference spectrum of diclofenac. Related substances Examine by liquid chromatography (Appendix 5.3) with the mobile phase and the chromatographic system as described in the Assay. Test solution: Shake a quantity of the powdered tablets containing 50 mg of diclofenac sodium with 70 ml of the mobile phase for 30 min, add sufficient of the mobile phase to produce 100.0 ml. Mix well, centrifuge and use the supernatant liquid. Reference solution: Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 5.0 ml of the resulting solution to 25.0 ml with the mobile phase. Resolution solution: A solution containing 0.0005% of diclofenac sodium RS and 0.0005% of diclofenac impurity A RS in the mobile phase. Procedure: The run time is 1.5 times the retention time of diclofenac. System suitability: Inject the resolution solution, the retention time of the impurity A peak is about 12 min, of the diclofenac peak is about 25 min. The resolution factor between the two peaks is not less than 6.5.

VP V

Inject the test solution and the reference solution. In the chromatogram obtained with the test solution, the area of any secondary peak is not greater than that of the principal peak in the chromatogram obtained with the reference solution (0.2%). The sum of the areas of all secondary peaks is not greater than 2.5 times of the area of the principal peak in the chromatogram obtained with the reference solution (0.5%). Disregard any peak with an area less than 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (0.05%) and any peak with retention times relative to the principal peak of 0.67 and 0.1.

Dissolution (Appendix 11.4) Acid stage Apparatus: Paddle. Medium: 900 ml of 0.1 M hydrochloric acid R. Rotation speed: 50 rpm. Time: 2 h. Procedure: After the specified time, remove each tablet from the medium and turn to the test in the Buffer stage. To the medium remaining in each vessel, add 20 ml of 5 M sodium hydroxide R, stir and filter if neccessary. Measure the absorbance of this solution (Appendix 4.1) at the maximum wavelength at about 276 nm, using a mixture of 0.1 M hydrochloric acid R and 5 M sodium hydroxide R (900 : 20) as blank. Compare with a reference solution prepared as follows: Transfer about 68 mg of diclofenac sodium RS, accurately weighed, into a 100-ml volumetric flask, add 10 ml of 0.1 M sodium hydroxide R, dilute with water to volume, and mix. Transfer 2.0 ml of this solution to another 100-ml volumetric flask, dilute with the blank to volume, and mix. Tolerance: Not more than 10% of the labeled amount of diclofenac sodium, C14H10Cl2NNaO2, is dissolved in 2 h. Buffer stage Apparatus: Paddle. Medium: 900 ml of phosphate buffer pH 6.8 R. Phosphate buffer pH 6.8: Dissolve 76 g of tribasic sodium phosphate R in water to obtain 1000 ml of solution. Mix 250 ml of this solution with 750 ml of 0.1 M hydrochloric acid R, and adjust the pH to 6.8 ± 0.1 with 2 M hydrochloric acid R or 2 M sodium hydroxide R. Rotation speed: 50 rpm. Time: 60 min. Procedure: After the specified time, withdraw a sample of the medium and filter, discarding the first 20 ml of the filtrate. Dilute the filtrate with the phosphate buffer pH 6.8 to obtain a solution having a known concentration of diclofenac sodium about 0.02 mg/ml. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum of about 276 nm in a 1-cm cell, using the phosphate buffer pH 6.8 as the blank. Compare with a reference solution prepared as follows: Transfer about 68 mg of diclofenac sodium RS, accurately weighed, to a 100-ml volumetric flask, add 10 ml

DICLOFENAC GASTRO-RESISTANT TABLETS

of 0.1 M sodium hydroxide R, dilute with water to volume, and mix. Transfer 3.0 ml of this solution to another 100-ml volumetric flask, dilute with phosphate buffer pH 6.8 to volume, and mix. Tolerances: Not less than 80% (Q) of the labeled amount of diclofenac sodium, C14H10Cl2NNaO2, is dissolved in two stages.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Mixture A - methanol (34 : 66). Mixture A: 1000 ml of a solution containing 0.5 g of phosphoric acid R and 0.8 g of sodium dihydrogen phosphate R, adjust the pH to 2.5 with phosphoric acid R. Test solution: Weigh 20 tablets, calculate the average mass and finely powder. Weigh accurately a quantity of the powder containing about 50 mg of diclofenac sodium, transfer into a 100-ml volumetric flask, add 70 ml of the mobile phase, sonicate for 5 min and dilute to volume with the mobile phase, mix well, filter. Dilute 5.0 ml of the filtrate to 50.0 ml with the mobile phase, mix well. Reference solution: Weigh accurately about 50 mg of diclofenac sodium RS and dissolve in sufficient mobile phase to produce 100.0 ml. Dilute 5.0 ml of the resulting solution to 50.0 ml with the mobile phase. Resolution solution: A solution containing 0.0005% of diclofenac sodium RS and 0.0005% of diclofenac impurity A RS in the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with end-capped octylsilyl silica gel for chromatography (5 μm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the resolution solution, the retention time of the impurity A peak is about 12 min, and of diclofenac peak is about 25 min. The resolution factor between two peaks is not less than 6.5 Inject separately the reference solution, the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject the reference solution and the test solution. Calculate the content of diclofenac sodium, C14H10Cl2NNaO2, in the tablets using the areas of principal peaks in the chromatograms obtained with the test solution, the reference solution and the declared content of C14H10Cl2NNaO2 in diclofenac sodium RS. Storage Store in an airtight container, in a cool place, protected from light. Action and use Non steroidal anti-inflammatory. Usual strength 25 mg, 50 mg. 339

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DICLOXACILLIN SODIUM

DICLOXACILLIN SODIUM Dicloxacillinum natricum

C19H16Cl2N3NaO5S,H2O 

C. Place about 2 mg of the substance to be examined in a test-tube about 150 mm long and about 15 mm in diameter. Moisten with 0.05 ml of water and add 2 ml of sulfuric acid- formaldehyde reagent R. Mix the contents of the tube by swirling; the solution is slightly greenish-yellow. Place the test-tube in a water-bath for 1 min; a yellow colour develops. D. It gives reaction of sodium (Appendix 8.1).

M. 510.3

Dicloxacillin sodium is sodium (2S,5R,6R)-6-[[[3-(2,6dichlorophenyl)-5-methylisoxazol-4- yl]carbonyl]amino]3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2carboxylate monohydrate. It contains not less than 95.0% and not more than 102.0% of C19H16Cl2N3NaO5S, calculated with reference to the anhydrous substance. Semi-synthetic product derived from a fermentation product.

Characters White or almost white, hygroscopic, crystalline powder. Freely soluble in water, soluble in ethanol (96%) and in methanol. Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of dicloxacillin sodium RS. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silanised silica gel. Mobile phase: Acetone - 15.4% solution of ammonium acetate R adjusted to pH 5.0 with glacial acetic acid R (30 : 70). Test solution: Dissolve 25 mg of the substance to be examined in 5 ml of water. Reference solution (1): Dissolve 25 mg of dicloxacillin sodium RS in 5 ml of water. Reference solution (2): Dissolve 25 mg of cloxacillin sodium RS, 25 mg of dicloxacillin sodium RS and 25 mg of flucloxacillin sodium RS in 5 ml of water. Procedure: Apply separately to the plate 1 µl of each solution. Develop over a path of 15 cm. Dry the plate in air and expose to iodine vapour until the spots appear and examine in daylight. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with the reference solution (2) shows 3 clearly separated spots. 340

Appearance of solution Solution S: Dissolve 2.50 g in carbon dioxide-free water R and dilute to 25.0 ml with the same solvent. Solution S is clear (Appendix 9.2) and its absorbance (Appendix 4.1) at 430 nm is not greater than 0.04. pH 5.0 to 7.0 (Appendix 6.2). Determined on solution S. Specific optical rotation +128° to +143°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.250 g in water and dilute to 25.0 ml with the same solvent. Related substances Examine by liquid chromatography (Appendix 5.3) Mobile phase: A 0.27% solution of potassium dihydrogen phosphate adjusted to pH 5.0 with dilute sodium hydroxide solution - acetonitrile (75 : 25). Test solution (1): Dissolve 50.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Test solution (2): Dilute 5.0 ml of test solution (1) to 50.0 ml with the mobile phase. Reference solution (1): Dissolve 50.0 mg of dicloxacillin sodium RS in the mobile phase and dilute to 50.0 ml with the mobile phase. Dilute 5.0 ml of this solution to 50.0 ml with the mobile phase. Reference solution (2): Dilute 5.0 ml of test solution (2) to 50.0 ml with the mobile phase. Reference solution (3): Dissolve 5 mg of flucloxacillin sodium RS and 5 mg of dicloxacillin sodium RS in the mobile phase, then dilute to 50.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.0 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 225 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Inject test solution (1) and reference solutions (2) and (3). The run time is 5 times the retention time of dicloxacillin. Retention time: Dicloxacillin = about 10 min.

VP V

System suitability: In the chromatogram obtained with reference solution (3), the resolution between the peaks due to flucloxacillin (1st peak) and dicloxacillin (2nd peak) is at least 2.5. Limits: Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (1%). The sum of the peak areas of all impurities is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (5%). Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Note: Impurity A: (4S)-2-[carboxy[[[3-(2,6-dichlorophenyl)-5-methylisoxazol-4-yl]carbonyl]amino]methyl]-5,5-dimethyl-thiazolidine-4-carboxylic acid (penicilloic acids of dicloxacillin), Impurity B: (2RS,4S)-2-[[[[3-(2,6-dichlorophenyl)-5-methylisoxazol-4-yl]carbonyl]amino]methyl]-5,5-dimethyl-thiazolidine -4-carboxylic acid (penilloic acids of dicloxacillin), Impurity C. (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia-1azabicyclo[3.2.0]heptane-2- carboxylic acid (6-aminopenicillanic acid), Impurity D: 3-(2,6-dichlorophenyl)-5-methylisoxazole-4-carboxylic acid.

N,N-Dimethylaniline Not more than 20 ppm (Appendix 10.16, method 2). 2-Ethylhexanoic acid Not more than 0.8% m/m (Appendix 10.17). Water 3.0% to 4.5% (Appendix 10.3). Determined on 0.300 g. Pyrogens If intended for use in the manufacture of parenteral preparations without a further appropriate procedure for the removal of pyrogens, it complies with the test for pyrogens. Inject per kilogram of the rabbit's mass 1 ml of a solution in water for injections R containing 20 mg of the substance to be examined per millilitre. Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. Inject test solution (2) and reference solution (1). System suitability: In the chromatogram obtained with reference solution (1), the relative standard deviation of the peak areas for 6 replicate injections is not more than 1.0%. Calculate the content of C19H16Cl2N3NaO5S, using the areas of the peaks in the chromatograms obtained with test solution (2), reference solution (1) and the declared content of C19H16Cl2N3NaO5S in dicloxacillin sodium RS.

DIETHYL PHTHALATE

Storage In an airtight container, at a temperature not exceeding 25 °C. If the substance is sterile, store in a sterile, airtight, tamper-proof container. Action and use Penicillin antibacterial. Preparations Capsules, injection powder. DIETHYL PHTHALATE Diethylis phthalas

C12H14O4 M. 222.2 Diethyl phthalate is diethyl benzene-1,2-dicarboxylate. It contains not less than 99.0% and not more than 101.0% (m/m) of C12H14O4.

Characters A clear, oily liquid, colourless or very slightly yellow. Practically insoluble in water, miscible with ethanol (96%) and with ether. Identification Apply one of the two following identifications: First identification: A, C. Second identification: B, D, E. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum obtained with diethyl phthalate RS. Examine as thin films. B. Relative density: 1.117 to 1.121 (Appendix 6.5). C. Refractive index: 1.500 to 1.505 (Appendix 6.1). D. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Heptane - ether (30 : 70). Test solution: Dissolve 50 mg of the substance to be examined in ether R and dilute to 10 ml with the same solvent. Reference solution: Dissolve 50 mg of diethyl phthalate RS in ether R and dilute to 10 ml with the same solvent. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm. After removal of the plate, allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. 341

VP V

DILTIAZEM HYDROCHLORIDE

E. To about 0.1 ml add 0.25 ml of sulfuric acid R and 50 mg of resorcinol R. Heat on a water bath for 5 min. Allow to cool. Add 10 ml of water and 1 ml of a 42% solution of sodium hydroxide R. The solution becomes yellow or brownish-yellow and shows green fluorescence.

Appearance of solution The substance to be examined is clear (Appendix 9.2) and not more intensely coloured than reference solution Y6 (Appendix 9.3, method 2). Acidity Dissolve 20.0 g in 50 ml of ethanol (96%) R previously neutralised to phenolphthalein solution R. Titrate with 0.1 N sodium hydroxide VS. Not more than 0.1 ml of 0.1 N sodium hydroxide VS is required to change the colour of the indicator to pink. Related substances Examine by gas chromatography (Appendix 5.2), using naphthalene R as the internal standard. Internal standard solution: Dissolve 60 mg of naphthalene R in methylene chloride R and dilute to 20.0 ml with the same solvent. Test solution (1): Dissolve 1.0 g of the substance to be examined in methylene chloride R and dilute to 20.0 ml with the same solvent. Test solution (2): Dissolve 1.0 g of the substance to be examined in methylene chloride R, add 2.0 ml of the internal standard solution and dilute to 20.0 ml with methylene chloride R. Reference solution: To 1.0 ml of test solution (1) add 10.0 ml of the internal standard solution and dilute to 100.0 ml with methylene chloride R. Chromatographic system: Aglass column (2 m × 2 mm) packed with silanised diatomaceous earth for gas chromatography R (150 µm to 180 µm) impregnated with 3% (m/m) of polymethylphenylsiloxane R. Carrier gas: Nitrogen for chromatography R. Flow rate: 30 ml/min. Detector: A flame-ionisation detector. Maintaining the temperature of the column at 150 °C and that of the injection port and of the detector at 225 °C. Volume of injection: 1 µl. Procedure: Inject the reference solution, the substances are eluted in the following order: Naphthalene and diethyl phthalate. Adjust the sensitivity of the detector so that the height of the peak due to naphthalene is not less than 50% of the full scale of the recorder. The test is not valid unless the resolution between the peaks due to naphthalene and diethyl phthalate is at least 10. Inject the test solution (1), in the chromatogram obtained, verify that there is no peak with the same retention time as the internal standard. 342

Inject separately the test solution (2) and the reference solution. Continue the chromatography for three times the retention time of diethyl phthalate. From the chromatogram obtained with the reference solution, calculate the ratio (R) of the area of the peak due to diethyl phthalate to the area of the peak due to the internal standard. From the chromatogram obtained with test solution (2), calculate the ratio of the sum of the areas of any peaks, apart from the principal peak and the peaks due to the internal standard and the solvent, to the area of the peak due to the internal standard; this ratio is not greater than R (1.0%).

Water Not more than 0.2% (Appendix 10.3). Determined on 5.0 g. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Introduce 0.750 g into a 250 ml borosilicate glass flask. Add 25.0 ml of 0.5 N alcoholic potassium hydroxide VS and a few glass beads. Boil on a water bath under a reflux condenser for 1 h. Add 1 ml of phenolphthalein solution R and titrate immediately with 0.5 N hydrochloric acid VS. Carry out a blank titration. Each ml of 0.5 N alcoholic potassium hydroxide VS is equivalent to 55.56 mg of C12H14O4. Storage Store in an airtight container, at cool and dry place. Action and use Treat scabies, itch. Preparation DEP ointment. DILTIAZEM HYDROCHLORIDE Diltiazemi hydrochloridum

C22H26N2O4S,HCl  



M. 451.0

Diltiazem hydrochloride is (2S,3S)-5-[2-(dimethylamino) ethyl]-2-(4-methoxyphenyl)-4-oxo-2,3,4,5-tetrahydro1,5-benzothiazepin-3-yl acetate hydrochloride. It contains not less than 98.5% and not more than 101.0% of C22H26N2O4S,HCl, calculated with reference to the dried substance.

VP V

Characters A white, crystalline powder. Freely soluble in water, in methanol and in methylene chloride, slightly soluble in ethanol. It melts at about 213 °C with decomposition. Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of diltiazem hydrochloride RS. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Acetic acid - water - methylene chloride ethanol (1 : 3 : 10 : 12). Test solution: Dissolve 0.10 g of the substance to be examined in methylene chloride R and dilute to 10 ml with the same solvent. Reference solution: Dissolve 0.10 g of diltiazem hydrochloride RS in methylene chloride R and dilute to 10 ml with the same solvent. Procedure: Apply to the plate 10 µl of each solution. Develop over a path of 10 cm. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. C. Dissolve 50 mg in 5 ml of water. Add 1 ml of ammonium reineckate solution R. A pink precipitate is produced. D. It gives reaction (A) of chlorides (Appendix 8.1). Appearance of solution Solution S: Dissolve 1.00 g in carbon-dioxide free water R and dilute to 20.0 ml with the same solvent. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). pH Dilute 2.0 ml of solution S to 10.0 ml with carbon dioxide-free water R. The pH of the solution is 4.3 to 5.3 (Appendix 6.2). Specific optical rotation +115º to +120º, calculated with reference to the dried substance (Appendix 9.2). Dilute 5.0 ml of solution S to 25.0 ml with water. Related substances Examine by liquid chromatography (Appendix 5.3). Buffer solution pH 4.5: Dissolve 6.8 g of potassium dihydrogen phosphate in 1 litre of water, add 0.1 ml of N,N-dimethyloctylamine R, adjust to pH 4.5 using dilute phosphoric acid R. Mobile phase: Ethanol - acetonitrile - buffer solution pH 4.5 (5 : 25 : 70).

DILTIAZEM HYDROCHLORIDE

Test solution: Dissolve 50.0 mg of the substance to be examined in the mobile phase and dilute to 200.0 ml with the same solvent. Reference solution (1): Dissolve 50.0 mg of diltiazem hydrochloride RS in the mobile phase and dilute to 200.0 ml with the same solvent. Reference solution (2): Dissolve 3 mg of diltiazem impurity A RS in the mobile phase and dilute to 10 ml with the mobile phase. To 1.0 ml of this solution, add 1.2 ml of reference solution (1) and dilute to 100.0 ml with the mobile phase. Reference solution (3): Dilute 0.3 ml of the test solution to 100.0 ml with the mobile phase. Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase C (3 µm). Detector: A spectrophotometer set at 240 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl Proceduce: Inject reference solution (3). Adjust the sensitivity of the detector so that the height of the principal peak in the chromatogram obtained with reference solution (3) is at least 10% of the full scale of the recorder. Inject reference solution (2). The resolution between the peaks due to diltiazem impurity A and diltiazem hydrochloride is at least 4.0 and the symmetry factors are not more than 2.0. If necessary, adjust the concentration of N, N-dimethyloctylamine in the mobile phase. Inject test solution. Continue the chromatography for at least 5 times the retention time of the principal peak. In the chromatogram obtained with the test solution, the sum of the areas of any peaks, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (3) (0.3%). Disregard any peak with an area less than 0.025 times that of the peak in the chromatogram obtained with reference solution (3).

Note: Impurity A: (2R,3S)-5-[2-(dimethylamino)ethyl]-2-(4-methoxyphenyl)-4-oxo-2,3,4,5-tetrahydro-1,5-benzothiazepin-3-yl acetate.

Heavy metals Not more than 10 ppm (Appendix 9.4.8). Dissolve 2.0 g in water and dilute to 20.0 ml with the same solvent. 12 ml of the solution complies with limit test 1 for heavy metals (10 ppm). Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 100 °C - 105 °C; 2 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. 343

DILTIAZEM TABLETS

Assay Dissolve 0.400 g in a mixture of 2 ml of anhydrous formic acid R and 60 ml of acetic anhydride R and titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 45.1 mg of C22H26N2O4S,HCl. Storage Store in an airtight container, protected from light. Action and use Calcium antagonist, treatment of angina pectoris and antihypertensive. Preperations Tablets, capsules, modified-release tablets. DILTIAZEM TABLETS Tabellae Diltiazemi Diltiazem tablets contain diltiazem hydrochloride. They may be coated. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements:

Content of diltiazem hydrochloride, C22H26N2O4S,HCl, 90.0% to 110.0% of the stated amount. Identification A. Shake a quantity of the powdered tablets, equivalent to 1 tablet, with 10 ml of 0.1 M hydrochloric acid R, filter. Add 2 ml of ammonium cobalthiocyanate solution to 2 ml of the filtrate and add 5 ml of chloroform R. Mix well, a blue color develops in the chloroform layer. Ammonium cobalthiocyanate solution: Transfer 17.4 mg of ammonium thiocyanate R and 8.0 g of cobalt chloride R to a 100 ml volumetric flask, dissolve in water. Dilute with water to volume and mix well. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to the retention time of diltiazem hydrochloride peak in the chromatogram obtained with the reference solution. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase, Resolution solution and Chromatographic system: Proceed as directed in the Assay. Test solution: Dissolve a quantity of the powdered tablets in ethanol (96%) R to obtain a solution containing 1 mg of diltiazem per ml. Reference solution: Dilute 1.0 ml of the test solution to 200.0 ml with ethanol (96%) R. 344

VP V

Procedure: Inject the reference solution. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained is not less than 20% of the full scale of the recorder. Separately inject the reference solution and the test solution, continue the chromatography for 2 times the retention time of the principal peak. In the chromatogram obtained with the test solution, the area of any secondary peak is not greater than the area of the principal peak in the chromatogram obtained with the reference solution (0.5%); the sum of areas of any secondary peaks is not more than 2 times the area of the principal peak in the chromatogram obtained with the reference solution (1.0%).

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of water. Rotation speed: 75 rpm. Time: 30 min and 180 min. Procedure: After the specified time (30 min and 180 min), withdraw 10 ml of the medium, replace the sample drawn at the time point of 30 min with 10 ml of the fresh dissolution medium. Filter and dilute the filtrate with water to obtain a solution containing about 8 µg/ml (test solution). Measure the absorbance (Appendix 4.1) of the test solution at the wavelength of 240 nm, in a 1 cm cell, using water as the blank. Calculate the content of diltiazem hydrochloride, C22H26N2O4S,HCl, dissolved using the absorbance of reference solution of diltiazem hydrochloride RS having the same concentration in the same medium. Tolerance: Not more than 60% (Q) of the stated amount of diltiazem hydrochloride, C22H26N2O4S,HCl, is dissolved in 30 min. Not less than 75% (Q) of the stated amount of diltiazem hydrochloride, C22H26N2O4S,HCl, is dissolved in 180 min. Acceptance for the 30 min time point: S1 stage: the content of diltiazem hydrochloride dissolved from each tablet is not more than Q. S2 stage: the average content of diltiazem hydrochloride dissolved of 12 tablets is not more than Q and no tablet is more than Q+10%. S3 stage: the average content of diltiazem hydrochloride dissolved of 24 tablets is not more than Q and not more than 2 tablets are more than Q+10%, no tablet is more than Q+25% Assay Examine by liquid chromatography (Appendix 5.3). Solution A: Dissolve 1.16 g of d-camphor sulfonic acid in 0.01 M sodium acetate, dilute to 1000 ml with the same solvent, adjust to pH 6.2 with 0.1 M sodium hydroxide R. Mobile phase: Solution A - acetonitrile - methanol (50 : 25 : 25). Make an adjustment if necessary. Reference solution: Dissolve an accurately weighed quantity of diltiazem hydrochloride RS in ethanol (96%) R to obtain a solution containing 0.1 mg/ml of diltiazem.

VP V

DIMENHYDRINATE

Resolution solution: To 5.0 ml of the reference solution add 2 drops of 0.1 N sodium hydroxide R, shake for 1 min, add 2 drops of 0.1 M hydrochloric acid R and mix well. Test solution: Weigh 20 tablets (with the coating layer removed), calculate the average mass and powder finely. Transfer an accurately weighed quantity of the powdered tablets containing the equivalent of about 10 mg of diltiazem hydrochloride to a 100 ml volumetric flask, add 50 ml of ethanol (96%) R, sonicate for 10 min, dilute to volume with ethanol (96%) R, mix well and filter. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 μm). Detector: A spectrophotometer set at 240 nm. The flow rate: 1.6 ml/min. Volume of injection: 20 μl. Procedure: System suitability: Inject the resolution solution, the column efficiency, determined on the diltiazem peak, is not less than 1200 theoretical plates, the resolution factor between the peaks due to diltiazem and desacetyl diltiazem (the relative retention time compared to diltiazem peak is about 0.65) is more than 2.5. Inject the reference solution, the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of diltiazem hydrochloride, C22H26N2O4S,HCl, in the tablets using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C22H26N2O4S,HCl in the diltiazem hydrochloride RS.

Storage Pack in aluminum blister or airtight bottle. Store in a cool and dry place, at a temperature not exceeding 30 °C, protected from humidity and light. Action and use Calcium channel blocker, treatment of heart attack and high blood pressure. Usual strength 60 mg.

Characters White or almost white, crystalline powder or colourless crystals. Slightly soluble in water, freely soluble in ethanol (96%). Identification Apply one of the two following identifications: First identification: A. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of dimenhydrinate RS. B. Melting point: 102 °C to 106 °C (Appendix 6.7). C. Dissolve 0.1 g of the substance to be examined in a mixture of 3 ml of water and 3 ml of ethanol (96%) R, add 6 ml of water and 1 ml of dilute hydrochloric acid R and cool in iced water for 30 min, if necessary, scratching the wall of the tube with a glass rod to initiate crystallisation. Dissolve about 10 mg of the precipitate obtained in 1 ml of hydrochloric acid R, add 0.1 g of potassium chlorate R and evaporate to dryness in a porcelain dish. A reddish residue is obtained that becomes violet-red when exposed to ammonia vapour. D. Dissolve 0.2 g of the substance to be examined in 10 ml of ethanol (96%) R. Add 10 ml of a 1% solution of picric acid R and initiate crystallisation by scratching the wall of the tube with a glass rod. The precipitate, washed with water and dried at 100 °C to 105 °C, melts at 130 °C to 134 °C (Appendix 6.7). Appearance of solution Dissolve 1.0 g in ethanol (96%) R and dilute to 20 ml with the same solvent. The solution is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). pH To 0.4 g add 20 ml of carbon dioxide-free water, shake for 2 min and filter. pH 7.1 to 7.6 for the filtrate (Appendix 6.2).

DIMENHYDRINATE Dimenhydrinatum

C17H21NO,C7H7ClN4O2 

Dimenhydrinate is diphenhydramine [2-(diphenylmethoxy)N,N-dimethylethanamine]8- chlorotheophylline (8-chloro1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione).It contains not less than 53.0% and not more than 55.5% of diphenhydramine (C17H21NO; Mr 255.4) and not less than 44.0% and not more than 46.5% of 8-chlorotheophylline (C7H7ClN4O2; Mr 214.6), calculated with reference to the dried substance.

M: 470.0  

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Dissolve 10.0 g of triethylamine R1 in 950 ml of water, adjust to pH 2.5 with phosphoric acid R and dilute to 1000 ml with water. Mobile phase B: Acetonitrile R1. Solvent mixture: Acetonitrile R - water (18 : 82). Test solution: Dissolve 0.100 g of the substance to be examined in the solvent mixture and dilute to 100.0 ml with the solvent mixture. 345

VP V

DIMENHYDRINATE

Reference solution (1): Dissolve 57 mg of diphenhydramine hydrochloride RS in the solvent mixture and dilute to 50.0 ml with the solvent mixture. Reference solution (2): Dilute 1.0 ml of reference solution (1) to 100.0 ml with the solvent mixture. Dilute 2.0 ml of this solution to 10.0 ml with the solvent mixture. Reference solution (3): Dissolve 5.0 mg of diphenhydramine impurity A RS (impurity F) in 5.0 ml of reference solution (1) and dilute to 50.0 ml with the solvent mixture. Reference solution (4): Dissolve the contents of a vial of dimenhydrinate for peak identification RS (containing impurities A and E) in 1.0 ml of the solvent mixture. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (5 µm). Column temperature: 30 °C. Detector: A spectrophotometer set at 225 nm. Volume of injection: 10 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

Flow rate (ml/min)

0-2

82

18

1.2

2 - 15

82 → 50

18 → 50

1.2

15 - 20

50 → 20

50 → 80

1.2 → 2.0

20 - 30

20

80

2.0

Identification of impurities: Use the chromatogram supplied with dimenhydrinate for peak identification RS and the chromatogram obtained with reference solution (4) to identify the peaks due to impurities A and E; use the chromatogram obtained with reference solution (3) to identify impurity F. Relative retention with reference to diphenhydramine (retention time = about 13 min): Impurity A = about 0.3; impurity E = about 0.7; impurity F = about 0.95. System suitability: In the chromatogram obtained with reference solution (3), the resolution between the peaks due to impurity F and diphenhydramine is at least 1.5. Limits: In the chromatogram obtained with the test solution: Impurities A, F: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.2%). Impurity E: The area of the peak due to impurity E is not more than 0.75 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.15%). Any other impurity: For each impurity, the area is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.10%). The sum of the peak areas of all impurities is not more than 2.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.5%). 346

Disregard any peak with an area less than 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Note: Impurity A: 1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione (theophylline). Impurity C: 1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione (caffeine). Impurity D: N-[2-(diphenylmethoxy)ethyl]-N,N’,N’- trimethylethane1,2-diamine. Impurity E: 8-chloro-1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6dione (8-chlorocaffeine). Impurity F: 2-(diphenylmethoxy)-N-methylethanamine (diphenhydramine impurity A). Impurity G: N,N-dimethyl-2-[(RS)-(4-methylphenyl) (phenyl) methoxy]ethanamine (4-methyldiphenhydramine). Impurity H: 2-[(RS)-(4-bromophenyl)-(phenyl)methoxy]-N,Ndimethylethanamine (4-bromodiphenhydramine). Impurity I: Diphenylmethanol (benzhydrol). Impurity J: Diphenylmethanone (benzophenone). Impurity K: [oxybis(methanetriyl)]tetrabenzene.

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; in vacuo). Sulfated ash Not more than 0.2% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Diphenhydramine Dissolve 0.200 g of the substance to be examined in 60 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 25.54 mg of C17H21NO. 8-Chlorotheophylline Add 50 ml of water, 3 ml of dilute ammonia R and 0.6 g of ammonium nitrate R to 0.800 g of the substance to be examined and heat on a water-bath for 5 min. Add 25.0 ml of 0.1 N silver nitrate VS and continue heating on a waterbath for 15 min with frequent swirling. Cool, add 25 ml of dilute nitric acid R and dilute to 250.0 ml with water. Filter and discard the first 25 ml of the filtrate. Using 5 ml of a 10% solution of ammonium iron (III) sulfate R as indicator, titrate 100.0 ml of the filtrate with 0.1 N ammonium thiocyanate VS until a yellowish-brown colour is obtained. 1 ml of 0.1 N silver nitrate VS is equivalent to 21.46 mg of C7H7ClN4O2.

Storage Store in an airtight container, protected from light.

VP V

Action and use Histamine H1 receptor antagonist. Preparation Tablets. DIMENHYDRINATE TABLETS Tabellae Dimenhydrinati Dimenhydrinate tablets contain dimenhydrinate. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of dimenhydrinate, C17H21NO,C7H7ClN4O2, 90.0% to 110.0% of the stated amount. Content of 8-chlorotheophylline, C7H7ClN4O2, 43.4% to 47.9% of the content of dimenhydrinate. Identification In the Assay, the retention times of the two principal peaks in the chromatogram obtained with the test solution correspond to the retention times of 8-chlorotheophylline peak and diphenhydramine peak in the chromatogram obtained with the reference solution. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of water. Rotation speed: 50 rpm. Time: 45 min. Procedure: After the specified time (45 min), withdraw a sample of the medium, filter. Dilute the filtrate with water to obtain a solution having a concentration of about 14 µg/ml, if necessary. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum at about 276 nm, using water as a blank, compared to a reference solution having the same concentration in water. Tolerance: Not less than 75% (Q) of the stated amount of dimenhydrinate, C17H21NO,C7H7ClN4O2, is dissolved in 45 min. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase, chromatographic system, test solution, resolution solution, system suitability: Proceed as directed in the Assay. Reference solution: Dilute 2.0 ml of the test solution to 100.0 ml with the mobile phase. Procedure: Inject the reference solution. Adjust the sensitivity of the system so that the height of 8-chlorotheophylline peak in the chromatogram obtained is about 10% of the full scale of the recorder.

DIMENHYDRINATE TABLETS

Inject alternately the reference solution and the test solution, continue the chromatography for twice the retention time of diphenhydramine peak. Limits: In the chromatogram obtained with the test solution, the area of theophyllin peak is not more than 0.75 times the area of 8-chlorotheophyllin peak in the chromatogram obtained with the reference solution. The sum of areas of any secondary peaks is not greater than the area of 8-chlorotheophyllin peak in the chromatogram obtained with the reference solution.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol - triethylamine buffer pH 3.2 (1 : 1). Triethylamine buffer pH 3.2: Dissolve a mixture of 8 ml of phosphoric acid R and 14 ml of triethylamine R in 1000 ml of water, adjust to pH 3.2 with triethylamine R, add 500 ml of water and mix. Reference solution: Dissolve an accurately weighed quantity of dimenhydrinate RS in the mobile phase to obtain a solution having a concentration of about 0.3 mg/ml. Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Transfer an accurately weighed quantity of the powdered tablets containing the equivalent of 30 mg of dimenhydrinate to a 100 ml volumetric flask, add a volume of the mobile phase and dissolve by sonicating. Dilute to volume with the mobile phase, mix and filter. Resolution solution: Dissolve a quantity of theophyllin RS and dimenhydrinate RS in the mobile phase to obtain a solution containing 20 µg/ml each of those substances. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 μm). Detector: A spectrophotometer set at 225 nm. The flow rate: 1.5 ml/min. Volume of injection: 10 μl. Procedure: System suitability: Inject the resolution solution, the order of the elution is theophyllin, 8-chlorotheophylline and diphenhydramine; the resolution between theophyllin and 8-chlorotheophylline is at least 1.5; the theoretical plates of the peak due to diphenhydramine is not less than 2000. Inject the reference solution, the relative standard deviation of the peak areas for replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of dimenhydrinate, C17H21NO, C7H7ClN4O2 and 8-chlorotheophylline, C7H7ClN4O2, in the tablets using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C17H21NO,C7H7ClN4O2 in dimenhydrinate RS. Storage Store in a well-closed container, in a cool place, protected from light. 347

VP V

DIMERCAPROL

Action and use Antihistamine (H1).

Halides Dissolve 2.0 g in 25 ml of alcoholic potassium hydroxide solution R and boil under a reflux condenser for 2 h. Eliminate the ethanol by evaporation in a stream of hot air, add 20 ml of water and cool. Add 40 ml of water and 10 ml of strong hydrogen peroxide solution R, boil gently for 10 min, cool and filter rapidly. Add 10 ml of dilute nitric acid R, 5.0 ml of 0.1 M silver nitrate VS and 2 ml of ammonium iron (III) sulfate solution R. Titrate with 0.1 M ammonium thiocyanate VS until a reddish-yellow colour is obtained. Carry out a blank titration. The difference between the titration volumes is not greater than 1.0 ml.

Usual strength 15 mg, 25 mg, 50 mg. DIMERCAPROL Dimercaprolum B.A.L

C3H8OS2

M. 124.2

Dimercaprol is (2RS)-2,3-disulfanylpropan-1-ol. It contains not less than 98.5% and not more than 101.5% of C3H8OS2.

Characters A clear, colourless or slightly yellow liquid with odour of garlic. Soluble in water and in arachis oil, miscible with ethanol (96%) and with benzyl benzoate. Identification A. Dissolve 0.05 ml in 2 ml of water. Add 1 ml of 0.1 N iodine VS. The colour of the iodine is discharged immediately. B. Dissolve 0.1 ml in 5 ml of water, add 2 ml of 12.5% solution of copper sulfate R. A bluish-black precipitate is formed which quickly becomes dark grey. C. In a ground-glass-stoppered tube, suspend 0.6 g of sodium bismuthate R, previously heated to 200 °C for 2 h, in a mixture of 2.8 ml of dilute phosphoric acid R and 6 ml of water. Add 0.2 ml of the substance to be examined, mix and allow to stand for 10 min with frequent shaking. To 1 ml of the supernatant liquid add 5 ml of a 0.4% solution of chromotropic acid, sodium salt R in sulfuric acid R and mix. Heat for 15 min in a water bath. A violet-red colour develops. Apparance of solution The substance to be examined is clear (Appendix 9.2) and not more intensely coloured than reference solution B6 or BY6 (Appendix 9.3, method 2). Acidity or alkalinity Dissolve 0.2 g in carbon dioxide-free water R and dilute to 10 ml with the same solvent. Add 0.25 ml of bromocresol green solution R1 and 0.3 ml of 0.01 N hydrochloric acid VS. The solution is yellow. Not more than 0.5 ml of 0.01 N sodium hydroxide VS is required to change the colour of the indicator to blue. Refractive index 1.568 to 1.574 (Appendix 6.1). 348

Assay Dissolve 0.100 g in 40 ml of methanol R. Add 20 ml of 0.1 N hydrochloric acid VS and 50.0 ml of 0.1 N iodine VS. Allow to stand for 10 min and titrate with 0.1 N sodium thiosulfate VS. Carry out a blank titration in the same manner. Each ml of 0.1 N iodine VS is equivalent to 6.21 mg of C3H8OS2. Storage Store in a well-filled, airtight container, protected from light, at a temperature of 2°C to 8°C. Action and use Antidote to arsenic, gold and mercury. Preparation Injection. DIMERCAPROL INJECTION Injectio Dimercaproli Dimercaprol injection is a sterile solution of dimercaprol in a mixture of benzyl benzoate and vegetable oil. The injection complies with the general requirements prescribed under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Content of dimercaprol, C3H8OS2, 95.0% to 105.0% of the stated amount. Characters A clear, colourless or light yellow liquid, with an unpleasant odour. Identification Shake a volume of the injection containing about 30 mg of dimercaprol with a mixture of a drop of a 0.5% solution of cobalt chloride R and 5 ml of water, a brownish yellow colour is produced. pH Shake a volume of the injection with an equal volume of water for 2 minutes and allow to separate. Filter the

VP V

DIPHENHYDRAMINE HYDROCHLORIDE

aqueous layer through a neutral filter paper, the pH of the obtained filtrate is 4.5 to 6.5 (Appendix 6.2).

Sterility Perform the membrane filtration method (Appendix 13.7) Assay Weigh accurately a quantity of the injection containing the equivalent of about 0.1 g of dimercaprol in a conical flask, add 50 ml of a mixture of methanol and ether (3 : 1), shake well. Titrate with 0.1 N iodine solution VS until a yellow colour persists. Perform a blank determination, and make any necessary correction. Each ml of 0.1 N iodine solution VS is equivalent to 6.21 mg of C3H8OS2. Determine density (g/ml) of the injection (Appendix 6.5) to calculate the content of dimercaprol in the percentage of weight per volume. Storage Store in closed container, in a cool place, protected from light. Action and use Treatment of heavy metal poisoning. Usual strength 5 g/100 ml; 10 g/100 ml. DIPHENHYDRAMINE HYDROCHLORIDE Diphenhydraminum hydrochloridum

C17H21NO,HCl  

M. 291.8

Diphenhydramine hydrochloride is 2-(diphenylmethoxy)N,N-dimethylethanamine hydrochloride. It contains not less than 99.0% and not more than 101.0% of C17H21NO,HCl, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder. Very soluble in water, freely soluble in ethanol (96%). Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of diphenhydramine hydrochloride RS. Examine the substances prepared as discs.

B. Dissolve 50 mg of the substance to be examined in ethanol (96%) R and dilute to 100.0 ml with the same solvent. The ultraviolet absorption spectrum (Appendix 4.1) of the solution in the range 230 nm to 350 nm exhibits 3 absorption maxima at 253 nm, 258 nm and 264 nm. The ratio of the absorbance measured at the maximum at 258 nm to that measured at the maximum at 253 nm is 1.1 to 1.3. The ratio of the absorbance measured at the maximum at 258 nm to that measured at the maximum at 264 nm is 1.2 to 1.4. C. Melting point: 168 °C to 172 °C (Appendix 6.7). D. It gives the reactions of chlorides (Appendix 8.1).

Appearance of solution Solution S: Dissolve 1.0 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent. Solution S and a fivefold dilution of solution S are clear (Appendix 6.7). Solution S is not more intensely coloured than reference solution BY6 (Appendix 9.3, method 2). Acidity or alkalinity To 10 ml of solution S add 0.15 ml of methyl red solution R and 0.25 ml of 0.01 N hydrochloric acid VS. The solution is pink. Not more than 0.5 ml of 0.01 N sodium hydroxide VS is required to change the colour of the indicator to yellow. Related substances Examine by liquid chromatography (Appendix 5.3) Mobile phase: Acetonitrile - a 0.54% solution of potassium dihydrogen phosphate adjusted to pH 3.0 using phosphoric acid R (35 : 65). Test solution: Dissolve 70 mg of the substance to be examined in the mobile phase and dilute to 20.0 ml with the mobile phase. Dilute 2.0 ml of the solution to 10.0 ml with the mobile phase. Reference solution (1): Dilute 1.0 ml of the test solution to 10.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 20.0 ml with the mobile phase. Reference solution (2): Dissolve 5 mg of diphenhydramine impurity A RS and 5 mg of diphenylmethanol R in the mobile phase and dilute to 10.0 ml with the mobile phase. To 2.0 ml of this solution add 1.5 ml of the test solution and dilute to 10.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase base-deactivated octylsilyl silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 220 nm. Flow rate: 1.2 ml/min. Volume of injection: 10 µl. Procedure: The run time is 7 times the retention time of diphenhydramine. Relative retention with reference to diphenhydramine (retention time = about 6 min): impurity A = about 0.9; 349

VP V

DIPHENHYDRAMINE ORAL SOLUTION

impurity B = about 1.5; impurity C = about 1.8; impurity D = about 2.6; impurity E = about 5.1. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to diphenhydramine and to impurity A is at least 2.0. Limits: Correction factor: for the calculation of content, multiply the peak area of impurity D by 0.7, Impurity A: The area of the peak due to impurity A is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). Any other impurity: For each impurity, the area is not more than 0.6 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.3%). The sum of the peak areas of all impurities is not more than twice times the area of the principal peak in the chromatogram obtained with reference solution (1) (1.0%). Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A: 2-(diphenylmethoxy)-N-methylethanamine. Impurity B: 2-[(RS)-(4-methylphenyl)phenylmethoxy]-N,Ndimethylethanamine. Impurity C: 2-[(RS)-(4-bromophenyl)phenylmethoxy]-N,Ndimethylethanamine. Impurity D: Diphenylmethanol (benzhydrol). Impurity E: Diphenylmethanone (benzophenone).

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.250 g of the substance to be examined in 50 ml of ethanol (96%) R and add 5.0 ml of 0.01 N hydrochloric acid VS. Carry out a potentiometric titration (Appendix 10.2), using 0.1 N sodium hydroxide VS. Read the volume added between the 2 points of inflexion. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 29.18 mg of C17H22ClNO. Storage Protected from light. Action and use Histamine H1 receptor antagonist; antihistamine. Preparations Tablets, capsules, injection powder, cream, oral solution.

350

DIPHENHYDRAMINE ORAL SOLUTION Solutio Diphenhydramini Diphenhydramine oral solution is a solution of diphenhydramine hydrochloride in a suitable favoured and colourized vehicle. The solution complies with the requirements stated under “Oral liquids” (Appendix 1.3) and with the following requirements.

Content of diphenhydramine hydrochloride, C17H21NO, HCl, 90.0% to 110.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Ethanol (96%) - glacial acetic acid - water (50 : 30 : 20). Test solution: Acidify a quantity of the solution containing 50 mg of diphenhydramine hydrochloride with 2 M hydrochloric acid R, shake with three 20 ml quantities of ether R, discard the ether, extract the aqueous layer with two 20 ml quantities of chloroform R. Shake the combined chloroform extracts with anhydrous sodium sulphate R. Filter, evaporate the filtrate to dryness. Dissolve the cooled residue in 5 ml of chloroform R. Reference solution: Contains 1% diphenhydramine hydrochloride RS in chloroform R. Procedure: Apply separately to the plate 5 µl of each of the above solutions. Develop the chromatograph until the solvent front has moved about 15 cm. After removal of the plate, allow it to dry in air and spray with potassium iodobismuthate solution R and 5% of potassium iodide R. The principal spot in the chromatogram obtained with the test solution corresponds in colour and position to that in the chromatogram obtained with the reference solution. B. Evaporate to dryness 1 ml of the test solution obtained in test A, dissolve the residue in 0.15 ml of water, add 2 ml of sulfuric acid R, a yellow colour is produced. Add 0.5 ml of nitric acid R, the colour changes to red. Add 15 ml of water, cool, add 5 ml of chloroform R, shake and allow to separate; the chloroform layer is violet. Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel H. Mobile phase: Chloroform - methanol (80 : 20). Test solution: Use the test solution described under test A for Identification Reference solution: Dilute 1 ml of the test solution to 100 ml with chloroform R. Procedure: Apply separately to the plate 5 µl of each of the above solutions. Develop the chromatograph until the solvent front has moved about 15 cm. After removal of the plate, allow it to dry in air and spray with potassium

VP V

iodobismuthate solution R. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the principal spot in the chromatogram obtained with the reference solution.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - water - triethylamine (50 : 50 : 0.5), adjust by glacial acetic acid R to pH 6.5. Adjust the proportions of the constituents of the mobile phase if necessary. Test solution: Transfer an accurate volume of the solution containing about 50 mg of diphenhydramine hydrochloride to a 100 ml volumetric flask, dilute to volume with water, mix well. Reference solution: Dissolve an accurately weighed quantity of diphenhydramine hydrochloride RS in water to obtain a solution with concentration of 0.5 mg/ml. Resolution solution: Dissolve about 5 mg of benzophenone in 5 ml of acetonitrile R, dilute to 100 ml with water, shake well. Transfer 1.0 ml of the resulting solution and 5 mg of diphenhydramine hydrochloride RS into a 10 ml volumetric flask, add water to volume, mix well. Chromatographic system: A column (25 cm × 4.6 mm) packed with silica gel chemically modified by the bonding of cyano groups (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject the resolution solution. The test is not valid unless the resolution factor between the peaks due to benzophenone and diphenhydramine is at least 2. Inject the reference solution, the relative standard deviation of the peak areas for replicate injections is not more than 2.0%. The symmetry factor of the peak due to diphenhydramine is not more than 2.0. Inject alternately the reference solution and the test solution test solution. Calculate the content of diphenhydramine hydrochloride, C17H21NO,HCl, in the solution using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C17H21NO,HCl in diphenhydramine hydrochloride RS. Storage Store protected from light. Action and use Antihistamine. Usual strength 12.5 mg/5 ml.

DIPHENHYDRAMINE TABLETS

DIPHENHYDRAMINE TABLETS Tabellae Diphenhydramini Diphenhydramine tablets contain diphenhydramine hydrochloride. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements:

Content of diphenhydramine hydrochloride, C17H21NO, HCl, from 90.0% to 110.0% of the stated amount. Identification A. To a quantity of powdered tablets containing 50 mg of diphenhydramine hydrochloride, extracts with two 10-ml portions of chloroform R. Filter and evaporate the filtrate to dryness. Dissolve about 5 mg of the residue in 0.15 ml of water, add 2 ml of sulfuric acid R, a yellow is produced. Further add 0.5 ml of nitric acid R the colour changes to red. Add 15 ml of water, allow to cool, add 5 ml of chloroform R, shake and allow to separate, chloroform layer is violet. B. In the Assay, the retention time of principal peak in the chromatogram obtained with the test solution is similar to that of diphenhydramine peak in the chromatogram obtained with the reference solution. Related substances Examine by Thin layer chromatography (Appendix 5.4). Coating substance: Silica gel H. Mobile phase: Chloroform - methanol - diethylamine (80 : 20 : 1). Test solution: To a quantity of the powdered tablets containing about 100 mg of diphenhydramine hydrochloride, extract with three 10-ml portions of chloroform R. Combine the extracts, filter and evaporate almost to dryness. Dissolve the residue in 5 ml of chloroform R. Reference solution: Dilute 1 ml of the test solution to 100 ml with chloroform R. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 10 cm. Remove the plate, allow it to dry in air for 5 min and spray with sulfuric acid R and heat at 120 °C for 10 min or until spots appear. Examine in daylight. In the chromatogram obtained with the test solution, any secondary spot is not more intense than the spot in the chromatogram obtained with the reference solution (1.0%). Dissolution Apparatus: Basket. Medium: 500 ml of water. Rotation speed: 100 rpm. Time: 30 min. Procedure: After the specified time, withdraw a sample of the medium and filter, discard the first 20 ml of the filtrate. 351

VP V

DOMPERIDONE MALEATE

Reference solution: Prepare a solution of diphenhydramine hydrochloride RS in water having the same concentration as the test solution. Determine the dissolved content of diphenhydramine hydrochloride by the liquid chromatography (Appendix 5.3). The mobile phase and chromatographic system are described in the Assay. The volume of injection is 50 µl. Tolerance: Not less than 70% (Q) of the stated amount of diphenhydramine hydrochloride, C17H21NO,HCl, is dissolved in 30 min.

Assay Examine by liquid chromatography (Appendix 5.3) Mobile phase: Acetonitrile - water - triethylamine (50 : 50 : 0.5), adjust the pH to 6.5 with acetic acid glacial R. Make adjustments if necessary. Test solution: Weigh 20 tablets, calculate the average mass and finely powder. Weigh a quantity of powdered tablets containing 50 mg of diphenhydramine hydrochloride, dissolve in water and add sufficient water to 100.0 ml, filter. Reference solution: Dissolve an accurately weighed quantity of diphenhydramine hydrochloride RS in water to obtain a solution containing 0.5 mg per ml. Resolution solution: Dissolve about 5 mg of benzophenone in 5 ml of acetonitrile R, dilute with water to obtain 100 ml, shake well. Dissolve 5 mg of diphenhydramine hydrochloride in water, add 1.0 ml of benzophenone solution above and dilute to 10 ml with water, shake well. Chromatographic system: A column (25 cm × 4.6 mm) packed with silica gel chemically modified by the bonding of cyano groups (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject the resolution solution, the test is not valid unless in the chromatogram obtained the resolution factor between the peaks due to benzophenone and diphenhydramine is at least 2. Inject the reference solution, the relative standard deviation of the peak areas due to diphenhydramine for replicate injections is not more than 2.0%. The tailing factor of diphenhydramine peak is not more than 2.0. Inject the reference solution and the test solution. Calculate the content of diphenhydramine hydrochloride, C17H21NO,HCl, in tablets using the peak areas in the chromatograms obtained with the reference solution and the test solutions and the declared content of C17H21NO,HCl in diphenhydramine hydrochloride RS. Storage Store in well-closed container. Action and use Antihistamine. 352

Usual strength 25 mg; 50 mg. DOMPERIDONE MALEATE Domperidoni maleas

C22H24ClN5O2,C4H4O4

M. 542.0

Domperidone maleate is 5-chloro-1-[1-[3-(2-oxo-2,3dihydro-1H-benzimidazol-1-yl)propyl]piperidin-4yl]-1,3-dihydro-2H-benzimidazol-2-one hydrogen (z)butenedioate. It contains not less than 99.0% and not more than 101.0% of C22H24ClN5O2,C4H4O4, calculated with reference to the dried substance.

Characters A white or almost white powder. Sparingly soluble in dimethylformamide, slightly soluble in methanol, very slightly soluble in water and in ethanol (96%). Identification Apply one of the two following identifications: First identification: A. Second identification: B and C. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum obtained with domperidone maleate RS. If the spectra obtained show differences, dissolve the substance to be examined and the reference substance separately in the minimum volume of isopropanol R, evaporate to dryness on a water bath and record new spectra using the residues. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Octadecylsilyl silica gel. Mobile phase: Ammonium acetate solution - dioxan - methanol (20 : 40 : 40). Test solution: Dissolve 20 mg of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Reference solution (1): Dissolve 20 mg of domperidone maleate RS in methanol R and dilute to 10 ml with the same solvent. Reference solution (2): Dissolve 20 mg of domperidone maleate RS and 20 mg of droperidol RS in methanol R and dilute to 10 ml with the same solvent. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm. Dry the plate in a current of hot air for about 15 min and expose it to iodine vapour until the spots appear. Examine in daylight. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (1).

VP V

The test is not valid unless the chromatogram obtained with reference solution (2) shows two clearly separated spots. C. Mix 0.1 g with a mixture of 1 ml of 40% solution of sodium hydroxide R and 3 ml of water. Shake with three quantities, each of 5 ml, of ether R. Discard the ether layer. To 0.1 ml of the aqueous layer add a solution of 10 mg of resorcinol R in 3 ml of sulfuric acid R. Heat on a water bath for 15 min. No colour develops. To the remainder of the aqueous layer add 2 ml of bromine solution R. Heat on a water bath for 15 min and then heat to boiling. Cool. To 0.1 ml of the solution add a solution of 10 mg of resorcinol R in 3 ml of sulfuric acid R. Heat on a water bath for 15 min. A violet colour develops.

Appenrance of solution Dissolve 0.20 g in dimethylformamide R and dilute to 20.0 ml with the same solvent. The solution is clear (Appendix 9.2) and not more intensely coloured than reference solution Y6 (Appendix 9.3, method 2). Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol - 0.5% solution of ammonium acetate (3 : 7), changing the mobile phase composition by linear gradient to methanol over 10 min, followed by elution with methanol for 2 min. Test solution: Dissolve 0.10 g of the substance to be examined in dimethylformamide R and dilute to 10.0 ml with the same solvent. Reference solution: Dilute 1.0 ml of the test solution to 100.0 ml with dimethylformamide R. Dilute 5.0 ml of this solution to 20.0 ml with dimethylformamide R. Resolution solution: Dissolve 10.0 mg of domperidone maleate RS and 15.0 mg of droperidol RS in dimethylformamide R and dilute to 100.0 ml with the same solvent. Chromatographic system: A column (0.1 m × 4.6 mm) packed with base-deactivated octadecylsilyl silica gel for chromatography (3 µm). Detector: A spectrophotometer set at 280 nm. Flow rate: 1.5 ml/min. Volume of injection: 10 µl. Procedure: Equilibrate the column for at least 30 min with methanol R and then equilibrate at the initial mobile phase composition for at least 5 min. Inject the reference solution. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with the reference solution is at least 50% of the full scale of the recorder. Inject the resolution solution. The retention times are: domperidone maleate, about 6.5 min and droperidol, about 7 min. The test is not valid unless the resolution between the peaks due to domperidone maleate and droperidol is at least 2.0. If necessary, adjust the concentration of methanol in the mobile phase or adjust the time programme for the linear gradient.

DOMPERIDONE TABLETS

Inject dimethylformamide R as a blank. Inject the test solution and the reference solution. Limits: In the chromatogram obtained with the test solution: The area of any peak, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with the reference solution (0.25%). The sum of the areas of all peaks, apart from the principal peak, is not greater than twice the area of the principal peak in the chromatogram obtained with the reference solution (0.5%). Disregard any peak in the chromatogram obtained with the blank run and any peak with an area less than 0.2 times that of the principal peak in the chromatogram obtained with the reference solution.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 4. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 100 °C to 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.400 g in 50 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS until the colour changes from orange-yellow to green, using 0.2 ml of naphtholbenzein solution R as indicator. Each ml of 0.1 N perchloric acid VS is equivalent to 54.20 mg of C22H24ClN5O2,C4H4O4. Storage Store in a well-closed container, protected from light. Action and use Treat vomiting, dopamine antagonist. Preparation Tablets. DOMPERIDONE TABLETS Tabellae Domperidoni Domperidone tablets contain domperidone maleate. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of domperidone, C22H24ClN5O2, 95.0% to 105.0% of the stated amount. 353

VP V

DOXYCYCLINE HYDROCHLORIDE

Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Sodium acetate solution pH 4.7: Dissolve 1.36 g of sodium acetate R in 50 ml of water, adjust the pH to 4.7 with dilute acetic acid R and add water to 100 ml. Mobile phase: Sodium acetate solution pH 4.7 - methanol - methylene chloride - ethyl acetate (5 : 18 : 23 : 54). Test solution: Shake a quantity of the powdered tablets containing 10 mg of domperidone with 10 ml of a mixture of equal volumes of dichloromethane R and methanol R and filter through a glass microfibre filter (Whatman GF/C is suitable). Reference solution: A 0.127% solution of domperidone maleate RS in a mixture of equal volumes of dichloromethane R and methanol R. Procedure: Apply separately to the plate 10 µl of each solution. After removal of the plate, allow it to dry in air and examine under ultraviolet light at 254 nm. Spray the plate with potassium iodobismuthate solution R and examine again. Using each method of visualisation the principal spot in the chromatogram obtained with the test solution is similar in position and colour to the principal spot in the chromatogram obtained with the reference solution. B. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the principal peak in the chromatogram obtained with the reference solution.

dissolve with the mobile phase and add the mobile phase to volume, mix. Dilute 5.0 ml of the solution to 50.0 ml with the mobile phase. Test solution: Weigh 20 tablets, calculate the average mass and finely powder. Weigh accurately a quantity of the powdered tablets containing 25 mg of domperidone in a 50 ml volumetric flask, dissolve with the mobile phase and add the mobile phase to volume. Mix and filter. Dilute 5.0 ml of the filtrate to 50.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Column temperature: 40 °C. Detector: A spectrophotometer set at 280 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: System suitability: The relative standard deviation of the peak areas for replicate injections of the reference solution is not greater than 2.0%. Inject the test solution and the reference solution. Calculate the content of domperidone, C22H24ClN5O2, in each tablet using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C22H24ClN5O2, in domperidone maleate RS. 1 mg of domperidone maleate is equivalent to 0.7858 mg of domperidone.

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of 0.1 M hydrochloric acid R. Rotation speed: 50 rpm. Time: 45 min. Procedure: After the specified time, withdraw a sample of the medium and filter, discarding the first portion of the filtrate, dilute the filtrate with 0.1 M hydrochloric acid R if necessary. Measure the absorbance at the maximum at 286 nm (Appendix 4.1), using a 1-cm cell and 0.1 M hydrochloric acid R in the reference cell. At the same time, measure the absorbance of a 0.001% solution of domperidone maleate RS in 0.1 M hydrochloric acid R. Calculate the content of domperidone, C22H24ClN5O2, dissolved using the absorbances of the test solution, the reference and declared content of C22H24ClN5O2 in domperidone maleate RS. Tolerance: Not less than 70% (Q) to of the stated amount of domperidone C22H24ClN5O2, is dissolved in 45 min.

Storage Store in a well-closed container, protected from light.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Water - acetonitrile - glacial acetic acid triethylamine (500 : 500 : 5 : 5). Reference solution: Weigh accurately 63.5 mg of domperidone maleate RS in a 100 ml volumetric flask, 354

Action and use Antiemetic. Usual strength 10 mg. DOXYCYCLINE HYDROCHLORIDE Doxycyclini hydrochloridum Doxycycline hyclate

, HCl , 1/2 C2H5-­OH , ½ H2O

C22H24N2O8,HCl,½C2H6O,½H2O

M. 512.9

Doxycycline hydrochloride is (4S,4aR,5S,5aR,6R,12aS)-4(dimethylamino)-3,5,10,12,12a-pentahydroxy-6-methyl1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2carboxamide hydrochloride hemiethanol hemihydrate. The substance obtained by semi-synthesis from oxytetracycline or metacycline or by any other means. It contains not less

VP V

than 95.0% and not more than 102.0% of C22H24N2O8,HCl, calculated with reference to the anhydrous and ethanolfree substance.

Characters Yellow, crystalline powder, hygroscopic. Freely soluble in water and in methanol, sparingly soluble in ethanol (96%). It dissolves in solutions of alkali hydroxides and carbonates. Identification A. In the test for Assay, the principal peak in the chromatogram obtained with the test solution is similar in retention time to the principal peak in the chromatogram obtained with reference solution (2). B. To about 2 mg add 5 ml of sulfuric acid R. A yellow colour develops. C. It gives reaction A of chlorides (Appendix 8.1). pH Dissolve 0.1 g in carbon dioxide-free water R and dilute to 10 ml with the same solvent. The pH of the solution is 2.0 to 3.0 (Appendix 6.2). Specific optical rotation -105° to -120°, calculated with reference to the anhydrous, ethanol-free substance (Appendix 6.4). Dissolve 0.250 g in a mixture of 1 volume of 1 M hydrochloric acid R and 99 volumes of methanol R and dilute to 25.0 ml with the same mixture of solvents. Carry out the measurement within 5 min of preparing the solution. Specific absorbance The specific absorbance determined at the maximum at 349 nm is 300 to 335, calculated with reference to the anhydrous, ethanol-free substance (Appendix 4.1). Dissolve 25.0 mg in a mixture of 1 volume of 1 M hydrochloric acid R and 99 volumes of methanol R and dilute to 25.0 ml with the same mixture of solvents. Dilute 1.0 ml of the solution to 100.0 ml with the same mixture of solvents. Carry out the measurement within 1 h of preparing the solution. Light-absorbing impurities Dissolve 0.10 g in a mixture of 1 volume of 1 M hydrochloric acid R and 99 volumes of methanol R and dilute to 10.0 ml with the same mixture of solvents. Carry out the measurement within 1 h of preparing the solution, determined at 490 nm (Appendix 4.1). The absorbance is not more than 0.07 (calculated with reference to the anhydrous and ethanol-free substance). Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Weigh 60.0 g of tert-butanol R into a beaker and transfer to a 1000 ml volumetric flask with the aid of 200 ml of water. Add 400 ml of buffer solution pH 8.0 R, 50 ml of a 1% solution of tetrabutylammonium hydrogen

DOXYCYCLINE HYDROCHLORIDE

sulphate R adjusted to pH 8.0 with 2 M sodium hydroxide R and 10 ml of a 4% solution of sodium edetate R adjusted to pH 8.0 with 2 M sodium hydroxide solution R; dilute to 1000.0 ml with water. Test solution: Dissolve 20.0 mg of the substance to be examined in 0.01 M hydrochloric acid R and dilute to 25.0 ml with the same acid. Reference solution (1): Dissolve 20.0 mg of doxycycline hydrochloride RS in 0.01 M hydrochloric acid R and dilute to 25.0 ml with the same acid. Reference solution (2): Dissolve 20.0 mg of 6-epidoxycycline hydrochloride RS in 0.01 M hydrochloric acid R and dilute to 25.0 ml with the same acid. Reference solution (3): Dissolve 20.0 mg of metacycline hydrochloride RS in 0.01 M hydrochloric acid R and dilute to 25.0 ml with the same acid. Reference solution (4): Mix 4.0 ml of reference solution (1), 1.5 ml of reference solution (2) and 1.0 ml of reference solution (3) and dilute to 25.0 ml with 0.01 M hydrochloric acid R. Reference solution (5): Mix 2.0 ml of reference solution (2) and 2.0 ml of reference solution (3) and dilute to 100.0 ml with 0.01 M hydrochloric acid R. Chromatographic system: Acolumn (25 cm × 4.6 mm) packed with styrene-divinylbenzene copolymer (8 µm). Column temperature: 60 °C. Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Inject the test solution, reference solution (4) and reference solution (5). Relative retention with reference to doxycycline: Impurity E = about 0.2; impurity D = about 0.3; impurity C = about 0.5; impurity F = about 1.2. System suitability: In the chromatogram obtained with reference solution (4), the resolution between the peaks due to impurity B (metacycline, 1st peak) and impurity A (6-epidoxycycline, 2nd peak) is at least 1.25. The resolution between the peaks due to impurity A and doxycycline (3rd peak) is at least 2.0 (if necessary, adjust the tert-butanol content in the mobile phase). The symmetry factor of the peak due to doxycycline is not more than 1.25. Limits: In the chromatogram obtained with the test solution: The area of the peak due to impurity A is not more than the area of the corresponding peak in the chromatogram obtained with reference solution (5) (2.0%). The area of the peak due to impurity B is not more than the area of the corresponding peak in the chromatogram obtained with reference solution (5) (2.0%). The area of the peak due to any other impurity is not more than 0.25 times the area of the peak due to impurity A in the chromatogram obtained with reference solution (5) (0.5%). Disregard any peak with an area less than 0.05 times the area of the peak due to impurity A in the chromatogram obtained with reference solution (5) (0.1%). 355

VP V

DOXYCYCLINE CAPSULES Note: Impurity A: (4S,4aR,5S,5aR,6S,12aS)-4-(dimethylamino)-3,5,10, -12,12a-pentahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,12aoctahydrotetracene-2-carboxamide(6-epidoxycycline). Impurity B: (4S,4aR,5S,5aR,12aS)-4-(dimethylamino)-3,5,10,12,12apentahydroxy-6-methylene-1,11-dioxo-1,4,4a,5,5a,6,11,12aoctahydrotetracene-2-carboxamide (metacycline). Impurity C: (4R,4aR,5S,5aR,6R,12aS)-4-(dimethylamino)-3,5,10, -12,12a-pentahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,12aoctahydrotetracene-2-carboxamide (4-epidoxycycline). Impurity D: (4R,4aR,5S,5aR,6S,12aS)-4-(dimethylamino)-3,5,10, -12,12a-pentahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,12aoctahydrotetracene-2-carboxamide(4-epi-6-epidoxycycline). Impurity E: Oxytetracycline. Impurity F: (4S,4aR,5S,5aR,6R,12aS)-2-acetyl-4-(dimethylamino)-3,5,10,12,12a-pentahydroxy-6-methyl-4a,5a,6,12a -tetrahydrotetracene-1,11(4H,5H)-dione(2-acetyl-2-decar -bamoyldoxycycline).

Sulfated ash Not more than 0.4% (Appendix 9.9, method 2). Determined on 1.0 g.

Ethanol 4.3% to 6.0%. Examine by gas chromatography (Appendix 5.2). Internal standard solution: Dilute 0.50 ml of propanol R to 1000.0 ml with water. Test solution (1): Dissolve 0.10 g of the substance to be examined in water and dilute to 10.0 ml with the same solvent. Test solution (2): Dissolve 0.10 g of the substance to be examined in the internal standard solution and dilute to 10.0 ml with the internal standard solution. Reference solution: Dilute 0.50 ml of ethanol R to 100.0 ml with the internal standard solution. Dilute 1.0 ml of this solution to 10.0 ml with the internal standard solution. Chromatographic system: A column (1.5 m × 4.0 mm) packed with ethylvinylbenzenedivinylbenzene copolymer (150 µm to 180 µm). Carrier gas: Nitrogen for chromatography. Flow rate: 60 ml/min. Detector: A flame-ionisation detector. Maintaining the temperature of the column at 135 °C, that of the injection port and of the detector at 150 °C. Procedure: Inject test solutions (1), (2), and the reference solution. Calculate the content of ethanol using the ratio of the area of ethanol peak to the area of propanol peak in the chromatograms obtained with test solution (2) and the reference solution. Relative density of ethanol at 20 °C is 0.790 g/ml.

Storage In an airtight container, protected from light. If the substance is sterile, store in a sterile, airtight, tamper-proof container.

Heavy metals Not more than 50 ppm (Appendix 9.4.8). 0.5 g complies with limit test for heavy metals, method 3. Prepare the standard using 2.5 ml of lead standard solution (10 ppm Pb) R. Water 1.4% to 2.8% (Appendix 10.3). Determined on 1.20 g. 356

Bacterial endotoxins Less than 1.14 EU/mg (Appendix 13.2), if intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins. Assay Examine by liquid chromatography (Appendix 13.2) as described in the test for Related substances. Inject the test solution and reference solution (1). Calculate the content of doxycycline due to the corresponding peaks. Molecular mass of C22H24N2O8,HCl is 480.9.

Labelling The label states: where applicable, that the substance is free from bacterial endotoxins. Action and use Antibacterial. Preparation Capsules. DOXYCYCLINE CAPSULES Capsulae Doxycyclini Doxycycline hard capsules contain doxycycline hydrochloride (doxycycline hyclate). The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of doxycycline, C22H24N2O8, 95.0% to 110.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel H. Spray the plate evenly with a 10% solution of disodium edetate the pH of which has been adjusted to 9.0 with 10 M sodium hydroxide R (about 10 ml for a 100 mm × 200 mm plate). Allow the plate to dry in a horizontal position for at least 1 h. Immediately before use dry it at 110 °C for 1 h. Mobile phase: Water - methanol - dichloromethane (6 : 35 : 59).

VP V

Test solution: Shake a quantity of the capsule contents containing the equivalent of 50 mg of doxycycline with 100 ml of methanol R for 1 min to 2 min, centrifuge and use the supernatant liquid. Reference solution (1): Dissolve a quantity of doxycycline hydrochloride RS, equivalent to 50 mg of doxycycline, in 100 ml of methanol R. Reference solution (2): Dissolve 50 mg of tetracycline hydrochloride RS and 50 mg of doxycycline hydrochloride RS in 100 ml of methanol R. Procedure: Apply separately to the plate 1 ml of each solution. Develop over a path of 15 cm. After removal of the plate, allow it to dry in air at room temperature, and examine under ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to that in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) shows two clearly separated spots. B. To a quantity of the contents of the capsules, equivalent to 0.5 mg of doxycycline, add 2 ml of sulfuric acid R. A yellow colour is produced. C. Shake a quantity of the contents of the capsules, equivalent to 10 mg of doxycycline, with 10 ml of water, and filter. The filtrate yields the reactions characteristic of chlorides (Appendix 8.1).

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of water. Rotation speed: 75 rpm. Time: 30 min. Procedure: After the specified time, withdraw a sample of the medium and filter, discarding the first portion of the filtrate. Measure the absorbance (Appendix 4.1) of the filtrate, diluted with the dissolution medium if necessary, at the maximum at 276 nm in a 1 cm cell, using the dissolution medium in the reference cell. Measure the absorbance of a suitable solution of doxycycline hydrochloride RS in the dissolution medium. Calculate the total content of doxycycline, C22H24N2O8, in the medium using the absorbances of the test solution, the reference solution and the declared content of C22H24N2O8 in doxycycline hydrochloride RS. Tolerance: Not less than 80% (Q) of the labelled amount of doxycycline, C22H24N2O8, is dissolved in 30 min. Water Not more than 8.5% (Appendix 10.3). Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Transfer 2.72 g of potassium dihydrogen phosphate R, 0.74 g of sodium hydroxide R, 0.5 g of tetrabutylammonium hydrogen sulfate R and 0.4 g of sodium

DOXYCYCLINE CAPSULES

edetate R to a 1000 ml volumetric flask. Add about 850 ml of water, and stir to dissolve. Add 60 g of tert-butanol R with the aid of water, shake and dilute with water to volume, and adjust with 1 M sodium hydroxide R to a pH of 8.0 ± 0.1. Filter through a filter having a porosity of 0.5 µm or finer, and degas before using. Make adjustments if necessary. Decreasing the proportion of tertiary butyl alcohol results in a longer retention time of doxycycline and improved separation of doxycycline from the related substances. Diluent: 0.01 M hydrochloric acid R. Test solution: Weigh 20 capsules, calculate the average weight of the capsule contents and finely powder. Transfer an accurately weighed portion of the powder, equivalent to 0.12 g of doxycycline, to a 100 ml volumetric flask, add about 75 ml of the diluent, sonicate for 5 min, shake for 15 min. Allow to cool, and dilute with the diluent to volume, and mix. Filter, discard the first portion of the filtrate. Reference solution: Transfer about 60 mg of doxycycline hydrochloride RS, accurately weighed, to a 50 ml volumetric flask, add about 30 ml of the diluent, sonicate to completely dissolve (about 5 min), dilute with the diluent to volume, and mix. Resolution solution: Prepare a solution of doxycycline hydrochloride RS in the diluent containing about 6 mg of doxycycline per ml. Transfer 5 ml of this solution to a 25 ml volumetric flask, heat on a water bath for 60 min, and evaporate to dryness on a hot plate, taking care not to char the residue. Dissolve the residue in 0.01 M hydrochloric acid R, dilute to volume with the diluent, and mix. Filter through a filter having a porosity of 0.5 mm or finer. Use the filtrate as the resolution solution. This solution contains a mixture of 4-epidoxycycline, 6-epidoxycycline and doxycycline. When stored in a refrigerator, this solution may be used for 14 days. Note: Protect the reference solution and the test solution from light.

Chromatographic system: A column (25 cm × 4.6 mm) packed with rigid, spherical styrene divinylbenzene copolymers (5 to 10 µm). Column temperature: 60 °C ± 1 °C. Detector: A spectrophotometer set at 270 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the resolution solution, the relative retention times are about 0.4 for 4-epidoxycycline (the main degradation product), 0.7 for 6-epidoxycycline, and 1.0 for doxycycline; the resolution between the 4-epidoxycycline peak and doxycycline peak is not less than 3.0, and the tailing factor for the doxycycline peak is not more than 2.0. Inject the reference solution: The relative standard deviation for 6 replicate injections is not more than 2.0%. 357

VP V

EFAVIRENZ

Inject separately the reference solution and the test solution. The run time is 1.7 times the retention time of doxycycline, and measure the areas for the pricipal peaks. Calculate the content of doxycycline, C22H24N2O8, in the capsules using the areas of the principal peaks in the chromatograms obtained with the test solution and the reference solution, and the declared content of C22H24N2O8 in doxycycline hydrochloride RS.

Storage Store in an airtight container or in blisters in a cool place, protected from light. Action and use Antibacterial. Usual strength 100 mg. EFAVIRENZ Efavirenzum

C14H9ClF3NO2

M: 315.7

Efavirenz is (4S)-6-chloro-4-(2-cyclopropylethynyl)-4(trifluoromethyl)-1,4-dihydro-2H-3,1-benzoxazin-2-one. It contains not less than 97.0% and not more than 103.0% of C14H9ClF3NO2, calculated with reference to the dried substance.

Characters White or slightly pink powder. Freely soluble in methanol, practically insoluble in water. Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of efavirenz RS. If the spectra obtained show differences, dissolve separately the substance to be examined and efavirenz RS in a small amount of methanol R, evaporate to dryness and record new spectra using the residues. B. The ultraviolet absorptiom spectrum (Appendix 4.1) of the test solution in Assay, in the range between 210 nm and 300 nm of a 1 cm layer of the solutions, use methanol R 358

as a blank solution, exhibits one absorption maximum at 247 nm. The specific absorbances A (1%, 1 cm) at the absorption maximum is 526 to 574. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel F254. Mobile phase: Dichloromethane - methanol - glalic acetic acid (90 : 10 : 3). Test solution: Dissolve 25 mg of the substance to be examined in 5 ml methanol R. Reference solution: A 5 mg/ml solution of efavirenz RS in methanol R. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 3/4 of the plate. Dry the plate in air or in cool air, examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, appearance and intensity to the principal spot in the chromatogram obtained with reference solution. D. Specific optical rotation (Appendix 6.4). From -89° to -100°, calculated with reference to the dried substance. Use a 0.3% solution of the substance to be examined in methanol R.

Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use. The solutions are protected from light, use the polypropylene vital to avoid decomposition caused by some kind of glass. Mobile phase A: A 0.05% solution of trifluoroacetic acid methanol (90 : 10). Mobile phase B: A 0.05% solution of trifluoroacetic acid methanol (10 : 90). Solvent mixture: A mixture of equal volumes of acetonitrile R and water. Test solution: Dissolve 25 mg of the substance to be examined in solvent mixture and dilute to 25.0 ml with the same solvent. Reference solution: Dilute 1.0 ml of the test solution to 50.0 ml with solvent mixture. Dilute 5.0 ml of the solution to 100.0 ml with solvent mixture. Resolution solution: Dissolve 1 mg of efavirenz impurity B RS in 10 ml solvent mixture. Dilute 1 ml of the solution to 25 ml with the same solvent. Dissolve 1 mg of efavirenz RS in 10 ml of the solution. Chromatographic system: A column (15 cm × 4.6 mm) packed with particles of silica gel, the surface of which has been modified with chemicallybonded cyanopropyl groups (nitril column, 3.5 μm). Detector: A spectrophotometer set at 250 nm. Flow rate: 1.5 ml/min. Volume of injection: 35 µl. Procedure: Carry out a linear gradient elution using the following conditions:

VP V

EFAVIRENZ CAPSULES

Time (min)

Mobile phase A (v/v)

Mobile phase B (v/v)

Comments

0 - 16

60 → 50

40 → 50

Linear gradient

16 - 23

50 → 35

50 → 65

Linear gradient

23 - 28

35 → 30

65 → 70

Linear gradient

28 - 29

30 → 20

70 → 80

Linear gradient

29 - 31

20

80

Isocratic

31 - 32

20 → 60

80 → 40

Return to initial composition

32 - 40

60

40

Re-equilibration

Inject the resolution solution, retention time of efavirenz is about 20 min, relative retention of impurity B with reference to efavirenz is about 0.9. The test is not valid unless the resolution between the peaks due to impurity B and efavirenz is at least 3. Inject the test solution and the reference solution. Limits: In the chromatogram obtained with the test solution. The area of the peak due to impurity B is not more than 4 times the area of the principal peak in the chromatogram obtained with the reference solution (0.4%). The area of any other peak, apart from the principal peak and impurity B peak, is not more than twice the area of the principal peak in the chromatogram obtained with the reference solution (0.2%) and the area of not more than three such peaks is more than the area of the principal peak in the chromatogram obtained with the reference solution (0.1%). The sum of the areas of all impurity peaks is not more than 8 times the area of the principal peak in the chromatogram obtained with the reference solution (0.8%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with the solution reference (0.05%).

Note: Impurity A:  N-{4-chloro-2-[(2S)-4-cyclopropyl-1,1,1-trifluoro2-hydroxybut-3-yn-2-yl]phenyl}-4-methoxy benzamide. Impurity B: (4S)-6-chloro-4-[(1E)-2-cyclopropyleth-1-en-1-yl]4-(trifluoromethyl)-1,4-dihydro-2H-3,1-benzoxazin-2-one. Impurity C: (4S)-6-chloro-4-(pent-1-yn-1-yl)-4-(trifluoromethyl) -1,4-dihydro-2H-3,1-benzoxazin-2-one. Impurity D: (4S)-6-chloro-4-(2-cyclopropyleth-1-yn-1-yl)-1[(4-methoxyphenyl)methyl]-4-(trifluoromethyl)-1,4-dihydro2H-3,1-benzoxazin-2-one. Impurity E: (2S)-2-(2-amino-5-chlorophenyl)-4-cyclopropyl-1,1,1 -trifluorobut-3-yn-2-ol. Impurity F: 6-chloro-2-cyclopropyl-4-(trifluoromethyl)quinoline. Impurity G: (4S)-6-chloro-4-[2-(2-methylcyclopropyl)eth-1-yn1-yl]-4-(trifluoromethyl)-1,4-dihydro-2H-3,1-benzoxazin-2-one (mixture of four stereoisomers). Impurity H: methyl {4-chloro-2-[(2S)-4-cyclopropyl-1,1,1-trifluoro -2-hydroxybut-3-yn-1-yl]phenyl}carbamate. Impurity I: (2S)-2-(5-chloro-2-{[(4-methoxyphenyl)methyl] amino}phenyl)-4-cyclopropyl-1,1,1-trifluorobut-3-yn-2-ol.

Impurity J: (2RS,4S)-6-chloro-4-(2-cyclopropyleth-1-yn-1-yl)2-(4-methoxyphenyl)-4-(trifluoromethyl)-1,4-dihydro-2H-3,1benzoxazine.

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C; 4 h). Sulfated ash Not more than 0.2% (Appendix 9.9, method 1). Determined on 1.0 g. Assay Dissolve about 25 mg of substance to be examined, accurately weighed, in methanol R to produce 50.0 ml. Dilute 2.0 ml of this solution to 100.0 ml with the same solvent. Measure the ultraviolet absorptiom spectrum (Appendix 4.1) of a 1 cm layer of the solution at the maximum at 247 nm, use methanol R as a blank solution. Calculate the amount of efavirenz, C14H9CIF3NO2, taking 550 as the value of A(1%, 1 cm) at the maximum at 247 nm. Storage Store in an airtight container, protected from light. Action and use Antiretroviral. Preparations Tablets, capsules, siro. EFAVIRENZ CAPSULES Capsulae Efavirenzi Efavirenz capsules contain efavirenz. The capsules comply with the requirements stated under Capsules (Appendix 1.13) and with the following requirements:

Content of efavirenz, C14H9ClF3NO2, 90.0% to 110.0% of the stated amount. Identification A. Shake a quantity of the capsule contents containing about 0.05 g of efavirenz with 100 ml of methanol R, filter and dilute 1 ml of the filtrate to 50 ml with methanol R. The ultra violet spectrum (Appendix 4.1) of the obtained solution in the range 220 nm to 350 nm is concordant with that of the reference solution at the same concentration in the same solvent. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of the efavirenz peak in the chromatogram obtained with the reference solution. Dissolution (Appendix 11.4) Apparatus: Paddle. 359

VP V

EMETINE HYDROCHLORIDE

Medium: 900 ml of a 1% solution of sodium lauryl sulfate R. Rotation speed: 50 rpm. Time: 45 min. Procedure: Test solution: After the specified time, withdraw a sample of the medium, and filter. Dilute the filtrate with the medium to obtain a 0.01 mg/ml solution of efavirenz. Reference solution: Weigh accurately a quantity about 20 mg of efavirenz RS, transfer into a 100-ml volumetric flask, add 10 ml of methanol R to dissolve and dilute to volume with the medium, mix well. Dilute 5.0 ml of the obtained solution to 100.0 ml with the medium. Measure the absorbance of the reference solution and test solution (Appendix 4.1) at the maximum wavelength at about 247 nm, in a 1-cm cell and using the medium as the blank. Calculate the content of efavirenz, C14H9ClF3NO2, dissolved in each capsule using the absorbances of the reference solution, the test solution and the declared content of C14H9ClF3NO2 in efavirenz RS. Tolerance: Not less than 70% (Q) of the labeled amount of efavirenz, C14H9ClF3NO2, is dissolved in 45 min.

Related substances Examine by liquid chromatography (Appendix 5.3). Phosphate buffer solution pH 3.0, mobile phase, reference solution, test solution and chromatographic system are described in the Assay. Procedure: Inject the test solution, record the chromatogram for twice the retention time of the efavirenz peak. The content of any secondary peak in the chromatogram obtained with the test solution is calculated by using normalisation procedure (Appendix 5). Limits: Each impurity is not more than 1.0%; total impurities is not more than 2.0%. Disregard any peak due to the blank and any peak with an area less than 0.05%. Assay Examine by liquid chromatography (Appendix 5.3). Phosphate buffer solution pH 3.0: Dissolve 8.6 g of ammonium dihydrogen phosphate R in 1000 ml water, adjust the pH to 3.0 ± 0.05 with phosphoric acid R. Mobile phase: Acetonitrile - phosphate buffer solution pH 3.0 (50 : 50). Reference solution: Dissolve an accurate quantity of efavirenz in methanol R to obtain a solution having a concentration of about 1.2 mg/ml. Test solution: Weigh 20 capsules, calculate the average mass of the capsules contents. Weigh accurately a quantity of powder containing about 60 mg of efavirenz. Transfer into a 50-ml volumetric flask, add 40 ml of methanol R, sonicate for 20 min. Dilute to volume with methanol R, shake and filter. 360

Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 252 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution, in the chromatogram obtained, the column efficiency, determined on the efavirenz peak, is not less than 6000 theoretical plates, the symmetry factor of the efavirenz peak is not more than 2.0. The relative standard deviation of peak areas for six replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of efavirenz, C14H9ClF3NO2, in each capsule using the peak areas in the chromatograms obtained with the reference solution, the test solution and the declared content of C14H9ClF3NO2 in efavirenz RS.

Storage Store in a well-closed container, in a cool place not exceed 30 °C, protected from light. Action and use Antiviral. Usual strength 50 mg, 100 mg and 200 mg. EMETINE HYDROCHLORIDE Emetini hydrochloridum

C29H40N2O4,2HCl,7H2O   

M:679.7 

Emetine hydrochloride is (2S,3R,11bS)-2-[[(1R)-6,7dimethoxy-1,2,3,4-tetrahydroisoquinolin-1-yl]methyl]3-ethyl-9,10-dimethoxy-1,3,4,6,7,11b-hexahydro-2Hbenzo[a]quinolizine dihydrochloride heptahydrate. It contains not less than 98.0% and not more than 102.0% of C29H40N2O4,2HCl, calculated with reference to the dried substance.

Characters A white or slightly yellowish, crystalline powder, freely soluble in water and in ethanol 96%. Identification Apply one of the two following identification: First identification: A, E. Second identification: B, C, D and E.

VP V

A. The infrared absorption spectrum (Apppendix 4.2) of the substance to be exanined is concordant with the spectrum of emetine hydrochloride RS. B. In the test for Related substances, the principal spot in the chromatogram obtained with the test solution is similar in position, fluorescence and size to the spot in the chromatogram obtained with reference solution (3). C. Dissolve about 10 mg in 2 ml of dilute hydrogen peroxide solution R, add 1 ml of hydrochloric acid R and heat. An orange colour develops. D. Sprinkle about 5 mg on the surface of 1 ml of 0.5% solution of ammonium molybdate R in sulfuric acid R. A bright-green colour develops. E. It gives reaction (A) of chlorides (Appendix 8.1).

Appearance of of solution Solution S: Dissolve 1.25 g in carbon dioxide-free water R and dilute to 25 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution Y5 or BY5 (Appendix 9.3, method 2). pH Dilute 4 ml of solution S to 10 ml with carbon dioxide-free water R. The pH of the solution is 4.0 to 6.0 (Appendix 6.2). Specific optical rotation +16° to +19°, calculated with reference to the dried substance. (Appendix 6.4). Dissolve in water a quantity of the substance to be examined corresponding to 1.250 g of dried substance and dilute to 25.0 ml with the same solvent. Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Chloroform - 2-methoxyethanol - methanol - water - diethylamine (200 : 40 : 10 : 4 : 1). Prepare the solutions immediately before use. Solvent: Methanol R containing 1% v/v of 2 M ammonia R. Test solution: A solution containing 0.5 mg of the substance to be examined per ml in the solvent. Reference solution (1): A solution containing 0.01 mg of isoemetine hydrobromide RS per ml in the solvent. Reference solution (2): A solution containing 0.01 mg of cephaeline hydrochloride RS per ml in the solvent. Reference solution (3): A solution containing 0.5 mg of emetine hydrochloride RS per ml in the solvent. Reference solution (4): Dilute 1 ml of reference solution (3) to 100 ml with the solvent. Reference solution (5): To 1 ml of reference solution (1) add 1 ml of reference solution (2) and 1 ml of reference solution (3). Procedure: Apply to the plate 10 µl of the test solution and each of reference solutions (1), (2), (3) and (4) and 30 µl of reference solution (5). Develop over a path of 15 cm. Allow

ENALAPRIL MALEATE

the plate to dry in air until the solvent has evaporated. Spray a 0.5% solution of iodine R in chloroform R and heat at 60 °C for 15 min. Examine in ultraviolet light at 365 nm. In the chromatogram obtained with the test solution, any spots corresponding to isoemetine and cephaeline are not more intense than the spots in the chromatograms obtained with reference solutions (1) and (2), respectively (2.0%); any spot, apart from the principal spot and the spots corresponding to isoemetine and cephaeline, is not more intense than the spot in the chromatogram obtained with reference solution (4) (1.0%). The test is not valid unless the chromatogram obtained with reference solution (5) shows three clearly separated spots.

Loss on drying 15.0% to 19.0% (Appendix 9.6). (1.000 g; 105 °C; 3 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.200 g in a mixture of 5.0 ml of 0.01 M hydrochloric acid R and 50 ml of ethanol (96%) R. Carry out a potentiometric titration (Appendix 10.2), using 0.1 M sodium hydroxide R. Read the volume added between the 2 points of inflexion. 1 ml of 0.1 M sodium hydroxide is equivalent to 27.68 mg of C29H42Cl2N2O4. Storage Store protected from light, in a well-close container. Action and use Antiprotozoal. Preparation Injection. ENALAPRIL MALEATE Enalaprili maleas

C20H28N2O5,C4H4O4

M. 492.5

Enalaprili maleate is (2S)-1-[(2S)-2-[[(1S)-1-(ethoxycarbonyl)-3-phenylpropyl]amino]propanoyl]pyrrolidine2-carboxylic acid (Z)-butenedioate. It contains not less than 98.5% and not more than 101.5% of C20H28N2O5,C4H4O4, calculated with reference to the dried substance. 361

VP V

ENALAPRIL MALEATE

Characters White or almost white, crystalline powder. Sparingly soluble in water, freely soluble in methanol, practically insoluble in dichloromethane. It dissolves in dilute solutions of alkali hydroxides. Melting point: About 144 °C (Appendix 6.7). Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of enalapril maleate RS. Appearance of solution Solution S: Dissolve 0.25  g in  carbon dioxide-free water R and dilute to 25.0 ml with the same solvent. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). pH 2.4 to 2.9 for solution S (Appendix 6.2). Specific optical rotation -51° to -48°, calculated with reference to the dried substance (Appendix 6.4). Determined on solution S. Related substances Examine by liquid chromatography (Appendix 5.3). Buffer solution A: Dissolve 2.8  g of  sodium dihydrogen phosphate monohydrate  R  in 950  ml of  water. Adjust to pH 2.5 with phosphoric acid R and dilute to 1000 ml with water. Buffer solution B: Dissolve 2.8  g of  sodium dihydrogen phosphate monohydrate R in 950 ml of water. Adjust to pH 6.8 with 40% solution of sodium hydroxide  R and dilute to 1000 ml with water. Solvent mixture: Acetonitrile R1 - buffer solution A (50 : 950). Mobile phase A: Acetonitrile R1 - buffer solution B (50 : 950). Mobile phase B: Buffer solution B - acetonitrile R1 (340 : 660). Test solution: Dissolve 30  mg of the substance to be examined in the solvent mixture and dilute to 100.0 ml with the same solvent. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with the solvent mixture. Reference solution (2): Dissolve 3  mg of  enalapril for system suitability RS (containing impurity A) in the solvent mixture and dilute to 10.0 ml with the same solvent. Reference solution (3):  Dissolve the contents of a vial of  enalapril  impurity mixture RS  (impurities B, C, D, E and H) in 1.0 ml of the solvent mixture. Chromatographic system: A column (15 cm × 4.1 mm) packed with stationary phase styrene-divinylbenzene copolymer R (5 µm). Column temperature: 70 °C. Detector: A spectrophotometer set at 215 nm. Flow rate: 1.0 ml/min. Volume of injection: 50 µl. 362

Procedure: Carry out a linear gradient elution using the following conditions (make adjustment if necssary): Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 20

95 → 40

5 → 60

20 - 25

40

60

Inject the test solution and the reference solutions. Identification of impurities: Use the chromatogram supplied with enalapril impurity mixture RS and the chromatogram obtained with reference solution (3) to identify the peaks due to impurities B, C, D, E and H; use the chromatogram obtained with reference solution (2) to identify the peak due to impurity A. Relative retention with reference to enalapril (retention time  =  about 11 min): Impurity C  =  about 0.2; impurity B = about 0.8; impurity A = about 1.1; impurity H = about 1.3; impurity E = about 1.5; impurity D = about 2.1. System suitability: In the chromatogramobtained with reference solution (2), peak-to-valley ratio (Hp/Hv) is at least 10, where Hp =height above the baseline of the peak due to impurity A and Hv = height above the baseline of the lowest point of the curve separating this peak from the peak due to enalapril. Limits: In the chromatogram obtained with the test solution: Impurity A: The area of the peak due to impurity A is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (1.0%). Impurities B, C, D, E, H: For each impurity, the area is not more than 0.3 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.3%); Any other impurity: For each impurity, the area is not more than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). The sum of the areas of all the impurity peaks (other than impurity A) is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (1.0%). Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%); disregard the peak due to maleic acid. Note: Impurity A: (2S)-1-[(2S)-2-[[(1R)-1-(ethoxycarbonyl)-3phenylpropyl]amino]propanoyl]pyrrolidine-2-carboxylic acid. Impurity B: (2S)-2-[[(1S)-1-(ethoxycarbonyl)-3-phenylpropyl] amino]propanoic acid. Impurity C: (2S)-1-[(2S)-2-[[(1S)-1-carboxy-3-phenylpropyl] amino]propanoyl]pyrrolidine-2-carboxylic acid. Impurity E: (2S)-1-[(2S)-2-[[(1S)-3-phenyl-1-[(2phenylethoxy) carbonyl]propyl]amino]propanoyl]pyrrolidine-2-carboxylic acid.

VP V Impurity F: (2S)-1-[(2S)-2-[[(1S)-1-(butoxycarbonyl)-3-phenylpropyl] amino]propanoyl]pyrrolidine-2-carboxylic acid. Impurity D: Ethyl (2S)-2-[(3S,8aS)-3-methyl-1,4-dioxooctahydropyrrolo [1,2-a]pyrazin-2-yl]-4-phenylbutanoate. Impurity G: (2S)-2-[[(1S)-3-cyclohexyl-1-(ethoxycarbonyl)propyl] amino]propanoic acid. Impurity H:  (2S)-1-[(2S)-2-[[(1S)-3-cyclohexyl-1-(ethoxycarbonyl) propyl]amino]propanoyl]pyrrolidine-2-carboxylic acid. Impurity I: 1H-imidazole.

Heavy metals Not more than 10 ppm (Appendix 9.4.8). 2.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 1.0% (Appendix 9.6). (1.000 g; 105 °C; 3 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.100 g of the substance to be examined in carbon dioxide-free water  R and dilute to 30 ml with the same solvent. Titrate with 0.1 N sodium hydroxide VS determining the end-point potentiometrically (Appendix 10.2). Titrate to the 2nd point of inflexion. 1 ml of 0.1  N  sodium hydroxide  VS is equivalent to 16.42 mg of C20H28N2O5,C4H4O4. Storage Store in an airtight container, protected from light. Action and use Angiotensin converting enzyme inhibitor. Preparation Tablets. ENALAPRIL TABLETS Tabellae Enalaprili Enalapril tablets contain enalapril maleate. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of enalapril maleate, C20H28N2O5,C4H4O4, 90.0% to 110.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel G.

ENALAPRIL TABLETS

Mobile phase: Butan-1-ol - water - glacial acetic acid (60 : 25 : 15). Visualization solution: Dissolve 0.85 g of bismuth nitrate base R in a mixture of 10 ml of glacial acetic acid R and 40 ml of water. Mix equal volumes of the obtained solution and a 40% solution of potassium iodide R. Dilute 10 volumes of this mixture with 20 volumes of glacial acetic acid R and 70 volumes of water immediately before use. Test solution: Weigh a quantity of the powdered tablets containing 20 mg of enalapril maleate, add 10 ml of ethanol (90%) R, shake for 10 min, centrifuge. Use the supernatant. Reference solution: A 0.2% solution of enalapril maleate RS in ethanol (90%) R. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm. Remove the plate, allow it to dry in air, spray with the visualization solution and then with dilute hydrogen peroxide solution R. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to that in the chromatogram obtained with the reference solution. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of the enalapril maleate peak in the chromatogram obtained with the reference solution.

Related substances Examine by liquid chromatography (Appendix 5.3). Solution A, mobile phase, test solution, resolution solution and chromatographic system are described in the Assay. Reference solution: Dilute 1.0 ml of the test solution to 100 volumes with solution A. Procedure: System suitability: Proceed as directed in the Assay. Inject alternately the test solution and the reference solution. In the chromatogram obtained with the test solution, the area of any peak corresponding to enalaprilat is not greater than twice the area of the principal peak in the chromatogram obtained with the reference solution, the area of any peak corresponding to enalapril diketopiperazine is not greater than the area of the principal maleate peak in the chromatogram obtained with the reference solution. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of water. Rotation speed: 50 rpm. Time: 45 min. Procedure: Examine by liquid chromatography (Appendix 5.3). Mobile phase, chromatographic system and procedure: Proceed as directed in the Assay. Test solution: After the specified time, withdraw a sample of the medium, and filter. Dilute the filtrate with the medium to obtain a solution containing about 0.00028% of enalapril maleate. 363

VP V

EPHEDRINE HYDROCHLORIDE

Reference solution: A 0.00028% solution of enalapril maleate RS in the medium. Perform the chromatographic procedure as directed in the Assay. Calculate the content of enalapril maleate, C20H28N2O5,C4H4O4, in a tablet dissolved in the medium. Tolerance: Not less than 70% (Q) of the labeled amount of enalapril maleate, C20H28N2O5,C4H4O4, is dissolved in 45 min.

standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of enalapril maleate, C20H28N2O5,C4H4O4, in the tablets using the areas (or height) of the enalapril peaks in the chromatograms obtained with the test solution, the reference solution and the declared content of C20H28N2O5,C4H4O4 in enalapril maleate RS.

Assay Examine by liquid chromatography (Appendix 5.3). Solution A: Dissolve 1.56 g of sodium dihydrogen phosphate R in 800 ml of water, adjust the pH to 2.2 with phosphoric acid R, add sufficient water to produce 1000 ml. Mobile phase: A mixture of acetonitrile R and solution A (25 : 75). Test solution: Weigh 20 tablets, calculate the average mass and finely powder. Weigh accurately a quantity of the powdered tablets containing 10 mg of enalapril maleate, add 50 ml of solution A, sonicate for 15 min, shake by mechanical means for 30 min and dilute to 100.0 ml with solution A. Continue to sonicate for further 15 min then filter through a 0.45 µm membrane. Enalaprilat stock solution: A solution containing 0.1 mg/ml of enalaprilat RS in water. Reference solution: Weigh accurately 10 mg of enalapril maleate RS and transfer into a 100-ml volumetric flask, add 50 ml of solution A, sonicate for 15 min. Allow to cool, add 1.0 ml of the enalaprilat stock solution and add sufficient solution A to volume. The resulting solution contains 0.1 mg/ml of enalapril maleate and 0.001 mg/ml of enalaprilat. Enalapril diketopiperazine solution: Add about 20 mg of enalapril maleate RS in a 100-ml beaker to form a mound on the bottom off the beaker. Place the beaker on stove and heat until the sample is changed to yellow, remove the beaker from the hot place, allow to cool. (Note: Avoid rising to a brown colour). Add 50 ml of acetonitrile R, sonicate to dissolve. Resolution solution: Dilute 1 ml of enalapril diketopiperazine solution to 50 ml with the reference solution. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase B (5 µm) Column temperature: 50 °C. Detector: A spectrophotometer set at 215 nm. Flow rate: 2.0 ml/min. Volume of injection: 50 µl. Procedure: System suitability: Inject the resolution solution, in the chromatogram obtained, the relative retention times are 0.3 for maleic acid, 0.5 for enalaprilat, 1.0 for enalapril and 1.5 for enalapril diketopiperazine; the resolution between the peaks due to enalapril and enalapril diketopiperazine is at least 2.0. Inject the reference solution, the symmetry factor calculated on the enalapril peak is not greater than 2.0. The relative

Storage Store in a dry and cool place, protected from light.

364

Action and use Antihypertensive. Usual strength 5 mg; 10 mg; 20 mg. EPHEDRINE HYDROCHLORIDE Ephedrini hydrochloridum

C10H15NO,HCl  

M. 201.7

Ephedrine hydrochloride is (1R,2S)-2-(methylamino)1-phenylpropan-1-ol hydrochloride. It contains not less than 99.0% and not more than 101.0% of C10H15NO,HCl, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder or colourless crystals. Freely soluble in water, soluble in ethanol (96%). Melting point: About 219 °C (Appenidx 6.7). Identification Apply one of the two following identifications: First identification: A, E. Second identification: B, C, D, E. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of ephedrine hydrochloride RS. A. Thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Methylene chloride - ammonia - 2-propanol (5 : 15 : 80). Test solution: Dissolve 20 mg of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Reference solution  Dissolve 10 mg of ephedrine hydrochloride RS in methanol R and dilute to 5 ml with the same solvent. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of two thirds of the plate. Dry

VP V

the plate in air and spray with ninhydrin solution R; heat at 110 °C for 5 min. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. C. It complies with the test for Specific optical rotation. D. To 0.1 ml of solution S (see Appearance of solution) add 1 ml of water, 0.2 ml of 12.5% solution of copper sulfate R and 1 ml of a 42% solution of sodium hydroxide R. A violet colour is produced. Add 2 ml of methylene chloride R and shake. The lower (organic) layer is dark grey and the upper (aqueous) layer is blue. E. To 5 ml of solution S (see Appearance of solution) add 5 ml of water. The solution gives reaction (A) of chlorides (Appedix 8.1).

Appearance of solution Solution S: Dissolve 5.00 g in distilled water R and dilute to 50.0 ml with the same solvent. Solution S is clear (Appedix 9.2) and colourless (Appedix 9.3, method 2). Acidity or alkalinity To 10 ml of solution S add 0.1 ml of methyl red solution R and 0.2 ml of 0.01 N sodium hydroxide VS. The solution is yellow. Add 0.4 ml of 0.01 N hydrochloric acid VS. The solution is red. Specific optical rotation -33.5° to -35.5°, calculated with reference to the dried substance (Appendix 6.4). Dilute 12.5 ml of solution S to 25.0 ml with water. Related substances Examine by liquid chromatography (Appendix 5.3) Mobile phase: Methanol - 1.16% solution of ammonium acetate R adjusted to pH 4.0 with glacial acetic acid R (6 : 94). Test solution: Dissolve 75 mg of the substance to be examined in the mobile phase and dilute to 10 ml with the mobile phase. Reference solution (1): Dilute 2.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (2): Dissolve 5 mg of the substance to be examined and 5 mg of pseudoephedrine hydrochloride RS in the mobile phase and dilute to 50 ml with the mobile phase. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase spherical phenylsilyl silica gel for chromatography R (3 µm). Detector: A spectrophotometer set at 257 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: The run time is 2.5 times the retention time of ephedrine.

EPHEDRINE HYDROCHLORIDE

Relative retention with reference to ephedrine (retention time = about 8 min): impurity B = about 1.1; impurity A = about 1.4. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to ephedrine and impurity B is at least 2.0. Limits: Correction factor: for the calculation of content, multiply the peak area of impurity A by 0.4; Impurity A: The corrected area of the peak due to impurity A is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%). Any other impurity: For each impurity, the area is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%). The sum of the peak areas of all impurities other than impurity A is not more than 2.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). Disregard any peak with an area less than 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A: (-)-(1R)-1-hydroxy-1-phenylpropan-2-one. Impurity B: (1S,2S)-2-(methylamino)-1-phenylpropan-1-ol (pseudoephedrine).

Sulfates Not more than 100 ppm (Appendix 9.4.14). Determined on 15 ml of solution S. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.150 g of the substance to be examined in 50 ml of ethanol (96%) R and add 5.0 ml of 0.01 N hydrochloric acid VS. Carry out a potentiometric titration (Appendix 10.2), using 0.1 N sodium hydroxide VS. Read the volume added between the 2 points of inflexion. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 20.17 mg of C10H16ClNO. Storage Protected from light. Action and use Adrenoceptor agonist. Preparations Elixir, nasal drops, tablets, injection. 365

VP V

EPHEDRINE HYDROCHLORIDE INJECTION

EPHEDRINE HYDROCHLORIDE INJECTION Injectio Ephedrini hydrochloridi Ephedrine hydrochloride injection is a sterile solution of ephedrine hydrochloride in water for injections. The injection complies with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Content of ephedrine hydrochloride, C10H15NO,HCl, 95.0% to 105.0% of the stated amount. Characters A clear, colourless solution. Identification A. To a volume of the injection containing about 0.1 g of ephedrine hydrochloride add 2 ml of 2 M hydrochloric acid R. Shake with two 20 ml quantities of chloroform R and discard the chloroform layers. Make the aqueous layer alkaline with 5 M ammonia R and extract with two 30 ml quantities of a mixture of 3 volumes of chloroform R and 1 volume of ethanol R. Dry the combined chloroform extracts over anhydrous sodium sulphate R, filter and evaporate to dryness at a pressure of 2 kPa, heating gently to remove the last traces of solvent R. The infrared absorption spectrum (Appendix 4.2) is concordant with the reference spectrum of ephedrine. B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (2) is similar in position, colour and size to that in the chromatogram obtained with reference solution (2). pH 4.0 to 7.0 (Appendix 6.2) Related substances Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel G. Mobile phase: Propan-2-ol - ammonia - chloroform (80 : 15 : 5). Test solution (1): Dilute a volume of the injection with water to obtain a solution containing 0.5% of ephedrine hydrochloride. Test solution (2): Dilute 1 volume of test solution (1) to 5 volumes with methanol R. Reference solution (1): Dilute 1 volume of solution (1) to 200 volumes with methanol R. Reference solution (2): A 0.1% solution of ephedrine hydrochloride RS in methanol R. Procedure: Apply separately to the plate 20 µl of each solution. After removal of the plate, allow it to dry in air, spray with 0.2% solution of ninhydrin R and heat at 100 °C for 5 minutes. Any secondary spot in the chromatogram obtained with test solution (1) is not more intense than the spot in the chromatogram obtained with reference solution (1). Disregard any spot of lighter colour than the background. 366

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: A 0.005M solution of dioctyl sodium sulphosuccinate R in a mixture of 65 volumes of methanol R, 35 volumes of water and 1 volume of glacial acetic acid R. Test solution: Dilute an accurately measured volume of the injection with methanol (80%) R to obtain a solution containing 0.1% of ephedrine hydrochloride. Reference solution: A 0.1% solution of ephedrine hydrochloride RS in methanol (65%) R. Chromatographic system: A column (20 cm × 4.6 mm) packed with stationary phase C (10 µm) (Nucleosil C18 is suitable). Detector: A spectrophotometer set at 263 nm. Flow rate: 2.0 ml/min. Volume of injection: 20 µl. Procedure: Separately inject the reference solution and the test solution. Calculate the content of ephedrine hydrochloride, C10H15NO,HCl, in the injection using the areas of the principal peaks in the chromatograms obtained with the test solution and the reference solution, and the declared content of C10H15NO,HCl in ephedrine hydrochloride RS. Storage Store in a cool place, protected from light. Action and use Beta-adrenoceptor agonist. Usual strength 1%. EPHEDRINE HYDROCHLORIDE TABLETS Tabellae Ephedrini hydrochloridi Ephedrine hydrochloride tablets contain ephedrine hydrochloride. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of ephedrine hydrochloride, C10H15NO,HCl, 92.5% to 107.5% of the stated amount. Identification A. Shake a quantity of the powdered tablets containing 0.1 g of ephedrine hydrochloride with 20 ml of 0.1 M hydrochloric acid R, filter, wash the filtrate with two 20 ml quantities of chloroform R and discard the chloroform layers. Make the aqueous layer alkaline with 5 M ammonia R and extract with two 30 ml quantities of a mixture of 3 volumes of chloroform R and 1 volume of ethanol (96%) R. Dry the combined chloroform extracts over anhydrous

VP V

ERGOCALCIFEROL

sodium sulfate R, filter and evaporate to a low volume at a pressure of 2 kPa. Prepare a disc using 0.3 g of potassium bromide R, apply the chloroform solution to the surface of the disc and heat at 50 °C for 2 min. The infrared absorption spectrum (Appendix 4.2) is concordant with the reference spectrum of ephedrine. B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (2) is similar in position, colour and size to that in the chromatogram obtained with reference solution (2). C. Triturate a quantity of the powdered tablets containing 0.4 g of ephedrine hydrochloride with two 10 ml quantities of chloroform R and discard the chloroform. Macerate the residue with 30 ml of warm ethanol (96%) R for 20 min, filter, evaporate the filtrate to dryness on a water bath and dry the residue at 80 °C. Dissolve about 10 mg of the residue in 1 ml of water and add 0.1 ml of 10% solution of copper sulfate R followed by 1 ml of 5 M sodium hydroxide R, a violet colour is produced. Add 1 ml of ether R and shake, the ether layer is purple and the aqueous layer is blue.

a 50 ml volumetric flask, wash the glass-fibre paper by water. Transfer the washing into the flask and add water to volume, mix well. Reference solution: A 0.1% solution of ephedrine hydrochloride RS in methanol (60%) R. Chromatographic system: A column (20 cm × 4.6 mm) packed with stationary phase C (10 µm) (Nucleosil C18 is suitable). Detector: A spectrophotometer set at 263 nm. Flow rate: 2.0 ml/min. Volume of injection: 20 µl. Procedure: Inject separately the reference solution and the test solution. Calculate the content of ephedrine hydrochloride, C10H15NO,HCl, in the tablets using the areas of the principal peaks in the chromatograms obtained with the test solution and the reference solution, and the declared content of C10H15NO,HCl in ephedrine hydrochloride RS.

Related substances Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel G. Mobile phase: Propan-2-ol - ammonia - chloroform (80 : 15 : 5). Test solution (1): Extract a quantity of the powdered tablets containing 0.10 g of ephedrine hydrochloride with 5 ml of methanol R and filter. Test solution (2): Dilute 1 volume of test solution (1) to 10 volumes with methanol R. Reference solution (1): Dilute 1 volume of test solution (1) to 200 volumes with methanol R. Reference solution (2): A 0.2% solution of ephedrine hydrochloride RS in methanol R. Procedure: Apply separately to the plate 10 µl of each solution. After removal of the plate, allow it to dry in air, spray with 0.2% solution of ninhydrin R and heat at 110°C for 5 minutes. Any secondary spot in the chromatogram obtained with test solution (1) is not more intense than the spot in the chromatogram obtained with reference solution (1). Disregard any spot of lighter colour than the background.

Action and use Angiotensin-converting enzyme inhibitor.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: A 0.005M solution of dioctyl sodium sulfosuccinate R in a mixture of 65 volumes of methanol R, 35 volumes of water and 1 volume of glacial acetic acid R. Test solution: Weigh 20 tablets, calculate the average mass, and finely powder. Shake an accurately weighed quantity of the powder, equivalent to about 50 mg of ephedrine hydrochloride, with 30 ml of methanol R for 10 min. Filter through glass-fibre paper (Whatman GF/C is suitable) into

Storage Store in an airtight container, protected from light.

Usual strength 10 mg. ERGOCALCIFEROL Ergocalciferolum Calciferol, vitamin D2

C28H44O

M. 396.7

Ergocalciferol is (5Z,7E,22E)-9,10-secoergosta-5,7,10(19), 22-tetraen-3β-ol. It contains not less than 97.0% and not more than 103.0% of C28H44O.

Characters A white or slightly yellowish, crystalline powder or white or almost white crystals. It is sensitive to air, heat and light. A reversible isomerisation to pre-ergocalciferol takes place in solution, depending on temperature and time. 367

ERGOCALCIFEROL

Freely soluble in ethanol (96%), soluble in fatty oils, practically insoluble in water. Solutions in volatile solvents are unstable and are to be used immediately.

Identification Apply one of the two following identifications: First identification: B, C. Second identification: A, C. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum obtained with ergocalciferol RS. B. In the test for Ergosterol, the principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). C. Melting point: 112 °C to 117 °C (Appendix 6.7). Do not grind substance into powder and dry when determining. Specific optical rotation +103° to +107° (Appendix 6.4). Dissolve 0.200 g rapidly and without heating in aldehydefree ethanol R and dilute to 25.0 ml with the same solvent. Determine within 30 min of preparing the solution, using the polarimeter tube 2 dm long. Ergosterol Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Cyclohexane - peroxide-free ether (1 : 1), the mixture containing 0.01% of butylhydroxytoluene R. Dissolution mixture: Ethylene chloride R containing 1.0% of squalane R and 0.01% of butylhydroxytoluene R. Test solution: Dissolve 0.25 g of the substance to be examined in the dissolution mixture and dilute to 5 ml with the same solvent. Reference solution (1): Dissolve 0.10 g of ergocalciferol RS in the dissolution mixture and dilute to 2 ml with the same solvent. Reference solution (2): Dissolve 5 mg of ergosterol RS in the dissolution mixture and dilute to 50 ml with the same solvent. Reference solution (3): Mix equal volumes of reference solution (1) and reference solution (2). Prepare all the above solutions immediately before use. Procedure: Apply separately to the plate 10 µl of each solution, and 20 µl of reference solution (3). Develop immediately, protected from light, over a path of 15 cm. After removal of the plate, allow the plate to dry in air. Spray three times with antimony trichloride solution R. Examine the chromatograms for 3 min to 4 min after spraying. The principal spot in the chromatogram obtained with the test solution is initially orange-yellow and then becomes brown. In the chromatogram obtained with the test solution, any slowly appearing violet spot (corresponding to ergosterol) below the principal spot is not more intense than the spot 368

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in the chromatogram obtained with reference solution (2) (0.2%). There is no spot in the chromatogram obtained with the test solution that does not correspond to one of the spots in the chromatograms obtained with reference solutions (1) and (2). The test is not valid unless the chromatogram obtained with reference solution (3) shows two clearly separated spots.

Reducing substances Not more than 20 ppm. Dissolve 0.1 g in aldehyde-free ethanol R and dilute to 10.0 ml with the same solvent. Add 0.5 ml of a 0.5% solution of tetrazolium blue R in aldehyde-free ethanol R and 0.5 ml of dilute tetramethylammonium hydroxide solution R. Allow to stand for exactly 5 min and add 1.0 ml of glacial acetic acid R. Prepare a reference solution at the same time and in the same manner using 10.0 ml of a solution containing 0.2 µg/ml of hydroquinone R in aldehyde-free ethanol R. Measure the absorbance (Appendix 4.1) of the two above solutions at 525 nm using as the compensation liquid 10.0 ml of aldehyde-free ethanol R treated in the same manner. The absorbance of the test solution is not greater than that of the reference solution. Assay Carry out the operations as rapidly as possible, avoiding exposure to actinic light and air. Examine by liquid chromatography (Appendix 5.3). Mobile phase: Hexane - pentanol (997 : 3). Test solution: Dissolve 10.0 mg of the substance to be examined without heating in 10.0 ml of toluene R and dilute to 100.0 ml with the mobile phase. Standard solution: Dissolve 10.0 mg of ergocalciferol RS without heating in 10.0 ml of toluene R and dilute to 100.0 ml with the mobile phase. Resolution solution: Dissolve 0.5 g of cholecalciferol for performance test RS in 2 ml of toluene R and dilute to 10.0 ml with the mobile phase; heat under a reflux condenser in a water bath at 90 °C for 45 min and cool. Chromatographic system: A column (25 cm × 4.6 mm) packed with a suitable silica gel (5 µm to 10 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 2 ml/min. Procedure: Inject a suitable volume of the resolution solution. Adjust the sensitivity of the system so that the height of the peak corresponding to cholecalciferol is at least 50% of the full scale of the recorder. Inject the resolution solution 6 times. When the chromatograms are recorded in the prescribed conditions, the relative retention times are about 0.4 for pre-cholecalciferol, 0.5 for trans-cholecalciferol and 1.0 for cholecalciferol. The relative standard deviation of the areas of cholecalciferol peaks is not greater than 1% and the resolution between the peaks corresponding to pre-

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ERGOCALCIFEROL TABLETS

cholecalciferol and trans-cholecalciferol is not less than 1.0. If necessary adjust the proportions of the constituents and the flow rate of the mobile phase to obtain this resolution. Inject a suitable volume of the standard solution. Adjust the sensitivity of the system so that the height of the peak corresponding to ergocalciferol is at least 50% of the full scale of the recorder. Inject the same volume of the test solution and record the chromatogram in the same manner. Calculate the percentage content of C28H44O from the expression:

100 × W2 × S1 W1 × S 2

In which: W1: Mass of the substance to be examined in the test solution, in milligrams. W2: Mass of ergocalciferol RS in the standard solution, in milligrams. S1: Area (or height) of the peak due to ergocalciferol in the chromatogram obtained with the test solution. S2: Area (or height) of the peak due to ergocalciferol in the chromatogram obtained with the standard solution.

Storage Store in an airtight container, under nitrogen, protected from light, at a temperature between 2 °C and 8 °C. The contents of an opened container are to be used immediately. Action and use Vitamin D. Preparation Tablets. ERGOCALCIFEROL TABLETS Tabellae Ergocalciferoli Ergocalciferol tablets contain ergocalciferol. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of ergocalciferol, C28H44O, 90.0% to 125.0% of the stated amount. Identification A. Triturate a quantity of the powdered tablets containing the equivalent of about 0.5 mg of ergocalciferol with 10 ml of chloroform R and filter. Add to the filtrate 0.3 ml of anhydride acetic R and 0.1 ml of sulfuric acid R, shake vigorously. A bright red colour is produced, which turns quickly to violet, blue and green. B. In the test for Uniformity of content, the principal peak in the chromatogram obtained with the test solution has the same retention time as the peak due to ergocalciferol in the chromatogram obtained with the reference solution.

Uniformity of content (Appendix 11.2) Examine by liquid chromatography (Appendix 5.3). Carry out the following procedure protected from light Mobile phase: Hexane - pentan-1-ol (992 : 8). Reference solution: Prepare a solution of ergocalciferol RS in hexane R having the same concentration as the test solution. Test solution: For tablets containing more than 0.25 mg of ergocalciferol: Add 4 ml of water to one tablet, disperse the tablet completely with the aid of ultrasound. Add 12 ml of dimethyl sulphoxide R, mix, extract with 100 ml of hexane R by shaking by mechanical means for 30 minutes. Centrifuge the hexane layer and use the clear supernatant liquid. For tablets containing equal to or less than 0.25 mg of ergocalciferol: Cary out the same procedure but using 2 ml of water, 6 ml of dimethyl suphoxide R and 25 ml of hexane R. Resolution solution: Dissolve (without heating) 50.0 mg of colecalciferol RS in 10 ml of toluene R, dilute to 100.0 ml with the mobile phase, mix well. Dilute 5.0 ml of the solution to 50.0 ml with mobile phase. Heat 5.0 ml of the resulting solution under a reflux condenser with a current of nitrogen in a water-bath for 60 minutes. Cool, dilute to 50.0 ml with the mobile phase, mix well. Chromatographic system: A column (20 cm × 4.6 mm) packed with stationary phase A (5 µm) (Partisil is suitable). Detector: A spectrophotometer set at 254 nm. Flow rate: 2.0 ml/min. Procedure: Inject a suitable volume of the resolution solution and record the chromatogram. Adjust the sensitivity of the system so that the height of the peak due to colecalciferol is more than 50% of full-scale deflection. In the chromatogram obtained with the prescribed conditions, approximate retention times relative to colecalciferol are 0.4 for precolecalciferol and 0.5 for trans-colecalciferol. The test is not valid unless the resolution between the peaks corresponding to precolecalciferol and trans-colecalciferol is at least 1.0. If necessary, adjust the proportions of the constituents and the flow rate of the mobile phase to obtain this resolution. Inject a suitable volume of the reference solution. Adjust the sensitivity of the system so that the height of the peak due to ergocalciferol is more than 50% of full-scale deflection. Inject the same volume of the test solution. Calculate the content of ergocalciferol, C28H44O, in each tablet using heights of the peaks due to ergocalciferol in the chromatograms obtained with the test solution and the reference solution and the declared content of C28H44O in ergocalciferol RS. Each µg of ergocalciferol is equivalent to 40 IU of vitamin D. Assay Use the average of the 10 individual results obtained in the test for Uniformity of content. 369

ERYTHROMYCIN

Storage Store in a cool and dry place, protected from light. Action and use Vitamin D. Usual strength 1.25 mg. ERYTHROMYCIN Erythromycinum

Erythromycin is a mixture of macrolide antibiotics produced by a strain of Streptomyces erythreus, the main component being (3R,4S,5S,6R,7R,9R,11R,12R,13S,14R)-4-[(2,6dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyl) oxy]-14-ethyl-7,12,13-trihydroxy-3,5,7,9,11,13hexamethyl-6-[(3,4,6-trideoxy-3-dimethylamino-β-Dxylo-hexopyranosyl)-oxy]oxacyclotetradecane-2,10-dione (erythromycin A).

Content Sum of the contents of erythromycin A, erythromycin B and erythromycin C: 93.0% to 102.0%, calculated with reference to the anhydrous substance. Erythromycin B: Not more than 5.0%. Erythromycin C: Not more than 5.0%. Characters White or slightly yellow powder or colourless or slightly yellow crystals, slightly hygroscopic. Slightly soluble in water (the solubility decreases as the temperature rises), freely soluble in ethanol (96%), soluble in methanol. Identification Apply one of the two following identifications: First identification: A. 370

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Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum obtained with erythromycin RS. Disregard any band in the region from 1980 cm-1 to 2050 cm-1. If the spectra obtained show differences, dissolve 50 mg of the substance to be examined and of the reference substance separately in 1.0 ml of methyle chloride R, dry at 60 °C at a pressure not exceeding 670 Pa for 3 h and record new spectra using the residues. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: 2-Propanol - a 15% solution of ammonium acetate previously adjusted to pH 9.6 with ammonia - ethyl acetate (4 : 8 : 9). Allow to settle and use the upper layer. Test solution: Dissolve 10 mg of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Reference solution (1): Dissolve 10 mg of erythromycin A RS in methanol R and dilute to 10 ml with the same solvent. Reference solution (2): Dissolve 20 mg of spiramycin RS in methanol R and dilute to 10 ml with the same solvent. Procedure: Apply separately to the plate 10 µl of each solution. Develop over 2/3 of the plate. Allow the plate to dry in air and spray with anisaldehyde solution in ethanol R. Heat at 110 °C for 5 min. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1) and its position and colour are different from those of the spots in the chromatogram obtained with reference solution (2). C. To about 5 mg add 5 ml of a 0.02% solution of xanthydrol R in a mixture of 1 volume of hydrochloric acid R and 99 volumes of acetic acid R and heat on a water-bath. A red colour develops. D. Dissolve about 10 mg in 5 ml of 7 M hydrochloric acid R and allow to stand for 10 min to 20 min. A yellow colour develops.

Specific optical rotation -71° to -78°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 1.00 g in ethanol (96%) R and dilute to 50.0 ml with the same solvent. The specific optical rotation is determined at least 30 min after preparing the solution. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: To 50 ml of a 3.5% solution of dipotassium hydrogen phosphate R adjusted to pH 9.0 ± 0.05 with dilute phosphoric acid R, add 400 ml of water, 165 ml of 2-methyl-2-propanol R and 30 ml of acetonitrile R, and dilute to 1000 ml with water. Dissolution mixture: A mixture of 1 volume of methanol R and 3 volumes of phosphate buffer solution pH 7.0 R.

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Test solution: Dissolve 40.0 mg of the substance to be examined in the dissolution mixture and dilute to 10.0 ml with the same solvent mixture. Reference solution (1): Dissolve 40.0 mg of erythromycin A RS in the dissolution mixture and dilute to 10.0 ml with the same solvent mixture. Reference solution (2): Dissolve 10.0 mg of erythromycin B RS and 10.0 mg of erythromycin C RS in the dissolution mixture and dilute to 50.0 ml with the same solvent mixture. Reference solution (3): Dissolve 5 mg of N-demethylerythromycin A RS in reference solution (2). Add 1.0 ml of reference solution (1) and dilute to 25 ml with reference solution (2). Reference solution (4): Dilute 3.0 ml of reference solution (1) to 100.0 ml with the dissolution mixture. Reference solution (5): Transfer 40 mg of erythromycin A RS to a glass vial and spread evenly such that it forms a layer not more than about 1 mm thick. Heat at 130 °C for 4 h. Allow to cool, dissolve in the dissolution mixture and dilute to 10 ml with the same solvent mixture. Chromatographic system: A column (25 cm × 4.6 mm) packed with styrenedivinylbenzene copolymer for chromatography R (8 µm) with a pore size of 100 nm. Column temperature: 70 °C using a water-bath for the column and at least one-third of the tubing preceding the column. Detector: A spectrophotometer set at 215 nm. Flow rate: 2.0 ml/min. Volume of injection: 100 µl. Procedure: Inject the test solution, reference solution (3), (4) and (5). The run time is 5 times the retention time of erythromycin A. Relative retentions with reference to erythromycin A (retention time = about 15 min) are: Impurity A = about 0.3; impurity B = about 0.45; erythromycin C = about 0.5; impurity C = about 0.9; impurity D = about 1.4; impurity F = about 1.5; erythromycin B = about 1.8 and impurity E = about 4.3. System suitability: In the chromatogram obtained with reference solution (3), the resolution between the peaks due to impurity B and to erythromycin C is at least 0.8; between the peaks due to impurity B and to erythromycin A is at least 5.5. If necessary, adjust the concentration of 2-methyl-2-propanol in the mobile phase or reduce the flow rate to 1.5 ml or 1.0 ml/min. Limits: Correction factors: For the calculation of contents, multiply the peak areas of the following impurities (use the chromatogram obtained with reference solution (5) to identify them) by the corresponding correction factor: impurity E = 0.09; impurity F = 0.15. The corrected area, if necessary, of any impurity peak is not more than the area of the principal peak in the chromatogram obtained with reference solution (4) (3.0%).

ERYTHROMYCIN

Sum of the areas of all the impurity peaks are not more than 2.3 times the area of the principal peak in the chromatogram obtained with reference solution (4) (7.0%). Disregard any peak with an area less than 0.02 times the area of the principal peak in the chromatogram obtained with reference solution (4) (0.06%); disregard the peaks due to erythromycin B and to erythromycin C. Note: Impurity A: Erythromycin F. Impurity B: N-demethylerythromycin A. Impurity C: Erythromycin E. Impurity D: Anhydroerythromycin A. Impurity E: Erythromycin A enol ether. Impurity F: Pseudoerythromycin A enol ether.

Thiocyanate Not more than 0.3%. Prepare the solutions immediately before use and protect from actinic light. Blank solution: Dilute 1.0 ml of a 9% solution of ferric chloride R to 50.0 ml with methanol R. Test solution: Dissolve 0.100 g (m g) of the substance to be examined in 20 ml of methanol R, add 1.0 ml of a 9% solution of ferric chloride R and dilute to 50.0 ml with methanol R. Prepare 2 independent reference solutions: Reference solution: Dissolve 0.100 g of potassium thiocyanate R, previously dried at 105 °C for 1 h in methanol R and dilute to 50.0 ml with the same solvent. Dilute 5.0 ml to 50.0 ml with methanol R. To 5.0 ml of the solution, add 1.0 ml of a 9% solution of ferric chloride R and dilute to 50.0 ml with methanol R. Measure the absorbances (Appendix 4.1) of each reference solution (D1, D2) and of the test solution (D) at the maximum at about 492 nm. Suitability value: m  D  S =  1  ×  2   m1   D2  In which: m1, m2 = mass in grams of the potassium thiocyanate used to prepare the respective reference solutions. The test is not valid unless S is 0.985 to 1.015. Calculate the percentage content of thiocyanate from the expression:  D   m   58.08  D   ×   (0.5) ×  1  ×  2  m  97.18   m1   D2  58.08 = relative molecular mass of the thiocyanate moiety. 97.18 = relative molecular mass of potassium thiocyanate.

Water Not more than 6.5% (Appendix 10.3). Determine on 0.200 g. Use a 10% solution of imidazole R in anhydrous methanol R as the solvent. 371

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ERYTHROMYCIN TABLETS

Sulfated ash Not more than 0.2% (Appendix 9.9). Determined on 1.0 g.

Loss on drying Not more than 5.0% (Appendix 9.6). (0.1 g of the powdered tablets; in vacuum at 60 °C for 3 hours).

Assay Examine by liquid chromatography (Appendix 5.3). Chromatographic system as described in the test for Related substances. System suitability: Inject reference solution (1) six times, the relative standard deviation of the areas of the peaks due to erythromycin A is not more than 1.2%. Inject the test solution and reference solutions (1) and (2). Calculate the percentage content of erythromycin A in the test solution using the chromatogram obtained with reference solution (1). Calculate the percentage contents of erythromycin B and erythromycin C in the test solution using the chromatogram obtained with reference solution (2).

Assay Weigh and finely powder 20 coating-removed tablets. Transfer an accurately weighed quantity of the powder containing 25 mg of erythromycin to a 100 ml volumetric flask, add 50 ml of methanol R, shake well, dilute to volume with methanol R, and mix. Carry out the biological assay of antibiotics for erythromycin (Appendix 13.9). Calculate the content of erythromycin, C37H67NO13, in the tablets, taking each 1000 IU found to be equivalent to 1 mg of C37H67NO13.

Storage Store in a well-closed container, protected from light. Action and use Macrolide antibiotics. Preparations Tablets, capsules, coated tablets, injection, eye ointment, lotion, cream. ERYTHROMYCIN TABLETS Tabellae Erythromycini

Storage Store in an airtight container or in blisters, in a cool place, protected from light. Action and use Antibacterial. Usual strength 250 mg; 500 mg. ERYTHROMYCIN ETHYL SUCCINATE Erythromycini ethyl succinas

Erythromycin tablets contain erythromycin. They are made gastro-resistant by enteric-coating. The tablets comply with the requirements stated for enteric-coated tablets under “Tablets” (Appendix 1.20) and with the following requirements.

Content of erythromycin, C37H67NO13, 90.0% to 110.0% of the stated amount. Identification A. Shake a quantity of the powdered tablets containing about 0.1 g of erythromycin with 5 ml of chloroform R, decolourise, if necessary, with activated charcoal R. Filter the extract and evaporate to dryness. The infrared absorption spectrum (Appendix 4.2) of the residue is concordant with the spectrum of erythromycin RS. B. Shake a quantity of the powdered tablets containing about 3 mg of erythromycin with 2 ml of acetone R and add 2 ml of hydrochloric acid R, an orange colour is produced which changes to red and then to deep violetred. Add 2 ml of chloroform R and shake, the chloroform layer becomes violet. Disintegration The tablets comply with the disintegration test for entericcoated tablets (Appendix 11.7), test for 60 min in the acid medium. 372

Erythromycin

Molecular Formula

R1

R2

M.

A B C

C43H75NO16

OH

CH3

862

C43H75NO15

H

CH3

846

C42H73NO16

OH

H

848

Main component of erythromycin ethyl succinate is (3R,4S,5S,6R,7R,9R,11R,12R,13S,14R)-4-[(2,6-dideoxy3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyl) oxy]-14-ethyl-7,12,13-trihydroxy-3,5,7,9,11,13hexamethyl-6-[[3,4,6-trideoxy-3-(dimethylamino)-2O-(4-ethoxy-4-oxobutanoyl)-β-D-xylo-hexopyranosyl] oxy]oxacyclotetradecane-2,10-dione (erythromycin A ethylsuccinate).

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Content Sum of erythromycin A, erythromycin B and erythromycin C is not less than 78.0%, calculated with reference to the anhydrous substance. Erythromycin B: Not more than 5.0%, calculated with reference to the anhydrous substance. Erythromycin C: Not more than 5.0%, calculated with reference to the anhydrous substance. Characters White, crystalline powder, hygroscopic. Practically insoluble in water, freely soluble in acetone, in ethanol and in methanol. Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum obtained with erythromycin ethyl succinate RS. Specific optical rotation -70° to -82°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.100 g in acetone R and dilute to 10.0 ml with the same solvent. Measure the angle of rotation at least 30 min after preparing the solution. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: To 50 ml of a 3.5% solution of dipotassium hydrogen phosphate R adjusted to pH 8.0 with 10% solution of phosphoric acid R, add 400 ml of water, 165 ml of 2-methyl-2-propanol R and 30 ml of acetonitrile R, and dilute to 1000 ml with water. Filter and degas. Hydrolysis solution: A 2.0% solution of dipotassium hydrogen phosphate R adjusted to pH 8.0 with phosphoric acid R. Test solution: Dissolve 0.115 g of the substance to be examined in 25 ml of methanol R. Add 20 ml of the hydrolysis solution, mix and allow to stand at room temperature for at least 12 h. Dilute to 50.0 ml with the hydrolysis solution. Reference solution (1): Dissolve 40.0 mg of erythromycin A RS in 10 ml of methanol R and dilute to 20.0 ml with the hydrolysis solution. Reference solution (2): Dissolve 10.0 mg of erythromycin B RS and 10.0 mg of erythromycin C RS in 50 ml of methanol R. Add 5.0 ml of reference solution (1) and dilute to 100.0 ml with the hydrolysis solution. Reference solution (3): Dissolve 2 mg of N-demethylerythromycin A RS (impurity B) in 20 ml of reference solution (2). Reference solution (4): Dilute 3.0 ml of reference solution (1) to 100.0 ml with a mixture of equal volumes of methanol R and the hydrolysis solution. Reference solution (5): Dissolve 40 mg of erythromycin A RS, previously heated at 130 °C for 3 h, in 10 ml of methanol R and dilute to 20 ml with the hydrolysis solution.

ERYTHROMYCIN ETHYL SUCCINATE

Chromatographic system: A column (25 cm × 4.6 mm) packed with styrenedivinylbenzene copolymer for chromatography R (8 µm) with a pore size of 100 nm. Column temperature: 70 °C using a water-bath for the column and at least one-third of the tubing preceding the column. Detector: A spectrophotometer set at 215 nm. Flow rate: 2.0 ml/min. Volume of injection: 200 µl. Procedure: Inject the test solution, reference solution (1), reference solution (3), reference solution (4) and reference solution (5). Record the chromatogram for 5 times the retention time of erythromycin A; begin integration after the hydrolysis peak. Relative retentions with reference to erythromycin A (retention time = about 15 min) are: hydrolysis peak = less than 0.3; impurity B = about 0.45; erythromycin C = about 0.5; impurity C (erythromycin E) = about 0.9; impurity G (erythromycin N-ethyl succinate) = about 1.3; impurity D (anhydroerythomycin A) = about 1,4; impurity F (pseudoerythromycin A enol ether) = about 1.5; erythromycin B = about 1.8; and impurity E (erythromycin A enol ether) = about 4.3. System suitability: In the chromatogram obtained with reference solution (3), the resolution between the peaks due to impurity B and to erythromycin C is at least 0.8; between the peaks due to impurity B and to erythromycin A is at least 5.5. Limits: Correction factors: For the calculation of contents, multiply the peak areas of the following impurities by the corresponding correction factor: impurity E = 0.09; impurity F = 0.15; impurity G = 0.14; use the chromatogram obtained with reference solution (5) to identify the peaks due to impurities E and F. The area of any impurity peak is not more than the area of the principal peak in the chromatogram obtained with reference solution (4) (3.0%). The sum of the areas of all impurity peaks is not more than 1.67 times the area of the principal peak in the chromatogram obtained with reference solution (4) (5.0%). Disregard any peak with an area less than 0.02 times the area of the principal peak in the chromatogram obtained with reference solution (4) (0.06%).

Free erythromycin Not more than 6.0%. Examine by liquid chromatography (Appendix 5.3). Mobile phase: Mix 35 volumes of acetonitrile R and 65 volumes of a solution containing 0.34% of potassium dihydrogen phosphate R and 0.20% of triethylamine R, adjusted to pH 3.0 with 2 M phosphoric acid R. Filter and degas. 373

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ERYTHROMYCIN STEARATE

Test solution: Dissolve 0.250 g of the substance to be examined in acetonitrile R and dilute to 50.0 ml with the same solvent. Reference solution: Dissolve 75.0 mg of erythromycin A RS in acetonitrile R and dilute to 50.0 ml with the same solvent. Dilute 5.0 ml of the solution to 25.0 ml with acetonitrile R. Chromatographic system: A column (25 cm × 4.6 mm) packed with B (5 µm). Detector: A spectrophotometer set at 195 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Procedure: Inject the test solution, record the chromatogram for twice the retention time of erythromycin ethyl succinate (retention time = about 24 min). Inject the reference solution, record the chromatogram for twice the retention time of erythromycin A (retention time = about 8 min). Limit: The area of the peak corresponding to free erythromycin is not more than the area of the principal peak in the chromatogram obtained with reference solution.

Water Not more than 3.0% (Appendix 10.3). Determined on 0.30 g. Use a 10.0% solution of imidazole R in anhydrous methanol R as the solvent. Sulfated ash Not more than 0.3% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase, test solution, reference solution (1), reference solution (2) and chromatographic system as described in the test for Related substances. System suitability: The relative standard deviation of the peak areas for six injections of reference solution (1) is not more than 1.2%. Calculate the percentage content of erythromycin A using the chromatogram obtained with reference solution (1). Calculate the contents of erythromycin B and erythromycin C using the chromatogram obtained with reference solution (2). Storage Store in an airtight container, protected from light. Action and use Macrolide antibiotic. Preparations Oral suspension; tablets.

374

ERYTHROMYCIN STEARATE Erythromycini stearas

Erythromycin stearate is a mixture of the stearates of erythromycin and stearic acid. The main component is the octadecanoate of (3R,4S,5S,6R,7R,9R,11R,12R,13S,14R)4-[(2,6-dideoxy-3-C-methyl-3-O-methyl-α-L-ribohexopyranosyl)oxy]-14-ethyl-7,12,13-trihydroxy-3,5,7,9,11,13hexamethyl-6-[[3,4,6-trideoxy-3-(dimethylamino)-β-Dxylo-hexopyranosyl]oxy]oxacyclotetradecane-2,10-dione (erythromycin A stearate). Fermentation product.

Content Sum of the contents of erythromycin A, erythromycin B and erythromycin C: minimum 60.5% (calculated with reference to the anhydrous substance). Erythromycin B: Not more than 5.0%. Erythromycin C: Not more than 5.0%. Characters White or almost white, crystalline powder. Practically insoluble in water, soluble in acetone and in methanol. Solutions may be opalescent. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of erythromycin stearate RS. A. Thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Mix well a mixture of 2-propanol - a 15% solution of ammonium acetate previously adjusted to pH 9.6 with ammonia - ethyl acetate (4 : 8 : 9). Allow to settle and use the upper layer. Test solution: Dissolve 28 mg of the substance to be examined in methanol R and dilute to 10 ml with the same solvent.

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Reference solution (1): Dissolve 20 mg of erythromycin A RS in methanol R and dilute to 10 ml with the same solvent. Reference solution (2): Dissolve 10 mg of stearic acid R in methanol R and dilute to 10 ml with the same solvent. Procedure: Apply separately to the plate 5 µl of each solution. Develop over 2/3 of the plate. Dry the plate in air. Detection A: Spray with a solution containing 0.2 g/L of dichlorofluorescein R and 0.1 g/L of rhodamine B R in ethanol (96%) R. Maintain the plate for a few seconds in the vapour above a water-bath. Examine in ultraviolet light at 365 nm. The chromatogram obtained with the test solution shows 2 spots, one of which corresponds in position to the principal spot in the chromatogram obtained with reference solution (1) and the other to the principal spot in the chromatogram obtained with reference solution (2). Detection B: Spray the plate with anisaldehyde solution R1. Heat at 110 °C for 5 min and examine in daylight. The spot in the chromatogram obtained with the test solution corresponds in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1).

Free stearic acid Not more than 14.0% of C18H36O2, calculated with reference to the anhydrous substance. Dissolve 0.400 g of the substance to be examined in 50 ml of methanol R. Titrate with 0.1 N sodium hydroxide VS, determining the end-point potentiometrically (Appendix 10.2). Calculate the volume of 0.1 N sodium hydroxide VS required per gram of the substance to be examined (n1 ml). Dissolve 0.500 g of the substance to be examined in 30 ml of methylene chloride R. If the solution is opalescent, filter and shake the residue with 3 quantities, each of 25 ml, of methylene chloride R. Filter, if necessary, and rinse the filter with methylene chloride R. Reduce the volume of the combined filtrate and rinsings to 30 ml by evaporation on a water-bath. Add 50 ml of glacial acetic acid R and titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). Calculate the volume of 0.1 N perchloric acid VS required per gram of the substance to be examined (n2 ml). Calculate the percentage content of C18H36O2 from the expression: 100 2.845(n1 − n2 ) × 100 − h Where: h = percentage water content (%). Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: To 50 ml of a 3.5% solution of dipotassium hydrogen phosphate R adjusted to pH 9.0 ± 0.05 with 2 M phosphoric acid R, add 400 ml of water, 165 ml of 2-methyl-2-propanol R and 30 ml of acetonitrile R, and dilute to 1000 ml with water. Test solution: Dissolve 55.0 mg of the substance to be examined in 5.0 ml of methanol R and dilute to 10.0 ml

ERYTHROMYCIN STEARATE

with buffer solution pH 8.0 R1. Centrifuge and use the clear solution. Reference solution (1): Dissolve 40.0 mg of erythromycin A RS in 5.0 ml of methanol R and dilute to 10.0 ml with buffer solution pH 8.0 R1. Reference solution (2): Dissolve 10.0 mg of erythromycin B RS and 10.0 mg of erythromycin C RS in 25.0 ml of methanol R and dilute to 50.0 ml with buffer solution pH 8.0 R1. Reference solution (3): Dissolve 5 mg of N-demethylerythromycin A RS in reference solution (2). Add 1.0 ml of reference solution (1) and dilute to 25 ml with reference solution (2). Reference solution (4): Dilute 3.0 ml of reference solution (1) to 100.0 ml with a mixture of equal volumes of methanol R and buffer solution pH 8.0 R1. Reference solution (5): Transfer 40 mg of erythromycin A RS to a glass vial and spread evenly such that it forms a layer not more than about 1 mm thick. Heat at 130 °C for 4 h. Allow to cool and dissolve in a mixture of methanol - buffer solution pH 8.0 R1 (1 : 3) and dilute to 10 ml with the same mixture of solvents. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase styrene-divinylbenzene copolymer R (8 µm) with a pore size of 100 nm. Column temperature: 70 °C using a water-bath for the column and at least one-third of the tubing preceding the column. Detector: A spectrophotometer set at 215 nm. Flow rate: 2.0 ml/min. Volume of injection: 100 µl. Procedure: Inject the test solution and reference solutions (3), (4) and (5). The run time is 5 times the retention time of erythromycin A. Relative retention with reference to erythromycin A (retention time = about 15 min): impurity A = about 0.3; impurity B = about 0.45; erythromycin C = about 0.5; impurity C = about 0.9; impurity D = about 1.4; impurity F = about 1.5; erythromycin B = about 1.8; impurity E = about 4.3. System suitability: In the chromatogram obtained with reference solution (3), the resolution between the peaks due to impurity B and erythromycin C is at least 0.8; between the peaks due to impurity B and erythromycin A is at least 5.5. If necessary, adjust the concentration of 2-methyl-2-propanol in the mobile phase or reduce the flow rate to 1.5 ml/min or 1.0 ml/min. Limits: Correction factors: for the calculation of contents, multiply the peak areas of the following impurities (use the chromatogram obtained with reference solution (5) to identify them) by the corresponding correction factor: impurity E = 0.09; impurity F = 0.15; 375

VP V

ERYTHROMYCIN STEARATE CAPSULES

Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (4) (3%). The sum of the areas of all the impurity peaks is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (4) (6%). Disregard any peak with an area less than 0.02 times the area of the principal peak in the chromatogram obtained with reference solution (4) (0.06%), disregard the peaks due to erythromycin B and erythromycin C.

Note: Impurity A: (3R,4S,5S,6R,7R,9R,11R,12R,13S,14R)-4-[(2,6-dideoxy3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyl)oxy]-14-ethyl7,12,13-trihydroxy-3-(hydroxymethyl)-5,7,9,11,13-pentamethyl-6[[3,4,6-trideoxy-3-(dimethylamino)-β-D-xylo-hexopyranosyl]oxy] oxacyclotetradecane-2,10-dione (erythromycin F). Impurity B: (3R,4S,5S,6R,7R,9R,11R,12R,13S,14R)-4-[(2,6-dideoxy3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyl)oxy]-14-ethyl7,12,13-trihydroxy-3,5,7,9,11,13-hexamethyl-6-[[3,4,6-trideoxy-3(methylamino)-β-D-xylo-hexopyranosyl]oxy]oxacyclotetradecane2,10-dione (3’’-N- desmethylerythromycin A). Impurity C: (2S,4aR,4’R,5’S,6’S,7R,8S,9R,10R,12R,14R,15R,16S)7-ethyl-5’,8,9,14-tetrahydroxy-4’-methoxy-4’,6’,8,10,12,14,16heptamethyl-15-[[3,4,6-trideoxy-3-(dimethylamino)-b-D-xylohexopyranosyl]oxy]hexadecahydrospiro[5H,11H-1,3-dioxino[5,4-c] oxacyclotetradecin-2,2’-pyrane]-5,11-dione (erythromycin E). Impurity D: (1S,2R,3R,4S,5R,8R,9S,10S,11R,12R,14R)-9-[(2,6dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyl)oxy]-5ethyl-3-hydroxy-2,4,8,10,12,14-hexamethyl-11-[[3,4,6-trideoxy3-(dimethylamino)-β-D-xylo-hexopyranosyl]oxy]-6,15,16trioxatricyclo[10.2.1.11,4]hexadecan-7-one(anhydroerythromycin A). Impurity E: (2R,3R,4S,5R,8R,9S,10S,11R,12R)-9-[(2,6-dideoxy3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyl)oxy]-5ethyl-3,4-dihydroxy-2,4,8,10,12,14-hexamethyl-11-[[3,4,6trideoxy-3-(dimethylamino)-β-D-xylo-hexopyranosyl]oxy]-6, 15-dioxabicyclo[10.2.1]pentadec-1(14)-en-7-one (erythromycin A enol ether), Impurity F: (2R,3R,6R,7S,8S,9R,10R)-7-[(2,6-dideoxy-3-C-methyl -3-O-methyl-α-L-ribo-hexopyranosyl)oxy]-3-[(1R,2R)-1,2dihydroxy-1-methylbutyl]-2,6,8,10,12-pentamethyl-9-[[3,4,6trideoxy-3-(dimethylamino)-β-D-xylo-hexopyranosyl]oxy]-4, 13-dioxabicyclo[8.2.1]tridec-1(12)-en-5-one (pseudoerythromycin A enol ether).

Water Not more than 4.0% (Appendix 10.3). Determined on 0.300 g. Use a 100 g/L solution of imidazole R in anhydrous methanol R as the solvent. Sulfated ash Not more than 0.5% (Appendix 9.9, method 2). Determined on 1.0 g. 376

Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. Inject the test solution and reference solutions (1) and (2). System suitability: In the chromatogram obtained with reference solution (1): the symmetry factor of the peak due to erythromycin A is not more than 5, the relative standard deviation of the peak areas for 6 replicate injections is not more than 1.2%. Calculate the content of erythromycin A using the chromatogram obtained with reference solution (1). Calculate the contents of erythromycin B and erythromycin C using the chromatogram obtained with reference solution (2). Storage Store in an airtight container. Action and use Macrolide antibacterial. Preparation Tablets. ERYTHROMYCIN STEARATE CAPSULES Capsulae Erythromycini stearatis Erythromycin stearate capsules contain erythromycin stearate. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of erythromycin, C37H67NO13, 90.0% to 110.0% of the stated amount. Identification Take the contents of 10 capsules, and finely powder. A. To a quantity of the powder containing the equivalent of 0.1 g of erythromycin stearate add 10 ml of water and shake well. Decant the supernatant liquid and discard. Extract the residue by shaking with 10 ml of methanol R, filter the extract and evaporate to dryness. The infrared absorption spectrum (Appendix 4.2) of the residue after drying at a pressure not exceeding 0.7 kPa is concordant with the reference spectrum of erythromycin stearate or with the spectrum of erythromycin stearate RS. B. Shake a quantity of the powder containing the equivalent of 3 mg of erythromycin with 2 ml of acetone R and add 2 ml of hydrochloric acid R, an orange colour is produced which changes to red and then to deep violet-red. Add 2 ml of chloroform R and shake, the chloroform layer becomes violet. C. Extract a quantity of the powder containing the equivalent of 50 mg of erythromycin with 10 ml of chloroform R, filter and evaporate to dryness. Heat 0.1 g

VP V

of the residue gently with 5 ml of 2 M hydrochloric acid R and 10 ml of water until the solution boils, oily globules rise to the surface. Cool, transfer the fatty layer to a test tube, heat it with 3 ml of 0.1 M sodium hydroxide R and allow to cool, the solution sets to a gel. Add 10 ml of hot water and shake, the solution froths. To 1 ml add 10% solution of calcium chloride R, a granular precipitate is produced which is insoluble in hydrochloric acid R.

Loss on drying Not more than 5.0% (Appendix 9.6). (100 mg of the contents of the capsules; in vacuum at 60 °C for 3 hours). Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of a 2.722% solution of sodium acetate R adjusted to pH 5.0 with glacial acetic acid R. Rotation speed: 50 rpm. Time: 45 min. Procedure: Test solution: After the specified time, withdraw a sample of the medium and filter, discard the first 20 ml of the filtrate. Transfer 5.0 ml of the filtrate to a 100 ml volumetric flask, add 40 ml of glacial acetic acid R and 10 ml of a 0.5% w/w solution of 4-dimethylaminobenzaldehyde R in glacial acetic acid R, and dilute to volume with a mixture of 35 volumes of glacial acetic acid R and 70 volumes of hydrochloric acid R. Allow to stand for 15 minutes. Reference solution: A suitable solution of erythromycin stearate RS in dissolution medium is prepared in a similar manner beginning at the words "Transfer 5.0 ml of the filtrate…". Measure the absorbances (Appendix 4.1) of the reference solution and the test solution at the maximum at 485 nm in a 1 cm cell, using in the reference cell the dissolution medium that has been subjected to the conditions of the test. Calculate the total content of erythromycin, C37H67NO13, in the medium using the declared content of C37H67NO13 in erythromycin stearate RS. Tolerance: Not less than 70% (Q) of the labelled amount of erythromycin, C37H67NO13, is dissolved in 45 minutes. Assay Weigh 20 capsules and calculate the average mass of the capsule contents and finely powder. Transfer an accurately weighed quantity of the powder containing the equivalent of 25 mg of erythromycin to a 100 ml volumetric flask, add 50 ml of methanol R, shake well, dilute to volume with methanol R, and mix. Carry out the biological assay of antibiotics for erythromycin (Appendix 13.9). Calculate the content of erythromycin, C37H67NO13, in the capsules, taking each 1000 IU found to be equivalent to 1 mg of C37H67NO13.

ERYTHROMYCIN STEARATE TABLETS

Storage Store in an airtight container or in blisters in a cool place, protected from light. Action and use Antibacterial. Usual strength 250 mg; 500 mg. ERYTHROMYCIN STEARATE TABLETS Tabellae Erythromycini stearatis Erythromycin stearate tablets contain erythromycin stearate. They may be coated. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of erythromycin, C37H67NO13, 90.0% to 110.0% of the stated amount. Identification Take 10 tables (if necessary, remove the coating), and finely powder. A. To a quantity of the powdered tablets containing the equivalent of 0.1 g of erythromycin stearate add 10 ml of water and shake well. Decant the supernatant liquid and discard. Extract the residue by shaking with 10 ml of methanol R, filter the extract and evaporate to dryness. The infrared absorption spectrum (Appendix 4.2) of the residue after drying at a pressure not exceeding 0.7 kPa is concordant with the reference spectrum of erythromycin stearate or with the spectrum of erythromycin stearate RS. B. Shake a quantity of the powdered tablets containing the equivalent of 3 mg of erythromycin with 2 ml of acetone R and add 2 ml of hydrochloric acid R, an orange colour is produced which changes to red and then to deep violetred. Add 2 ml of chloroform R and shake, the chloroform layer becomes violet. C. Extract a quantity of the powdered tablets containing the equivalent of 50 mg of erythromycin with 10 ml of chloroform R, filter and evaporate to dryness. Heat 0.1 g of the residue gently with 5 ml of 2 M hydrochloric acid R and 10 ml of water until the solution boils, oily globules rise to the surface. Cool, transfer the fatty layer to a test tube, heat it with 3 ml of 0.1 M sodium hydroxide R and allow to cool, the solution sets to a gel. Add 10 ml of hot water and shake, the solution froths. To 1 ml add a 10% solution of calcium chloride R, a granular precipitate is produced which is insoluble in hydrochloric acid R. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of a 2.722% solution of sodium acetate R, previously adjusted to pH 5.0 with glacial acetic acid R. 377

VP V

ERYTHROSINE

Rotation speed: 50 rpm Time: 45 min. Procedure: Test solution: After the specified time, withdraw a sample of the medium and filter, discard the first 20 ml of the filtrate. Transfer 5.0 ml of the filtrate to a 100 ml volumetric flask, add 40 ml of glacial acetic acid R and 10 ml of a 0.5% w/w solution of 4-dimethylaminobenzaldehyde R in glacial acetic acid R, and dilute to volume with a mixture of 35 volumes of glacial acetic acid R and 70 volumes of hydrochloric acid R. Allow to stand for 15 min. Reference solution: A suitable solution of erythromycin stearate RS in dissolution medium which has been prepared in a similar manner beginning at the words ‘Transfer 5.0 ml of the filtrate…”. Measure the absorbances (Appendix 4.1) of the reference solution and the test solution at the maximum at 485 nm in a 1 cm cell, using in the reference cell the dissolution medium that has been subjected to the conditions of the test. Calculate the total content of erythromycin, C37H67NO13, in the medium using the declared content of C37H67NO13 in erythromycin stearate RS. Tolerance: Not less than 70% (Q) of the labelled amount of erythromycin, C37H67NO13, is dissolved in 45 min.

Loss on drying Not more than 5.0% (Appendix 9.6). (100 mg of the powdered tablets; in vacuum at 60 °C for 3 hours). Assay Weigh 20 tablets (if necessary, remove the coating), calculate the average mass and finely powder. Transfer an accurately weighed quantity of the powder containing the equivalent of 25 mg of erythromycin to a 100 ml volumetric flask, add 50 ml of methanol R, shake well, dilute to volume with methanol R, and mix. Carry out the biological assay of antibiotics for erythromycin (Appendix 13.9). Calculate the content of erythromycin, C37H67NO13, in the tablets, taking each 1000 IU found to be equivalent to 1 mg of C37H67NO13. Storage Store in an airtight container or in blisters, in a cool place, protected from light. Action and use Antibacterial. Usual strength 250 mg; 500 mg.

378

ERYTHROSINE Erythrosinum

C20H6I4Na2O5 Or C20H6I4K2O5

M. 880

M. 912

Erythrosine is disodium or dipotassium 2-(2,4,5,7-tetraiodo3-oxo-6-oxido-3H-9-xanthenyl) benzoate. It contains not less than 85.0% of C20H6I4Na2O5 or C20H6I4K2O5, calculated with reference to the dried substance.

Characters Dark red powder. Freely soluble in water, soluble in ethanol, slightly soluble in acetone, practically insoluble in methylene chloride. A 0.1% solution of erythrosine shows orange-red colour and the traces on the wall of test tube show violet colour. At pH 2.5, a coloured precipitate is produced. Identification Solution S: Dissolve 50 mg of the substance to be examined in water and dilute to 50 ml with the same solvent. A. Dilute 1 ml of solution S to 100 ml with 0.1 M sodium hydroxide R. The light absorption spectrum (Appendix 4.1) of the solution in the range 230 nm to 550 nm exhibits three absorption maxima, at (262 ± 5) nm; (309 ± 5) nm and (525 ± 3) nm. B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (2) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). C. When heating, a violet vapour is produced. Appearance of solution Solution S is clear (Appendix 9.2). Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Ammonia - water - ethanol - butanol (10 : 25 : 25 : 50). Test solution (1): Dissolve 40 mg of the substance to be examined in a mixture of water - methanol (50 : 50) and dilute to 10 ml with the same mixture of solvents. Test solution (2): Dilute 2 ml of test solution (1) to 10 ml with a mixture of water - methanol (50 : 50). Reference solution (1): Dissolve 40 mg of erythrosine RS in a mixture of water - methanol (50 : 50) and dilute to 50 ml with the same mixture of solvents.

VP V

Reference solution (2): Dilute 5 ml of reference solution (1) to 100 ml with a mixture of water - methanol (50 : 50). Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm. After removal of the plate, allow the plate to dry in air. Examine in daylight. Any spot in the chromatogram obtained with test solution (1), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (2).

Ether-soluble substances Not more than 0.5%. Weigh into a 200 ml volumetric flask 2.0 g of the substance to be examined previously dried in vacuo at 60 °C, make up to the volume with anhydrous ether. Shake for 30 min in a mechanical shaker, filter. Evaporate 100 ml of the filtrate in vacuo at a temperature not over 20 °C. Dry the residue in a desiccator to constant mass. The mass of the residue is not more than 5 mg. Water-insoluble matter Not more than 0.2%. Dissolve 2.0 g in 200 ml of water by heating at about 90 °C. Cool and filter through a sintered-glass filter No.16, previously dried to constant mass and weighed. Wash the residue with water until the washings are colourless, dry the residue at 100  °C to 105 °C to constant mass. The residue weighs not more than 4 mg.

Primary aromatic amines Not more than 40 ppm. Dissolve the residue obtained in the test for Ether-soluble substances in 10 ml of toluene R. To 2.5 ml of this solution add 6 ml of water and 4 ml of 0.1 N hydrochloric acid R. Shake vigorously, allow to separate and discard the upper organic solvent layer, to the water layer add 0.4 ml of a 0.25% solution of sodium nitrite R. Mix well and allow to stand for 1 min. Add 0.8 ml of a 0.5% solution of ammonium sulfamate R and allow to stand for 1 min. Add 2 ml of 0.5% solution of naphthylethylenediamine dihydrochloride R. Allow to stand for 1 h. Any colour of the result solution is not more intensely coloured than the reference solution, which is prepared at the same time in the same manner, using 1 ml of 0.001% solution of naphthylamine R, 5 ml of water and 4 ml of 0.1 N hydrochloric acid R instead of the water phase. Soluble chromium compounds Not more than 50 ppm. Determine by atomic absorption spectrometry (Appendix 4.4). Test solution: Dissolve 0.500 g in 25 ml of water by heating at about 90 °C, allow to cool, and add water to 25 ml, filter through a sintered-glass filter No. 16.

ERYTHROSINE

Reference solutions: Prepare the reference solutions: 0.5 ppm, 1 ppm and 2 ppm; using chromium standard solution (100 ppm) R. Measure the absorbance at 357.9 nm using a chromium hollow-cathode lamp as source of radiation and an airacetylene flame.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Iodides Not more than 0.1%. Dissolve 0.10 g in 5 ml of water, add 0.5 ml of nitric acid R, filter, rinse the filter paper with water to obtain 10 ml of filtrate. Add 1 ml of a 0.5% solution of potassium chromate R and 5 ml of cyclohexane R, shake and allow to stand. Prepare at the same time and in the same manner a reference solution containing 1 ml of a 0.0131% solution of potassium iodide R. The colour of the organic solvent layer of the test solution is not more intense than that of the reference solution Assay Dissolve 75.0 mg of the substance to be examined, previously dried in vacuo at 60 °C to constant mass, in a 0.1542% solution of ammonium acetate freshly prepared and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml of this solution to 200.0 ml with a 0.1542% solution of ammonium acetate. Prepare simultaneously the standard solution, using 75.0 mg of erythrosine RS previously dried in vacuo at 60 °C to constant mass. Measure the visible absorption spectrum (Appendix 4.1) of the standard solution and the test solution, using a 0.1542% solution of ammonium acetate as the blank. The absorption spectrum of the test solution exhibits an absorption maximum at about 525 nm and the value obtained does not differ from that of the standard solution prepared at the same condition by more than 5 nm. Measure the absorbances (Appendix 4.1) of the standard solution and the tesst solution at the maximum wavelength, using a 0.1542% solution of amononium acetate as the blank. From the obtained absorbances and the concentration of the standard solution and the test solution calculate the content of C20H6I4Na2O5 or C20H6I4K2O5. Storage Store in an airtight container.

379

VP V

ESOMEPRAZOLE MAGNESIUM TRIHYDRATE

ESOMEPRAZOLE MAGNESIUM TRIHYDRATE Esomeprazolum magnesicum trihydricum

C34H36MgN6O6S2,3H2O 

M. 767.2

Esomeprazole magnesium trihydrate is magnesium bis[5methoxy-2-[(S)-[(4-methoxy-3,5-dimethylpyridin-2-yl) methyl]sulfinyl]-1H-benzimidazol-1-ide] trihydrate. It contains not less than 98.0% and not more than 102.0% of C34H36MgN6O6S2, calculated with reference to the anhydrous substance.

Characters White or slightly coloured powder, slightly hygroscopic. Slightly soluble in water, soluble in methanol, practically insoluble in heptane. Identification Apply one of the four following identifications: First identification: A, B, C. Second identification: A, C, E. Third identification: A, B, D. Fourth identification: A, D, E. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of esomeprazole magnesium trihydrate RS. B. Atomic absorption spectrometry (Appendix 4.4, method 1) as described in the test for Magnesium. The test solution shows the absorption maximum at 285.2 nm. C. Specific optical rotation: -137° to -155°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.250 g in methanol R and dilute to 25.0 ml with the same solvent. D. It complies with the test for Enantiomeric purity. E. Ignite about 0.5 g according to the procedure in the test for Sulfated ash (Appendix 9.9, method 2). Dissolve the residue in 10 ml of water. 2 ml of this solution gives the reaction of magnesium (Appendix 8.1). Absorbance Dissolve 0.500 g in methanol R and dilute to 25.0 ml with the same solvent. Filter the solution through a membrane filter (nominal pore size 0.45 µm). The absorbances measured at 440 nm (Appendix 4.1) is not more than 0.20. Related substances Examine by liquid chromatography (Appendix 5.3). Use the normalisation procedure. Use freshly prepared solutions. 380

Mobile phase: Acetonitrile - a 0.14% solution of disodium hydrogen phosphate previously adjusted to pH 7.6 with phosphoric acid (27 : 73). Test solution: Dissolve 3.5 mg of the substance to be examined in the mobile phase and dilute to 25.0 ml with the mobile phase. Reference solution (1): Dissolve 1 mg of omeprazole RS and 1 mg of omeprazole impurity D RS in the mobile phase and dilute to 10.0 ml with the same solvent. Reference solution (2): Dissolve 3 mg of the omeprazole for peak identification RS (containing impurity E) in the mobile phase and dilute to 20.0 ml with the same solvent. Reference solution (3): Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Chromatographic system: A column (12.5 cm × 4.6 mm) packed with stationary phase B (5 µm). Detector: A spectrophotometer set at 280 nm. Flow rate: 1 ml/min. Volume of injection: 40 µl. Procedure: The run time is 5 times the retention time of esomeprazole. Identification of impurities: Use the chromatogram supplied with omeprazole for peak identification RS and the chromatogram obtained with reference solution (2) to identify the peak due to impurity E; use the chromatogram obtained with reference solution (1) to identify the peak due to impurity D. Relative retention with reference to esomeprazole (retention time = about 9 min): Impurity E = about 0.6; impurity D = about 0.8. System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to impurity C and omeprazole is at least 3.0. If necessary, adjust the pH of the aqueous part of the mobile phase or its proportion of acetonitrile; an increase in the pH will improve the resolution. Limits: Impurity D: Not more than 0.2%. Impurity E: Not more than 0.1%. Unspecified impurities: For each impurity, not more than 0.10%. Total: Not more than 0.5%. Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (3) (0.05%). Note: Impurity A: 5-methoxy-1H-benzimidazole-2-thiol. Impurity B: 2-[(RS)-[(3,5-dimethylpyridin-2-yl)methyl]sulfinyl] -5-methoxy-1H-benzimidazole. Impurity C: 5-methoxy-2-[[(4-methoxy-3,5-dimethylpyridin-2yl)methyl]sulfanyl]-1H-benzimidazole (ufiprazole).

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ESOMEPRAZOLE MAGNESIUM TRIHYDRATE

Impurity D: 5-methoxy-2-[[(4-methoxy-3,5-dimethylpyridin-2yl)methyl]sulfonyl]-1H-benzimidazole (omeprazole sulfone). Impurity E: 4-methoxy-2-[[(RS)-(5-methoxy-1H-benzimidazol2-yl)sulfinyl]methyl]-3,5-dimethylpyridine 1-oxide.

Enantiomeric purity Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - buffer solution pH 6.0 (65 : 435). Buffer solution pH 6.0: Mix 70 ml of a 15.6% solution of sodium dihydrogen phosphate R with 20 ml of a 17.91% solution of disodium hydrogen phosphate R. Dilute to 1000 ml with water, then dilute 250 ml of this solution to 1000.0 ml with water. Buffer solution pH 11.0: Mix 11 ml of a 9.5% solution of trisodium phosphate dodecahydrate R with 22 ml of a 17.91% solution of disodium hydrogen phosphate R, then dilute to 1000.0 ml with water. Test solution: Dissolve 40 mg of the substance to be examined in 5 ml of methanol R and dilute to 25 ml with buffer solution pH 11.0. Dilute 1.0 ml of this solution to 50.0 ml with buffer solution pH 11.0. Reference solution (1): Dissolve 2 mg of omeprazole RS in buffer solution pH 11.0 and dilute to 10.0 ml with the same solvent. Dilute 1.0 ml of this solution to 50.0 ml with buffer solution pH 11.0. Reference solution (2): Dilute 1.0 ml of reference solution (1) to 50.0 ml with buffer solution pH 11.0. Chromatographic system: A column (10 cm × 4.0 mm) packed with stationary phase silica gel AGP (α1-acid glucoprotein) for chiral chromatography R (5 µm). Detector: A spectrophotometer set at 302 nm. Flow rate: 0.6 ml/min. Volume of injection: 20 µl. Procedure: Elution order: Impurity F, esomeprazole. Retention time of esomeprazole is about 4 min. System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to impurity F and esomeprazole is at least 3.0. In the chromatogram obtained with reference solution (2), the signal-to-noise ratio of the peak due to impurity F is not less than 10. Calculate the percentage content of impurity F using the following expression:

100 ×

ri rs

Where: ri: area of the peak due to impurity F in the chromatogram obtained with the test solution. rs: sum of the areas of the peaks due to esomeprazole and impurity F in the chromatogram obtained with the test solution.

Limits: Impurity F: Not more than 0.2%. Note: Impurity F: 5-methoxy-2-[(R)-[(4-methoxy-3,5-dimethylpyridin2-yl) methyl]sulfinyl]-1H-benzimidazole((R)-omeprazole).

Magnesium 3.30% to 3.55%, calculated with reference to the anhydrous substance. Examine by atomic absorption spectrometry (Appendix 4.4, method 1). Test solution: Dissolve 0.250 g in 20 ml of 1 M hydrochloric acid R, adding the acid slowly, and dilute to 100.0 ml with water. Dilute 10.0 ml of this solution to 200.0 ml with water. To 10.0 ml of the solution obtained add 4 ml of lanthanum chloride solution R and dilute to 100.0 ml with water. Standard solutions: Prepare the standard solutions using magnesium standard solution (1000 ppm Mg) R, diluted as necessary with a mixture of 1 ml of 1 M hydrochloric acid R in 1000.0 ml of water. Measure the absorbance at 285.2 nm using an magnesium hollow-cathode lamp as a source of radiation and an airacetylene flame. Water 6.0% to 8.0% (Appendix 10.3). Determined on 0.200 g. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - a 0.14% solution of disodium hydrogen phosphate previously adjusted to pH 7.6 with phosphoric acid (35 : 65). Buffer solution pH 11.0: Mix 11 ml of a 9.5% solution of trisodium phosphate dodecahydrate R with 22 ml of a 17.91% solution of disodium hydrogen phosphate R, and dilute to 100.0 ml with water. Test solution: Dissolve 10.0 mg of the substance to be examined in about 10 ml of methanol R, add 10 ml of buffer solution pH 11.0 and dilute to 200.0 ml with water. Standard solution: Dissolve 10.0 mg of omeprazole RS in about 10 ml of methanol R, add 10 ml of buffer solution pH 11.0 and dilute to 200.0 ml with water. Chromatographic system: A column (12.5 cm × 4.0 mm) packed with stationary phase B (5 µm). Detector: A spectrophotometer set at 280 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Procedure: The run time is 1.5 times the retention time of esomeprazole. Retention time esomeprazole is about 4 min. Calculate the content of C34H36MgN6O6S2 using the areas of the peaks in the chromatograms obtained with 381

GASTRO-RESISTANT ESOMEPRAZOLE CAPSULES

the test solution, the standard solution and the content of C34H36MgN6O6S2 in omeprazole RS. 1 g of omeprazole is equivalent to 1.032 g of esomeprazole magnesium.

Storage Store in an airtight container, protected from light. Action and use Proton pump inhibitor. Preparations Tablets, capsules. GASTRO-RESISTANT ESOMEPRAZOLE CAPSULES Capsulae Esomeprazoli Gastro-resistant esomeprazole capsules contain esomeprazole magnesium, in form of pellets with gastro-resistant coating. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of esomeprazole, C17H19N3O3S, 90.0% to 110.0% of the stated amount. Identification Examine by liquid chromatography (Appendix 5.3). Phosphate buffer pH 6.0: A solution contains 2.66% of disosium hydrogen phosphate dihydrate R and 5.52% of sodium dihydrogen phosphate monohydrate R. Mobile phase: Mix 150 ml of acetonitrile R and 85 ml of phosphate buffer pH 6.0 and dilute to 1000 ml with water. Solvent: Dissolve 5.24 g of tribasic sodium phosphate R in water, add 110 ml of 0.5 M disodium hydrogen phosphate R and dilute to 1000 ml with water. Reference solution: Transfer an accurately weighed quantity of about 20 mg of omeprazole RS to a 100 ml volumetric flask, add 20 ml of ethanol (96%) R and shake to dissolve, dilute to volume with the solvent and mix well. Dilute 10.0 ml of the resulting solution to 100.0 ml with water and mix. Test solution: Transfer a quantity of the capsule contents containing the equivalent of 20 mg of esomeprazole to a 200 ml volumetric flask, add 120 ml of the solvent and shake for 20 minutes to dissolve the pellets, sonicate for a few minutes, if necessary, to dissolve completely. Add 40 ml of ethanol (96%) R and sonicate for a few minutes. Allow to cool and dilute to volume with the solvent, mix and filter. Dilute 10.0 ml of the filtrate to 100.0 ml with water and mix. Chromatographic system: A column (10 cm × 4 mm) packed with spherical silica particles immobilized α1-acid glycoprotein (5 μm) (equivalent to USP L 41 column). 382

VP V

Detector: A spectrophotometer set at 302 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 μl. Procedure: Inject the reference solution. The elution order: R-enantiomer followed by S-enantiomer which is esomeprazole. The test is not valid unless the resolution factor between the enantiomer peaks is at least 1.0. Inject the test solution. The ratio of the retention time of the esomeprazole peak in the test solution to that in the reference solution is 0.98 to 1.02.

Dissolution (Appendix 11.4) Acid stage Apparatus: Paddle. Medium: 300 ml of 0.1 M hydrochloric acid R. Rotation speed: 100 rpm. Time: 2 hours. Buffer stage Apparatus: Paddle. Medium: 1000 ml of phosphate buffer pH 6.8. After 2 hours, continue the test as follows: Add 700 ml of 0.086 M disodium hydrogen phosphate R to each vessel. Adjust to pH 6.8 ± 0.05 with 2 M hydrochloric acid R or 2 M sodium hydroxide R. Rotation speed: 100 rpm. Time: 30 min. Procedure: Examine by liquid chromatography (Appendix 5.3). Phosphate buffer pH 6.8: Mix 300 ml of 0.1 M hydrochloric acid R and 700 ml of 0.086 M disodium hydrogen phosphate R, adju st to pH 6.8 ± 0.05 with 2 M hydrochloric acid R or 2 M sodium hydroxide R. Mobile phase and chromatographic system: Proceed as directed in the Assay. Reference solution: Dissolve an accurately weighed quantity of omeprazole RS in ethanol (96%) R to obtain a solution having a known concentration of 2 mg per ml. Dilute a portion of this solution with phosphate buffer pH 6.8 to obtain a solution having a known concentrtion of L/1000 mg per ml (Where L is the labeled amount, in mg per capsule). Immediately add 2.0 ml of 0.25 M sodium hydroxide R to 10.0 ml of this solution. Note: Do not allow the solution to stand for too long before adding the sodium hydroxide solution.

Test solution: After 30 min in the buffer medium, withdraw a sample of the medium, filter. Transfer 5.0 ml of the filtrate to a test tube containing 1.0 ml of 0.25 M sodium hydroxide R, mix. Protect from light. Inject alternately the reference solution and the test solution. Calculate the content of esomeprazole, C17H19N3O3S, dissolved using the peak areas due to esomeprazole in the chromatograms obtained with the test solution and omeprazole in the chromatograms obtained with the

VP V

GASTRO-RESISTANT ESOMEPRAZOLE CAPSULES

reference solution and the declared content of C17H19N3O3S in omeprazole RS. Tolerance: Not less than 75% (Q) of the stated amount of esomeprazole, C17H19N3O3S, is dissolved for both stages (Appendix 11.4, item 4.3).

Related substances Examine by liquid chromatography (Appendix 5.3). Solvent: Prepare as directed in the Assay. Phosphate buffer pH 7.6: Mix 5.2 ml of 1 M sodium dihydrogen phosphate R and 63 ml of 0.5 M disodium hydrogen phosphate R. Dilute to 1000 ml with water. Mobile phase A: Mix 100 ml of acetonitrile R and 100 ml of phosphate buffer pH 7.6. Dilute to 1000 ml with water. Mobile phase B: Mix 800 ml of acetonitrile R and 10 ml of phosphate buffer pH 7.6. Dilute to 1000 ml with water. System suitability solution: Dissolve a quantity of omeprazole RS and omeprazole sulfone RS in methanol R to obtain a solution containing 1 mg of each substance per ml. Dilute 1.0 ml of the resulting solution to 100.0 ml with a mixture of the solvent and water (1 : 4). Dilute 1.0 ml of this solution to 10.0 ml with the same mixture. Test solution: Collect the pellets from 20 capsules and powder finely. Transfer an accurately weighed quantity of the powder containing the equivalent of about 20 mg of esomeprazole to a 200 ml volumetric flask, add 20 ml of methanol R and shake for 30 seconds. Add 40 ml of the solvent, shake for 30 seconds by hand and sonicate for a few minutes. Allow to cool and dilute to volume with water, mix and filter. Note: The solution is stable for 3 hours if protected from light. Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase C (3 μm). Detector: A spectrophotometer set at 302 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 μl. Procedure: Carry out the chromatography with the following gradient programme: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 10

100 → 80

0 → 20

10 - 30

80 → 0

20 → 100

30 - 31

0 → 100

100 → 0

31 - 45

100

0

Inject the system suitability solution, the relative retention time of the omeprazole sulfone peak with reference to omeprazole is 0.93. The test is not valid unless the resolution factor between the peaks due to omeprazole sulfone and omeprazole is at least 2.5. Inject the test solution. Calculate the content of any individual impurity using the peak area of each impurty (if

present) in comparision with the sum of all peak areas in the chromatogram obtained with the test solution. Limits: Omeprazol sunfone: Not more than 0.5%. Any other individual impurity: Not more than 0.2%. Total impurities: Not more than 2.0%.

Assay Examine by liquid chromatography (Appendix 5.3). Phosphate buffer pH 7.3: Mix 10.5 ml of 1 M sodium dihydrogen phosphate R and 60 ml of 0.5 M disodium hydrogen phosphate R, dilute to 1000 ml with water. Mobile phase: Mix 350 ml of acetonitrile R and 500 ml of phosphate buffer pH 7.3, dilute to 1000 ml with water. Solvent: Dissolve 5.24 g of tribasic sodium phosphate R in water, add 110 ml of 0.5 M disodium hydrogen phosphate R, dilute to 1000 ml with water. Reference solution: Transfer an accurately weighed quantity of about 10 mg of omeprazole RS in a 250 ml volumetric flask, dissolve in 10 ml ethanol R (96%), add 40 ml of the solvent and dilute to volume with water, mix. This solution contains about 0.04 mg of omeprazole per ml. Test solution: Weigh 20 capsules and calculate the average mass of the contents. Transfer an accurately weighed quantity of the capsule contents containing the equivalent of 20 mg of esomeprazole, to a 100 ml volumetric flask, add 60 ml of the solvent and shake for 20 minutes to dissolve the pellets, sonicate for a few minutes, if necessary, to dissolve completely. Add 20 ml of ethanol (96%) R and sonicate for a few minutes. Allow to cool and dilute to volume with the solvent, mix and filter. Dilute 10.0 ml of the filtrate to 50.0 ml with water, mix. Store this solution protected from light. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 μm). Detector: A spectrophotometer set at 302 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 μl. Procedure: Inject the reference solution, the test is not valid unless the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of esomeprazole, C17H19N3O3S, in the capsules using peak areas due to esomeprazole in the chromatograms obtained with the test solution and omeprazole in the chromatograms obtained with the reference solution, and the declared content of C17H19N3O3S in omeprazole RS. Storage Store in an airtight container, in a cool and dry place, protected from light. 383

GASTRO-RESISTANT ESOMEPRAZOLE TABLETS

Action and use Proton pump inhibitor, treament of peptic ulcer disease. Usual strength 20 mg, 40 mg. GASTRO-RESISTANT ESOMEPRAZOLE TABLETS Tabellae Esomeprazoli Gastro-resistant esomeprazole tablets contain esomeprazole magnesium. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements:

Content of esomeprazole, C17H19N3O3S, 90.0% to 110.0% of the stated amount. Identification Examine by liquid chromatography (Appendix 5.3). Phosphate buffer pH 6.0: A solution contains 2.66% of disosium hydrogen phosphate dihydrate R and 5.52% of sodium dihydrogen phosphate monohydrate R. Mobile phase: Mix 150 ml of acetonitrile R and 85 ml of phosphate buffer pH 6.0, dilute to 1000 ml with water. Solvent: Dissolve 5.24 g of tribasic sodium phosphate R in water, add 110 ml of 0.5 M disodium hydrogen phosphate R and dilute to 1000 ml with water. Reference solution: Transfer about 20 mg of omeprazole RS to a 100 ml volumetric flask, add 20 ml of ethanol (96%) R and shake to dissolve, dilute to volume with the solvent and mix well. Dilute 10.0 ml of the resulting solution to 100.0 ml with water and mix. Test solution: Transfer a quantity of the powdered tablets containing the equivalent of 20 mg of esomeprazole to a 200 ml volumetric flask, add 120 ml of the solvent and shake for 20 minutes to dissolve, sonicate for a few minutes, if necessary, to dissolve completely. Add 40 ml of ethanol (96%) R and sonicate for a few minutes. Allow to cool and dilute to volume with the solvent, mix and filter. Dilute 10.0 ml of the filtrate to 100.0 ml with water and mix. Chromatographic system: A column (10 cm × 4 mm) packed with spherical silica particles immobilized α1-acid glycoprotein (5 μm) (equivalent to USP L41 column). Detector: A spectrophotometer set at 302 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 μl. Procedure: Inject the reference solution. The elution order: R-enantiomer, followed by S-enantiomer which is esomeprazole. The test is not valid unless the resolution factor between the enantiomer peaks is at least 1.0. Inject the test solution. The ratio of the retention time of the esomeprazole peak in the test solution to that in the reference solution is 0.98 to 1.02. 384

VP V

Dissolution (Appendix 11.4) Acid stage Apparatus: Paddle. Medium: 300 ml of 0.1 M hydrochloric acid R. Rotation speed: 100 rpm. Time: 2 hours. Buffer stage Apparatus: Paddle. Medium: Phosphate buffer pH 6.8. After 2 hours, continue the test as follows: Add 700 ml of 0.086 M disodium hydrogen phosphate R to each vessel. Adjust to pH 6.8 ± 0.05 with 2 M hydrochloric acid R or 2 M sodium hydroxide R. Rotation speed: 100 rpm. Time: 30 min. Procedure: Examine by liquid chromatography (Appendix 5.3). Phosphate buffer pH 6.8: Mix 300 ml of 0.1 M hydrochloric acid R and 700 ml of 0.086 M disodium hydrogen phosphate R, adjust to pH 6.8 ± 0.05 with 2 M hydrochloric acid R or 2 M sodium hydroxide R. Mobile phase and chromatographic system: Proceed as directed in the Assay. Reference solution: Dissolve an accurately weighed quantity of omeprazole RS in ethanol (96%) R to obtain a solution having a known concentration of 2 mg per ml. Dilute with phosphate buffer pH 6.8 to obtain a solution having a known concentrtion of L/1000 mg per ml (Where L is the labeled amount, in mg per tablet). Immediately add 2.0 ml of 0.25 M sodium hydroxide to 10.0 ml of this solution and mix. Note: Do not allow the solution to stand for too long before adding the sodium hydroxide solution. Test solution: After 30 min in the buffer medium, withdraw a sample of the medium, filter. Transfer 5.0 ml of the filtrate to a test tube containing 1.0 ml of 0.25 M sodium hydroxide, mix. Protect from light. Inject alternately the reference solution and the test solution. Calculate the content of esomeprazole dissolved using the peak areas due to esomeprazole in the chromatograms obtained with the test solution and omeprazole in the chromatograms obtained with the reference solution and the declared content of C17H19N3O3S in omeprazole RS. Tolerance: Not less than 75% (Q) of the stated amount of esomeprazole, C17H19N3O3S, is dissolved (Appendix 11.4, item 4.3). Related substances Examine by liquid chromatography (Appendix 5.3). Solvent: Prepare as directed in the Assay. Phosphate buffer pH 7.6: Mix 5.2 ml of 1 M sodium dihydrogen phosphate R and 63 ml of 0.5 M disodium hydrogen phosphate R. Dilute to 1000 ml with water. Mobile phase A: Mix 100 ml of acetonitrile R and 100 ml of phosphate buffer pH 7.6. Dilute to 1000 ml with water.

VP V

ETHAMBUTOL HYDROCHLORIDE

Mobile phase B: Mix 800 ml of acetonitrile R and 10 ml of phosphate buffer pH 7.6. Dilute to 1000 ml with water. System suitability solution: Dissolve a quantity of omeprazole RS and omeprazole sulfone RS in methanol R to obtain a solution containing 1 mg of each substance per ml. Dilute 1.0 ml of the resulting solution to 100.0 ml with a mixture of the solvent and water (1 : 4), mix. Dilute 1.0 ml of this solution to 10.0 ml with the same mixture. Test solution: Transfer an accurately weighed quantity of the powdered tablets containing the equivalent to about 20 mg of esomeprazole to a 200 ml volumetric flask, add 20 ml of methanol R and shake for 30 seconds. Add 40 ml of the solvent, shake for 30 seconds by hand and sonicate for a few minutes. Allow to cool and dilute to volume with water, mix and filter. Note: The solution is stable for 3 hours if protected from light. Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase C (3 μm). Detector: A spectrophotometer set at 302 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 μl. Procedure: Carry out the chromatography with the following gradient programme: Time (min) 0 - 10 10 - 30 30 - 31 31 - 45

Mobile phase A (% v/v) 100 → 80 80 → 0 0 → 100 100

Mobile phase B (% v/v) 0 → 20 20 → 100 100 → 0 0

Inject the system suitability solution, the relative retention time of the omeprazole sulfone peak with reference to omeprazole is 0.93. The test is not valid unless the resolution factor between the peaks due to omeprazole sulfone and omeprazole is at least 2.5. Inject the test solution. Calculate the content of any individual impurity using the peak area of each impurty (if present) in comparision with the sum of all peak areas in the chromatogram obtained with the test solution. Limits: Omeprazol sunfone: Not more than 0.5%. Any other individual impurity: Not more than 0.2%. Total impurities: Not more than 2.0%.

Assay Examine by liquid chromatography (Appendix 5.3). Phosphate buffer pH 7.3: Mix 10.5 ml of 1 M sodium dihydrogen phosphate R and 60 ml of 0.5 M disodium hydrogen phosphate R, dilute to 1000 ml with water. Mobile phase: Mix 350 ml of acetonitrile R and 500 ml of phosphate buffer pH 7.3, dilute to 1000 ml with water. Solvent: Dissolve 5.24 g of tribasic sodium phosphate R in water, add 110 ml of 0.5 M disodium hydrogen phosphate R, dilute to 1000 ml with water.

Reference solution: Transfer an accurately weighed quantity of about 10 mg of omeprazole RS in a 250 ml volumetric flask, dissolve in 10 ml ethanol (96%) R, add 40 ml of the solvent and dilute to volume with water, mix. This solution contains about 0.04 mg of omeprazole per ml. Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Transfer an accurately weighed quantity of the powder containing the equivalent of about 20 mg of esomeprazole to a 100 ml volumetric flask, add 60 ml of the solvent and sonicate to dissolve. Add 20 ml of ethanol (96%) R and sonicate for a few minutes. Allow to cool and dilute to volume with the solvent, mix and filter. Dilute 10.0 ml of the filtrate to 50.0 ml with water, mix. Store this solution protected from light. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 μm). Detector: A spectrophotometer set at 302 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 μl. Procedure: Inject the reference solution, the test is not valid unless the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of esomeprazole, C17H19N3O3S, in the tablets using the peak areas due to esomeprazole in the chromatograms obtained with the test solution and omeprazole in the chromatograms obtained with the reference solution and the declared content of C17H19N3O3S in omeprazole RS.

Storage Store in an airtight container, in a cool and dry place, protected from light. Action and use Proton pump inhibitor, treament of peptic ulcer disease. Usual strength 20 mg, 40 mg. ETHAMBUTOL HYDROCHLORIDE Ethambutoli hydrochloridum

, 2 HCl

C10H24N2O2,2HCl

M. 277.2

Ethambutol hydrochloride is (2S,2′S)-2,2′-(ethylenediimino) dibutan-1-ol dihydrochloride. It contains not less than 99.0% and not more than 101.0% of C10H24N2O2.2HCl, calculated with reference to the dried substance. 385

VP V

ETHAMBUTOL HYDROCHLORIDE

Characters White or almost white, crystalline powder, hygroscopic. Freely soluble in water, soluble in ethanol (96%). Identification Apply one of the two following identifications: First identification: A, D, E. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of ethambutol hydrochloride RS. B. In the test for 2-Aminobutanol, the principal spot in the chromatogram obtained with test solution (2) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (2). C. Dissolve 0.1 g of the substance to be examined in 10 ml of water, add 0.2 ml of 12.5% solution of copper sulfate R. Add 0.5 ml of 2 M sodium hydroxide R, a blue colour is produced. D. It gives reaction (A) of chlorides (Appendix 8.1). E. It complies with the test for Related substances. pH 3.7 to 4.0 (Appendix 6.2). Dissolve 0.2 g in 10 ml of carbon dioxide-free water R. 2-Aminobutanol Not more than 1.0%. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Ammonia - water - methanol (10 : 15 : 75). Test solution (1): Dissolve 0.50 g of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Test solution (2): Dilute 1 ml of test solution (1) to 10 ml with methanol R. Reference solution (1): Dissolve 50.0  mg of  2-aminobutanol R (impurity A) in methanol R and dilute to 10.0 ml with the same solvent. Dilute 1.0  ml of this solution to 10.0 ml with methanol R. Reference solution (2): Dissolve 50 mg of  ethambutol  hydrochloride RS  and 5  mg of 2-aminobutanol R in methanol R and dilute to 10 ml with the same solvent. Procedure: Apply separately to the plate 2 µl of each solution. Develop over a path of two thirds of the plate. Allow the plate to dry in air, heat at 110 °C for 10 min. Allow to cool, spray with ninhydrin solution R1; heat at 110 °C for 5 min. Any spot corresponding to 2-aminobutanol in the chromatogram obtained with test solution (1) is not more intense than the spot in the chromatogram obtained with reference solution (1) (1.0%). The test is not valid unless the chromatogram obtained with reference solution (2) shows two clearly separated spots. 386

Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use. Mobile phase A: Methanol - water (50 : 50). Mobile phase B: Methanol R. Test solution: Suspend 4.0 mg of the substance to be examined in 4.0 ml of acetonitrile R1 and add 100 µl of triethylamine R. Sonicate the mixture for 5 min. Add 15  µl of  (R)-(+)-αmethylbenzyl isocyanate R and heat at 70 °C for 20 min. Reference solution (1): Dilute 0.50 ml of the test solution to 100.0 ml with acetonitrile R1. Reference solution (2): Treat 4.0 mg of ethambutol for system suitability RS (containing impurity B) as described for the test solution. Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (3 µm). Column temperature: 40 °C. Detector: A spectrophotometer set at 215 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 30

71

29

30 - 35

71 → 0

29 → 100

35 - 37

0

100

37 - 38

0 → 71

100 → 29

Inject the test solution and the reference solutions. Relative retention with reference to ethambutol (retention time = about 14 min): impurity B = about 1.3. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to ethambutol and impurity B is at least 4.0. Limits: In the chromatogram obtained with the test solution: Impurity B: The area of the peak due to impurity B is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (1) (1.0%). Unspecified impurities with a relative retention of 0.75 to 1.5 with reference to  ethambutol: For each impurity, the area is not more than 0.2 times the area of the peak due to ethambutol in the chromatogram obtained with reference solution (1) (0.10%). The sum of the areas of all the impurity peaks (impurity B and unspecified impurities with a relative retention of 0.75 to 1.5 with reference to ethambutol) is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (1) (1.0%). Disregard any peak with an area less than 0.1 times the area of the peak due to ethambutol in the chromatogram obtained with reference solution (1) (0.05%).

VP V Note: Impurity A: 2-aminobutan-1-ol. Impurity B: (2R,2′S)-2,2′-(ethylenediimino)dibutan-1-ol (mesoethambutol). Impurity C: (2R,2′R)-2,2′-(ethylenediimino)dibutan-1-ol ((R,R)ethambutol). Impurity D: 1,2-dichloroethane (ethylene chloride).

1,2-dichloroethane Not more than 5 ppm (Appendix 10.14). Heavy metals Not more than 10 ppm (Appendix 9.4.8). Dissolve 2.0 g in water and dilute to 20 ml with the same solvent. 12 ml of the solution complies with limit test for heavy metals, method 1. Prepare the standard using 10 ml of lead standard solution (1 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (0.500 g; 105 °C; 3 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.200 g of the substance to be examined in 50 ml of  water and add 1.0 ml of 0.1  N hydrochloric acid VS. Carry out a potentiometric titration (Appendix 10.2), using 0.1 N sodium hydroxide VS. Read the volume added between the 2 points of inflexion. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 27.72 mg of C10H26Cl2N2O2. Storage Store in an airtight container. Action and use Antituberculosis drug. Preparation Tablets. ETHAMBUTOL TABLETS Tabellae Ethambutoli Ethambutol tablets contain ethambutol hydrochloride. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of ethambutol hydrochloride, C10H24N2O2,2HCl, 95.0% to 105.0% of the stated amount.

ETHAMBUTOL TABLETS

Identification A. Extract a quantity of the powdered tablets containing about 50 mg of ethambutol hydrochloride with 5 ml of methanol R, filter and evaporate to dryness. The infrared absorption spectrum (Appendix 4.2) of the residue is concordant with the reference spectrum of ethambutol hydrochloride. B. Extract a quantity of the powdered tablets containing about 0.1 g of ethambutol hydrochloride with 10 ml of water, filter. Add to the filtrate 2 ml of a 1% solution of copper sulfate R followed by 1 ml of 1 M sodium hydroxide R. A blue colour is produced. 2-Aminobutanol Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel G. Mobile phase: 13.5 M ammonia - water - methanol (10 : 15 : 75). Test solution: Shake a quantity of the powdered tablets containing 0.50 g of ethambutol hydrochloride for 5 min with 10 ml of methanol R, filter. Reference solution: A solution containing 0.050% of 2-aminobutanol RS in methanol R. Procedure: Apply separately to the plate 2 µl of each solution. After development, remove the plate, allow it to dry in air, heat at 110 °C for 5 min, cool, spray with ninhydrin solution R1 and heat at 110 °C for 5 min. Any spot corresponding to 2-aminobutanol in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution (1%). Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of water. Rotation speed: 100 rpm. Time: 45 min. Mobile phase, reference solution, chromatographic system: Proceed as directed in the Assay. Test solution: After the specified time, withdraw a sample of the medium and filter. Dilute the filtrate if necessary with water to obtain a solution having a known concentration of about 0.3 mg/ml. Tolerance: Not less than 75% (Q) of ethambutol hydrochloride, C10H24N2O2,2HCl, of the stated amount is dissolved in 45 min. Assay Examine by liquid chromatography (Appendix 5.3). Buffer solution: Dilute 1 ml of triethylamine R with water to obtain 1000 ml, adjust the pH to 7.0 with phosphoric acid R. Mobile phase: Acetonitrile - buffer solution A (50 : 50). Adjust if necessary. Prepare the following solutions immediately before use. Reference solution: Dissolve a quantity of ethambutol hydrochloride RS in water to obtain a solution having a known concentration of about 0.3 mg/ml. 387

ETHAMBUTOL HYDROCHLORIDE AND ISONIAZID TABLETS

Test solution: Weigh 20 tablets, calculate the average mass and finely powder. Weigh accurately a quantity of the powdered tablets containing about 30 mg of ethambutol hydrochloride, transfer into a 100-ml volumetric flask, add 50 ml of water, sonicate for 15 min, dilute to volume with water, filter. Chromatographic system: A column (15 cm × 4.6 mm) packed with nitrile silica gel for chromatography (5 µm). Column temperature: 30 °C. Detector: A spectrophotometer set at 200 nm. Flow rate: 1.0 ml/min. Volume of injection: 50 µl. Procedure: System suitability: In the chromatogram obtained with the reference solution, the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject the reference solution and the test solution. Calculate the content of ethambutol hydrochloride, C10H24N2O2,2HCl, in the tablets using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C10H24N2O2,2HCl in ethambutol hydrochloride RS.

Storage Store in an airtight container. Action and use Antituberculosis. Usual strength 200 mg; 400 mg. ETHAMBUTOL HYDROCHLORIDE AND ISONIAZID TABLETS Tabellae Ethambutoli hydrochloridi et Isoniazidi Ethambutol hydrochloride and isoniazid tablets are filmcoated tablets containing ethambutol hydrochloride and isoniazid. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of ethambutol hydrochloride, C10H24N2O2, 2HCl, 90.0% to 110.0% of the stated amount. Content of isoniazid, C6H7N3O, 90.0% to 110.0% of the stated amount. Identification A. In the Assay for isoniazid, the retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of the principal peak in the chromatogram obtained with the reference solution. 388

VP V

B. In the Assay for ethambutol hydrochloride, the retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of the principal peak in the chromatogram obtained with the reference solution.

2-Aminobutanol Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Ammonia - water - methanol (10 : 15 : 75). Test solution: Shake a quantity of the powdered tablets containing 0.50 g of ethambutol hydrochloride for 5 min with 10 ml of methanol R, filter. Reference solution: A solution containing 0.050% of 2-aminobutanol RS in methanol R. Procedure: Apply separately to the plate 2 µl of each solution. After development, allow it to dry in air, heat at 110 °C for 5 min, cool, spray with ninhydrin solution R1 and heat at 110 °C for 5 min. Any spot corresponding to 2-aminobutanol in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution (1%). Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of water. Rotation speed: 100 rpm. Time: 45 min. Procedure: For ethambutol hydrochloride Examine by liquid chromatography (Appendix 5.3). Mobile phase, chromatographic system and procedure: Proceed as directed in theAssay for ethambutol hydrochloride. Test solution: After the specified time, withdraw a sample of the medium, and filter. Reference solution: A solution containing 0.44 mg/ml of ethambutol hydrochloride RS in water. Tolerance: Not less than 75% (Q) of the stated amount of ethambutol hydrochloride, C10H24N2O2,2HCl, is dissolved in 45 min. For isoniazid Examine by ultraviolet and visible absorption spectrophotometry (Appendix 4.1). Test solution: After the specified time, withdraw a sample of the medium, and filter. Dilute the filtrate with water to obtain a solution having the same concentration as the reference solution. Reference solution: A solution containing 0.015 mg/ml of isoniazid RS in water. Measure the absorbances of the reference solution and the test solution at the maximum wavelength at 263 nm in a 1-cm cell, using water as the blank. Tolerance: Not less than 75% (Q) of the stated amount of isoniazid, C6H7N3O, is dissolved in 45 min.

VP V

Assay Assay for ethambutol hydrochloride Examine by liquid chromatography (Appendix 5.3). Buffer solution: Dilute 1 ml of triethylamine R to 1000 ml with water, adjust the pH to 7.0 with phosphoric acid R. Mobile phase: Acetonitrile - buffer solution (50 : 50). Make adjustments if necessary. Diluent: Dissolve 1.4 g of anhydrous disodium hydrogen phosphate R in 1000 ml of water, and adjust the pH to 6.8 with 10% phosphoric acid solution R. Reference solution: Dissolve a quantity of ethambutol hydrochloride RS in the diluent to obtain a solution having a known concentration of about 0.6 mg/ ml. Test solution: Weigh 20 tablets, remove the coating if necessary, calculate the average mass and finely powder. Weigh accurate a quantity of the powder containing about 60 mg of ethambutol hydrochloride, transfer into a 100-ml volumetric flask, add 70 ml of the diluent, sonicate for 10 min, dilute to volume with the diluent, mix, and filter. Chromatographic system: A column (15 cm × 4.6 mm) packed with nitrile silica gel for chromatography (5 µm). Detector: A spectrophotometer set at 200 nm. Flow rate: 1.0 ml/min. Volume of injection: 50 µl. Procedure: System suitability: Inject the reference solution. The test is not valid unless, in the chromatogram obtained, the number of theoretical plates is not less than 1500, the symmetry factor for the ethambutol peak is not more than 3.0, and the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of ethambutol hydrochloride, C10H24N2O2,2HCl, in the tablets using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C10H24N2O2,2HCl in ethambutol hydrochloride RS. Assay for isoniazid Examine by liquid chromatography (Appendix 5.3). Buffer solution: Dissolve 1.4 g of anhydrous disodium hydrogen phosphate R in 1000 ml with water, and adjust to pH 6.8 with 10% phosphoric acid solution R. Mobile phase: Acetonitrile - buffer solution (4 : 96). Reference solution: Dissolve an accurately weighed quantity of about 40 mg of isoniazid RS in 50.0 ml of methanol R and dilute to volume with the buffer solution to produce 500.0 ml. Test solution: Weigh 20 tablets, remove the coating if necessary, calculate the average mass and finely powder. Weigh accurately a quantity of the powdered tablets containing about 40 mg of isoniazid, transfer into a 500-ml volumetric flask, add 50.0 ml of methanol R, sonicate for

ETHANOL

10 min to dissolve and dilute to volume with the buffer solution, mix. Filter. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 mm). Column temperature: 30 °C. Detector: A spectrophotometer set at 254 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution. The test is not valid unless the number of theoretical plates is not less than 1500, the symmetry factor for the isoniazid peak is not more than 2.0, and the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of isoniazid, C6H7N3O, in the tablets using the areas of the principal peaks in the chromatograms obtained with the test solution, the reference solution and the declared content of C6H7N3O in isoniazid RS.

Storage Store in an airtight container in a cool place, protected from light. Action and use Antituberculosis. Usual strength 400 mg of ethambutol and 150 mg of isoniazid. ETHANOL Ethanolum Absolute alcohol, dehydrated alcohol C2H6O

CH3-CH2-OH M. 46.07

Ethanol contains not less than 99.5% (v/v) or 99.2% (m/m) of C2H5OH, at 20 °C, calculated from the relative density using the alcoholimetric tables (Appendix 19).

Characters Colourless, clear, and volatile liquid, boiling at 78 °C; odour, characteristic and spirituos; flammable liquid, burning with a blue, smokeless flame; hygroscopic. Miscible with water, with chloroform and with ether. Identification; Appearance of solution; Acidity or alkalinity; Absorbance; Volatile impurities; Residue on evaporation complies with the requirements stated under Ethanol (96%). Relative density 0.790 to 0.793 (Appendix 6.5). 389

VP V

ETHANOL (96%)

Storage Store protected from moisture, at a temperature of 8 °C to 15 °C, flammable.

Volatile impurities Examine by gas chromatography (Appendix 5.2). Test solution (1): The substance to be examined. Test solution (2): Dissolve 150 µl of 4-methylpentan-2-ol R in 500.0 ml of the substance to be examined. ETHANOL (96%) Reference solution (1): Dilute 100 µl of anhydrous Ethanolum (96%) methanol R to 50.0 ml with the substance to be examined. Dilute 5.0 ml of the solution to 50.0 ml with the substance CH3-CH2-OH to be examined. C2H6O M. 46.07 Reference solution (2): Dilute 50 µl of anhydrous methanol R and 50 ml of acetaldehyde R to 50.0 ml with the Ethanol (96%) is a mixture of ethanol and water. It contains substance to be examined. Dilute 100 ml of the solution to from 92.6% (m/m) to 95.2% (m/m) or from 95.1% (v/v) 10.0 ml with the substance to be examined. to 96.9% (v/v) of C2H5OH, at 20 °C, calculated from Reference solution (3): Dilute 150 µl of acetal the relative density using the alcoholimetric tables (1,1-diethoxyethane) R to 50.0 ml with the substance to be (Appendix 19). examined. Dilute 100 µl of the solution to 10.0 ml with the substance to be examined. Characters Reference solution (4): Dilute 100 µl of benzene R to Colourless, clear, volatile liquid, with characteristic odour; 100.0 ml with the substance to be examined. Dilute 100 µl flammable, burning with a blue, smokeless flame. Miscible of the solution to 50.0 ml with the substance to be examined. with water, with chloroform, with ether and with glycerol. Chromatographic system: A fused silica column (30 m × 0.32 mm) coated with Identification A. Heat 1ml with 1 ml of glacial acetic acid R and add poly[(cyanopropyl)(phenyl)][dimethyl]siloxane R (film some drops of diluted sulfuric acid solution R. The odour thickness 1.8 µm). Carrier gas: Helium for chromatography R; linear velocity: of ethyl acetate is detectable. B. To 5 ml of a 10% (v/v) solution, add 1 ml of 1 M sodium 35 cm/s. Split ratio: 1 : 20. hydroxide R and then, slowly, 2 ml of a solution containing Temperature: 2% iodine R and 4% of potassium iodide R. The odour of Time Temperature iodoform is detectable and a yellow precipitate is produced. Appearance of solution It is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2) when compared with water. Dilute 1.0 ml to 20 ml with water. After standing for 5 minutes, the dilution remains clear (Appendix 9.2) when compared with water. Acidity or alkalinity To 20 ml add 20 ml of carbon dioxide-free water R and 0.1 ml of phenolphthalein solution R. The solution is colourless. Add 1.0 ml of 0.01 N sodium hydroxide VS. The solution is pink. Relative density 0.805 to 0.812 (Appendix 6.5). Absorbance Record the ultraviolet absorption spectrum of the substance to be examined in the range 235 nm to 340 nm, in a 5 cm cell using water as the blank solution. The maximum of absorbance at 240 nm is 0.40; the maximum of absorbance in the range 250 nm to 260 nm is 0.30; the maximum of absorbance in the range 270 nm to 340 nm is 0.10. The absorption curve is smooth (without noise) (Appendix 4.1). 390

Column

(min)

(°C)

0 - 12 12 - 32 32 - 42

40 40 → 240 240

Injection port

200

Detector

280

Detector: A flame-ionisation detector. Volume of injection: 1 µl. Procedure: System suitability: In the chromatogram obtained with reference solution (2), the resolution between the first peak (acetaldehyde) and the second peak (methanol) is at least 1.5. Limits: The area of the methanol peak in the chromatogram obtained with test solution (1): not more than half the area of the corresponding peak in the chromatogram obtained with reference solution (1) (200 ppm v/v). Sum of the areas of the acetaldehyde peak and the acetal peak: Not more than 10 ppm (v/v), expressed as acetaldehyde. Calculate the sum of the contents of acetaldehyde and acetal in parts per million (v/v) using the following expression: 10 × AE 30 × C E + Ar − AE Cr − C E

VP V

DILUTE ETHANOLS

In which: AE is area of the acetaldehyde peak in the chromatogram obtained with test solution (1). AT is area of the acetaldehyde peak in the chromatogram obtained with reference solution (2). CE is area of the acetal peak in the chromatogram obtained with test solution (1). CT is area of the acetal peak in the chromatogram obtained with reference solution (3). Benzene: Not more than 2 ppm v/v. Calculate the content of benzene in parts per million (v/v) using the following expression:

2 BE B T − BE

In which: BE is area of the benzene peak in the chromatogram obtained with the test solution (1). BT is area of the benzene peak in the chromatogram obtained with reference solution (4). If necessary, the identity of benzene can be confirmed using another suitable chromatographic system (stationary phase with a different polarity). Total peak area of other impurities in the chromatogram obtained with test solution (2): not more than the area of the peak due to 4-methylpentan-2-ol in the chromatogram obtained with test solution (2) (300 ppm). Disregard any peak with an area less than 0.03 times the area of the peak corresponding to 4-methylpentan-2-ol in the chromatogram obtained with test solution (2) (9 ppm).

Residue on evaporation Not more than 25 ppm (m/v). Evaporate 100 ml to dryness on a water-bath and dry at 100 °C to 105 °C for 1 h. The residue weighs not more than 2.5 mg. Storage Store protected from moisture, at a temperature of 8 °C to 15 °C, flammable. DILUTE ETHANOLS Dilutum ethanolum The official dilute ethanols contain 90%, 80%, 70%, 60%, 50%, 45%, 25% and 20% v/v respectively of C2H5OH. They may be prepared as described below, the final adjustment of volume being made at the same temperature, 20 °C, as that at which the ethanol (96%) is measured. Note: On mixing ethanol and water, contraction of volume and rise of temperature occur.

Identification; Appearance of solution; Acidity or alkalinity; Volatile impurities; Residue on evaporation: Comply with the requirements stated under Ethanol (96%).

Ethanol (90%) Alcohol (90%). Dilute 934 ml of ethanol (96%) to 1000 ml with water. Content of ethanol: 89.6% to 90.5% v/v. Apparent density (Appendix 6.5): 826.4 kg·m-3 to 829.4 kg·m-3. Ethanol (80%) Alcohol (80%). Dilute 831 ml of ethanol (96%) to 1000 ml with water. Content of ethanol: 79.5% to 80.3% v/v. Apparent density (Appendix 6.5): 857.4 kg·m-3 to 859.6 kg·m-3. Ethanol (70%) Alcohol (70%). Dilute 727 ml of ethanol (96%) to 1000 ml with water. Content of ethanol: 69.5% to 70.4% v/v. Apparent density (Appendix 6.5): 883.5 kg·m-3 to 885.8 kg·m-3. Ethanol (60%) Alcohol (60%). Dilute 623 ml of ethanol (96%) to 1000 ml with water. Content of ethanol: 59.7% to 60.2% v/v. Apparent density (Appendix 6.5): 907.6 kg·m-3 to 908.7 kg·m-3. Ethanol (50%) Alcohol (50%). Dilute 519 ml of ethanol (96%) to 1000 ml with water. Content of ethanol: 49.6% to 50.2% v/v. Apparent density (Appendix 6.5): 928.6 kg·m-3 to 929.8 kg·m-3. Ethanol (45%) Alcohol (45%). Dilute 468 ml of ethanol (96%) to 1000 ml with water. Content of ethanol: 44.7% to 45.3% v/v. Apparent density (Appendix 6.5): 938.0 kg·m-3 to 939.0 kg·m-3. Ethanol (25%) Alcohol (25%). Dilute 259 ml of ethanol (96%) to 1000 ml with water. Content of ethanol: 24.6% to 25.4% v/v. Apparent density (Appendix 6.5): 966.6 kg·m-3 to 967.5 kg·m-3. Ethanol (20%) Alcohol (20%). Dilute 207 ml of ethanol (96%) to 1000 ml with water. Content of ethanol: 19.5% to 20.5% v/v. Apparent density (Appendix 6.5): 972.0 kg·m-3 to 973.1 kg·m-3.

391

VP V

ETHER

ETHER Aether medicinalis H 3C

C4H10O

O

CH3



M. 74.1

Ether is diethyl ether. It contains not less than 96.0% and not more than 98.0% of C4H10O, and a little amount of ethanol and water.

Characters A clear, colourless, very mobile liquid; characteristic odour. Highly flammable, volatile. Mixtures of its vapour with air, oxygen or nitrous oxide in certain concentration are explosive. Soluble in 15 parts of water, miscible with ethanol, with benzene, with chloroform, with petroleum ether, with fatty oils and with volatile oils. Identification A. It complies with the test for Relative density. B. It complies with the test for Distillation range. Relative density 0.714 to 0.718 (Appendix 6.5). Distillation range Do not distil if the substance to be examined does not comply with the test for peroxides. It distils completely between 34 °C and 36 °C (Appendix 6.8). Carry out the test using a suitable heating device and taking care to avoid directly heating the flask above the level of the liquid. Acidity To 20.0 ml of ethanol (96%) R add 0.25 ml of bromothymol blue solution R and, dropwise, 0.02 N sodium hydroxide VS until a blue colour persists for 30 s. Add 25.0 ml of the substance to be examined, shake and add, dropwise, 0.02 N sodium hydroxide VS until the blue colour reappears and persists for 30 s. Not more than 0.4 ml of 0.02 N sodium hydroxide VS is required. Peroxides Place 8.0 ml of 10% solution of potassium iodide R in a 12 ml ground-glass-stoppered cylinder about 1.5 cm in diameter. Fill completely with the substance to be examined, shake vigorously and allow to stand protected from light for 30 min. Any yellow colour produced is not more intense than that of 0.5 ml of 0.0005 M iodine VS diluted with 8.0 ml of 10% solution of potassium iodide R. Aldehydes Shake 10.0 ml with 1 ml of alkaline potassium tetraiodomercurate solution R in a ground-glass-stoppered cylinder for 10 s and allow to stand protected from light for 5 min. The lower layer may show yellow or reddishbrown opalescence but not grey or black opalescence. 392

Foreign odour Drop 10.0 ml of the substance to be examined to a clean, odourless filter paper with 25 cm2 area and allow to evaporate in air. No foreign odour is detectable after the evaporation. Water Not more than 0.2% (m/v) (Appendix 10.3). Determined on 20 ml. Residue on evaporation Do not carry out this test unless the substance to be examined complies with the test for peroxides. Place accurately 50 ml into a tared glass beaker. Evaporate to dryness on a water-bath and dry the residue in an oven at 100 °C to 105 °C to constant mass. The residue weighs not more than 1.0 mg. Storage Store in an airtight container, protected from light, at a temperature not more than 15 °C and very flammable. Action and use Solvent. ANAESTHETIC ETHER Aether anaesthesicus H 3C

O

CH3

C4H10O

M. 74.1

Anaesthetic ether is diethyl ether which may contain a suitable non-volatile antioxidant at an appropriate concentration.

Characters A clear, colourless, very mobile liquid; characteristic odour. Highly flammable, volatile. Mixtures of its vapour with air, oxygen or nitrous oxide in certain concentration are explosive. Soluble in 15 parts of water, miscible with ethanol, with benzene, with chloroform, with petroleum ether, with fatty oils and with volatile oils. Identification A. It complies with the test for Relative density. B. It complies with the test for Distillation range. Relative density 0.714 to 0.716 (Appendix 6.5). Distillation range Do not distil if the substance to be examined does not comply with the test for peroxides. It distils completely between 34 °C and 35 °C (Appendix 6.8). Carry out the test using a suitable heating device and taking care to avoid directly heating the flask above the level of the liquid.

VP V

ETHINYLESTRADIOL

Anaesthetic ether complies with the requirements of purity tests stated under “Ether” and with the following requirements:

Peroxides Place 8.0 ml of potassium iodide and starch solution R in a 12 ml ground-glass-stoppered cylinder about 1.5 cm in diameter. Fill completely with the substance to be examined, shake vigorously and allow to stand protected from light for 30 min. No colour is produced. Acetone and aldehydes Shake 10.0 ml with 1 ml of alkaline potassium tetraiodomercurate solution R in a ground-glass-stoppered cylinder for 10 s and allow to stand protected from light for 5 min. The lower layer shows only a slight opalescence. If the substance to be examined does not comply with the test, distil 40.0 ml until only 5 ml remains. Collect the distillate in a receiver cooled by placing in a bath of iced water and repeat the test described above using 10.0 ml of the distillate (Do not redistil unless the substance to be examined complies with the test for peroxides). Storage Store in an airtight container, protected from light, at a temperature of 8 °C to 15 °C, very flammable. Note: Not to be used for anaesthesia if the container has been opened longer than 24 h. After 6 months storage the substance must be examined again.

Labelling The label states the name and concentration of any added non-volatile antioxidant; the suitable route of administration in anaesthesia. Action and use Anaesthesia. ETHINYLESTRADIOL Ethinylestradiolum

C20H24O2  

M. 296.4

Ethinylestradiol is 19-nor-17α-pregna-1,3,5(10)-trien20-yne-3,17-diol. It contains not less than 97.5% and not more than 102.0% of C20H24O2, calculated with reference to the dried substance.

Characters White or slightly yellowish-white, crystalline powder. It shows polymorphism. Practically insoluble in water,

freely soluble in ethanol (96%). It dissolves in dilute alkaline solutions.

Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of ethinylestradiol RS. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in methanol R, evaporate to dryness and record new spectra using the residues. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Ethanol (96%) - toluene (10 : 90). Solvent mixture: Methanol - methylene chloride (10 : 90). Test solution: Dissolve 25 mg of the substance to be examined in the solvent mixture and dilute to 25 ml with the solvent mixture. Reference solution: Dissolve 25 mg of ethinylestradiol RS in the solvent mixture and dilute to 25 ml with the solvent mixture. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of two thirds of the plate. Dry the plate in air until the solvent has evaporated. Heat at 110 °C for 10 min, spray the hot plate with a 20% solution of sulfuric acid R in ethanol 96% R and heat again at 110 °C for 10 min. Examine in daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, colour, fluorescence and size to the principal spot in the chromatogram obtained with the reference solution. Related substances Examine by liquid chromatography (Appendix 5.3) Mobile phase A: Acetonitrile R1 - water (30 : 70). Mobile phase B: Water - acetonitrile R1 (25 : 75). Solvent mixture: Water - acetonitrile R1 (40 : 60). Test solution: Dissolve 50.0 mg of the substance to be examined in 30 ml of acetonitrile R1 and dilute to 50.0 ml with water. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 10.0 ml with the solvent mixture. Reference solution (2): Dissolve 2 mg of estrone RS (impurity C) in 10.0 ml of the solvent mixture. Dilute 1.0 ml of the solution to 100.0 ml with the solvent mixture. Use 1.0 ml of this solution to dissolve the contents of a vial of ethinylestradiol for system suitability RS (containing impurities B, F, H, I and K). Reference solution (3): Dissolve 50.0 mg of ethinylestradiol RS in 30 ml of acetonitrile R1 and dilute to 50.0 ml with water. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase end-capped butylsilyl silica gel for chromatography R (5 µm). 393

VP V

ETHINYLESTRADIOL TABLETS

Column temperature: 30 °C. Detector: A spectrophotometer set at 220 nm. Flow rate: 1.5 ml/min. Volume of injection: 30 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 35

100

0

35 - 65

100 → 0

0 → 100

Inject the test solution and reference solutions (1) and (2). Identification of impurities: Use the chromatogram supplied with ethinylestradiol for system suitability RS and the chromatogram obtained with reference solution (2) to identify the peaks due to impurities B, C, F, H, I and K. Relative retention with reference to ethinylestradiol (retention time = about 35 min): impurity F = about 0.2; impurity H = about 0.5; impurity I = about 0.8; impurity B = about 0.88; impurity C = about 0.92; impurity K = about 1.3. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurities I and B is at least 1.2. Limits: Correction factors: for the calculation of content, multiply the peak areas of the following impurities by the corresponding correction factor: impurity B = 0.7; impurity I = 0.4; Impurity B: The corrected area of the peak due to impurity B is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). Impurities H, I, K: For each impurity, corrected if necessary, the area is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%). Impurities C, F: For each impurity, the area is not more than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.15%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). Total peak area of all impurities is not more than 8 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.8%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A: 19-norpregna-1,3,5(10)-trien-20-yne-3,17-diol (17b-ethinylestradiol). Impurity B: 19-nor-17α-pregna-1,3,5(10),9(11)-tetraen-20-yne3,17-diol. Impurity C: 3-hydroxyestra-1,3,5(10)-trien-17-one (estrone). Impurity D: estra-1,3,5(10)-triene-3,17β-diol (estradiol).

394

Impurity E: 19-nor-17α-pregna-1,3,5(10)-trien-20-yne-3,6α,17triol (6α-hydroxy-ethinylestradiol). Impurity F: 19-nor-17α-pregna-1,3,5(10)-trien-20-yne-3,6β,17triol (6β-hydroxy-ethinylestradiol). Impurity G: 3,17-dihydroxy-19-nor-17α-pregna-1,3,5(10)-trien20-yn-6-one (6-oxo-ethinylestradiol). Impurity H: 3,17-dihydroxy-19-nor-17α-pregna-1,3,5(10)-trien20-yn-16-one (16-oxo-ethinylestradiol). Impurity I: 19-nor-17α-pregna-1,3,5(10),6-tetraen-20-yne-3,17diol. Impurity J: 1-methyl-19-nor-17α-pregna-1,3,5(10)-trien-20yne-3,17-diol (1-methyl-ethinylestradiol). Impurity K: 4-methyl-19-nor-17α-pregna-1,3,5(10)-trien-20yne-3,17-diol (4-methyl-ethinylestradiol). Impurity L: estra-1,3,5(10)-triene-3,17α-diol (17α-estradiol). Impurity M: 2-methyl-19-nor-17α-pregna-1,3,5(10)-trien-20yne-3,17-diol (2-methyl-ethinylestradiol).

Loss on drying Not more than 1.0% (Appendix 9.6). (0.500 g; 105 °C; 3 h). Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for related substances. Inject the test solution and reference solution (3). Calculate the content of C20H24O2 using the areas of the peaks in the chromatograms obtained with the test solution, the reference solution (3) and the declared content of C20H24O2 in ethinylestradiol RS. Storage Protected from light. Action and use Estrogen. Preparations Tablets. Levonorgestrel and ethinylestradiol tablets. ETHINYLESTRADIOL TABLETS Tabellae Ethinylestradioli Ethinylestradiol tablets contain ethinylestradiol. They may be coated. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of ethinylestradiol, C20H24 O2, 90.0% to 110.0% of the stated amount. Identification Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Toluene - ethanol (96%) (90 : 10).

VP V

Test solution: Shake a quantity of the powdered tablets (remove the coating if necessary) containing the equivalent of about 0.25 mg of ethinylestradiol with four 20 ml quantities of acetone R. Filter each extract in turn, evaporate the combined filtrates to dryness on a water bath in a current of nitrogen. Dissolve the residue in 0.25 ml of acetone R. Reference solution: A 0.1% solution of ethinylestradiol RS in acetone R. Procedure: Apply separately to the plate 20 µl of each of the test and the reference solution. Develop the chromatograph in a saturated tank until the solvent front has moved about 15 cm. After removal of the plate, allow it to dry in air and spray with ethanolic sulfuric acid R and dry at 110 oC for 10 minutes. Examine under ultraviolet light at 365 nm and in daylight. By both method of visualisation, the principal spot in the chromatogram obtained with the test solution corresponds in position, colour and size to that in the chromatogram obtained with the reference solution. B. Triturate a quantity of the powdered tablets containing the equivalent of about 0.1 mg of ethinylestradiol with 0.5 ml of 0.1M sodium hydroxide R and 5 ml of water. Allow to stand for 5 minutes, filter, acidify the filtrate with 0.15 ml of sulfuric acid R, add 3 ml of ether R, shake and allow to separate. Evaporate the ether layer to dryness and heat the residue on a water bath for 5 minutes with 0.2 ml of glacial acetic acid R and 2 ml of phosphoric acid R. A pink colour with an intense orange fluorescence is produced. C. In the test for Uniformity of content, the principal peak in the chromatogram obtained with the test solution has the same retention time as the principal peak in the chromatogram obtained with the reference solution.

Uniformity of content (Appendix 11.2) Examine by liquid chromatography (Appendix 5.3). Mobile phase: Water - acetonitrile (40 : 60). Test solution: For tablets containing more than 50 µg of ethinylestradiol: Put one tablet into a suitable glass-stoppered conical flask. Add 10.0 ml of the mobile phase, allow to stand for complete disintegration, sonicate for 10 minutes. Centrifuge and use the clear supernatant liquid. For tablets containing equal to or less than 50 µg of ethinylestradiol: Put one tablet into a suitable glassstoppered conical flask. Add 5.0 ml of the mobile phase, allow to stand for complete disintegration, sonicate for 10 minutes. Centrifuge and use the clear supernatant liquid. Reference solution: Dissolve ethinylestradiol RS in the mobile phase to obtain a solution having the same concentration as the test solution. Chromatographic system: A column (20 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 280 nm. Flow rate: 1.5 ml/min. Volume of injection: 100 µl or 200 µl.

ETHYLCELLULOSE

Procedure: Inject alternately the test solution and the reference solution. Calculate the content of ethinylestradiol, C20H24O2, in each tablet using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C20H24O2 in ethinylestradiol RS.

Assay Use the average of the 10 individual results obtained in the test for Uniformity of content. Storage Store in a cool and dry place, protected from light. Action and use Estrogen. Usual strength 10 µg; 50 µg; 1 mg. ETHYLCELLULOSE Ethylcellulosum Ethylcellulose is partly O-ethylated cellulose. It contains not less than 44.0% and not more than 51.0% of ethoxy (-OC2H5) groups, calculated with reference to the dried substance.

Characters White or yellowish-white powder or granular powder, odourless or almost odourless. Soluble in methylene chloride and in a mixture of 20 g of ethanol (96%) and 80 g of toluene, slightly soluble in ethyl acetate and in methanol, practically insoluble in water, in glycerol (85%) and in propylene glycol. The solutions may show a slight opalescence. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of reference spectrum of ethylcellulose. B. It complies with the limits of the Assay. Acidity or alkalinity Add 25 ml of carbon dioxide-free water R to 0.5 g of the substance to be examined and shake for 15 min. Filter through a sintered-glass filter (40). To 10 ml of the solution, add 0.1 ml of phenolphthalein solution R and 0.5 ml of 0.01 N sodium hydroxide VS. The solution is pink. To 10 ml of the solution add 0.1 ml of methyl red solution R and 0.5 ml of 0.01 N hydrochloric acid VS. The solution is red. Viscosity 80.0% to 120.0% of that stated on the label for a nominal viscosity greater than 6 mPa·s. 395

VP V

ETHYLCELLULOSE

75.0% to 140.0% of that stated on the label for a nominal viscosity not greater than 6 mPa·s. Shake a quantity of the substance to be examined equivalent to 5.00 g of the dried substance with 95 g of a mixture of 20 g of ethanol (96%) R and 80 g of toluene R until the substance is dissolved. Determine the viscosity at 25 °C, using a capillary viscometer (Appendix 6.3).

Acetaldehyde Not more than 100 ppm. Introduce 3.0 g of the substance to be examined into a 250 ml conical flask with a ground-glass stopper, add 10 ml of water and stir mechanically for 1 h. Allow to stand for 24 h, filter and dilute the filtrate to 100.0 ml with water (solution A). Transfer 5.0 ml of the solution A to a 25 ml volumetric flask, add 5 ml of a 0.05% solution of methylbenzothiazolone hydrazone hydrochloride R and heat in a water-bath at 60 °C for 5 min. Add 2 ml of iron (III) chloride-sulfamic acid reagent R and heat again in a water-bath at 60 °C for 5 min. Cool and dilute to 25.0 ml with water. The solution is not more intensely coloured than a standard prepared at the same time and in the same manner using instead of the 5.0 ml of solution A, 5.0 ml of a reference solution prepared by diluting 3.0 ml of acetaldehyde standard solution (100 ppm C2H4O) in water to 100.0 ml with water. Chlorides Not more than 0.1% (Appendix 9.4.5). Disperse 0.250 g of the substance to be examined in 50 ml of water, heat to boiling and allow to cool, shaking occasionally. Filter and discard the first 10 ml of the filtrate. Dilute 10 ml of the filtrate to 15 ml with water. Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 3. Prepare the standard solution using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 3.0% (Appendix 9.6). (1.000 g; 105 °C; 2 h). Sulfated ash Not more than 0.5% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by gas chromatography (Appendix 5.2). Caution: Hydriodic acid and its reaction by-products are highly toxic. Perform all steps for preparation of the test and reference solutions in a properly functioning hood. Internal standard solution: Dilute 120 µl of toluene R to 10 ml with o-xylene R. 396

Test solution: Transfer 50.0 mg of the substance to be examined, 50.0 mg of adipic acid R and 2.0 ml of the internal standard solution into a suitable 5 ml thick-walled reaction vial with a pressure-tight septum-type closure. Cautiously add 2.0 ml of hydriodic acid R, immediately close the vial tightly and weigh the contents and the vial accurately. Shake the vial for 30 s, heat to 125 °C for 10 min, allow to cool for 2 min, shake again for 30 s and heat to 125 °C for 10 min. Afterwards allow to cool for 2 min and repeat shaking and heating for a 3rd time. Allow the vial to cool for 45 min and reweigh. If the loss is greater than 10 mg, discard the mixture and prepare another. Use the upper layer. Standard solution: Transfer 100.0 mg of adipic acid R, 4.0 ml of the internal standard solution and 4.0 ml of hydriodic acid R into a suitable 10 ml thick-walled reaction vial with a pressure-tight septum-type closure. Close the vial tightly and weigh the vial and contents accurately. Afterwards inject 50 µl of iodoethane R through the septum with a syringe, weigh the vial again and calculate the mass of iodoethane added, by difference. Shake well and allow the layers to separate. Use the upper layer. Chromatographic system: A column (5.0 m long and 2 mm in internal diameter) packed with diatomaceous earth for gas chromatography R (150 µm -180 µm) impregnated with 3% m/m of poly (dimethyl)siloxane R. Carrier gas: Nitrogen for chromatography. Flow rate: 15 ml/min. Detection: Flame ionisation. Temperature: Column: 80 °C, injection port and detector: 200 °C. Volume of injection: 1 µl. Procedure: Inject the standard solution. Relative retention with reference to toluene of iodoethane = about 0.6 and of o-xylene = about 2.3. The test is not valid unless the resolution between the peaks due to iodoethane and toluene is at least 2.0. Separately inject the test solution and the standard solution. Calculate the percentage content of ethoxy groups using the following expression:

Q1 × m2 × 45.1 × 100 × 100 2 × Q2 × m1 × 156.0 × (100 − d )

Where: Q1 ratio of iodoethane peak area to toluene peak area in the chromatogram obtained with the test solution. Q2 ratio of iodoethane peak area to toluene peak area in the chromatogram obtained with the standard solution. m1 mass of the substance to be examined used in the test solution (mg). m2 mass of iodoethane used in the standard solution (mg). d percentage loss on drying (%).

VP V

EUGENOL

Storage Store in an airtight container, protected from light. Action and use Excipients. Labelling The label states the nominal viscosity in millipascal seconds for a 5% m/m solution.

OH

OCH3

C10H12O2   Eugenol is 2-methoxy-4-(prop-2-enyl)phenol.

Relative density 1.066 to 1.070 (Appendix 6.5). Refractive index 1.540 to 1.542 (Appendix 6.1). Dimeric and oligomeric compounds Dissolve 0.150 g in ethanol R and dilute to 100.0 ml with the same solvent. The absorbance (Appendix 4.1) of the solution at 330 nm is not greater than 0.25.

EUGENOL Eugenolum

H 2C

D. Dissolve 0.05 ml in 2 ml of ethanol (96%) R and add 0.1 ml of 10.5% solution of ferric chloride R. A dark green colour is produced which changes to yellowish-green within 10 min.

M. 164.2

Characters Colourless or pale yellow, clear liquid, darkening on exposure to air. It has a strong odour of clove. Practically insoluble in water, freely soluble in ethanol (70%), practically insoluble in glycerol, miscible with ethanol (96%), with glacial acetic acid with ether, with methylene chloride and with fatty oils. Identification Apply one of the two following identifications: First identification: A. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of eugenol RS. B. It complies with the test for Refractive index. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel F254. Mobile phase: Ethyl acetate - toluene (10 : 90). Test solution: Dissolve 50 µl of the substance to be examined in ethanol (96%) R and dilute to 25 ml with the same solvent. Reference solution: Dissolve 50 µl of eugenol RS in ethanol (96%) R and dilute to 25 ml with the same solvent. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm. Dry the plate in a current of cold air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. Spray the place with anisaldehyde solution R and heat at 100 °C to 105 °C for 10 min. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution.

Related substances Examine by gas chromatography (Appendix 5.2), use the normalisation procedure. Test solution: Dissolve 1.00 g of the substance to be examined in ethanol R and dilute to 5.0 ml with the same solvent. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with ethanol R. Reference solution (2): Dissolve 50 mg of vanillin R (impurity H) in 1 ml of the test solution and dilute to 5 ml with ethanol R. Chromatographic system: A fused-silica capillary column (30 m long and 0.25 mm in internal diameter) coated with polymethylphenylsiloxane R (film thickness 0.25 µm). Carrier gas: Helium for chromatography. Flow rate: 1 ml/min. Split ratio: 1 : 40. Detector: A flame-ionisation detector. Volume of injection: 1 µl. Temperature programme:

Column

Time (min)

Temperature (°C)

0-2 2 - 27 27 - 47

80 80 → 280 280

Injection port

250

Detector

280

Procedure: System suitability: In the chromatogram obtained with reference solution (2), relative retention with reference to eugenol: impurity H = minimum 1.1. Limits: Any impurity: For each impurity, not more than 0.5%. Sum of impurities with a relative retention greater than 2.0 with reference to eugenol: Not more than 1.0%. Total: Not more than 3.0%. Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). 397

VP V

FAMOTIDINE Note: Impurity A: (1R,4E,9S)-4,11,11-trimethyl-8-methylenebicyclo [7.2.0]undec-4-ene (b- caryophyllene), Impurity B: (1E,4E,8E)-2,6,6,9-tetramethylcycloundeca-1,4,8triene (a-humulene, a- caryophyllene), Impurity C: (1R,4R,6R,10S)-4,12,12-trimethyl-9-methylene-5oxatricyclo[8.2.0.04,6]dodecane (b-caryophyllene oxide). Impurity D: 4-(prop-2-enyl)phenol. Impurity E: 1,2-dimethoxy-4-(prop-2-enyl)benzene (eugenol methyl ether). Impurity F(cis): 2-methoxy-4-[(Z)-prop-1-enyl]phenol (cisisoeugenol). Impurity G (trans): 2-methoxy-4-[(E)-prop-1- enyl]phenol (trans -isoeugenol). Impurity H: 4-hydroxy-3-methoxybenzaldehyde (vanillin). Impurity I: 2-methoxy-4-(prop-2- enyl)phenyl acetate (acetyleugenol). Impurity J: 1-(4-hydroxy-3-methoxyphenyl)prop-2-enone. Impurity K: (E)-3-(4-hydroxy-3-methoxyphenyl)prop-2-enal (trans-coniferyl aldehyde). Impurity L: 2-methoxy-4-[3-methyl-5-(prop-2-enyl)-2,3-dihydro benzofuran-2-yl]phenol (dehydrodi-isoeugenol), Impurity M: 3,3’-dimethoxy-5,5'-bis(prop-2-enyl)biphenyl2,2’-diol (dehydrodieugenol). Impurity N: O: 2 further unknown dimeric compounds. Impurity P: Toluene.

Hydrocarbons Dissolve 1 ml of the substance to be examined in 5 ml of 2 M sodium hydroxide R and add 30 ml of water in a stoppered test-tube. Examined immediately, the solution is yellow (Appendix 9.3) and clear (Appendix 9.2). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Storage In a well-filled container, protected from light. Action and use Local anaesthetic in dentistry. FAMOTIDINE Famotidinum

C8H15N7O2S3  

M. 337.4

Famotidine is 3-[[[2-[(diaminomethylidene)amino]thiazol4-yl]methyl]sulfanyl]-N’-sulfamoylpropanimidamide. It contains not less than 98.5% and not more than 101.5% of C8H15N7O2S3, calculated with reference to the dried substance. 398

Characters White or yellowish-white, crystalline powder or crystals. It shows polymorphism. Very slightly soluble in water, freely soluble in glacial acetic acid, very slightly soluble in anhydrous ethanol, practically insoluble in ethyl acetate. It dissolves in dilute mineral acids. Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of famotidine RS. Examine the substances prepared as discs. If the spectra obtained show differences, suspend 0.10 g of the substance to be examined and 0.10 g of the reference substance separately in 5 ml of water. Heat to boiling and allow to cool, scratching the wall of the tube with a glass rod to initiate crystallisation. Filter, wash the crystals with 2 ml of iced water and dry in an oven at 80 °C at a pressure not exceeding 670 Pa for 1 h. Record new spectra using the residues. Appearance of solution Dissolve 0.20 g in 0.5 M hydrochloric acid R, heating to 40 °C if necessary, and dilute to 20 ml with the same acid. The solution is clear (Appendix 9.2) and not more intensely coloured than reference solution BY7 (Appendix 9.3, method 2). Related substances Examine by liquid chromatography (Appendix 5.3) Mobile phase A: Mix 6 volumes of methanol R, 94 volumes of acetonitrile R and 900 volumes of a 1.882 g/L solution of sodium hexanesulfonate R previously adjusted to pH 3.5 with acetic acid R. Mobile phase B: Acetonitrile R. Test solution: Dissolve 12.5 mg of the substance to be examined in mobile phase A and dilute to 25.0 ml with mobile phase A. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with mobile phase A. Dilute 1.0 ml of this solution to 10.0 ml with mobile phase A. Reference solution (2): Dissolve 2.5 mg of famotidine impurity D RS in methanol R and dilute to 10.0 ml with the same solvent. To 1.0 ml of the solution add 0.50 ml of the test solution and dilute to 100.0 ml with mobile phase A. Reference solution (3): Dissolve 5.0 mg of famotidine for system suitability RS (containing impurities A, B, C, D, F and G) in mobile phase A and dilute to 10.0 ml with mobile phase A. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (5 µm). Column temperature: 50 °C. Detector: A spectrophotometer set at 265 nm. Volume of injection: 20 µl.

VP V

FAMOTIDINE TABLETS

Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

Flow rate (ml/min)

0 - 23

100 → 96

0→4

1

23 - 27

96

4

1→2

27 - 47

96 → 78

4 → 22

2

Identification of impurities: Use the chromatogram supplied with famotidine for system suitability RS and the chromatogram obtained with reference solution (3) to identify the peaks due to impurities A, B, C, F and G; use the chromatogram obtained with reference solution (2) to identify the peak due to impurity D. Relative retention with reference to famotidine (retention time = about 21 min): impurity D = about 1.1; impurity C = about 1.2; impurity G = about 1.4; impurity F = about 1.5; impurity A = about 1.6; impurity B = about 2.0. System suitability: Retention time of famotidine is from 19 min to 23 min in all the chromatograms; in the chromatogram obtained with reference solution (2), the resolution between the peaks due to famotidine and impurity D is at least 3.5. Limits: Correction factors: For the calculation of content, multiply the peak areas of the following impurities by the corresponding correction factor: impurity A = 1.9; impurity B = 2.5; impurity C = 1.9; impurity F = 1.7; impurity G = 1.4; Impurities C, D: For each impurity, the area, corrected if necessary, is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.3%). Impurities A, B, F, G: For each impurity, the corrected area is not more than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.15%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%). The sum of the areas of all the impurity peaks is not more than 8 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.8%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1). Note: Impurity A: 3-[[[2-[(diaminomethylene)amino]thiazol-4-yl] methyl]sulfanyl]propanimidamide. Impurity B: 3,5-bis[2-[[[2-[(diaminomethylene)amino]thiazol4-yl]methyl]sulfanyl]ethyl]-4H-1,2,4,6-thiatriazine 1,1-dioxide. Impurity C: 3-[[[2-[(diaminomethylene)amino]thiazol-4-yl] methyl]sulfanyl]-N-sulfamoylpropanamide. Impurity D: 3-[[[2-[(diaminomethylene)amino]thiazol-4-yl] methyl]sulfanyl]propanamide.

Impurity E: 2,2’-[disulfanediylbis(methylenethiazole-4,2-diyl)] diguanidine. Impurity F: 3-[[[2-[(diaminomethylene)amino]thiazol-4-yl]methyl] sulfanyl]propanoic acid. Impurity G: N-cyano-3-[[[2-[(diaminomethylene)amino]thiazol4-yl] methyl]sulfanyl]propanimidamide.

Heavy metals Not more than 10 ppm (Appendix 9.4.8). 0.5 g complies with limit test for heavy metals, method 8. Prepare the standard using 0.5 ml of lead standard solution (10 ppm Pb) R. Solvent mixture: Dimethylformamide - water (30 : 70). Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 80 °C; at a pressure not exceeding 0.67 kPa; 5 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.120 g of the substance to be examined in 60 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 16.87 mg of C8H15N7O2S3. Storage Store in an airtight container, protected from light. Action and use Histamine H2 receptor antagonist. Preparation Tablets. FAMOTIDINE TABLETS Tabellae Famotidini Famotidine tablets contain famotidine. They may be sugarcoated or film-coated. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of famotidine, C8H15N7O2S3, 90.0% to 110.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel GF254. Mobile phase: Ethyl acetate - methanol - toluene ammonia (40 : 25 : 20 : 2). 399

FAMOTIDINE TABLETS

Test solution: Shake a quantity of the powdered tablets, containing about 40 mg of famotidine, with 4 ml of glacial acetic acid R with the aid of ultrasound, dilute to 10 ml with the same solvent, centrifuge. Use the supernatant liquid. Reference solution: A 0.4% solution of famotidine RS in glacial acetic acid R. Procedure: Apply separately to the plate 10 µl of each solution. Allow the spots to dry in air, and develop over a path of about 15 cm (chromatographic chamber is equilibrated with the mobile phase for 1 h before use). Remove of the plate, allow it to dry in air, and examine under ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to that in the chromatogram obtained with the reference solution. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of the principal peak in the chromatogram obtained with the reference solution.

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase, diluent, test solution, resolution solution and chromatographic system: Proceed as directed in the Assay. Reference solution: Dilute 1.0 ml of the test solution to 100 ml with the diluent. Procedure: System suitability is performed as directed in the Assay. Inject alternately the test solution and reference solution. Limits: In the chromatogram obtained with the test solution. The area of the peak corresponding to famotidine sulfoxide is not greater than that of the principal peak in the chromatogram obtained with the reference solution (1.0%); the area of each of the peak due to impurity F and impurity C is not greater than 0.5% times the area of the principal peak in the chromatogram obtained with the reference solution (0.5%); the area of the peak corresponding to impurity D after dividing by 1.3 (relative response factor of impurity D) is not greater than 0.5 times the area of principal peak in the chromatogram obtained with the reference solution (0.5%). The sum of the areas of all secondary peaks is not greater than 1.5%. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of 0.1 M phosphate buffer pH 4.5 prepared by dissolving 13.6 g of potassium dihydrogen phosphate R in water, adjust the pH of the solution with phosphoric acid R or 1 M potassium hydroxide R as necessary and add sufficient water to produce 1000 ml. Rotation speed: 50 rpm. Time: 45 min (60 min for sugar-coated tablets). Procedure: After the specified time, withdraw a sample of the medium, and filter, dilute the filtrate if necessary with the medium to obtain a solution having a suitable 400

VP V

concentration. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum wavelength at 265 nm in a 1-cm cell, using the dissolution medium as blank. Perform simultaneously a reference solution of famotidine RS in the dissolution medium having the similar concentration as the test solution. Calculate the content of famotidine, C8H15N7O2S3, dissolved using the absorbances of the test solution, the reference solution and the declared content of C8H15N7O2S3 in famotidine RS. Tolerance: Not less than 75% (Q) of the labeled amount of famotidine, C8H15N7O2S3, is dissolved in 45 minutes (or 60 min for sugar-coated tablets).

Assay Examine by liquid chromatography (Appendix 5.3). Buffer solution: Dissolve 13.6 g of sodium acetate R in 750 ml of water, add 1 ml triethylamine R, adjust the pH to 6.0 with glacial acetic acid R, add sufficient water to produce 1000 ml and mix. Mobile phase: Acetonitrile - buffer solution (7 : 93). Diluent: Dissolve 6.8 g of potassium dihydrogen phosphate R in 750 ml of water, adjust the pH to 6.0 with 1 M potassium hydroxide R, add sufficient water to produce 1000 ml and mix. Resolution solution: Transfer 10 mg of famotidine RS into a 50-ml volumetric flask. Add 1 ml of 0.1N hydrochloric acid R and heat at 80 oC for 30 min, allow to cool to room temperature. Add 2 ml of 0.1N sodium hydroxide R and heat at 80 oC for 30 min, allow to cool to room temperature and neutralized with 1 ml of 0.1 N hydrochloric acid R. Add sufficient diluent to volume and mix. Transfer 10 ml of the resulting solution into a 50-ml volumetric flask containing 5 mg of famotidine RS dissolved in 8 ml of methanol R, add sufficient diluent to volume, mix. Dilute 25 ml of the resulting solution to 50 ml with the diluent (this solution is stable in one month). Mix 1 ml to 1.5 ml of the resulting solution with 1 drop of dilute hydrogen peroxide solution R in an suitable container. (Prepare fresh daily) Test solution: Weigh 20 tablets and finely powder. Weigh accurately a quantity of the powdered tablets containing about 80 mg of famotidine, transfer into a 200-ml volumetric flask, add 40 ml of the diluent, mix, add 40 ml of methanol R, sonicate for 5 min, and shake by mechanical means for 1 h. Dilute to volume with the diluent and filter. Dilute 5.0 ml of the filtrate to 20.0 ml with the diluent. Reference solution: Weigh accurately 10 mg of famotidine RS and transfer into a 100-volumetric flask, add 20 ml of methanol R and shake to dissolve, add sufficient diluent to volume and mix. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 mm). Column temperature: 40 °C. Flow rate: 1.4 ml/min.

VP V

FELODIPINE

Identification Apply one of the two following identifications: First identification: A. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of felodipine RS. Examine the substances prepared as discs. B. Dissolve 50 mg of the substance to be examined in methanol R and dilute to 100 ml with the same solvent. Dilute 3 ml of this solution to 100 ml with methanol R. The ultraviolet absorption spectrum (Appendix 4.1) of the this solution in the range between 220 nm and 400 nm, exhibits two absorption maxima at 238 nm and at 361 nm. The ratios of the absorbances measured at the maxima at 361 nm to that measured at the maximum at 238 nm is 0.34 to 0.36. A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica F254. Mobile phase: Ethyl acetate - cyclohexane (40 : 60). Test solution: Dissolve 10 mg of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Storage Reference solution (1): Dissolve 10 mg of felodipine RS Store in a cool place, protected from light. in methanol R and dilute to 10 ml with the same solvent. Reference solution (2): Dissolve 5 mg of nifedipine RS Action and use in reference solution (1) and dilute to 5 ml with reference Histamine H2-receptor antagonist. solution (1). Usual strength Procedure: Apply separately to the plate 5 µl of each 40 mg. solution. Develop over a path of 15 cm. Allow the plate to dry in air. Examine in ultravioletlight at 254 nm. The principal spot in the chromatogram obtained with the test FELODIPINE solution is similar in position, fluorescence and size to the Felodipinum principal spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with the reference solution (2) shows two clearly separated spots. D. Dissolve 0.150 g of the substance to be examined in a mixture of 25 ml of 2-methyl-2-propanol R and 25 ml of and enantiomer 1 M perchloric acid R. Add 10 ml of a 0.1 M solution of cerium sulfate R, allow to stand for 15 min, add 3.5 ml of a 42% solution of sodium hydroxide R and neutralise with dilute sodium hydroxide solution R. Shake with 25 ml of methylene chloride R. Evaporate the lower layer to dryness on a waterC18H19Cl2NO4  M. 384.3  bath under nitrogen (the residue is also used in the test for Felodipine is ethyl methyl (4RS)-4-(2,3-dichlorophenyl)- Related substances). Dissolve about 20 mg of the residue in 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate.It methanol R and dilute to 50 ml with the same solvent. Dilute 2 ml of this solution to 50 ml with methanol R. contains not less than 99.0% and not more than 101.0% of The ultraviolt absorptiom spectrum (Appendix 4.1) of the C18H19Cl2NO4,calculatedwithreferencetothedriedsubstance. this solution, in the range between 220 nm and 400 nm, exhibits absorption maximum at 273 nm. Characters White or light yellow, crystalline powder. Appearance of solution Practically insoluble in water, freely soluble in acetone, in Solution S: Dissolve 1.00 g in methanol R and dilute to anhydrous ethanol, in methanol and in methylene chloride. 20.0 ml with the same solvent. Solution S is clear (Appendix 9.2). Detector: A spectrophotometer set at 275 nm. Volume of injection: 50 µl. Procedure: System suitability: Inject the reference solution. The peaks are eluted in the following order: famotidine sulfoxide, impurity F, impurity C, famotidine, impurity D corresponding to the following relative retention times: 0.4; 0.7; 0.8; 1.0; 1.2, respectively. The resolution factor between impurity C peak and famotidine peak is not less than 1.3 and between famotidine peak and impurity D peak is not less than 1.3. The capacity factor, determined on famotidine peak, is not less than 2.0. Inject the reference solution, the relative standard deviation for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of famotidine, C8H15N7O2S3, in the tablets using the areas of the principal peaks in the chromatograms obtained with the test solution, the reference solution and the declared content of C8H15N7O2S3 in famotidine RS.

401

VP V

FENOFIBRATE

Absorbance Not more than 0.10 at 440 nm (Appendix 4.1). Determined on solution S. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol - acetonitrile - phosphate buffer solution pH 3.0 containing 0.08% of phosphoric acid R and 0.8% of sodium dihydrogen phosphate R (20 : 40 : 40). Test solution: Dissolve 25.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Reference solution (2): Dilute 1.0 ml of reference solution (1) to 10.0 ml with the mobile phase. Reference solution (3): Dissolve 50.0 mg of the residue obtained in identification test D (impurity A) and 25.0 mg of felodipine RS in the mobile phase, then dilute to 50.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of the solution to 10.0 ml with the mobile phase. Chromatographic system: A column (12.5 cm - 15 cm × 4 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Elution order: Impurity B, impurity A, felodipine, impurity C. Procedure: The run time is 2 times the retention time of felodipine. Retention time of felodipine is about 12 min. System suitability: In the chromatogram obtained with reference solution (3), the resolution between the peaks due to impurity A and felodipine is at least 2.5. Limits: In the chromatogram obtained with the test solution: Sum of impurities B and C: Sum of impurities B and C is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (1.0%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.10%). The sum of the areas of all the impurity peaks, other than B and C, is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.3%). Disregard any peak with an area less than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.02%). Note: ImpurityA: Ethyl methyl 4-(2,3-dichlorophenyl)-2,6-dimethylpyridine -3,5-dicarboxylate.

402

Impurity B: Dimethyl 4-(2,3-dichlorophenyl)-2,6-dimethyl-1,4 dihydropyridine-3,5-dicarboxylate. Impurity C: Diethyl 4-(2,3-dichlorophenyl)-2,6-dimethyl-1,4dihydropyridine-3,5-dicarboxylate.

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C; 3 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.160 g of the substance to be examined in a mixture of 25 ml of 2-methyl-2-propanol R and 25 ml of 1 M perchloric acid R. Add 0.05 ml of ferroin sulfate solution R. Titrate with 0.1 M cerium sulfate VS until the pink colour disappears. Titrate slowly towards the end of the titration. 1 ml of 0.1 M cerium sulfate VS is equivalent to 19.21 mg of C18H19Cl2NO4. Storage Protected from light. Action and use Calcium channel blocker. Preparation Tablets. FENOFIBRATE Fenofibratum

C20H21ClO4

M. 360.8

Fenofibrate is 1-methylethyl 2-[4-(4-chlorobenzoyl) phenoxy]-2-methylpropanoate. It contains not less than 98.5% and not more than 101.0% of C20H21ClO4, calculated with reference to the dried substance.

Characters A white or almost white, crystalline powder. Practically insoluble in water, very soluble in methylene chloride, slightly soluble in ethanol (96%). Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum obtained with fenofibrate RS. B. Melting point (Appendix 6.7): 79 °C to 82 °C.

VP V

Appearance of solution Dissolve 0.50 g in acetone R and dilute to 10.0 ml with the same solvent. The solution is clear (Appendix 9.2) and not more intensely coloured than reference solution BY6 (Appendix 9.3, method 2). Acidity Dissolve 1.0 g in 50 ml of ethanol (96%) R previously neutralised using 0.2 ml of phenolphthalein solution R. Not more than 0.2 ml of 0.1 N sodium hydroxide VS is required to change the colour of the indicator to pink. Related substances Examine by liquid chromatography (Appendix 5.3). Chromatographic system and the solutions as described under Assay. Volume of injection: 20 µl. Procedure: The run time of the test solution is twice the retention time of fenofibrate. Inject reference solution (2), adjust the sensitivity of the system so that the height of the peaks in the chromatogram obtained is at least 20% of the full scale of the recorder. The relative retention times are: impurity A = about 0.34, impurity B = about 0.36, impurity C = about 0.50, impurity D = about 0.65, impurity E = about 0.80, impurity F = about 0.85 and impurity G = about 1.35. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks corresponding to impurity A and impurity B is at least 1.5. Limits: In the chromatogram obtained with the test solution: The area of any peak corresponding to impurity A, impurity B or impurity G is not greater than that of the corresponding peak in the chromatogram obtained with reference solution (2) (0.1% for impurities A and B and 0.2% for impurity G). The area of any peak, apart from the principal peak and any peaks corresponding to impurities A, B and G, is not greater than the area of the peak corresponding to fenofibrate in the chromatogram obtained with reference solution (2) (0.1%). The sum of the areas of all the peaks, apart from the principal peak, is not greater than five times the area of the peak corresponding to fenofibrate in the chromatogram obtained with reference solution (2) (0.5%). Disregard any peak with an area less than 0.1 times that of the peak due to fenofibrate in the chromatogram obtained with reference solution (2). Note: Impurity A: (4-chlorophenyl)(4-hydroxyphenyl) methanone. Impurity B: 2-[4-(4-chlorobenzoyl)phenoxy]-2-methylpropanoic acid (fenofibric acid). Impurity C: (3RS)-3-[4-(4-chlorobenzoyl)phenoxy] butan-2-one.

FENOFIBRATE Impurity D: Methyl 2-[4-(4-chlorobenzoyl)phenoxy]-2-methylpropanoate. Impurity E: Ethyl 2-[4-(4-chlorobenzoyl)phenoxy]-2-methylpropanoate. Impurity F: (4-chlorophenyl)[4-(1methylethoxy) phenyl]methanone. Impurity G: 1-methylethyl 2-[[2-[4-(4-chlorobenzoyl) phenoxy]2-methylpropanoyl]oxy]-2-methylpropanoate.

Halides expressed as chlorides Not more than 0.01% (Appendix 9.4.5). Solution S: Dissolve 5.0 g in 25 ml of distilled water and heat at 50 °C for 10 min. Cool and dilute to 50.0 ml with distilled water R. Filter. To 5 ml of solution S add 10 ml of distilled water R. The solution complies with the limit test for chlorides. Sulfates Not more than 0.01% (Appendix 9.4.14). 15 ml of solution S complies with the limit test for sulfates. Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; in vacuo; 60 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Water acidified to pH 2.5 with phosphoric acid - acetonitrile (30 : 70). Test solution: Dissolve 0.100 g of the substance to be examined in the mobile phase and dilute to 100.0 ml with the mobile phase. Reference solution (1): Dissolve 25.0 mg of fenofibrate RS in the mobile phase and dilute to 25.0 ml with the mobile phase. Reference solution (2): Dissolve 5.0 mg of fenofibrate RS, 5.0 mg of fenofibrate impurity A RS, 5.0 mg of fenofibrate impurity B RS and 10.0 mg of fenofibrate impurity G RS in the mobile phase and dilute to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 50.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 286 nm. Flow rate: 1 ml/min. Volume of injection: 5 µl. Procedure: Inject reference solution (2). Adjust the sensitivity of the system so that the height of the peaks in 403

FENOFIBRATE CAPSULES

the chromatogram is at least 50% of the full scale of the recorder. Inject reference solution (1) six times. The assay is not valid unless the relative standard deviation of the peak areas due to fenofibrate is not more than 1.0%. Inject the test solution and reference solution (1). Calculate the content of fenofibrate, C20H21ClO4, using the areas of principal peaks in the chromatograms obtained with the test solution, reference solution (1) and the declared content of C20H21ClO4 in fenofibrate RS.

Storage Store protected from light. Action and use Hypolipidaemic. FENOFIBRATE CAPSULES Capsulae Fenofibratis Fenofibrate capsules are hard capsules containing fenofibrate. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements:

Content of fenofibrate, C20H21ClO4, 90.0% to 110.0% of the stated amount. Identification In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of the fenofibrate peak in the chromatogram obtained with the reference solution. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase, chromatographic system are described in the Assay. Test solution: Weigh a quantity of the powdered capsule contents containing about 40 mg of fenofibrate, transfer into a 100-ml volumetric flask, add 70 ml of the mobile phase, sonicate for 15 min, allow to cool. Add to volume with the mobile phase, shake well, filter. Reference solution: Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Procedure: Inject the test solution and the reference solution. The run times is twice the retention time of the principal peak. Limits: In the chromatogram obtained with the test solution: The area of any secondary peak, is not more than 0.5 times the area of the principal peak in the chromatogram obtained with the reference solution (0.5%). The sum of areas of all secondary peaks is not more than the area of the principal peak in the chromatogram obtained with the reference solution (1.0%). Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with the reference solution. 404

VP V

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 1000 ml of a 1% solution of sodium lauryle sulfate R in water. Rotation speed: 100 rpm. Time: 60 min. Procedure: Test solution: After the specified time, withdraw a sample of the medium and filter. If necessary, dilute the filtrate with the medium to obtain a solution having a concentration of fenofibrate of 0.1 mg per ml. Reference solution: Weigh accurately a quantity of fenofibrate RS, dissolve in the medium to obtain a solution having a concentration of fenofibrate similar to that expected in the test solution. Examine by liquid chromatography (Appendix 5.3) with mobile phase, chromatographic system as described in the Assay. Calculate the content of fenofibrate, C20H21ClO4, dissolved using the peak areas in the chromatograms obtained with the reference solution, the test solution and the declared content of C20H21ClO4 in fenofibrate RS. Tolerance: Not less than 70% (Q) of the labeled amount of fenofibrate, C20H21ClO4, is dissolved in 60 min. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - water adjusted the pH to 2.5 with phosphoric acid R (70 : 30). Test solution: Weigh 20 capsules, calculate the average weight of the capsule contents and grind to fine powder. Weigh accurately a quantity of the powdered contents containing about 0.1 g of fenofibrate. Transfer into a 100-ml volumetric flask, add 70 ml of the mobile phase, sonicate for 15 min. Dilute to volume with the mobile phase, shake well and filter. Dilute 10.0 ml of the obtained solution with the mobile phase to 100.0 ml. Reference solution: Dissolve a quantity of fenofibrate RS in the mobile phase to obtain a solution having a concentration of fenofibrate of about 0.1 mg/ml . Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 286 nm. Flow rate: 1 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject the reference solution for six times, the relative standard deviation of peak areas is not more than 2.0%. The column efficieny, determined on the fenofibrate peak, is not less than 3000 theoretical plates. Inject the reference solution and the test solution. Calculate the content of fenofibrate, C20H21ClO4, in the capsules using the peak areas in the chromatograms obtained with the reference solution, the test solution and the declared content of C20H21ClO4 in fenofibrate RS.

VP V

FERRIC OXIDE

Storage Store in a well-closed container, protected from light. Action and use Lipid-regulating drug. Usual strength 100 mg, 200 mg.

Arsenic Not more than 3 ppm (Appendix 9.4.2). Dissolve 0.5 g of the substance to be examined in several ml of hydrochloric acid R with the aid of heat, and dilute to 15.0 ml with hydrochloric acid R, and mix. 10.0 ml of the solution complies with limit test for arsenic, method A.

FERRIC OXIDE Ferrici oxydum Fe2O3

turbitity, centrifuge for 15 minutes. Record the spectra (Appendix 4.1) of the filtrates, in the range between 350 nm and 750 nm, in a 1-cm cell, using respective solvents in the reference cell. No peak, above the noise level, with a slope greater than +0.001 absorbance unit per nm is found.



M.159.7

Ferric oxide contains not less than 97.0% and not more than 100.5% of Fe2O3, calculated with reference to the ignited substance.

Characters Powder exhibiting three basic colours (red, yellow and black), or other shades producing on blending the basic colours. Insoluble in water and in organic solvents, dissolves in concentrated hydrochloric acid upon warming. Identification Dissolve 0.5 g of the substance to be examined in 50 ml of hydrochloric acid R and dilute to 200 ml with water. The solution gives the reactions of iron (III) (Appendix 8.1). Water-soluble substances Not more than 1.0%. To 2.0 g of the substance to be examined add 100 ml of water on a boiling water bath for 2 h. Filter and wash the filter with water. Evaporate the filtrate and washings, and dry the residue at 105 °C for 1 hour. The weight of the dried residue is not more than 20 mg. Acid-insoluble substances Not more than 0.1%. To 2.0 g of the substance to be examined add 25 ml hydrochloric acid R by boiling for 20 min. Add 100 ml of hot water and filter through a tared filtering crucible, wash the crucible with hot water until the filtrate tests negative for chloride. Dry the crucible and contents at 105 °C for 1 h. The weight of the dried residue is not more than 2 mg. Organic colours and lakes Weight 1.0 g of the substance to be examined in each of 3 beakers, and add 25 ml of each of the following reagents, respectively: 1-chloronaphthalene R, ethanol (96%) R and chloroform R. Heat the beakers containing ethanol and chloroform just to boiling. Heat the other beaker on a boiling water bath for 15 minutes, with occasional swirling. Filter the contents of the beakers through retentive, solventresistant filter papers. If any of the filtrates show visible

Lead Not more than 10 ppm. Determined by atomic absorption spectrometry (Appendix 4.4, method 1). Test solution: Weigh 2.5 g of the substance to be examined, add 35 ml of 0.1 N hydrochloric acid R, and stir for 1 hour. Filter, collecting the filtrate in a 50 ml volumetric flask, and dilute to volume with 0.1 N hydrochloric acid R, and mix. Reference solution: Transfer 5.0 ml of lead standard solution (1000 ppm Pb) R to a 100 ml volumetric flask, add 10 ml of 1 N hydrochloric acid R and dilute to volume with water, and mix. Transfer 1.0 ml of this solution to a 100 ml volumetric flask, add 10 ml of 1 N hydrochloric acid R and dilute to volume with water, and mix. This solution contains 0.5 µg of lead/ml. Measure the absorbance at 217.0 nm using a lead hollowcathode lamp as the source of radiation and an air-acetylene flame. The absorbance of the test solution does not exceed that of the reference solution. Mercury Not more than 3 ppm. Determined by atomic absorption spectrometry (Appendix 4.4). Method Mercury detection instrument: Use any suitable atomic absorption spectrophotometer equipped with a fastresponse recorder and capable of measuring the radiation absorbed by mercury vapors at the mercury resonance line of 253.6 nm. (Note: Wash all glassware associated with the test with 10% nitric acid solution R, and rinse thoroughly with water before use). Aeration apparatus: The apparatus consists of a flowmeter capable of measuring flow rates from 500 to 1000 ml/min, connected via a three-way stopcock fitted with a polytef plug to an aeration vessel (250 ml gas washing bottle), followed by a trap, a drying tube packed with magnesium perchlorate, a 10 cm × 25 mm flow-through cell with quartz windows, and terminating with a vent to a fume hood. Connections are glass or polyvinyl chloride.

405

VP V

FERROUS FUMARATE C

A

B E

F H

D G

Fig.1 - Mercury aeration apparatus A: Air or nitrogen B: Flowmeter C: Three-way stopcock D: Aeration vessel E: Trap F: Drying tube packed with Mg(ClO4)2 G: 10-cm cell quartz windows H: Vent to hood Reagent 5% potassium permanganate solution: Dissolve 5 g of potassium permanganate R in 100 ml of water. Hydroxylamine hydrochloride solution: Dissolve 10 g of hydroxylamine hydrochloride R in 100 ml of water. Stannous chloride solution: Dissolve 10 g of SnCl2,2H2O in 20 ml of hydrochloric acid R, and add 80 ml of water. Prepare fresh each week. Mercury 1000 µg/ml stock solution. Reference solution: Dilute the mercury 1000 µg/ml stock solution with water to obtain a reference solution having the equivalent of 1 µg of mercury/ml. Pipet 2.0 ml of the reference solution into a 100 ml conical flask, and add 35 ml of water, 3 ml of sulfuric acid R, and 1 ml of 5% potassium permanganate solution. Cover the conical flask with a watch glass, boil for a few seconds, and cool. Test solution: Weigh 0.67 g of the substance to be examined a 100 ml conical flask, and add 35 ml of 0.5 M hydrochloric acid R. Stir, and boil. Allow to cool, add 2 drops of phenolphthalein solution R, and, as necessary, slowly neutralize with constant stirring, using 1 M sodium hydroxide R or 1 M sulfuric acid R. Add 3 ml of sulfuric acid R and 1 ml of 5% potassium permanganate solution. Cover the conical flask with a watch glass, boil for a few seconds, and cool. Procedure Assemble the Aeration apparatus as shown in the accompanying diagram, with the aeration vessel and the trap empty, and the stopcock in the bypass position. Connect the apparatus to the absorption cell, and adjust the air or nitrogen flow rate so that, in the following procedure, maximum absorption and reproducibility are obtained without excessive foaming in the test solution. Obtain 406

a smooth baseline reading at 253.6 nm, following the manufacturer’s instructions for operating the instrument. Treat the reference solution and the test solution similarly, as follows. Destroy the excess permanganate by adding hydroxylamine hydrochloride solution, dropwise, until the solution is colorless. Immediately wash the solution into the aeration vessel with water, and dilute with water to 100 ml. Add 2 ml of stannous chloride solution, and immediately reconnect the aeration vessel to the aeration apparatus. Turn the stopcock from the bypass position to the aerating position, and continue the aeration until the absorption peak has been passed and the recorder pen returns to the baseline. Disconnect the aeration vessel from the apparatus, and wash with water after each use. After correcting for any reagent blank, any absorbance produced by the test solution does not exceed that produced by the reference solution.

Assay To about 1.500 g, accurately weighed, of the substance to be examined in 25 ml of hydrochloric acid R on a water bath until dissolved. Add 10 ml of hydrogen peroxide solution (10 volumes) R, and evaporate on a water bath almost to dryness in order to volatilize all hydrogen peroxide. Dissolve the residue by warming with 5 ml of hydrochloric acid R, add 25 ml of water, filter into a 250 ml volumetric flask, washing the filter with water, dilute to volume with water, and mix. Transfer 50.0 ml of the solution to a glassstoppered flask, add 3 g of potassium iodide R and 5 ml of hydrochloric acid R, and insert the stopper in the flask. Allow the mixture to stand for 15 minutes, add 50 ml of water, and titrate the liberated iodine with 0.1 N sodium thiosulphate VS, using starch solution R as indicator. Carry out a blank test. 1 ml of 0.1 N sodium thiosulphate VS is equivalent to 7.985 mg of Fe2O3. Ignite about 2 g of the substance to be examined at 800 ± 25°C to constant weight to enable calculation of content of Fe2O3 with reference to the ignited substance. Storage Store in an airtight container. Action and use Excipients. FERROUS FUMARATE Ferrosi fumaras -O C 2

CO2-

Fe2+

C4H2FeO4 M.169.9 Ferrous fumarate is iron (II) (E)-butendioate. It contains not less than 93.0% and not more than 101.0% of C4H2FeO4, calculated with reference to the dried substance.

VP V

Characters A fine, reddish-orange or reddish-brown powder. Slightly soluble in water, very slightly soluble in ethanol (96%). Identification A. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel GF254 Mobile phase: Anhydrous formic acid - methylene chloride - n-butanol - heptane (12 : 16 : 32 : 44). Test solution: To 1.0 g of the substance to be examined add 25 ml of a mixture of equal volumes of hydrochloric acid R and water and heat on a water-bath for 15 minutes. Cool and filter. Use the filtrate for identification test C. Wash the residue with 50 ml of a mixture of 2 M hydrochloric acid R and water (1 : 9). Dry the residue at 100 - 105 °C. Dissolve 20 mg of the residue in acetone R and dilute to 10 ml with the same solvent, and mix. Reference solution: Dissolve 20 mg of fumaric acid RS in acetone R and dilute to 10 ml with the same solvent, and mix. Procedure: Apply separately to the plate 5 µl of each solution. Develop in an unsaturated tank over a path of 10 cm. Remove the plate, dry it at 105 °C for 15 minutes and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. B. Mix 0.5 g of the substance to be examined with 1 g of resorcinol R. To 0.5 g of the mixture in a crucible add 0.15 ml of sulfuric acid R and heat gently. A dark red semi-solid mass is formed. Add the mass, with care, to 100 ml of water. An orange-yellow colour develops and the solution shows no fluorescence. C. The filtrate obtained for the test solution in identification test A gives reaction of iron (II) (Appendix 8.1). Sulfates Not more than 0.2% (Appendix 9.4.14). Heat 0.15 g of the substance to be examined with 8 ml of dilute hydrochloric acid R and 20 ml of water. Cool in iced water, filter and dilute to 30 ml with water, and mix. 15 ml of this solution complies with the limit test for sulfates. Arsenic Not more than 5 ppm (Appendix 9.4.2). Mix 1.0 g of the substance to be examined with 15 ml of water and 15 ml of sulfuric acid R. Warm to precipitate the fumaric acid completely. Cool and add 30 ml of water, and filter. Wash the precipitate with water. Dilute the combined filtrate and washings to 100 ml with water, and mix. 20 ml of the solution complies with limit test for arsenic, method A. Ferric ion Not more than 2.0%. In a flask with a ground-glass stopper, dissolve 3.0 g of the substance to be examined in a mixture of 10 ml of hydrochloric acid R and 100 ml of water by heating rapidly to boiling. Boil for 15 seconds. Cool rapidly, add 3 g of

FERROUS FUMARATE

potassium iodide R, stopper the flask and allow to stand protected from light for 15 minutes. Add 2 ml of starch solution R as indicator. Titrate the liberated iodine with 0.1 N sodium thiosulfate VS. Carry out a blank test. The difference between the volumes used in the two titrations corresponds to the volumes of iodine liberated by ferric ion. 1 ml of 0.1 N sodium thiosulfate VS is equivalent to 5.585 mg of ferric ion.

Solution S Dissolve 2.0 g of the substance to be examined in a mixture of 10 ml of lead-free hydrochloric acid R and 80 ml of water, heating slightly if necessary. Allow to cool, filter if necessary and dilute to 100.0 ml with water, and mix. Cadmium Not more than 10 ppm. Determined by atomic absorption spectrometry (Appendix 4.4, method 1). Test solution: Use solution S. Reference solutions: Prepare the solutions using cadmium standard solution (0.1% Cd) R and diluting with a 10% v/v solution of lead-free hydrochloric acid R. Measure the absorbance at 228.8 nm using a cadmium hollow-cathode lamp as source of radiation and an airacetylene flame. Chromium Not more than 200 ppm. Determined by atomic absorption spectrometry (Appendix 4.4, method 1). Test solution: Use solution S. Reference solutions: Prepare the solutions using chromium standard solution (0.1% Cr) R and diluting with a 10% v/v solution of lead-free hydrochloricacid R. Measure the absorbance at 357.9 nm using a chromium hollow-cathode lamp as source of radiation and an airacetylene flame. Lead Not more than 20 ppm. Determined by atomic absorption spectrometry (Appendix 4.4, method 1). Test solution: Use solution S. Reference solutions: Prepare the solutions using lead standard solution (10 ppm Pb) R and diluting with a 10% v/v solution of lead-free hydrochloric acid R. Measure the absorbance at 283.3 nm using a lead hollowcathode lamp as the source of radiation and an air-acetylene flame. Mercury Not more than 1 ppm. Determined by atomic absorption spectrometry (Appendix 4.4, method 1). Test solution: Use solution S. 407

FERROUS FUMARATE AND FOLIC ACID TABLETS

Reference solutions: Prepare the solutions using mercury standard solution (10 ppm Hg) R and diluting with a 25% v/v solution of lead-free hydrochloricacid R. Following the recommendations of the manufacturer, introduce 5 ml of solution S or 5 ml of the reference solutions into the reaction vessel of the cold-vapour mercury assay accessory, add 10 ml of water and 1 ml of stannous chloride solution R1. Measure the absorbance at 253.7 nm using a mercury hollow-cathode lamp as the source of radiation.

Nickel Not more than 200 ppm. Determined by atomic absorption spectrometry (Appendix 4.4, method 1). Test solution: Use solution S. Reference solutions: Prepare the solutions using nickel standard solution (10 ppm Ni) R and diluting with a 10% v/v solution of lead-free hydrochloric acid R. Measure the absorbance at 232 nm using a nickel hollowcathode lamp as source of radiation and an air-acetylene flame. Zinc Not more than 500 ppm. Determined by atomic absorption spectrometry (Appendix 4.4, method 1). Test solution: Use solution S diluted ten times. Reference solutions: Prepare the solutions using zinc standard solution (10 ppm Zn) R and diluting with a 1% v/v solution of lead-free hydrochloric acid R. Measure the absorbance at 213.9 nm using a zinc hollowcathode lamp as source of radiation and an air-acetylene flame. Loss on drying Not more than 1.0% (Appendix 9.6). (1.000 g ; 100 °C - 105 °C). Assay Dissolve with slight heating 0.150 g, accurately weighed, of the substance to be examined in 7.5 ml of 10% solution of sulfuric acid R. Cool and add 25 ml of water. Add 0.1ml of ferroin sulfate solution R. Titrate immediately with 0.1 M cerium sulfate VS until the colour changes from orange to light bluish-green. 1ml of 0.1 M cerium sulfate VS is equivalent to 16.99 mg of C4H2FeO4. Storage Store in an airtight container, protected from light. Action and use Used in prevention and treatment of ferrous deficiencies, anemia. Preparations Tablets, capsules, in combination with folic acid. 408

VP V

FERROUS FUMARATE AND FOLIC ACID TABLETS Tabellae Ferrosi fumaratis et Acidi folici Ferrous fumarate and folic acid tablets contain ferrous fumarate and folic acid. They are film-coated. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) section Coated tablets and with the following requirements.

Content of ferrous fumarate, C4H2FeO4, 90.0% to 105.0% of the stated amount. Content of folic acid, C19H19N7O6, 90.0% to 115.0% of the stated amount. Carry out the tests avoiding exposure to actinic light. Identification A. In the Assay for folic acid, the chromatogram obtained with the test solution shows a peak with the same retention time as folic acid peak in the chromatogram obtained with the reference solution. B. Heat a quantity of the powdered tablets containing 0.8 g of ferrous fumarate with 25 ml of a mixture of equal volumes of hydrochloric acid R and water on a water bath for 15 minutes, cool and filter. Retain the residue for test C. The filtrate yields reaction characteristic of iron (II) salts (Appendix 8.1). C. Wash the residue reserved in test B with several 5 ml quantities of 0.2 M hydrochloric acid R until the washing is no longer yellow, dry at 105 °C. Suspend 0.1 g of the residue in 2 ml of 10% sodium carbonate solution R and add 2 - 3 drops of 5% potassium permanganate solution R. A brownish solution is produced. Ferric iron Not more than 5.0% in ferrous fumarate. Weigh accurately a quantity of the powdered tablets containing 1.5 g of ferrous fumarate, add a mixture of 100 ml of water and 10 ml of hydrochloric acid R, shake well and heat rapidly to the boiling point. Boil for 15 seconds, cool rapidly, add 3 g of potassium iodide R, stopper, allow to stand in the dark for 15 minutes and titrate the liberated iodine with 0.1 N sodium thiosulfate VS using starch solution R as indicator. Repeat the operation without the substance being examined. The difference between the titrations is not more than 13.4 ml Uniformity of content for folic acid Tablets containing less than 2 mg of folic acid comply with the test for Uniformity of content (Appendis 11.2). Carry out the method for liquid chromatography (Appendix 5.3). Mobile phase: A mixture of 135 ml of methanol R and 800 ml of a solution containing 0.938% of sodium perchlorate R

VP V

and 0.075% of potassium dihydrogen orthophosphate R adjusted to pH 7.2 with 0.1 M potassium hydroxide R and diluted to 1000 ml with water. Solvent mixture: A mixture contains 800 volumes of a 0.57% solution of dipotassium hydrogen orthophosphate R and 135 volumes of methanol R. Reference solution: A 0.0007% solution of folic acid RS in the solvent mixture. Test solution: Place one tablet in a 50 ml volumetric flask, add 40 ml of the solvent mixture, sonicate for 5 minutes, shake for a further 15 minutes, dilute to volume with solvent mixture, mix and filter. Dilute the filtrate, if necessary, with the solvent mixture to obtain a concentration of folic acid equivalent to that of the reference solution. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 277 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution, the relative standard deviation of the peak area of folic acid in replicate injections is not more than 2.0%. Inject separately the reference solution and the test solution. Calculate the content of folic acid, C19H19N7O6, in each tablet using the areas of the principal peaks in the chromatograms obtained with the test solution and the reference solution and the declared content of C19H19N7O6 in folic acid RS.

Assay Weigh 20 tablets (with coating removed), calculate the average mass of tablet core and finely powder. For ferrous fumarate Weigh accurately a quantity of the powdered tablets containing 0.3 g of ferrous fumarate, add 7.5 ml of 1 M sulfuric acid R, shake with gentle heating. Cool, add 25 ml of water and titrate immediately with 0.1 M ammonium cerium(IV) sulfate VS using ferroin solution R as indicator. 1 ml of 0.1 M ammonium cerium(IV) sulfate VS is equivalent to 16.99 mg of C4H2FeO4. For folic acid For tablets containing less than 2 mg of folic acid, use the average of the ten individual results obtained in the test for Uniformity of content. For tablets containing 2 mg or more of folic acid: Carry out the method for liquid chromatography (Appendix 5.3). Mobile phase, solvent mixture, reference solution, chromatographic system, procedure: Proceed as directed in the test for Uniformity of content for folic acid.

FERROUS SULFATE

Test solution: Weigh accurately a quantity of the powdered tablets containing 0.35 mg of folic acid in a 50 ml volumetric flask, add 40 ml of solvent mixture, sonicate for 5 minutes, shake for a further 15 minutes, dilute to volume with solvent mixture, mix and filter. Calculate the content of folic acid, C19H19N7O6, in each tablet using the areas of the principal peaks in the chromatograms obtained with the test solution and the reference solution and the declared content of C19H19N7O6 in folic acid RS.

Storage Store in a well closed container, protected from light. Action and use Used in prevention and treatment of anaemias. Usual strength 200 mg of ferrous fumarate and 1 mg of folic acid. FERROUS SULFATE Ferrosi Sulfas FeSO4,7H2O  

M. 278.0

Ferrous sulfate contains not less than 98.0% and not more than 105.0% of FeSO4,7H2O.

Characters Light green, crystalline powder or bluish-green crystals, efflorescent in air and oxidised in moist air, becoming brown. Freely soluble in water, very soluble in boiling water, practically insoluble in ethanol (96%). Identification A. It gives the reactions of sulfates (Appendix 8.1). B. It gives reaction (A) of iron (II) (Appendix 8.1). C. It complies with the limits of the assay. pH 3.0 to 4.0 (Appendix 6.2). Dissolve 1.0 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent. Chlorides Not more than 0.02% (Appendix 9.4.5). Solution S: Dissolve 4.0 g of the substance to be examined in a 5% v/v solution of lead-free nitric acid R and dilute to 100.0 ml with the same solution. Dilute 5 ml of solution S to 10 ml with water and add 5 ml of 2 M nitric acid R. Prepare the standard with a mixture of 2 ml of water, 5 ml of 2 M nitric acid R and 8 ml of chloride standard solution (5 ppm Cl R). Use 0.15 ml of 1.7% solution of silver nitrate R in this test. Chromium Not more than 50 ppm. 409

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FERROUS SULFATE TABLETS

Examine by atomic absorption spectrometry (Appendix 4.4, method 2). Test solution: Solution S. Reference solutions: Prepare the reference solutions using chromium standard solution (100 ppm Cr) R, diluting with a 5% v/v solution of lead-free nitric acid R. Measure the absorbance at 357.9 nm using a chromium hollow-cathode lamp using a transmission band preferably of 1 nm as a source of radiation and an air-acetylene flame.

Copper Not more than 50 ppm. Examine by atomic absorption spectrometry (Appendix 4.4, method 2). Test solution: Solution S. Reference solutions: Prepare the reference solutions using copper standard solution (100 ppm Cu) R, diluting with a 5% v/v solution of lead-free nitric acid R. Measure the absorbance at 324.7 nm using a copper hollow-cathode lamp using a transmission band preferably of 1 nm as a source of radiation and an air-acetylene flame. Ferric ions Not more than 0.3%. In a ground-glass-stoppered flask, dissolve 5.00 g of the substance to be examined in a mixture of 10 ml of hydrochloric acid R and 100 ml of carbon dioxide-free water R. Add 3 g of potassium iodide R, close the flask and allow to stand in the dark for 5 min. Titrate the liberated iodine with 0.1 N sodium thiosulfate VS, using 0.5 ml of starch solution R, added towards the end of the titration, as indicator. Carry out a blank test in the same conditions. Not more than 2.7 ml of 0.1 N sodium thiosulfate VS is used, taking into account the blank titration. Manganese Not more than 0.1%. Examine by atomic absorption spectrometry (Appendix 4.4, method 2). Test solution: Dilute 1.0 ml of solution S to 20.0 ml with a 5% v/v solution of lead-free nitric acid R. Reference solutions: Prepare the reference solutions using manganese standard solution (1000 ppm Mn) R, diluting with a 5% v/v solution of lead-free nitric acid R. Measure the absorbance at 279.5 nm using a manganese hollow-cathode lamp using a transmission band preferably of 1 nm as a source of radiation and an air-acetylene flame. Nickel Not more than 50 ppm. Examine by atomic absorption spectrometry (Appendix 4.4, method 2). Test solution: Solution S. Reference solutions: Prepare the reference solutions using nickel standard solution (10 ppm Ni) R, diluting with a 5% v/v solution of lead-free nitric acid R. 410

Measure the absorbance at 232.0 nm using a nickel hollowcathode lamp using a transmission band preferably of 1 nm as a source of radiation and an air-acetylene flame.

Zinc Not more than 50 ppm. Examine by atomic absorption spectrometry (Appendix 4.4, method 2). Test solution: Solution S. Reference solutions: Prepare the reference solutions using zinc standard solution (100 ppm Zn) R, diluting with a 5% (v/v) solution of lead-free nitric acid R. Measure the absorbance at 213.9 nm using a zinc hollowcathode lamp using a transmission band preferably of 1 nm as a source of radiation and an air-acetylene flame. Assay Dissolve 2.5 g of sodium hydrogen carbonate R in a mixture of 150 ml of water and 10 ml of sulfuric acid R. When the effervescence ceases add to the solution 0.500 g of the substance to be examined and dissolve with gentle swirling. Add 0.1 ml of ferroin R and titrate with 0.1 M ammonium cerium nitrate VS until the red colour disappears. 1 ml of 0.1 M ammonium cerium nitrate VS is equivalent to 27.80 mg of FeSO4,7H2O. Storage In an airtight container. Action and use Used in prevention and treatment of anaemias. Preparation Tablets. FERROUS SULFATE TABLETS Tabellae Ferrosi sulfatis Ferrous sulfate tablets contain ferrous sulfate. They are sugar-coated. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) section Coated tablets and with the following requirements.

Content of ferrous sulfate, FeSO4,7H2O, 95.0% to 105.0% of the stated amount. Identification To a quantity of the powdered tablets equivalent to about 0.25 g ferrous sulfate, add 20 ml of 2 M hydrochloric acid R, shake and filter. The filtrate yields the reaction characteristic of ferrous salts and sulfates (Appendix 8.1). Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of 0.1 M hydrochloric acid R.

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DRIED FERROUS SULFATE

Rotation speed: 50 rpm. Time: 45 min. Procedure: Determine the content of ferrous sulfate dissolved by atomic absorption spectrophotometry at a wavelength of about 248.3 nm (Appendix 4.4). Reference solution: A reference solution of ferrous sulfate diluted with 0.1 M hydrochloric acid R to obtain an appropriate concentration. Test solution: After the specified time, withdraw a sample of the medium and filter, discarding the first portion of the filtrate. Dilute a suitable volume of the filtrate with 0.1 M hydrochloric acid R to obtain an appropriate concentration. Tolerances: Not less than 75% (Q) of the stated amount of ferrous sulfate, FeSO4,7H2O, is dissolved in 45 minutes.

pH 3.0 to 4.0 (Appendix 6.2). Dissolve 1.0 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent.

Assay Weigh 20 tablets (with coating removed), calculate the average mass of tablet core, and finely powder. Weigh accurately a quantity of the powdered tablets equivalent to about 0.5 g of ferrous sulfate in a 250 ml conical flask, add 25 ml of 10% sulfuric acid solution R and 50 ml of freshly boiled and cooled water, shake well to dissolve. Titrate immediately with 0.1 M ammonium cerium (IV) sulfate VS using ferroin solution R as indicator. 1 ml of 0.1 M ammonium cerium (IV) sulfate VS is equivalent to 27.80 mg of FeSO4,7H2O.

Chromium Not more than 100 ppm. Examine by atomic absorption spectrometry (Appendix 4.4, method 2). Solution S: Dissolve 2.00 g of the substance to be examined in a 5% v/v solution of lead-free nitric acid R and dilute to 100.0 ml with the same solution. Test solution: Solution S. Reference solutions: Prepare the reference solutions using chromium standard solution (100 ppm Cr) R, diluting with a 5% v/v solution of lead-free nitric acid R. Measure the absorbance at 357.9 nm using a chromium hollow-cathode lamp using a transmission band preferably of 1 nm as a source of radiation and an air-acetylene flame.

Storage Store in a cool and dry place, protected from light. Action and use Ferrous supplement, used in prevention and treatment of anaemias. Usual strength 200 mg. DRIED FERROUS SULFATE Ferrosi sulfas siccum FeSO4  

M. 151.9

Hydrated ferrous sulfate from which part of the water of hydration has been removed by drying. It contains not less than 86.0% and not more than 90.0% of FeSO4.

Characters Greyish-white powder. Slowly but freely soluble in water, very soluble in boiling water, practically insoluble in ethanol (96%). It is oxidised in moist air, becoming brown. Identification A. It gives the reactions of sulfates (Appendix 8.1). B. It gives reaction (A) of iron (II) (Appendix 8.1). C. It complies with the limits of the assay.

Chlorides Not more than 0.03% (Appendix 9.4.5). Dissolve 2.5 g in water, add 0.5 ml of dilute sulfuric acid R and dilute to 50 ml with water. Dilute 3.3 ml of this solution to 10 ml with water and add 5 ml of 2 M nitric acid R. Prepare the standard using a mixture of 10 ml of chloride standard solution (5 ppm Cl) R and 5 ml of 2 M nitric acid R. Use 0.15 ml of 1.7% solution of silver nitrate R in this test.

Copper Not more than 50 ppm. Examine by atomic absorption spectrometry (Appendix 4.4, method 2). Test solution: Solution S. Reference solutions: Prepare the reference solutions using copper standard solution (100 ppm Cu) R, diluting with a 5% v/v solution of lead-free nitric acid R. Measure the absorbance at 324.7 nm using a copper hollow-cathode lamp using a transmission band preferably of 1 nm as a source of radiation and an air-acetylene flame. Ferric ions Not more than 0.5%. In a ground-glass-stoppered flask, dissolve 5.00 g of the substance to be examined in a mixture of 10 ml of hydrochloric acid R and 100 ml of carbon dioxide-free water R. Add 3 g of potassium iodide R, close the flask and allow to stand in the dark for 5 min. Titrate the liberated iodine with 0.1 N sodium thiosulfate VS, using 0.5 ml of starch solution R as indicator, added towards the end of the titration. Carry out a blank test in the same conditions. Not more than 4.5 ml of 0.1 N sodium thiosulfate VS is used, taking into account the blank titration. 411

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FEXOFENADINE HYDROCHLORIDE

Manganese Not more than 0.1%. Examine by atomic absorption spectrometry (Appendix 4.4, method 2). Test solution: Dilute 1.0 ml of solution S to 20.0 ml with a 5% v/v solution of lead-free nitric acid R. Reference solutions: Prepare the reference solutions using manganese standard solution (1000 ppm Mn) R, diluting with a 5% v/v solution of lead-free nitric acid R. Measure the absorbance at 279.5 nm using a manganese hollow-cathode lamp using a transmission band preferably of 1 nm as a source of radiation and an air-acetylene flame.

FEXOFENADINE HYDROCHLORIDE Fexofenadini hydrochloridum

Nickel Not more than 100 ppm. Examine by atomic absorption spectrometry (Appendix 4.4, method 2). Test solution: Solution S. Reference solutions: Prepare the reference solutions using nickel standard solution (10 ppm Ni) R, diluting with a 5% (v/v) solution of lead-free nitric acid R. Measure the absorbance at 232.0 nm using a nickel hollowcathode lamp using a transmission band preferably of 1 nm as a source of radiation and an air-acetylene flame.

C32H39NO4,HCl

Zinc Not more than 100 ppm. Examine by atomic absorption spectrometry (Appendix 4.4, method 2). Test solution: Solution S. Reference solutions: Prepare the reference solutions using zinc standard solution (100 ppm Zn) R, diluting with a 5% (v/v) solution of lead-free nitric acid R. Measure the absorbance at 213.9 nm using a zinc hollowcathode lamp using a transmission band preferably of 1 nm as a source of radiation and an air-acetylene flame.

Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum obtained with fexofenadine RS. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in methanol R, evaporate to dryness and record new spectra using the residues. B. Dissolve 30 mg of the substance to be examined in a mixture of equal volumes of methanol R and water; sonicate if necessary and dilute to 2 ml with the same mixture of solvents. The solution gives reaction (A) of chlorides (Appendix 8.1).

Assay Dissolve 2.5 g of sodium hydrogen carbonate R in a mixture of 150 ml of water and 10 ml of sulfuric acid R. When the effervescence ceases add to the solution 0.140 g of the substance to be examined and dissolve with gentle swirling. Add 0.1 ml of ferroin R and titrate with 0.1 M ammonium cerium nitrate VS until the red colour disappears. 1 ml of 0.1 M ammonium cerium nitrate VS is equivalent to 15.19 mg of FeSO4. Storage In an airtight container. Action and use Used in prevention and treatment of anaemias. Preparation Tablets. 412



M.538.1

Fexofenadine hydrochloride is (2-[4-[(1RS)-1-hydroxy-4-[4(hydroxydiphenylmethyl)piperidin-1-yl]butyl]phenyl]-2methylpropanoic acid hydrochloride . It contains not less than 98.0% and not more than 102.0% of C32H39NO4,HCl, calculated with reference to the anhydrous substance.

Characters A white or almost white powder. It shows polymorphism. Slightly soluble in water, freely soluble in methanol, very slightly soluble in acetone.

Impurity B Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - buffer solution (20 : 80). Buffer solution: To 1.15 ml of glacial acetic acid R add 900 ml of water, adjust to pH 4.0 ± 0.1 with dilute ammonia R and dilute to 1000 ml with water. Test solution: Dissolve 50 mg of the substance to be examined in the mobile phase and dilute to 100.0 ml with the mobile phase. Reference solution: Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1 volume of this solution to 10 volumes with the mobile phase. Resolution solution: Dissolve the contents of a vial of fexofenadine impurity B RS in the test solution and dilute to 2.0 ml with the test solution. Chromatographic system: A column (25 cm × 4.6 mm) packed with silica gel BC for chiral chromatography.

VP V

Detector: A spectrophotometer set at 220 nm. Flow rate: 0.5 ml/min. Volume of injection: 20 µl. Procedure: Run time: 1.2 times the retention time of fexofenadine. Inject the resolution solution, the relative retention time of impurity B with reference to fexofenadine (retention time = about 20 min) is about 0.7. The test is not valid unless the resolution between the peaks due to fexofenadine and impurity B is at least 3.0. Inject separately the test solution and reference solution, in the chromatogram obtained with the test solution, the area of the peak corresponding to impurity B multiply by 1.3 is not more than the area of the principal peak in the chromatogram obtained with the reference solution (0.1%).

Related substances Examine by liquid chromatography (Appendix 5.3) Mobile phase: Acetonitrile - buffer solution - triethylamine (350 : 650 : 3). Buffer solution: Dissolve 6.64 g of sodium dihydrogen phosphate monohydrate R and 0.84 g of sodium perchlorate R in water, adjust to pH 2.0 ± 0.1 with phosphoric acid R and dilute to 1000 ml with water. Solvent mixture: Mix equal volumes of acetonitrile R and the buffer solution. Test solution (1): Dissolve 25.0 mg of the substance to be examined in 25.0 ml of the solvent mixture. Test solution (2): Dilute 3.0 ml of the test solution (1) to 50.0 ml with the mobile phase. Reference solution (1): Dissolve 25.0 mg of fexofenadine hydrochloride RS with the solvent mixture and dilute to 25.0 ml with the same solvent. Dilute 3.0 ml of this solution to 50.0 ml with the mobile phase. Reference solution (2): Dilute 1.0 ml of the test solution (1) to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (3): Dissolve 1.0 mg of fexofenadine impurity A RS and 1.0 mg of fexofenadine impurity C RS in 20.0 ml the reference solution (1) and dilute to 200.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with phenylsilyl silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 220 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: Run time: 6 times the retention time of fexofenadine for test solution (1) and reference solution (3), 2 times the retention time of fexofenadine for reference solution (2). System suitability: Inject the reference solution (3), the resolution between the peaks due to fexofenadine and impurity A is at least 10. The relative retention with reference to fexofenadine (retention time = about 9 min): impurity A = about 1.7, impurity D = about 2.3, impurity C = about 3.2.

FEXOFENADINE HYDROCHLORIDE

Correction factor: for the calculation of content, multiply the peak area of impurity A by 1.4. Limits: Impurities A, C, D: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.1%). Unspecified impurities: for each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.1%). Total peak area of all impurities is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.3%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Note: Impurity A: 2-[4-[4-[4-(hydroxydiphenylmethyl)piperidin-1-yl] butanoyl]phenyl]-2-methylpropanoic acid, Impurity B: 2-[3-[(1RS)-1-hydroxy-4-[4-(hydroxydiphenylmethyl) piperidin-1-yl]butyl]phenyl]-2-methylpropanoic acid. Impurity C: (1RS)-4-[4-(hydroxydiphenylmethyl)piperidin-1-yl]-1 -[4-(1 methylethyl)phenyl]butan-1-ol. Impurity D: methyl 2-[4-[(1RS)-1-hydroxy-4-[4-(hydroxydiphenylmethyl)piperidin-1-yl]butyl]phenyl]-2-methylpropanoate.

Heavy metals Not more than 10 ppm (Appendix 9.4.8). Dissolve 1.0 g in a mixture of 15 volumes of water and 85 volumes of methanol R and dilute to 20 ml with the same mixture of solvents. 12 ml of the test solution complies with limit test for heavy metals, method 2. Prepare the the standard solution using 5.0 ml of lead standard solution (1 ppm Pb) R. Water Not more than 0.5% (Appendix 10.3). Dissolve 1.000 g in anhydrous methanol R and dilute to 5.0 ml with the same solvent. Use 1.0 ml of this solution. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase, buffer solution, solvent mixture, chromatographic system and preparation of the solutions: as described in the test for Related substances. Procedure: Inject test solution (2) and reference solution (1). The run time is 2 times the retention time of fexofenadine. Calculate the percentage content of fexofenadine, C32H39NO4,HCl, using the areas of the peaks in the chromatograms obtained with test solution (2) and reference solution (1) and the declared content of C32H39NO4,HCl in fexofenadine hydrochloride RS. 413

FEXOFENADINE TABLETS

Storage Protected from light, at a temperature not exceeding 30 °C. Action and use Antihistamine. Preparation Tablets. FEXOFENADINE TABLETS Tabellae Fexofenadini Fexofenadine tablets contain fexofenadine hydrochloride. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of fexofenadine hydrochloride, C32H39NO4,HCl, from 95.0% to 105.0% of the stated amount. Identification A. Dissolve a quantity of the powdered tablets containing about 30 mg of fexofenadine hydrochloride in 80 ml of 0.001 M hydrochloric acid R, dilute to 100 ml with the same solvent, shake well and filter. The ultraviolet absorption spectrum (Appendix 4.1) of the resulting solution in the range 230 nm to 350 nm exhibits a maximum at 259 ± 1 nm and similar to the spectrum of the reference solution having the same concentration as the test solution in 0.001 M hydrochloric acid R. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of the principal peak in the chromatogram obtained with the reference solution. Dissolution (Appendix 11.4). Apparatus: Paddle. Medium: 900 ml of 0.001 M hydrochloride acid R. Rotation speed: 50 rpm. Time: 30 min. Procedure: Examine by liquid chromatography (Appendix 5.3) Acid solution, buffer solution, diluent, mobile phase, reference stock solution and chromatographic system are described in the Assay. Reference solution: Dilute the reference stock solution in the mobile phase to obtain a solution having the same concentration of fexofenadine hydrochloride as the test solution. Test solution: After the specified time, withdraw a sample of the medium and filter, discard the first 20 ml of the filtrate. If necessary dilute the filtrate to obtain a solution containing about 0.06 mg of fexofenadine hydrochloride per ml. 414

VP V

Inject separately the reference solution and test solution. Calculate the content of fexofenadine hydrochloride, C32H39NO4,HCl, dissolved using the areas of the principal peaks in the chromatograms obtained with the reference solution, the test solution and the declared content of fexofenadine hydrochloride RS. Tolerance: Not less than 75% (Q) of the labeled amount of fexofenadine hydrochloride, C32H39NO4,HCl, is dissolved in 30 min.

Related substances Examine by liquid chromatography (Appendix 5.3) Acid solution, buffer solution, diluent, mobile phase, test stock solution and chromatographic system are described in the Assay. Test solution: Use the test stock solution in the Assay. Reference solution: Dilute 1.0 ml of test solution to 100.0 ml with the mobile phase, shake well. Dilute 5.0 ml of the resulting solution to 50.0 ml with the mobile phase, shake well. Procedure: Inject the reference solution and the test solution. In the chromatogram obtained with the test solution, the relative retention times comparing with fexofenadine peak are about 1.6 for impurity A and 6.7 for decarboxylated degradant. Limits: In the chromatogram obtained with the test solution: The area of peak due to impurity A is not more than 4 times the area of the principal peak in the chromatogram obtained with the reference solution (0.4%). Decarboxylated degradant: The area of the peak due to decarboxylated degradant after divided with 1.1 is not more than 1.5 times the area of the principal peak in the chromatogram obtained with the reference solution (0.15%). The area of each other impurity is not more than twice the area of the principal peak in the chromatogram obtained with the reference solution (0.2%). The sum of all secondary peaks is not more than 5 times the area of the principal peak in the chromatogram obtained with the reference solution (0.5%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with the reference solution (0.05%). Assay Examine by liquid chromatography (Appendix 5.3). Acid solution: Dilute 17 ml of glacial acetic acid R to 1000 ml of water, mix well. Dilute 100 ml of the resulting solution to 1000 ml with water, mix well. Buffer solution: Dilute 15 ml of a mixture of acetonitrile and triethylamine (1 : 1) to 1000 ml with the acid solution, adjust the pH to 5.25 with phosphoric acid R. Diluent: Acetonitrile - acid solution (75 : 25). Mobile phase: Buffer solution - acetonitrile (64 : 36), adjust if necessary. Reference stock solution: Weigh accurately about 30 mg of fexofenadine hydrochloride RS, transfer into a 100-ml

VP V

FLUCLOXACILLIN SODIUM

volumetric flask, dissolve and dilute to volume with diluent, shake well. Reference solution: Dilute 5.0 ml of the reference stock solution to 100.0 ml with mobile phase, shake well. Test stock solution: Transfer 10 tablets into a 200-ml volumetric flask, add 40 ml of acid solution, shake for 30 min to disintegrate completely. Add 120 ml of acetonitrile R, sonicate for 60 min, allow to cool and dilute to volume with the diluent, shake well, filter. Dilute if necessary with the diluent to obtain a solution containing about 1.2 mg of fexofenadine hydrochloride per ml. Test solution: Dilute 5.0 ml of the test stock solution to 20.0 ml with the diluent. Dilute 5.0 ml of the resulting solution to 100.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase phenylsilyl silica gel for chromatography (5 µm). Column temperature: 35 °C. Detector: A spectrophotometer set at 220 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: Inject the reference solution. The test is not valid unless the tailing factor of the peak due to fexofenadine hydrochloride is not more than 2.0 and the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject separately the reference solution and the test solution. Calculate the content of fexofenadine hydrochloride, C32H39NO4,HCl, in the tablets using the peak areas in the chromatograms obtained with the reference solution, the test solution and the declared content of C32H39NO4,HCl in fexefenadine hydrochloride RS.

Storage Store in a cool place, protected from light. Action and use Antihistamine. Usual strength 60 mg, 120 mg, 180 mg. FLUCLOXACILLIN SODIUM Flucloxacillinum natricum

Flucloxacillin sodium is sodium (2S,5R,6R)-6-[[[3-(2chloro-6-fluorophenyl)-5-methylisoxazol-4-yl]carbonyl] amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0] heptane-2-carboxylate. It contains not less than 95.0% and not more than 102.0% of C19H16ClFN3NaO5S, calculated with reference to the anhydrous substance.

Characters A white or almost white, crystalline powder, hygroscopic. Freely soluble in water and in methanol, soluble in ethanol (96%). Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum obtained with flucloxacillin sodium RS. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silanised silica gel H. Mobile phase: Acetone - a 15.4% solution of ammonium acetate adjusted to pH 5.0 with glacial acetic acid (30 : 70). Test solution: Dissolve 25 mg of the substance to be examined in 5 ml of water. Reference solution (1): Dissolve 25 mg of flucloxacillin sodium RS in 5 ml of water. Reference solution (2): Dissolve 25 mg of cloxacillin sodium RS, 25 mg of dicloxacillin sodium RS and 25 mg of flucloxacillin sodium RS in 5 ml of water. Procedure: Apply separately to the plate 1 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in air and expose it to iodine vapour until the spots appear. Examine in daylight. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) shows 3 clearly separated spots. C. Place about 2 mg in a test-tube about 15 cm long and 1.5 cm in diameter. Moisten with 0.05 ml of water and add 2 ml of sulfuric acid-formaldehyde reagent R. Mix the contents of the tube by swirling; the colour of the solution is slightly greenish-yellow. Place the test-tube in a waterbath for about 1 min; the solution becomes yellow. D. It gives reaction (A) of sodium (Appendix 8.1). Appearance of solution Solution S: Dissolve 2.50 g in carbon dioxide-free water R and dilute to 25.0 ml with the same solvent. Solution S is clear (Appendix 9.2). The absorbance (Appendix 4.1) of solution S measured at 430 nm is not greater than 0.04.

C19H16ClFN3NaO5S,H2O

M. 493.9

pH The pH of solution S is 5.0 to 7.0 (Appendix 6.2). 415

VP V

FLUCLOXACILLIN CAPSULES

Specific optical rotation +158° to +168°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.250 g in water and dilute to 25.0 ml with the same solvent. Related substances Examine by liquid chromatography (Appendix 5.3). Chromatographic system as prescribed under Assay. Inject test solution (1), the reference solution. The run time of the test solution is six times the retention time of the principal peak. In the chromatogram obtained with test solution (1): the area of any peak, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with the reference solution (1%); the sum of the areas of all peaks, apart from the principal peak, is not greater than five times the area of the principal peak in the chromatogram obtained with the reference solution (5%). Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with the reference solution. N,N-Dimethylaniline Not more than 20 ppm (Appendix 10.16, method 2). 2-Ethylhexanoic acid Not more than 0.8% m/m (Appendix 10.17). Water 3.0% to 4.5% (Appendix 10.3). Determined on 0.300 g. Pyrogens If intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of pyrogens, it complies with the test for pyrogens (Appendix 13.4). Inject per kilogram of the rabbit’s mass 1 ml of a solution in water for injections containing 20 mg of the substance to be examined per millilitre. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - a 0.27% solution of potassium dihydrogen phosphate adjusted to pH 5.0 with dilute sodium hydroxide solution R (25 : 75). Test solution (1): Dissolve 50.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Test solution (2): Dilute 5.0 ml of test solution (1) to 50.0 ml with the mobile phase. Standard solution (1): Dissolve 50.0 mg of flucloxacillin sodium RS in the mobile phase and dilute to 50.0 ml with the mobile phase. Dilute 5.0 ml of the solution to 50.0 ml with the mobile phase. 416

Satndard solution (2): Dissolve 5 mg of flucloxacillin sodium RS and 5 mg of cloxacillin sodium RS in the mobile phase and dilute to 50.0 ml with the mobile phase. Reference solution: Dilute 5.0 ml of standard solution (1) to 50.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 225 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Procedure: Inject standard solution (2). Adjust the sensitivity of the system so that the heights of the principal peaks in the chromatogram obtained are at least 50% of the full scale of the recorder. The test is not valid unless the resolution between the peaks due to cloxacillin (the first peak) and flucloxacillin (the second peak) is at least 2.5. Inject standard solution (1) six times. The test is not valid unless the relative standard deviation of the peak area for flucloxacillin is not more than 1.0%. Inject alternately test solution (2) and standard solution (1). Calculate the content of C19H16ClFN3NaO5S using the peak areas for flucloxacillin in the chromatograms obtained with test solution (2) and standard solution (1).

Storage Store in an airtight container, at a temperature not exceeding 25 °C. If the substance is sterile, store in a sterile, airtight, tamper-proof container. Action and use Antibacterial. Preparations Capsules; suspension. FLUCLOXACILLIN CAPSULES Capsulae Flucloxacillini Flucloxacillin capsules contain flucloxacillin sodium. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of flucloxacillin, C19H17ClFN3O5S, 92.5% to 110.0% of the stated amount. Identification A. The infrared absorption spectrum (Appendix 4.2) of the contents of the capsules is concordant with the reference spectrum of flucloxacillin sodium. B. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the principal peak in the chromatogram obtained with the reference solution.

VP V

FLUCONAZOLE

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - a 0.27% solution of potassium dihydrogen phosphate adjusted to pH 5.0 with 2 M sodium hydroxide (25 : 75). Test solution: Transfer a quantity of the contents of the capsules equivalent to about 0.1 g of flucloxacillin, accurately weighed, to a 100 ml volumetric flask, add about 80 ml of the mobile phase, shake for about 15 minutes to dissolve, dilute to volume with the mobile phase, mix well and filter. Reference solution: Dilute 1 ml of the test solution to 100 ml with the mobile phase. Resolution solution: Containing 0.01% each of flucloxacillin sodium RS and cloxacillin sodium RS in the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm), Hypersil 5 ODS is suitable. Detector: A spectrophotometer set at 225 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: System suitability: In the chromatogram obtained with the resolution solution, the resolution factor between the peaks due to cloxacillin and flucloxacillin is at least 2.5. Inject the test solution and allow the chromatography to proceed for six times the retention time of the principal peak (flucloxacillin). In the chromatogram obtained with the test solution, the area of any secondary peak is not greater than the area of the principal peak in the chromatogram obtained with the reference solution (1%) and the sum of the areas of any such peaks is not greater than 5 times the area of the principal peak in the chromatogram obtained with the reference solution (5%). Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with the reference solution (0.05%).

Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm), Hypersil 5 ODS is suitable. Detector: A spectrophotometer set at 225 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: System suitability: In the chromatogram obtained with the resolution solution, the resolution factor between the peaks due to cloxacillin and flucloxacillin is at least 2.5. Calculate the content of flucloxacillin, C19H17ClFN3O5S, in the capsules using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C19H17ClFN3O5S in flucloxacillin sodium RS. Each mg of flucloxacillin sodium is equivalent to 0.9538 mg of flucloxacillin C19H17ClFN3O5S.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - a 0.27% solution of potassium dihydrogen phosphate adjusted to pH 5.0 with 2 M sodium hydroxide (25 : 75). Test solution: Weigh 20 capsules, calculate the average mass of the capsule contents, powder finely. Transfer a quantity of the powder equivalent to about 50 mg of flucloxacillin, accurately weighed, to a 50 ml volumetric flask, add about 40 ml of the mobile phase, shake for about 15 minutes to dissolve, dilute to volume with the mobile phase, mix well and filter. Dilute 5.0 ml of the filtrate to 50.0 ml with the mobile phase. Reference solution: Containing 0.011% of flucloxacillin sodium RS in the mobile phase. Resolution solution: Containing 0.01% each of flucloxacillin sodium RS and cloxacillin sodium RS in the mobile phase.

C13H12F2N6O  

Storage Store in well-closed container, in a cool and dry place. Action and use Penicillin antibacterial. Usual strength 250 mg, 500 mg. FLUCONAZOLE Fluconazolum

M. 306.3 

Fluconazole is 2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4triazol-1-yl)propan-2-ol. It contains not less than 99.0% and not more than 101.0% of C13H12F2N6O, calculated with reference to the dried substance.

Characters White or almost white, hygroscopic, crystalline powder. It shows polymorphism. Slightly soluble in water, freely soluble in methanol, soluble in acetone. Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of fluconazole RS. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference 417

VP V

FLUCONAZOLE

substance separately in the minimum volume of methylene chloride R, evaporate to dryness on a water-bath and record new spectra using the residues.

Appearance of solution Dissolve 1.0 g in methanol R and dilute to 20 ml with the same solvent. The solution is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). Related substances Examine by liquid chromatography (Appendix 5.3) Mobile phase: Acetonitrile - 0.063% solution of ammonium formate (14 : 86). Test solution: Dissolve 0.100 g of the substance to be examined in the mobile phase, sonicate if necessary, and dilute to 10.0 ml with the mobile phase. Reference solution (1): Dilute 5.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (2): Dissolve 5 mg of fluconazole for peak identification RS (containing impurity A) in the mobile phase, sonicate if necessary, and dilute to 10.0 ml with the mobile phase. Reference solution (3): Dissolve 3.0 mg of fluconazole impurity B RS in the mobile phase, sonicate if necessary, and dilute to 100.0 ml with the mobile phase. Reference solution (4): Dissolve 2.0 mg of fluconazole impurity C RS in the mobile phase and dilute to 20.0 ml with the mobile phase. To 1.0 ml of this solution add 1.0 ml of the test solution and dilute to 10.0 ml with the mobile phase. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 µm). Column temperature: 40 °C. Detector: A spectrophotometer set at 260 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: The run time is 3.5 times the retention time of fluconazole. Identification of impurities: Use the chromatogram supplied with fluconazole for peak identification RS and the chromatogram obtained with reference solution (2) to identify the peak due to impurity A; use the chromatogram obtained with reference solution (3) to identify the peak due to impurity B and the chromatogram obtained with reference solution (4) to identify the peak due to impurity C. Relative retention with reference to fluconazole (retention time = about 11 min): impurity B = about 0.4; impurity A = about 0.5; impurity C = about 0.8. System suitability: In the chromatogram obtained with reference solution (4), the resolution between the peaks due to impurity C and fluconazole is at least 3.0. Limits: Impurity A: The area of the peak due to impurity A is not more than 0.8 times the area of the principal peak in the 418

chromatogram obtained with reference solution (1) (0.4%). Impurity B: The area of the peak due to impurity B is not more than the area of the principal peak in the chromatogram obtained with reference solution (3) (0.3%). Impurity C: The area of the peak due to impurity C is not more than the area of the corresponding peak in the chromatogram obtained with reference solution (4) (0.1%). Any other impurity: For each impurity, the area is not more than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). Total peak area of all impurities is not more than 1.2 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.6%). Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A: (2RS)-2-(2,4-difluorophenyl)-1-(1H-1,2,4-triazol1-yl)-3-(4H-1,2,4-triazol-4- yl)propan-2-ol. Impurity B: 2-[2-fluoro-4-(1H-1,2,4-triazol-1-yl)phenyl]-1,3-bis (1H-1,2,4-triazol-1-yl)propan- 2-ol. Impurity C: 1,1’-(1,3-phenylene)di-1H-1,2,4-triazole. Impurity D: 2-(4-fluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl) propan-2-ol. Impurity E: 1-(2,4-difluorophenyl)-2-(1H-1,2,4-triazol-1-yl)ethanone. Impurity F: (2RS)-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1yl)propane-1,2-diol. Impurity G: 1-[[(2RS)-2-(2,4-difluorophenyl)oxiran-2-yl]methyl] -1H-1,2,4-triazole. Impurity H: (2RS)-1-bromo-2-(2,4-difluorophenyl)-3-(1H-1,2,4 -triazol-1-yl)propan-2-ol. Impurity I: 4-amino-1-[(2RS)-2-(2,4-difluorophenyl)-2-hydroxy-3 (1H-1,2,4-triazol-1-yl)propyl]-4H-1,2,4-triazolium.

Heavy metals

Not more than 10 ppm (Appendix 9.4.8). Dissolve 2.0 g of the substance to be examined in a mixture of water and methanol R (15 : 85) and dilute to 20.0 ml with the same solvent. 12 ml of the solution complies with limit test for heavy metals, method 2. Prepare the standard using lead standard solution (1 ppm Pb) R.

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.125 g of the substance to be examined in 60 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2).

VP V

1 ml of 0.1 N perchloric acid VS is equivalent to 15.32 mg of C13H12F2N6O.

Storage In an airtight container. Action and use Antifungal. Preparations Tablets, capsules. FLUCONAZOLE CAPSULES Capsulae Fluconazoli Fluconazole capsules contain fluconazole. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of fluconazole, C13H12F2N6O, 90.0% to 110.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel GF254 Mobile phase: Dichloromethane - methanol - ammonia (80 : 20 : 1). Test solution: Shake well a quantity of the capsule contents, equivalent to about 0.1 g of fluconazole with 10 ml of methanol R, and filter. Use the filtrate. Reference solution: Dissolve 0.1 g of fluconazole RS in 10 ml of methanol R. Procedure: Apply separately to the plate 10 ml of each solution. After removal of the plate, allow it to dry in air, and examine under ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and colour to that in the chromatogram obtained with the reference solution. B. The ultraviolet absorption spectrum (Appendix 4.1) of the test solution obtained in the Assay exhibits two maxima at 261 nm and 267 nm, and a minimum at 264 nm. Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 500 ml of 0.1 M hydrochloric acid R. Rotation speed: 100 rpm. Time: 45 min. Procedure: Test solution: After the specified time, withdraw a sample of the medium and filter, discarding the first portion of filtrate. Reference solution: Dissolve an accurately weighed quantity of fluconazole RS in 0.1 M hydrochloric acid R to obtain a solution having a concentration equivalent to that in the test solution.

FLUOCINOLONE ACETONIDE

Capsule solution: Prepare the capsule solution in the same manner as the test solution but using an empty capsule. Measure the absorbance (Appendix 4.1) of the test solution, capsule solution and the reference solution at the maximum at 261 nm in a 1-cm cell, using the dissolution medium in the reference cell. Calculate the total content of fluconazole, C13H12F2N6O, in the medium. Absorbance of the sample is the difference between absorbance of the test solution and absorbance of the cappsule solution. Tolerance: Not less than 80% (Q) of the labelled amount of fluconazole, C13H12F2N6O, is dissolved in 45 minutes.

Assay Test solution: Weigh 20 capsules and calculate the average mass of the capsule contents and finely powder. Transfer an accurately weighed portion of the powder, equivalent to 50 mg of fluconazole, to a 100 ml volumetric flask, add 70 ml of 0.1 M hydrochloric acid R, shake well to dissolve fluconazole, dilute with 0.1 M hydrochloric acid R to volume, and mix. Filter, discarding the first 20 ml of the filtrate. Dilute 10.0 ml of the filtrate to 25.0 ml with 0.1 M hydrochloric acid R, and mix. Reference solution: Dissolve an accurately weighed quantity of fluconazole RS in 0.1 M hydrochloric acid R to obtain a solution containing about 200 µg/ml. Measure the absorbance (Appendix 4.1) of the test solution and the reference solution at the maximum at 261 nm in a 1-cm cell, using 0.1 M hydrochloric acid R in the reference cell. Calculate the content of fluconazole, C13H12F2N6O, in the capsules using the measured absorbances of the test solution, the reference solution, and the declared content of C13H12F2N6O in fluconazole RS. Storage Store in an airtight container in a cool place. Action and use Antifungal. Usual strength 50 mg; 100 mg; 150 mg. FLUOCINOLONE ACETONIDE Fluocinolonum acetonidum

C24H30F2O6  

M. 452.5  419

VP V

FLUOCINOLONE ACETONIDE

Fluocinolone acetonide is 6α,9-difluoro-11β,21-dihydroxy -16α,17-(1-methylethylidenedioxy)pregna-1,4-diene3,20-dione. It contains not less than 97.0% and not more than 103.0% of C24H30F2O6, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder. It shows polymorphism. Practically insoluble in water, soluble in acetone and in ethanol. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of fluocinolone acetonide RS. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in ethanol R, evaporate to dryness and record new spectra using the residues. B. In the test for related substances, the principal peak in the chromatogram obtained with the reference solution (2) is similar in retention time to the peak due to fluocinolone acetonide RS in the chromatogram obtained with the reference solution (1). Specific optical rotation +100° to +104°, calculated with reference to the dried substance (Appendix 6.4). Dissolve 0.100 g of the substance to be examined in ethanol R and dilute to 10.0 ml with the same solvent. Related substances Examine by liquid chromatography (Appendix 5.3). Carry out the test protected from light. Mobile phase: Mix 450 ml of acetonitrile R with 500 ml of water and allow to equilibrate; adjust the volume to 1000.0 ml with water and mix again. Test solution: Dissolve 25.0 mg of the substance to be examined in acetonitrile R and dilute to 10.0 ml with the same solvent. Reference solution (1): Dissolve 2.5 mg of fluocinolone acetonide RS and 2.5 mg of triamcinolone acetonide R in 45 ml of acetonitrile R and dilute to 100.0 ml with water. Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with acetonitrile R. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase base-deactivated end-capped octadecylsilyl silica gel for chromatography (5 µm). Detector: A spectrophotometer set at 238 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: The run time is 4 times the retention time of fluocinolone acetonide. Retention times: Triamcinolone acetonide = about 8.5 min; fluocinolone acetonide = about 10 min. 420

System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to triamcinolone acetonide and fluocinolone acetonide is at least 3.0. Limits: In the chromatogram obtained with the test solution: Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (1%) and not more than 1 such peak has an area greater than half the area of the principal peak in the chromatogram obtained with reference solution (2) (0.5%). The sum of the peak areas of all impurities is not more than 2.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (2.5%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Note: Impurity A: 6α,9-difluoro-11β-hydroxy-16α,17-(1-methylethylidenedioxy) -3,20-dioxopregna-1,4-dien-21-oic acid. Impurity B: 6α,9-difluoro-11β-hydroxy-16α,17- (1-methylethylidenedioxy)-3-oxoandrosta-1,4-diene-17β-carboxylic acid. Impurity D: 6α,9-difluoro-11β-hydroxy-16α,17-(1-methylethylidenedioxy)- 3,20-dioxopregna-1,4-dien-21-al, Impurity C: 6α,9-difluoro-11β,16α,17,21-tetrahydroxypregna1,4-diene-3,20-dione (fluocinolone). Impurity E: 9,11β-epoxy-6α-fluoro-21-hydroxy-16α,17-(1methylethylidenedioxy)-9β-pregna-1,4-diene-3,20-dione. ImpurityF:6α-fluoro-21-hydroxy-16α,17-(1-methylethylidenedioxy) pregn-4- ene-3,20-dione. ImpurityG:6α-fluoro-11β-hydroxy-16α,17-(1-methylethylidenedioxy) -3,20-dioxopregn-4-en-21-yl acetate.

Loss on drying Not more than 1.0% (Appendix 9.6). (1.000 g; 105 °C; 3 h). Assay Protect the solutions from light throughout the assay. Dissolve 50.0 mg of the substance to be examined in ethanol (96%) R and dilute to 50.0 ml with the same solvent. Dilute 2.0 ml of this solution to 100.0 ml with ethanol (96%) R. Measure the absorbance at the maximum at 238 nm. Calculate the content of C24H30F2O6 taking the specific absorbance at 238 nm to be 355. Storage Store in an airtight container, protected from light. Action and use Glucocorticoid. Preparations Cream, ointment.

VP V

FLUOCINOLONE ACETONIDE DIHYDRATE

FLUOCINOLONE ACETONIDE DIHYDRATE Fluocinolone acetonidum dihydricum

C24H30F2O6,2H2O 

M. 488.5 (anhydrous)

Fluocinolone acetonide dihydrate is 6α,9α-difluoro11β,21-dihydroxy-16α-,17α- isopropylidenedioxypregna1,4-diene-3,20-dione dihydrate. It contains not less than 96.0% and not more than 104.0% of C24H30F2O6, calculated with reference to the anhydrous substance.

Characters A white or almost white, crystalline powder. Freely soluble in acetone, soluble in absolute ethanol, sparingly soluble in dichloromethane and in methanol, practically insoluble in water and in hexane. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of fluocinolone acetonide dihydrate RS. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Siliagel G. Impregnate the dry plate with formamide - acetone (1 : 9) by placing it in a tank containing a shallow layer of the specified impregnating solvent, allowing the solvent to ascend to the top, removing the plate from the tank and allowing the solvent to evaporate; use within 2 hours. Mobile phases: Toluene - chloroform - cyclohexane (29 : 56 : 115). Test solution: Dissolve 25 mg of the substance to be examined in chloroform - methanol (9 : 1) and dilute to 10 ml with the same solvent. Reference solution (1): Dissolve 25 mg of fluocinolone acetonide dihydrate RS in chloroform - methanol (9 : 1) and dilute to 10 ml with the same solvent. Reference solution (2): Mix 1.0 ml the test solution and 1.0 ml the reference solution (1). Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm. Allow the solvent to evaporate, heat at 120 °C for 15 minutes and spray the hot plate with 20% solution of sulfuric acid in ethanol. Heat at 120 °C for a further 10 minutes, allow to cool and examine in daylight and under ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, colour in daylight,

fluorescence in ultraviolet light at 365 nm and size to that in the chromatogram obtained with reference solution (1). The principal spot in the chromatogram obtained with reference solution (2) appears as a single, compact spot. C. Using the conditions specified in test B but using solutions prepared in the following manner. Test solution: Dissolve 10 mg of the substance to be examined in 1.5 ml of glacial acetic acid in a separating funnel, add 0.5 ml of a 2% w/v solution of chromium(VI) oxide R and allow to stand for 30 min. Add 5 ml of water and 2 ml of dichloromethane R and shake vigorously for 2 min. Allow to separate and use the lower layer. Prepare reference solution in the same manner but using 10 mg of fluocinolone acetonide RS.

Specific optical rotation +92° to +96°, calculated with reference to the anhydrous substance (Appendix 6.4). Determine on a 1% solution of the substance to be examined in 1,4-dioxan R. Light absorption Dissolve 15.0 mg of the substance to be examined in sufficient ethanol R to produce 100.0 ml. Dilute 10.0 ml of the solution to 100.0 ml with ethanol R. The A (1%, 1 cm) of the resulting solution at the maximum at 239 nm is 345 to 375, calculated with reference to the anhydrous substance. Related substances Examine by liquid chromatography (Appendix 5.3). Carry out the test protected from light. Mobile phase: Acetonitrile - water (45 : 55). Test solution: Dissolve 25.0 mg of the substance to be examined in acetonitrile R and dilute to 10.0 ml with the same solvent. Reference solution (1): Dissolve 2.5 mg of fluocinolone acetonide RS and 2.5 mg of triamcinolone acetonide RS in 45% m/v of acetonitrile R and dilute to 10.0 ml with the same solvent. Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with acetonitrile R. Reference solution (3): Dilute 1.0 ml of reference solution (2) to 20.0 ml with acetonitrile R. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase base- deactivated end-capped octadecylsilyl silica gel for chromatography (5 µm). Detector: A spectrophotometer set at 238 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Procedure: The run time is 4 times the retention time of the principal peak. System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to triamcinolone acetonide and fluocinolone acetonide 421

FLUOCINOLONE CREAM

is at least 3.0. In the chromatogram obtained with reference solution (3), the signal-to-noise ratio of the principal peak is not less than 10. Limits: In the chromatogram obtained with the test solution: The area of any secondary peak is not greater than the area of the principal peak in the chromatogram obtained with reference solution (2) (1%). The area of not more than one secondary peak is greater than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.5%). The sum of the areas of any secondary peaks is not greater than 2.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (2.5%). Disregard any peak with an area less than the area of the principal peak in the chromatogram obtained with reference solution (3) (0.05%).

Water 7.0% to 8.5% (Appendix 10.3). Determined on 0.5 g. Assay Carry out the tetrazolium assay of steroids (Appendix 10.8). Calculate the content of C24H30F2O6 from the absorbances obtained by the test solution, standard solution and the declared content of C24H30F2O6 in fluocinolone acetonide RS. Storage Store in an airtight container, protected from. Action and use Glucocorticoid. Preparations Cream, ointment. FLUOCINOLONE CREAM Cremoris Fluocinoloni acetonidi Fluocinolone cream contains fluocinolone acetonide or fluocinolone acetonide dihydrate. The cream complies with the requirements stated under “Topical semi-solid preparations” (Appendix 1.12) and with the following requirements.

Content of fluocinolone acetonide, C24H30F2O6, 90.0% to 110.0% of the stated amount. Characters A white or off-white, homogeneous cream. Identification A. In the Assay, the chromatogram obtained with the test solution shows a peak with the same retention time as the peak due to fluocinolone acetonide in the chromatogram obtained with the reference solution. 422

VP V

B. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel G. Mobile phase: n-Hexane - chloroform - methanol - triethylamine (60 : 40 : 10 : 1). Test solution: Shake a quantity of the cream containing about 0.25 mg of fluocinolone acetonide in 2 ml of chloroform R, add 10 ml of methanol R, shake vigorously. Cool in ice for 15 minutes, centrifuge at 3000 r/min for 15 min, decant the clear, supernatant liquid, evaporate to dryness on a water bath in a current of nitrogen. Dissolve the residue in 1 ml of chloroform R. Reference solution: A 0.025% solution of fluocinolone acetonide RS in chloroform R. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of about 15 cm. After removal of the plate, allow it to dry in air, heat at 105 °C for 5 min, and spray whilst hot with tetrazolium blue solution R. The principal spot in the chromatogram obtained with the test solution is similar in position and colour to that in the chromatogram obtained with the reference solution.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: n-Hexane - chloroform - methanol - acetic acid (58 : 40 : 2 : 0.1). Solution A: To 20 ml of a 25% solution of lithium chloride R add 80 ml of methanol R, and mix. Prepare solutions: For creams containing 0.025% to 0.20% of fluocinolone acetonide Test solution (1a): To an accurately weighed quantity of the cream containing about 2.5 mg of fluocinolone acetonide add 60 ml of solution A, and disperse by skaking vigorously. Add 100 ml of cyclohexane R, shake gently for 2 min, and separate the lower, aqueous methanolic layer, taking care to exclude any solid matter that separates the interface. Repeat the extraction using a further 25 ml of solution A. To the combined extracts add a solution containing 11 g of aluminium potassium sulphate dodecahydrate R in 214 ml of water, following 50 ml of chloroform R. Shake vogorously for 3 min, allow the layers to separate and filter the chloroform extract through filter paper (Whatman No.1 filters are suitable), previously moistened with chloroform R. Repeat the extraction with 50 ml and 10 ml quantities of chloroform R, filtering the extracts as before. Evaporate the combined filtered extracts to dryness on a water bath. Dissolve the obtained residue in 5 ml of chloroform R, and dilute to 10.0 ml with the same solvent. Test solution (2): Prepare as directed for Test solution (1a), but adding 1.0 ml of a 0.050% solution of phenacetin in chloroform R to the chloroform solution before dilution to 10.0 ml.

VP V

Reference solution: A solution containing 0.025% of fluocinolone acetonide RS and 0.005% phenacetin (internal standard) in chloroform R. For creams containing 0.01% of fluocinolone acetonide Test solution (1): Prepare as directed for Test solution (1a), using an accurately weighed quantity of the cream containing about 1 mg of fluocinolone acetonide. Test solution (2): Prepare as directed for Test solution (1), but adding 1.0 ml of a 0.02% solution of phenacetin in chloroform R to the chloroform solution before dilution to 10.0 ml. Reference solution: A solution containing 0.01% of fluocinolone acetonide RS and 0.002% phenacetin (internal standard) in chloroform R. For creams containing 0.00625% of fluocinolone acetonide Test solution (1): Prepare as directed for Test solution (1a), using an accurately weighed quantity of the cream containing about 0.62 mg of fluocinolone acetonide. Test solution (2): Prepare as directed for Test solution (1), but adding 1.0 ml of a 0.0125% solution of phenacetin in chloroform R to the chloroform solution before dilution to 10.0 ml. Reference solution: A solution containing 0.00625% of fluocinolone acetonide RS and 0.00125% phenacetin (internal standard) in chloroform R. For creams containing 0.0025% of fluocinolone acetonide Test solution (1): Prepare as directed for Test solution (1a), using an accurately weighed quantity of the cream containing about 0.25 mg of fluocinolone acetonide. Test solution (2): Prepare as directed for Test solution (1a), but adding 1.0 ml of a 0.005% solution of phenacetin in chloroform R to the chloroform solution before dilution to 10.0 ml. Reference solution: A solution containing 0.0025% of fluocinolone acetonide RS and 0.0005% phenacetin (internal standard) in chloroform R. Chromatographic system: A column (20 cm × 5 mm) packed with stationary phase C (5 µm) (Spherisorb ODS 1 is suitable). Detector: A spectrophotometer set at 243 nm. Flow rate: 1.8 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution, the test is not valid unless the resolution factor (Rs) between the peaks due to fluocinolone acetonide and phenacetin is more than 2, and the capacity factors (k’) of fluocinolone acetonide and phenacetin are about 3 and 2, respectively. If these conditions are not achieved, adjust the concentration of methanol in the mobile phase, increasing its concentration to reduce the value of k’ and reducing its concentration to increase the value of k’. If the correct values can not be

FOLIC ACID

obtained by this means, the column is faulty and must be repacked. If, when the correct k’ values have been obtained, the value of Rs is less than 2, reduce the concentration of chloroform in the mobile phase by 5% to obtain an increased retention time for both fluocinolone acetonide and phenacetin and re-adjust the k’ values to the specified values by increasing the concentration of methanol. Repeat the adjustment of chloroform and methanol concentrations until correct values for both Rs and k’ have been obtained. Separately inject the reference solution and the test solution. Calculate the content of fluocinolone acetonide, C24H30F2O6, in the cream using the areas for the principal peaks in the chromatograms obtained with the test solution, the reference solution and the declared content of C24H30F2O6 in fluocinolone acetonide RS.

Storage Store in an airtight container in cool place, protected from light. Action and use Corticosteroid for external use. Usual strength 0.01%; 0.025%; 0.05%. FOLIC ACID Acidum folicum

C19H19N7O6

M. 441.4

Folic acid is (2S)-2-[[4-[[(2-amino-4-oxo-1,4dihydropteridin-6-yl) methyl] amino] benzoyl] amino] pentanedioic acid. It contains not less than 96.0% and not more than 102.0% of C19H19N7O6, calculated with reference to the anhydrous substance.

Characters Yellowish or orange, crystalline powder. Practically insoluble in water and in most organic solvents. It dissolves in dilute acids and in alkaline solutions. Identification Apply one of the two following identifications. First identification: A, B. Second identification: A, C A. Specific optical rotation (Appendix 6.4): +18° to +22°, calculated with reference to the anhydrous substance. 423

VP V

FOLIC ACID

Dissolve 0.25 g in 0.1 M sodium hydroxide R and dilute to 25.0 ml with the same solvent. B. In the test for Assay, the principal peak in the chromatogram obtained with the test solution is similar in retention time to the principal peak in the chromatogram obtained with reference solution (1). C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Concentrated ammonia - propanol – ethanol (96%) (20 : 20 : 60). Test solution: Dissolve 50 mg of the substance to be examined in a mixture of 2 volumes of concentrated ammonia R and 9 volumes of methanol R and dilute to 100 ml with the same mixture of solvents. Reference solution: Dissolve 50 mg of folic acid RS in a mixture of 2 volumes of concentrated ammonia R and 9 volumes of methanol R and dilute to 100 ml with the same mixture of solvents. Procedure: Apply separately to the plate 2 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in air and examine in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, fluorescence and size to the principal spot in the chromatogram obtained with the reference solution.

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol - a solution containing 1.116% of potassium dihydrogen phosphate and 0.550% of dipotassium hydrogen phosphate solution (12 : 88). Test solution: Dissolve 0.100 g of the substance to be examined in 5 ml of a 1.06% solution of sodium carbonate R and dilute to 100.0 ml with the mobile phase. Dilute 2.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (1): Dissolve 0.100 g of folic acid RS in 5 ml of a 1.06% solution of sodium carbonate R and dilute to 100.0 ml with the mobile phase. Dilute 2.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (2): Dissolve 20 mg of pteroic acid R in 5 ml of a 1.06% solution of sodium carbonate R and dilute to 100.0 ml with the mobile phase. Mix 1.0 ml of this solution with 1.0 ml of reference solution (1) and dilute to 100.0 ml with the mobile phase. Reference solution (3): Dilute 2.0 ml of the test solution to 20.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 20.0 ml with the mobile phase. Reference solution (4): Dissolve 10.0 mg of N-(4aminobenzoyl)-L-glutamic acid R in 1 ml of a 1.06% solution of sodium carbonate R and dilute to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 100.0 ml with the mobile phase. Reference solution (5): Dissolve 12.0 mg of pteroic acid R in 1 ml of a 1.06% solution of sodium carbonate R and dilute to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 100.0 ml with the mobile phase. 424

Chromatographic system: A column (25 cm × 4.0 mm) packed with spherical octylsilyl silica gel for chromatography R (5 µm) with a specific surface of 350 m2/g, a pore size of 10 nm and a carbon loading of 12.5%. Detector: A spectrophotometer set at 280 nm. Flow rate: 0.6 ml/min. Volume of injection: 5 µl. Procedure: Inject the test solution and reference solutions (3), (4) and (5). Continue the chromatography for 3 times the retention time of folic acid. Relative retention times with reference to folic acid (retention time = about 8.5 min) are: impurity A = about 0.5; impurity B = about 0.6; impurity C = about 0.9; impurity E = about 1.27; impurity D = about 1.33; impurity F = about 2.2. System suitability: Inject the reference solution (2). The resolution between the peaks due to folic acid and to pteroic acid (impurity D) not less than 4.0. Limit: In the chromatogram obtained with the test solution: The area of any peak, apart from the principal peak, corresponding to impurity A, is not more than the area of the principal peak in the chromatogram obtained with reference solution (4) (0.5%). The area of any peak, apart from the principal peak, corresponding to impurity D, is not more than the area of the principal peak in the chromatogram obtained with reference solution (5) (0.6%). The area of any other impurity, apart from the principal peak, and peaks corresponding to impurities A and D is not more than the area of the principal peak in the chromatogram obtained with reference solution (3) (0.5%). The sum of the areas of other impurities, apart from the principal peak, and peaks corresponding to impurities A and D is not more than 2 times the area of the principal peak in the chromatogram obtained with reference solution (3) (1.0%). Disregard solvent peak and any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (3) (0.05%) Note: Impurity A: (2S)-2-[(4-aminobenzoyl)amino] pentanedioic acid (N-(4-aminobenzoyl)-L-glutamic acid). Impurity B: 2,5,6-triaminopyrimidin-4(1H)-one. Impurity C: (2S)-2-[[4-[[(2-amino-4-oxo-1,4-dihydropteridin-7yl)methyl]amino]benzoyl]amino] pentanedioic acid (isofolic acid) Impurity D: 4-[[(2-amino-4-oxo-1,4-dihydropteridin-6-yl)methyl] amino]benzoic acid (pteroic acid). Impurity E: (2S)-2-[[4-[bis[(2-amino-4-oxo-1,4-dihydropteridin-6yl)methyl]amino]benzoyl]amino] pentanedioic acid (6-pterinylfolic acid). Impurity F: 2-amino-7-(chloromethyl)pteridin-4(1H)-one.

Water 5.0% to 8.5% (Appendix 10.3). Determined on 0.150 g.

VP V

Sulfated ash Not more than 0.2% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. Inject the test solution and reference solution (1). Calculate the content of C19H19N7O6 using the peak areas of folic acid in the chromatograms obtained with the test solution, reference solution (1) and the content of folic acid RS. Storage Store in a well-closed container, protected from light. Action and use Prevent and treat anemia. Preparation Tablets. FOLIC ACID TABLETS Tabellae Acidi folici Folic acid tablets contain folic acid. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of folic acid, C19H19N7O6, 90.0% to 110.0% of the stated amount. Identification Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel G. Mobile phase: 13.5 M ammonia - propan-1-ol - ethanol (96%) (20 : 20 : 60). Test solution: Extract a quantity of the powdered tablets containing 0.5 mg of folic acid with 1 ml of a mixture of 1 volume of 13.5 M ammonia R and 9 volumes of methanol R , centrifuge and use the supernatant liquid. Reference solution: A 0.05% solution of folic acid RS in a mixture of 1 volume of 13.5 M ammonia R and 9 volumes of methanol R. Procedure: Apply separately to the plate 2 µl of each solution. Develop over a path of about 15 cm. After removal of the plate, allow it to dry in air and examine under ultraviolet light (365 nm). The principal spot in the chromatogram obtained with the test solution is similar in position, fluorescence and size to that in the chromatogram obtained with the reference solution. Hydrolysis products Examine by liquid chromatography (Appendix 5.3).

FOLIC ACID TABLETS

Carry out the test protected from light. Mobile phase: A 0.05 M solution of potassium dihydrogen phosphate R, adjusted to pH 5.5 with 5 M sodium hydroxide R. Filter and degas. Reference solution: A solution in the mobile phase containing 0.5 mg/ml of 4-aminobenzoic acid R and 2.0 mg/ml of N-(4-aminobenzoyl)-L-glutamic acid R . Test solution: Shake a quantity of the powdered tablets containing 5.0 mg of folic acid with 50.0 ml of the mobile phase, centrifuge and use the supernatant liquid. Chromatographic system: A column (20 cm × 4.6 mm) packed with stationary phase C (10 µm) (Spherisorb ODS1 is suitable). Detector: A spectrophotometer set at 269 nm. Flow rate: 2 ml/min. Volume of injection: 10 µl. Procedure: Inject the reference solution and test solution. In the chromatogram obtained with the reference solution the resolution factor between the two peaks, due to N-(4aminobenzoyl)-L-glutamic acid and 4-aminobenzoic acid, is at least 3.0. The areas of the peaks due to 4-aminobenzoic acid and N-(4-aminobenzoyl)-L-glutamic acid in the chromatogram obtained with the test solution are not greater than the areas of any corresponding peaks in the chromatogram obtained with the reference solution.

Uniformity of content Tablets containing less than 2 mg of folic acid comply with the requirements stated under Uniformity of content (Appendix 11.2). Examine by liquid chromatography (Appendix 5.3). Carry out the test protected from light. Mobile phase: A mixture of 0.05 M potassium dihydrogen phosphate R and acetonitrile R (93 : 7) adjusted to pH 6.0 with 5 M sodium hydroxide R. Reference solution: Add 1 ml of 0.5 M hydrochloric acid R to 5.0 ml of a 0.0020% solution of folic acid RS in 0.1 M sodium hydroxide R and dilute to 10.0 ml with the mobile phase. Test solution: Shake one tablet with 5 ml of 0.1 M sodium hydroxide R, add sufficient mobile phase to produce a solution containing 0.001% w/v of folic acid, centrifuge and use the supernatant liquid. Chromatographic system: A column (20 cm × 4.6 mm) packed with stationary phase C (10 µm) (Spherisorb ODS1 is suitable). Detector: A spectrophotometer set at 283 nm. Flow rate: 2 ml/min. Volume of injection: 10 µl. Procedure: Inject the reference solution and test solution. Calculate the content of C19H19N7O6 in each tablet using the areas for the principal peaks in the chromatograms 425

VP V

FORMALDEHYDE SOLUTION

obtained with the test solution and the reference solution, and the declared content of C19H19N7O6 in folic acid RS.

Assay Examine by liquid chromatography (Appendix 5.3). Carry out the assay protected from light. For tablets containing 2 mg or more of folic acid Mobile phase and Chromatographic system: As described under Uniformity of content. Reference solution: Dilute 5.0 ml of a 0.020% w/v solution of folic acid RS in 0.1 M sodium hydroxide R to 100.0 ml with the mobile phase, and mix. Test solution: Weigh 20 tablets, calculate the average mass, and powder finely. Shake an accurately weighed quantity of the powdered tablets containing about 20 mg of folic acid with 50 ml of 0.1 M sodium hydroxide R, dilute to 100.0 ml with the same solvent, centrifuge and dilute 5.0 ml of the supernatant liquid to 100.0 ml with the mobile phase. Calculate the content of C19H19N7O6 in the tablets using the areas for the principal peaks in the chromatogram obtained with the test solution and the reference solution, and the declared content of C19H19N7O6 in folic acid RS. For tablets containing less than 2 mg of folic acid Use the average of the 10 individual results obtained in the test for Uniformity of content.

Storage Store in an airtight container at cool place, protected from light. Action and use Vitamin. Usual strength 0.4 mg; 0.8 mg; 1 mg; 5 mg. FORMALDEHYDE SOLUTION Formaldehydi solutio Formalin CH2O

M. 30.03

Formaldehyde solution (35%) contains not less than 34.5% (m/m) and not more than 38.0% (m/m) of formaldehyde (CH2O), with methanol as stabiliser.

Characters A clear, colourless liquid. Miscible with water and with ethanol (96%). It may be cloudy after storage. Identification A. Dilute 1 ml of solution S to 10 ml with water. To 0.05 ml of the solution add 1 ml of a 1.5% solution of chromotropic acid sodium salt R, 2 ml of water and 8 ml 426

of sulfuric acid R. A violet-blue or violet-red colour develops within 5 minutes. B. To 0.1 ml of solution S add 10 ml of water. Add 2 ml of a 1.0% solution of phenylhydrazine hydrochloride R, prepared immediately before use, 1 ml of 5% solution of potassium ferricyanide R and 5 ml of hydrochloric acid R. An intense red colour is formed. C. Mix 0.5 ml with 2 ml of water and 2 ml of 2% solution of silver nitrate R in a test-tube. Add 2 M ammonia R until slightly alkaline. Heat on a water-bath. A grey precipitate or a silver mirror is formed. D. It complies with the limits of the assay.

Colour of solution Solution S: Dilute 10 ml, filtered if necessary, to 50 ml with carbon dioxide-free water R. Solution S is colourless (Appendix 9.3, method 2). Acidity To 10 ml of solution S add 1 ml of phenolphthalein solution R. Not more than 0.4 ml of 0.1 N sodium hydroxide VS is required to change the colour of the indicator to red. Methanol 9.0% to 15.0% (v/v). Examine by gas chromatography (Appendix 5.2). Internal standard solution: Dilute 10 ml of ethanol R to 100 ml with water (use ethanol containing less than 0.005% (v/v) of methanol as the internal standard). Reference solution: To 1.0 ml of methanol R add 10.0 ml of the internal standard solution and dilute to 100.0 ml with water. Test solution: To 10.0 ml of the solution to be examined add 10.0 ml of the internal standard solution and dilute to 100.0 ml with water. Chromatographic system: A glass column 1.5 m to 2.0 m long and 2 mm to 4 mm in internal diameter, packed with ethylvinylbenzenedivinylbenzene copolymer (150 µm to 180 µm). Carrier gas: Nitrogen for chromatography R, at a flow rate of 30 ml to 40 ml per minute. Detector: A flame-ionisation detector. Temperature: Maintain the temperature of the column at 120 °C and that of the injection port and of the detector at 150 °C. Volume of injection: 1 µl. Procedure: Inject the reference solution. Adjust the sensitivity of the detector so that the heights of the peaks in the chromatogram obtained are at least 50% of the full scale of the recorder. The test is not valid unless the resolution between the peaks due to methanol and ethanol is at least 2.0. Inject separately the test solution and the reference solution. Calculate the percentage content of methanol.

VP V

FUROSEMIDE

Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determine on 1.0 g. Assay Into a 100 ml volumetric flask containing 2.5 ml of water and 1 ml of 2 M sodium hydroxide R, weigh 1.000 g of the solution to be examined, shake and dilute to 100.0 ml with water. To 10.0 ml of the solution add 30.0 ml of 0.1 N iodine VS. Mix and add 10 ml of 2 M sodium hydroxide R. After 15 minutes, add 25 ml of a 10% solution of sulfuric acid R and 2 ml of starch solution R. Titrate with 0.1 N sodium thiosulfate VS. Each ml of 0.1 N iodine VS is equivalent to 1.501 mg of CH2O. Storage Store in an airtight container, protected from light, at a temperature of 15 °C to 25 °C. FUROSEMIDE

Furosemidum

O H 2N

S

Cl

C12H11ClN2O5S

O CO2H N H

O

M. 330.7

Furosemide is 4-chloro-2-[(furan-2-ylmethyl)amino]-5sulfamoylbenzoic acid. It contains not less than 98.5% and not more than 101.0% of C12H11ClN2O5S, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder. Soluble in acetone, sparingly soluble in ethanol (96%), practically insoluble in water and methylene chloride. It dissolves in dilute solutions of alkali hydroxides. Melting point: About 210 °C, with decomposition. Identification Apply one of the two following identifications: First identification: A. Second identification: B, C. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of furosemide RS. If the spectia obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in acetone R, evaposate to dryness and record new spectia using the residues. B. Dissolve 50 mg in 0.1 M sodium hydroxide R and dilute to 100 ml with the same solution. Dilute 1 ml of this solution to 100 ml with 0.1 M sodium hydroxide R.

The ultraviolet absorption spectrum (Appendix 4.1) of the solution in the range 220 nm to 350 nm exhibits 3 absorption maxima at 228 nm, 270 nm and 333 nm. The ratio of the absorbances measured at 270 nm to that measured at 228 nm is 0.52 to 0.57. C. Dissolve about 25 mg in 10 ml of ethanol (96%) R. Mix 5 ml of the solution and 10 ml of water. To 0.2 ml of this solution add 10 ml of 2 M hydrochloric acid R and heat under a reflux condenser for 15 min. Allow to cool and add 18 ml of 1 M sodium hydroxide R and 1 ml of a 0.5% solution of sodium nitrite R. Allow to stand for 3 min, add 2 ml of a 2.5% solution of sulfamic acid R and mix. Add 1 ml of a 0.5% solution of naphthylethylenediamine dihydrochloride R. A violet-red colour develops.

Appearance of solution Dissolve 0.5 g in 0.5 M sodium hydroxide R and dilute to 10.0 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution BY5 (Appendix 9.3, method 2). Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use and protect from light. Mobile phase: Dissolve 2.0 g of potassium dihydrogen phosphate R and 2.5 g of cetrimide R in 700 ml of water; adjust to pH 7.0 with ammonia R and add 300 ml of propanol R. Test solution: Dissolve 50.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Reference solution (1): Dissolve 2 mg of furosemide impurity A RS in the mobile phase, add 2.0 ml of the test solution and dilute to 20.0 ml with the mobile phase. Dilute 0.5 ml of this solution to 20.0 ml with the mobile phase. Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (3): Dissolve 2 mg of furosemide for peak identification RS (containing impurities C and D) in 2.0 ml of the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase end-capped octylsilyl silica gel for chromatography (5 µm). Detector: A spectrophotometer set at 238 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: The run time is 3 times the retention time of furosemide. Identification of impurities: Use the chromatogram obtained with reference solution (1) to identify the peak due to impurity A; use the chromatogram supplied with furosemide for peak identification RS and the chromatogram obtained with reference solution (3) to identify the peaks due to impurities C and D. Relative retention with reference to furosemide (retention time = about 9 min): impurity C = about 0.5; impurity A = about 0.8; impurity D = about 1.5. 427

VP V

FUROSEMIDE TABLETS

System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to impurity A and furosemide is at least 4.0. Limits: Correction factors: for the calculation of content, multiply the peak areas of the following impurities by the corresponding correction factor: impurity C = 1.4; impurity D = 2.0; Impurity C: The corrected area of the peak due to impurity C is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (2) (0.2%). Impurity D: The corrected area of the peak due to impurity D is not more than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.15%). Unspecified impurities: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.10%). The sum of the peak areas of all impurities is not more than 5 times the area of the peak due to impurity A in the chromatogram obtained with reference solution (2) (0.5%). Disregard any peak with an area less than 0.5 times the area of the peak due to impurity A in the chromatogram obtained with reference solution (2) (0.05%). Note: Impurity A: 2-chloro-4-[(furan-2-ylmethyl)amino]-5-sulfamoylbenzoic acid. Impurity B: 2,4-dichloro-5-sulfamoylbenzoic acid. Impurity C: 2-amino-4-chloro-5-sulfamoylbenzoic acid. Impurity D: 2,4-bis[(furan-2-ylmethyl)amino]-5-sulfamoylbenzoic acid. Impurity E: 2,4-dichlorobenzoic acid. Impurity F: 4-chloro-5-sulfamoyl-2-[[((2RS)-tetrahydrofuran-2-yl) methyl]amino]benzoic acid.

Chlorides Not more than 0.02% (Appendix 9.4.5). To 0.5 g of the substance to be examined add a mixture of 30 ml of water and 0.2 ml of nitric acid R, shake for 5 min. Allow to stand for 15 min and filter. Use 15 ml of the filtrate. Sulfates Not more than 0.03% (Appendix 9.4.14). To 1.0 g of the substance to be examined add a mixture of 0.2 ml of acetic acid R and 30 ml of water, shake for 5 min. Allow to stand for 15 min and filter. Use 15 ml of the filtrate. Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 8. Prepare the standard using 0.5 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). 428

Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.250 g of the substance to be examined in 40 ml of dimethylformamide R. Titrate with 0.1 N sodium hydroxide VS. Determine the end-point potentiometrically (Appendix 10.2). Carry out a blank titration. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 33.07 mg of C12H11ClN2O5S. Storage Protected from light. Action and use Loop diuretic. Preparations Injection, tablets. FUROSEMIDE TABLETS Tabellae Furosemidi Furosemide tablets contain furosemide. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of furosemide, C12H11ClN2O5S, 90.0% to 110.0% of the stated amount. Identification A. The ultraviolet absorption spectrum (Appendix 4.1) of the test solution obtained in the Assay, in the range of 220 nm to 360 nm, exhibits three maxima at 228 nm, 271 nm and 333 nm. B. Shake a quantity of the powdered tablets containing 25 mg of furosemide with 10 ml of ethanol R, filter and evaporate the filtrate to dryness on a water bath. Dissolve the residue in 2.5 ml of ethanol R, add 2 ml dimethylamino benzaldehyde solution R. A green colour is produced and then becomes deep red. Related substances Examined by the liquid chromatography (Appendix 5.3) Mobile phase: Propanol - buffer solution (30 : 70). Buffer solution: Dissolve 2 g of potassium dihydrogen phosphate R and 2.5 g of cetrimide R in 700 ml of water, adjust the pH to 7.0 with ammonia R. Prepare the following solutions immediately before use. Test solution: Weigh accurately a quantity of the powdered tablets containing 20 mg of furosemide and dissolve in the mobile phase to produce 50.0 ml, mix and filter. Reference solution: Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase.

VP V

Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase B (5 µm). Flow rate: 1.0 ml/min. Detector: A spectrophotometer set at 238 nm. Volume of injection: 100 µl. Procedure: Inject the test solution and the reference solution. The run time is 3 times the retention time of the principal peak. In the chromatogram obtained with the test solution, the area of any secondary peak is not greater than the area of the principal peak in the chromatogram obtained with the reference solution (1.0%). The sum of areas of all secondary peaks is not greater than 1.5 times the area of the principal peak in the chromatogram obtained with the reference solution (1.5%).

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of phosphate buffer pH 5.8 R Rotation speed: 50 rpm. Time: 45 min. Procedure: After the specified time, withdraw a sample of the medium and filter, discard the first 20 ml of the filtrate. Dilute with the dissolution medium to obtain a solution having a concentration of 0.001% of furosemide if necessary. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum wavelength at 277 nm in a 1-cm cell, using the phosphate buffer pH 5.8 R as the blank. Measure simultaneously the absorbance of a 0.001% solution of furosemide RS in the phosphate buffer pH 5.8 R. Calculate the total content of furosemide, C12H11ClN2O5S, dissolved using the declared content of C12H11ClN2O5S in furosemide RS. Tolerance: Not less than 70% (Q) of the labeled amount of furosemide, C12H11ClN2O5S, is dissolved in 45 min. Assay Weigh 20 tablets, calculate the average mass and finely powder. Weigh accurately a quantity of the powdered tablets containing 20 mg of furosemide, transfer into a 100-ml volumetric flask, add 60 ml of 0.1 M sodium hydroxide R, shake for 10 min, dilute to volume with 0.1 M sodium hydroxide R, and mix. Filter, discard the first 20 ml of the filtrate. Dilute 5.0 ml of the filtrate to 100.0 ml with 0.1 M sodium hydroxide R, and mix. Measure the absorbance (Appendix 4.2) of the solution at the maximum wavelength at 271 nm in a 1-cm cell, using 0.1 M sodium hydroxide R as blank. Calculate the content of furosemide, C12H11ClN2O5S, in the tablets taking 580 as the value of A (1%, 1 cm) at the maximum wavelength at 271 nm. Storage Store in an airtight containers or in blisters in cool place, protected from light.

GABAPENTIN

Action and use Diuretic. Usual strength 20 mg; 40 mg. GABAPENTIN Gabapentinum H 2N

C9H17NO2  

CO2H

M: 171.2

Gabapentin is [1-(aminomethyl)cyclohexyl]acetic acid. It contains not less than 97.5% and not more than 102.0% of C9H17NO2, calculated with reference to the anhydrous substance.

Characters White or almost white, crystalline powder, it shows polymorphism. Sparingly soluble in water, slightly soluble in ethanol (96%), practically insoluble in methylene chloride. It dissolves in dilute acids and dilute solutions of alkali hydroxides. Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of gabapentin RS. If the spectra obtained with the substance to be examined and gabapentin RS show differences, dissolve separately the substance to be examined and the reference substance in methanol R, evaporate to dryness and record new spectra using the residues. Appearance of solution Dissolve 1.50 g in a mixture of 0.5 ml of acetic acid R, 19.5 ml of methanol R and 30 ml of water. The solution is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). pH 6.5 to 7.5 (Appendix 6.2). Dissolve 1.0 g in carbon dioxide-free water R and dilute to 50 ml with the same solvent. Related substances A. Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use. Solution A: Dissolve 2.32 g of ammonium dihydrogen phosphate R in 950 ml of water, adjust to pH 2.0 with phosphoric acid R, and dilute to 1000 ml with water. Buffer solution: Dissolve 0.58 g of ammonium dihydrogen phosphate R and 1.83 g of sodium perchlorate R in 950 ml 429

VP V

GABAPENTIN

of water, adjust to pH 1.8 with perchloric acid R, and dilute to 1000 ml with water. Mobile phase: Acetonitrile R1 - buffer solution (24 : 76). Test solution: Dissolve 0.140 g of the substance to be examined in solution A and dilute to 10.0 ml with the same solvent. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with solution A. Dilute 1.0 ml of this solution to 10.0 ml with solution A. Reference solution (2): Dissolve 7.0 mg of gabapentin impurity A RS and 10 mg of gabapentin impurity B RS in methanol R1 and dilute to 50.0 ml with the same solvent. Dilute 1.0 ml of the solution to 10.0 ml with solution A. Reference solution (3): Dissolve 0.140 g of gabapentin RS in solution A and dilute to 10.0 ml with the same solvent. Reference solution (4): Dissolve 7.0 mg of gabapentin impurity D RS in 25 ml of methanol R1 and dilute to 100.0 ml with solution A. Dilute 1.0 ml of this solution to 10.0 ml with solution A. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase end-capped octadecylsilyl amorphous organosilica polymer R (5 µm). Column temperature: 40 °C. Detector: A spectrophotometer set at 215 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Inject the blank, reference solutions (1), (2) and the test solution. The run time of the test solution is 4 times the retention time of gabapentin. Identification of impurities: Use the chromatogram obtained with reference solution (2) to identify the peaks due to impurities A and B. Relative retention with reference to gabapentin (retention time = about 4 min): Impurity A = about 2.4; impurity B = about 2.8. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurities A and B is at least 2.3. To avoid memory effects between 2 chromatograms, the column may be washed using acetonitrile R1. Limits: In the chromatogram obtained with the test solution: Impurity A: The area of the peak due to impurity A is not more 1.5 times than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.15%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). B. Examine by liquid chromatography (Appendix 5.3) as described in related substances test A with the following modifications. 430

Mobile phase: Methanol R1 - acetonitrile R1 - buffer solution (30 : 35 : 35). Injection the test solution and reference solution (4). The run time is 1.2 times the retention time of impurity D. Retention time of impurity D is about 10 min. Limits: In the chromatogram obtained with the test solution: Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (4) (0.05%). Disregard any peak with an area less than 0.6 times the area of the principal peak in the chromatogram obtained with reference solution (4) (0.03%), disregard any peak with a relative retention of not more than 0.4 with reference to impurity D. Total of all impurities for tests A and B: Not more than 0.5%. Note: Impurity A: 2-azaspiro[4.5]decan-3-one. Impurity B: (1-cyanocyclohexyl)acetic acid. Impurity D: [1-[(3-oxo-2-azaspiro[4.5]dec-2-yl)methyl]cyclohexyl] acetic acid. Impurity E: 1-(carboxymethyl)cyclohexanecarboxylic acid. Impurity G: [1-(2-aminoethyl)cyclohexyl]acetic acid.

Chlorides Not more than 100 ppm (Appendix 9.4.5). Dissolve 1.5 g of the substance to be examined in a mixture of 0.5 ml of acetic acid R, 19.5 ml of methanol R and 30 ml of water. Titrate with 0.001 N silver nitrate VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.001 N silver nitrate VS is equivalent to 0.03545 mg of chlorides. Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 6. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Water Not more than 0.3% (Appendix 10.3). Determined on 1.000 g. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by liquid chromatography (Appendix 5.3) as described in test A for Related substances. Inject the test solution and reference solution (3). System suitability: In the chromatogram obtained with the reference solution (3), the symmetry factor for the peak due to gabapentin is not more than 5.0. Calculate the percentage content of gabapentin, C9H17NO2 using the peak areas of gabapentin in the chromatograms obtained with the test solution and reference solution (3) and the content of C9H17NO2 in gabapentin RS.

VP V

Storage Store in an airtight container, protected from light. Action and use Antiepileptic. Preparations Tablets, capsules. GABAPENTIN CAPSULES Capsulae Gabapentini Gabapentin capsules are hard capsules containing gabapentin. The capsules comply with the requirements stated under "Capsules" (Appendix 1.13) and with the following requirements:

Content of gabapentin, C9H17NO2, 90.0% to 110 .0% of the stated amount. Identification A. Empty at least 10 capsules, mix the capsule contents and grind to fine powder, use a quantity of the powder containing about 2 mg of gabapentin and 200 mg of potassium bromide R to make a disc and measure the IR spectrum (Appendix 4.2). The infrared spectrum obtained is concordant with the spectrum of gabapentin RS. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of the gabapentin peak in the chromatogram obtained with the reference solution. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of 0.06 M hydrochloric acid R. Rotation speed: 50 rpm. Time: 20 min. Procedure: Test solution: After the specified time, withdraw a sample of the medium and filter. Reference solution: Weigh accurately a quantity of gabapentin RS, dissolve in the medium to obtain a solution at a concentration of gabapentin similar to that expected in the test solution. Examined by liquid chromatography (Appendix 5.3) with the mobile phase and the chromatographic system as described in the Assay. The injection volume is 100 µl. Calculate the content of gabapentin, C9H17NO2, dissolved using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C9H17NO2 in gabapentin RS. Tolerance: Not less than 80% (Q) of the stated amount of gabapentin, C9H17NO2, is dissolved in 20 min. Related substances Examine by liquid chromatography (Appendix 5.3).

GABAPENTIN CAPSULES

Diluent: As described in the Assay. Mobile phase A: Dissolve 1.2 g of potassium dihydrogen phosphate R in 940 ml of water, adjust to pH 6.9 with 5 M potassium hydroxide solution R. Add 60 ml of acetonitrile R and mix well. Mobile phase B: Dissolve 1.2 g of potassium dihydrogen phosphate R in 700 ml of water, adjust to pH 6.9 with 5 M potassium hydroxide solution R. Add 200 ml of acetonitrile R and mix well. Test solution: Weigh 20 capsules, calculate the average mass of the capsule contents and grind to fine powder. Weigh a quantity of the fine powder containing 500 mg of gabapentin and transfer into a 25-ml volumetric flask. Add 15 ml of the diluent and sonicate for about 30 s (if necessary) to dissolve gabapentin, add the diluent to volume, mix and filter. Reference solution: Weigh accurately a quantity of gabapentin RS and gabapentin impurity A RS and dissolve in the diluent to obtain a solution having a concentration of gabapentin of 0.04 mg/ml and of gabapentin impurity A of 0.04 mg/ml. Chromatographic system: A column (250 mm × 4.6 mm) packed with stationary phase B (5 µm). Detector: A spectrophotometer set at 210 nm. Flow rate: 1.5 ml/min. Volume of injection: 50 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0.0 - 4.0

100

0

4.0 - 45.0

100 → 0

0 → 100

45.0 - 45.1

0 → 100

100 → 0

45.1 - 50.0

100

0

System suitability: In the chromatogram obtained with the reference solution, the symmetry factor of the gabapentin peak is not more than 2.0; the relative standard deviation of the peak areas of gabapentin and of gabapentin impurity A obtained from 6 replicate injections are not more than 5.0%. Inject the test solution. Calculate the content of the impurity A using the peak areas of impurity A obtained with the test solution, the reference solution and the concentration of the impurity A in the reference solution. Calculate the contents of other impurities using the peak areas obtained with the test solution, the reference solution and the concentration of gabapentin in the reference solution. Limits: The content of gabapentin impurity A is not more than 0.4%. The content of any other impurity is not more than 0.1%. The content of total impurities is not more than 1.0%. Note: Impurity A: 2-azaspiro [4.5]decane-3-one.

431

GABAPENTIN TABLETS

VP V

Assay Examine by liquid chromatography (Appendix 5.3). Diluent: Dissolve 1.2 g of potassium dihydrogen phosphate R in 1000 ml of water, adjust to pH 6.9 with 5 M potassium hydroxide R. Mobile phase: Dissolve 1.2 g of potassium dihydrogen phosphate R in 940 ml of water, adjust to pH 6.9 with 5 M potassium hydroxide R. Add 60 ml of acetonitrile R and mix well. Reference solution: Prepare a 4.0 mg/ml solution of gabapentin RS in the diluent. Test solution: Weigh 20 capsules, calculate the average mass of the capsule contents and grind to fine powder. Weigh a quantity of the fine powder containing 200 mg of gabapentin, transfer into a 50-ml volumetric flask. Add 30 ml of the diluent, sonicate for about 60 s (if necessary) to dissolve gabapentin, dilute to volume with the diluent, mix well and filter. Chromatographic system: A column (250 × 4.6 mm) packed with stationary phase B (5 µm). Detector: A spectrophotometer set at 210 nm. Flow rate: 1.2 ml/min. Volume of injection: 50 µl. Procedure: System stability: In the chromatograms obtained with the reference solution, the column efficieny, determined on the gabapentin peak, is not less than 7000 theoretical plates; the symmetry factor is not more than 2.0; the relative standard deviation of the peak areas of gabapentin for six replicate injections is not more than 2.0%. Inject the reference solution and the test solution. Calculate the content of gabapentin, C9H17NO2, in the capsules using the peak areas in the chromatograms obtained with the reference solution, the test solutions and the declared content of C9H17NO2 in gabapentin RS.

Identification A. Grind 10 tablets to a fine powder. Use an amount of the powdered tablets equivalent to 2 mg of gabapentin and 200 mg of potassium bromide R. Carefully grind the mixture and force to produce a disc. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum obtained with gabapetin RS. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution correspond to that in the chromatogram obtained with the reference solution.

Storage Store in a tight container, in a dry and cool place.

Related substances Examine by liquid chromatography (Appendix 5.3). Diluent: Proceed as directed in the Assay. Mobile phase A: Dissolve 1.2 g of potassium dihydrogen phosphate R in 940 ml of water, adjust to pH 6.9 with 5 M potassium hydroxide. Add 60 ml of acetonitrile R and mix. Mobile phase B: Dissolve 1.2 g of potassium dihydrogen phosphate R in 700 ml of water, adjust to pH 6.9 with 5 M potassium hydroxide R. Add 200 ml of acetonitrile R and mix. Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Transfer an accurately weighed quantity of the powdered tablets containing the equivalent of 500 mg of gabapentin to a 25 ml volumetric flask, add 15 ml of the diluent and dissolve by sonicating for 30 seconds (if necessary). Dilute to volume with the diluent, mix and filter. Reference solution: Dissolve an accurately weighed quantity of gabapentin RS and gabapentin related

Action and use Antiepileptic, treatment of neuropathic pain. Preparations 100 mg, 300 mg and 400 mg. GABAPENTIN TABLETS Tabellae Gabapentini Gabapentin tablets contain gabapentin. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements:

Content of gabapentin, C9H17NO2, 90.0% to 110.0% of the stated amount. 432

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of 0.06 M hydrochloric acid R. Rotation speed: 50 rpm. Time: 45 min. Procedure: Test solution: After the specified time, withdraw a sample of the medium, filter. Reference solution: Dissolve an accurately weighed quantity of gabapetin RS in the medium to obtain a solution having the same concentration of gabapetin to that in the test solution. Examine by liquid chromatography (Appendix 5.3). Mobile phase, chromatographic system: Proceed as directed in the Assay. For the tablets containing not more than 400 mg of gabapentin, volume of injection is 100 μl. Calculate the content of gabapentin, C9H17NO2, dissolved using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C9H17NO2 in gabapentin RS. Tolerance: Not less than 80% (Q) of the stated amount of gabapentin, C9H17NO2, is dissolved in 45 minutes.

VP V

GELATIN

compound A RS in the diluent to obtain a solution containing 0.04 mg/ml of gabapentin and 0.04 mg/ml of gabapentin related compound A. Chromatographic system: A column (250 mm × 4.6 mm) packed with stationary phase B (5 μm). Detector: A spectrophotometer set at 210 nm. Flow rate: 1.5 ml/min. Volume of injection: 50 μl. Procedure: Carry out a linear gradient elution using the following conditions. Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0.0 - 4.0

100

0

4.0 - 45.0

100 → 0

0 → 100

45.0 - 45.1

0 → 100

100 → 0

45.1 - 50.0

100

0

System suitability: Inject the reference solution, the tailing factor of gabapentin peak is not more than 2.0. The relative standard deviation of the peak areas due to gabapentin peak and gabapentin related compound A peak for six replicate injections is not more than 5.0%. Inject the test solution. Calculate the content of gabapentin related compound A in the tablets using the peak areas in the chromatograms obtained with the test solution and the reference solution and the concentration of gabapentin related compound A in the reference solution. Calculate the contents of other unspecified impurities in the tablets using the peak areas in the chromatograms obtained with the test solution and the reference solution and the concentration of gabapentin in the reference solution. Limits: Gabapentin related compound A: Not more than 0.4% Any individual unspecified impurity: Not more than 0.1% Total impurities: Not more than 1.0% Note: Gabapentin related compound A: 2-Azaspiro[4.5]decan-3-one.

Assay Examine by liquid chromatography (Appendix 5.3). Diluent: Dissolve 1.2 g of potassium dihydrogen phosphate R in 1000 ml of water, adjust to pH 6.9 with 5 M potassium hydroxide R. Mobile phase: Dissolve 1.2 g of potassium dihydrogen phosphate R in 940 ml of water, adjust to pH 6.9 with 5 M potassium hydroxide. Add 60 ml of acetonitrile R and mix. Reference solution: Dissolve an accurately weighed quantity of gabapentin RS in the diluent to obtain a solution containing 4.0 mg/ml of gabapentin. Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Transfer an accurately weighed quantity of the powdered tablets containing the equivalent

of 200 mg of gabapentin to a 50 ml volumetric flask, add 30 ml of the diluent and dissolve by sonicating for 60 seconds if necessary. Dilute to volume with the diluent, mix and filter. Chromatographic system: A column (250 mm × 4.6 mm) packed with stationary phase B (5 μm). Detector: A spectrophotometer set at 210 nm. Flow rate: 1.2 ml/min. Volume of injection: 50 μl. Procedure: System suitability: Inject the reference solution, the column efficiency, determined on gabapentin peak is not less than 7000 theoretical plates; the tailing factor is not more than 2.0. The relative standard deviation of the peak areas for six replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of gabapentin, C9H17NO2, in the tablets using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C9H17NO2 in gabapentin RS.

Storage Store in a well-closed container, in a cool and dry place, at a temperature not exceeding 30 °C. Action and use Antiepileptic. Treament of neuropathic pain. Usual strength 100 mg, 300 mg, 400 mg, 600 mg and 800 mg. GELATIN Gelatinum Gelatin is purified protein obtained either by partial acid hydrolysis (type A), partial alkaline hydrolysis (type B) or enzymatic hydrolysis of collagen from animals (including fish and poultry); it may also be a mixture of different types. The hydrolysis leads to gelling or non-gelling product grades. Both product grades are covered by this monograph. Gelatin described in this monograph is not suitable for parenteral administration or for other special purposes.

Characters Faintly yellow or light yellowish-brown, solid, usually occurring as translucent sheets, shreds, granules or powder. Solubility Practically insoluble in common organic solvents; gelling grades swell in cold water and give on heating a colloidal solution which on cooling forms a more or less firm gel. The isoelectric point is a relevant quality parameter for use of gelatin in different applications: for type A gelatin it is 433

GELATIN

typically between pH 6.0 and pH 9.5 and for type B gelatin is typically between pH 4.7 and pH 5.6. These ranges cover a variety of different gelatins and for specific applications a narrower tolerance is usually applied. Different gelatins form aqueous solutions that vary in clarity and colour. For a particular application, a suitable specification for clarity and colour is usually applied.

Identification A. Solution S: Dissolve 1.00 g of the substance to be examined in carbon dioxide-free water R at about 55 °C, dilute to 100 ml with the same solvent and keep the solution at this temperature to carry out the tests. Add 0.05 ml of 12.5% solution of copper sulfate R to 2 ml of solution S. Mix and add 0.5 ml of dilute sodium hydroxide solution R. A violet colour is produced. B. To 0.5 g in a test-tube add 10 ml of water. Allow to stand for 10 min, heat at 60 °C for 15 min and keep the tube upright at 0 °C for 6 h. Invert the tube; the contents immediately flow out for non-gelling grades and do not flow out immediately for gelling grades. pH 3.8 to 7.6 (Appendix 6.2). Determined on solution S. Conductivity Maximum 1 mS·cm-1, determined on a 1.0% solution at 30 °C ± 1.0 °C. Sulfur dioxide Not more than 50 ppm (Appendix 7.9, method 2). Peroxides Not more than 10 ppm, determined using peroxide test strips R. Peroxidase transfers oxygen from peroxides to an organic redox indicator which is converted to a blue oxidation product. The intensity of the colour obtained is proportional to the quantity of peroxide and can be compared with a colour scale provided with the test strips, to determine the peroxide concentration. Suitability test: Dip a test strip for 1 s into hydrogen peroxide standard solution (10 ppm H2O2) R, such that the reaction zone is properly wetted. Remove the test strip, shake off excess liquid and compare the reaction zone after 15 s with the colour scale provided with the test strips used. The colour must match that of the 10 ppm concentration, otherwise the test is invalid. Test: Weigh 20.0 g ± 0.1 g of the substance to be tested in a beaker and add 80.0 ± 0.2 ml of water. Stir to moisten all gelatin and allow the sample to stand at room temperature for 1-3 h. Cover the beaker with a watch-glass. Place the beaker for 20 ± 5 min in a water bath at 65 ± 2 °C to dissolve the sample. Stir the contents of the beaker with a glass rod to achieve a homogeneous solution. Dip a test strip for 1 s into the test solution, such that the reaction 434

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zone is properly wetted. Remove the test strip, shake off excess liquid and compare the reaction zone after 15 s with the colour scale provided with the test strips used. Multiply the concentration read from the colour scale by a factor of 5 to calculate the concentration in parts per million of peroxide in the test substance.

Gel strength (Bloom value) 80% to 120% of the labelled nominal value. The gel strength is expressed as the mass in grams necessary to produce the force which, applied to a plunger 12.7 mm in diameter, makes a depression 4 mm deep in a gel having a concentration of 6.67% m/m and matured at 10 °C. Apparatus: Texture analyser or gelometer with: A cylindrical piston 12.7 ± 0.1 mm in diameter with a plane pressure surface with a sharp bottom edge, A bottle 59 ± 1 mm in internal diameter and 85 mm high. Adjust the apparatus according to the manufacturer's manual. Settings are: distance 4 mm, test speed 0.5 mm/s. Method: Perform the test in duplicate. Place 7.5 g of the substance to be tested in each bottle. Add 105 ml of water, place a watch-glass over each bottle and allow to stand for 1 h to 4 h. Heat in a water-bath at 65 °C ± 2 °C for 15 min. While heating, gently stir with a glass rod. Ensure that the solution is uniform and that any condensed water on the inner walls of the bottle is incorporated. Allow to cool at room temperature for 15 min and transfer the bottles to a thermostatically controlled bath at 10.0 °C ± 0.1 °C, and fitted with a device to ensure that the platform on which the bottles stand is perfectly horizontal. Close the bottles with a rubber stopper and allow to stand for 16 h to 18 h. Remove the sample bottles from the bath and quickly wipe the water from the exterior of the bottle. Centre consecutively the 2 bottles on the platform of the apparatus so that the plunger contacts the sample as nearly at its midpoint as possible and start the measurement. Report the result as the average of the 2 measurements. Iron Not more than 30 ppm. Examine by Atomic absorption spectrometry (Appendix 4.4). Test solution: To 5.00 g of the substance to be examined, in a conical flask, add 10 ml of hydrochloric acid R. Close the flask and place in a water-bath at 75 °C to 80 °C for 2 h. Allow to cool and adjust the content of the flask to 100.0 g with water. Reference solutions: Prepare the reference solutions using iron standard solution (8 ppm Fe) R, diluted as necessary with water. Wavelength: 248.3 nm. Chromium Not more than 10 ppm. Examine by Atomic absorption spectrometry (Appendix 4.4).

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HARD GELATIN CAPSULE SHELLS

Test solution: Test solution described in the test for Iron. Reference solutions: Prepare the reference solutions using chromium standard solution (100 ppm Cr) R, diluted if necessary with water. Wavelength: 357.9 nm.

is rounded and closed and the other is open. They are uncoloured or coloured, if coloured, of identical or different colours; transparent or opaque, partially or completely; printed or unprinted or bear other surface markings. The cap overlaps the body and maintains a tight friction closure.

Zinc Not more than 30 ppm. Examine by Atomic absorption spectrometry (Appendix 4.4). Test solution: Test solution described in the test for Iron. Reference solutions: Prepare the reference solutions using zinc standard solution (10 ppm Zn) R, diluted if necessary with water. Wavelength: 213.9 nm.

Characteristic The shells are smooth and uniform in size, shape and colour.

Loss on drying Not more than 15.0% (Appendix 9.6). (5.000 g; 105 °C, 16 h). Microbial contamination Total aerobic microbial count is not more than 103 CFU/g. Total combined yeasts/moulds count is not more than 102 CFU/g (Appendix 13.6). Absence of Escherichia coli, Salmonella (Appendix13.6). Storage Protect from heat and moisture. Labelling The label states the gel strength (Bloom value) or that it is a non-gelling grade. HARD GELATIN CAPSULE SHELLS Hard gelatin capsule shells are soluble containers to contain drug substances, ensuring the dosage units and protecting the drug substances. They are considered as a component of the dosage form. The contents in hard capsules are usually in solid form (powder, granules, pellets…) and are commonly intended for oral administration. The shells disintegrate in the presence of digestive fluids and then the filled contents are released. The capsule shells may be prepared by a special process designed to release the drug substances in the intestine.  The main components of the gelatin capsule shells are gelatin and water. They are also composed of other additives such as plasticizers, opacifying agents, colouring agents, antimicrobial agents…Each component of the capsule shells should meet the compendial requirements and/or in-house specifications, and should not interact with drug substances or excipients filled in the capsules. 

Appearance Hard gelatin capsule shells consist of two prefabricated cylindrical sections (cap and body), one end of which

Odour Transfer 100 capsule shells into a stoppered bottle, allow to stand for 24 hours at a temperature between 30 °C - 40 °C. No foreign odour is perceptible. Dimensions The dimensions of hard gelatin capsule shells may vary depending on humidity, storage conditions and environmental exposure. The composition of the shells also, in some extent, influences on the dimensions of which exposed to heat and moisture. The conventional dimensions (outside diameter, length and double wall thickness) of the capsule shells of sizes 0 to 4 are provided in the table 1, 2 và 3 for the guidance of users. It should be noted that any measurement of reasonable accuracy can be made only under controlled conditions of temperature and humidity. A temperature between 20 °C and 25 °C and a relative humidity between 45% and 55% are recommended. Table 1 - External diameter Size

Cap (mm)

Body (mm)

0

7.57 - 7.69

7.26 - 7.38

1

6.85 - 6.97

6.56 - 6.68

2

6.28 - 6.40

6.01 - 6.13

3

5.75 - 5.87

5.50 - 5.62

4

5.25 - 5.37

5.00 - 5.12

Note: Measure at a position 3 mm from the cutting end of the shell.

Table 2 - Length Size

Cap (mm)

Body (mm)

0

10.68 - 11.68

18.22 - 19.22

1

9.51 - 10.51

16.22 - 17.22

2

8.67 - 9.67

14.84 - 15.84

3

7.73 - 8.73

12.98 - 13.98

4

6.97 - 7.97

11.84 - 12.84

Table 3 - Double wall thickness Size

Cap (mm)

Body (mm)

0

0.187 - 0.223

0.177 - 0.213

1

0.182 - 0.218

0.175 - 0.211

2

0.180 - 0.216

0.173 - 0.209

3

0.178 - 0.214

0.170 - 0.206

4

0.176 - 0.212

0.164 - 0.200

Note: Measure at a position 3 mm from the cutting end of the shell.

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HARD GELATIN CAPSULE SHELLS

Average weight Capsule shells are of various sizes as specified in Table 4, ranging from 0 to 4, which are the common sizes available. Requirements for other sizes may be decided upon mutually between the manufacturer of the capsule shells and the user. Procedure: Weigh individually 100 capsules, calculate the average mass. The requirements are shown in Table 4. Table 4 - Average weight Size

Target weight (mg)

Tolerance (%)

0

96

± 10

1

76

± 10

2

63

± 10

3

50

± 10

4

40

± 10

Note: In order to ensure that the quality of the shells is not affected by temperature and humidity; the capsule shells should be conditioned at a temperature of 25° ± 2° and a relative humidity of 50 ± 5% for not less than 12 hours before conducting the test for “Average weight”.

Identification Boil 1 capsule shell with 20 ml water, allow to cool and centrifuge. Take the supernatant liquid (solution A) to carry out the following tests: A. To 5 ml of solution A, add 1 ml of saturated picric acid solution R, a light yellow precipitate is produced. B. To 5 ml of solution A, add 1 ml of a 5% solution of tannin R. A precipitate is formed. pH (Appendix 6.2) Solution S: Dissolve 1.0 g of the capsule shells in carbon dioxide-free water R at about 55 °C, dilute to 100 ml with the same solvent and keep the solution at this temperature to carry out the test. pH of solution S is from 3.8 to 7.6. Disintegration (Appendix 11.6) Not more than 15 min, using discs. Sulfur dioxide Not more than 200 ppm Test solution: Transfer 5.0 g of the capsule shell into a round-bottomed flask with a long neck and dissolve in 150 ml of hot water. Add 5 ml of phosphoric acid R and 1 g of sodium bicarbonate R. Then immediately attach the flask with a reflux condenser (Note: a few drops of suitable anti-foaming agents such as silicon oil may be added to avoid foaming). Distil, collect 50 ml of the distillate into below the surface of 15 ml of 0.1 N iodine R. Dilute the resulting solution to 100.0 ml with water. Evaporate 50 ml of the solution on a water-bath with occasionally adding water and continue evaporating until the solution should be almost colorless. Dilute this colorless solution to 40 ml with water. Neutralise with hydrochloric acid R, filter if necessary. Transfer the solution into a Nessler cylinder, add 2 ml of dilute hydrochloric acid R. 436

Reference solution: Dilute 10 ml of sulfate standard solution (1000 ppm SO4) R to 100.0 ml with water. Transfer 7.5 ml of the resulting solution into a Nessler cylinder and dilute to 40 ml with water, add 2 ml of diluted hydrochloric acid R. Mix. Procedure: To each cylinder containing test solution and reference solution, add 5 ml of a 25% solution of barium chloride R, dilute to 50.0 ml with water. Allow to stand for 10 min. Examine the solutions by viewing vertically against a black background. Any opalescence in the test solution is not more intense than that in the reference solution.

Arsenic (Appendix 9.4.2). Not more than 2 ppm. Weigh accurately about 5.0 g of the capsule shell, add 10 ml of water, allow to stand for 1 hour. Warm gently to dissolve, add 10 ml of hydrochloric acid R and a slight excess of bromine solution R. Add 2 ml of stannous hydrochloric acid R, boil under a reflux condenser for 1 hour. Allow to cool. Use water to transfer the resulting solution into a 100 ml flask. Dilute to volume with water and mix. 10 ml of this solution complies with limit test A for Arsenic. Residue on ignition Not more than 7.0%. Weigh accurately about 5.0 g of the capsule shell, add 1.5 g to 2.0 g of parafin oil (to avoid loss of the sample due to swelling) and heat untill no more fumes are produced. Ignite at 550 oC to a constant mass. Heavy metal (Appendix 9.4.8) Not more than 50 ppm To the residue obtained in the test for “Residue on ignition”, add 2 ml of hydrochloric acid R and 0.5 ml of nitric acid R. Evaporate to dryness on a water-bath. Add 1 ml of 1 N hydrochloric acid R and 15 ml of water to the obtained residue after evaporation. Warm gently in several minutes. Filter and wash with sufficient water so that the combination of the filtrate and washings obtains 100.0 ml. Dilute 20.0 ml of this solution to 25.0 ml with water. 12 ml of the solution complies with the limits for method 1. Prepare the reference solution using lead standard solution (2 ppm Pb) R. Loss on drying 12.5% to 16.0% (Appendix 9.6). (1.000 g, 105 °C). Microbial contamination It complies with the limit for Microbial contamination (Appendix 13.6, using the plate-count method). Storage Store protected from moisture and at a temperature not exceeding 30 °C.

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GENTAMICIN SULFATE Gentamicini sulfas

Gentamicin sulfate is a mixture of the sulfates of antimicrobial substances produced by Micromonospora purpurea. It contents not less than 590 µg/mg of gentamicin, calculated with reference to the dried substance.

Characters White or almost white powder. Freely soluble in water, practically insoluble in ethanol (96%). Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of gentamicin sulfate RS. B. It gives reaction A of sulfates (Appendix 8.1). pH 3.5 to 5.5 (Appendix 6.2). Dissolve 1.0 g in carbon dioxide-free water R and dilute to 25.0 ml with the same solvent. Specific optical rotation +107° to +121°, calculated with reference to the dried substance (Appendix 6.4). Determined on a 10 mg/ml solution of the substance to be examined in water. Composition Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 5.5 g of sodium heptanesulfonate R in a mixture of 50 ml of glacial acetic acid R, 250 ml of water and 700 ml of methanol R. Test solution: Dissolve 0.10 g of the substance to be examined in water and dilute to 100.0 ml with the same solvent. To 10.0 ml of this solution add 5 ml of methanol R and 4 ml of phthalaldehyde reagent R. Mix and dilute to

GENTAMICIN SULFATE

25.0 ml with methanol R. Heat in a water-bath at 60 °C for 15 minutes and cool to room temperature. If the solution is not used immediately, cool to 0°C and use within 4 hours. Reference solution: Prepare as prescribed for the test solution using 0.10 g of gentamicin sulfate RS instead of the substance to be examined. Chromatographic system: A column (10 cm to 12.5 cm × 4.6 mm to 5 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 330 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: Elution order: gentamicin C1, gentamicin C1a, gentamicin C2a, gentamicin C2. System suitability: In the chromatogram obtained with reference solution, the resolution between any two peaks is at least 1.25. The capacity factor of the peak due to gentamicin C1 is 2 to 7. The column efficiency determined from the gentamicin C2 peak is not less than 1200 theoretical plates. The relative standard deviation for 6 replicate injections of the reference solution is not more than 2.0%. Calculate contents of gentamicin C1, gentamicin C1a, gentamicin C2a, gentamicin C2 from the expression: 100 rf/rs Where: rf peak area response corresponding to the particular gentamicin in the test solution. rs sum of the area responses of four gentamicin peaks. Limits: Gentamicin C1: 25.0% to 50.0%. Gentamicin C1a: 10.0% to 35.0%. Gentamicin C2 and gentamicin C2a: 25.0% to 55.0%.

Methanol Not more than 1.0% Examine by gas chromatography (Appendix 5.2). Internal standard solution:  A 0.50% v/v solution of n-propanol R in water. Reference solution:  A 0.25% v/v solution of each of methanol R and n-propanol R in water. Control solution:  Dissolve 0.50 g of the substance to be examined in 2.0 ml of water. Test solution:  Dissolve 0.50 g of the substance to be examined in 1.0 ml of the internal standard solution and add 1.0 ml of water, mix well. Chromatographic system: A column (1.5 m × 4 mm) packed with copolymer ethylvinylbenzene-divinylbenzene, with a specific surface area of 500 m2/g to 600 m2/g, a pore size of 0.0075 µm. Carrier gas: Helium for chromatography. Flow rate: 30 to 40 ml/min. 437

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GENTAMICIN EYE DROPS

Temperature: Column:  Constant temperature between 120 °C and 140 °C. Injector and detector: Constant temperature at least 50 °C higher than the column temperature. Detector: A flame-ionisation detector. Volume of injection: 2 µl. Procedure: Inject the reference solution, the resolution between the peaks due to n-propanol and methanol is at least 1.0. Chromatograph the control solution, and examine the chromatogram: if any peak is observed at a retention time corresponding to that of n-propanol, use the response of that peak to correct the n-propanol peak response of the test solution. Inject the reference solution and the test solution. Calculate the percentage of methanol from the expression: (RU/RS) × (CS/CU) × D × F In which: RU: Peak area response of methanol to n-propyl alcohol (corrected for any peak at the locus of the n-propanol peak in the control solution) from the test solution. RS: Peak area response of methanol to n-propanol ol from the standard solution. CS: Percentage of methanol in the standard solution. CU: Concentration of Gentamicin sulfate in the sample solution (mg/ml). D: Density of methanol (g/ml). F: Conversion factor, 1000 mg/g.

Loss on drying Not more than 18.0% (Appendix 9.6). (1.0 g; 110 °C; in vacuo at a pressure not exceeding 5 mmHg, 3 h). Sulfated ash Not more than 1.0% (Appendix 9.9, method 2). Determine on 0.50 g. Bacterial endotoxins Less than 0.71 EU/mg (Appendix 13.2). If intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins. Sterility If intended for use in the manufacture of parenteral or ophthalmic dosage forms without a further appropriate sterilisation procedure, it complies with the test for sterility (Appendix 13.7). Assay Carry out the microbiological assay of antibiotics (Appendix 13.9). Storage Store in an airtight container. If the substance is sterile, store in a sterile, airtight, tamper-proof container. 438

Labelling The label states: where applicable, that the substance is sterile and free from bacterial endotoxins. Action and use Aminoglycoside antibacterial. Preparations Cream, eye drops, injection, ointment. GENTAMICIN EYE DROPS Collyrium Gentamicini Gentamicin eye drops are a sterile solution of gentamicin sulfate in purified water. They may contain suitable excipients. The eye drops comply with the requirements stated under “Eye Preparations” (Appendix 1.14) and with the following requirements.

Content of gentamicin, 90.0% to 120.0% of the stated amount. Characters A clear, colourless solution. pH 6.5 to 7.5 (Appendix 6.2). Identification A. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel G. Mobile phase: The lower layer obtained by shaking together equal volumes of 13.5 M ammonia R, chloroform R and methanol R and allowing to separate. Test solution: Dilute a volume of the eye drops with water to obtain a solution having a known concentration of about 1 mg of gentamicin sulfate per ml. Reference solution: Dissolve a quantity of gentamicin sulfate RS in water to obtain a solution having a known concentration of about 1 mg per ml. Procedure: Apply separately to the plate 20 µl of each solution. Allow the drops to dry in air. Develop over a path of 15 cm. After removal of the plate, allow it to dry in air. Expose the plate in iodine vapour or spray the plate with a 0.3% solution of ninhydrin in ethanol R and dry it at 105 °C for 5 minutes. The three principal spots in the chromatogram obtained with the test solution correspond to the three principal spots in the chromatogram obtained with the reference solution. B. In the test for Composition of gentamicin sulfate, the retention times of the four principal peaks in the chromatogram obtained with the test solution correspond to those of the four principal peaks in the chromatogram obtained with the reference solution.

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Composition of gentamicin sulfate Examine by liquid chromatography (Appendix 5.3). Mobile phase: A 0.025 M solution of sodium heptanesulfonate monohydrate R in a mixture of methanol R, water and glacial acetic acid R (70 : 25 : 5), filter through a 0.45 µm filter. Reference solution: Add 5 ml of methanol R to 10 ml of a 0.065% solution of gentamicin sulfate RS, swirl and add 4 ml of phthalaldehyde reagent R, mix, add sufficient methanol R to produce 25.0 ml, heat in a water bath at 60 °C for 15 minutes and cool. If the solution is not used immediately, store at 0 °C and use within 4 hours. Test solution: Prepare in the same manner as the reference solution, but using 10 ml of a solution prepared by diluting a suitable volume of the eye drops with water to contain the equivalent of 0.045% of gentamicin. Chromatographic system: A column (10 cm to 12.5 cm × 4.6 mm to 5.0 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 330 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: System suitability: When the chromatograms obtained with the reference solution are recorded under the prescribed conditions the retention time of component C2 is 10 to 20 minutes. The retention times relative to component C2 are: about 0.13 (reagent); about 0.27 (component C1); about 0.65 (component C1a); about 0.85 (component C2a). Adjust the sensitivity so that the height of the peak due to component C1 is about 75% of full-scale deflection. Plot a horizontal baseline on the chromatogram from the level portion of the curve immediately prior to the reagent peak. Measure the peak height above this baseline for each component. Repeat the procedure with the test solution. The test is not valid unless, in the chromatogram obtained, the resolution between the peaks due to components C2a and C2 is at least 1.3. From the peak heights in the chromatogram obtained with the test solution and the reference solution, and the known proportions of equivalent components in the reference solution, calculate the percentage content of components C1, C1a, C2 and C2a in the eye drops. The proportions are within the following limits: Gentamicin C1: 25.0% to 50.0%. Gentamicin C1a: 10.0% to 35.0%. Gentamicin C2 + C2a: 25.0% to 55.0%. Assay Carry out the microbiological assay of antibiotics (Appendix 13.9). Calculate the content of gentamicin in the eye drops, taking each 1000 IU found to be equivalent to 1 mg of gentamicin.

GENTAMICIN INJECTION

Storage Store in cool place, protected from light. Action and use Antibacterial. Usual strength 0.3%. GENTAMICIN INJECTION Injectio Gentamicini Gentamicin injection is a sterile solution of gentamicin sulphate in water for injections. The injection complies with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Content of gentamicin, 95.0% to 110.0% of the stated amount. Characters A clear, colourless solution. pH 3.0 to 5.5 (Appendix 6.2). Identification A. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel G. Mobile phase: The lower layer obtained by shaking together equal volumes of 13.5 M ammonia R, chloroform R and methanol R and allowing to separate. Test solution: Dilute a volume of the injection with water to obtain a solution having a known concentration of about 1 mg of gentamicin sulphate per ml. Reference solution: Dissolve 10 mg of gentamicin sulphate RS in 10 ml of water, and mix. Procedure: Apply separately to the plate 20 ml of each of the solutions. Allow the drops to dry in air. Develop over a path of 15 cm. After removal of the plate, allow it to dry in air. Expose the plate in iodine vapour or spray the plate with a 0.3% solution of ninhydrin in ethanol R and dry it at 105 °C for 5 minutes. The three principal spots in the chromatogram obtained with the test solution correspond to the three principal spots in the chromatogram obtained with the reference solution. B. In the test for Composition of gentamicin sulphate, the retention times of the four principal peaks in the chromatogram obtained with the test solution correspond to those of the four principal peaks in the chromatogram obtained with the reference solution. Composition of gentamicin sulphate Examine by liquid chromatography (Appendix 5.3). 439

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GLIBENCLAMIDE

Mobile phase: A 0.025 M solution of sodium heptanesulphonate monohydrate R in a mixture of methanol R, water and glacial acetic acid R (70 : 25 : 5), filter through a 0.45 µm filter. Reference solution: Add 5 ml of methanol R to 10 ml of a 0.065% solution of gentamicin sulphate RS, swirl and add 4 ml of phthalaldehyde reagent R, mix, add sufficient methanol to produce 25.0 ml, heat in a water bath at 60 °C for 15 minutes and cool. If the solution is not used immediately, cool to 0 °C and use within 4 hours. Test solution: Prepare in the same manner as the reference solution, but using 10 ml of a solution prepared by diluting a suitable volume of the injection with water to contain the equivalent of 0.045% of gentamicin. Chromatographic system: A column (10 cm to 12.5 cm × 4.6 mm to 5.0 mm) packed with stationary phase C (5 µm) (Hypersil ODS is suitable). Detector: A spectrophotometer set at 330 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: System suitability: When the chromatograms obtained with the reference solution are recorded under the prescribed conditions the retention time of component C2 is 10 to 20 minutes. The retention times relative to component C2 are: about 0.13 (reagent); about 0.27 (component C1); about 0.65 (component C1a); about 0.85 (component C2a). Adjust the sensitivity so that the height of the peak due to component C1 is about 75% of full-scale deflection. Plot a horizontal baseline on the chromatogram from the level portion of the curve immediately prior to the reagent peak. Measure the peak height above this baseline for each component. Repeat the procedure with the test solution. The test is not valid unless, in the chromatogram obtained, the resolution between the peaks due to components C2a and C2 is at least 1.3. From the peak heights in the chromatogram obtained with the test solution and the reference solution, and the known proportions of equivalent components in the reference solution, calculate the percentage content of components C1, C1a, C2 and C2a in the eye drops. The proportions are within the following limits: Gentamicin C1: 25.0 to 50.0% Gentamicin C1a: 10.0 to 35.0% Gentamicin C2 + C2a: 25.0 to 55.0%.

Bacterial endotoxins Carry out the test for bacterial endotoxins (Appendix 13.2). Dilute the injection, if necessary, with water BET to give a solution containing the equivalent of 10 mg of gentamicin per ml (solution A). The endotoxin limit concentration of solution A is 16.7 IU per ml. Carry out the test using the maximum valid dilution of solution A calculated from the declared sensitivity of the lysate used in the test. 440

Assay Carry out the microbiological assay of antibiotics (Appendix 13.9). Calculate the content of gentamicin in the eye drops, taking each 1000 IU found to be equivalent to 1 mg of gentamicin. Storage Store in a cool place, protected from light. Action and use Antibacterial. Usual strength 80 mg/2 ml, 40 mg/1 ml and 40 mg/2 ml. GLIBENCLAMIDE Glibenclamidum

C23H28ClN3O5S



M. 494.0

Glibenclamide is 1-[[4-[2-[(5-chloro-2-methoxybenzoyl) amino]ethyl]phenyl]sulfonyl]-3-cyclohexylurea. It contains not less than 99.0% and not more than 101.0% of C23H28ClN3O5S, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder. Practically insoluble in water, sparingly soluble in methylene chloride, slightly soluble in ethanol (96%) and in methanol. Identification Apply one of the two following identifications: First identification: A, B. Second identification: B, C, D, E. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum obtained with glibenclamide RS. If the spectra obtained show differences, moisten separately the substance to be examined and the reference substance with methanol R, triturate, dry at 100 °C to 105 °C and record the spectra again. B. Melting point: 169 °C to 174 °C (Appendix 6.7). C. Dissolve 50.0 mg in methanol R, with the aid of ultrasound if necessary, and dilute to 50.0 ml with the same solvent. To 10.0 ml of the solution add 1.0 ml of a 1 M hydrochloric acid R and dilute to 100.0 ml with methanol

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R. Examined between 230 nm and 350 nm (Appendix 4.1), the solution shows absorption maxima at 300 nm and at 275 nm. The A (1%, 1 cm) at the maxima are 61 to 65 and 27 to 32, respectively. D. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Ethanol (96%) - glacial acetic acid cyclohexane - methylene chloride (5 : 5 : 45 : 45). Test solution: Dissolve 10 mg of the substance to be examined in a mixture of equal volumes of methanol R and methylene chloride R and dilute to 10 ml with the same mixture of solvents. Reference solution: Dissolve 10 mg of glibenclamide RS in a mixture of equal volumes of methanol R and methylene chloride R and dilute to 10 ml with the same mixture of solvents. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 10 cm. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. E. Dissolve 20 mg in 2 ml of sulfuric acid R. The solution is colourless and shows blue fluorescence in ultraviolet light at 365 nm. Dissolve 0.1 g of chloral hydrate R in the solution. Within about 5 min, the colour changes to deep yellow and, after about 20 min, develops a brownish tinge.

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Mix 20 ml of a 10.18% solution of freshly distilled triethylamine R adjusted to pH 3.0 using phosphoric acid R, and 50 ml of acetonitrile R; dilute to 1000 ml with water. Mobile phase B: Mobile phase A - water - acetonitrile (20 : 65 : 915). Test solution: Dissolve 25.0 mg of the substance to be examined in methanol R and dilute to 10.0 ml with the same solvent. Prepare immediately before use. Reference solution (1): Dissolve 5.0 mg of glibenclamide impurity A RS and 5.0 mg of glibenclamide impurity B RS in methanol R and dilute to 100.0 ml with the same solvent. Dilute 5.0 ml of the solution to 20.0 ml with methanol R. Reference solution (2): Dilute 2.0 ml of the test solution to 100.0 ml with methanol R. Dilute 5.0 ml of this solution to 50.0 ml with methanol R. Reference solution (3): Dissolve 5 mg of gliclazide RS in methanol R, add 2 ml of the test solution and dilute to 100 ml with methanol R. Dilute 1 ml of this solution to 10 ml with methanol R. Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase C (3 µm). Temperature of the column: 35 °C. Detector: A spectrophotometer set at 230 nm.

GLIBENCLAMIDE

Flow rate: 0.8 ml/min. Volume of injection: 10 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 15

45

55

15 - 30

45 → 5

55 → 95

30 - 40

5

95

40 - 41

5 → 45

95 → 55

41 - 55

45

55

Relative retention times with reference to glibenclamide (retention time = about 5 min) are: impurity A = about 0.5, impurity B = about 0.6. System suitability: In the chromatogram obtained with the reference solution (3), the resolution between the peaks corresponding to glibenclamide and gliclazide is at least 5.0. Limits: In the chromatogram obtained with the test solution: The area of any peak corresponding to impurity A is not greater than that of the corresponding peak in the chromatogram obtained with reference solution (1) (0.5%). The area of any peak corresponding to impurity B is not greater than that of the corresponding peak in the chromatogram obtained with reference solution (1) (0.5%). The area of any other impurity peak is not greater than that of the principal peak in the chromatogram obtained with reference solution (2) (0.2%) and not more than 2 such peaks have an area greater than half the area of the principal peak in the chromatogram obtained with reference solution (2) (0.1%). The sum of the areas of all other impurity peaks, apart from the peaks corresponding to impurities A and B, is not greater than 2.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.5%). Disregard any peak with an area less than 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Note: Impurity A: 5-chloro-2-methoxy-N-[2-(4-sulfamoylphenyl) ethyl] benzamide. Impurity B: Methyl [[4-[2-[(5-chloro-2-methoxybenzoyl) amino] ethyl]phenyl]sulfonyl] carbamate.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 4. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 1.0% (Appendix 9.6). (1.000 g; 100 °C to 105 °C). 441

GLIBENCLAMIDE TABLETS

Sulfated ash Not more than 0.1% (Appendix 9.9). Determined on 1.0 g. Assay Dissolve 0.400 g with heating in 100 ml of ethanol (96%) R. Titrate with 0.1 N sodium hydroxide VS, using 1.0 ml of phenolphthalein solution R as indicator, until a pink colour is obtained. Each ml of 0.1 N sodium hydroxide VS is equivalent to 49.40 mg of C23H28ClN3O5S. Storage Store in an airtight container. Action and use Hypoglycaemic. Preparation Tablets. GLIBENCLAMIDE TABLETS Tabellae Glibenclamidi Glibenclamide tablets contain glibenclamide. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of glibenclamide, C23H28ClN3O5S, 95.0% to 105.0% of the stated amount. Identification A. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the principal peak in the chromatogram obtained with the reference solution. B. In the test for Related substances, the principal spot in the chromatogram obtained with the test solution corresponds in position and size to that in the chromatogram obtained with reference solution (2). Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Chloroform - cyclohexane - ethanol (96%) - glacial acetic acid (45 : 45 : 5 : 5). Test solution: Weigh a quantity of the powdered tablets containing the equivalent of about 20 mg of glibenclamide, extract with four quantities, each of 5 ml, of a mixture of dichloromethane - acetone (2 : 1). Evaporate the combined extracts to dryness at a temperature not exceeding 40 °C at a pressure of 2 kPa. Dissolve the residue in 4 ml of a mixture of equal volumes of chloroform R and methanol R. Reference solution (1): A 0.01% solution of glibenclamide RS in a mixture of equal volumes of chloroform R and methanol R. 442

VP V

Reference solution (2): A 0.5% solution of glibenclamide RS in a mixture of equal volumes of chloroform R and methanol R. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm. After removal of the plate, allow it to dry in air and examine under ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (1).

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of a solution containing 0.8134% of anhydrous disodium hydrogen phosphate R and 0.1350% of potassium dihydrogenphosphate R. Rotation speed: 100 rpm. Time: 45 min. Procedure: Examine by liquid chromatography (Appendix 5.3). Mobile phase: 0.1 M potassium dihydrogen phosphate adjusted to pH 3.0 with phosphoric acid - acetonitrile (40 : 60). Test solution: After the specified time, withdraw a sample of the medium and filter. Reference solution: Dissolve glibenclamide RS in a minimum volume of methanol R and dilute with the dissolution medium to obtain a solution having the same concentration as the test solution. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm) (Spherisorb ODS is suitable). Detector: A spectrophotometer set at 225 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Inject alternately the test solution and the reference solution. Calculate the content of glibenclamide, C23H28ClN3O5S, dissolved using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C23H28ClN3O5S in glibenclamide RS. Tolerance: Not less than 70% (Q) of the stated amount of glibenclamide, C23H28ClN3O5S, is dissolved in 45 min. Uniformity of content Tablets containing 5 mg or less of glibenclamide comply with the requirements stated under the test for Uniformity of content (Appendix 11.2). Test solution: Powder one tablet finely, add a mixture of 2.0 ml of water and 20.0 ml of methanol R, mix with the aid of ultrasound until fully dispersed. Filter through a 0.2-µm membrane filter (Anatop LC is suitable). Reference solution: Add 2.0 ml of water to 20.0 ml of a 0.025% solution of glibenclamide RS in methanol R, mix with the aid of ultrasound until fully dispersed and filter (0.2-µm Anatop LC membrane is suitable).

VP V

Examine by liquid chromatography (Appendix 5.3). Use the chromatographic system and procedure as directed in the Assay.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - 1.36% solution of potassium dihydrogen phosphate adjusted to pH 3.0 with phosphoric acid (47 : 53). Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Weigh accurately a quantity of powdered tablets containing the equivalent of about 5 mg of glibenclamide, add a mixture of 2.0 ml of water and 20.0 ml of methanol R, mix with the aid of ultrasound until fully dispersed. Filter through a 0.2-µm membrane filter (Anatop LC is suitable). Reference solution: Dissolve 50 mg of glibenclamide RS in 10 ml of methanol R with the aid of ultrasound for 20 minutes, add sufficient methanol R to produce 50.0 ml and dilute this solution 4-fold in methanol R. To 20.0 ml of the obtained solution add 2.0 ml of water, mix well and filter (0.2-µm Anatop LC membrane is suitable). Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase C (5 µm) (Spherisorb ODS 1 is suitable). Detector: A spectrophotometer set at 300 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Inject alternately the test solution and the reference solution. Calculate the content of glibenclamide, C23H28ClN3O5S, in the tablets using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C23H28ClN3O5S in glibenclamide RS. Storage Store at room temperature, in a dry place, protected from light. Action and use Treatment of diabetes mellitus. Usual strength 2.5 mg; 5 mg. GLIBENCLAMIDE AND METFORMIN TABLETS Tabellae Glibenclamidi et Metformini Glyburide and metformin tablets Glibenclamide and metformin tablets contain glibenclamide and metformin hydrochloride. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

GLIBENCLAMIDE AND METFORMIN TABLETS

Content of glibenclamide, C23H28ClN3O5,HCl, from 90.0% to 110.0% of the stated amount Content of metformin hydrochloride, C4H11N5O4,HCl, from 90.0% to 110.0% of the stated amount. Identification A. In the Assay for glibenclamide, the retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of the principal peak in the chromatogram obtained with the reference solution. B. In the Assay for metformin hydrochloride, the retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of the principal peak in the chromatogram obtained with the reference solution. Dissolution (Appendix 11.4) Glibenclamide Apparatus: Paddle. Medium: 500 ml of a solution of boric acid and 0.05 M potassium chloride prepared by dissolving 3.09 g of boric acid R and 3.73 g of potassium chloride R in about 250 ml of water, adjust to pH 9.5 with 1 M sodium hydroxide R, add sufficient water to produce 1000 ml. Rotation speed: 75 rpm. Time: 30 min. Procedure: Determine the content of glibenclamide dissolved by the liquid chromatography (Appendix 5.3). Mobile phase: Ammonium phosphate buffer - acetonitrile (50 : 50), adjust to pH 5.3 with 1 M sodium hydroxide R. Adjust the mobile phase if necessary. Ammonium phosphate buffer solution: Dissolve 28.8 g ammonium dihydrogen phosphate R in water and dilute to 1000 ml with the same solvent. Test solution: After the specified time, withdraw a sample of the medium, filter, discard the first portion of the filtrate. Filter again through a membrane filter (nominal pore size: 0.45 µm or 1 µm). Reference solution: Weigh accurately about 25 mg of glibenclamide RS, transfer into a 100-ml volumetric flask. Add 20 ml of acetonitrile R, sonicate to dissolve and dilute to volume with the medium, shake well. Dilute the solution with the medium to obtain a solution having a concentration similar to that expected in the test solution. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase B (5 µm). Column temperature: 30 °C. Detector: A spectrophotometer set at 230 nm. Flow rate: 1.5 ml/min. Volume of injection: 200 µl. Procedure: Inject the reference solution, in the chromatogram obtained, the column efficiency determined on the glibenclamide peak is not less than 5000 theoretical 443

GLIBENCLAMIDE AND METFORMIN TABLETS

plates, the tailing factor is 0.8 to 2.0 and the relative standard deviation of the areas of the principal peaks for six replicate injections is not more than 2.0%. Inject the test solution, calculate the content of glibenclamide, C23H28ClN3O5, dissolved using the peak areas in the chromatogram obtained with the reference solution, the test solution and the declared content of glibenclamide RS. Tolerance: Not less than 85% (Q) of the labeled amount of glibenclamide, C23H28ClN3O5, is dissolved in 30 min. Metformin hydrochloride Apparatus: Paddle. Medium: 1000 ml of 0.05 M phosphate buffer pH 6.8 prepared by dissolving 6.8 g of potassium phosphate monobasic R in 1000 ml of water, adjust to pH 6.8 ± 0.1 with 0.2 M sodium hydroxide R. Rotation speed: 50 rpm. Time: 30 min. Procedure: Test solution: After the specified time, withdraw a sample of the medium and filter, discard the first portion of filtrate. Filter again through a membrane filter (nominal pore size: 0.45 µm or 1 µm). Dilute the filtrate with the medium if necessary. Reference solution: Dissolve a quantity of metformin hydrochloride RS in the medium to obtain a solution having a known concentration similar to that of the test solution. Measure the absorbances (Appendix 4.1) of the solutions at the maximum at 232 nm, in a 1-cm cell using the medium as blank. Calculate the content of metformin hydrochloride, C4H11N5O4,HCl, dissolved, using the absorbances of the reference solution, the test solution and the declared content of metformin hydrochloride RS. Tolerance: Not less than 85% (Q) of the labeled amount of metformin hydrochloride, C4H11N5O4,HCl, is dissolved in 30 min.

Uniformity of content of glibenclamide (Appendix 11.2) Examine by liquid chromatography (Appendix 5.3). The diluent, ammonium phosphate buffer solution, reference solution, mobile phase and chromatographic system are described in the Assay of glibenclamide. Test solution: Transfer a tablet into a 100-ml volumetric flask, add 2 ml of water, shake to disintegrate completely, add 70 ml of diluent and sonicate for about 30 min. Allow to cool and dilute to volume with the diluent, shake well. Centrifuge at 3000 rpm for 10 min, use the clear supernatant. If necessary, dilute with the diluent to obtain a solution containing 0.025 mg of glibenclamide per ml. Repeat the procedure with further 9 tablets. Procedure: Inject alternatively the reference solution, the test solutions and record the chromatograms. Calculate the content of glibenclamide in each tablet using the peak areas in the chromatograms obtained with the reference solution, the test solution and the declared content of glibenclamide RS. 444

VP V

Assay Glibenclamide Examine by liquid chromatography (Appendix 5.3). Diluent: Acetonitrile - water (1 : 1). Ammonium phosphate buffer solution: Dissolve 28.8 g ammonium phosphate monobasic R in water and dilute to 1000 ml with the same solvent, mix well. Mobile phase: Ammonium phosphate buffer - acetonitrile (60 : 40), adjust the pH to 5.3 with 1 M sodium hydroxide R and make adjustments if necessary. Reference solution: Weigh accurately a quantity about 25 mg of glibenclamide RS, transfer into a 100-ml volumetric flask. Add 50 ml of acetonitrile R, sonicate to dissolve. Add sufficient of water and shake well. Dilute the solution with the diluent to obtain a solution having a known concentration of about 0.025 mg per ml. Test solution: Weigh 20 tablets (remove the coating if necessary), calculate the average mass and finely powder. Weigh accurately a quantity of the powdered tablets containing about 2.5 mg of glibenclamide and transfer into a 100-ml volumetric flask, add about 70 ml of diluent, sonicate for bout 30 min to dissolve. Add the diluent to volume and shake well. Centrifuge at 3000 rpm for 10 min, use the clear supernatant. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase B (5 µm). Column temperature: 40 ºC. Detector: A spectrophotometer set at 230 nm. Flow rate: 1.2 ml/min. Volume of injection: 100 µl. Procedure: In the chromatogram obtained with the reference solution, the relative standard deviation of the peak areas for 6 replicate injections is not more than 1.5%, the column efficiency determined on the glibenclamide peak is not less than 3000 theoretical plates. Inject the test solution, calculate the content of glibenclamide in the tablets using the peak areas in the chromatograms obtained with the reference solution, the test solution and the declared content of glibenclamide RS. Metformin hydrochloride Examine by liquid chromatography (Appendix 5.3). Diluent: A 2.5% solution of acetonitrile R (v/v) in water. Buffer solution: Dissolve 1.0 g of sodium heptanesulfonate R and 1.0 g of sodium chloride R in 1800 ml of water. Adjust the pH to 3.85 with 0.06 M phosphoric acid R. Add sufficient of water to produce 1000 ml, mix well. Mobile phase: Buffer solution - acetonitrile (9 : 1), adjust if necessary. Reference solution: Dissolve a quantity of metformin hydrochloride RS in the diluent to obtain a solution having a known concentration of about 0.05 mg/ml.

VP V

GLICLAZIDE

Test solution: Dilute the test solution in the assay of glibenclamide to obtain a solution having a concentration of about 0.05 mg of metformin hydrochloride per ml in water. Chromatographic system: A column (30 cm × 3.9 mm) packed with stationary phase C (5 µm). Column temperature: 30 °C. Detector: A spectrophotometer set at 218 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Procedure: In the chromatogram obtained with the reference solution, the tailing factor of the peak due to metformin hydrochloride is 0.8 to 2.0 and the relative standard deviation of the peak areas for 6 replicate injections is note more than 1.5%. Inject the test solution, calculate the content of metformin hydrochloride in tablets using the peak areas in the chromatograms obtained with the reference solution, the test solution and the declared content of metformin hydrochloride RS.

Storage Store in a cool place, protected from light. Action and use Treatment of diabetes mellitus. Usual strength Metformin hydrochloride 500 mg and glibenclamide 5 mg. Metformin hydrochloride 500 mg and glibenclamide 2.5 mg. GLICLAZIDE Gliclazidum

C15H21N3O3S

M. 323.4

Gliclazide is 1-(hexahydrocyclopenta[c]pyrrol-2(1H)yl)-3-[(4-methylphenyl)sulfonyl]urea. It contains not less than 99.0% and not more than 101.0% of C15H21N3O3S, calculated with reference to the dried substance.

Characters A white or almost white powder. Practically insoluble in water, freely soluble in methylene chloride, sparingly soluble in acetone, slightly soluble in ethanol (96%). Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum obtained with gliclazide RS.

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Triethylamine - trifluoroacetic acid acetonitrile - water (0.1 : 0.1 : 45 : 55). Prepare the solutions immediately before use. Test solution: Dissolve 50.0 mg of the substance to be examined in 23 ml of acetonitrile R and dilute to 50.0 ml with the same solvent. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with a mixture of acetonitrile - water (45 : 55). Dilute 10.0 ml of the solution to 100.0 ml with the same mixture of solvents. Resolution solution: Dissolve 5 mg of the substance to be examined and 15 mg of gliclazide impurity F RS in 23 ml of acetonitrile R and dilute to 50 ml with the same solvent. Dilute 1.0 ml of the solution to 20 ml with a mixture of acetonitrile - water (45 : 55). Reference solution (2): Dissolve 10.0 mg of gliclazide impurity F RS in 45 ml of acetonitrile R and dilute to 100.0 ml with water. Dilute 1.0 ml of the solution to 100.0 ml with a mixture of acetonitrile - water (45 : 55). Chromatographic system: A column (0.25 m × 4 mm) packed with stationary phase B (5 µm). Detector: A spectrophotometer set at 235 nm. Flow rate: 0.9 ml/min. Volume of injection: 20 µl. Procedure: The run time is twice the retention time of gliclazide. Inject the resolution solution. Adjust the sensitivity of the system so that the heights of the 2 principal peaks in the chromatogram obtained with the resolution solution are at least 50% of the full scale of the recorder. The test is not valid unless in the chromatogram obtained the resolution between the two principal peaks is at least 1.8. Limits: In the chromatogram obtained with the test solution: The area of any peak corresponding to impurity F is not greater than the area of the corresponding peak in the chromatogram obtained with reference solution (2) (0.1%). The area of any peaks, apart from the principal peak and the peak due to impurity F, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%). The sum of the areas of any such peaks is not greater than twice the area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%). Disregard any peak with an area less than 0.2 times that of the principal peak in the chromatogram obtained with reference solution (1). Note: Impurity E: 1-[(4-methylphenyl)sulfonyl]-3-(3,3a,4,6a-tetrahydrocyclopenta[c]pyrrol-2(1H)-yl)urea. Impurity F: 1-(hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-3-[(2methylphenyl)sulfonyl]urea.

445

VP V

GLICLAZIDE TABLETS

Impurity B Not more than 2 ppm. Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. Test solution: Dissolve 0.400 g in 2.5 ml of dimethyl sulfoxide R and dilute to 10.0 ml with water. Stir for 10 min, store at 4 °C for 30 min and filter. Reference solution (1): Dissolve 20.0 mg of gliclazide impurity B RS (2-nitroso-octahydrocyclopenta [c]pyrrole in dimethyl sulfoxide R and dilute to 100.0 ml with the same solvent. To 1.0 ml of the solution, add 12 ml of dimethyl sulfoxide R and dilute to 50.0 ml with water. Reference solution (2): To 1.0 ml of reference solution (1), add 12 ml of dimethyl sulfoxide R and dilute to 50.0 ml with water. Procedure: Inject 50 ml of the test solution and 50 µl of reference solution (2). In the chromatogram obtained with the test solution, the area of any peak corresponding to impurity B is not greater than the area of the corresponding peak in the chromatogram obtained with reference solution (2). Heavy metals Not more than 10 ppm (Appendix 9.4.8). 1.5 g complies with limit test for heavy metals, method 6. Prepare the standard using 1.5 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.25% (Appendix 9.6). (1.000 g; 100 °C to 105 °C; 2 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.250 g in 50 ml of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid VS, Determine the endpoint potentiometrically (Appendix 10.2). Each ml of 0.1 M perchloric acid VS is equivalent to 32.34 mg of C15H21N3O3S. Storage Store in an airtight container. Action and use Hypoglycaemic. Preparation Tablets.

446

GLICLAZIDE TABLETS Tabellae Gliclazidi Gliclazide tablets contain gliclazide. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of gliclazide, C15H21N3O3S, 93.0% to 107.0% of the stated amount. Identification Shake a quantity of the powdered tablets containing about 0.4 g of gliclazide with 10 ml of dichlromethane R, filter and evaporate the filtrate to dryness. The infrared absorption spectrum of the residue (Appendix 4.2) is concordant with the reference spectrum of gliclazide. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of phosphate buffer pH 7.4 R. Rotation speed: 100 rpm. Time: 45 min. Procedure: Test solution: After the specified time, withdraw a sample of the medium and filter. Dilute the filtrate, if necessary, with the dissolution medium to obtain a solution having a concentration of about 12.5 µg of gliclazide per ml. Reference solution: Dissolve 62.0 mg of gliclazide RS in 20 ml of methanol R, dilute to 100.0 ml with the dissolution medium and mix. Dilute 2.0 ml of the obtained solution to 100.0 ml with the dissolution medium and mix. Measure the absorbance (Appendix 4.1) of the test solution and the reference solution at the maximum wavelength at 226 nm and 290 nm in a 1-cm cell, using the dissolution medium as blank. Correct the absorbance obtained at 226 nm by subtracting the absorbance obtained at 290 nm. Calculate the content of gliclazide, C15H21N3O3S, dissolved using the corrected absorbances of the test solution, the reference solution and the declared content of C15H21N3O3S in gliclazide RS. Tolerance: Not less than 70% (Q) of the labeled amount of gliclazide, C15H21N3O3S, is dissolved in 45 min. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase, diluent and chromatographic system: Proceed as directed in the Assay. Prepare the solutions immediately before use. Test solution: Weigh accurately a quantity of the powdered tablets containing about 100 mg of gliclazide, transfer into a 100 ml volumetric flask, add 40 ml of acetonitrile R, shake to dissolve. Dilute to volume with the diluent and filter.

VP V

GLIMEPIRIDE

Reference solution: Transfer accurately 2.0 ml of the test solution into a 100-ml volumetric flask, add sufficient diluent to volume and mix. Dilute 5.0 ml of the filtrate to 50.0 ml with the diluent. Procedure: System suitability: Inject the reference solution. The number of theoretical plates is not less than 3000. The run time of the test solution is about twice the retention time of gliclazide. In the chromatogram obtained with the test solution, the area of any secondary peak is not greater than that of the principal peak in the chromatogram obtained with the reference solution (0.2%). The sum of the areas of all secondary peaks is not greater than twice the area of the principal peak in the chromatogram obtained with the reference solution (0.4%).

Action and use Treatment of diabetes mellitus.

Assay Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use. Mobile phase: Triethylamine - trifluoroacetic acid acetonitrile - water (0.1 : 0.1 : 40 : 60). Make adjustments if necessary. Diluent: Water - acetonitrile (60 : 40). Test solution: Weigh 20 tablets, calculate average mass and finely powder. Weigh accurately a quantity of the powder containing about 100 mg of gliclazide, transfer into a 50-ml volumetric flask, add 30 ml of the diluent, sonicate for 15 min. Dilute to volume with the diluent and filter. Dilute 5.0 ml of the filtrate to 50.0 ml with the diluent, mix. Reference solution: Dissolve an accurately weighed quantity of about 10 mg of gliclazide RS in 20 ml of acetonitrile R, dilute to 50.0 ml with the water. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase B (5 µm). Detector: A spectrophotometer set at 235 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution. The resolution factor between famotidine peak and the adjacent impurity peak (if any) is not less than 1.5, the relative standard deviation of peak areas due to gliclazide for replicate injections is not more than 2.0%, the number of theoretical plates is not less than 3000. Inject alternately the reference solution and the test solution. Calculate the content of gliclazide, C15H21N3O3S, in the tablets using the areas of the peaks in the chromatograms obtained with the test solution, the reference solution and the declared content of C15H21N3O3S in gliclazide RS.

C24H34N4O5S   

Storage Store in a dry and cool place, protected from light.

Usual strength 80 mg. GLIMEPIRIDE Glimepiridum

M: 490.6

Glimepiride is 1-[[4-[2-(3-ethyl-4-methyl-2-oxo-3-pyrroline-1-carboxamido)-ethyl]phenyl]sulfonyl]-3-trans -(4-methylcyclohexyl)urea. It contains not less than 97.0% and not more than 102.0% of C24H34N4O5S, calculated with reference to the anhydrous substance.

Characters White or almost white powder, it shows polymorphism. Practically insoluble in water, soluble in dimethylformamide, slightly soluble in methylene chloride, very slightly soluble in methanol. Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of glimepiride RS. If the spectra obtained with the substance to be examined and glimepiride RS show differences, dissolve separately the substance to be examined and the reference substance in dimethylformamide R, evaporate to dryness and record new spectra using the residues. Related substances Examine by liquid chromatography (Appendix 5.3). Store the solutions at a temperature not exceeding 12 °C and for not more than 15 h. Mobile phase: Dissolve 0.5 g of sodium dihydrogen phosphate R in 500 ml of water for chromatography R and adjust to pH 2.5 with phosphoric acid R. Add 500 ml of acetonitrile for chromatography R. Solvent mixture: Water for chromatography - acetonitrile for chromatography (1 : 4). Test solution: Dissolve 20.0 mg of the substance to be examined in the solvent mixture and dilute to 100.0 ml with the solvent mixture. Reference solution (1): Dissolve the contents of a vial of glimepiride for system suitability RS (containing impurities B, C and D) in 2.0 ml of the test solution. 447

GLIMEPIRIDE

Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 10.0 ml with the solvent mixture. Reference solution (3): Dissolve 20.0 mg of glimepiride RS in the solvent mixture and dilute to 100.0 ml with the solvent mixture. Chromatographic system: A column (25 cm × 4.0 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (4 µm). Detector: A spectrophotometer set at 228 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: The run time is 2.5 times the retention time of glimepiride. Relative retention with reference to glimepiride (retention time = about 17 min): Impurity B = about 0.2; impurity C = about 0.3; impurity D = about 1.1. System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to impurities B and C is at least 4.0. Limits: In the chromatogram obtained with the test solution: Impurity B: The area of the peak due to impurity B is not more than 4 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.4%). Impurity D: The area of the peak due to impurity D is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (2) (0.2%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.10%). The sum of the peak areas of all impurities, other than impurity B, is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.5%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Note: Impurity B: 3-ethyl-4-methyl-2-oxo-N-[2-(4-sulfamoylphenyl) ethyl]-2,3-dihydro-1H-pyrrole-1- carboxamide. Impurity C: methyl [[4-[2-[[(3-ethyl-4-methyl-2-oxo-2,3dihydro-1H-pyrrol-1- yl)carbonyl]amino]ethyl]phenyl] sulfonyl] carbamate. Impurity D: 1-[[3-[2-[[(3-ethyl-4-methyl-2-oxo-2,3-dihydro-1Hpyrrol-1- yl)carbonyl]amino]ethyl]phenyl] sulfonyl]-3-(trans-4-methylcyclohexyl)urea. Impurity E: 3-ethyl-4-methyl-2-oxo-N-[2-(3-sulfamoylphenyl) ethyl]-2,3-dihydro-1H-pyrrole-1- carboxamide, Impurity F: methyl [[2-[2-[[(3-ethyl-4-methyl-2-oxo-2,3dihydro-1H-pyrrol-1- yl)carbonyl]amino]ethyl] phenyl]sulfonyl] carbamate.

448

VP V Impurity G: methyl [[4-[2-[[(3-ethyl-4-methyl-2-oxo-2,3dihydro-1H-pyrrol-1- yl)carbonyl]amino]ethyl]phenyl] sulfonyl] methylcarbamate. Impurity H: 1-[[4-[2-[[(3-ethyl-4-methyl-2-oxo-2,3-dihydro1H-pyrrol-1- yl)carbonyl]amino]ethyl]phenyl] sulfonyl]-3-(4-methylphenyl)urea. Impurity I: 1-[[2-[2-[[(3-ethyl-4-methyl-2-oxo-2,3-dihydro-1Hpyrrol-1- yl)carbonyl]amino]ethyl]phenyl] sulfonyl]-3-(trans-4-methylcyclohexyl)urea. Impurity J: 1-[[4-(2-aminoethyl)phenyl]sulfonyl]-3-(trans-4methylcyclohexyl)urea.

Impurity A Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use. Mobile phase: Anhydrous acetic acid - 2-propanol heptane (1 : 100 : 899). Test solution: Dissolve 10.0 mg of the substance to be examined in 5 ml of methylene chloride R and dilute to 20.0 ml with the mobile phase. Reference solution (1): Dilute 0.8 ml of the test solution to 100.0 ml with the mobile phase. Reference solution (2): Dissolve 2.0 mg of glimepiride RS (containing impurity A) in 1 ml of methylene chloride R and dilute to 4.0 ml with the mobile phase. Chromatographic system: A column (15 cm × 3.0 mm) packed with stationary phase diol silica gel for chromatography (5 µm). Detector: A spectrophotometer set at 228 nm. Flow rate: 0.5 ml/min. Volume of injection: 10 µl. Procedure: The run time is 1.5 times the retention time of glimepiride. Identification of impurities: Use the chromatogram supplied with glimepiride RS and the chromatogram obtained with reference solution (2) to identify the peak due to impurity A. Relative retention with reference to glimepiride (retention time = about 14 min): Impurity A = about 0.9. System suitability: In the chromatogramobtained with reference solution (2), peak-to-valley ratio (Hp/Hv) is at least 2.0, where Hp =height above the baseline of the peak due to impurity A and Hv = height above the baseline of the lowest point of the curve separating this peak from the peak due to glimepiride. Limit: In the chromatogram obtained with the test solution: Impurity A: The area of the peak due to impurity A is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.8%). Note: Impurity A: 1-[[4-[2-[[(3-ethyl-4-methyl-2-oxo-2,3-dihydro1H-pyrrol-1- yl)carbonyl]amino]ethyl]phenyl] sulfonyl]-3-(cis4-methylcyclohexyl)urea.

VP V

Water Not more than 0.5% (Appendix 10.3). Dissolve 0.250 g in dimethylformamide R and dilute to 5.0 ml with the same solvent. Carry out the test on 1.0 ml of the resulting solution. Carry out a blank test. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. Inject the test solution and reference solution (3). Calculate the content of glimepiride, C24H34N4O5S, using the areas for the principal peaks in the chromatograms obtained with the test solution, reference solution (3) and the declared content of C24H34N4O5S in glimepiride RS. Storage Store in an airtight container, in cool place, protected from light. Action and use Treatment of diabetes. Preparation Tablets. GLIMEPIRIDE TABLETS Tabellae Glimepridi Glimepiride tablets contain glimepiride. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements:

Content of glimepiride, C24H34N4O5S, 90.0% to 110.0% of the stated amount. Identification In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that of the pricipal peak in the chromatogram obtained with the reference solution. Related substances Examine by liquid chromatography (Appendix 5.3). Chromatographic system, mobile phase, diluent: Proceed as directed in the Assay, with the following modifications: Test solution: Transfer an accurately weighed quantity of the powdered tablets containing the equivalent of 10 mg of glimepiride to a 100 ml volumetric flask, moisten with 0.5 ml of water and then add 70 ml of the diluent, dissolve by sonicating for 10 min at a temperature not exceeding 20 °C.

GLIMEPIRIDE TABLETS

Dilute to volume with the diluent, mix and filter. Reference solution: Dilute 1.0 ml of the test solution to 100.0 ml with the diluent. Chromatographic system: The flow rate is adjusted so that the retention time of glimepiride is about 12 min. Volume of injection: 10 μl. Procedure: System suitability: Inject the reference solution, the theoretical plates of glimepiride peak is not less than 6000 and the tailing factor is not more than 1.5. The relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject the test solution and continue the chromatography for 3 times the retention time of glimepiride peak. The area of any peak corresponding to the peak with the retention time of 0.3 relative to glimepiride is not more than 2.6 times the area of the principal peak in the chromatogram obtained with the reference solution. The area of any other secondary peak, apart from the principal peak and the secondary peak mentioned above, is not greater than 0.3 times the area of the principal peak in the chromatogram obtained with the reference solution. The sum of the areas of any other secondary peaks, apart from the principal peak and the secondary peak mentioned above, is not greater than the area of the principal peak in the chromatogram obtained with the reference solution. The sum of the areas of any secondary peaks, apart from the principal peak, is not more than 3 times the area of the principal peak in the chromatogram obtained with the reference solution. Disregard any peak with area less than 0.1 times the area of the principal peak in the chromatogram obtained with the reference solution.

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of disodium hydrogen phosphate - citric acid buffer pH 7.5 prepared as follows: Add a volume of a 0.525% solution of citric acid R to 1000 ml of 0.05 M disodium hydrogen phosphate to obtain a solution having a pH of 7.5. Rotation speed: 50 rpm. Time: 30 min. Procedure: Examine by liquid chromatography (Appendix 5.3). Chromatographic system and mobile phase: Proceed as directed in the Assay. Except for volume of injection is 100 μl. Test solution: After the specified time (30 min), withdraw a sample of the medium, filter. Reference solution: Dilute solution A in the Assay with the medium to obtain a solution having the same concentration of glimepiride to that in the test solution. 449

VP V

GLIMEPIRIDE AND METFORMIN TABLETS

Tolerance: Not less than 70% (Q) of the stated amount of glimepiride, C24H34N4O5S, is dissolved in 30 min.

Uniformity of content Tablets containing 5 mg or less of glimepiride must comply with the requirements stated under test for Uniformity of content (Appendix 11.2). Test solution: Transfer one tablet to a 100 ml volumetric flask, add 5 ml of water and allow to disintegrate. Add 70 ml of the diluent, dissolve by sonicating for 10 min at a temperature not exceeding 20 °C. Dilute to volume with the diluent, mix and filter. Reference solution: Dilute solution A in the Assay with the diluent to obtain a solution having the same concentration of glimepiride to that in the test solution. Examine by liquid chromatography (Appendix 5.3). Chromatographic system, mobile phase and diluent: Proceed as directed in the Assay. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 0.5 g of sodium dihydrogen phosphate R in 500 ml of water, add 500 ml of acetonitrile R, adjust to pH 3.5 with a 10% solution of phosphoric acid R. Diluent: Acetonitrile - water (4 : 1). Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Transfer an accurately weighed quantity of the powdered tablets containing the equivalent of 4 mg of glimepiride to a 100 ml volumetric flask, moisten with 5 ml of water, add 60 ml of the diluent, dissolve by sonicating for 10 min at a temperature not exceeding 20 °C. Dilute to volume with the diluent, mix and filter. Reference solution: Transfer an accurately weighed quantity of about 50 mg of glimepiride RS to a 50 ml volumetric flask, add 30 ml of the diluent, dissolve by sonicating and dilute to volume with the same solvent, mix (Solution A). Dilute 5.0 ml of solution A to 50.0 ml with the diluent. Resolution solution: Transfer an accurately weighed quantity of about 50 mg of butyl parahydroxy benzoate to a 50 ml volumetric flask, dissolve in a volume of the diluent and dilute to volume with the same solvent. Transfer 5.0 ml of this solution to a 50 ml volumetric flask, add 5.0 ml of solution A, dilute to volume with the diluent, mix well. Chromatographic system: A column (12.5 cm × 4.6 mm) packed with stationary phase C (5 μm), maintained at 25 °C. Detector: A spectrophotometer set at 228 nm. The flow rate is adjusted so that the retention time of glimepiride is about 10 min. Volume of injection: 20 μl. Procedure: System suitability: Inject the resolution solution, the resolution factor between the peaks due to butyl parahydroxy benzoate and glimepiride is not less than 6. 450

Inject the reference solution, the relative standard deviation of the peak areas for six replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of glimepiride, C24H34N4O5S, in the tablets using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C24H34N4O5S in glimepiride RS.

Storage Store in a well-closed container, in a cool and dry place, protected from light. Action and use Treatment of diabetes mellitus. Usual strength 4 mg. GLIMEPIRIDE AND METFORMIN TABLETS Tabellae Glimepiridi et Metformini Glimepiride and metformin tablets contain glimepiride and metformin hydrochloride. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements:

Content of glimepiride, C24H34N4O5S, 90.0% to 110.0% of the stated amount. Content of metformin hydrochloride, C4H11N5,HCl, 95.0% to 105.0% of the stated amount. Identification In the Assay, the retention times of the two principal peaks in the chromatogram obtained with the test solution are the same as those of two principal peaks in the chromatogram obtained with the reference solution. Dissolution for glimepiride Apparatus: Paddle. Medium: 500 ml of a 0.5% solution of sodium laurylsulfate R. Rotation speed: 100 rpm. Time: 45 min. Procedure: Test solution: After the specified time, withdraw a sample of the medium, filter. Reference stock solution: Transfer an accurately weighed quantity of about 20 mg of glimepiride RS to a 100 ml volumetric flask. Add about 80 ml of methanol R, sonicate to dissolve. Dilute to volume with methanol R, mix. Dilute 5.0 ml of this solution to 50.0 ml with the medium. Reference solution: Transfer an accurately weighed quantity of about 25 mg or 50 mg of metformin hydrochloride RS (corresponding to the tablets labelled 250 mg or 500 mg of metformin hydrochloride, respectively) to a 50 ml

VP V

volumetric flask, add 30 ml of the medium, sonicate to dissolve. Add 5.0 ml or 10.0 ml of the reference stock solution (corresponding to the tablets labelled 1 mg or 2 mg of glimepiride, respectively). Dilute to volume with the medium, mix. Procedure: Examine by liquid chromatography (Appendix 5.3). Chromatographic system: Proceed as directed in the Assay. Inject the reference solution. In the chromatogram obtained, the number of theoretical plates is not less than 4000; the tailing factor for glimepiride peak is not more than 2.0; the relative standard deviation of the peak areas due to glimepiride for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of glimepiride, C24H34N4O5S, dissolved using the areas of the glimepiride peak in the chromatograms obtained with the test solution and the reference solution and the declared content of C24H34N4O5S in glimepiride RS. Tolerance: Not less than 70% (Q) of the stated amount of glimepiride, C24H34N4O5S, is dissolved in 45 minutes.

Dissolution for metformin Apparatus: Basket. Medium: 900 ml of a 0.68% solution of potassium dihydrogen phosphate R adjusted to pH 6.8 with 1 M sodium hydroxide R. Rotation speed: 100 rpm. Time: 45 min. Procedure: Test solution: After the specified time, withdraw a sample of the medium, filter. Dilute 10.0 ml of the filtrate to 100.0 ml with water. Dilute 10.0 ml of the resulting solution to 100.0 ml with water. Measure the absorbance (Appendix 4.1) of the test solution at 233 nm, in a 1 cm cell, using water as a blank. Calculate the content of metformin hydrochloride, C4H11N5.HCl, in the medium taking 806 as the value of A (1%, 1 cm) at the maximum at 233 nm. Tolerance: Not less than 70% (Q) of the stated amount of metformin hydrochloride, C4H11N5,HCl, is dissolved in 45 minutes. 1-Cyanoguanidine Mobile phase: A 1.7% solution of ammonium dihydrogen phosphate R adjusted to pH 3.0 with phosphoric acid R. Reference solution: Transfer an accurately weighed quantity of about 10 mg of 1-cyanoguanidine RS to a 50 ml volumetric flask. Add about 30 ml of water and sonicate to dissolve. Dilute to volume with water, mix. Dilute 1.0 ml of the resulting solution to 200.0 ml with the mobile phase. Test solution: Transfer an accurately weighed quantity of the powdered tablets containing the equivalent of about 500 mg of metformin hydrochloride to a 100 ml volumetric

GLIMEPIRIDE AND METFORMIN TABLETS

flask, add about 80 ml of the mobile phase and sonicate for 30 min. Allow to cool and dilute to volume with the mobile phase, mix and filter. Chromatographic system: A column (12.5 cm × 4.6 mm) packed with porous silica gel to which benzens sulfonic acid groups have been chemicaaly bonded (Partisphere 5 µ SCX is suitable). Detector: A spectrophotometer set at 218 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 μl. Procedure: Inject the reference solution. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained is not less than 50% of the full scale of the recorder. Inject the test solution. In the chromatogram obtained, the area of the peak corresponding to 1-cyanoguanidine is not greater than that of the 1-cyanoguanidine peak in the chromatogram obtained with the reference solution (0.02%).

Uniformity of content for glimepiride (Appendix 11.2) Examine by liquid chromatography (Appendix 5.3). Mobile phase, chromatographic system, glimepiride reference stock solution, metformin reference stock solution and mixed reference solution: Prepare as directed in the Assay. Test solution: Transfer one tablet to a 100 ml volumetric flask, add about 80 ml of the mobile phase, sonicate for 60 min to dissolve. Allow to cool. Dilute to volume with the mobile phase, mix and filter. Centrifuge at 3500 rpm for 15 min. Dilute 5.0 ml of the supernatant liquid to 25.0 ml with the mobile phase, mix. Procedure: Inject the mixed reference solution. Adjust the ratio of the components in the mobile phase, if necessary, so that the system suitability as directed in the Assay is met. Inject alternately the test solutions and the mixed reference solution. Calculate the contents of glimepiride, C24H34N4O5S, in each tablet using the peak areas in the chromatograms obtained with the test solutions and the reference solution, and the declared content of C24H34N4O5S in glimepiride RS. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - 0.001 M ammonium acetate triethylamine (500 : 500 : 0.1). Adjust to pH 6.1 ± 0.1 with a 10% solution of acetic acid R. Metformin reference stock solution: Transfer an accurately weighed quantity of about 25 mg of metformin hydrochloride RS to a 100 ml volumetric flask. Add 80 ml of the mobile phase and sonicate to dissolve. Dilute to volume with the mobile phase, mix. Glimepiride reference stock solution: Transfer an accurately weighed quantity of about 20 mg of glimepiride RS to a 100 ml volumetric flask. Add 80 ml of the mobile phase and sonicate to dissolve. Dilute to volume with the 451

VP V

GLIPIZIDE

mobile phase, mix. Dilute 5.0 ml of the solution to 50.0 ml with the mobile phase. Mixed reference solution: Transfer 5.0 ml of metformin reference stock solution and 5.0 ml or 10.0 ml of glimepiride reference stock solution (corresponding to 1 mg or 2 mg strength of glimepiride, respectively) to a 50 ml volumetric flask, dilute to volume with the mobile phase. Sample stock solution: Weigh 20 tablets, calculate the average mass and powder finely. Transfer an accurately weighed quantity of the powder, equivalent to one tablet, to a 100 ml volumetric flask, add about 80 ml of the mobile phase, sonicate for 30 min to dissolve. Dilute to volume with the same solvent, mix. Centrifuge at 3500 rpm for 15 min. Glimepiride test solution: Dilute 5.0 ml of the sample stock solution to 25.0 ml with the mobile phase. Metformin test solution: Dilute 5.0 ml of the sample stock solution to 100.0 ml with the mobile phase. Dilute 5.0 of the resulting solution to 50.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 μm). Detector: A spectrophotometer set at 229 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 μl. Procedure: System suitability: Inject the mixed reference solution. In the chromatogram obtained, the number of theoretical plates for the peaks due to metformin and glimepiride is not less than 2000 and 4000, respectively. The tailing factor for the peaks due to metformin and glimepiride is not more than 2.5 and 2.0, respectively. The resolution factor between the peaks due to metformin and glimepiride is at least 8.0. The relative standard deviation of the peak areas due to metformin and glimepiride for 6 replicate injections is not more than 2.0%. Inject alternately the test solution and the reference solution. Calculate the contents of glimepiride, C24H34N4O5S, and metformin hydrochloride, C4H11N5,HCl, in the tablets using the peak areas in the chromatograms obtained with the test solution and the reference solution, and the declared contents of C24H34N4O5S and C4H11N5,HCl in glimepiride RS and metformin hydrochloride RS, respectively.

Storage Store in an airtight container, in a cool and dry place, protected from light. Action and use Treament of diabetes mellitus. Usual strength Glimepiride/metformin hydrochloride: 1 mg/250 mg; 1 mg/500 mg; 2 mg/250 mg; 2 mg/500 mg.

452

GLIPIZIDE Glipizidum

C21H27N5O4S  

M: 445.5

Glipizide is 1-cyclohexyl-3-[[4-[2-[[(5-methylpyrazin-2-yl) carbonyl]amino]ethyl]phenyl]sulfonyl]urea. It contains not less than 98.0% and not more than 102.0% of C21H27N5O4S, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder. Practically insoluble in water and in ethanol (96%), very slightly soluble in methylene chloride and in acetone, dissolves in dilute solutions of alkali hydroxides. Identification Apply one of the two following identifications: First identification: A. Second identification: B, C. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of glipizide RS. B. Dissolve about 2 mg in methanol R and dilute to 100 ml with the same solvent. The ultraviolet absorption spectrum (Appendix 4.1) of the solution in the range 220 nm to 350 nm, exhibits two absorption maxima at 266 nm and 274 nm. The ratios of the absorbances A226/A274 is 2.0 to 2.4. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Anhydrous formic acid - ethyl acetate methylene chloride (25 : 25 : 50). Test solution: Dissolve 10 mg of the substance to be examined in a mixture of equal volumes of methanol R and methylene chloride R and dilute to 10 ml with the same mixture of solvents. Reference solution: Dissolve 10 mg of glipizide RS in a mixture of equal volumes of methanol R and methylene chloride R and dilute to 10 ml with the same mixture of solvents. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 2/3 the plate. Allow the plate to dry in air and examine in ultravioletlight at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution.

VP V

Related substances Examine by liquid chromatography (Appendix 5.3) as described in the test for Assay. Test solution: Dissolve 25 mg of the substance to be examined in a 50 ml flask, add 25.0 ml of methanol R, shake vigorously to dissolve and dilute to 50.0 ml with 0.1 M sodium dihydrogen phosphate. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Reference solution (2): Dissolve 12.5 mg of glipizide impurity A RS in methanol R and dilute to 50 ml with methanol R. Dilute 1.0 ml of the solution to 100.0 ml with the mobile phase. Procedure: Inject reference solution (1), adjust system to the height peak due to pricipal peak in the chromatogram is at least 25% range scale. Inject the test solution, reference solution (1), (2) with the run time is twice the retention time of principal peak. Limits: In the chromatogram obtained with the test solution: Impurity A: The area of the peak due to impurity A is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.5%). The area of any other peak, apart from the principal peak and impurity A, is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). Sum of the areas of all second peaks, other than the principal peak and impurity A, is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (1.0%). Note: Impurity A: 5-methyl-N-[2-(4-sulfamoylphenyl)ethyl]pyrazine2-carboxamide.

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.2% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: 0.1 M sodium dihydrogen phosphate previously adjusted to pH 6.00 ± 0.05 with 2 M sodium hydroxide - methanol (55 : 45). Test solution: Dissolve 25.0 mg of the substance to be examined in methanol R, dilute to 50.0 ml with the same solution. To 5.0 ml the solution in 50 ml flask, add 20.0 ml of methanol R and dilute to 50.0 ml with 0.1 M sodium dihydrogen phosphate. Reference solution: Dissolve 25.0 mg glipizide RS in methanol R, dilute to 50.0 ml with the same solution. To

GLIPIZIDE TABLETS

5.0 ml the solution in 50 ml flask, add 20.0 ml of methanol R and dilute to 50 ml with 0.1 M sodium dihydrogen phosphate. Resolution solution: Dissolve a quantity of glipizide RS and impurity A glipizide RS in methanol R to obtain a solution containing 0.5 mg/ml of glipizide and 2.5 µg/ml of impurity A. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 225 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: System suitability: In the chromatogram obtained with resolution solution, the column efficiency determined on the glipizide peak is not less than 2000 theoretical plates and the resolution between the peaks due to glipizide and impurity A is at least 1.5. Inject the test solution and the reference solution. Calculate the content of glipizide, C21H27N5O4S, using the peak areas of glipizide in the chromatograms obtained with the test solution, the reference solution and the declared content of C21H27N5O4S in glipizide RS.

Storage Store in an airtight container, protected from light. Action and use Treatment of diabetes. Preparation Tablets. GLIPIZIDE TABLETS Tabellae Glipizidi Glipizide tablets contain glipizide. They may be coated. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements:

Content of glipizide, C21H27N5O4S, 90.0% to 110.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix5.4). Coating substance: Silica gel G. Mobile phase: Toluene - ethyl acetate - 98% formic acid (5 : 3 : 2). Test solution: Transfer a quantity of finely powdered tablets, equivalent to 10 mg of glipizide, to a glassstoppered centrifuge tube, add 10 ml of methanol R and shake. Centrifuge the mixture and use the clear supernatant. 453

GLIPIZIDE TABLETS

Reference solution: A 1 mg/ml solution of glipizide RS in methanol R. Procedure: Apply separately to the plate 20 μl of each solution. Allow the spots to dry in air. After developing over a path of 3/4 of the plate, remove the plate, allow it to dry in air and then dry the plate at 80 °C for 30 min. Allow the plate to cool, spray it with a 0.5% solution of sodium hypochlorite R and allow the plate to dry in air. Spray the plate with ethanol 96% R, allow it to dry in air and spray with a freshly prepared mixture of soluble starch solution - 1% potassium iodide solution (1 : 1). The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to that in the chromatogram obtained with the reference solution. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that of the glipizide peak in the chromatogram obtained with the reference solution.

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of the medium prepared as follows: Dissolve 6.8 g of potassium dihydrogen phosphate R in 250 ml of water, add 77 ml of 0.2 M sodium hydroxide R and 500 ml of water. Adjust to pH 6.8 ± 0.1 with 0.2 M sodium hydroxide R or 0.2 M hydrochloric acid R. Dilute with water to 1000 ml. Rotation speed: 50 rpm. Time: 45 min. Procedure: Test solution: After the specified time (45 min), withdraw a sample of the medium, filter and dilute the filtrate with the medium if necessary. Reference solution: Transfer an accurately weighed quantity of about 10 mg of glipizide RS to a 100 ml volumetric flask, add 5 ml of methanol R, dissolve by sonicating. Dilute to volume with the medium and mix. Dilute this solution with the medium to obtain a solution having the same concentration of glipizide to that in the test solution. Measure the absorbances (Appendix 4.1) of the test solution and the reference solution at the maximum at about 276 nm, in a 1 cm cell, using the medium as a blank. Calculate the content of glipizide, C21H27N5O4S, dissolved using the absorbances of the reference solution and the test solution and the content of C21H27N5O4S in glipizide RS. Tolerance: Not less than 80% (Q) of the stated amount of glipizide, C21H27N5O4S, is dissolved in 45min. Uniformity of content (Appendix 11.2) Examine by liquid chromatography (Appendix 5.3). Chromatographic system, buffer, mobile phase, reference solution: Prepare as directed in the Assay. Test solution: Transfer one tablet to an appropriate volumetric flask, add a volume of the buffer equal to about one-half 454

VP V

of the total flask volume, shake by mechanical means for 10 min to allow the tablet to disintegrate completely. Add a volume of methanol R to 4/5 of the total flask volume and sonicate for another 15 min. Dilute to volume with methanol R and mix to obtain a solution having a concentration of about 0.05 mg/ml of glipizide. Filter. 9 other tablets are prepared as the same procedure above. Procedure: Inject alternately the reference solution and the test solution. Calculate the content of glipizide, C21H27N5O4S, in the tablet using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C21H27N5O4S in glipizide RS.

Related substances Examine by liquid chromatography (Appendix 5.3). Chromatographic system, buffer, mobile phase: Prepare as directed in the Assay. Reference stock solution: Dissolve an accurately weighed quantity of glipizide related compound A RS in methanol R to obtain a solution having a known concentration of about 50 µg/ml. Reference solution: Transfer an accurately weighed quantity of 20 mg of glipizide RS to a 200 ml volumetric flask, dissolve in a volume of methanol R, add 2.0 ml of the reference stock solution. Dilute to volume with methanol R and mix. Dilute 25.0 ml to 50.0 ml with the buffer, mix. Test solution: Use the test solution in the Assay. Procedure: System suitability: Inject the reference solution, the relative retention times are 0.2 for glipizide related compound A and 1.0 for glipizide. The relative standard deviation of the peak areas due to glipizide related compound A for 6 replicate injections is not more than 5.0%. Inject alternately the reference solution and the test solution. From the peak areas of glipizide related compound A in the chromatograms obtained with the test solution and the reference solution and the concentration of glipizide related compound A in the reference solution, calculate the content of glipizide related compound A if detectable, in the tablets, relative to the content of glipizide determined in the Assay. Limit: The content of glipizide related compound A is not more than 2.0%. Note: Glipizide related compoundA: 5-methyl-N-[2-(4-sulfamoylphenyl) ethyl]pyrazine-2-carboxamide.

Assay Examine by liquid chromatography (Appendix 5.3). Buffer: Dissolve 13.8 mg of sodium dihydrogen phosphate R in 1000 ml of water. Adjust to pH 6.0 ± 0.05 with 2 M sodium hydroxide R.

VP V

Mobile phase: Buffer - methanol (55 : 45). Make adjustments if necessary. Reference solution: Dissolve an accurately weighed quantity of glipizide RS in methanol R to obtain a solution having a known concentration of about 0.1 mg/ml. Dilute 25.0 ml of this solution to 50.0 ml with the buffer. Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Transfer an accurately weighed quantity of the powdered tablets containing the equivalent of 5 mg of glipizide to a 100 volumetric flask, add 50 ml of methanol R, sonicate for 15 min. Dilute to volume with the buffer and mix, sonicate for another 15 min and filter. Chromatographic system: A column (15 cm × 3.9 mm) packed with stationary phase C (5 μm). Detector: A spectrophotometer set at 225 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 μl. Procedure: Inject the reference solution, the relative standard deviation of the peak areas for 6 replicate injections is not more than 1.0%. Inject the test solution. Calculate the content of glipizide, C21H27N5O4S, in the tablets, using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C21H27N5O4S in glipizide RS.

Storage Store in an airtight container, in a cool and dry place, protected from light. Action and use Treatment of diabetes mellitus. Usual strength 5 mg, 10 mg. GLIPIZIDE AND METFORMIN TABLETS Tabellae Glipizide et Metformini Glipizide and metformin tablets contain glipizide and metformin hydrochloride. They may be coated. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements:

Content of glipizide, C21H27N5O4S, 90.0% to 110.0% of the stated amount. Content of metformin hydrochloride, C4H11N5,HCl, 90.0% to 110.0% of the stated amount. Identification A. Transfer 10 tablets (with the coating layer removed, if necessary), previously powdered, to a 100 ml conical flask.

GLIPIZIDE AND METFORMIN TABLETS

Add 20 ml of water and shake for 1 hour, filter. Transfer the solution to a separating funnel and extract twice with 10 ml of chloroform R and shake for 5 min for each. Transfer the chloroform layer into a baker containing 3 to 4 g of anhydrous magnesium sulfate R, swirl for 1 minute. Filter, evaporate the solvent under vacuum and dry the residue under vacuum at 105 oC for 4 hours. The infrared absorption spectrum (Appendix 4.2) of the obtained residue is concordant with the spectrum of glipizide RS. B. In the Assay for glipizide, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that of glipizide peak in the chromatogram obtained with the reference solution. C. In the Assay for metformin hydrochloride, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that of metformin peak in the chromatogram obtained with the reference solution.

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 1000 ml of 0.05 M phosphate buffer pH 6.8 ± 0.05, prepared as follows: Dissolve 12.96 g of potassium dihydrogen phosphate R and 1.66 g of sodium hydroxide R in about 400 ml of water. Dilute to 2000 ml with water. Adjust to pH 6.8 ± 0.05 with 0.2 M sodium hydroxide R if necessary. Rotation speed: 50 rpm. Time: 45 min. For glipizide Examine by liquid chromatography (Appendix 5.3). Solution A: Dissolve 3.4 g of potassium dihydrogen phosphate R in about 800 ml of water. Adjust to pH 6.0 ± 0.1 with 10 M sodium hydroxide R. Dilute to 1000 ml with water. Mobile phase: Methanol - solution A (52 : 48). Reference solution: Transfer an accurately weighed quantity of about 50 mg of glipizide RS to a 1000 ml volumetric flask. Add 100 ml of methanol R, dissolve by sonicating for 5 min. Dilute to volume with the medium, mix. Dilute this solution with the medium to obtain a solution having a known concentration equivalent to that in the test solution. Test solution: After the specified time (45 min), withdraw a sample of the medium, filter. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 μm). Detector: A spectrophotometer set at 220 nm. The flow rate: 1.0 ml/min. Volume of injection: 50 μl. Procedure: System suitability: Inject the reference solution, the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. 455

VP V

GLIPIZIDE AND METFORMIN TABLETS

Inject the test solution. Calculate the content of glipizide, C21H27N5O4S, dissolved using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C21H27N5O4S in glipizide RS. Tolerance: Not less than 80% (Q) of the stated amount of glipizide, C21H27N5O4S, is dissolved in 45 min. For metformin hydrochloride After the specified time (45 min), withdraw a sample of the medium, filter. Measure the absorbance (Appendix 4.1) of the resulting solution (suitably diluted with the medium, if necessary) at maximum at about 233 nm, in a 1 cm cell, using the medium as a blank. Calculate the content of metformin hydrochloride, C4H11N5,HCl, dissolved using the absorbances of the test solution and a reference solution of metformin hydrochloride RS having a known concentration in the same medium. Tolerance: Not less than 80% (Q) of the stated amount of metformin hydrochloride, C4H11N5,HCl, is dissolved in 45 min.

Uniformity of content for glipizide (Appendix 11.2) Examine by liquid chromatography (Appendix 5.3). Mobile phase, chromatographic system, diluent, resolution solution, reference solution and procedure: Proceed as directed in the Assay for glipizide. Test solution: Transfer one tablet to a 100 ml volumetric flask, add about 50 ml of the diluent, sonicate for 30 min. Shake by mechanical means for another 30 min to dissolve. Dilute to volume with water, mix and filter. Dilute the filtrate with the diluent and water to obtain a solution having a ratio of the organic solvent volume and a known concentration of glipizide equivalent to those in the reference solution. Glipizide related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase, chromatographic system, diluent, resolution solution, reference solution, test solution and procedure: Proceed as directed in the Assay for glipizide. Calculate the content of impurities, if found, in the chromatogram obtained with the test solution using the normalisation procedure (Appendix 5). Calculate the content of glipizide related compound A (relative retention time of 0.92) by dividing the area of the corresponding peak by 1.4. Limits: Glipizide related compound A: Not more than 2.0%. Other individual impurities that elute after 8 min: Not more than 0.5%. The sum of other impurities, apart from glipizide related compound A: Not more than 1.0%. 456

Disregard the peak due to metformin that elutes before 8 min. Disregard any peak observed in the blank and disregard any peak less than 0.05%. Note: Glipizide related compoundA: 5-methyl-N-[2-(4-sulfamoylphenyl) ethyl]pyrazine-2-carboxamide.

Metformin related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase, chromatographic system, diluent, resolution solution, reference solution, test solution and procedure: Proceed as directed in the Assay for metformin. Calculate the content of impurities, if found, in the chromatogram obtained with the test solution using the normalisation procedure (Appendix 5). Acceptance criteria: Individual impurities: Not more than 0.1%. Total impurities: Not more than 0.5%. Disregard any peak observed in the blank, glipizide peak and disregard any peak less than 0.05%. Note: Metformin related compound A: Cyanoguanidine.

Assay for glipizide Examine by liquid chromatography (Appendix 5.3). Solution A: Dissolve 2.6 g of ammonium phosphate R in 1000 ml of water, adjust to pH 8.0 with ammonium hydroxide R. Solution B: Acetonitrile - water - solution A (5 : 70 : 25). Solution C: Acetonitrile - water - solution A (50 : 25 : 25). Diluent: Acetonitrile - water (60 : 40). Reference solution: Transfer an accurately weighed quantity of about 10 mg of glipizide RS to a 100 ml brown volumetric flask, add 60 ml of acetonitrile R, sonicate for 20 min. Dilute to volume with water and mix. Transfer 25.0 ml of this solution to a 200 ml brown volumetric flask, add 75 ml of the diluent and dilute to volume with water, mix. Resolution solution: Transfer an accurately weighed quantity of about 5 mg of glipizide related compound A to a 500 ml volumetric flask, add about 250 ml of acetonitrile R and sonicate for 30 min. Dilute to volume with acetonitrile R and mix. Dilute 1.0 ml of this solution to 50.0 ml with the reference solution. Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Transfer an accurately weighed quantity of the powdered tablets containing the equivalent of 25 mg of glipizide to a 500 ml volumetric flask, add 300 ml of the diluent, sonicate for 30 min and shake by mechanical means for another 30 min. Dilute to volume with the diluent, mix and filter. Transfer 25.0 ml of the filtrate to a 100 ml volumetric flask, add 25 ml of the diluent, dilute to volume with water, mix. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase B (5 μm).

VP V

GLUCOSAMINE HYDROCHLORIDE

Detector: A spectrophotometer set at 223 nm. Flow rate: 1.0 ml/min. Volume of injection: 50 μl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min) 0-3

Solution B (% v/v)

Solution C (% v/v)

100

0

3 - 18

100 → 0

0 → 100

20

0

100

20 - 22

0 → 100

30

100

100 → 0 0

System suitability: Inject the resolution solution and the reference solution, the relative retention times are 0.92 for glipizide related compound A and 1.0 for glipizide. Resolution factor between glipizide related compound A peak and glipizide peak is not less than 1.2. The relative standard deviation of the peak areas for 6 replicate injections of the reference solution is not more than 2.0%. Inject the test solution. Calculate the content of glipizide, C21H27N5O4S, in the tablets using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C21H27N5O4S in glipizide RS.

Assay for metformin hydrochloride Examine by liquid chromatography (Appendix 5.3). Solution A: Dissolve 9.41 g of sodium hexanesulfonate R in 1000 ml of water. Adjust to pH 2.0 with trifluoroacetic acid R (obtaining 50 mM hexanesulfonic acid solution). Solution B: Acetonitrile - water (40 : 60). Mobile phase: Solution A - solution B - water (30 : 20 : 50). Diluent: Acetonitrile - solution A - water (7 : 30 : 63). Reference solution: A 0.1 mg/ml solution of metformin hydrochloride RS in the diluent. Resolution solution: Dissolve a quantity of metformin related compound A in water to obtain a solution having a known concentration of 5 µg/ml. Dilute 0.5 ml of this solution to 50.0 ml with the reference solution. Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Transfer an accurately weighed quantity of the powdered tablets containing the equivalent of about 100 mg of metformin hydrochloride to a 100 ml volumetric flask, add about 70 ml of the diluent, shake to dissolve. Dilute to volume with the diluent, mix and filter. Dilute the filtrate with the diluent to obtain a solution containing 0.1 mg/ml of metformin hydrochloride. Chromatographic system: A column (15 cm × 4.6 mm) packed with particles of silica the surfaces of which has been modified by chemically bonded phenyl groups (3.5 μm).

Detector: A spectrophotometer set at 218 nm. The flow rate: 1.0 ml/min. Volume of injection: 25 μl. Procedure: System suitability: Inject the resolution solution and the reference solution, the relative retention times are 0.26 for metformin related compound A and 1.0 for metformin. The resolution factor between two peaks due to metformin related compound A and metformin is not less than 3.0. The relative standard deviation of the peak areas due to metformin for 6 replicate injections of the reference solution is not more than 2.0%. Inject the test solution and continue the chromatography for 4 times the retention time of metformin peak. Calculate the content of metformin hydrochloride, C4H11N5,HCl, in the tablets using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C4H11N5,HCl in metformin hydrochloride RS.

Storage Store in a well-closed container, in a cool and dry place, protected from light. Action and use Treatment of diabetes mellitus. Usual strength 2.5 mg of glipizide and 250 mg of metformin hydrochloride. 2.5 mg of glipizide and 500 mg of metformin hydrochloride. 5.0 mg of glipizide and 500 mg of metformin hydrochloride. GLUCOSAMINE HYDROCHLORIDE Glucosamini hydrochloridum HO O OH

OH HO

C6H13NO5,HCl   

, HCl

NH2

M. 215.6

Glucosamine hydrochloride is 2-amino-2-deoxy-Dglucopyranose hydrochloride. It contains not less than 98.0% and not more than 102.0% of C6H13NO5,HCl, calculated with reference to the anhydrous substance.

Characters White crystalline powder. Freely soluble in water. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of glucosamine hydrochloride RS. 457

VP V

GLUCOSAMINE SULFATE POTASSIUM CHLORIDE

B. It gives reaction (A) of chlorides (Appendix 8.1). In the test for Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is concordant with retention time of the glucosamine peak in the chromatogram obstained with the reference solution.

Specific optical rotation +70.0° to +73.0°, calculated with reference to the dried substance (Appendix 6.4). Prepare a 25 mg/ml solution of the substance to be examined in water. Examine 3 h after preparation of solution S at 25 °C. pH 3.0 to 5.0 (Appendix 6.2). Determined on a 20.0 mg/ml solution of the substance to be examined in carbon dioxide-free water R. Loss on drying Not more than 1.0% (Appendix 9.6). (0.500 g; 105 °C; 2 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Sulfate Not more than 0.24% (Appendix 9.4.14). Prepare test solution: Dissolve 0.420 g in 100 ml sufficient water. Arsenic Not more than 3 ppm (Appendix 9.4.2). Weigh 0.330 g of the substance to be examined, add 5 ml sulfuric acid R and boil to ash. Cautiously add, dropwise on the sides, 30% hydrogen peroxide solution R untill the solution become clear. Cool, add 10 ml of water and again evaporate to strong fuming to remove hydrogen peroxide. Cool, dilute with water to 25 ml and use the method A. Prepare the standard in the same manner using 1 ml of arsenic standard solution (1 ppm As) R. Assay Examine by liquid chromatography (Appendix 5.3). Buffer phosphate solution: Dissolve 3.5 g of dipotassium hydrogenphosphate in water. Add 0.25 ml of ammonium hydroxide R, dilute to 1000 ml with water. Adjust to pH 7.5 with phosphoric acid R. Mobile phase:  Buffer phosphate solution - acetonitrile (25 : 75). Solvent mixture: Acetonitrile - water (50 : 50). Test solution: Dissolve 380 mg of the substance to be examined in solvent mixture and dilute to 100.0 ml with the same solvent. Reference solution: A 3.8 mg/ml solution of glucosamine hydrochloride RS in solvent mixture. 458

Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase amminopropylsilyl silica gel for chromatography (5 µm). Column temperature: 35 °C. Detector: A spectrophotometer set at 195 nm. Flow rate: 1.5 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject the reference solution, the retention time of the peak due to glucosamine is about 10 min. The chromatogram shows a large additional peak near the void volume, due to the chloride ion. The symmetry factor of the peak due to glucosamine is not more than 2.0; the relative standard deviation of the peak due to glucosamine for 6 replicate injections of the reference solution is not more than 2.0%. The column efficiency (determined on the peak due to glucosamine) is not less than 1500 theoretical plates. Inject the test solution and the reference solution. Calculate the content of C6H13NO5,HCl, using the peak areas in the chromatograms obtained with the test solution, the reference solution and the content of C6H13NO5,HCl in glucosamine hydrochloride RS.

Storage Store in an airtight container, protected from light. Action and use Therapy for osteoarthritis.  Preparations Tablets, capsules. GLUCOSAMINE SULFATE POTASSIUM CHLORIDE Glucosamini sulfas kalii chloridum

(C6H14NO5)2SO4,2KCl

M. 605.5

Glucosamine sulfate potassium chloride is bis(2-amino-2deoxy-β-D-glucopyranose) sulfate potassium chloride. It contains not less than 98.0% and not more than 102.0% of (C6H14NO5)2SO4,2KCl, calculated with reference to the dried substance.

Characters White crystalline powder. Very soluble in water.

VP V

Identification A. Transfer 50 mg of the substance to be examined to a centrifuge tube, add in 2 ml of water and shake to dissolve. Add 0.5 ml 12% solution of barium chloride R, shake and centrifuge. Collect the supernatant, and evaporate to dryness. Dry the residue at 105 °C for 2 h. The infrared absorption spectrum (Appendix 4.2) of the residue is concordant with the spectrum of glucosamine sulfate potassium chloride RS repared in the same manner without adding 12% solution of barium chloride R. B. In the test for Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is concordant with retention time of the glucosamine peak in the chromatogram obtained with the reference solution. C. It gives reaction of sodium, potassium and sulfates (Appendix 8.1). pH 3.0 to 5.0 for a 20 mg/ml solution of the substance to be examined in carbon dioxide-free water R (Appendix 6.2). Specific optical rotation +47.0° to +53.0°, calculated with reference to the dried substance (Appendix 6.4). Test solution: A 35 mg/ml solution of of the substance to be examined in water. Examine 3 h after preparation of the test solution. Sulfate 15.5% to 16.5%. Transfer 1 g of the substance to be examined to a 250-ml beaker, add in 100 ml of water and stir to dissolve completely. Add 4 ml of 6 N hydrochloric acid R. Heat the solution to boiling, and add, with constant stirring, sufficient boiling 12% solution of barium chloride R to completely precipitate the sulfate. Add an additional 2 ml of 12% solution of barium chloride R, and heat on a water-bath for 1 h. Pass the mixture through ashless filter paper, and wash the residue with hot water until no precipitate is obtained when 1 ml of 0.1 N silver nitrate R is added to 5 ml of washing. Transfer the paper containing the residue to a tared crucible. Char the paper, without burning, and ignite the crucible and its contents to constant weight. Calculate the content of sulfate by multiplying the weight obtained by 0.4116. Arsenic Not more than 3 ppm (Appendix 9.4.2). Weigh 0.330 g of the substance to be examined, add 5 ml sulfuric acid R and boil to ash. Cautiously add, dropwise on the sides, 30% hydrogen peroxide solution R untill the solution become clear. Cool, add 10 ml of water and again evaporate to strong fuming to remove hydrogen peroxide. Cool dilute with water to 25 ml and use the method A. Prepare the standard in the same manner using 1 ml of arsenic standard solution (1 ppm As) R.

GLUCOSAMINE SULFATE POTASSIUM CHLORIDE

Sodium A 10% solution of the substance to be examined in water tested on a platinum wire, does not impart a pronounced yellow color to a nonluminous flame. Loss on drying Not more than 1.0% (Appendix 9.6). (0.500 g; 105 °C; 2 h). Sulfated ash 26.5% to 31.0% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by liquid chromatography (Appendix 5.3). Buffer phosphate solution: Dissolve 3.5 g of dipotassium hydrogenphosphate in water. Add 0.25 ml of ammonia R, dilute to 1000 ml with water. Adjust to pH 7.5 with phosphoric acid R. Mobile phase:  Acetonitrile - buffer phosphate solution (75 : 25). Solvent mixture: Acetonitrile - water (50 : 50). Test solution: Weigh accurately 263 mg of the substance to be examined a 50-ml volumetric flask. Add 30 ml of solvent mixture, shake by mechanical means to dissolve completely. Dilute with solvent mixture to volume. Reference solution: A 3.8 mg/ml solution of glucosamine hydrochloride RS in solvent mixture. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase amminopropylsilyl silica gel for chromatography (5 µm). Column temperature: 35 °C. Detector: A spectrophotometer set at 195 nm. Flow rate: 1.5 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject the reference solution, the retention time of the peak due to glucosamine is about 10 min. The chromatogram shows a large additional peak near the void volume, due to the chloride ion. The symmetry factor of the peak due to glucosamine is not more than 2.0; The column efficiency (determined on the peak due to glucosamine) is not less than 1500 theoretical plates; the relative standard deviation of the peak areas due to glucosamine for replicate injections of the reference solution is not more than 2.0%. Inject the test solution and the reference solution. Calculate the content of glucosamine using the peak areas in the chromatograms obtained with the test solution, the reference solution and the content of glucosamine hydrochloride RS. Calculate the content of glucosamine sulfate potassium chloride, (C6H14NO5)2SO4,2KCl, by multiplying glucosamine hydrochloride with 605.52/431.26, where: 605.52 is molecular weight of (C6H14NO5)2SO4, 2KCl and 459

VP V

GLUCOSAMINE SULFATE SODIUM CHLORIDE

431.26 is twice the molecular weight of glucosamine hydrochloride.

Storage Store in an airtight container, protected from light. Action and use Therapy for osteoarthritis.  Preparations Tablets, capsules. GLUCOSAMINE SULFATE SODIUM CHLORIDE Glucosamini sulfas natrii chloridum (C6H14NO5)2SO4,2NaCl  

M. 573.3

Glucosamine sulfate sodium chloride is bis(2-amino-2deoxy-β-D-glucopyranose) sulfate sodium chloride. It contains not less than 98.0% and not more than 102.0% of (C6H14NO5)2SO4,2NaCl, calculated with reference to the dried substance.

Characters White crystalline powder. Very soluble in water. Identification A. Transfer 50 mg of the substance to be examined to a centrifuge tube, add in 2 ml of water and shake to dissolve. Add 0.5 ml of a 12% solution of barium chloride R, shake and centrifuge. Collect the supernatant, and evaporate to dryness. Dry the residue at 105 °C for 2 h. The infrared absorption spectrum (Appendix 4.2) of the residue is concordant with the spectrum of glucosamine sulfate potassium chloride RS repared in the same manner without adding a 12% solution of barium chloride R. B. In the test for Assay, the retention time of the principal peak in the chromatogram obtained with test solution is concordant with retention time of the glucosamine peak in the chromatogram obtained with standard solution. C. It gives reactions of sodium, potassium and sulfates (Appendix 8.1). pH 3.0 to 5.0 for a 20 mg/ml solution of the substance to be examined in carbon dioxide-free water R (Appendix 6.2). Specific optical rotation +50.0° to +55.0°, calculated with reference to the dried substance (Appendix 6.4). Test solution: A 35 mg/ml solution of of the substance to be examined in water. Examine 3 h after preparation of the test solution. Sulfate 16.3% to 17.3%. Transfer 1 g of the substance to be examined to a 250-ml beaker, add in 100 ml of water and stir to dissolve completely. 460

Add 4 ml of 6 N hydrochloric acid R. Heat the solution to boiling, and add, with constant stirring, sufficient boiling 12% solution of barium chloride R to completely precipitate the sulfate. Add an additional 2 ml of a 12% solution of barium chloride R, and heat on a water-bath for 1 h. Pass the mixture through ashless filter paper, and wash the residue with hot water until no precipitate is obtained when 1 ml of 0.1 N silver nitrate R is added to 5 ml of washing. Transfer the paper containing the residue to a tared crucible. Char the paper, without burning, and ignite the crucible and its contents to constant weight. Calculate the content of sulfate by multiplying the weight obtained by 0.4116.

Arsenic Not more than 3 ppm (Appendix 9.4.2). Weigh 0.330 g of the substance to be examined, add 5 ml sulfuric acid R and boil to ash. Cautiously add, dropwise on the sides, 30% hydrogen peroxide solution R untill the solution become clear. Cool, add 10 ml of water and again evaporate to strong fuming to remove hydrogen peroxide. Cool dilute with water to 25 ml and use the method A. Prepare the standard in the same manner using 1 ml of arsenic standard solution (1 ppm As) R. Potassium Acidify 5 ml of a 5% solution of the substance to be examined in water with 6 M acetic acid R, and add 5 drops of a 20% solution of sodium cobaltinitrite R. No precipitate is formed. Loss on drying Not more than 1.0% (Appendix 9.6). (0.500 g; 105 °C; 2 h). Sulfated ash 22.5% to 26.0% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by liquid chromatography (Appendix 5.3). Buffer phosphate solution: Dissolve 3.5 g of dipotassium hydrogenphosphate R in water. Add 0.25 ml of ammonium hydroxide R, dilute to 1000 ml with water. Adjust to pH 7.5 with phosphoric acid R. Mobile phase:  Acetonitrile - buffer phosphate solution (75 : 25). Solvent mixture: Acetonitrile - water (50 : 50). Test solution: Weigh accurately 250 mg of the substance to be examined a 50-ml volumetric flask. Add 30 ml of solvent mixture, shake by mechanical means to dissolve completely. Dilute with solvent mixture to volume. Reference solution: A 3.8 mg/ml solution of glucosamine hydrochloride RS in solvent mixture. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase amminopropylsilyl silica gel for chromatography (5 µm). Column temperature: 35 °C. Detector: A spectrophotometer set at 195 nm.

VP V

Flow rate: 1.5 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject the reference solution, the retention time of the peak due to glucosamine is about 10 min. The chromatogram shows a large additional peak near the void volume, due to the chloride ion. The symmetry factor of the peak due to glucosamine is not more than 2.0; the column efficiency (determined from the peak due to glucosamine) is not less than 1,500 theoretical plates; the relative standard deviation of the peak due to glucosamine for replicate injections of the reference solution is not more than 2.0%. Inject the test solution and the reference solution. Calculate the content of glucosamine using the areas in the chromatograms obtained with the test solution, the reference solution and the content of glucosamine hydrochloride RS. Calculate the content of glucosamine sulfate sodium chloride, (C6H14NO5)2SO4,2NaCl, by multiplying glucosamine hydrochloride with 573.31/431.26, where: 573.31 is molecular weight of (C6H14NO5)2SO4,2NaCl and 431.26 is twice the molecular weight of glucosamine hydrochloride.

Storage Store in an airtight container, protected from light. Action and use Therapy for osteoarthritis.  Preparations Tablets, capsules. GLUCOSAMINE TABLETS Tabellae Glucosamini Glucosamine tablets contain glucosamine hydrochloride, or glucosamine sulfate sodium chloride, or glucosamine sulfate potassium chloride, or a mixture of any of them. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of glucosamine, C6H13NO5, 90.0% to 110.0% of the stated amount. Identification A. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of glucosamine peak in the chromatogram obtained with the reference solution. B. Shake a quantity of the powdered tablets containing about 60 mg of glucosamine with 10 ml of water, and filter. 2 ml of the filtrate gives reaction A of chlorides (Appendix 8.1).

GLUCOSAMINE TABLETS

C. Shake a quantity of the powdered tablets containing about 50 mg of glucosamine with 10 ml of water, and filter. 5 ml of the filtrate gives the reactions of sulfates (Appendix 8.1) (only for the tablets containing glucosamine sodium sulfate or glucosamine sulfate potassium chloride).

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of water. Rotation speed: 75 rpm. Time: 60 min. Procedure: Examine by liquid chromatography (Appendix 5.3). Phosphate buffer: Mix 1.0 ml of phosphoric acid R with 2000 ml of water, adjust the pH to 3.0 with 30% potassium hydroxide solution R. Mobile phase: Phosphate buffer - acetonitrile (3 : 2). Reference solution: A solution of glucosamine hydrochloride RS in water having the same concentration as expected in the test solution. Test solution: Withdraw a sample of the medium and filter (discarding the first portion of the filtrate). Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase B (5 mm). Detector: A spectrophotometer set at 195 nm. Flow rate: 0.6 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject the reference solution. The symmetry factor of the glucosamine hydrochloride peak is not more than 2.0, the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of glucosamine, C6H13NO5, dissolved using the areas of the principal peaks in the chromatograms obtained with the test solution, the reference solution and the declared content of C6H13NO5 in glucosamine RS. Tolerance: Not less than 75% (Q) of the labeled amount of glucosamine, C6H13NO5, is dissolved in 60 min. Assay Examine by liquid chromatography (Appendix 5.3). Buffer solution: Dissolve 3.5 g of dipotassium hydrogen phosphate R in 900 ml of water, add 0.25 ml of ammonia R, dilute with water to produce 1000 ml, mix. Adjust the pH to 7.5 with phosphoric acid R. Mobile phase: Buffer solution - acetonitrile (25 : 75). Make adjustments if necessary. Diluent: Water - acetonitrile (50 : 50). Test solution: Weigh 20 tablets, calculate the average mass and finely powder. Weigh accurately a quantity of the powder containing about 300 mg of glucosamine, transfer into a 100-ml volumetric flask, add 60 ml of diluent, sonicate for 10 min. Shake by mechanical means for 15 min, dilute to volume with diluent. Mix and filter. 461

VP V

GLUCOSE MONOHYDRATE

Reference solution: Dissolve a quantity of glucosamine hydrochloride RS in the diluent to obtain a solution having a known concentration of about 3.0 mg/ml. Chromatographic system A column (15 cm × 4.6 mm) packed with aminopropylsilyl silica gel for chromatography R (5 µm). Column temperature: 35 °C. Detector: A spectrophotometer set at 195 nm. Flow rate: 1.5 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject the reference solution. The retention time of the glucosamine peak is about 10 min. The chromatogram shows a large additional peak near the void volume due to chloride ion. The symmetry factor of the peak due to glucosamine is not greater than 2.0 and the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. The number of theoretical plates is not less than 1500. Inject alternately the reference solution and the test solution. Calculate the content of glucosamine, C6H13NO5, in the tablets using the areas of the principal peaks in the chromatograms obtained with the test solution, the reference solution and the declared content of C6H13NO5 in glucosamine hydrochloride RS.

Storage Store in an airtight container, in a cool place, protected from light. Action and use Anti-arthritic. Usual strength 250 mg; 500 mg.

M. 198.2

Glucose monohydrate is the monohydrate of D-(+)glucopyranose.

Characters A white, crystalline powder, odourless with a sweet taste. Freely soluble in water, sparingly soluble in ethanol (96%).

Identification

A. Dissolve 0.2 g in 5 ml of water, add 2 ml of Fehling reagent R, heat to boiling, a brownish-red precipitate is formed. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Water - methanol - anhydrous acetic acid ethylene chloride (10 : 15 : 25 : 50). The solvents should 462

Appearance of solution Dissolve 10.0 g of substance to be examined in 15 ml of water. The solution is clear (Appendix 9.2) and not more intensely coloured than reference solution BY7 (Appendix 9.3, method 2). Specific optical rotation +52.5° to +53.3°, calculated with reference to the anhydrous substance (Appendix 6.4). Determined on the solution prepared by dissolving 10.0 g of substance to be examined in 80 ml of water, adding 0.2 ml of a 10% solution of ammonia R, allowing to stand for 30 min and diluting to 100.0 ml with water.

GLUCOSE MONOHYDRATE Glucosum monohydricum Dextrose monohydrate C6H12O6,H2O

be measured accurately because a slight excess of water makes cloudiness. Test solution: Dissolve 10 mg of the substance to be examined in mixture of water - methanol (2 : 3), dilute to 20 ml with the same mixture of solvents. Reference solution (1): Dissolve 10 mg of glucose RS in a mixture of water - methanol (2 : 3) and dilute to 20 ml with the same mixture of solvents. Reference solution (2): Dissolve 10 mg of each of fructose RS, glucose RS, lactose RS and sucrose RS in a mixture of water - methanol (2 : 3) and dilute to 20 ml with the same mixture of solvents. Procedure: Apply separately to the plate 2 µl of each solution, dry carefully the starting points. Develop over a path of 15 cm. Removal of the plate and dry in a current of warm air. Repeat the development immediately, after renewing the mobile phase. Dry the plate in a current of warm air and spray evenly with a solution of 0.5 g of thymol R in 5 ml of a mixture of sulfuric acid - ethanol (96%) (5 : 95). Heat at 130 °C for 10 min. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) shows four clearly separated spots. C. It complies with the test for Specific optical rotation.

Acidity or alkalinity Dissolve 6.0 g of substance to be examined in 25 ml of carbon dioxide-free water R and add 0.3 ml of phenolphthalein solution R. The solution is colourless. Not more than 0.15 ml of 0.1 M sodium hydroxide VS is required to change the colour of the indicator to pink. Chlorides Not more than 0.0125% (Appendix 9.4.5) Solution S: Dissolve 10.0 g substance to be examined in water and dilute to 100 ml with the same solvent. 4 ml of solution S diluted to 15 ml with water complies with the limit test for chlorides. Sulfates Not more than 0.02% (Appendix 9.4.14).

VP V

ANHYDROUS GLUCOSE

7.5 ml of solution S diluted to 15 ml with water complies with the limit test for sulfates.

5 ml of solution S diluted to 15 ml with water complies with the limit test for calcium.

Arsenic Not more than 1 ppm (Appendix 9.4.2). 1.0 g complies with limit test A for arsenic.

Water 7.0% to 9.5% (Appendix 10.3). Determined on 0.500 g.

Heavy metals Not more than 5 ppm (Appendix 9.4.8). Dissolve 4.0 g substance to be examined in 20 ml of water. 12 ml of the resulting solution complies with the limit test for heavy metals, method 1. Prepare the standard using lead standard solution (1 ppm Pb) R.

Pyrogens If intended for use in large-volume preparations for parenteral use, it complies with the test for pyrogens (Appendix 13.4). Inject per kilogram of the rabbit’s mass 10 ml of a solution containing 55 mg per millilitre of the substance to be examined in water for injections R.

Less soluble sugars and dextrins Dissolve 1.0 g by boiling in 30 ml of ethanol (90%) R. Cool, the resulting solution is not more opalescent than 30 ml of ethanol (90%) R.

Storage Store in a well-closed container.

Soluble starch Dissolve 2.5 g substance to be examined in 25 ml of water, boil for 1 min. Cool, add 0.1 ml of 0.1 N iodine solution VS, the blue colour does not appear. Sulfites Not more than 15 ppm of SO2. Test solution: Dissolve 5.0 g in 40 ml of water, add 2.0 ml of 0.1 M sodium hydroxide and dilute to 50.0 ml with water. Reference solution: Dissolve 76 mg of sodium metabisulfite R in water and dilute to 50.0 ml with the same solvent. Dilute 5.0 ml of this solution to 100.0 ml with water. To 3.0 ml of this solution add 4.0 ml of 0.1 M sodium hydroxide R and dilute to 100.0 ml with water. Add to 10.0 ml of each test solution and reference solution 1 ml of a 310 g/l solution of hydrochloric acid R, 2.0 ml of decolorised fuchsin solution R and 2.0 ml of a 0.5% (v/v) solution of formaldehyde R. Allow to stand for 30 min and measure the absorbances at the maximum at 583 nm (Appendix 4.1). Use a solution prepared in the same manner using 10.0 ml of water as the blank. The absorbance of the test solution is not greater than that of the reference solution. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Dissolve 5.0 g substance to be examined in 5 ml of water, add 2 ml of sulfuric acid R, evaporate to dryness on a water-bath and ignite to constant mass. If necessary, repeat the heating with sulfuric acid R. Barium To 10 ml of solution S add 1 ml of 1 M sulfuric acid R. When examined immediately and after 1 h, any opalescence in the solution is not more intense than that in a mixture of 1 ml of water and 10 ml of solution S. Calcium Not more than 0.02% (Appendix 9.4.3).

Preparations Glucose intravenous infusion; Oral rehydration salts; Potassium chloride and glucose intravenous infusion Potassium chloride, sodium chloride and glucose intravenous infusion; Sodium chloride and glucose intravenous infusion. ANHYDROUS GLUCOSE Glucosum anhydricum Dextrose

C6H12O6

M. 180.2

Anhydrous glucose is D-(+)-glucopyranose.

Characters A white, crystalline powder, odourless with a sweet taste. Freely soluble in water, sparingly soluble in ethanol (96%). Identification A. Dissolve 0.2 g in 5 ml of water, add 2 ml of Fehling reagent R, heat to boiling, a brownish-red precipitate is formed. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Water - methanol - anhydrous acetic acid ethylene chloride (10 : 15 : 25 : 50). The solvents should be measured accurately because a slight excess of water makes cloudiness. Test solution: Dissolve 10 mg of the substance to be examined in mixture of water - methanol (2 : 3), dilute to 20 ml with the same mixture of solvents. Reference solution (1): Dissolve 10 mg of glucose RS in a mixture of water - methanol (2 : 3) and dilute to 20 ml with the same mixture of solvents. 463

ANHYDROUS GLUCOSE

Reference solution (2): Dissolve 10 mg of each of fructose RS, glucose RS, lactose RS and sucrose RS in a mixture of water - methanol (2 : 3) and dilute to 20 ml with the same mixture of solvents. Procedure: Apply separately to the plate 2 µl of each solution, dry carefully the starting points. Develop over a path of 15 cm. Removal of the plate and dry in a current of warm air. Repeat the development immediately, after renewing the mobile phase. Dry the plate in a current of warm air and spray evenly with a solution of 0.5 g of thymol R in 5 ml of a mixture of sulfuric acid - ethanol (96%) (5 : 95). Heat at 130 °C for 10 min. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) shows four clearly separated spots. C. It complies with the test for Specific optical rotation.

Appearance of solution Dissolve 10.0 g of substance to be examined in 15 ml of water. The solution is clear (Appendix 9.2) and not more intensely coloured than reference solution BY7 (Appendix 9.3, method 2). Specific optical rotation +52.5° to +53.3°, calculated with reference to the anhydrous substance (Appendix 6.4). Determined on the solution prepared by dissolving 10.0 g of substance to be examined in 80 ml of water, adding 0.2 ml of a 10% solution of ammonia R, allowing to stand for 30 min and diluting to 100.0 ml with water. Acidity or alkalinity Dissolve 6.0 g of substance to be examined in 25 ml of carbon dioxide-free water R and add 0.3 ml of phenolphthalein solution R. The solution is colourless. Not more than 0.15 ml of 0.1 N sodium hydroxide VS is required to change the colour of the indicator to pink. Chlorides Not more than 0.0125% (Appendix 9.4.5) Solution S: Dissolve 10.0 g substance to be examined in water and dilute to 100 ml with the same solvent. 4 ml of solution S diluted to 15 ml with water complies with the limit test for chlorides. Sulfates Not more than 0.02% (Appendix 9.4.14). 7.5 ml of solution S diluted to 15 ml with water complies with the limit test for sulfates. Arsenic Not more than 1 ppm (Appendix 9.4.2). 1.0 g complies with limit test A for arsenic. 464

VP V

Heavy metals Not more than 5 ppm (Appendix 9.4.8). Dissolve 4.0 g substance to be examined in 20 ml of water. 12 ml of the resulting solution complies with the limit test for heavy metals, method 1. Prepare the standard using lead standard solution (1 ppm Pb) R. Less soluble sugars and dextrins Dissolve 1.0 g by boiling in 30 ml of ethanol (90%) R. Cool, the resulting solution is not more opalescent than 30 ml of ethanol (90%) R. Soluble starch Dissolve 2.5 g substance to be examined in 25 ml of water, boil for 1 min. Cool, add 0.1 ml of 0.1 N iodine solution VS, the blue colour does not appear. Sulfites Not more than 15 ppm of SO2. Test solution: Dissolve 5.0 g in 40 ml of water, add 2.0 ml of 0.1 M sodium hydroxide and dilute to 50.0 ml with water. Reference solution: Dissolve 76 mg of sodium metabisulfite R in water and dilute to 50.0 ml with the same solvent. Dilute 5.0 ml of this solution to 100.0 ml with water. To 3.0 ml of this solution add 4.0 ml of 0.1 M sodium hydroxide R and dilute to 100.0 ml with water. Add to 10.0 ml of each test solution and reference solution 1 ml of a 310 g/l solution of hydrochloric acid R, 2.0 ml of decolorised fuchsin solution R and 2.0 ml of a 0.5% (v/v) solution of formaldehyde R. Allow to stand for 30 min and measure the absorbances at the maximum at 583 nm (Appendix 4.1). Use a solution prepared in the same manner using 10.0 ml of water as the blank. The absorbance of the test solution is not greater than that of the reference solution. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Dissolve 5.0 g substance to be examined in 5 ml of water, add 2 ml of sulfuric acid R, evaporate to dryness on a water-bath and ignite to constant mass. If necessary, repeat the heating with sulfuric acid R. Water Not more than 1.0% (Appendix 10.3). Determined on 0.500 g of the substance being examined. Barium To 10 ml of solution S add 1 ml of 1 M sulfuric acid R. When examined immediately and after 1 h, any opalescence in the solution is not more intense than that in a mixture of 1 ml of water and 10 ml of solution S. Calcium Not more than 0.02% (Appendix 9.4.3). 5 ml of solution S diluted to 15 ml with water complies with the limit test for calcium.

VP V

Pyrogens If intended for use in large-volume preparations for parenteral use, it complies with the test for pyrogens (Appendix 13.4). Inject per kilogram of the rabbit’s mass 10 ml of a solution containing 50 mg per millilitre of the substance to be examined in water for injections R. Storage Store in a well-closed container. Preparations Glucose intravenous infusion; Oral rehydration salts; Potassium chloride and glucose intravenous infusion Potassium chloride, sodium chloride and glucose intravenous infusion; Sodium chloride and glucose intravenous infusion. GLUCOSE INJECTION Injectio Glucosi

GLUCOSE INTRAVENOUS INFUSION

Assay To an measured volume containing 2 g to 5 g of glucose, C6H12O6, add 0.2 ml of 5 M ammonia R and sufficient water to produce 100.0 ml. Mix well, allow to stand for 30 minutes and determine the optical rotation in a 2 dm tube (Appendix 6.4). The observed rotation in degrees multiplied by 0.9477 represents the weight in g of glucose, C6H12O6, in the volume taken for assay. Storage The preparation is usually packed 5 ml ampoules and is stored at a temperature not exceeding 25 °C. Usual strength 1.5 g/5 ml (calculated as anhydrous glucose). GLUCOSE INTRAVENOUS INFUSION Infusio Glucosi

Glucose injection is a sterile solution of anhydrous glucose or glucose monohydrate in water for injections. The injection contains no preservative and is sterilized immediately after preparation. The injection complies with the requirements stated under "Injections, infusions" (Appendix 1.19) and with the following requirements.

Glucose intravenous infusion is a sterile solution of anhydrous glucose or glucose monohydrate in water for injections. The infusion contains no preservative and is sterilized immediately after preparation. The intravenous infusion complies with the requirements stated under "Injections, infusions" (Appendix 1.19) and with the requirements stated under Glucose Injection, and with the following requirements.

Content of glucose, C6H12O6, 95.0% to 105.0% of the stated amount.

Content of glucose, C6H12O6, 95.0% to 105.0% of the stated amount.

Characters A clear, colourless or faintly yellowish solution.

Identification A. To 1 ml of the injection add 5 ml of Fehling reagent R, and boil. A red precipitate of copper (I) oxide is produced. B. The solution prepared as directed in the Assay is dextrorotatory.

Identification A. To 1 ml of the injection add 5 ml of Fehling reagent R, and boil. A red precipitate of copper (I) oxide is produced. B. The solution prepared as directed in the Assay is dextrorotatory. 5-Hydroxymethylfurfural and related substances Dilute a measured volume of the injection, equivalent to 1.0 g of glucose, C6H12O6, with water to 250.0 ml. The absorbance (Appendix 4.1) of the resulting solution at the maximum at 284 nm is not more than 0.25. pH 3.5 to 6.5 (Appendix 6.2). Use a solution prepared by diluting the injection, if necessary, with water to obtain a solution containing 5% of glucose, C6H12O6, and to which 0.30 ml of a saturated solution of potassium chloride R has been added for each 100 ml of solution.

5-Hydroxymethylfurfural and related substances Dilute a measured volume of the injection, equivalent to 1.0 g of glucose, C6H12O6, with water to 250.0 ml. The absorbance (Appendix 4.1) of the resulting solution at the maximum at 284 nm is not more than 0.25. pH 3.5 to 6.5 (Appendix 6.2). Use a solution prepared by diluting the injection, if necessary, with water to obtain a solution containing 5% of glucose, C6H12O6, and to which 0.30 ml of a saturated solution of potassium chloride R has been added for each 100 ml of solution. Assay To an measured volume containing 2 g to 5 g of glucose, C6H12O6, add 0.2 ml of 5 M ammonia R and sufficient water to produce 100.0 ml. Mix well, allow to stand for 465

GLUTATHIONE

30 minutes and determine the optical rotation in a 2 dm tube (Appendix 6.4). The observed rotation in degrees multiplied by 0.9477 represents the weight in g of glucose, C6H12O6, in the volume taken for assay.

Bacterial endotoxins Carry out the test for bacterial endotoxins (Appendix 13.2). Dilute the infusion, if necessary, with water BET to give a solution containing the equivalent of 50.0 mg of glucose, C6H12O6, per ml (solution A). The endotoxin limit concentration of solution A is 0.25 IU per ml. Carry out the test using the maximum valid dilution of solution A calculated from the declared sensitivity of the lysate used in the test.

VP V

Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2).

Specific optical rotation -15.5° to -17.5°, calculated with reference to the dried substance (Appendix 6.4). Dissolve 1.0 g in water and dilute to 25.0 ml with the same solvent.

Related substances Examine by capillary electrophoresis (Appendix 5.7). Prepare the solutions immediately before use. Internal standard solution (1): Dissolve 0.100 g of phenylalanine R in the electrolyte solution and dilute to 50.0 ml with the same solution. Internal standard solution (2): Dilute 10.0 ml of internal Particulate contamination When supplied in a container with a nominal volume of standard solution (1) to 100.0 ml with the electrolyte more than 100 ml, it complies with Test A. Sub-visible solution. Test solution (1): Dissolve 0.200 g of the substance to be particles under Limit Test for Particles (Appendix 11.8). examined in the electrolyte solution and dilute to 10.0 ml Storage with the same solution. Store in a airtight container at a temperature not exceeding Test solution (2): Dissolve 0.200 g of the substance to be 25 °C, protected from light. examined in internal standard solution (2) and dilute to 10.0 ml with the same solution. Usual strength Reference solution (1): Dissolve 20.0 mg of the substance 5%; 10%; 20%; 30%. to be examined in internal standard solution (1) and dilute to 10.0 ml with the same solution. Reference solution (2): Dilute 5.0 ml of reference solution GLUTATHIONE (1) to 50.0 ml with the electrolyte solution. Glutathionum Reference solution (3): Dissolve 0.200 g of the substance to be examined in 5 ml of the electrolyte solution. Add 1.0 ml of internal standard solution (1), 0.5 ml of a 2 mg/ml solution of L-cysteine R (impurity B) in the electrolyte solution, 0.5 ml of a 2 mg/ml solution of oxidised L-glutathione R (impurity C) in the electrolyte solution and 0.5 ml of a C10H17N3O6S   M. 307.3  2 mg/ml solution of L-g-glutamyl-L-cysteine R (impurity Glutathione is L-γ-glutamyl-L-cysteinylglycine. It contains D) in the electrolyte solution. Dilute to 10.0 ml with the not less than 98.0% and not more than 101.0% of C10H17N3O6S, electrolyte solution. Capillary: calculated with reference to the dried substance. Fermentation product. Capillary column: uncoated fused silica. Size column: total length = 60 cm, length to the detector Characters cell = 50 cm, internal diameter = 75 µm. White or almost white, crystalline powder or colourless crystals. Column temperature: 25 °C. Freely soluble in water, very slightly soluble in ethanol Electrolyte solution: Dissolve 1.50 g of anhydrous sodium (96%) and in methylene chloride. dihydrogen phosphate R in 230 ml of water and adjust to pH 1.80 with phosphoric acid R. Dilute to 250.0 ml Identification A. The infrared absorption spectrum (Appendix 4.2) of the with water. Check the pH and if necessary, adjust with substance to be examined is concordant with the spectrum phosphoric acid R or dilute sodium hydroxide solution R. of glutathione RS. Preconditioning of a new capillary: Rinse the new B. It complies with the test for Specific optical rotation capillary before the first injection with 0.1 M hydrochloric (Appendix 6.4). acid R at 138 kPa for 20 min and with water at 138 kPa for 10 min; for complete equilibration, condition the capillary Appearance of solution Solution S: Dissolve 5.0 g in distilled water R and dilute to with the electrolyte solution at 350 kPa for 40 min, and subsequently at a voltage of 20 kV for 60 min. 50 ml with the same solvent.

466

VP V

Preconditioning of the capillary: Rinse the capillary with the electrolyte solution at 138 kPa for 40 min. Between-run rinsing: Rinse the capillary with water at 138 kPa for 1 min, with 0.1 M sodium hydroxide R at 138 kPa for 2 min, with water at 138 kPa for 1 min, with 0.1 M hydrochloric acid R at 138 kPa for 3 min and with the electrolyte solution at 138 kPa for 10 min. Detection: Spectrophotometer at 200 nm. Migration: Apply a voltage of 20 kV. Injection: Under pressure 3.45 kPa for 5 s. Run time: 45 min. Procedure: Injec test solutions (1) and (2), reference solutions (2) and (3) and the electrolyte solution (blank solution). Relative migration with reference to the internal standard (about 14 min): Impurity A = about 0.77; impurity B = about 1.04; impurity E = about 1.2; impurity C = about 1.26; impurity D = about 1.3. System suitability: In the chromatogram obtained with reference solution (3), the resolution between the peaks due to the internal standard and impurity B is at least 1.5. If necessary, increase the pH with a 8.5% soluion of sodium hydroxide R. In the chromatogramobtained with reference solution (3), peak-to-valley ratio (Hp/Hv) is at least 2.5, where Hp =height above the baseline of the peak due to impurity D and Hv = height above the baseline of the lowest point of the curve separating this peak from the peak due to glutathione. If necessary, lower the pH with phosphoric acid R. Check that in the electropherogram obtained with test solution (1) there is no peak with the same migration time as the internal standard (in such case correct the area of the phenylalanine peak). Limits: In the chromatogram obtained with test solution (2): Corrected areas: Divide all the peak areas by the corresponding migration times. Correction factors: For the calculation of content, multiply the ratio of time-corrected peak areas of impurity and the internal standard by the corresponding correction factor: impurity B = 3.0; impurity D = 1.4. Impurity C: The ratio of the area of the peak due to impurity C to the area of the peak due to the internal standard is not more than 1.5 times the ratio of the area of the peak due to glutathione to the area of the peak due to the internal standard in the electropherogram obtained with reference solution (2) (1.5%). Impurity D: The ratio of the area of the peak due to impurity D to the area of the peak due to the internal standard is not more than the ratio of the area of the peak due to glutathione to the area of the peak due to the internal standard in the electropherogram obtained with reference solution (2) (1.0%). Impurities A, B and E: For each impurity, the ratio of the area of the peak due to each impurity to the area of the peak due to the internal standard is not more than 0.5 times the

GLUTATHIONE

ratio of the area of the peak due to glutathione to the area of the peak due to the internal standard in the electropherogram obtained with reference solution (2) (0.5%). Any other impurity: For each impurity, the ratio of the area of the peak due to each impurity to the area of the peak due to the internal standard is not more than 0.2 times the ratio of the area of the peak due to glutathione to the area of the peak due to the internal standard in the electropherogram obtained with reference solution (2) (0.2%). Total of the ratio of the area of the peak due to all impurity to the area of the peak due to the internal standard is not more than 2.5 times the ratio of the area of the peak due to glutathione to the area of the peak due to the internal standard in the electropherogram obtained with reference solution (2) (2.5%). Disregard limit: The ratio of the area of the peak due to all impurity to the area of the peak due to the internal standard is less than or equal 0.05 times the ratio of the area of the peak due to glutathione to the area of the peak due to the internal standard in the electropherogram obtained with reference solution (2) (0.05%). Note: Impurity A: L-cysteinylglycine. Impurity B: (2R)-2-amino-3-sulfanylpropanoic acid (cysteine). Impurity C: Bis (L-g-glutamyl-L-cysteinylglycine) disulfide (L-glutathione oxidised). Impurity D: L-g-glutamyl-L-cysteine. Impurity E: Unknown structure (product of degradation).

Chlorides Not more than 200 ppm (Appendix 9.4.5). Dilute 2.5 ml of solution S to 15 ml with water. Sulfates Not more than 300 ppm (Appendix 9.4.14). Dilute 5 ml of solution S to 15 ml with distilled water R. Ammonium Not more than 200 ppm (Appendix 9.4.1). 50 mg complies with method B. Prepare the standard using 0.1 ml of ammonium standard solution (100 ppm NH4) R. Iron Not more than 10 ppm (Appendix 9.4.13). Dissolve 1.0 g in 10 ml of dilute hydrochloric acid R. Shake with 3 quantities, each of 10 ml, of methyl isobutyl ketone R1, shaking for 3 min each time. To the combined organic layers, add 10 ml of water and shake for 3 min. The aqueous layer complies with the test. Heavy metals Not more than 10 ppm (Appendix 9.4.8). 12 ml of solution S complies with limit test for heavy metals, method 1. Prepare the standard using lead standard solution (1 ppm Pb) R. 467

VP V

GLYCEROL

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C; 3 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay In a ground-glass-stoppered flask, dissolve 0.500 g of the substance to be examined and 2 g of potassium iodide R in 50 ml of water. Cool the solution in iced water and add 10 ml of a 25% solution of hydrochloric acid R and 20.0 ml of 0.1 N iodine VS. Stopper the flask and allow to stand in the dark for 15 min. Titrate with 0.1 N sodium thiosulfate VS using 1 ml of starch solution R, added towards the end of the titration, as indicator. Carry out a blank titration. 1 ml of 0.1 N iodine VS is equivalent to 30.73 mg of C10H17N3O6S. Storage Store in an airtight container, protected from light. Action and use Amino acid.

GLYCEROL Glycerinum Glycerin OH OH

C3H8O3  

M. 92.1 

Glycerin is propane-1,2,3-triol. It contains not less than 98.0% and not more than 101.0% of C3H8O3 (m/m), calculated with reference to the anhydrous substance.

Characters Syrupy liquid, unctuous to the touch, colourless or almost colourless, clear, very hygroscopic. Miscible with water and with ethanol (96%), slightly soluble in acetone, practically insoluble in fatty oils and in essential oils. Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D. A. To 5 ml of the substance to be examined, add 1 ml of water and mix carefully. The infrared absorption spectrum (Appendix 4.2) of the solution is concordant with the reference spectrum of glycerin (85%). 468

Appearance of solution Solution S: Dilute 100.0 g of the substance to be examined to 200 ml with carbon dioxide-free water R. Solution S is clear (Appendix 9.2). Dilute 10 ml of solution S to 25 ml with water. The solution is colourless (Appendix 9.3, method 2). Acidity or alkalinity To 50 ml of solution S add 0.5 ml of phenolphthalein solution R. The solution is colourless. Not more than 0.2 ml of 0.1 M sodium hydroxide R is required to change the colour of the indicator to pink. Keep the last solution to the test for Esters. Refractive index 1.470 to 1.475 (Appendix 6.1).

Preparations Tablets, capsules, injection.

HO

B. Mix 1 ml with 0.5 ml of nitric acid R. Superimpose 0.5 ml of 10% solution of potassium dichromate R. A blue ring develops at the interface of the liquids. Within 10 min, the blue colour does not diffuse into the lower layer. C. Heat 1 ml with 2 g of potassium hydrogen sulfate R in an evaporating dish. Vapours (acrolein) are evolved which blacken filter paper impregnated with alkaline potassium tetraiodomercurate solution R. D. It complies with the test for Refractive index.

Impurity A and related substances Examine by gas chromatography (Appendix 5.2). Test solution: Dilute 10.0 ml of solution S to 100.0 ml with water. Reference solution (1): Dilute 10.0 g of glycerin R1 to 20.0 ml with water. Dilute 10.0 ml of the solution to 100.0 ml with water. Reference solution (2): Dissolve 1.000 g of diethylene glycol R in water and dilute to 100.0 ml with the same solvent. Reference solution (3): Dilute 1.0 ml of reference solution (2) to 10.0 ml with reference solution (1). Dilute 1.0 ml of this solution to 20.0 ml with reference solution (1). Reference solution (4): Mix 1.0 ml of the test solution and 5.0 ml of reference solution (2) and dilute to 100.0 ml with water R. Dilute 1.0 ml of this solution to 10.0 ml with water. Reference solution (5): Dilute 5.0 ml of reference solution (2) to 100.0 ml with water. Chromatographic system: A column (30 m × 0.53 mm) coated with 6% of polycyanopropylphenyl siloxane and 94% of polydimethylsiloxane. Carrier gas: Helium for chromatography. Linear velocity: 38 cm/s. Split ratio: 1 : 10. Temperature programme:

VP V

GLYCEROL

Column

Time (min)

Temperature (°C)

0 0 - 16 16 - 20

100 100 → 220 220

Injection port

220

Detector

250

Detector: A flame-ionisation detector. Volume of injection: 0.5 µl. Elution order: Impurity A, glycerin. Procedure: System suitability: In the chromatogram obtained with reference solution (4), the resolution between the peaks due to impurity A and glycerin is at least 7.0. Limits: Impurity A: The area of the peak due to impurity A is not more than the area of the corresponding peak in the chromatogram obtained with reference solution (3) (0.1%). Any other impurity with a retention time less than the retention time of glycerin: For each impurity, the area is not more than the area of the peak due to impurity A in the chromatogram obtained with reference solution (3) (0.1%). Total of all impurities with retention times greater than the retention time of glycerin: not more than 5 times the area of the peak due to impurity A in the chromatogram obtained with reference solution (3) (0.5%). Disregard any peak with an area less than 0.05 times the area of the peak due to impurity A in the chromatogram obtained with reference solution (5) (0.05%). Note: Impurity A: 2,2’-oxydiethanol (diethylene glycol). Impurity B: ethane-1,2-diol (ethylene glycol). Impurity C: (RS)-propane-1,2-diol (propylene glycol).

Aldehydes Not more than 10 ppm. Place 7.5 ml of solution S in a ground-glass-stoppered flask and add 7.5 ml of water and 1.0 ml of decolorised pararosaniline solution R. Close the flask and allow to stand for 1 h at a temperature of 25 °C ± 1 °C. The absorbance (Appendix 4.1) of the solution measured at 552 nm is not greater than that of a standard prepared at the same time and in the same manner using 7.5 ml of formaldehyde standard solution (5 ppm CH2O) R and 7.5 ml of water. The test is not valid unless the standard is pink. Esters Add 10.0 ml of 0.1 N sodium hydroxide VS to the final solution obtained in the test for Acidity or alkalinity. Boil under a reflux condenser for 5 min. Cool. Add 0.5 ml of phenolphthalein solution R and titrate with 0.1 N hydrochloric acid VS. Not less than 8.0 ml of 0.1 N hydrochloric acid VS is required to change the colour of the indicator.

Halogenated compounds Not more than 35 ppm. To 10 ml of solution S add 1 ml of 2 M sodium hydroxide R, 5 ml of water and 50 mg of halogen-free nickel-aluminium alloy R. Heat on a water-bath for 10 min, allow to cool and filter. Rinse the flask and the filter with water until 25 ml of filtrate is obtained. To 5 ml of the filtrate add 4 ml of ethanol (96%) R, 2.5 ml of water, 0.5 ml of nitric acid R and 0.05 ml of 1.7% solution of silver nitrate R and mix. Allow to stand for 2 min. Any opalescence in the solution is not more intense than that in a reference prepared at the same time. Reference solution: Mixing 7.0 ml of chloride standard solution (5 ppm Cl) R, 4 ml of ethanol (96%) R, 0.5 ml of water, 0.5 ml of nitric acid R and 0.05 ml of 1.7% solution of silver nitrate R. Chlorides Not more than 10 ppm (Appendix 9.4.5). 1 ml of solution S diluted to 15 ml with water complies with the limit test for chlorides. Prepare the standard using 1 ml of chloride standard solution (5 ppm Cl) R diluted to 15 ml with water. Sugars To 10 ml of solution S add 1 ml of 1 M sulfuric acid R and heat on a water-bath for 5 min. Add 3 ml of 2 M sodium hydroxide R in carbon dioxide-free water R, mix and add dropwise 1 ml of freshly prepared a 12.5% solution of copper sulfate R. The solution is clear and blue. Continue heating on the water-bath for 5 min. The solution remains blue and no precipitate is formed. Heavy metals Not more than 5 ppm (Appendix 9.4.8). Dilute 8 ml of solution S to 20 ml with water. 12 ml of the solution complies with limit test for heavy metals, method 1. Prepare the standard using lead standard solution (1 ppm Pb) R. Water Not more than 2.0% (Appendix 10.3). Determined on 1.000 g. Sulfated ash Not more than 0.01% (Appendix 9.9, method 2). Determined on 5.0 g after heating to boiling and ignition. Assay Thoroughly mix 0.075 g of the substance to be examined with 45 ml of water. Add 25.0 ml of a mixture of 0.1 M sulfuric acid - 0.1 M sodium periodate (1 : 20). Allow to stand protected from light for 15 min. Add 5.0 ml of a 50% solution of ethylene glycol R in water and allow to stand protected from light for 20 min. Using 0.5 ml of phenolphthalein solution R as indicator, titrate with 0.1 N sodium hydroxide VS. Carry out a blank titration. 469

VP V

GLYCEROL MONOSTEARATE 40 - 55

1 ml of 0.1 N sodium hydroxide VS is equivalent to 9.21 mg of C3H8O3.

Storage Store in an airtight container, protected from light. Action and use Laxative, solvent, excipients. Preparations Suppositories solution. GLYCEROL MONOSTEARATE 40 - 55 Glyceroli monostearas 40 - 55 Glycerol monostearate 40 - 55 is a mixture of monoacylglycerols, mainly monostearoylglycerol, together with variable quantities of di- and triacylglycerols. It contains 40.0% to 55.0% of monoacylglycerols, 30.0% to 45.0% of diacylglycerols and 5.0% to 15.0% of triacylglycerols, obtained by partial glycerolysis of vegetable oils mainly containing triacylglycerols of palmitic or stearic acid or by esterification of glycerol with stearic acid 50 (type I), stearic acid 70 (type II) or stearic acid 95 (type III). The fatty acids may be of vegetable or animal origin.

Characters A hard, waxy mass or unctuous powder or flakes, white or almost white, practically insoluble in water, soluble in ethanol (96%) at 60 °C. Identification A. Melting point (Appendix 6.7): 54 °C to 64 °C. Introduce the melted substance into the capillary tubes and allow to stand for 24 h in a well-closed container . B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel R. Mobile phase: Hexane - ether (30 : 70). Test solution: Dissolve 1.0 g of the substance to be examined in methylene chloride R, with gentle heating, and dilute to 20 ml with the same solvent. Reference solution: Dissolve 1.0 g of glycerol monostearate 40-55 RS in methylene chloride R, with gentle heating, and dilute to 20 ml with the same solvent. Procedure: Apply to the plate 10 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in air. Spray with a 0.1 g/l solution of rhodamine B R in ethanol (96%) R and examine in ultraviolet light at 365 nm. The spots in the chromatogram obtained with the test solution are similar in position to those in the chromatogram obtained with the reference solution. C. It complies with the test for Composition of fatty acids according to the type stated on the label (see Tests). D. It complies with the limits of theAssay (monoacylglycerol content). 470

Acid value Not more than 3.0 (Appendix 7.2), Determined on 1.0 g, using a mixture of equal volumes of ethanol (96%) R and toluene R as solvent and heating gently. Iodine value Not more than 3.0 (Appendix 7.5). Saponification value 158 to 177 (Appendix 7.7). Determined on 2.0 g. Carry out the titration with heating. Free glycerol Not more than 6.0%, determined as described under Assay. Composition of fatty acids Examine by gas chromatography (Appendix 5.2). Test solution: Dissolve 0.10 g of the substance to be examined in 5 ml of 2.0% solution of sodium hydroxide in methanol in a 25-ml conical flask and boil under a reflux condenser for 30 min. Add 5.0 ml of boron trifluoridemethanol solution R through the condenser and boil for 30 min. Add 4 ml of heptane R through the condenser and boil for 5 min. Cool and add 10.0 ml of saturated sodium chloride solution R, shake for about 15 s and add a quantity a saturated sodium chloride solution R such that the upper phase is brought into the neck of the flask. Collect 2 ml of the upper phase, wash with 2 ml of water and dry over anhydrous sodium sulfate R. Reference solution (1): Dissolve 0.50 g of a mixture of the following substances: Methyl laurate, methyl myristate, methyl palmitate, methyl stearate, methyl arachidate, methyl oleate (each substance less than 100 mg) in heptane R and dilute to 50.0 ml with the same solvent. Reference solution (2): Dilute 1.0 ml of reference solution (1) to 10.0 ml with heptane R. Reference solution (3): Dissolve 0.50 g of a mixture of methyl palmitate and methyl stearate in heptane R and dilute to 50.0 ml with the same solvent. Chromatographic system: A fused silica, glass or quartz column, 10 - 30 m long and 0.2 - 0.8 mm diameter, coated with poly [(cyanopropyl) (phenyl)][(phenyl)(methyl)]siloxane R or macrogol 20000 (film thickness 0.1 - 0.5 mm or some other suitable stationary phase). Carrier gas: Helium for chromatography R or nitrogen for chromatography R Flow rate: 1.3 ml/min (for a column 0.32 mm diameter). Split ratio: 1 : 100 or less, according to the internal diameter of the column used (1 : 50 when 0.32 mm diameter). Temperature: Column: 160 ºC to 200 ºC, according to the length and type of column used (200 ºC for a column 30 m long and coated with a layer of macrogol 20 000 ); if necessary of

VP V

GLYCEROL MONOSTEARATE 40 - 55

where prescribed, raise the temperature of the column at a rate of 3 ºC/min from 170 ºC to 230 ºC (for the macrogol 20 000 column). Injection port: 250 ºC. Detector: 250 ºC. Detector: A flame-ionisation detector. Volume of injection: 1 µl. System suitability: The resolution between the peaks due to methyl oleate and methyl stearate in the chromatogram obtained with reference solution (1) is at least 1.8. The signal-to-noise ratio of the peak due to methyl myristate and methyl stearate in the chromatogram obtained with reference solution (2) is at least 5. Number of theoretical plates: minimum 30 000 calculated for the peak due to methyl stearate in the chromatogram obtained with reference solution (1). Assessment of chromatograms Qualitative analysis: Identify the peaks in the chromatogram obtained reference solution (3). Quantitative analysis: The normalisation procedure is used in which the sum of the areas of the peaks in the chromatogram, except that of the solvent, is set at 100%. The content of a constituent is calculated by determining the area of the corresponding peak as a percentage of the sum of the areas of all the peaks. Disregard any peak with an area less than 0.05% of the total area. The fatty acid fraction of the substance to be examined has the following composition: Fatty acid used for production by esterification

Composition of fatty acids

Glycerol monostearate 40-50 type I

Stearic acid 50

Stearic acid: 40.0% to 60.0% Sum of the contents of palmitic and stearic acids: Not less than 90.0%

Glycerol monostearate 40-50 type II

Stearic acid 70

Stearic acid: 60.0% to 80.0% Sum of the contents of palmitic and stearic acids: Not less than 90.0%

Glycerol monostearate 40-50 type III

Stearic acid 95

Stearic acid: 80.0% to 99.0% Sum of the contents of palmitic and stearic acids: Not less than 96.0%

Nickel Not more than 1 ppm (Appendix 9.4.11). Water Not more than 1.0% (Appendix 10.3).

Determined on 1.0 g. Use pyridine R as the solvent and heat gently.

Total ash Not more than 0.1% (Appendix 9.8). Determined on 1.0 g. Assay Determine the free glycerol content and the mono-, di- and triacylglycerol contents by size-exclusion chromatography (Appendix 5.5). Mobile phase: Tetrahydrofuran R. Test solution: Weigh accurately about 0.2 g (m), to the nearest 0.1 mg into a 15 ml flask. Add 5 ml of tetrahydrofuran R and shake to dissolve. Reweigh the flask and calculate the total mass of solvent and substance (M). Reference solutions: Weigh accurately, respectively weigh, to the nearest 0.1 mg, about 2.5 mg, 5 mg, 10 mg and 20 mg of glycerol R into four 15 ml flasks. Add 5 ml of tetrahydrofuran R and shake to dissolve. Weigh the flasks again and calculate the concentration of glycerol (mg/g) for each reference solution. Chromatographic system: Column: a gel-permeation column 0.6 m long and 7 mm in internal diameter packed with styrene-divinylbenzene copolymer R (particle diameter 5 µm and porosity 10 nm), Flow rate: 1 ml/min. Detector: A differential refractive index detector. Injection volume: 40 µl. Procedure: Inject of each solution into the chromatographer. When the chromatograms are recorded in the prescribed conditions, the retention times relative to glycerol are about 0.86 for the monoacylglycerols, about 0.81 for the diacylglycerols and about 0.77 for the triacylglycerols. From the calibration curve obtained with the reference solutions determine the concentration C (mg/g) of glycerol in the test solution. Calculate the percentage content of free glycerol in the substance to be examined using the following expression: C × M m × 10 Calculate the percentage content of mono-, di- and triacylglycerols in the substance to be examined by the normalisation procedure.

Labelling The label states the type of glycerol monostearate 40-55. Storage Store in a well-closed container, protect from light. Action and use Pharmaceutical aid. 471

VP V

GLYCERYL TRINITRATE SOLUTION

Colour of solution If necessary dilute the solution to be examined to a concentration of 1% with ethanol R. The solution is not more intensely coloured than reference solution Y7 (Appendix 9.3, method 2).

GLYCERYL TRINITRATE SOLUTION Solutio Glycerylis trinitras

C3H5N3O9

M. 227.1

Glyceryl trinitrate solution is a 1% (m/m) to 10% (m/m) solution of propane-1,2,3-triyl trinitrate in ethanol (96%). It contains not less than 96.5% and not more than 102.5% of the declared content of glyceryl trinitrate stated on the label.

Characters A clear, colourless or slightly yellow solution. Miscible with acetone and with ethanol (96%). Pure glyceryl trinitrate is practically insoluble in water, freely soluble in ethanol (96%), miscible with acetone. Identification Apply one of the two following identifications: First identification: A, C. Second identification: B, C. Upon diluting glyceryl trinitrate solution, care must be taken to always use anhydrous ethanol, otherwise droplets of pure glyceryl trinitrate may precipitate from the solution. After examination, the residues and the solutions obtained in both the identification and the test sections must be heated on a water-bath for 5 min with dilute sodium hydroxide solution R. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum obtained with glyceryl trinitrate RS. Preparation: Place 50 µl of a solution diluted, if necessary, with ethanol R, to contain 1% of glyceryl trinitrate, on a disc of potassium bromide and evaporate the solvent in vacuo. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Ethyl acetate - toluene (20 : 80). Test solution: Dilute a quantity of the substance to be examined corresponding to 50 mg of glyceryl trinitrate to 100 ml with acetone R. Reference solution: Dilute 0.05 ml of glyceryl trinitrate solution R to 1 ml with acetone R. Procedure: Apply separately to the plate 5 µl of each solution. Develop over 2/3 of the plate. After removal of the plate, allow the plate to dry in air. Spray the plate with freshly prepared potassium iodide-starch solution R. Expose the plate to ultraviolet light at 254 nm for 15 minutes. Examine in daylight. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. C. It complies with the limits of the assay. 472

Inorganic nitrates Not more than 0.5% of the content of glyceryl trinitrate calculated as potassium nitrate. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel. Mobile phase: Toluene - acetone - glacial acetic acid (60 : 30 : 15). Test solution: If necessary dilute the solution to be examined to a concentration of 1% with ethanol R. Reference solution: Dissolve 5 mg of potassium nitrate R in 1 ml of water and dilute to 100 ml with ethanol R. Procedure: Apply separately to the plate 10 µl of each solution. Develop over 2/3 of the plate. After removal of the plate, allow the plate to dry in a current of air until the acetic acid is completely removed. Spray the plate with freshly prepared potassium iodide - starch solution R. Expose the plate to ultraviolet light at 254 nm for 15 min. Examine in daylight. Any spot corresponding to the nitrate ion in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - water (50 : 50). Test solution: Dissolve a quantity of the substance to be examined corresponding to 2 mg of glyceryl trinitrate in the mobile phase and dilute to 20.0 ml with the mobile phase. Reference solution (1): Dissolve 0.10 g of glyceryl trinitrate solution RS and a quantity of diluted pentaerythrityl tetranitrate RS equivalent to 1.0 mg of pentaerythrityl tetranitrate in the mobile phase and dilute to 100.0 ml with the mobile phase. Sonicate and filter if necessary. Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with octadecylsilyl silica gel for chromatography (5 µm). Detector: A spectrophotometer set at 210 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Inject reference solution (1). Record the chromatogram for 3 times the retention time of the principal peak. The test is not valid unless in the chromatogram obtained with reference solution (1), the resolution between the peaks due to glyceryl trinitrate and to pentaerythrityl tetranitrate is at least 2.0. In the chromatogram obtained with the test solution:

VP V

GLYCERYL TRINITRATE TABLETS

The area of any peak, apart from the principal peak, is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (1%, calculated as glyceryl trinitrate); the sum of the areas of all peaks, apart from the principal peak, is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (2) (3%, calculated as glyceryl trinitrate) (0.5%). Disregard any peak with an area less than 0.1 times that of the principal peak in the chromatogram obtained with reference solution (2) (0.1%).

Assay Test solution: Weigh accurately a quantity of the substance to be examined equivalent to 1.0 mg of glyceryl trinitrate, dissolve in methanol R in a 250 ml volumetric flask, dilute to volume with methanol R. Reference solution: Weigh accurately about 70.0 mg of sodium nitrite R, dissolve in methanol R in a 250 ml volumetric flask, dilute to volume with methanol R. Dilute 5.0 ml of the solution to 500.0 ml with methanol R. Into three 50 ml volumetric flasks introduce 10.0 ml of the test solution, 10.0 ml of the reference solution and 10.0 ml of methanol R as the blank. To each flask add 5 ml of dilute sodium hydroxide solution R, close the flask, mix and allow to stand at room temperature for 30 minutes. Add 10 ml of sulphanilic acid solution R and 10 ml of dilute hydrochloric acid R and mix. After exactly 4 minutes, add 10 ml of naphthylethylenediamine dihydrochloride solution R, dilute to volume with water and mix. After 10 minutes, measure the absorbances (Appendix 4.1) of the test solution and the reference solution at 540 nm using the blank solution (methanol solution) as the compensation liquid. Calculate the amount of glyceryl trinitrate (mg) in the test solution from the following expression: AT × mS × C AR × mT × 60.8 × 100 In which: AT: Absorption of the test solution. mT: Mass of the substance to be examined, in milligrams. C: Percentage content of sodium nitrite used as reference. AR: Absorption of the reference solution. mS: Mass of sodium nitrite, in milligrams.

Storage Store diluted solutions (1%) protected from light, at a temperature of 2 °C to 15 °C. Store more concentrated solutions protected from light, at a temperature of 15 °C to 20 °C. Action and use Cardio vascular drug. Preparation Tablets.

GLYCERYL TRINITRATE TABLETS Tabellae Glicerylis trinitratis Glyceryl trinitrate tablets are sublingual tablets prepared by adding glyceryl trinitrate solution of an appropriate concentration to dried granules of mannitol, mixing intimately, drying at a temperature not exceeding 50 °C or without heating for not more than 4 hours and compressing. Caution: Undiluted glyceryl trinitrate can be exploded by percussion or excessive heat. Appropriate precautions should be exercised and only exceedingly small amounts should be isolated. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of glyceryl trinitrate, C3H5N3O9, 85.0% to 115.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Toluene. Test solution: Extract a quantity of the powdered tablets containing 0.5 mg of glyceryl trinitrate with 1 ml of acetone R, centrifuge. Reference solution: Dilute a quantity of glyceryl trinitrate solution RS with sufficient water to produce a solution containing 0.05% of glyceryl trinitrate. Procedure: Apply separately to the plate 20 µl of each solution. Develop the chromatograph. After removal of the plate, dry it in a stream of air and spray with a 1% solution of diphenylamine R in sulfuric acid R and irradiate for 15 minutes with ultraviolet light (365 nm). Examine the plate in the daylight. The spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. B. Extract a quantity of the powdered tablets containing 3 mg of glyceryl trinitrate with 5 ml of ether R and filter. Evaporate the ether and dissolve the residue in 0.2 ml of sulfuric acid R containing a trace of diphenylamine R. An intense blue colour is produced. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - water (40 : 60), adjust the ratio if necessary. Test solution: Mix a quantity of the powdered tablets containing 2.5 mg of glyceryl trinitrate with 10 ml of acetonitrile R, sonicate to dissolve, filter through a 4-µm filter and dilute one volume of the filtrate with an equal volume of water. Reference solution: Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Resolution solution: Dilute a quantity of glyceryl trinitrate solution RS with sufficient 1 M hydrochloric acid R to 473

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GRISEOFULVIN

produce a solution containing 0.05% of glyceryl trinitrate and heat in a reaction vial at 100 °C for 30 min. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm) (Nucleosil ODS is suitable). Flow rate: 1.0 ml/min. Detector: A spectrophotometer set at 210 nm. Volume of injection: 50 µl. Procedure: Inject alternately the above solutions; allow the chromatography to proceed for three times the retention time of the principal peak. The test is not valid unless the chromatogram obtained with the resolution solution resembles the reference chromatogram supplied with glyceryl trinitrate solution RS in that it shows a principal peak due to glyceryl trinitrate and two clearly separated peaks due to the dinitrate impurities with retention times relative to glyceryl trinitrate of approximately 0.5. In the chromatogram obtained with the test solution, the area of any secondary peak is not greater than the area of the principal peak in the chromatogram obtained with the reference solution (1%) and the sum of the areas of any such peaks is not greater than three times the area of the principal peak in the chromatogram obtained with the reference solution (3%). Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with the reference solution (0.1%).

Uniformity of content (Appendix 11.2) Examine by liquid chromatography (Appendix 5.3) with the chromatographic conditions described under the test for Related substances. Test solution: Add 2 ml of acetonitrile R to one tablet, mix with the aid of ultrasound for 5 minutes, filter through a 4-µm membrane filter and dilute 1 volume of the filtrate with an equal volume of water. Reference solution: Dilute glyceryl trinitrate solution RS with the mobile phase to produce a solution containing the equivalent amount of glyceryl trinitrate as that expected for the test solution. Resolution solution: Dilute a quantity of glyceryl trinitrate solution RS with sufficient 1M hydrochloric acid R to produce a solution containing 0.05% of glyceryl trinitrate and heat in a reaction vial at 100 °C for 30 min. Procedure: Inject alternately the above solutions. The test is not valid unless the chromatogram obtained with the reference solution resembles the reference chromatogram supplied with glyceryl trinitrate solution RS in that it shows a principal peak due to glyceryl trinitrate and two clearly separated peaks due to the dinitrate impurities with retention times relative to glyceryl trinitrate of approximately 0.5. Calculate the content of C3H5N3O9 in each tablet using the peak areas of glyceryl trinitrate peaks in the chromatograms obtained with the test solution and the reference solution 474

and the declared content of C3H5N3O9 in glyceryl trinitrate solution RS.

Assay Use the average of the 10 individual results obtained from the test for Uniformity of content. Storage Glyceryl trinitrate tablets should be protected from light and stored in a glass container closed by means of a screw closure lined with aluminium or tin foil. Additional packing that absorbs glyceryl trinitrate should be avoided. Glyceryl trinitrate tablets should be issued for patients in containers of not more than 100 tablets. Action and use Cardiovascular drug. Preventing angina pectoris. GRISEOFULVIN Griseofulvinum

C17H17ClO6

M. 352.8

Griseofulvin is (1’S,6’R)-7-chloro-2’,4,6-trimethoxy-6’methylspiro[benzofuran-2(3H),1’-[2]cyclohexene]-3,4’dione, a substance produced by the growth of certain strains of Penicillium griseofulvum or obtained by any other means. It contains not less than 97.0% and not more than 102.0% of C17H17ClO6, calculated with reference to the dried substance.

Charaters A white or yellowish-white, microfine powder, the particles of which are generally up to 5 µm in maximum dimension although larger particles which may occasionally exceed 30 µm may be present, tasteless, practically insoluble in water, freely soluble in dimethylformamide and in tetrachloroethane, slightly soluble in ethanol and in methanol. It melts at about 220 °C. Identification A.The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of griseofulvin RS. B. Dissolve about 5 mg in 1 ml of sulfuric acid R and add about 5 mg of powdered potassium dichromate R. A winered colour develops. Appearance of solution Dissolve 0.75 g in dimethylformamide R and dilute to 10 ml with the same solvent. The solution is clear (Appendix 9.2)

VP V

and not more intensely coloured than reference solution Y4 (Appendix 9.3, method 2).

Acidity Suspend 0.25 g in 20 ml of ethanol (96%) R and add 0.1 ml of phenolphthalein solution R. Not more than 1.0 ml of 0.02 M sodium hydroxide VS is required to change the colour of the indicator. Specific optical rotation +354º to +364°, calculated with reference to the dried substance (Appendix 6.4). Determined on the solution prepared by dissolving 0.250 g in dimethylformamide R and diluting to 25.0 ml with the same solvent. Related substances Examine by gas chromatography (Appendix 5.2). Internal standard solution: Dissolve 0.2 g of diphenylanthracene R in acetone R and dilute to 100.0 ml with the same solvent. Test solution (1): Dissolve 0.10 g of the substance to be examined in acetone R and dilute to 10.0 ml with the same solvent. Test solution (2): Dissolve 0.10 g of the substance to be examined in acetone R, add 1.0 ml of the internal standard solution and dilute to 10.0 ml with acetone R. Reference solution: Dissolve 5.0 mg of griseofulvin RS in acetone R, add 1.0 ml of the internal standard solution and dilute to 10.0 ml with acetone R. Chromatographic system: A glass column (1 m × 4 mm) packed with diatomaceous earth for gas chromatography impregnated with 1% (m/m) of poly[(cyanopropyl) (methyl)][(phenyl)(methyl)] siloxane. Carrier gas: nitrogen for chromatography at a flow rate of 50 ml to 60 ml per minute. Detector: A flame-ionisation detector. Maintain the temperature of the column at 250 °C, that of the injection port at 270 °C and that of the detector at 300 °C. Procedure: Continue the chromatography for three times the period of time required for the appearance of the peak corresponding to griseofulvin which is about 11 min. Inject the reference solution, determine the ratio of the area of the peak corresponding to griseofulvin to the area of the peak corresponding to the internal standard (Rs). Inject test solution (2), determine the ratio of the area of the peak corresponding to dechloro-griseofulvin (related retention time about 0.6 compared to that of griseofulvin) to the area of the peak corresponding to the internal standard (R1), determine also the ratio of the area of the peak corresponding to dehydrogriseofulvin (related retention time about 1.4 compared to that of griseofulvin) to the area of the peak corresponding to the internal standard (R2).

GRISEOFULVIN TABLETS

The ratios calculated from the chromatogram obtained with test solution (2) divided by the ratio calculated from the chromatogram obtained with the reference solution are less than 0.6 for dechlorogriseofulvin (R1/Rs 420 nm) and protect them from light. Identification Apply one of the two following identifications: First identification: A. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of nifedipine RS. B. Melting point: 171 °C to 175 °C (Appendix 6.7). C. Thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Ethyl acetate - cyclohexane (40 : 60). Test solution: Dissolve 10 mg of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Reference solution: Dissolve 10  mg of  nifedipine  RS in methanol R and dilute to 10 ml with the same solvent. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in air and examine in ultraviolet light at 254 nm.

VP V

The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. D. To 25 mg of the substance to be examined in a test tube, add 10  ml of a mixture of 1.5 volumes of  hydrochloric acid  R, 3.5 volumes of water  and 5 volumes of  ethanol 96%  R  and dissolve with gentle heating. Add 0.5  g of  zinc  R  in granules and allow to stand for 5 min with occasional swirling. Filter into a second test tube, add 5  ml of a 1% solution of  sodium nitrite  R  to the filtrate and allow to stand for 2 min. Add 2 ml of a 5% solution of ammonium sulfamate R, shake vigorously with care and add 2 ml of a 0.5% solution of naphthylethylenediamine dihydrochloride R. An intense red colour develops which persists for not less than 5 min.

Impurity D and other basic impurities Not more than 0.14%. Transfer 4 g to of the subsance to be examined a 250 ml conical flask and dissolve in 160 ml of glacial acetic acid R using an ultrasonic bath. Titrate with 0.1 N perchloric acid VS (Appendix 10.6), using 0.25 ml of 1-naphtholbenzein solution R  as indicator until the colour changes from brownish-yellow to green. Not more than 0.48 ml of 0.1 N perchloric acid VS is required. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - methanol - water (9 : 36 : 55). Test solution: Dissolve 0.200  g of the substance to be examined in 20 ml of methanol R  and dilute to 50.0  ml with the mobile phase. Reference solution (1): Dissolve 10  mg of  nifedipine  impurity A RS in methanol R and dilute to 25.0 ml with the same solvent. Reference solution (2): Dissolve 10  mg of  nifedipine  impurity B RS in methanol R and dilute to 25.0 ml with the same solvent. Reference solution (3): Mix 1.0 ml of reference solution (1), 1.0 ml of reference solution (2) and 0.1 ml of the test solution and dilute to 20.0 ml with the mobile phase. Dilute 2.0 ml of this solution to 10.0 ml with the mobile phase. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 235 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Inject the test solution and reference solution (3). The run time of the test solution is twice the retention time of nifedipine.

NIFEDIPINE

Elution order: Impurity A, impurity B, nifedipine. Retention time of nifedipine is about 15.5 min. System suitability: In the chromatogram obtained with reference solution (3), the resolution between the peaks due to impurity A and impurity B is at least 1.5; the resolution between the peaks due to impurity B and nifedipine is at least 1.5. Limits: In the chromatogram obtained with the test solution: Impurities A, B: For each impurity, the area is not more than the area of the corresponding peak in the chromatogram obtained with reference solution (3) (0.1%). Any other impurity: For each impurity, other than the principal peak and impurity A, B, the area is not more than the area of the peak due to nifedipine in the chromatogram obtained with reference solution (3) (0.1%). Total: Not more than 0.3%. Disregard any peak with an area less than 0.1 times the area of the peak due to  nifedipine in the chromatogram obtained with reference solution (3) (0.01%). Note: Impurity A: Dimethyl 2,6-dimethyl-4-(2-nitrophenyl)pyridine3,5-dicarboxylate (nitrophenylpyridine analogue). Impurity B: Dimethyl 2,6-dimethyl-4-(2-nitrosophenyl)pyridine-3, 5-dicarboxylate (nitrosophenylpyridine analogue). Impurity C: Methyl 2-(2-nitrobenzylidene)-3-oxobutanoate. Impurity D: Methyl 3-aminobut-2-enoate.

Loss on drying

Not more than 0.5% (Appendix 9.6). (1.000 g; 100 °C to 105 °C; 2 h).

Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.1300  g in a mixture of 25  ml of  2-methyl-2propanol R and 25 ml of  1 M perchloric acid R. Titrate with 0.1 M cerium sulfate VS using 0.1 ml of ferroin R as indicator, until the pink colour disappears. Titrate slowly towards the end of the titration. Carry out a blank titration. 1 ml of 0.1 M cerium sulfate VS is equivalent to 17.32 mg of C17H18N2O6. Storage Protected from light. Action and use Treatment of hypertension. Preparations Tablets, capsules.

681

NIFEDIPINE TABLETS

NIFEDIPINE TABLETS Tabellae Nifedipini Nifedipine tablets are the film-coated tablets containing nifedipine. The tablets comply with the requirements stated under Tablets (Appendix 1.20), Coated tablets section and with the following requirements.

Content of nifedipine, C17H18N2O6, 95.0% to 105.0% of the stated amount. Carry out all following procedures in the condition protected from light or under long-wavelength light (greater than 420 nm). Prepare solutions immediately before use and protect them from light. Use the brown glassware. Identification A. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel GF254 (Merck silica gel F254 plate is suitable). Mobile phase: Ethyl acetate - cyclohexane (40 : 60). Diluent: Dichloromethane - methanol (50 : 50). Test solution: Shake a quantity of the finely powdered tablets (remove the coating if necessary) containing about 20 mg of nifedipine with 100 ml of the diluent. Reference solution (1): A 0.02% solution of nifedipine RS in the diluent. Reference solution (2): Mix an equal volume of the test solution and reference solution (1). Procedure: Apply separately to the plate 20 µl of each solution. Develop in a non-equilibrated chamber over a path of about 15 cm. Remove the plate, allow it to dry in air, and examine under ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to that in the chromatogram obtained with reference solution (1) and there is only one spot in the chromatogram obtained with reference solution (2). B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that of nifedipine peak in the chromatogram obtained with the reference solution. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - methanol - water (9 : 36 : 55). Test solution: Weigh a quantity of the powdered tablets containing the equivalent to 50 mg of nifedipine into a 25-ml volumetric flask, add 15 ml of methanol R, shake well, add sufficient methanol R to volume, filter. Mix equal volumes of the filtrate and the mobile phase. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Then dilute 5.0 ml of the resulting solution to 25.0 ml with the mobile phase. 682

VP V

Reference solution (2): A 0.0005% solution of the nifedipine impurity A in the mobile phase. Reference solution (3): A 0.0005% solution of the nifedipine impurity B in the mobile phase. Reference solution (4): Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Mix equal volumes of the test solution, reference solution (2) and reference solution (3). Reference solution (5): Dilute 5.0 ml of the reference solution (1) to 20.0 ml with the mobile phase. Chromatographic system: A stainless steel column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 235 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Inject the reference solutions and the test solution. In the chromatogram obtained with the reference solution (4), the resolution between the peaks due to impurity A and impurity B is not less than 1.5; between the peaks due to impurity B and nifedipine is not less than 1.5. Limits: In the chromatogram obtained with the test solution: The area of any peak corresponding to nifedipine impurity A is not more than the area of nifedipine impurity A peak in the chromatogram obtained with reference solution (2) (0.5%). The area of any peak corresponding to nifedipine impurity B is not more than the area of nifedipine impurity B peak in the chromatograms obtained with reference solution (3) (0.5%). The area of any other secondary peak is not more than the area of the principal peak in the chromatograms obtained with reference solution (1) (0.2%). The sum of the areas of all other secondary peaks is not more than 5 times the area of the principal peak in the chromatograms obtained with reference solution (1) (1.0%). Disregard any peak with an area less than the area of the principal peak in the chromatogram obtained with reference solution (5) (0.05%). Note: Impurity A: Dimethyl-2,6-dimethyl-4-(2-nitrophenyl)pyridine3,5-dicarboxylate. Impurity B: Dimethyl-2,6-dimethyl-4-(2-nitrosophenyl)pyridine-3, 5-dicarboxylate.

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of 0.1 M hydrochlorid acid R. Rotation speed: 50 rpm. Time: 45 min. Test solution: After the specified time, withdraw a sample of the medium, filter, discard the first portion of the filtrate. Reference solution: Dilute a 0.025% solution of nifedipine RS in methanol R with the medium to obtain a solution

VP V

NIFUROXAZIDE

having the same concentration of nifedipine as in the test solution. Determine the content of nifedipine dissolved by the liquid chromatography (Appendix 5.3) as described in the test for Related substances. Calculate the content of nifedipine, C17H18N2O6, is dissolved in each tablet. Tolerance: Not less than 70% (Q) of the stated amount of nifedipine, C17H18N2O6, is dissolved in 60 min.

NIFUROXAZIDE Nifuroxazidum

Assay Examine by liquid chromatography (Appendix 5.3) with the mobile phase and chromatographic system are described in the test for Relative substances. Reference solution: Weigh accurately about 20 mg of nifedipine RS, dissolve in a minimum portion of methanol R and dilute to 100.0 ml with the mobile phase. Test solution: Weigh 20 tablets, remove the coating (if necessary), calculate the average mass and finely powder. Weigh accurately a quantity of the powdered tablets containing the equivalent of 50 mg of nifedipine, transfer into a 100-ml volumetric flask, add 70 ml of methanol R, sonicate to dissolve, dilute to volume with methanol R, mix and filter. Dilute 10.0 ml of the resulting solution to 25.0 ml with the mobile phase. Resolution solution: A solution containing 0.0003% of nifedipine RS, 0.0002% of nifedipine impurity A RS and 0.0002% nifedipine impurity B RS in the mobile phase. Procedure: Inject the resolution solution. In the chromatogram obtained, the resolution between the peaks due to impurity A and impurity B is not less than 1.5 and between the peaks due to impurity B and nifedipine is not less than 1.5. Inject the reference solution, the relative standard deviation of the peak areas due to nifedipine for 6 replicate injections is not more than 2.0%. Separately inject the reference solution and the test solution. Calculate the content of nifedipine, C17H18N2O6, in the tablets using the areas of the principal peaks in the chromatograms obtained with the test solution, the reference solution and the declared content of C17H18N2O6 in nifedipine RS.

Nifuroxazide is (E)-4-hydroxy-N’-[(5-nitrofuran-2-yl) methylidene]benzohydrazide. It contains not less than 98.5% and not more than 101.5% of C12H9N3O5, calculated with reference to the dried substance.

Storage Store in cool and dry place, protected from light. Action and use Treatment of hypertension and angina pectoris. Usual strength 10 mg.

C12H9N3O5  

M. 275.2

Characters Bright yellow, crystalline powder. Practically insoluble in water and methylene chloride and slightly soluble in ethanol (96%). Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of nifuroxazide RS. Specific absorbance Dissolve 10.0 mg in 10.0 ml of ethylene glycol monomethyl ether R and dilute to 100.0 ml with methanol R, protected from light. Dilute 5.0 ml of this solution to 100.0 ml with methanol R. The specific absorbance of the solution at the absorption maximum at 367 nm (Appendix 4.1) is 940 to 1000. Impurity A Not more than 0.05%. Test solution (1): Dissolve 1.0 g of the substance to be examined in dimethyl sulfoxide R and dilute to 10.0 ml with the same solvent. Test solution (2): To 5.5 ml of test solution (1) add 50.0 ml of water while stirring. Allow to stand for 15 min and filter. Reference solution: To 0.5 ml of test solution (1) add 5.0 ml of a 50 mg/L solution of 4-hydroxybenzohydrazide (impurity A) in dimethyl sulfoxide R. Add 50.0 ml of water while stirring. Allow to stand for 15 min and filter. Add 0.5 ml of phosphomolybdotungstic reagent R and 10.0 ml of a 10.6% solution of sodium carbonate R separately to 10.0 ml of test solution (2) and to 10.0 ml of the reference solution. Allow to stand for 1 h. Measure the light absorbance (Appendix 4.1) of the 2 solutions at 750 nm. The absorbance of the solution obtained with test solution (2) is not greater than that obtained with the reference solution. Related substances Examine by liquid chromatography (Appendix 5.3). Use amber volumetric flasks, unless otherwise specified. Mobile phase A: Tetrahydrofuran - water (5 : 95). 683

VP V

NIFUROXAZIDE

Mobile phase B: Acetonitrile R. Solvent mixture: Acetonitrile - water (40 : 60). Test solution: Dissolve 10.0 mg of the substance to be examined in the solvent mixture, using sonication for not more than 5 min, and dilute to 100.0 ml with the solvent mixture. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 10.0 ml with the solvent mixture. Reference solution (2): In order to prepare impurity E in situ, dissolve 5 mg of the substance to be examined in the solvent mixture in a colourless volumetric flask, using sonication for 5 min and dilute to 50 ml with the solvent mixture. Allow to stand for 1 h. Reference solution (3): Dissolve 5.0 mg of methyl parahydroxybenzoate RS (impurity B) in the solvent mixture and dilute to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 100.0 ml with the solvent mixture. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm, sphericity). Column temperature: 10 °C. Detector: A spectrophotometer set at 280 nm. Flow rate: 1.0 ml/min. Volume of injection: 50 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 10

67

33

10 - 30

67 → 43

33 → 57

Inject the test solution and reference solutions (1), (2) and (3). Relative retention with reference to nifuroxazide (retention time = about 8 min): Impurity A (keto-enol tautomers) = about 0.36 and 0.39; impurity E = about 0.9; impurity B = about 1.2; impurity C = about 2.6; impurity D = about 3.4. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurity E and nifuroxazide is at least 2.0. Limits: Impurity E: The area of the peak due to impurity E is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.3%). Impurities B, C, D: For each impurity, the area is not more than 0.6 times the area of the principal peak in the chromatogram obtained with reference solution (3) (0.3%); and not more than 1 such peak has an area more than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (3) (0.1%). 684

Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). The sum of the peak areas of all impurities other than E, is not more than the area of the principal peak in the chromatogram obtained with reference solution (3) (0.5%). Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (3) (0.05%); disregard the peaks due to impurity A. Note: Impurity A: 4-hydroxybenzohydrazide (p-hydroxybenzohydrazide). Impurity B: Methyl 4-hydroxybenzoate (methyl parahydroxybenzoate). Impurity C: (5-nitrofuran-2-yl)methylidene diacetate. Impurity D: (E,E)-N,N’-bis[(5-nitrofuran-2-yl)methylidene] hydrazine (5-nitrofurfural azine). Impurity E: (Z)-4-hydroxy-N’-[(5-nitrofuran-2-yl)methylidene] benzohydrazide.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 4. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C; 3 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.200 g of the substance to be examined, with heating if necessary, in 30 ml of dimethylformamide R and add 20 ml of water. Titrate with 0.1 N sodium hydroxide VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N sodium hydroxide VS is equivalent to 27.52 mg of C12H9N3O5. Storage Protected from light. Action and use Antibacterial. Preparation Capsules.

VP V

NIKETHAMIDE SOLUTION

NIKETHAMIDE Nikethamidum O N

N

CH3 CH3

C10H14N2O

M. 178.2

Nikethamide is N,N-diethylpyridine-3-carboxamide. It contains not less than 99.0% and not more than 101.0% of C10H14N2O, calculated with reference to the anhydrous substance.

Characters An oily liquid or a crystalline mass, colourless or slightly yellowish. Miscible with water, with chloroform, with ethanol (96%) and with ether at any ratio. Identification Apply one of the two following identifications: First identification: A, B. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of nikethamide RS. B. The ultraviolet absorption spectrum (Appendix 4.1) between 230 nm and 350 nm in a 2 cm cell of a 0.0015% solution of the substance to be examined in 0.01 M hydrochloric acid R shows a single absorption maximum at 263 nm and the A(1%, 1 cm) at the maximum at 263 nm is about 285. C. Heat 0.1 g with 1 ml of 2 M sodium hydroxide R. Diethylamine is evolved and is recognisable by its characteristic odour and by its turning red litmus paper blue. D. To 2 ml of a 0.1% solution of the substance to be examined add 2 ml of cyanogen bromide solution R and 3 ml of a 2.5% solution of aniline R and shake. A yellow colour develops. pH The pH of a 25% solution of the substance to be examined is 6.0 to 7.8 (Appendix 6.2). Appearance of solution The substance to be examined, in liquid form or liquefied by slight heating, is clear (Appendix 9.2) and not more intensely coloured than reference solution Y5 (Appendix 9.3, method 2). Refractive index 1.524 to 1.526 (Appendix 6.1). Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254.

Mobile phase: Propanol - chloroform (25 : 75). Test solution: Dissolve 0.4 g of the substance to be examined in methanol R and dilute to 10.0 ml with the same solvent. Reference solution (1): Dissolve 40 mg of ethylnicotinamide RS in methanol R and dilute to 100.0 ml with the same solvent. Reference solution (2): Dilute 1.0 ml of reference solution (1) to 10.0 ml with methanol R. Procedure: Apply separately to the plate 10 µl of each solution. Develop the chromatogram over a path of 15 cm. After removal of the plate and allow it to dry in air. Examine in ultraviolet light at 254 nm. In the chromatogram obtained with the test solution, any spot corresponding to ethylnicotinamide is not more intense than the spot in the chromatogram obtained with reference solution (1) and any spot, apart from the principal spot and the spot corresponding to ethylnicotinamide, is not more intense than the spot in the chromatogram obtained with reference solution (2).

Heavy metals Not more than 10 ppm (Appendix 9.4.8). 12 ml of a 10% solution of the substance to be examined in water complies with limit test 1 for heavy metals. Prepare the standard using lead standard solution (1 ppm Pb) R. Water Not more than 0.3% (Appendix 10.3). Determined on 2.000 g. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.150 g in a mixture of 20 ml of anhydrous acetic acid R and 5 ml of acetic anhydride R. Titrate with 0.1 N perchloric acid VS, determine the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 17.82 mg of C10H14N2O. Storage Store in a well-closed container, protected from light. Action and use Central nervous system stimulant. NIKETHAMIDE SOLUTION Solutio Nikethamidi Nikethamide solution is a solution containing nikethamide. The preparation complies with the general requirements stated under “Solution” (Appendix 1.3) and with the following requirements: 685

VP V

NITRAZEPAM

Content of nikethamide, C10H14N2O, 95.0% to 105.0% of the stated amount. Characters A clear and colourless or yellowish solution. Identification A. Make 1 ml of the solution alkaline with 5 M sodium hydroxide R, extract with 5 ml of dichloromethane R and evaporate to dryness on a water bath under a stream of nitrogen R. The infrared absorption spectrum (Appendix 4.2) of the residue is concordant with the spectrum of nikethamide RS. B. In the Assay, the ultraviolet absorption spectrum (Appendix 4.1) of the resulting solution in the range 230 nm to 350 nm, in a 1 cm cell, exhibits an only absorption maximum at 263 nm. Assay Dilute 5.0 ml of the preperation to 500.0 ml with water. To exactly 5.0 ml of this solution add 5 ml of 1 M hydrochloric acid R and dilute to 500.0 ml with water. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum at 263 nm, in a 1 cm cell, using 0.01 M hydrochloric acid R as a blank. Calculate the content of nikethamide, C10H14N2O, taking 282 as the value of A (1%, 1 cm) at the maximum at 263 nm. Storage Store in a well-closed container, in a cool and dry place, protected from light. Action and use Central nervous system stimulant. Usual strength 25% solution. NITRAZEPAM Nitrazepamum

C15H11N3O3 

M. 281.3

Nitrazepam is 7-nitro-5-phenyl-1,3-dihydro-2H-1,4benzodiazepin-2-one. It contains not less than 99.0% and not more than 101.0% of C15H11N3O3, calculated with reference to the dried substance. 686

Characters White or yellow, crystalline powder. Practically insoluble in water, slightly soluble in ethanol (96%). Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of nitrazepam RS. Related substances Examine by liquid chromatography (Appendix 5.3). Carry out the test protected from light. Mobile phase A: A 7.8 g/L solution of sodium dihydrogen phosphate R adjusted to pH 3.0 with phosphoric acid R. Mobile phase B: Acetonitrile R. Test solution: Dissolve 50 mg of the substance to be examined in acetonitrile R and dilute to 20.0 ml with the same solvent. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with acetonitrile R. Dilute 1.0 ml of this solution to 10.0 ml with acetonitrile R. Reference solution (2): Dissolve 2 mg of clonazepam RS in acetonitrile R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of this solution to 10.0 ml with the test solution. Chromatographic system: A column (25 cm × 4.0 mm) packed with stationary phase B (5 µm). Column temperature: 40 °C. Detector: A spectrophotometer set at 270 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0-3

65

35

3 - 10

65 → 50

35 → 50

10 - 20

50

50

Relative retention with reference to nitrazepam (retention time = about 9 min): Clonazepam = about 1.1. System suitability: In the chromatogramobtained with reference solution (2), peak-to-valley ratio (Hp/Hv) is at least 4.0, where Hp = height above the baseline of the peak due to clonazepam and Hv = height above the baseline of the lowest point of the curve separating this peak from the peak due to nitrazepam. Limits: Any impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%).

VP V

NITROFURANTOIN

The sum of the peak areas of all impurities is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A: 3-amino-6-nitro-4-phenylquinolin-2(1H)-one. Impurity B: (2-amino-5-nitrophenyl)phenylmethanone. Impurity C: 2-bromo-N-[4-nitro-2-(phenylcarbonyl)phenyl] acetamide. Impurity D: 2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-N-[4-nitro -2- (phenylcarbonyl)phenyl]acetamide.

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C; 4 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.250 g of the substance to be examined in 25 ml of acetic anhydride R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 28.13 mg of C15H11N3O3. Storage Protected from light. Action and use Tranquilizer.

Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Using silica gel HF254. Mobile phase: Methanol - nitromethane (10 : 90). Test solution: Dissolve 0.25 g of the substance to be examined in a minimum portion of dimethylformamide R and dilute to 10 ml with acetone R. Reference solution: Dilute 1 ml of the test solution to 100 ml with acetone R. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in air and heat at 100 °C to 105 °C for 5 min. Examine in ultraviolet light at 254 nm. Spray with phenylhydrazine hydrochloride solution R. Heat the plate at 100 °C to 105 °C for a further 10 min. When examined in ultraviolet light at 254 nm and after spraying, any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with the reference solution (1.0%). Loss on drying Not more than 1.0% (Appendix 9.6). (1.000 g; 100 °C to 105 °C).

Preparation Tablets.

Sulfated ash Not more than 0.1% (Appendix 9.9, method 2) Determined on 1.0 g.

NITROFURANTOIN Nitrofurantoinum

C8H6N4O5  

Identification A. The ultraviolet absorption spectrum (Appendix 4.1) of the solution prepared for the assay in the range 220 nm to 400 nm exhibits two absorption maxima, at 266 nm and 367 nm. The ratio of the absorbance at the maximum at 367 nm to that at the maximum at 266 nm is 1.36 to 1.42. Carry out the test protected from bright light. B. Dissolve about 10 mg in 10 ml of dimethylformamide R. To 1 ml of the solution add 0.1 ml of 0.5 M alcoholic potassium hydroxide R. A brown colour develops.

M. 238.2

Nitrofurantoin is 1-[[(5-nitrofuran-2-yl)methylene] amino] imidazolidine-2,4-dione. It contains not less than 98.0% and not more than 102.0% of C8H6N4O5, calculated with reference to the dried substance.

Characters A yellow, crystalline powder or yellow crystals. Odourless or almost odourless. Very slightly soluble in water and in ethanol (96%), soluble in dimethylformamide.

Assay Carry out the assay protected from bright light. Dissolve 0.120 g in 50 ml of dimethylformamide R and dilute to 1000.0 ml with water. Dilute 5.0 ml of the solution to 100.0 ml with a solution containing 1.8% of sodium acetate R and 0.14% v/v of glacial acetic acid R. Measure the absorbance (Appendix 4.1) at the absorption maximum at 367 nm, using the sodium acetate solution described above as compensation liquid. Calculate the content of C8H6N4O5, taking 765 as the value of A (1%, 1 cm) at 367 nm. Storage Protected from light, at a temperature below 25 °C. Action and use Antibacterial. 687

VP V

NITROFURANTOIN TABLETS

NITROFURANTOIN TABLETS Tabellae Nitrofurantoini Nitrofurantoin tablets contain nitrofurantoin. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of nitrofurantoin, C8H6N4O5, 90.0% to 110.0% of the stated amount. Identification The ultraviolet absorption spectrum (Appendix 4.1) of the test solution obtained in the Assay in the range 220 nm to 400 nm, exhibits two maxima, at 266 nm and 367 nm. Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel HF254. Mobile phase: Methanol - nitromethane (10 : 90). Test solution: Shake a quantity of the powdered tablets containing the equivalent of about 0.1 g of nitrofurantoin with 10 ml of mixture of dimethylformamide - acetone (1 : 9), filter. Reference solution: Dilute 1 ml of the test solution to 100 ml with acetone R. Procedure: Apply separately to the plate 10 µl of each of the above solutions. After removal of the plate, allow it to dry in air, heat at 100 °C to 105 °C for 5 min and examine under ultraviolet light at 254 nm. Spray the plate with phenylhydrazine hydrochloride solution R and heat it at 100 °C to 105 °C for a further 10 min. By each method of visualisation, any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution (1%). Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 900 ml of phosphate buffer pH 7.2 R. Rotation speed: 100 rpm. Time: 60 min, 120 min. Procedure: Test solution: After the specified time, withdraw a sample of the medium and filter, discard the first 20 ml of the filtrate. Dilute with the dissolution medium to obtain a solution having the same concentration as the reference solution. Reference solution: Weigh accurately about 50 mg of nitrofurantoin RS, add 25 ml of dimethylformamide R, shake well to dissolve, dilute with the dissolution medium to 500 ml, mix well and dilute further a suitable volume of the resulting solution with the dissolution medium to obtain a solution with concentration of about 10 µg/ml. Measure the absorbance (Appendix 4.1) of the test and the reference solution at the maximum at about 375 nm, 688

using the dissolution medium as the blank. Calculate the content of nitrofurantoin, C8H6N4O5, dissolved at 60-min and 120-min time-points, using the absorbances of the test solution, the reference solution and the declared content of C8H6N4O5 in nitrofurantoin RS. Tolerance: Not less than 25% (Q) and 85% (Q) of the stated amount of nitrofurantoin, C8H6N4O, is dissolved in 60 minutes and 120 minutes, respectively.

Assay Carry out the following procedure in subdued light. Weigh 20 tablets, calculate the average mass, and powder finely. Weigh a quantity of the powder containing the equivalent of about 0.12 g of nitrofurantoin, add 50 ml of dimethylformamide R, shake for 5 min, add sufficient water to produce 1000 ml and mix well. Dilute 5 ml to 100 ml with a solution containing 1.8% of sodium acetate R and 0.14% (v/v) of glacial acetic acid R and filter. Measure the absorbance of the filtrate at the maximum at 367 nm (Appendix 4.1), in a 1-cm cell, using the sodium acetate-acetic acid solution as blank. Calculate the content of nitrofurantoin, C8H6N4O5, taking 765 as the value of A(1%, 1 cm) at 367 nm. Storage Store in well-closed container, protected from light, in a cool and dry place. Action and use Antibacterial. Usual strength 50 mg; 100 mg. NORFLOXACIN Norfloxacinum

C16H18FN3O3  

M. 319.3

Norfloxacin is 1-ethyl-6-fluoro-4-oxo-7-(piperazin-1-yl)1,4-dihydroquinoline-3-carboxylic acid. It contains not less than 99.0% and not more than 101.0% of C16H18FN3O3, calculated with reference to the dried substance.

Characters White or pale yellow, hygroscopic, photosensitive, crystalline powder. Very slightly soluble in water, slightly soluble in acetone and in ethanol (96%).

VP V

NORFLOXACIN

Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of norfloxacin RS. Appearance of solution Dissolve 0.5 g of the substance to be examined in a previously filtered 0.1 M sodium hydroxide R in methanol R and dilute to 50 ml with the same solution. The solution is not more opalescent than reference suspension II (Appendix 9.2) and not more intensely coloured than reference solution B7 (Appendix 9.3, method 2). Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Water adjusted to pH 2.0 with phosphoric acid R. Mobile phase B: Acetonitrile R. Solution A: Mobile phase A - mobile phase B (95 : 5). Test solution: Dissolve 20 mg of the substance to be examined in 25 ml of solution A. Sonicate for 5 min and dilute to 50.0 ml with solution A. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with solution A. Dilute 1.0 ml of this solution to 10.0 ml with solution A. Reference solution (2): Dissolve 4 mg of norfloxacin for system suitability RS (containing impurities A, E and H) in 5 ml of solution A. Sonicate for 5 min and dilute to 10 ml with solution A. Reference solution (3): Dissolve 4 mg of norfloxacin for peak identification RS (containing impurity K) in 5 ml of solution A. Sonicate for 5 min and dilute to 10 ml with solution A. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase endcapped hexadecylamidylsilyl silica gel for chromatography R (5 µm). Column temperature: 60 °C. Detector: A spectrophotometer set at 265 nm. Flow rate: 1.4 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0-5

95

5

5-7

95 → 93

5→7

7 - 10

93 → 87

7 → 13

10 - 15

87 → 47

13 → 53

15 - 20

47 → 10

53 → 90

Identification of impurities: Use the chromatogram supplied with norfloxacin for system suitability RS and

the chromatogram obtained with reference solution (2) to identify the peaks due to impurities A, E and H. Use the chromatogram supplied with norfloxacin for peak identification RS and the chromatogram obtained with reference solution (3) to identify the peak due to impurity K. Relative retention with reference to norfloxacin (retention time = about 11 min): impurity K = about 0.6; impurity E = about 0.97; impurity A = about 1.5; impurity H = about 1.6. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurities A and H is at least 3.0. Peak-to-valley ratio (Hp/Hv) is at least 5, where Hp = height above the baseline of the peak due to impurity E and Hv = height above the baseline of the lowest point of the curve separating this peak from the peak due to norfloxacin. Limits: Impurities E, K: For each impurity, the area is not more than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.15%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). The sum of the peak areas of all impurities is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A: 7-chloro-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline3-carboxylic acid. Impurity B: 7-[(2-aminoethyl)amino]-1-ethyl-6-fluoro-4-oxo1,4-dihydroquinoline-3- carboxylic acid. Impurity C: 1-ethyl-4-oxo-6,7-bis(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid. Impurity D: 1-ethyl-6-fluoro-7-(piperazin-1-yl)quinolin-4(1H)one. Impurity E: 7-chloro-1-ethyl-4-oxo-6-(piperazin-1-yl)-1,4dihydroquinoline-3-carboxylic acid. Impurity F: 6-chloro-1-ethyl-4-oxo-7-(piperazin-1-yl)-1,4dihydroquinoline-3-carboxylic acid. Impurity G: 1-ethyl-6-fluoro-7-(4-formylpiperazin-1-yl)-4-oxo1,4-dihydroquinoline-3- carboxylic acid, Impurity H: 7-[4-(ethoxycarbonyl)piperazin-1-yl]-1-ethyl-6fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid. Impurity I: 7-chloro-6-[4-(ethoxycarbonyl)piperazin-1-yl]-1ethyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid. Impurity J: 6,7-bis[4-(ethoxycarbonyl)piperazin-1-yl]-1-ethyl4-oxo-1,4-dihydroquinoline-3-carboxylic acid. Impurity K: 6-fluoro-1-methyl-4-oxo-7-(piperazin-1-yl)-1,4dihydroquinoline-3-carboxylic acid.

Heavy metals Not more than 15 ppm (Appendix 9.4.8). 689

NORFLOXACIN TABLETS

2.0 g complies with limit test for heavy metals, method 4. Prepare the standard using 3 ml of lead standard solution (10 ppm Pb) R.

Loss on drying Not more than 1.0% (Appendix 9.6). (1.000 g; 105 °C; in vacuo; 2 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g in a platinum crucible. Assay Dissolve 0.240 g of the substance to be examined in 80 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 31.93 mg of C16H18FN3O3. Storage In an airtight container, protected from light. Action and use Fluoroquinolone antibacterial. Preparations Tablets, eye drops. NORFLOXACIN TABLETS Tabellae Norfloxacini Norfloxacin tablets contain norfloxacin. They are film coated tablets. The tablets comply with the requirements stated under “Tablets” Appendix 1.20) and with the following requirements.

Content of norfloxacin, C16H18FN3O3, 95.0% to 105.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4), protected from light. Coating substance: Silica gel GF254. The plate is previously developed with methanol R and allowed to dry in air. Mobile phase: Water - diethylamine - toluene - chloroform methanol (8 : 14 : 20 : 40 : 40). Solvent mixture: A mixture of 50 volumes of dichloromethane R and 50 volumes of a solution prepared by adding 9 ml of hydrochloric acid R to 1000 ml of methanol R. Test solution: Add 2 ml of water to a quantity of the powdered tablets containing 0.4 g of norfloxacin and disperse by sonicating. Add 100 ml of the solvent mixture, mix well. Sonicate until a uniform suspension is produced, dilute to 200 ml with the same solvent. Centrifuge 25 ml of the final solution. 690

VP V

Reference solution: Dissolve by sonicating 50 mg of norfloxacin RS in 15 ml of the solvent mixture and dilute to 25 ml with the same solvent. Procedure: Apply separately to the plate 50 µl of each solution. Develop over a path of 15 cm. After removal of the plate, allow it to dry in air. Examine under ultraviolet light at 254 nm and 365 nm. By each method of visualisation, the principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to that in the chromatogram obtained with the reference solution. B. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the principal peak in the chromatogram obtained with the reference solution.

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 750 ml of an acetate buffer solution prepared in the following manner: Add 14.3 ml of glacial acetic acid R to 4 500 ml of water, mix well, add slowly with stirring 2.5 ml of a 50% solution of sodium hydroxide and add sufficient water to produce 5 000 ml. If necessary, adjust pH to 4.0 with glacial acetic acid R or a 50% solution of sodium hydroxide. Rotation speed: 50 rpm. Time: 30 min. Procedure: After the specified time, withdraw a sample of the medium, filter, discard the first 20 ml of the filtrate. Dilute a portion of the filtrate with the medium to obtain a solution containing 0.0016% of norfloxacin. Measure the absorbance (Appendix 4.1) at the maximum at 313 nm in a 1 cm cell, using the medium as a blank, in comparison with the reference solution of norfloxacin RS having the same concentration in the same medium. Calculate the content of norfloxacin, C16H18FN3O3, dissolved using the absorbances obtained and the declared content of C16H18FN3O3 in norfloxacin RS. Tolerance: Not less than 80% (Q) of the stated amount of norfloxacin, C16H18FN3O3, is dissolved in 30 minutes. Assay Examine by liquid chromatography (Appendix 5.3), protected from light. Mobile phase: Acetonitrile - 0.1% v/v solution of phosphoric acid (150 : 850). Test solution: Weigh 20 tablets (with the coating removed), calculate the average mass and powder finely. Transfer an accurately weighed quantity of the powdered tablets containing the equivalent of about 0.1 g of norfloxacin to a 200 ml volumetric flask, add 80 ml of the mobile phase and sonicate for at least 5 minutes, add the mobile phase to volume and filter. Dilute 10.0 ml of the filtrate to 25.0 ml with the mobile phase. Reference solution: The solution contains 0.02% of norfloxacin RS in the mobile phase.

VP V

NYSTATIN

Chromatographic system: A column (30 cm × 3.9 mm) packed with stationary phase C (10 µm). Column temperature: 40 °C. Detector: A spectrophotometer set at 275 nm. Flow rate: 2 ml/min. Volume of injection: 10 µl. Procedure: Precondition the column using 0.01 M sodium dihydrogen phosphate R, adjusted to pH 4.0 with phosphoric acid R, at a flow rate of 0.5 ml per minute for 8 hours. Equilibrate the column with the mobile phase for about 30 minutes before starting the chromatography. System suitability: Inject the reference solution. The retention time of norfloxacin is about 5 minutes. The test is not valid unless the symmetry factor of the norfloxacin peak in the chromatogram obtained with the reference solution is less than 2.0 and the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of norfloxacin, C16H18FN3O3, in the tablets using the areas of norfloxacin peaks in the chromatograms obtained with the test solution, the reference solution and the declared content of C16H18FN3O3 in norfloxacin RS.

Storage Store in a well-closed container, in a cool and dry place, protected from light. Action and use Quinolone antibiotic. Usual strength 200 mg, 400 mg. NYSTATIN Nystatinum

C47H75NO17   

M. 926

Nystatin is antifungal substance obtained by fermentation using certain strains of Streptomyces noursei as the production micro-organism. It contains mainly tetraenes, the principal component being (1S,3R,4R,7R,9R,11R,15S ,16R,17R,18S,19E,21E,25E,27E,29E,31E,33R,35S,36R,3 7S)-33-[(3-amino-3,6-dideoxy-β-D-mannopyranosyl)oxy]1,3,4,7,9,11,17,37-octahydroxy-15,16,18-trimethyl-13-oxo14,39-dioxabicyclo[33.3.1]nonatriaconta-19,21,25,27,29,31hexaene-36-carboxylic acid (nystatin A1). It contains not less than 4400 IU/mg, calculated with reference to the dried substance and not more than 5000 IU/mg, calculated with reference to the dried substance, if intended for oral administration.

Production If nystatin is not intended for cutaneous administration, the method of manufacture is validated to demonstrate that the product, if tested, would comply with the following test: Abnormal toxicity (Appendix 13.5) Inject intraperitoneally into each mouse a quantity equivalent to not less than 600 IU suspended in 0.5 ml of a 0.5% solution of acacia R. Characters Yellow or slightly brownish powder, hygroscopic. Practically insoluble in water, freely soluble in dimethylformamide and in dimethyl sulfoxide, slightly soluble in methanol, practically insoluble in alcohol. Identification Apply one of the two following identifications: First identification: A, E. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of nystatin RS. B. The ultraviolet absorptiom spectrum (Appendix 4.1) of the solution prepared in the test for Absorbance shows 4 absorption maxima at 230 nm, 291 nm, 305 nm and 319 nm, and a shoulder at 280 nm. The ratios of the absorbances at the absorption maxima at 291 nm and 319 nm to the absorbance at the absorption maximum at 305  nm are 0.61 to 0.73 and 0.83 to 0.96, respectively. The ratio of the absorbance measured at the absorption maximum at 230 nm to that measured at the shoulder at 280 nm is 0.83 to 1.25. C. Add 0.1 ml of hydrochloric acid R to about 2 mg of the substance to be examined. A brown colour develops. D. Add 0.1  ml of  sulfuric acid  R to about 2 mg of the substance to be examined. A brown colour develops that becomes violet on standing. E. In the test for Composition nystatin, the principal peak in the chromatogram obtained with the test solution corres ponds to the principal peak in the chromatogram obtained with reference solution (1). 691

VP V

NYSTATIN OITMENT

Absorbance Dissolve 0.10  g of the substance to be examined in a mixture of 5.0  ml of  glacial acetic acid  R  and 50  ml of  methanol R  and dilute to 100.0 ml with methanol  R. Dilute 1.0 ml of the solution to 100.0 ml with methanol R. The absorbance (Appendix 4.1) of the solution at the maximum at 305 nm within 30 min of preparation of the solution is not less than 0.60. Composition nystatin Examine by liquid chromatography (Appendix 5.3), use the normalisation procedure. Carry out the test protected from light. Mobile phase A:  Acetonitrile  - a 0.385% solution of ammonium acetate (29 : 71). Mobile phase B: Acetonitrile  - a 0.385% solution of ammonium acetate (60 : 40). Test solution: Dissolve 20  mg of the substance to be examined in dimethyl sulfoxide R and dilute to 50 ml with the same solvent. Reference solution (1): Dissolve 20 mg of nystatin RS in dimethyl sulfoxide  R  and dilute to 50  ml with the same solvent. Reference solution (2): Dissolve 20 mg of the substance to be examined in 25 ml of methanol R and dilute to 50 ml with water R. To 10.0 ml of the solution add 2.0 ml of dilute hydrochloric acid R. Allow to stand at room temperature for 1 h. Reference solution (3): Dilute 1.0 ml of reference solution (1) to 100.0 ml with dimethyl sulfoxide R. Dilute 1.0 ml of this solution to 10.0 ml with dimethyl sulfoxide R. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase base-deactivated end-capped octadecylsilyl silica gel for chromatography R (5 µm). Column temperature: 30 °C. Detector: A spectrophotometer set at 305 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 25

100

0

25 - 35

100 → 0

0 → 100

35 - 45

0

100

45 - 50

0 → 100

100 → 0

50 - 55

100

0

System suitability: In the chromatogram obtained with reference solution (2), the resolution between the 2 principal peaks (retention time = about 13 min and 19 min) is at least 3.5. 692

Inject the test solution and reference solutions (1), (3). In the chromatogram obtained with the test solution, the area of the peak due to nystatin A1 (retention time is about 14 min) is not less than 85% of total peak area. Any other impurity: For each impurity, the area is not more than 4% of total peak area Disregard any peak with an area less than the area of the principal peak in the chromatogram obtained with reference solution (3), disregard any peak with a retention time of less than 2 min.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 5.0% (Appendix 9.6). (1.000 g; diphosphorus pentoxide; 60 °C; pressure not exceeding 0.1 kPa, 3 h). Sulfated ash Not more than 3.5% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Carry out the microbiological assay of antibiotics (Appendix 13.9). Protect the solutions from light throughout the assay. Standard solution: Dissolve 75 mg of nystatin  RS in dimethyl-formamide R and dilute to 50 ml with the same solution. Dilute the solution with buffer solution No.19 to obtain working solutions. Test solution: Prepare as the standard solution. Storage Store in an airtight container, protected from light. The label states where applicable, that the substance is only for cutaneous use. Action and use Antifungal. Preparations Tablets, ointment, cream, oral suspension. NYSTATIN OITMENT Unguentum Nystatini Nystatin ointment for cutaneous application is a dispersion of nystatin in microfine powder in a suitable emulsifying basis. The ointment complies with the requirements stated under “Topical semi-solid preparations” (Appendix 1.12) and with the following requirements.

VP V

Content of nystatin, C47H75NO17, 90.0% to 130.0% of the stated amount. Characters Ointment should be smooth, homogeneous and not show any discoloration or strange odour. Identification Disperse a quantity of the ointment containing 25,000 IU in 10 ml of chloroform R, add 40 ml of methanol R, shake well and filter. Dilute 1 ml of the filtrate to 25 ml with methanol R. Measure the ultraviolet absorption spectrum (Appendix 4.1) of the resulting solution, in the range 250 to 350 nm. Use a solution prepared in exactly the same manner but omitting the preparation being examined as a blank. The ultraviolet absorption spectrum exhibits three maxima, at 291, 305 and 319 nm. The ratios of the absorbances at the maxima at 291 nm and 319 nm to the absorbance at the maximum at 305 nm are 0.61 to 0.73 and 0.83 to 0.96, respectively. Assay Carry out the following procedure protected from light. Take an accurately weighed quantity of the ointment containing 200,000 IU of nystatin to 100 ml volumetric flask, add sufficient dimethylformamide R to volume and shake for 15 min. Dilute the solution with buffer solution No 19 to obtain test solutions. Carry out the biological assay of antibiotics (Appendix 13.9). Storage Store in a well-closed container, protected from light. Action and use Antifungal. Usual strength 100,000 IU/g. NYSTATIN TABLETS Tabellae Nystatini Nystatin tablets are coating tablets containing nystatin. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of nystatin, C47H75NO17, 90.0% to 130.0% of the stated amount. Identification To a quantity of the powdered tablets containing 300,000 IU of nystatin add a mixture of 5 ml of glacial acetic acid R and 50 ml of methanol R, shake well, add sufficient methanol R to produce 100 ml and filter. Dilute 1 ml of the filtrate to 100 ml with methanol R. Measure the ultraviolet

NYSTATIN VAGINAL TABLETS

absorption spectrum (Appendix 4.1) of the resulting solution, in the range 250 to 350 nm. Use a solution prepared in the same manner but omitting the preparation being examined as a blank. The ultraviolet absorption spectrum exhibits three maxima at 291, 305 and 319 nm. The ratios of the absorbances at the maxima at 291 nm and 319 nm to the absorbance at the maximum at 305 nm are 0.61 to 0.73 and 0.83 to 0.96, respectively.

Disintegration (Appendix 11.6) Not more than 30 min. Medium: 2.5% hydrochloric acid solution R. If the tablets fail to disintegrate, wash them rapidly by immersion in water and continue the test using phosphate buffer pH 6.8 R. The tablets then disintegrate within a further 30 min. Loss on drying Not more than 5.0% (Appendix 9.6). (Use 1.000 g of the powdered tablets, dry at 60 °C at a pressure not exceeding 0.7 kPa for 3 h using phosphorus pentoxide). Assay Carry out the following procedure protected from light. Weigh 20 tablets (with coating layer removed), calculate the average weight and powder finely. Weigh accurately a quantity of the powder containing 200,000 IU of nystatin, transfer into a 50-ml volumetric flask. Add 40 ml of dimethyl formamide R and shake vigorously for 1 h. Centrifuge and use the supernatant liquid. Dilute the clear supernatant liquid with buffer solution No 19 (Appendix 13.9) to obtain the test solutions. Carry out the biological assay of antibiotics (Appendix 13.9). Storage Store in a well-closed container, protected from light and humidity. Action and use Antifungal. Usual strength 500,000 IU. NYSTATIN VAGINAL TABLETS Tabellae vaginalia Nystatini Nystatin vaginal tablets contain nystatin. The vaginal tablets comply with the requirements stated under “Suppositories” (Appendix 1.10) and with the following requirements.

Content of nystatin, C47H75NO17, 90.0% to 130.0% of the stated amount. 693

VP V

OFLOXACIN

Identification To a quantity of the powdered tablets containing 300,000 IU add a mixture of 5 ml of glacial acetic acid R and 50 ml of methanol R, shake, add sufficient methanol R to produce 100 ml, filter. Dilute 1 ml of the filtrate to 100 ml with methanol R. Measure the ultraviolet absorption spectrum (Appendix 4.1) of the resulting solution, in the range 250 to 350 nm. Use a solution prepared in exactly the same manner but omitting the preparation being examined as a blank. The ultraviolet absorption spectrum exhibits three maxima, at 291, 305 and 319 nm. The ratios of the absorbances at the maxima at 291 nm and 319 nm to the absorbance at the maximum at 305 nm are 0.61 to 0.73 and 0.83 to 0.96, respectively. Loss on drying Not more than 5.0% (Appendix 9.6). (1.000 g, 105 °C). Assay Carry out the following procedure protected from light. Weigh 20 vaginal tablets, calculate the average mass and powder finely. Take an accurately weighed quantity of the powdered tablets containing 200,000 IU of nystatin to a 50 ml volumetric flask, add 40 ml of dimethylformamide R and shake vigorously for 1 hour. Add dimethylformamide R to volume and shake. Centrifuge and use the clear supernatant liquid. Dilute the solution with buffer solution No 19 to obtain test solutions. Carry out the biological assay of antibiotics (Appendix 13.9). Storage Store in a well-closed container, protected from light and moisture. Action and use Antifungal. Usual strength 100,000 IU. OFLOXACIN Ofloxacinum

than 99.0% and not more than 101.0% of C18H20FN3O4, calculated with reference to the dried substance.

Characters Pale yellow or bright yellow, crystalline powder. Slightly soluble in water, soluble in glacial acetic acid, slightly soluble or soluble in methylene chloride, slightly soluble in methanol. Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of ofloxacin RS. Optical rotation -0.10° to +0.10° (Appendix 6.4). Dissolve 0.300 g of the substance to be examined in a mixture of methanol - methylene chloride (1 : 4) and dilute to 10.0 ml with the same mixture of solvents. Absorbance Dissolve 0.5 g in 0.1 M hydrochloric acid R and dilute to 100.0 ml with the same solvent. The light absorption (Appendix 4.1) of the resulting solution at 440 nm is not more than 0.25. Impurity A Not more than 0.2%. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254 R (2 µm - 10 µm). Mobile phase: Glacial acetic acid - water - ethyl acetate (10 : 10 : 20). Solvent mixture: Methanol - methylene chloride (10 : 40). Test solution: Dissolve 0.250 g of the substance to be examined in the solvent mixture and dilute to 5.0 ml with the solvent mixture. Reference solution: Dissolve 10.0 mg of ofloxacin impurity A RS in the solvent mixture and dilute to 100.0 ml with the solvent mixture. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of two thirds of the plate. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot due to impurity A is not more intense than the corresponding spot in the chromatogram obtained with the reference solution. Note: Impurity A: (3RS)-9,10-difluoro-3-methyl-7-oxo-2,3-dihydro7H-pyrido[1,2,3-de]-1,4- benzoxazine-6-carboxylic acid (FPA).

C18H20FN3O4  

M. 361.4

Ofloxacin is (3RS)-9-fluoro-3-methyl-10-(4-methylpiperazin-1-yl)-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de]1,4-benzoxazine-6-carboxylic acid. It contains not less 694

Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use. Mobile phase: Dissolve 4.0 g of ammonium acetate R and 7.0 g of sodium perchlorate R in 1300 ml of water R; adjust to pH 2.2 with phosphoric acid R and add 240 ml of acetonitrile R.

VP V

Solvent mixture: Acetonitrile - water (10 : 60). Test solution: Dissolve 10 mg of the substance to be examined in the solvent mixture and dilute to 50.0 ml with the solvent mixture. Reference solution (1): Dilute 1.0 ml of the test solution to 50.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 10.0 ml with the solvent mixture. Reference solution (2): Dissolve 10 mg of ofloxacin impurity E RS in the solvent mixture and dilute to 100.0 ml with the solvent mixture. Mix 10 ml of the solution and 5 ml of the test solution and dilute to 50.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 50.0 ml with the solvent mixture. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 µm). Column temperature: 45 °C. Detector: A spectrophotometer set at 294 nm. Flow rate: Adjust so that a retention time of about 20 min is obtained for ofloxacin. Volume of injection: 10 µl. Procedure: The run time is 2.5 times the retention time of ofloxacin. Identification of impurities: Use the chromatogram obtained with reference solution (2) to identify the peak due to impurity E. Relative retention with reference to ofloxacin (retention time = about 20 min): impurity B = about 0.3; impurity C = about 0.5; impurity D = about 0.7; impurity E = about 0.9; impurity F = about 1.6. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurity E and ofloxacin is at least 2.0. Limits: Impurities B, C, D, E, F: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%). Any other impurity: For each impurity, the area is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). The sum of the peak areas of all impurities is not more than 2.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). Disregard any peak with an area less than 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity B: (3RS)-9-fluoro-3-methyl-10-(4-methylpiperazin-1yl)-2,3-dihydro-7H-pyrido[1,2, 3-de]-1,4-benzoxazin-7-one. Impurity C: (3RS)-3-methyl-10-(4-methylpiperazin-1-yl)-7-oxo2,3-dihydro-7H-pyrido[1,2,3-de]-1,4-benzoxazine-6-carboxylic acid. Impurity D: (3RS)-10-fluoro-3-methyl-9-(4-methylpiperazin-1yl)-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de]-1,4-benzoxazine-6carboxylic acid.

OFLOXACIN CAPSULES Impurity E: (3RS)-9-fluoro-3-methyl-7-oxo-10-(piperazin-1-yl)2,3-dihydro-7H-pyrido[1,2,3-de]-1,4-benzoxazine-6-carboxylic acid. Impurity F: 4-[(3RS)-6-carboxy-9-fluoro-3-methyl-7-oxo-2,3 -dihydro-7H-pyrido[1,2,3-de]-1,4-benzoxazin-10-yl]-1-methylpiperazine 1-oxide.

Heavy metals Not more than 10 ppm (Appendix 9.4.8). 2.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.2% (Appendix 9.6). (1.000 g; 105 °C; 4 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.300 g of the substance to be examined in 100 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 36.14 mg of C18H20FN3O4. Storage In an airtight container, protected from light. Action and use Fluoroquinolone antibacterial. Preparations Tablets, capsules, eye drops. OFLOXACIN CAPSULES Capsulae Ofloxacini Ofloxacin capsules contain ofloxacin. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of ofloxacin, C18H20FN3O4, 90.0% to 110.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Chloroform - methanol - 0.45 M ammonia solution (150 : 75 : 15). Test solution: Dissolve a quantity of the contents of the capsules containing the equivalent of 3 mg of ofloxacin with 10 ml of a mixture of chloroform - methanol (1 : 1), filter. 695

VP V

OFLOXACIN EYE DROPS

Reference solution: The solution of ofloxacin RS in a mixture of chloroform - methanol (1 : 1) contains about 0.3 mg per ml. Procedure: Apply separately to the plate 2 µl of each of the above solutions. Develop over a path of about three fourths of the length of the plate. After removal of the plate, allow it to dry in air. Examine under ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution corresponds in position, colour and size to that in the chromatogram obtained with the reference solution. B. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the peak due to ofloxacin in the chromatogram obtained with the reference solution.

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of 0.1 M hydrochloric acid R. Rotation speed: 50 rpm. Time: 30 min. Procedure: After the specified time, withdraw a sample of the medium and filter, discard the first 20 ml of the filtrate. Dilute the filtrate with 0.1 M hydrochloric acid R to obtain a solution containing about 5 µg of ofloxacin per ml. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum at 293 nm, in a 1 cm cell, using 0.1 M hydrochloric acid R as a blank, in comparison with a reference solution of ofloxacin RS having the same concentration in the same medium. Calculate the content of ofloxacin, C18H20FN3O4, dissolved using the absorbances obtained and the declared content of C18H20FN3O4 in ofloxacin RS. Tolerance: Not less than 80% (Q) of the stated amount of ofloxacin is dissolved in 30 minutes. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: 0.24% solution of sodium lauryl sulfate acetonitrile - glacial acetic acid (580 : 400 : 20). Reference solution: Dissolve ofloxacin RS in 0.05 M hydrochloric acid R to obtain a solution containing about 0.06 mg per ml. Test solution: Weigh 20 capsules, calculate the average weight of the capsule contents and powder finely. Transfer an accurately weighed quantity of the powder equivalent to about 30 mg of ofloxacin to a 100 ml volumetric flask, add 75 ml of 0.05 M hydrochloric acid R and sonicate for 10 min. Dilute to volume with 0.05 M hydrochloric acid R and mix well. Filter, discard the first 20 ml of the filtrate. Transfer 10.0 ml of the filtrate to a 50 ml volumetric flask, dilute to volume with 0.05 M hydrochloric acid R and mix well. Resolution solution: Dissolve ofloxacin RS and propylparaben RS in acetonitrile R to obtain a solution having a concentration of about 0.1 mg and 2.4 mg per ml, respectively. 696

Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Column temperature: 35 °C. Detector: A spectrophotometer set at 294 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the resolution solution. The resolution between the peaks due to ofloxacin and propylparaben is not less than 2.0. Inject the reference solution 6 times, the symmetry factor of ofloxacin peak is not more than 3.0, the relative standard deviation of ofloxacin peak areas for replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of ofloxacin, C18H20FN3O4, in the capsules using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C18H20FN3O4 in ofloxacin RS. Storage Store in a well-closed container, in a cool and dry place, protected from light. Action and use Antibacterial. Usual strength 100 mg, 200 mg. OFLOXACIN EYE DROPS Collyrium Ofloxacini Ofloxacin eye drops are a sterile solution of ofloxacin in water. The eye drops comply with the requirements stated under “Eye drops” (Appendix 1.14) and with the following requirements.

Content of ofloxacin, C18H20FN3O4, 90.0% to 110.0% of the stated amount. Characters A clear and colourless solution. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Chloroform - methanol - 0.45 M ammonia solution (150 : 75 : 15). Test solution: Dilute a volume of the eye drops with a mixture of chloroform - methanol (1 : 1) to obtain a solution containing about 0.3 mg of ofloxacin per ml. Reference solution: A solution of ofloxacin RS in a mixture of chloroform - methanol (1 : 1) containing about 0.3 mg per ml.

VP V

Procedure: Apply separately to the plate 2 µl of each of the above solutions. Develop over a path of about three fourths of the length of the plate. After removal of the plate, allow it to dry in air. Examine under ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution corresponds in position, colour and size to that in the chromatogram obtained with the reference solution. B. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the peak due to ofloxacin in the chromatogram obtained with the reference solution.

pH 6.0 to 6.8 (Appendix 6.2). Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: 0.24% solution of sodium lauryl sulfate acetonitrile - glacial acetic acid (580 : 400 : 20). Test solution: Dilute an accurately measured volume of the eye drops with 0.05 M hydrochloric acid R to obtain a solution containing about 0.06 mg of ofloxacin per ml. Reference solution: Dissolve ofloxacin RS in 0.05 M hydrochloric acid R to obtain a solution containing about 0.06 mg per ml. Resolution solution: Dissolve ofloxacin RS and propylparaben in acetonitrile R to obtain a solution having a concentration of about 0.1 mg and 2.4 mg per ml, respectively. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Clumn temperature: 35 °C. Detector: A spectrophotometer set at 294 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the resolution solution. The resolution between ofloxacin and propylparaben peak is not less than 2.0. Inject the reference solution 6 times, the symmetry factor of ofloxacin peak is not more than 3.0, the relative standard deviation of ofloxacin peak areas for replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of ofloxacin, C18H20FN3O4, in the eye drops using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C18H20FN3O4 in ofloxacin RS. Storage Store in a cool and dry place, protected from light. Action and use Antibacterial. Usual strength 0.3%.

OFLOXACIN TABLETS

OFLOXACIN TABLETS Tabellae Ofloxacini Ofloxacin tablets contain ofloxacin. They are tablets or film-coated tablets. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of ofloxacin, C18H20FN3O4, 90.0% to 110.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Chloroform - methanol - 0.45 M ammonia solution (150 : 75 : 15). Test solution: Dissolve a quantity of the powdered tablets containing the equivalent of 3 mg of ofloxacin with 10 ml of a mixture of chloroform - methanol (1 : 1), filter. Reference solution: A solution of ofloxacin RS in a mixture of chloroform - methanol (1 : 1) containing about 0.3 mg per ml. Procedure: Apply separately to the plate 2 µl of each solution. Develop over a path of about three fourths of the length of the plate. After removal of the plate, allow it to dry in air. Examine under ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to that in the chromatogram obtained with the reference solution. B. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the peak due to ofloxacin in the chromatogram obtained with the reference solution. Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 900 ml of 0.1 M hydrochloric acid R. Rotation speed: 50 rpm. Time: 30 min. Procedure: After the specified time, withdraw a sample of the medium and filter, discard the first 20 ml of the filtrate. Dilute the filtrate with 0.1 M hydrochloric acid R to obtain a solution containing about 5 µg of ofloxacin per ml. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum at 293 nm, in a 1 cm cell, using 0.1 M hydrochloric acid R as a blank, in comparison with the reference solution of ofloxacin RS having the same concentration in the same medium. Calculate the content of ofloxacin, C18H20FN3O4, dissolved using the absorbances obtained and the declared content of C18H20FN3O4 in ofloxacin RS. Tolerance: Not less than 80% (Q) of the stated amount of ofloxacin is dissolved in 30 minutes. 697

VP V

OMEPRAZOLE

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: A mixture of a 0.24% solution of sodium lauryl sulfate - acetonitrile - glacial acetic acid (580 : 400 : 20). Reference solution: Dissolve ofloxacin RS in 0.05 M hydrochloric acid R to obtain a solution containing about 0.06 mg per ml. Test solution: Weigh 20 tablets (remove the coating in case of the coated tablets), calculate the average mass and powder finely. Transfer an accurately weighed quantity of powdered tablets equivalent to about 30 mg of ofloxacin to a 100 ml volumetric flask, add 75 ml of 0.05 M hydrochloric acid R and sonicate for 10 min. Dilute to volume with 0.05 M hydrochloric acid R and mix well. Filter, discard the first 20 ml of the filtrate. Transfer 10.0 ml of the filtrate to a 50 ml volumetric flask, dilute to volume with 0.05 M hydrochloric acid R and mix well. Resolution solution: Dissolve ofloxacin RS and propylparaben RS in acetonitrile R to obtain a solution having a concentration of about 0.1 mg and 2.4 mg per ml, respectively. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Column temperature: 35 °C. Detector: A spectrophotometer set at 294 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the resolution solution. The resolution between the peaks due to ofloxacin and propylparaben is not less than 2.0. Inject the reference solution 6 times, the symmetry factor of ofloxacin peak is not more than 3.0, the relative standard deviation of ofloxacin peak areas for replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of ofloxacin, C18H20FN3O4, in the tablets using the peak areas in the chromatograms obtained with the test solution, the reference solution, the declared content of C18H20FN3O4 in ofloxacin RS. Storage Store in a well-closed container, in a cool and dry place, protected from light. Action and use Antibacterial. Usual strength 100 mg, 200 mg.

698

OMEPRAZOLE Omeprazolum

and enantiomer

C17H19N3O3S  

M. 345.4

Omeprazole is 5-methoxy-2-[(RS)-[(4-methoxy-3,5dimethylpyridin-2-yl)methyl]sulfinyl]-1H-benzimidazole. It contains not less than 99.0% and not more than 101.0% of C17H19N3O3S, calculated with reference to the dried substance.

Characters White or almost white powder, it shows polymorphism. Very slightly soluble in water, soluble in methylene chloride, sparingly soluble in ethanol (96%) and in methanol. It dissolves in dilute solutions of alkali hydroxides. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of omeprazole RS. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in methanol R, evaporate to dryness and record new spectra using the residues. Appearance of solution Solution S: Dissolve 0.50 g in methylene chloride R and dilute to 25 ml with the same solvent. Solution S is clear (Appendix 9.2). Impurities F and G Not more than 350 ppm for the sum of the contents. The absorbance (Appendix 4.1) of solution S determined at 440 nm is not greater than 0.10. Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use. Mobile phase: Acetonitrile - a 0.14% solution of disodium hydrogen phosphate  R  previously adjusted to pH 7.6 with phosphoric acid R (27 : 73). Test solution: Dissolve 3 mg of the substance to be examined in the mobile phase and dilute to 25.0 ml with the mobile phase. Reference solution (1): Dissolve 1 mg of omeprazole RS and 1 mg of omeprazole impurity D RS in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase.

VP V

Reference solution (3): Dissolve 3 mg of omeprazole for peak identification RS  (containing impurity E) in the mobile phase and dilute to 20.0 ml with the mobile phase. Chromatographic system: A column (12.5 cm × 4.6 mm) packed with stationary phase B (5 µm). Detector: A spectrophotometer set at 280 nm. Flow rate: 1 ml/min. Volume of injection: 40 µl. Procedure: The run time of the test solution is 5 times the retention time of omeprazole. Inject the reference solution. Identification of impurities: Use the chromatogram obtained with reference solution (1) to identify the peak due to impurity D; use the chromatogram supplied with  omeprazole  for peak identification RS and the chromatogram obtained with reference solution (3) to identify the peak due to impurity E. Relative retention with reference to omeprazole (retention time = about 5 min): Impurity E = about 0.6; impurity D = about 0.8. System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to impurity D and omeprazole is at least 3.0. If necessary, adjust the pH of the aqueous part of the mobile phase or the concentration of acetonitrile; an increase in the pH will improve the resolution. Limits: In the chromatogram obtained with the test solution: Impurities D, E: For each impurity, the area is not more than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.15%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.10%). The sum of the peak areas of all impurities is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.5%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Note: Impurity A: 5-methoxy-1H-benzimidazole-2-thiol. Impurity B: 2-[(RS)-[(3,5-dimethylpyridin-2-yl)methyl] sulfinyl]-5-methoxy-1H- benzimidazole. Impurity C: 5-methoxy-2-[[(4-methoxy-3,5-dimethylpyridin-2yl)methyl]sulfanyl]-1H- benzimidazole (ufiprazole). Impurity D: 5-methoxy-2-[[(4-methoxy-3,5-dimethylpyridin-2yl)methyl]sulfonyl]-1H- benzimidazole (omeprazole sulfone). Impurity E: 4-methoxy-2-[[(RS)-(5-methoxy-1H-benzimidazol2-yl)sulfinyl]methyl]-3,5- dimethylpyridine 1-oxide. Impurity F: 8-methoxy-1,3-dimethyl-12-thioxopyrido[1’,2’:3,4] imidazo[1,2-a]benzimidazol- 2(12H)-one. Impurity G: 9-methoxy-1,3-dimethyl-12-thioxopyrido [1’,2’:3,4]imidazo[1,2-a]benzimidazol- 2(12H)-one. Impurity H: 2-[(RS)-[(4-chloro-3,5-dimethylpyridin-2-yl) methyl]sulfinyl]-5-methoxy-1H- benzimidazole.

GASTRO-RESISTANT OMEPRAZOLE CAPSULES Impurity I: 4-methoxy-2-[[(5-methoxy-1H-benzimidazol-2-yl) sulfonyl]methyl]-3,5- dimethylpyridine 1-oxide.

Loss on drying Not more than 0.2% (Appendix 9.6). (1.000 g; 60 °C; in vacuo; 4 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.250  g of the substance to be examined in a mixture of 10 ml of water and 40 ml of ethanol (96%) R. Titrate with 0.1 N sodium hydroxide VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1  N  sodium hydroxide VS  is equivalent to 34.54 mg of C17H19N3O3S. Storage Store in an airtight container, protected from light at a temperature of 2 °C to 8 °C. Action and use Proton pump inhibitor; treatment of peptic ulcer disease. Preparations Gastro-resistant capsules, gastro-resistant tablets, suspension. GASTRO-RESISTANT OMEPRAZOLE CAPSULES Capsulae Omeprazoli Gastro-resistant omeprazole capsules contain omeprazole. They are covered with a gastro-resistant coating or prepared from granules or particles covered with a gastro-resistant coating. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of omeprazole, C17H19N3O3S, 90.0% to 110.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Methylene chloride saturated with ammonia - methylene chloride - isopropanol (2 : 2 : 1). Solvent mixture: Methylene chloride - methanol (1 : 1). Test solution: Take the contents of at least 5 capsules, powder finely and mix well. Transfer a quantity of the contents of the capsules containing about 10 mg of omeprazole to a suitable flask, add 2 ml of the solvent mixture, sonicate for 5 min and allow to stand for 20 min before apply to the plate. 699

GASTRO-RESISTANT OMEPRAZOLE CAPSULES

Reference solution: Dissolve a quantity of omeprazole RS in the solvent mixture to obtain a solution containing about 5 mg per ml. Procedure: Apply separately to the plate 10 µl of each solution. Develop the chromatograph over a path of about three fourths of the length of the plate. Allow it to dry in air and examine under ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to that in the chromatogram obtained with the reference solution. B. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time the principal peak in the chromatogram obtained with the reference solution.

Dissolution (Appendix 11.4) Acid stage Apparatus: Basket. Medium: 500 ml of 0.1 M hydrochloric acid R. Rotation speed: 100 rpm. Time: 2 h. Procedure: Examine by liquid chromatography (Appendix 5.3). Mobile phase, phosphate buffer pH 7.6 and chromatographic system: Proceed as directed under Buffer stage. Reference solution: Transfer an accurately weighed about 50 mg of omeprazole RS to a 250 ml volumetric flask, dissolve with 50 ml of ethanol R, dilute with 0.01 M sodium tetraborate solution R to volume and mix. Transfer 10.0 ml of this solution into a 100 ml volumetric flask, add 20 ml of ethanol R, dilute with 0.01 M sodium borate solution R to volume and mix. Test solution: After 2 h, filter the medium containing the pellets through a sieve with an aperture of not more than 0.2 mm. Collect the pellets on the sieve, and rinse them with water, carefully transfer the pellets to a 100 ml volumetric flask using about 60 ml of 0.01 M sodium tetraborate solution R. Sonicate for about 20 min until the pellets are broken up, add 20 ml of ethanol R to the flask, dilute with 0.01 M sodium tetraborate solution to volume, and mix well. Dilute an appropriate volume of this solution with 0.01 M sodium tetraborate solution R to obtain a solution having a concentration of about 0.02 mg per ml. Chromatograph alternately the reference solution and the test solution. Calculate the content of omeprazole dissolved in the acid medium by subtracting the undissolved amount from the stated amount, using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C17H19N3O3S in the omeprazole RS. Tolerance: Level L1: The requirements are met if no value exceeds 15% of the stated amount dissolved. Level L2: If the results do not conform at L1, continue testing 6 capsules. The average of L2 capsules is not more 700

VP V

than 20% of the stated amount dissolved, and no capsule is greater than 35% of the stated amount dissolved. Level L3: If the results do not conform at L1 and L2, continue testing 12 capsules. The average of 24 capsules is not more than 20% of the stated amount dissolved, not more than 2 capsules are greater than 35% of the stated amount dissolved, and no capsule is greater than 45% of the stated amount dissolved. Buffer stage Apparatus: Basket. Medium: 900 ml of phosphate buffer pH 6.8. Proceed as directed for Acid stage with a new set of the capsules in the same batch. After 2 h, add 400 ml of 0.235 M disodium hydrogen phosphate pH 10.4 to each vessel. Adjust with 2 M hydrochloric acid R or 2 M sodium hydroxide R to pH 6.8 ± 0.05. Preparing the buffers: 0.235 M disodium hydrogen phosphate pH 10.4: Dissolve 33.36 g of anhydrous disodium hydrogen phosphate R in 1000 ml of water, and adjust to pH 10.4 ± 0.1 with 2 M sodium hydroxide R. Phosphate buffer pH 6.8: Add 400 ml of 0.1 M hydrochloric acid R to 320 ml of 0.235 M disodium hydrogen phosphate pH 10.4 and adjust to pH 6.8 ± 0.05 with 2 M hydrochloric acid R or 2 M sodium hydroxide R. Phosphate buffer pH 7.6: Dissolve 0.718 g of sodium dihydrogen phosphate R and 4.49 g of disodium hydrogen phosphate R in 1000 ml of water. Adjust to pH 7.6 ± 0.1 with 2 M hydrochloric acid R or 2 M sodium hydroxide R, if necessary. Dilute 250 ml of this solution with water to 1000 ml. Rotation speed: 100 rpm. Time: 30 min. Procedure: Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - phosphate buffer pH 7.6 (34 : 66), adjust the ratio if necessary. Reference solution (1) (for capsules stated 10 mg): Dissolve an accurately quantity of omeprazole RS in ethanol R to obtain a solution having a concentration of about 2 mg/ml. Dilute with phosphate buffer pH 6.8 to obtain a solution having a concentration of about 0.01 mg/ml. Immediately add 2.0 ml of 0.25 M sodium hydroxide to 10.0 ml of this solution, and mix. Reference solution (2) (for capsules stated 20 mg and 40 mg): Proceed as directed for reference solution (1), except to obtain a solution having a concentration of about 0.02 mg/ml before mixing with 2.0 ml of 0.25 M sodium hydroxide. Test solution (1) (for capsules stated 10 mg and 20 mg): Transfer 5.0 ml of the solution under test to a test tube containing 1.0 ml of 0.25 M sodium hydroxide. Mix well, and filter through a membrane filter having a 1.2 µm or finer porosity. Protect from light.

VP V

GASTRO-RESISTANT OMEPRAZOLE CAPSULES

Test solution (2) (for capsules stated 40 mg): Transfer 5.0 ml of the solution under test to a test tube containing 2.0 ml of 0.25 M sodium hydroxide and 5.0 ml of phosphate buffer pH 6.8. Mix well, and filter through a membrane filter having a 1.2 µm or finer porosity. Protect from light. Chromatographic system: A column (12.5 cm × 4.0 mm) packed with stationary phase B (5 µm). Detector: A spectrophotometer set at 280 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the appropriate reference solution. The test is not valid unless the column efficiency is not less than 2000 theoretical plates and the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of omeprazole, C17H19N3O3S, dissolved using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C17H19N3O3S in the omeprazole RS. Tolerance: For capsules stated 10 mg and 20 mg: Not less than 75% (Q) of the stated amount of omeprazole, C17H19N3O3S, is dissolved in 30 min. For capsules stated 40 mg: Not less than 70% of the stated amount of omeprazole, C17H19N3O3S, is dissolved in 30 min.

Chromatographic purity Solvent mixture, solution A, solution B, reference solution, test solution, mobile phase, and chromatographic system: Proceed as directed in the Assay. Procedure: Separately inject 10 µl of the reference solution and the test solution. Record the chromatograms. Calculate the percentage of each impurity in the portion of capsules taken by the formula: P (%) = (10C/FA)( ri/rs)

in which: C is the concentration of omeprazole RS in the reference solution (in µg/ml); F is the relative response factor (F = 1.6 for peak having the relative retention time about 0.33; F = 3.1 for peak having the relative retention time about 0.64; F = 1 for any other impurity); A is the quantity of omeprazole (in mg) in the portion of the content of the capsules taken, as determined in the Assay; ri is the peak area for each impurity obtained from the test solution; rs is the peak response for omeprazole obtained from the reference solution; Tolerance: Each impurity is not more than 0.5%; and the total impurities is not more than 2.0%.

Assay Examine by liquid chromatography (Appendix 5.3). Solvent mixture: Dissolve 7.6 g of sodium tetraborate R in about 800 ml of water. Add 1.0 g sodium edetate R, and adjust with a 50% solution of sodium hydroxide to pH 11.0 ± 0.1. Transfer this solution to a 2000 ml volumetric flask, add 400 ml of ethanol R, and dilute with water to volume. Mobile phase A: Dissolve 6.0 g of glycine in 1500 ml of water, adjust with a 50% solution of sodium hydroxide to pH 9.0, and dilute with water to 2000 ml, filter. Mobile phase B: Acetonitrile - methanol (85 : 15). Mobile phase: A mixture of solution A and solution B as directed under chromatographic system (adjust the ratio if necessary). Reference solution: Dissolve, by sonicating, an accurately weighed quantity of omeprazole RS in the solvent mixture to obtain a solution having a concentration of about 0.2 mg/ml. Test solution: Weigh 20 capsules, calculate the average weight of the capsule contents and powder finely. Transfer an accurately weighed quantity of the contents of the capsules, equivalent to about 20 mg of omeprazole, to a 100 ml volumetric flask, add 50 ml of the solvent mixture, and sonicate for 15 min, dilute with the solvent mixture to volume and filter. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase B (5 µm). Detector: A spectrophotometer set at 305 nm. Flow rate: 1.2 ml/min. Volume of injection: 20 µl. A gradient programme uses following condition. Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 20

88 → 40

12 → 60

20 - 21

40 →88

60 → 12

21 - 25

88

12

Procedure: System suitability: Inject the reference solution. The test is not valid unless the column efficiency is not less than 20,000 theoretical plates; the symmetry factor is not less than 0.8 and not more than 2; and the relative standard deviation of omeprazole peak areas for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of omeprazole, C17H19N3O3S, in the capsules using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of omeprazole RS.

Storage Store in a well-closed container, in a cool and dry place, protected from light. 701

VP V

ORESOL

Action and use Proton pump inhibitor, used in treatment of gastrointestinal ulcer. Usual strength 20 mg. ORESOL Sales perorales ad rehydratationem Oral rehydration salts Oresol is oral powder containing either glucose or anhydrous glucose, sodium chloride, potassium chloride and either sodium citrate or sodium hydrogen carbonate. After being dissolved in the requisite volume of water they are intended for the prevention and treatment of dehydration due to diarrhoea, including maintenance therapy. Oresol may contain suitable flavouring agents. Where necessary, they may also contain suitable flow agents in the minimum quantity required to achieve a satisfactory product.

Formula: The formula given below is for one sachet. In use, the whole contents of one sachet are dissolved in sufficient water to produce 1 litre.

Composition

The weight (g) ORS ORS hydrocarbonate citrate (B) (C)

Sodium chloride

3.5

3.5

Potassium chloride

1.5

1.5

Sodium citrate

0

2.9

Sodium hydrogen carbonate

2.5

0

Anhydrous glucose (or glucose monohydrate )

20.0 22.0

20.0 22.0

Total weights

27.5 g (or 29.5 g)

27.9 g (or 29.9 g)

If sodium hydrogen carbonate is packed separately, glucose monohydrate may be used. The powder complies with the requirements stated under “Powders” (Appendix 1.7) and with the following requirements.

Content of potassium, K; sodium, Na; bicarbonate, HCO3; chloride, Cl, and citrate, C6H5O7, 90.0% to 110.0% of the stated amount. Content of anhydrous glucose, C6H12O6, or of glucose monohydrate C6H12O6,H2O, 90.0% to 110.0% of the stated amount. 702

Characters A white or almost white dry powder without aggregate, with sweetish salty taste. The solution of the contents of one sachet in 1 litre of water is clear and almost colourless. Identification A. When heated gently to melt, the preparation turns from yellow to brown and swells. And then it burns with a burn sugar odour evolved. Dissolve the contents of one sachet in 250 ml of water and test as follows: B. It yields reaction characteristic of sodium salts (Appendix 8.1). C. It yields reaction (B) characteristic of potassium salts (Appendix 8.1). D. It yields reaction (A) characteristic of chlorides (Appendix 8.1). E. For preparation containing sodium citrate, it yields reactions characteristic of citrates (Appendix 8.1). G. For preparation containing sodium hydrogen carbonate, it yields reactions characteristic of bicarbonates (Appendix 8.1). H. Heat 1 ml of this solution with 5 ml of Fehling reagent R. A red precipitate of copper (I) oxide is produced. pH (for preparation containing citrate only) Dissolve 1.4 g of the preparation in 50 ml of water. The pH of the resulting solution is from 7.0 to 8.8 (Appendix 6.2). Assay For glucose Dissolve about 8 g of the preparation, accurately weighed, in 60 ml of water, add 0.2 ml of a 10% solution of ammonia R and sufficient water to produce 100.0 ml. Mix well and allow to stand for 30 min. Filter, if nessecery, to obtained a clearly solution. Determine the optical rotation α of the resulting solution in a 2 dm tube at 25 °C. Calculate the weight, in g, of anhydrous glucose, C6H12O6, in the weight by multiplying the observed rotation by 0.9477. For citrate Dissolve about 1 g of the preparation, accurately weighed, in 40 ml of anhydrous acetic acid R by warming gently at 50 °C, allow to cool and carry out non-aqueous titration (Appendix 10.6) with 0.1 N perchloric acid VS using a 0.2% solution of 1-naphtholbenzein in anhydrous acetic acid R as indicator. Carry out a blank determination. Each ml of 0.1 N perchloric acid VS is equivalent to 6.303 mg of citrate (C6H5O7). Each g of sodium citrate is equivalent to 0.643 g of C6H5O7. For chloride To 25.0 ml of solution A in the Assay for sodium add 20 ml of water. Titrate with 0.1 N silver nitrate VS using a 5% solution of potassium chromate R as indicator.

VP V

ORESOL

Each ml of 0.1 N silver nitrate VS is equivalent to 3.545 mg of chloride (Cl-). Each g of sodium chloride or potassium chloride is equivalent to 0.6066 g or 0.4765 g of Cl-, respectively. For hydrocarbonate Dissolve about 2 g, accurately weighed, of the preparation (if sodium hydrogen carbonate and other components are packed together) or 0.15 g, accurately weighed, of sodium hydrogen carbonate (if it is packed separately) in 70 ml of water. Titrate with 0.1 N hydrochloric acid VS using methyl orange solution R as indicator. Each ml of 0.1 N hydrochloric acid VS is equivalent to 6.101 mg of hydrocarbonate (HCO3). Each g of sodium hydrogen carbonate is equivalent to 0.7263 mg of HCO3. For sodium Examine by atomic absorption spectrophotometry (Appendix 4.4, method 1). Sodium stock solution: A solution containing 1000 µg of sodium per ml. Potassium chloride solution (4%): Dissolve 4 g of potassium chloride R in ion exchanged water R and dilute to 100 ml with the same solvent. Reference solutions of sodium: Dilute 10.0 ml of sodium stock solution (1000 µg/ml) with sufficient ion exchanged water R to produce 100.0 ml containing 100 µg of sodium per ml. Prepare the reference solutions of sodium with concentrations 2.0, 4.0, 6.0 and 8.0 µg/ml as directed in the table below: Reference Potassium Concentration solution chloride of sodium of sodium solution (µg/ml) (100 µg/ml) (4%) (ml) (ml)

Ion exchanged water sufficient (ml)

0 (blank)

0

10

100

2.0

2.0

10

100

4.0

4.0

10

100

6.0

6.0

10

100

8.0

8.0

10

100

Test solution: Transfer 10 g of the preparation, accurately weighed, to a 500 ml volumetric flask, add 300 ml of ion exchanged water R, shake to dissolve and add ion exchanged water R to volume and mix (solution A). Dilute solution A with ion exchanged water R to obtain a solution of sodium containing about 4 µg/ml. Add potassium chloride solution (4%) to the final solution with the ratio 1 in 10. Procedure: Use an atomic absorption spectrophotometer with a sodium hollow-cathode lamp and an air-acetylene flame. Measure the absorbances of the reference solutions

and the test solution at sodium emission line at 589.0 nm. From the absorbances of the reference solutions and the test solution, plot a calibration curve, representing the correlation between the absorbance and the concentration of sodium. Calculate the concentration of sodium in the test solution from the calibration curve.

For potassium Examine by atomic absorption spectrophotometry (Appendix 4.4, method 1). Sodium chloride solution (4%): Dissolve 4 g of sodium chloride R in ion exchanged water R and dilute to 100 ml with the same solvent. Potassium stock solution: A solution containing 1000 µg of potassium per ml. Reference solutions: Dilute 10.0 ml of potassium stock solution (1000 µg/ml) with sufficient ion exchanged water R to produce 100.0 ml containing 100 µg of sodium per ml. Prepare the reference solutions of potassium with concentrations of 1.0, 2.0, 3.0 and 4.0 µg/ml as directed in the table below: Concentration of potassium (µg/ml)

Reference solution of potassium (100 µg/ml) (ml)

Sodium chloride solution (4%) (ml)

Ion exchanged water sufficient (ml)

0 (blank)

0

10

100

1.0

1.0

10

100

2.0

2.0

10

100

3.0

3.0

10

100

4.0

4.0

10

100

Test solution: Dilute solution A in the Assay for sodium with ion exchanged water R to obtain a solution containing about 2 µg of potassium per ml. Add sodium chloride solution (4%) to the final solution with the ratio 1 in 10. Procedure: Use an atomic absorption spectrophotometer with a potassium hollow-cathode lamp and an air-acetylene flame. Measure the absorbances of the reference solutions and the test solution at potassium emission line at 776.5 nm. From the absorbances of the reference solutions and the test solution, plot a calibration curve, representing the correlation between the absorbance and the concentration of potassium. Calculate the concentration of potassium in the test solution from the calibration curve.

Storage Store in the well-closed sachets, protected from humidity, preferably made of aluminum foil bag. It should be packed for one unit dose dispensing or one day dose dispensing Store at a temperature not exceeding 30 °C. Action and use Used for treatment of hydration and electrolyte deficiencies. 703

VP V

OSELTAMIVIR PHOSPHATE

OSELTAMIVIR PHOSPHATE Oseltamiviri phosphas

C16H28N2O4,H3PO4

M: 410.4

Oseltamivir phosphate is ethyl (3R,4R,5S)-4-acetamido-5amino-3-(1-ethylpropoxy)-cyclohex-1-ene-1- carboxylate phosphate. It contains not less than 98.0% and not more than 102.0% of C16H28N2O4,H3PO4, calculated with reference to the anhydrous substance.

Characters White or almost white powder, it shows polymorphism. Freely soluble in water and in methanol, practically insoluble in methylene chloride. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of oseltamivir phosphate (impurity B-free) RS. If the spectra obtained in the solid state with the substance to be examined and the reference substance show differences, dissolve separately the substance to be examined and the oseltamivir phosphate (impurity B-free) RS in methanol R, evaporate to dryness and record new spectra using the residues. B. It complies with the test for Specific optical rotation. C. Dissolve 200 mg of the substance to be examined in 10 ml of water. It gives reaction (B) of phosphates (Appendix 8.1). Specific optical rotation -30.7° to -32.6°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.50 g of the substance to be examined in water and dilute to 50.0 ml with the same solvent, measured at 25 °C. Impurity B Examine by liquid chromatography (Appendix 5.3) coupled with mass spectrometry (Appendix 4.5). Mobile phase: A 0.154% solution of ammonium acetate R in water for chromatography R - acetonitrile R1 - water for chromatography R (10 : 30 : 60). Test solution: Dissolve 0.100 g of the substance to be examined in water for chromatography R and dilute to 10.0 ml with the same solvent. Reference solution (1): Dissolve 2.5 mg of oseltamivir impurity B RS in 5.0 ml of anhydrous ethanol R and dilute to 704

50.0 ml with water for chromatography R. Dilute 2.0 ml of the solution to 100.0 ml with water for chromatography R. Reference solution (2): Dissolve 50.0 mg of oseltamivir phosphate (impurity B-free) RS in reference solution (1) and dilute to 5.0 ml with the same solution. Chromatographic system: A column (5 cm × 3.0 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (5 µm). Column temperature: 40 °C. Mass detector: The detector settings so as to comply with the system suitability criterion. For example: Ionisation: ESI-positive; Detection m/z: 356.2; Dwell: 580 ms; Gain EMV: 1; Fragmentator voltage: 120 V; Gas temperature: 350 °C; Drying gas flow: 13 L/min; Nebuliser pressure: 345 kPa; Capillary voltage (Vcap): 3 kV. Flow rate: 1.5 ml/min. Volume of injection: 1 µl. Post-column split ratio: Use a split ratio suitable for the mass detector (e.g. 1 : 3). Run time: 3 min. Procedure: System suitability: In the chromatogram obtained with reference solution (2), the relative standard deviation of the peak areas due to oseltamivir phosphate for 6 replicate injections is not more than 15%. Limit: The area of the peak due to impurity B in the chromatogram obtained with the test solution is not more than the area of the corresponding peak in the chromatogram obtained with reference solution (2) (100 ppm). Note: Impurity B: Ethyl (1R,2R,3S,4R,5S)-4-acetamido-5-amino-2azido-3(1-ethylpropoxy)cyclohexanecarboxylate.

Impurity H Examine by gas chromatography (Appendix 5.2). Silylation reagent: Mix 1.0 ml of chlorotrimethylsilane R, 2.0 ml of hexamethyldisilazane R and 10.0 ml of anhydrous pyridine R. Test solution: Accurately weigh 15.0 mg of the substance to be examined into a 2 ml vial and add 1.0 ml of the silylation reagent. Close the vial, shake and heat at 60 °C for 20 min. Centrifuge and discard the precipitate. Reference solution: Accurately weigh 15.0 mg of oseltamivir impurity H RS into a 2 ml vial and add 1.0 ml of anhydrous pyridine R. Close the vial and shake (solution A). Note, impurity H is hygroscopic. Accurately weigh

VP V

OSELTAMIVIR PHOSPHATE

15.0 mg of the substance to be examined into another 2 ml vial and add 1.0 ml of the silylation reagent. Close the vial, shake and heat at 60 °C for 20 min. Centrifuge and discard the precipitate (solution B). Introduce 10.0 µl of solution A and 10.0 µl of solution B into a volumetric flask and dilute to 10.0 ml with anhydrous pyridine R. Chromatographic system: A fused-silica column (30 m long and 0.32 mm in internal diameter) coated with poly(dimethyl)siloxane for chromatography R (film thickness 0.25 µm). Carrier gas: Helium for chromatography. Flow rate: 1.2 ml/min. Split ratio: 1 : 50. Detector: A flame-ionisation detector. Temperature programme:

Column

Time (min)

Temperature (°C)

0-2 2 - 11 11 - 21

180 180 → 250 250

Injection port

260

Detector

260

Volume of injection: 1 µl. Procedure: Relative retention with reference to oseltamivir phosphate (retention time = about 10 min): Impurity H = about 0.5. System suitability: In the chromatogram obtained with reference solution, the relative standard deviation of the peak areas due to impurity H for 6 replicate injections is not more than 5%. Limit: The area of the peak due to impurity H in the chromatogram obtained with test solution is not more than 1.5 times the area of the corresponding peak in the chromatogram obtained with reference solution (0.15%). Note: Impurity H: Tributylphosphane oxide.

Related substances Examine by liquid chromatography (Appendix 5.3). Solution A: Dissolve 6.8 g of potassium dihydrogen phosphate R in 980 ml of water for chromatography R, adjusted to pH 6.0 with 1 M potassium hydroxide R and dilute to 1000 ml with water for chromatography R. Mobile phase: Acetonitrile R1 - methanol R2 - solution A (135 : 245 : 620). Solvent mixture: Acetonitrile R1 - methanol R2 - water for chromatography R (135 : 245 : 620). Test solution: Dissolve 50.0 mg of the substance to be examined in the solvent mixture and dilute to 50.0 ml with the solvent mixture. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 10.0 ml with the solvent mixture.

Reference solution (2): Dissolve 5 mg of oseltamivir impurity A RS and 5.0 mg of oseltamivir impurity C RS in the solvent mixture and dilute to 50.0 ml with the solvent mixture. Dilute 1.0 ml of the solution to 10.0 ml with the solvent mixture. Reference solution (3): Dissolve 50.0 mg of oseltamivir phosphate (impurity B-free) RS in the solvent mixture and dilute to 50.0 ml with the solvent mixture. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase end-capped octylsilyl silica gel for chromatography R (5 µm). Column temperature: 50 °C. Detector: A spectrophotometer set at 207 nm. Flow rate: 1.2 ml/min. Volume of injection: 15 µl. Procedure: The run time is twice the retention time of oseltamivir phosphate. Relative retention with reference to oseltamivir phosphate (retention time = about 17 min): Impurity A = about 0.16; impurity C = about 0.17. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurities A and C is at least 1.5. Limits: In the chromatogram obtained with the test solution: Impurity C: The area of the peak due to impurity C is not more than 0.3 times the area of the corresponding peak in the chromatogram obtained with reference solution (2) (0.3%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). The sum of the peak areas of all impurities is not more than 7 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.7%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A: (3R,4R,5S)-5-acetamido-4-amino-3-(1-ethylpropoxy) cyclohex-1-ene-1-carboxylic acid. Impurity C: (3R,4R,5S)-4-acetamido-5-amino-3-(1-ethylpropoxy) cyclohex-1-ene-1-carboxylic acid.

Water Not more than 0.5% (Appendix 10.3). Determined on 0.500 g. Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. Inject the test solution and reference solution (3). Calculate the content of oseltamivir phosphate, C16H28N2O4,H3PO4, using the areas of the oseltamivir phosphate peaks in the chromatograms obtained with the test solution, reference solution (3) and the declared content of C16H28N2O4,H3PO4 in oseltamivir phosphate (impurity B-free) RS. 705

VP V

OSELTAMIVIR CAPSULES

Storage Protected from light.

Tolerance: Not less than 75% (Q) of the stated amount of oseltamivir, C16H28N2O4, is dissolved in 20 minutes.

Action and use Treatment of influenza.

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase, chromatographic system, reference solution and test solution: Prepare as directed in the Assay. Procedure: Inject alternately the reference solution and the test solution. In the chromatogram obtained with the test solution, the percentage of each individual impurity, if present, is calculated by the following expression:

Preparation Capsules. OSELTAMIVIR CAPSULES Capsulae Oseltamiviri Oseltamivir capsules contain oseltamivir phosphate. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of oseltamivir, C16H28N2O4, 90.0% to 110.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel GF254. Mobile phase: Methanol - ethyl acetate - toluene ammonium hydroxide (8 : 6 : 4 : 2). Test solution: Shake a quantity of the powder in the capsules containing the equivalent of 15 mg of oseltamivir, with 10 ml of methanol R, filter. Reference solution: Dissolve about 20 mg of oseltamivir phosphate RS in 10 ml of methanol R. Procedure: Apply separately to the plate 10 μl of each solution. After developing over 3/4 of the plate. Remove the plate, allow it to dry in air and examine under ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, shape and size to that in the chromatogram obtained with the reference solution. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that of the oseltamivir peak in the chromatogram obtained with the reference solution. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of 0.1 M hydrochloric acid R. Rotation speed: 50 rpm. Time: 20 min. Procedure: After the specified time (20 min), withdraw a sample of the medium, filter. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum at about 240 nm, in a 1 cm cell, using the medium as a blank. Calculate the content of oseltamivir, C16H28N2O4, dissolved using the absorbance of a reference solution of oseltamivir phosphat RS having the same concentration in the medium. 706

A1 × mc × 2 × a × M × 312.4 1 × × 100 F Ac × m1 × L × 410.4

In which: Ac: Peak area of the principal peak from the reference solution. Ai: Peak area of each individual impurity from the test solution. mc: Weight of oseltamivir phosphate RS (mg). mt: Weight of the powder in the test solution (mg). a: The declared content of oseltamivir phosphate RS (%). M: Average mass of the contents (mg) L: The stated amount of oseltamivir on the label (mg). 312,4; 410,4: Molecular weight of oseltamivir and oseltamivir phosphate, respectively. F: Relative response factor (As directed in table 1). Table 1 - Relative retention time, relative response factor and acceptance criteria Relative retention time

Relative response factor F

Acceptance criteria, NMT (%)

Impurity Aa

0.18

1.4

2.0

Bb

Impurity Oseltamivir

0.49

2.7

0.3

1.00

1.0

Cc

1.45

0.9

0.5

Individual unidentified impurity

1.0

0.2

Total unidentified impurities

1.0

0.5

Total impurities

1.0

3.0

Impurity

a:

(3R,4R,5S)-4-Acetylamino-5-amino-3-(1-ethylpropoxy) -1-cyclohexene-1-carboxylic acid. b: 4-Acetylamino-3-hydroxybenzoic acid ethyl ester. c: (3R,4R,5S)-4-Amino-5-acetylamino-3-(1-ethylpropoxy) -1-cyclohexene-1-carboxylic acid ethyl ester.

Assay Examine by liquid chromatography (Appendix 5.3). Phosphate buffer pH 6.0: Dissolve 6.8 g of potassium dihydrogen phosphate R in 980 ml of water, adjust to pH 6.0 with 1 M potassium hydroxide R. Dilute to 1000 ml with water.

VP V

OUABAIN

Mobile phase: Acetonitrile - methanol - phosphate buffer pH 6.0 (135 : 245 : 620). Solvent mixture: Acetonitrile - methanol - 0.01 N phosphoric acid (135 : 245 : 620). Reference solution: Dissolve an accurately weighed quantity of oseltamivir phosphate RS containing the equivalent of 50 mg of oseltamivir in 50.0 ml of the solvent. Test solution: Weigh 20 capsules and calculate the average mass of the capsule contents, and powder finely. Transfer an accurately weighed quantity of the powder containing the equivalent of about 100 mg of oseltamivir, to a 100 ml volumetric flask, add 40 ml of the solvent mixture and sonicate for 20 min. Allow to cool and dilute to volume with the solvent mixture, mix well. Centrifuge at 3000 rpm for 5 min, and use the supernatant liquid. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase B (5 μm), maintained at 50 oC. Detector: A spectrophotometer set at 207 nm. Flow rate: 1.2 ml/min. Volume of injection: 15 μl. Procedure: System suitability: Inject the reference solution for six times; the relative standard deviation of the peak areas is not more than 2.0%. The tailing factor is not more than 2.0. Inject alternately the reference solution and the test solution. Calculate the content of oseltamivir, C16H28N2O4, in the capsules using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C16H28N2O4 in oseltamivir phosphate RS.

Storage Store in an airtight container, in a cool and dry place, at a temparature not exceeding 30 °C, protected from light. Action and use Treatment of influenza. Usual strength 45 mg, 75 mg and 150 mg. OUABAIN Ouabainum

, 8 H2O

C29H44O12,8H2O 



M: 729 

Ouabain is 3β-[(6-deoxy-α-L-mannopyranosyl)oxy]-1β, 5,11α,14,19-pentahydroxy-5β,14β-card-20(22)-enolide. It contains not less than 96.0% and not more than 104.0% of C29H44O12, calculated with reference to the anhydrous substance.

Characters A white or almost white crystalline powder or colourless crystals, sparingly soluble in water and in ethanol, practically insoluble in ethyl acetate. Identification A. In the test for Related substances, the principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). B. Dissolve 2 mg to 3 mg in 2 ml of sulfuric acid R; a pink colour develops which quickly changes to red. The solution shows green fluorescence in ultraviolet light. C. Dissolve about 1 mg in 1 ml of a 1% solution of dinitrobenzene in ethanol (96%) R and add 0.2 ml of dilute sodium hydroxide solution R. An intense blue colour develops. D. Dissolve 0.1 g in 5 ml of a 15% solution of sulfuric acid R and boil for a few minutes. The solution becomes yellow and turbid. Filter and add to the filtrate 5 ml of a 12% solution of sodium hydroxide R and 3 ml of Fehling reagent R. Heat. A red precipitate is formed. Appearance of solution Solution S: Dissolve 0.20 g in 15 ml of water, heating on a water-bath. Allow to cool and dilute to 20.0 ml with the same solvent. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). Specific optical rotation -30° to -33°, determined on solution S and calculated with reference to the anhydrous substance (Appendix 6.4). Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Water - methanol - dimethyl sulfoxide chloroform (4 : 15 : 15 : 70). Test solution: Dissolve a quantity of the substance to be examined corresponding to 20 mg of the anhydrous substance in 1.0 ml of a mixture of 32 volumes of water, 100 volumes of chloroform R and 100 volumes of methanol R Reference solution (1): Dissolve a quantity of ouabain RS corresponding to 20 mg of the anhydrous substance in 1.0 ml of a mixture of 32 volumes of water, 100 volumes of chloroform R and 100 volumes of methanol R. Reference solution (2): Dissolve a quantity of ouabain RS corresponding to 10 mg of the anhydrous substance in a mixture of 32 volumes of water, 100 volumes of chloroform R, 100 volumes of methanol R and dilute to 25 ml with the same mixture of solvents. 707

VP V

OXACILLIN SODIUM MONOHYDRATE

Reference solution (3): Dilute 2.5 ml of reference solution (2) to 10 ml with a mixture of 32 volumes of water, 100 volumes of chloroform R and 100 volumes of methanol R. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 13 cm. Dry the plate immediately at 140 °C for 30 min in a ventilated drying oven. Allow to cool, spray with alcoholic sulfuric acid solution R and heat at 140 °C for 15 min. Any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (2) (2.0%). The test is not valid unless the principal spot in the chromatogram obtained with reference solution (1) and the principal spot in the chromatogram obtained with the test solution migrate over a distance sufficient to give unequivocal separation of the secondary spots and the spot in the chromatogram obtained with reference solution (3) is clearly visible.

Alkaloids and strophanthin-K To 5.0 ml of solution S add 0.5 ml of a 10% solution of tannic acid R. No precipitate is formed. Water 18.0% to 22.0% (Appendix 10.3). Determined on 0.100 g. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g.

OXACILLIN SODIUM MONOHYDRATE Oxacillininum natricum monohydricum

C19H18N3NaO5S,H2O

M. 441.4 

Oxacillin sodium monohydrate is sodium (2S,5R,6R)-3,3dimethyl-6-[[(5-methyl-3-phenylisoxazol-4-yl)carbonyl] amino]-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2carboxylate monohydrate. It contains not less than 95.0% and not more than 102.0% of C19H18N3NaO5S, calculated with reference to the anhydrous substance. Semi-synthetic product derived from a fermentation product.

Characters White or almost white powder. Freely soluble in water, soluble in methanol, practically insoluble in methylene chloride. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of oxacillin sodium monohydrate RS. B. It gives reaction (A) of sodium (Appendix 8.1) .

Assay Dissolve 40.0 mg in ethanol (96%) R and dilute to 50.0 ml with the same solvent. Dilute 5.0 ml of the solution to 100.0 ml with ethanol (96%) R. Prepare a reference solution in the same manner using 40.0 mg of ouabain RS. To 5.0 ml of each solution add 3.0 ml of alkaline sodium picrate solution R, allow to stand protected from bright light for 30 min and measure the absorbance (Appendix 4.1) of each solution at the maximum at 495 nm using as the compensation liquid a mixture of 5.0 ml of ethanol (96%) R and 3.0 ml of alkaline sodium picrate solution R prepared at the same time. Calculate the content of C29H44O12 from the absorbances measured and the concentrations of the solutions.

Appearance of solution Dissolve 2.50 g of the substance to be examined in water and dilute to 25.0 ml with the same solvent. The solution is clear (Appendix 9.2) and its absorbance (Appendix 4.1) at 430 nm is not greater than 0.10.

Storage Store protected from light.

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Mix 25 volumes of acetonitrile R and 75 volumes of a 0.27% solution of potassium dihydrogen phosphate R previously adjusted to pH 5.0 with dilute sodium hydroxide solution R. Test solution: Dissolve 50.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase.

Action and use For the treatment of heart failure.

708

pH 4.5 to 7.5 (Appendix 6.2) . Dissolve 0.30 g in carbon dioxide-free water and dilute to 10 ml with the same solvent. Specific optical rotation +196° to +212°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.250 g in water and dilute to 25.0 ml with the same solvent.

VP V

Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Reference solution (2): Dissolve 5 mg of cloxacillin sodium RS (impurity E) and 5 mg of oxacillin sodium monohydrate RS in the mobile phase and dilute to 50.0 ml with the mobile phase. Reference solution (3): In order to prepare impurities B and D in situ, dissolve 25 mg of the substance to be examined in 1 ml of 0.05 M sodium hydroxide R, allow to stand for 3 min, then dilute to 100 ml with the mobile phase. Inject immediately. Reference solution (4): Dissolve 5 mg of oxacillin for peak identification RS (containing impurities E, F, G, I and J) in 5 ml of the mobile phase. Chromatographic system: A column (25 cm × 4.0 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 225 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. The run time is 7 times the retention time of oxacillin. Procedure: Inject the blank, the test solution and the reference solutions. Identification of impurities: In the chromatogram obtained with reference solution (3), the 2 peaks eluting before the principal peak are due to impurities B and D respectively. Use the chromatogram supplied with oxacillin for peak identification RS and the chromatogram obtained with reference solution (4) to identify the peaks due to impurities E, F, G, I and J. Relative retention with reference to oxacillin (retention time = about 5 min): Impurity A = about 0.3; impurity B (isomer 1) = about 0.4; impurity B (isomer 2) = about 0.5; impurity C = about 0.65; impurity D (2 isomers) = about 0.9; impurity E = about 1.5; impurity F = about 1.9; impurity G = about 2.1; impurity H = about 3.5; impurity I = about 3.8; impurity J = about 5.8. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to oxacillin and impurity E is at least 2.5. The chromatogram obtained with reference solution (4) is similar to the chromatogram supplied with oxacillin for peak identification RS. Limits: In the chromatogram obtained with the test solution: Impurity B: The sum of the areas of the 2 isomer peaks of impurity B is not more than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (1.5%). Impurity E: The area of the peak due to impurity E is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (1.0%).

OXACILLIN SODIUM MONOHYDRATE

Impurities D (sum of the 2 isomers), F, G, I, J: The sum of the areas of the 2 isomer peaks of impurity D, for each impurity F, G, I, J is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%); Any other impurity: For each impurity, the area is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). The sum of the peak areas of all impurities is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (1) (3.0%). Disregard limit: Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A: (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia-1azabicyclo[3.2.0]heptane-2-carboxylic acid (6-aminopenicillanic acid). Impurity B: (4S)-2-[carboxy[[(5-methyl-3-phenylisoxazol-4-yl) carbonyl]amino]methyl]-5,5- dimethylthiazolidine -4-carboxylic acid (penicilloic acids of oxacillin). Impurity C: 5-methyl-3-phenylisoxazole-4-carboxylic acid. Impurity D: (2RS,4S)-5,5-dimethyl-2-[[[(5-methyl-3-phenylisoxazol -4-yl)carbonyl]amino]methyl]thiazolidine-4-carboxylic acid (penilloic acids of oxacillin). Impurity E: (2S,5R,6R)-6-[[[3-(2-chlorophenyl)-5-methylisoxazol -4-yl]carbonyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo [3.2.0]heptane-2-carboxylic acid (cloxacillin). Impurity F: (2R,5R,6R)-3,3-dimethyl-6-[[(5-methyl-3phenylisoxazol-4-yl)carbonyl]amino]-7-oxo-4-thia-1-azabicyclo [3.2.0]heptane-2-carbothioic acid (thiooxacillin). Impurity G: (2S,5R,6R)-6-[[[3-(chlorophenyl)-5-methylisoxazol4-yl]carbonyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo [3.2.0]heptane-2-carboxylic acid (cloxacillin isomer). Impurity I: (2S,5R,6R)-6-[[(2S,5R,6R)-3,3-dimethyl-6-[[(5methyl-3-phenylisoxazol-4- yl)carbonyl]amino]-7-oxo-4-thia1-azabicyclo[3.2.0]heptane-2-carbonyl]amino]-3, 3-dimethyl7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid (6APA oxacillin amide). Impurity J: (2S,5R,6R)-6-[[(2R)-[(2R,4S)-4-carboxy-5,5dimethylthiazolidin -2-yl][[(5-methyl- 3-phenylisoxazol-4-yl) carbonyl]amino]acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1azabicyclo[3.2.0]heptane-2-carboxylic acid (ozolamide of 6-APA dimer).

Ethyl acetate and butyl acetate Examine by head-space gas chromatography (Appendix 5.2). Test solution: Dissolve 0.200 g of the substance to be examined in 6.0 ml of water. Reference solution: Dissolve 83 mg of butyl acetate R and 83 mg of ethyl acetate R in water and dilute to 250.0 ml with the same solvent. Use 6.0 ml of this solution. 709

VP V

OXYGEN

Close the vials immediately with a rubber membrane stopper coated with polytetrafluoroethylene and secured with an aluminium crimped cap. Mix to obtain a homogeneous solution. Chromatographic system: A fused-silica capillary column (50 m long and 0.32 mm in internal diameter) coated with a film poly(dimethyl) siloxane R (film thickness 5 µm). Carrier gas: Helium for gas chromatography. Flow rate: 2 ml/min. Static head-space conditions: Equilibration temperature: 80 °C. Equilibration time: 60 min. Transfer-line temperature: 140 °C. Pressurisation time: 30 s. Temperature programme:

Column

Time (min)

Temperature (°C)

0-6

70

6 - 16

70 → 220

16 - 18

220

Injection port

140

Detector

250

Detector: A flame-ionisation detector. Retention time: Ethyl acetate = about 10 min; butyl acetate = about 15.5 min. Limits: Butyl acetate: Not more than 1.0%. Ethyl acetate: Not more than 1.0%.

N,N-Dimethylanilin Not more than 20 ppm (Appendix 10.16, method 2). 2-Ethylhexanoic acid Not more than 0.8% (Appendix 10.17). Water 3.5% to 5.0% (Appendix 10.3). Determined on 0.300 g. Bacterial endotoxins Less than 0.20 EU/mg (Appendix 13.2). If intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase and chromatographic system as described in the test for Related substances. Test solution: Dissolve 50.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Dilute 5.0 ml of this solution to 50.0 ml with the mobile phase. 710

Reference solution: Dissolve 50.0 mg of oxacillin sodium monohydrate RS in the mobile phase and dilute to 50.0 ml with the mobile phase. Dilute 5.0 ml of this solution to 50.0 ml with the mobile phase. Procedure: Injection the test solution and reference solution. Calculate the content of oxacillin sodium, C19H18N3NaO5S, using the peak areas in the chromatograms obtained with the test solution, the reference solution and using the declared content of C19H18N3NaO5S in oxacillin sodium monohydrate RS.

Action and use Penicillin antibacterial. Storage Store in an airtight container. If the substance is sterile, store in a sterile, airtight container. Preparations Capsules, injection powder. OXYGEN Oxygenium O2



M. 32.00

Oxygen contains not less than 99.5% of O2 (v/v).

Characters A colourless gas; odourless. One volume dissolves in about 32 volumes of water and in about 7 volumes of ethanol, both at a pressure of 101.3 kPa and 20°C. Identification A. Place a glowing splinter of wood into the test gas; the splinter bursts into flame. B. Shake with alkaline pyrogallol R; the test gas is absorbed and the solution becomes dark brown. Oxygen labelled as having been produced by the airliquefaction process may be exempted from the requirements of the tests for carbon monoxide and carbon dioxide. Carbon monoxide Not more than 5 ppm (v/v). Examine by appendix 9.5 “Limit test for carbon monoxide in medicinal gases”. Pass 7.5 litres of the test gas from the container through the apparatus at flow rate: 4 L/h. Titrate the liberated iodine with 0.002 M sodium thiosulfate VS. Repeat the procedure using 7.5 litres of argon R. For the following tests - "carbon monoxide", "Carbon dioxide", and "Oxidizing substances", - pass the test gas through the appropriate reagent contained in a hermetically closed flat-bottomed glass cylinder (with dimensions such that 50 ml of liquid reaches a height of 12 cm to 14 cm.

VP V

Carbon dioxide Not more than 0.03% (v/v). Pass 1.0 L of the test gas through 50 ml of a clear 0.15 M barium hydroxide R. Similarly prepare a reference solution by adding 1.0 ml of a 0.11% solution of sodium hydrogen carbonate R in carbon dioxide-free water R to 50 ml of 0.15 M barium hydroxide R. Any turbidity in the solution after the passage of the test gas is not more intense than that of the reference solution. Acidity and alkalinity Test solution: Pass 2.0 L of the test gas through a mixture of 0.10 ml of 0.01 N hydrochloric acid VS and 50 ml of carbon dioxide-free water R. Reference solution (1): 50 ml of carbon dioxide-free water R. Reference solution (2): A mixture of 0.20 ml of 0.01 N hydrochloric acid VS and 50 ml of carbon dioxide-free water R. To each solution add 0.1 ml of 0.02% solution of methyl red in ethanol (70%); the intensity of the colour in the solution of the test gas is between those of reference solutions 1 and 2.

OXYGEN

the upper tap is used to introduce the absorbent solution. Wash the burette with water and dry. Open the two taps. Connect the nozzle to the container of the test gas and set the flow rate to 1 L/min. Flush the burette by passing the gas through it for 1 min. Close the upper tap of the burette and immediately afterwards the lower tap. Rapidly disconnect the burette from the container of the test gas, and give a half turn to the upper tap to eliminate any excess pressure in the burette. Keeping the burette vertical, fill the funnel with a freshly prepared mixture of 21 ml of 56% solution of potassium hydroxide R and 130 ml of 20% solution of sodium dithionite R. Open the upper tap slowly. The solution absorbs the oxygen and enters the burette. Allow to stand for 10 min without shaking. Read the level of the liquid meniscus on the graduated part of the burette; the figure represents the content of oxygen as a percentage in v/v.

Oxidizing substances To two cylinders add 50 ml of freshly prepared potassium iodide starch R and about 0.2 ml of glacial acetic acid R. Protect the cylinders from light. Pass 5.0 L of the test gas into one of the solutions and compare the colour produced. The solutions in both cylinders remain colourless. Water The apparatus consists either of an electrolytic hygrometer as described below, an appropriate humidity detector tube, or a capacity hygrometer. The measuring cell consists of a thin film of phosphoric anhydride placed between two coiled platinum wires that act as electrodes. The water vapour in the test gas is absorbed by the phosphoric anhydride to form phosphoric acid, which acts as an electrical conductor. Before introducing the test gas into the device, allow the gas to stabilize at room temperature and make sure that the temperature is constant throughout the apparatus. Apply a continuous voltage across the electrodes to produce electrolysis of the water and regeneration of phosphoric anhydride. Measure the resulting electrical current, which is proportional to the water content in the test gas. (This is a self-calibrating system that obeys Faraday's law.) Calculate the content of water; not more than 60 μg/l. Assay For the determination use a 25-ml capacity gas burette (Fig. 1) in the form of a chamber with at its upper end, a tube graduated in 0.2% between 95 and 100, and isolated at each end by a tap with a conical barrel. The lower tap is joined to a tube with an olive-shaped nozzle and is used to introduce the test gas into the apparatus. A cylindrical funnel above

Figure 1 - Burette used for the assay of oxygen.

Storage.  Oxygen should be kept as compressed gas or liquid at cryogenic temperature, in appropriate containers complying with the safety regulations of the national authority. Valves or taps should not be lubricated with oil or grease. 711

VP V

OXYMETAZOLINE HYDROCHLORIDE

Appearance of solution Solution S: Dissolve 2.5 g in water and dilute to 50 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution BY7 (Appendix 9.3, method 2).

OXYMETAZOLINE HYDROCHLORIDE Oxymetazolini hydrochloridum

C16H24N2O,HCl 

M. 296.8

Oxymetazoline hydrochloride is 3-[(4,5-dihydro-1Himidazol-2-yl)methyl]-6-(1,1-dimethylethyl)-2,4dimethylphenol hydrochloride. It contains not less than 99.0% and not more than 101.0% of C16H24N2O,HCl, calculated with reference to the anhydrous substance.

Characters White or almost white, crystalline powder. Freely soluble in water and in ethanol (96%). Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of oxymetazoline hydrochloride RS. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Diethylamine - cyclohexane - anhydrous ethanol (6 : 15 : 79). Test solution: Dissolve 20 mg of the substance to be examined in a mixture of ethyl acetate R - methanol R (1 : 1) and dilute to 5 ml with the same mixture of solvents. Reference solution: Dissolve 20 mg of oxymetazoline hydrochloride RS in a mixture of ethyl acetate R - methanol R (1 : 1) and dilute to 5 ml with the same mixture of solvents. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of two thirds of the plate. Dry the plate in a current of warm air for 5 min, then allow to cool. Spray with a freshly prepared 0.5% solution of potassium ferricyanide R in 1.3% solution of ferric chloride R; examine in daylight. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. C. Dissolve about 2 mg of the substance to be examined in 1 ml of water, then add 0.2 ml of a 5% solution of sodium nitroprusside R and 0.2 ml of dilute sodium hydroxide solution R. Allow to stand for 10 min. Add 2 ml of 4.2% solution of sodium hydrogen carbonate R. A violet colour develops. D. It gives reaction (A) of chlorides (Appendix 8.1). 712

Acidity or alkalinity Dissolve 0.25 g of the substance to be examined in carbon dioxide-free water R and dilute to 25 ml with the same solvent. Add 0.1 ml of methyl red solution R and 0.2 ml of 0.01 M hydrochloric acid VS. The solution is red. Not more than 0.4 ml of 0.01 M sodium hydroxide (VS) is required to change the colour of the indicator to yellow. Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use. Mobile phase A: A 0.136% solution of potassium dihydrogen phosphate R adjusted to pH 3.0 with phosphoric acid R. Mobile phase B: Acetonitrile R1. Test solution: Dissolve 50.0 mg of the substance to be examined in water and dilute to 50.0 ml with the same solvent. Reference solution (1): Dilute 5.0 ml of the test solution to 100.0 ml with water. Dilute 2.0 ml of this solution to 100.0 ml with water. Reference solution (2): Dissolve 5.0 mg of oxymetazoline impurity A RS and 5 mg of the substance to be examined in water R and dilute to 50.0 ml with the same solvent. Dilute 10.0 ml of the solution to 50.0 ml with water. Reference solution (3): Dilute 1.0 ml of reference solution (2) to 20.0 ml with water. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography with polar incorporated groups R (5 µm). Detector: A spectrophotometer set at 220 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0-5

70

30

5 - 20

70 → 15

30 → 85

20 - 35

15

85

Relative retention with reference to oxymetazoline (retention time = about 5.0 min): impurity A = about 0.9. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurity A and oxymetazoline is at least 4.0.

VP V

Limits: Impurity A: The area of the peak due to impurity A is not more than 1.5 times the area of the corresponding peak in the chromatogram obtained with reference solution (3) (0.15%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). Total peak area of all impurities is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A: N-(2-aminoethyl)-2-[4-(1,1-dimethylethyl)-3-hydroxy -2,6- dimethylphenyl]acetamide, Impurity B. 2-[[4-(1,1-dimethylethyl)-2,6-dimethylphenyl] methyl]-4,5-dihydro-1H-imidazole (xylometazoline). Impurity C: 2-[4-(1,1-dimethylethyl)-3-hydroxy-2,6-dimethylphenyl]acetamide. Impurity D: 2-[4-(1,1-dimethylethyl)-3-hydroxy-2,6-dimethylphenyl]acetic acid. Impurity E: 2-[4-(1,1-dimethylethyl)-3-hydroxy-2,6-dimethylphenyl]acetonitrile.

Water Not more than 0.3% (Appendix 10.3). Determined on 1.00 g. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.200 g of the substance to be examined in a mixture of 20 ml of acetic anhydride R and 20 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 29.68 mg of C16H25ClN2O. Storage Store in an airtight container. Action and use Alpha-adrenoceptor agonist; decongestant. OXYMETAZOLINE NASAL DROPS Nasalia Oxymetazolini Oxymetazoline nasal drops are a solution of oxymetazoline hydrochloride in water. It may also contain suitable excipients. The nasal drops comply with the requirements stated under “Nasal drops” (Appendix 1.15) and with the following requirements.

OXYMETAZOLINE NASAL DROPS

Content of oxymetazoline hydrochloride, C16H24N2O, HCl, 90.0% to 110.0% of the stated amount. Characters A clear and colourless solution. pH 4.0 to 6.5 (Appendix 6.2). Identification A. Transfer a volume of the nasal drops containing about 2.5 mg of oxymetazoline hydrochloride to a 60 ml separating funnel, add water to produce 10 ml. Add 2 ml of a 10% solution of sodium carbonate R. Extract the solution with 10 ml of chloroform R. Transfer the chloroform layer to another separating funnel and extract with 10 ml of 0.1 M hydrochloric acid R. Allow to separate and discard the chloroform layer. Transfer 8 ml of the acidic aqueous layer to the test tube, neutralize with a few drops of 1 M sodium hydroxide R, add 1 drop of 1 M sodium hydroxide R in excess and mix. And then add a few drop of a 5% solution of sodium nitroprusside R and 2 drops of a 15% solution of sodium hydroxide R, allow to stand for 10 min. Add 0.1 M hydrochloric acid R until pH is between 8 and 9. Allow to stand for 10 min, a violet colour is produced. B. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the peak due to oxymetazoline hydrochloride in the chromatogram obtained with the reference solution. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol - ammonium nitrate buffer solution pH 9.5 (9 : 1). Test solution: Dilute 2.0 ml of the nasal drops with methanol R to 20.0 ml. Filter. Reference solution: Dissolve a quantity of oxymetazoline hydrochloride RS, accurately weighed, in water to obtain a solution containing the same concentration as the nasal drops. Dilute 2.0 ml of the resulting solution with methanol R to 20.0 ml. Chromatographic system: A column (250 mm × 4 mm) packed with stationary phase A (5 µm). Detector: A spectrophotometer set at 280 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: Inject alternately the reference solution and the test solution. Calculate the content of oxymetazoline hydrochloride, C16H24N2O,HCl, in the nasal drops using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C16H24N2O,HCl in oxymetazoline hydrochloride RS. 713

VP V

OXYTETRACYCLINE DIHYDRATE

Storage Store in a cool place, protected from light.

B. To about 2 mg of the substance to be examined, add 5 ml of sulfuric acid R. A deep red colour develops. Add further 2.5 ml of water to the solution. The colour becomes yellow. C. Dissolve about 10 mg of the substance to be examined in a mixture of 1 ml of dilute nitric acid R and 5 ml of water. Shake and add 1 ml of 0.1 N silver nitrate VS. Any opalescence in the solution is not more intense than that in a mixture of 1 ml of dilute nitric acid R, 5 ml of a 0.0021% solution of potassium chloride and 1 ml of 0.1 N silver nitrate VS .

Usual strength 0.025% and 0.05%. OXYTETRACYCLINE DIHYDRATE Oxytetracyclinum dihydratum

, 2H2O

C22H24N2O9, 2H2O

M. 496.4

Oxytetracycline dihydrate is (4S,4aR,5S,5aR,6S,12aS)-4(dimethylamino)-3,5,6,10,12,12a-hexahydroxy-6-methyl1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2carboxamide dihydrate. It contains not less than 95.0% and not more than 102.0% of C22H24N2O9, calculated with reference to the anhydrous substance. Substance produced by the growth of certain strains of Streptomyces rimosus or obtained by any other means.

Characters Yellow, crystalline powder. Very slightly soluble in water, dissolves in dilute acid and alkaline solutions. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Octadecylsilyl silica gel F254. Mobile phase: Acetonitrile - methanol - a 6.3% solution of oxalic acid previously adjusted to pH 2.0 with concentrated ammonia R (20 : 20 : 60). Test solution: Dissolve 5 mg of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Reference solution (1): Dissolve 5 mg of oxytetracycline RS in methanol R and dilute to 10 ml with the same solvent. Reference solution (2): Dissolve 5 mg of oxytetracycline RS, 5 mg of tetracycline hydrochloride RS and 5 mg of minocycline hydrochloride RS in methanol R and dilute to 10 ml with the same solvent. Procedure: Apply separately to the plate 1 µl of each solution. Develop over a path of 15 cm. Dry the plate in a current of air. Examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (1). The test is invalid unless in the chromatogram obtained with reference solution (2) shows three clearly separated spots. 714

pH 4.5 to 7.5 (Appendix 6.2). Suspend 0.1 g of the substance to be examined in 10 ml of carbon dioxide-free water R. Specific optical rotation -203° to -216°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.250 g of the substance to be examined in 0.1 M hydrochloric acid R and dilute to 25.0 ml with the same solvent. Specific absorbance From 290 to 310 determined at 353 nm, calculated with reference to the anhydrous substance (Appendix 4.1). Dissolve 20.0 mg of the substance to be examined in buffer solution pH 2.0 R and dilute to 100.0 ml with the same buffer solution. Dilute 10.0 ml of the solution to 100.0 ml with buffer solution pH 2.0 R. Measure absorbance at 353 nm. Light-absorbing impurities Carry out the measurements within 1 h of preparing the solutions. Dissolve 20.0 mg of the substance to be examined in a mixture of 1 M hydrochloric acid - methanol (1 : 99) and dilute to 10.0 ml with the same solvent. The absorbance (Appendix 4.1) of the solution, determined at 430 nm has a maximum of 0.25 (calculated with reference to anhydrous substance). Dissolve 0.100 g of the substance to be examined in a mixture of 1 M hydrochloric acid - methanol (1 : 99) and dilute to 10.0 ml with the same solvent. The absorbance (Appendix 4.1) of the solution, determined at 490 nm has a maximum of 0.20 (calculated with reference to anhydrous substance). Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Weigh 60.0 g of 2-methyl-2-propanol R and transfer to a 1000 ml volumetric flask with the aid of 200 ml of water; add 60 ml of 0.33 M phosphate buffer solution pH 7.5, 50 ml of a 1% solution of tetrabutylammonium hydrogen sulfate R adjusted to pH 7.5 with dilute sodium hydroxide solution R. Add 10 ml of

VP V

a 0.04% solution of sodium edetate R adjusted to pH 7.5 with dilute sodium hydroxide solution R; dilute to 1000 ml with water. Filter and degas before use. Test solution: Dissolve 20.0 mg of the substance to be examined in 0.01 M hydrochloric acid R and dilute to 25.0 ml with the same solvent. Reference solution (1): Dissolve 20.0 mg of oxytetracycline RS in 0.01 M hydrochloric acid R and dilute to 25.0 ml with the same solvent. Reference solution (2): Dissolve 20.0 mg of 4-epioxytetracycline RS in 0.01 M hydrochloric acid R and dilute to 25.0 ml with the same solvent. Reference solution (3): Dissolve 20.0 mg of tetracycline hydrochloride RS in 0.01 M hydrochloric acid R and dilute to 25.0 ml with the same solvent. Resolution solution: Mix 1.5 ml of reference solution (1), 1.0 ml of reference solution (2) and 3.0 ml of reference solution (3) and dilute to 25.0 ml with 0.01 M hydrochloric acid R. Reference solution (4): Mix 1.0 ml of reference solution (2) and 4.0 ml of reference solution (3) and dilute to 200.0 ml with 0.01 M hydrochloric acid R. Chromatographic system: A column (25 cm × 4.6 mm) packed with styrene-divinyl benzene copolymer R (8 µm). Column temperature: 60 °C. Detector: A spectrophotometer at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the resolution solution. The resolution between the peaks due to 4-epioxytetracycline (1st peak) and oxytetracycline (2nd peak) is at least 4.0. The resolution between the peaks and due to oxytetracycline and tetracycline (3rd peak) is at least 5.0. Adjust the content of 2-methyl-2-propanol in the mobile phase if necessary. Symmetry factor of the peak due to oxytetracycline is not more than 1.25. Inject the test solution and reference solution (4). Limits: 4-epioxytetracycline: The area of the peak due to 4-epioxytetracycline in the chromatogram obtained with the test solution is not more than the area of the corresponding peak in the chromatogram obtained with reference solution (4) (0.5%). Tetracycline: The area of the peak due to teracycline in the chromatogram obtained with the test solution is not more than the area of the corresponding peak in the chromatogram obtained with reference solution (4) (2.0%). 2-Acetyl-2-decarbamoyloxytetracycline (eluting on the tail of the principal peak): The area of the peak due to 2-acetyl2-decarbamoyloxytetracycline in the chromatogram obtained with the test solution is not more than 4 times the area of the peak due to 4-epioxytetracycline in the chromatogram obtained with reference solution (4) (2.0%).

OXYTETRACYCLINE HYDROCHLORIDE

Disregard any peak whose area is less than 0.02 times the area of the peak due to oxytetracycline in the chromatogram obtained with resolution solution (0.1%).

Heavy metals Not more than 50 ppm (Appendix 9.4.8). Take 0.5 g of the substance to be examined and carry out the method 6. Prepare the standard using 2.5 ml of lead standard solution (10 ppm Pb) R. Water 6.0% to 9.0% (Appendix 10.3). Determined on 0.250 g. Sulfated ash Not more than 0.5% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by liquid chromatography (Appendix 5.3). Chromatographic system is the same as described in the test for Related substances. Carry out with the test solution and reference solution (1). Calculate the content of C22H24N2O9, using the areas of the principal peaks in the chromatograms obtained with the test solution and the reference solution (1). Storage Store in an airtight container, protected from light. Action and use Antibacterial. Preparation Tablets. OXYTETRACYCLINE HYDROCHLORIDE Oxytetracyclinum hydrochloridum

C22H24N2O9,HCl 

M. 496.9

Oxytetracycline hydrochloride is (4S,4aR,5S,5aR,6S,12aS)-4(dimethylamino)-3,5,6,10,12,12a-hexahydroxy-6- methyl1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2carboxamide hydrochloride. It contains not less than 95.0% and not more than 102.0% of C22H24N2O9,HCl, calculated with reference to the anhydrous substance. Substance produced by the growth of certain strains of Streptomyces rimosus or obtained by any other means. 715

OXYTETRACYCLINE HYDROCHLORIDE

Characters A yellow, crystalline powder, hygroscopic. Freely soluble in water, sparingly soluble in ethanol (96%). Solutions in water become turbid on standing, owing to the precipitation of oxytetracycline. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Octadecylsilyl silica gel F254. Mobile phase: Acetonitrile - methanol - 63 g/L solution of oxalic acid previously adjusted to pH 2.0 with concentrated ammonia (20 : 20 : 60). Test solution: Dissolve 5 mg of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Reference solution (1): Dissolve 5 mg of oxytetracycline hydrochloride RS in methanol R and dilute to 10 ml with the same solvent. Reference solution (2): Dissolve 5 mg of oxytetracycline hydrochloride RS, 5 mg of tetracycline hydrochloride RS and 5 mg of minocycline hydrochloride RS in methanol R and dilute to 10 ml with the same solvent. Procedure: Apply separately to the plate 1 µl of each of the test solution, reference solution (1) and reference solution (2) . Develop over a path of three fourths of the plate. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution (1). The test is not valid unless the chromatogram obtained with the reference solution (2) shows 3 clearly separated spots. B. To about 2 mg add 5 ml of sulfuric acid R. A deep red colour develops. Add the solution to 2.5 ml of water. The colour becomes yellow. C. It gives reaction (A) of chlorides (Appendix 8.1). pH 2.3 to 2.9 (Appendix 6.2). Dissolve 0.1 g in 10 ml of carbon dioxide-free water. Specific optical rotation -188° to -200°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.250 g in 0.1 M hydrochloric acid R and dilute to 25.0 ml with the same acid. Specific absorbance 270 to 290, calculated with reference to the anhydrous substance (Appendix 4.1). The specific absorbance determined at the maximum at 353 nm Dissolve 20.0 mg in buffer solution pH 2.0 R and dilute to 100.0 ml with the same buffer solution. Dilute 10.0 ml of the solution to 100.0 ml with buffer solution pH 2.0 R. 716

VP V

Light-absorbing impurities Carry out the measurements within 1 h of preparing the solutions (Appendix 4.1). Dissolve 20.0 mg in a mixture of 1 volume of 1 M hydrochloric acid R and 99 volumes of methanol R and dilute to 10.0 ml with the same mixture of solvents. The absorbance determined at 430 nm has a maximum of 0.50, calculated with reference to the anhydrous substance. Dissolve 0.100 g in a mixture of 1 volume of 1 M hydrochloric acid R and 99 volumes of methanol R and dilute to 10.0 ml with the same mixture of solvents. The absorbance determined at 490 nm has a maximum of 0.20, calculated with reference to the anhydrous substance. Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use. Mobile phase A: Weigh 30.0 g of 2-methyl-2-propanol R and transfer separately to 1000 ml volumetric flasks with the aid of 200 ml of water; to each flask add 60 ml of 0.33 M phosphate buffer solution pH 7.5 R, 50 ml of a 10 g/L solution of tetrabutylammonium hydrogen sulfate R adjusted to pH 7.5 with dilute sodium hydroxide solution R and 10 ml of a 0.4 g/L solution of sodium edetate R adjusted to pH 7.5 with dilute sodium hydroxide solution R; dilute solution to 1000 ml with water. Filter and degas before using. Mobile phase B: Repeat the procedure as mobile phase A using 100.0 g of 2-methyl-2-propanol R. Test solution: Dissolve 20.0 mg of the substance to be examined in 0.01 M hydrochloric acid and dilute to 25.0 ml with the same acid. Reference solution (1): Dissolve 20.0 mg of oxytetracycline RS in 0.01 M hydrochloric acid and dilute to 25.0 ml with the same acid. Reference solution (2): Dissolve 20.0 mg of 4-epioxytetracycline RS in 0.01 M hydrochloric acid and dilute to 25.0 ml with the same acid. Reference solution (3): Dissolve 20.0 mg of tetracycline hydrochloride RS in 0.01 M hydrochloric acid and dilute to 25.0 ml with the same acid. Reference solution (4): Dissolve 8.0 mg of α-apooxytetracycline RS in 5 ml of 0.01 M sodium hydroxide and dilute to 100.0 ml with 0.01 M hydrochloric acid R. Reference solution (5): Dissolve 8.0 mg of β-apooxytetracycline RS in 5 ml of 0.01 M sodium hydroxide and dilute to 100.0 ml with 0.01 M hydrochloric acid R. Reference solution (6): Mix 1.5 ml of reference solution (1), 1.0 ml of reference solution (2), 3.0 ml of reference solution (3), 3.0 ml of reference solution (4) and 3.0 ml of reference solution (5) and dilute to 25.0 ml with 0.01 M hydrochloric acid.

VP V

OXYTETRACYCLINE HYDROCHLORIDE

Reference solution (7): Mix 1.0 ml of reference solution (2), 4.0 ml of reference solution (3) and 40.0 ml of reference solution (5) and dilute to 200.0 ml with 0.01 M hydrochloric acid R. Chromatographic system: A column (25 cm × 4.6 mm) packed styrene-divinylbenzene copolymer R (8 µm). Column temperature: 60 °C. Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 15

70

30

15 - 30

30

70

30 - 45

70

30

System suitability: Inject reference solution (6), the resolution between the peaks due to impurity A (1st peak) and oxytetracycline (2nd peak) is minimum 4.0. The resolution between the peaks due to oxytetracycline and impurity B (3rd peak) is minimum 5.0. The resolution between the peaks due to impurity D (4th peak) and impurity E (5th peak) is minimum 3.5. If necessary, adapt the ratio mobile phase A: mobile phase B and/or adjust the time programme used to produce the 1-step gradient elution. Symmetry factor: maximum 1.25 for the peak due to oxytetracycline. Inject test solution and reference solution (7). Limits: In the chromatogram obtained with the test solution: Impurity A: The area of the peak due to impurity A is not more than the area of the corresponding peak in the chromatogram obtained with reference solution (7) (0.5%). Impurity B: The area of the peak due to impurity B is not more than the area of the corresponding peak in the chromatogram obtained with reference solution (7) (2.0%). Impurity C (eluting on the tail of the main peak): the area is not more than 4 times the area of the peak due to impurity A in the chromatogram obtained with reference solution (7) (2.0%). Total of impurities D, E and F (eluting between the latter two) is not more than the area of the peak due to impurity E in the chromatogram obtained with reference solution (7) (2.0%). Disregard any peak with an area less than 0.02 times the area of the principal peak in the chromatogram obtained with reference solution (6) (0.1%). Note: Impurity A: (4R,4aR,5S,5aR,6S,12aS)-4-(dimethylamino)-3,5,6, 10,12,12a-hexahydroxy-6-methyl-1,11-dioxo-1,4,4a, 5,5a,6,11,12aoctahydrotetracene-2-carboxamide (4-epioxytetracycline).

Impurity C: (4S,4aR,5S,5aR,6S,12aS)-2-acetyl-4-(dimethylamino) -3,5,6,10,12,12a-hexahydroxy-6-methyl-4a,5a,6,12atetrahydrotetracene-1, 11(4H,5H)-dione (2-acetyl-2-decarbamoyloxytetracycline). Impurity B: (4S,4aS,5aS,6S,12aS)-4-(dimethylamino)-3,6,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a, 6,11,12a-octahydrotetracene-2-carboxamide (tetracycline). Impurity D: (3S,4S,5S)-4-[(1R)-4,5-dihydroxy-9-methyl-3oxo-1,3-dihydronaphtho[2,3-c]furan-1-yl]-3-(dimethylamino)2,5-dihydroxy-6-oxocyclohex-1-enecarboxamide(a-apooxytetracycline). Impurity E: (3S,4S,5R)-4-[(1R)-4,5-dihydroxy-9-methyl-3-oxo -1,3-dihydronaphtho[2,3-c]furan-1-yl]-3-(dimethylamino)2,5-dihydroxy-6-oxocyclohex-1-enecarboxamide(b-apooxytetracycline). Impurity F: (4S,4aR,5R,12aS)-4-(dimethylamino)-3,5,10,11,12apentahydroxy-6-methyl-1,12-dioxo-1,4,4a,5,12,12a-hexahydrotetracene-2-carboxamide (anhydro-oxytetracycline).

Heavy metals Not more than 50 ppm (Appendix 9.4.8). 0.5 g complies with limit test for heavy metals, method 6. Prepare the reference solution using 2.5 ml of lead standard solution (10 ppm Pb) R. Water Not more than 2.0% (Appendix 10.3). Determined on 0.500 g. Sulfated ash Not more than 0.5% (Appendix 9.9, method 2). Determined on 1.0 g. Bacterial endotoxins Less than 0.4 EU/mg, if intended for use in the manufacture of parenteral preparations without a further appropriate procedure for the removal of bacterial endotoxins. Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. Inject the test solution and reference solution (1). Calculate the content of C22H25ClN2O9 using the areas of the principal peaks in the chromatograms obtained with the test solution and reference solution (1). 1 mg of oxytetracycline is equivalent to 1.079 mg of oxytetracycline hydrochloride. Storage Store in an airtight container, protected from light. If the substance is sterile, store in a sterile, airtight, tamper-proof container. Action and use Antibacterial. Preparation Capsules. 717

OXYTETRACYCLINE CAPSULES

OXYTETRACYCLINE CAPSULES Capsulae Oxytetracyclini Oxytetracycline capsules contain oxytetracycline hydrochloride. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of oxytetracycline hydrochloride, C22H24N2O9,HCl, 95.0% to 110.0% of the stated amount. Identification Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel H. Prepare a 10% solution of sodium edetate R adjusted to pH 7.0 with 10 M sodium hydroxide R, spray evenly on the plate (about 10 ml for a plate 100 mm × 200 mm). Allow the plate to dry in a horizontal position for at least 1 hour. Before use, dry the plate at 110 °C for 1 hour. Mobile phase: Water - methanol - dichloromethane (6 : 35 : 59). Test solution: Shake a quantity of the contents of the capsules containing 10 mg of oxytetracycline hydrochloride with 20 ml of methanol R, centrifuge and use the clear supernatant liquid. Reference solution (1): Dissolve 5 mg of oxytetracycline hydrochloride RS in 10 ml of methanol R. Reference solution (2): Dissolve 5 mg of oxytetracycline hydrochloride RS and 5 mg of demeclocycline hydrochloride RS in 10 ml of methanol R. Procedure: Apply separately to the plate 1 µl of each solution. Develop over a path of 15 cm. After removal of the plate, allow it to dry in air and examine under ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to that in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) shows two clearly separated spots. B. To a quantity of the contents of the capsules containing 0.5 mg of oxytetracycline hydrochloride add 2 ml of sulfuric acid R; a deep crimson colour is produced. Add 1 ml of water; the colour changes to yellow. C. Shake thoroughly a quantity of the contents of the capsules containing 20 mg of oxytetracycline hydrochloride with 10 ml of water, filter, the filtrate gives reaction of chlorides (Appendix 8.1). Loss on drying Not more than 5.0% (Appendix 9.6). (1.000 g of the contents of the capsules, pressure not exceeding 0.7 kPa, 60 °C, 3 hours). Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 900 ml of 0.1 M hydrochloric acid R. Rotation speed: 100 rpm. 718

VP V

Time: 45 min. Procedure: After the specified time, withdraw a sample of the medium and filter, discard the first 10 ml of the filtrate, dilute if necessary. Measure the absorbance (Appendix 4.1) at the maximum at about 353 nm in a 1-cm cell, using 0.1 M hydrochloric acid R as the blank. Calculate the content of oxytetracycline hydrochloride, C22H24N2O9,HCl, dissolved, taking 282 as the value of A(1%, 1 cm) at the maximum at 353 nm. Tolerance: Not less than 70% (Q) of the stated amount of oxytetracycline hydrochloride, C22H24N2O9,HCl, is dissolved in 45 minutes.

Light-absorbing impurities Dissolve a portion of the mixed contents of five capsules as completely as possible in sufficient of a mixture of 1 M hydrochloric acid R and methanol R (1 : 99) to produce two solutions containing 0.20% and 1.0% of oxytetracycline hydrochloride. Filter each solution. The absorbance of the filtrate obtained from 0.20% solution at about 430 nm is not more than 0.75 (Appendix 4.1), calculated with reference to the dried capsule contents. The absorbance of the filtrate obtained from 1.0% solution at 490 nm is not more than 0.40, calculated with reference to the dried capsule contents. Assay Examine by liquid chromatography (Appendix 5.3). 0.1 M methanolic hydrochloric acid: Dilute 1 volume of 1 M hydrochloric acid R to 10 volumes by methanol R. Mobile phase: Dissolve 50.0 g of 2-methyl-propan-2-ol R in 200 ml of water, add 60 ml of 0.33 M phosphate buffer pH 7.5 R, 50 ml of a 1.0% solution of tetrabutylammonium hydrogen sulfate R previously adjusted to pH 7.5 with 2 M sodium hydroxide and 10 ml of a 0.04% solution of sodium edetate R previously adjusted to pH 7.5 with 2 M sodium hydroxide R and dilute to 1 litre with water. Test solution: Weigh 20 capsules, calculate the average weight of the contents of the capsules, powder finely. Transfer a quantity of the contents of the capsules equivalent to about 0.1 g of oxytetracycline hydrochloride, accurately weighed, to a 100 ml volumetric flask, add about 80 ml of 0.1 M methanolic hydrochloric acid, shake with the aid of ultrasound to dissolve, dilute to volume with the same solvent, mix well and filter. Dilute 1.0 ml of the filtrate to 20.0 ml with the same solvent. Reference solution (1): Containing 0.1% of oxytetracycline RS in the same solvent. Reference solution (2): Containing 0.005% of oxytetracycline RS in the same solvent. Reference solution (3): Containing 0.1% of 4-epioxytetracycline RS in the same solvent. Reference solution (4): Containing 0.1% of tetracycline hydrochloride RS in the same solvent. Resolution solution: Dilute a mixture containing 1.5 ml of reference solution (1), 1 ml of reference solution (3)

PANTOPRAZOLE SODIUM SESQUIHYDRATE

VP V

and 3 ml of reference solution (4) to 25 ml with the same solvent. Chromatographic system: A column (25 cm × 4.6 mm) packed with styrenedivinylbenzen copolymer (8 to 10 µm), maintained at 60 °C. Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the resolution solution. The test is not valid unless the resolution factor between the first peak (4-epioxytetracycline) and the second peak (oxytetracycline) is at least 4.0; the resolution factor between the second peak (oxytetracycline) and the third peak (tetracycline) is at least 5.0 (if necessary, reduce the content of 2-methylpropan-2-ol in the mobile phase to increase the resolution); and the symmetry factor of the peak due to oxytetracycline is not more than 1.25. Calculate the content of oxytetracycline hydrochloride, C22H24N2O9.HCl in the capsules using the peak areas in the chromatogram obtained with reference solution (2), the test solution and the declared content of C22H24N2O9 in oxytetracycline RS. Each mg of C22H24N2O9 is equivalent to 1.079 mg of C22H24N2O9.HCl.

with a suitable test that demonstrates its sesquihydrate nature (for example near-infrared spectrophotometry or X- ray powder diffraction).

Storage Store in well-closed container, in a dry place, protected from light.

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: A 1.74 g/l solution of dipotassium hydrogen phosphate R adjusted to pH 7.00 ± 0.05 with a 330 g/l solution of phosphoric acid R. Mobile phase B: Acetonitrile for chromatography. Solvent mixture: Acetonitrile for chromatography - 0.001 M sodium hydroxide (50 : 50). Test solution: Dissolve 23 mg of the substance to be examined in the solvent mixture and dilute to 50.0 ml with the solvent mixture. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 10.0 ml with the solvent mixture. Reference solution (2): Dissolve the contents of a vial of pantoprazole for system suitability RS (containing impurities A, B, C, D and E) in 1.0 ml of the solvent mixture. Chromatographic system: A column (12.5 cm × 4.0 mm) packed with stationary C (5 µm). Column temperature: 40 °C. Detector: A spectrophotometer set at 290 nm and for impurity C at 305 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions:

Action and use Antibacterial. Usual strength 100 mg. PANTOPRAZOLE SODIUM SESQUIHYDRATE Pantoprazolum natricum sesquihydricum

C16H14F2N3NaO4S,1½ H2O 

M: 432.4

Pantoprazole sodium sesquihydrate is sodium 5-(difluoromethoxy)-2-[(RS)-[(3,4-dimethoxypyridin-2yl)methyl]sulfinyl]benzimidazol-1-ide sesquihydrate. It contains not less than 99.0% and not more than 101.0% of C16H14F2N3NaO4S, calculated with reference to the anhydrous substance. It is produced by methods of manufacture designed to guarantee the proper hydrate form and it complies, if tested,

Characters White or almost white powder. Freely soluble in water and in ethanol (96%), practically insoluble in hexane. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of pantoprazole sodium sesquihydrate RS. B. It gives reaction of sodium (Appendix 8.1). Appearance of solution Dissolve 0.20 g in water and dilute to 20.0 ml with the same solvent. The solution is clear (Appendix 9.2) and not more intensely coloured than reference solution B6 (Appendix 9.3, method 2). Optical rotation -0.4° to +0.4° (Appendix 6.4). Dissolve 0.2 g in 10 ml of water, adjust to pH 11.5 to 12.0 with 0.2 M sodium hydroxide R and dilute to 20.0 ml with water.

719

VP V

GASTRO-RESISTANT PANTOPRAZOLE TABLETS

Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 40

80 → 20

20 → 80

40 - 45

20 → 80

80 → 20

Inject the blank, the test solution and reference solutions (1), (2). Identification of impurities: Use the chromatogram supplied with pantoprazole for system suitability RS and the chromatogram obtained with reference solution (2) to identify the peaks due to impurities A, B, C, D + F and E. Relative retention with reference to pantoprazole (retention time = about 11 min): Impurity C = about 0.6; impurity A = about 0.9; impurities D and F = about 1.2; impurity E = about 1.3; impurity B = about 1.5. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurities E and D + F is at least 1.5. The chromatogram obtained is similar to the chromatogram supplied with pantoprazole for system suitability RS. Limits: In the chromatogram obtained with the test solution: Correction factor: For the calculation of content, multiply the peak area of impurity C by 0.3. Impurity A: The area of the peak due to impurity A is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%). Sum of impurities D and F is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%). Impurities B, C, E: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). The sum of the peak areas of all impurities is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A: 5-(difluoromethoxy)-2-[[(3,4-dimethoxypyridin-2yl)methyl]sulfonyl]-1H-benzimidazole. Impurity B: 5-(difluoromethoxy)-2-[[(3,4-dimethoxypyridin-2yl)methyl]sulfanyl]-1H- benzimidazole. Impurity C: 5-(difluoromethoxy)-1H-benzimidazole-2-thiol. Impurity D: 5-(difluoromethoxy)-2-[(RS)-[(3,4-dimethoxypyridin-2yl)methyl]sulfinyl]-1-methyl-1H-benzimidazole. ImpurityF:6-(difluoromethoxy)-2-[(RS)-[(3,4-dimethoxypyridin-2yl)methyl]sulfinyl]-1-methyl-1H-benzimidazole. Impurity E: Mixture of the stereoisomers of 6,6’-bis(difluoromethoxy)-2,2’-bis[[(3,4- dimethoxypyridin-2-yl) methyl]sulfinyl]-1H,1’H-5,5’-bibenzimidazolyl.

720

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2.0 ml of lead standard solution (10 ppm Pb) R. Water 5.9% to 6.9% (Appendix 10.3). Determined on 0.150 g. Assay Dissolve 0.200 g of the substance to be examined in 80 ml of anhydrous acetic acid R, add 5 ml of acetic anhydride R and mix for at least 10 min. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 20.27 mg of C16H14F2N3NaO4S. Storage Protected from light. Action and use Proton pump inhibitor; treatment of peptic ulcer disease. Preparations Tablets, capsules, injection powder. GASTRO-RESISTANT PANTOPRAZOLE TABLETS Tabellae Pantoprazoli Gastro-resistant pantoprazole tablets contain pantoprazole sodium. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements:

Content of pantoprazole, C16H15F2N3O4S, 90.0% to 110.0% of the stated amount Identification In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that of the principal peak in the chromatogram obtained with the reference solution. Dissolution (Appendix 11.4) Acid stage Apparatus: Paddle. Medium: 1000 ml of 0.1 M hydrochloric acid R. Rotation speed: 75 rpm. Time: 2 hours. Procedure: Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - triethylamine - water (40 : 1 : 60). Adjust to pH 7,0 ± 0,05 with phosphoric acid R.

VP V

Reference stock solution: Transfer an accurately weighed quantity of pantoprazole sodium RS, equivalent to 20 mg of pantoprazole, to a 50 ml volumetric flask, add 30 ml of 0.02 M sodium hydroxide R and sonicate to dissolve completely. Add 2 ml of acetonitrile R and dilute to volume with 0.02 M sodium hydroxide R, mix. Reference solution: Transfer 1.0 ml of reference stock solution to a suitable flask and dilute with a mixtute of 0.1 M hydrochloric acid - 0.5 M sodium hydroxide (1 : 1) to obtain a solution having the same concentration of pantoprazole as in the test solution. Test solution: After 2 hours, withdraw a sample of the medium, filter. Dilute 10.0 ml of the filtrate to 20.0 ml with 0.5 M sodium hydroxide. Chromatographic system: A column (7.5 cm × 4.6 mm) packed with stationary phase C (3 μm). Column temperature: 30 °C. Detector: A spectrophotometer set at 290 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 μl. Procedure: Inject the reference solution. The test is not valid unless the tailing factor for pantoprazole peak is not more than 2.5 and the relative standard deviation of peak areas due to pantoprazole for six replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of pantoprazole dissolved using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C16H15F2N3O4S in pantoprazole sodium RS (the molecular weights of pantoprazole and pantoprazole sodium are 383.37 and 405.35, respectively). Tolerance: Not more than 10% of the stated amount of pantoprazole is dissolved in 2 hours (Appendix 11.4, item 4.3). Buffer stage Apparatus: Paddle. Medium: 1000 ml of phosphate buffer pH 6.8. Rotation speed: 75 rpm. Time: 30 min. Phosphate buffer pH 6.8: A mixture of 0.1 M hydrochloric acid R and 0.2 M tribasic sodium phosphate (3 : 1), adjust to pH 6.8 ± 0.05 with 2 M hydrochloric acid or 2 M sodium hydroxide, if needed. Reference solution: Dilute 1.0 ml of the reference stock solution in the acid stage with a mixture of phosphate buffer pH 6.8 and 0.5 M sodium hydroxide (1 : 1) to obtain a solution having the same concentration of pantoprazole as in the test solution. Test solution: Transfer each tablet in the Acid stage to the vessels contaning 1000 ml of phosphate buffer pH 6.8, previously held at 37 °C ± 0.5 °C. After 30 min, withdraw a sample of the medium, filter. Dilute 10.0 ml of the filtrate

GASTRO-RESISTANT PANTOPRAZOLE TABLETS

to 20.0 ml with 0.5 M sodium hydroxide. Procedure: Examine by liquid chromatography (Appendix 5.3). Chromatographic system: Proceed as directed in the acid stage. Tolerance: Not less than 75% (Q) of the stated amount of pantoprazole, C16H15F2N3O4S, is dissolved in both stages (Appendix 11.4, item 4.3).

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase, system suitability solution, test solution and chromatographic system: Prepare as directed in the Assay. Reference solution: Dilute an accurately measured volume of the reference solution, prepared as directed in the Assay, with 0.02 M sodium hydroxide to obtain a solution having a known concentration of about 0.0004 mg of pantoprazole per ml. Procedure: Inject the system suitability solution. The test is not valid unless the resolution factor between the peaks due to pantoprazole and pantoprazole related compound A is at least 3; the symmetry factor for pantoprazole peak is not more than 2.0. Inject the reference solution; the relative standard deviation of pantoprazole peak areas for 6 replicate injections is not more than 10.0%. The run time of the test solution is 3 times the retention time of the pantoprazole peak. Relative retention with reference to pantoprazole: Impurity D and F = about 1.2; impurity A = about 1.3; impurity B = about 2.7. Limits: In the chromatograms obtained with the test solution, Impurity A: The area of the impurity A peak is not greater than 2.5 times the area of the principal peak in the chromatogram obtained with the reference solution (0.5%). Impurities D and F (which are not fully separated and should be integrated together): The sum of the peak areas due to impurities D and F is not greater than 2.5 times the area of the principal peak in the chromatogram obtained with reference solution (0.5%). Impurity B: The area of the impurity B peak is not more than 1.5 times the area of the principal peak in the chromatogram obtained with the reference solution (0.3%). Any other impurity: The area of any other impurity peak is not greater than the area of the principal peak in the chromatogram obtained with the reference solution (0.2%). Total impurities: The sum of the areas of all impurity peaks is not greater than 5 times the area of the principal peak in the chromatogram obtained with the reference solution (1.0%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (0.05%).

721

VP V

PAPAVERINE HYDROCHLORIDE

Assay Examine by liquid chromatography (Appendix 5.3). Buffer solution pH 7.9: Dissolve 3.85 g of ammonium acetate R and 1.1 g of tetrabutylammonium hydrogensulfate R in 1000 ml of water, adjust to pH 7.9 with a 12.5% solution of ammonium hydroxide R. Mobile phase: Acetonitrile - buffer solution pH 7.9 (35 : 65). Solvent: Acetonitrile - 0.02 M sodium hydroxide (1 : 1). Reference solution: Transfer an accurately weighed quantity of pantoprazole sodium RS containing the equivalent of 20 mg of pantoprazole to a 100 ml volumetric flask, add 60 ml of 0.02 M sodium hydroxide and sonicate for 5 min to dissolve. Add 2 ml of acetonitrile R and dilute to volume with 0.02 M sodium hydroxide, mix. System suitability solution: Dissolve quantities of pantoprazole sodium RS and pantoprazole related compound A RS in 0.02 M sodium hydroxide to obtain a solution containing 0.2 mg of pantoprazole sodium and 0.0004 mg of pantoprazole related compound A per ml. Test solution: Weigh 20 tablets, remove the coating layer, calculate the average weight and powder finely. Transfer an accurately weighed quantity of the powdered tablets containing about 20 mg of pantoprazole to a 100 ml volumetric flask, add 60 ml of the solvent and sonicate for 15 min. Dilute to volume with the solvent, mix and filter. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 μm). Detector: A spectrophotometer set at 290 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 μl. Procedure: Inject the system suitability solution. The test is not valid unless the resolution factor between the peaks due to pantoprazole and pantoprazole related compound A is not less than 3; the symmetry factor for pantoprazole peak is not more than 2.0. Inject the reference solution; the relative standard deviation of peak areas for 6 replicate injections is not more than 2.0%. Calculate the content of pantoprazole in the tablets using the areas of the pantoprazole peaks in the chromatograms obtained with the test solution, the reference solution and the declared content of C16H15F2N3O4S in pantoprazole sodium RS. Storage Store in an airtight container, in a cool and dry place, protected from light. Action and use Proton pump inhibitor, treament of peptic ulcer disease. Usual strength 20 mg, 40 mg.

722

PAPAVERINE HYDROCHLORIDE Papaverini hydrochloridum

C20H21NO4,HCl  

M. 375.9

Papaverine hydrochloride is 1-(3,4-dimethoxybenzyl)-6,7dimethoxyisoquinoline hydrochloride. It contains not less than 99.0% and not more than 101.0% of C20H21NO4,HCl, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder, or white or almost white crystals. Sparingly soluble in water, slightly soluble in ethanol (96%). Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of papaverine hydrochloride RS. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254 Mobile phase: Diethylamine - ethyl acetate - toluene (10 : 20 : 70). Test solution: Dissolve 5 mg of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Reference solution: Dissolve 5 mg of papaverine hydrochloride RS in methanol R and dilute to 10 ml with the same solvent. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of two thirds of the plate. Dry the plate at 100 °C to 105 °C for 2 h. Examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. C. Add 5 ml of ammonia R dropwise to 10 ml of solution S (see Appearance of solution) and allow to stand for 10 min. The precipitate, washed and dried, melts at 146 °C to 149 °C (Appendix 6.7). D. It gives reaction (A) of chlorides (Appendix 8.1). Appearance of solution Solution S: Dissolve 0.4 g in carbon dioxide-free water R, heating gently if necessary, and dilute to 20 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution BY6 (Appendix 9.3, method 2).

PAPAVERINE HYDROCHLORIDE TABLETS

VP V

pH 3.0 to 4.0 (Appendix 6.2). Determined on solution S. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: A 0.34% solution of potassium dihydrogen phosphate R adjusted to pH 3.0 with dilute phosphoric acid R. Mobile phase B: Acetonitrile R. Mobile phase C: Methanol R. Solvent mixture: Acetonitrile - mobile phase A (20 : 80). Test solution: Dissolve 20.0 mg of the substance to be examined in the solvent mixture and dilute to 10.0 ml with the solvent mixture. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 10.0 ml with the solvent mixture. Reference solution (2): Dissolve 12 mg of noscapine RS in 1.0 ml of the test solution and dilute to 100.0 ml with the solvent mixture. Chromatographic system: A column (25 cm × 4.0 mm) packed with stationary phase base-deactivated octylsilyl silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 238 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

Mobile phase C (% v/v)

0-5

85

5

10

5 - 12

85 → 60

5

10 → 35

12 - 20

60

5

35

20 - 24

60 → 40

5 → 20

35 → 40

24- 27

40

20

40

27 - 32

40 → 85

20 → 5

40 → 10

Relative retention with reference to papaverine (retention time = about 24 min): impurity E = about 0.7; impurity C = about 0.75; impurity B = about 0.8; impurity A = about 0.9; impurity F = about 1.1; impurity D = about 1.2. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurity A and papaverine is at least 1.5. Limits: Correction factors: for the calculation of contents, multiply the peak areas of the following impurities by the corresponding correction factor: Impurity A = 6.2; impurity C = 2.7; impurity D = 0.5; Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%).

The sum of the peak areas of all impurities is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A: (3S)-6,7-dimethoxy-3-[(5R)-4-methoxy-6-methyl5,6,7,8-tetrahydro-1,3-dioxolo [4,5-g]isoquinolin-5-yl]isobenzofuran-1(3H)-one (noscapine). Impurity B: (RS)-(3,4-dimethoxyphenyl)(6,7-dimethoxyisoquinolin-1-yl)methanol (papaverinol). Impurity C: 1-(3,4-dimethoxybenzyl)-6,7-dimethoxy-3,4-dihydroisoquinoline (dihydropapaverine). Impurity D: (3,4-dimethoxyphenyl)(6,7-dimethoxyisoquinolin1-yl)methanone (papaveraldine). Impurity E: (1RS)-1-(3,4-dimethoxybenzyl)-6,7-dimethoxy1,2,3,4-tetrahydroisoquinoline (tetrahydropapaverine). Impurity F: 2-(3,4-dimethoxyphenyl)-N-[2-(3,4-dimethoxyphenyl)ethyl]acetamide.

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on the residue from the test for Loss on drying. Assay Dissolve 0.300 g of the substance to be examined in a mixture of 5.0 ml of 0.01 N hydrochloric acid VS and 50 ml of ethanol (96%) R. Carry out a potentiometric titration (Appendix 10.2), using 0.1 N sodium hydroxide VS. Read the volume added between the 2 points of inflexion. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 37.59 mg of C20H22ClNO4. Storage Store in an airtight container, protected from light. Action and use Phosphodiesterase inhibitor; smooth muscle relaxant. Preparations Tablets, capsules, injection. PAPAVERINE HYDROCHLORIDE TABLETS Tabellae Papaverini hydrochloridi Papaverine hydrochloride tablets contain papaverine hydrochloride. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

723

VP V

PARACETAMOL

Content of papaverine hydrochloride, C20H21O4N,HCl, 93.0% to 107.0% of the stated amount.

Action and use Antispasmodic.

Identification To a portion of the powdered tablets, equivalent to about 30 mg of papaverine hydrochloride, add 10 ml of 0.1 M hydrochloric acid R. Transfer the mixture to a separating funnel and extract with 10 ml of chloroform R, filter the chloroform layer through a paper-filter, evaporate the filtrate on a water bath to dryness, and dry the residue at 105 °C for 2 h. The infrared absorption spectrum (Appendix 4.2) of the residue is concordant with the reference spectrum of papaverine hydrochloride.

Usual strength 40 mg.

Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 900 ml of water. Rotation speed: 100 rpm. Time: 30 min. Procedure: After the specified time, withdraw a sample of the medium, filter, discard the first 20 ml of the filtrate and dilute with 0.1 M hydrochloric acid R to obtain a solution having a suitable concentration of papaverine hydrochloride. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum at 250 nm in comparison with a reference solution of papaverine hydrochloride RS having the same concentration in the same medium. Tolerance: Not less than 80% (Q) of the stated amount of papaverine hydrochloride, C20H21O4N,HCl, is dissolved in 30 min. Assay Weigh 20 tablets, calculate the average mass and powder finely. Transfer quantity of powdered tablets equivalent to about 30 mg of papaverine hydrochloride, accurately weighed, to a glass ground-stoppered conical flask, add about 100 ml of 0.1 M hydrochloric acid R, and shake by mechanical shaker for 15 min. Filter the mixture into a 200 ml volumetric flask, dilute with 0.1 M hydrochloric acid R to volume. Transfer 3.0 ml of this solution into a separating funnel, add 10 ml of water, and make alkaline with 6 M ammonium hydroxide R. Extract the alkaloid with five 5 ml quantities of chloroform R, and evaporate the combined extracts to dryness. Dissolve the residue in 0.1 M hydrochloric acid R and dilute with the same solvent to 100.0 ml. Measure the absorbances (Appendix 4.1) of the resulting solution at the maximum at 251 nm, in a 1 cm cell, using 0.1 M hydrochloric acid R as a blank. Prepare a reference solution of papaverine hydrochloride RS having a concentration of about 4.5 µg per ml at the same time. Calculate the content of papaverine hydrochloride, C20H21O4N,HCl, in the tablets using the absorbances of the test solution and the reference solution and the declared content of C20H21O4N,HCl in papaverine hydrochloride RS. Storage Store in a well-closed container, protected from light. 724

PARACETAMOL Paracetamolum OH

O H 3C

C8H9NO2

N H



M. 151.2

Paracetamol is N-(4-Hydroxyphenyl)acetamide. It contains not less than 99.0% and not more than 101.0% of C8H9NO2, calculated with reference to the dried substance. Characters A white, crystalline powder, odourless. Sparingly soluble in water, very slightly soluble in chloroform, in ether and in methylene chloride, freely soluble in alkali solutions and in ethanol (96%).

Identification Apply one of the two following identifications: First identification: A, C. Second identification: B, C, D, E. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of paracetamol RS. B. Dissolve 0.1 g of the substance to be examined in methanol R and dilute to 100.0 ml with the same solvent. To 1.0 ml of the solution add 0.5 ml of 0.1 M hydrochloric acid R and dilute to 100.0 ml with methanol R. Mix. Protect the solution from bright light and immediately measure the absorbance (Appendix 4.1) at the maximum at 249 nm. The A (1%, 1 cm) is 860 to 980. C. Melting point (Appendix 6.7): 168 °C to 172 °C. D. To 0.1 g of the substance to be examined add 1 ml of hydrochloric acid R, heat for 3 min, add 1 ml of water and cool in ice, no precipitate is formed. Add 0.05 ml of a 0.49% solution of potassium dichromate R. A violet colour develops which does not change to red. E. It gives the reaction of acetyl (Appendix 8.1). Heat over a naked flame. Related substances Examine by liquid chromatography (Appendix 5.3) Prepare the solutions immediately before use. Mobile phase: Mix 375 volumes of a 1.79% solution of disodium hydrogen phosphate R, 375 volumes of a 0.78% solution of sodium dihydrogen phosphate R and 250 volumes of methanol R containing 0.46% of a 40% solution of tetrabutylammonium hydroxide R.

PARACETAMOL

VP V

Test solution: Dissolve 0.200 g of the substance to be examined in 2.5 ml of methanol R containing 0.46% of a 40% solution of tetrabutylammonium hydroxide R and dilute to 10.0 ml with a mixture of equal volumes of a 1.79% solution of disodium hydrogen phosphate R and of a 0.78% solution of sodium dihydrogen phosphate R. Reference solution (1): Dilute 1.0 ml of the test solution to 50.0 ml with the mobile phase. Dilute 5.0 ml of this solution to 100.0 ml with the mobile phase. Reference solution (2): Dilute 1.0 ml of reference solution (1) to 10.0 ml with the mobile phase. Reference solution (3): Dissolve 5.0 mg of 4-aminophenol R, 5 mg of paracetamol RS and 5.0 mg of chloroacetanilide R in methanol R and dilute to 20.0 ml with the same solvent. Dilute 1.0 ml to 250.0 ml with the mobile phase. Reference solution (4): Dissolve 20.0 mg of 4-nitrophenol R in methanol R and dilute to 50.0 ml with the same solvent. Dilute 1.0 ml to 20.0 ml with the mobile phase. Chromatographic system: A column (25 cm x 4.6 mm) packed with stationary phase B (5 µm). Column temperature: 35 °C.

Detector: A spectrophotometer at 245 nm.   Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: Inject separately reference solutions. Relative retentions with reference to paracetamol (retention time is about 4 min): impurity K is about 0.8; impurity F is about 3; impurity J is about 7. The test is invalid unless in the chromatogram of reference solution (3): the resolution between the peaks due to impurity K and to paracetamol is at least 4.0 and signalto-noise ratio of the peak due to impurity J is at least 50. Inject the test solution and allow the chromatography to proceed for 12 times the retention time of paracetamol. Limits: In the chromatogram obtained with the test solution: The area of the peak due to impurity J is not more than 0.2 times the area of corresponding peak in the chromatogram obtained with reference solution (3) (10 ppm). The area of the peak due to impurity K is not more than the area of corresponding peak in the chromatogram obtained with reference solution (3) (50 ppm).

0.050 0.048 0.046 0.044 0.042 0.040 0.038 0.036

2

0.034 0.032 0.030 0.028 0.026

6

0.024 0.022

3

0.020 0.018

1

0.016

5

0.014 0.012 0.010 0.008

7 8

10

4

0.006

11

0.004

9

0.002 0.000 -0.002 0.000

5.00

10.00

15.00

1. impurity K 2. paracetamol 3. impurity B 4. impurity A

20.00

25.00 Minutes

5. impurity C 6. impurities E and D 7. impurity G 8. impurity H

30.00

35.00

40.00

45.00

50.00

9. impurity F 10. impurity I 11. impurity J

Fig.1 - Chromatogram for the test for related substaces: paracetamol solution spiked with the impuritie. 725

VP V

PARACETAMOL CAPSULES

The area of the peak due to impurity F is not more than half of the area of corresponding peak in the chromatogram obtained with reference solution (4) (0.05%). The area of any other impurity peak is not more than half of the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). The total of the areas of all impurity peaks is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%). Disregard any peak with the area less than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.01%). Comment: Impurity A: N-(2-hydroxyphenyl)acetamide. Impurity B: N-(4-hydroxyphenyl)propanamide. Impurity C: N-(3-chloro-4-hydroxyphenyl)acetamide. Impurity D: N-phenylacetamide. Impurity E: 1-(4-hydroxyphenyl)ethanone. Impurity F: 4-nitrophenol. Impurity G: 1-(4-hydroxyphenyl)ethanone oxime. Impurity H: 4-(acetylamino)phenyl acetate. Impurity I: 1-(2-hydroxyphenyl)ethanone. Impurity J: N-(4-chlorophenyl)acetamide (chloroacetanilide), Impurity K: 4-aminophenol.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). Dissolve 1.0 g of the substance to be examined in a mixture of water - acetone (15 : 85) and dilute to 20 ml with the same solvent. Take 12 ml of the solution and carry out method 2. Prepare the standard using lead standard solution (1 ppm Pb) obtained by diluting lead standard solution (100 ppm Pb) R with a mixture of water - acetone (15 : 85). Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 100 °C - 105 °C). Sulphated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.300 g of the substance to be examined in a mixture of 10 ml of water and 30 ml of dilute sulphuric acid R. Boil under a reflux condenser for 1 h, cool and dilute to 100.0 ml with water. Take 20.0 ml of the solution, add 40 ml of water, 40 g of ice, 15 ml of dilute hydrochloric acid R and 0.1 ml of ferroin solution R. Titrate with 0.1 M ammonium cerium sulphate VS until a greenish-yellow colour is obtained. Carry out a blank titration. 1 ml of 0.1 M ammonium cerium sulphate VS is equivalent to 7.56 mg of C8H9NO2. Storage Store in a well-closed container, protected from light. 726

Action and use Analgesic and antipyretic. Preparations Tablets, capsules, oral suspension, oral solution, effervescent tablets, suppositories, intravenous infusion. PARACETAMOL CAPSULES Capsulae Paracetamoli Paracetamol capsules contain paracetamol. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of paracetamol, C8H9NO2, 95.0% to 105.0% of the stated amount. Identification Shake a quantity of the contents of the capsules containing 0.5 g of paracetamol with 20 ml of acetone R, filter, and evaporate the filtrate to dryness. Dry the residue at 105 °C. The infrared absorption spectrum of the dried residue (Appendix 4.2) is concordant with the reference spectrum of paracetamol. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of phosphate buffer pH 5.8 R. Rotation speed: 50 rpm. Time: 45 min. Procedure: After the specified time, withdraw a sample of the medium, filter. Dilute the filtrate with 0.1 M sodium hydroxide R to obtain a solution containing about 7.5 µg of paracetamol per ml. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum wavelength at 257 nm, in a 1-cm cell, using 0.1 M sodium hydroxide R as a blank. Calculate the content of paracetamol dissolved taking 715 as the value of A (1%, 1 cm) at the maximum at 257 nm. Tolerance: Not less than 75% (Q) of the stated amount of paracetamol, C8H9NO2, is dissolved in 45 min. Related substances Examined by the liquid chromatography (Appendix 5.3). Prepare the following solutions immediately before use and protect from light. Mobile phase: A mixture of 250 volumes of methanol R containing 4.6 g/l of a 40% tetrabutyl ammonium hydroxide solution and 375 volumes of 0.05 M disodium hydrogen phosphate and 375 volumes of 0.05 M sodium dihydrogen phosphate. Test solution: Weigh accurately a quantity of the capsule contents containing 0.2 g of paracetamol and transfer into a 10-ml volumetric flask. Add 8 ml of the mobile phase, sonicate, add sufficient mobile phase, mix and filter.

VP V

Reference solution (1): Dilute 1.0 ml of the test solution to 20.0 ml with the mobile phase. Dilute 1.0 ml of the resulting solution to 20.0 ml with the mobile phase. Reference solution (2): A solution containing 0.002% of 4-aminophenol R and 0.002% of paracetamol RS in the mobile phase. Reference solution (3): Dilute a solution containing 0.02% of 4’-chloroacetanilide R in methanol R with the mobile phase to obtain a 0.00002% solution of 4’-chloroacetanilide. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase B (5 μm). Zorbax Rx C8 is suitable. Column temperature: 35 °C. Detector: A spectrophotometer set at 245 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject reference solution (2), the resolution factor between two peaks due to 4-aminophenol and paracetamol is not less than 4.0. Inject the test solution, record the chromatogram for 12 times the retention time of paracetamol. Limits: In the chromatogram obtained with the test solution, the area of the peak corresponding to 4-aminophenol is not greater than the area of the peak due to 4-aminophenol in the chromatogram obtained with reference solution (2) (0.1%). The area of the peak corresponding to 4’-chloroacetanilide is not greater than the area of the principal peak in the chromatogram obtained with reference solution (3) (10 ppm). The area of any other secondary peak is not greater than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.25%).

Assay Weigh 20 capsules, calculate the average weight of the contents of the capsules and powder finely. Weigh accurately a quantity of the capsule contents containing about 0.150 g of paracetamol, transfer into a 200-ml volumetric flask, add 50 ml of 0.1 M sodium hydroxide R and 100 ml of water, shake for 15 min. Dilute to volume with water and mix. Filter, discard the first 20 ml of the filtrate and dilute 10.0 ml of the filtrate with water to 100.0 ml and mix. Transfer 10.0 ml of this solution to a 100 ml volumetric flask, add 10 ml of 0.1 M sodium hydroxide R and dilute with water to volume. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum wavelength at 257 nm, in a 1-cm cell, using 0.01 M sodium hydroxide R as a blank. Calculate the content of paracetamol, C8H9NO2, taking 715 as the value of A (1%, 1 cm) at the maximum at 257 nm. Storage Store in a well-closed container, in a cool place, protected from light.

PARACETAMOL EFFERVESCENT TABLETS

Action and use Analgesic and antipyretic. Usual strength 500 mg. PARACETAMOL EFFERVESCENT TABLETS Effervescentis tabellae Paracetamoli Paracetamol effervescent tablets contain paracetamol. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of paracetamol, C8H9NO2, 95.0% to 105.0% of the stated amount. Identification A. In warm water, the tablets is dissolved with vigorous effervescence and produce a slightly opalescent solution. B. The ultraviolet absorption spectrum (Appendix 4.1) in the range 230 to 350 nm of the resulting solution obtained in the Assay exhibits an absorption maximum at 257 nm. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Toluene - acetone - chloroform (10 : 25 : 65). Test solution: Transfer a quantity of the powdered tablets containing 0.1 g of paracetamol, dissolve in sufficient ethanol (96%) R to produce 100 ml. Reference solution (1): A solution containing 0.1% paracetamol RS in ethanol (96%) R. Reference solution (2): Dissolve 0.25 g of 4’-chloroacetanilide R and 0.1 g of paracetamol RS in sufficient ethanol (96%) R to produce 100 ml. Procedure: Apply separately to the plate 40 µl of each solution. Pour the mobile phase into the unlined tank, immediately place the prepared plate in the tank and close the tank. Develop over a path of 15 cm. After removal of the plate, allow it to dry completely in air. Examine under ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to that in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) shows two clearly separated principal spots, the spot corresponding to 4’-chloroacetanilide having the higher Rf value. Related substances Examined by liquid chromatography (Appendix 5.3). Prepare the following solutions immediately before use and protect from light. Mobile phase: A mixture of 250 volumes of methanol R containing 4.6 g/l of 40% tetrabutyl ammonium hydroxide solution and 375 volumes of 0.05 M disodium hydrogen 727

VP V

PARACETAMOL INTRAVENOUS INFUSION

phosphate and 375 volumes of 0.05 M sodium dihydrogen phosphate. Test solution: Weigh accurately a quantity of the powdered tablets equivalent to 0.2 g of paracetamol and transfer into a 10-ml volumetric flask. Add 8 ml of the mobile phase, sonicate, add sufficient mobile phase, mix and filter. Reference solution (1): Dilute 1.0 ml of the test solution to 20.0 ml with the mobile phase. Dilute 1.0 ml of the resulting solution to 20.0 ml with the mobile phase. Reference solution (2): A solution containing 0.002% of 4-aminophenol R and 0.002% of paracetamol RS in the mobile phase. Reference solution (3): Dilute a solution containing 0.02% of 4’-chloroacetanilide R in methanol R with the mobile phase to obtain a 0.00002% solution of 4’-chloroacetanilide. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase B (5 μm). Zorbax Rx C8 is suitable. Column temperature: 35 °C. Detector: A spectrophotometer set at 245 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject reference solution (2), the resolution factor between the peaks due to 4-aminophenol and paracetamol is not less than 4.0. Inject the test solution, record the chromatogram for 12 times the retention time of paracetamol. Limits: In the chromatogram obtained with the test solution, the area of the peak corresponding to 4-aminophenol is not greater than the area of the peak due to 4-aminophenol in the chromatogram obtained with reference solution (2) (0.1%). The area of the peak corresponding to 4’-chloroacetanilide is not greater than the area of the principal peak in the chromatogram obtained with reference solution (3) (10 ppm). The area of any other secondary peak is not greater than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.25%).

Assay Weigh 20 tablets, calculate the average weight and powder finely. Transfer an accurately weighed quantity of the powdered tablets containing about 0.150 g of paracetamol to a 200-ml volumetric flask, add gradually 50 ml of 0.1 M sodium hydroxide R, wait until effervescence has stopped, add 100 ml of water, shake for 15 min. Dilute to volume with water and mix. Filter, discard the first 20 ml of the filtrate. Dilute 10.0 ml of the filtrate with water to 100.0 ml. Transfer accurately 10 ml of this solution into a 100-ml volumetric flask, add 10 ml of 0.1 M sodium hydroxide R and dilute with water to volume. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum wavelength at 257 nm, in a 1 cm cell, using 0.01 M sodium hydroxide R as a blank. Calculate the content of paracetamol, C8H9NO2, taking 715 as the value of A (1%, 1 cm) at the maximum wavelength at 257 nm. 728

Storage Store in a well-closed container, in a cool place and protected from light. Action and use Analgesic and antipyretic. Usual strength 500 mg. PARACETAMOL INTRAVENOUS INFUSION Injectio paracetamoli Paracetamol intravenous infusion is a sterile solution of paracetamol in a suitable solvent. The intravenous infusion complies with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements:

Content of paracetamol, C8­H9NO2, 95.0% to 105.0% of the stated amount. Characters A clear, colourless or almost colourless solution. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Methylene chloride - methanol (4 : 1). Test solution: Dilute a quantity of intravenous infusion with methanol R to obtain a solution containing 2 mg of paracetamol per ml. Reference solution: Dissolve 10 mg of paracetamol RS in 5 ml of methanol R. Procedure: Apply separately to the plate 10 μl of each solution. Develop over a path of about 15 cm. After removal of the plate, allow it to dry in air. Examine under ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution corresponds in position, colour and size to that in the chromatogram obtained with the reference solution. B. In the Assay, retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that of the principal peak in the chromatogram obtained with the reference solution. pH 5.0 to 6.0 (Appendix 6.2). 4-Aminophenol Not more than 0.1%. Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 1.6 g of sodium butanesulfonate R in 1000 ml of a mixture of water - methanol - formic acid (85 : 15 : 0.4). Reference solution: Transfer an accurately weighed quantity of about 20 mg of 4-aminophenol to a 100 ml

VP V

volumetric flask. Dissolve and dilute to volume with a mixture of water - methanol (15 : 85). Dilute 5.0 ml of the obtained solution to 100.0 ml with the same solvent. Test solution: The intravenous infusion. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (10 μm). Detector: A spectrophotometer set at 272 nm. Flow rate: 2 ml/min. Volume of injection: 20 μl. Procedure: Inject separately the test solution and the reference solution. In the chromatogram obtained with the test solution the area of any peak corresponding to 4-aminophenol is not greater than the area of the 4-aminophenol peak in the chromatogram obtained with the reference solution.

Bacterial endotoxins Not more than 0.17 EU per mg of paracetamol. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Water - methanol (3 : 1). Make an adjustment if necessary. Reference solution: Transfer an accurately weighed quantity of about 30 mg of paracetamol RS to a 100 ml volumetric flask. Dissolve in the mobile phase and dilute to volume with the same solvent, mix well. Dilute 5.0 ml of this solution to 100.0 ml with the mobile phase, mix well. Test solution: Withdraw an accurately volume of the intravenous infusion containing about 150 mg of paracetamol to a 100 ml volumetric flask. Add the mobile phase to volume, mix well. Dilute 1.0 ml of this solution to 100.0 ml with the mobile phase, mix well. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 μm to 10 µm). Detector: A spectrophotometer set at 243 nm. The flow rate: 1.5 ml/min. Volume of injection: 10 μl. Procedure: System suitability: Inject the reference solution, the theoretical plates for the paracetamol peak are not less than 1000, the symmetry factor is not more than 2.0 and the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of paracetamol, C8­H9NO2, in the intravenous infusion using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C8­H9NO2 in the paracetamol RS.

PARACETAMOL SUPPOSITORIES

Storage Store in a cool place, protected from light. Action and use Analgesic and antipyretic. Usual strength 10 mg/ml. Bottles of 50 ml or 100 ml. PARACETAMOL SUPPOSITORIES Suppositoria Paracetamoli Paracetamol suppositories contain paracetamol. The suppositories comply with the requirements stated under “Suppositories” (Appendix 1.10) and with the following requirements:

Content of paracetamol, C8H9NO2, 95.0% to 105.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Methanol - dichloromethane (1 : 4). Test solution: Add sufficient methanol R to 1 suppository to produce a solution containing 0.1% of paracetamol and warm on a water-bath until the suppository has melted. Allow it to cool, stir occasionally and filter. Reference solution: A solution containing 0.1% of paracetamol RS in methanol R. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of about 10 cm. After removal of the plate, allow it to dry in air and examine under ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution corresponds in position, colour and size to that in the chromatogram obtained with the reference solution. B. Cut into small pieces. To a quantity of the suppositories containing 50 mg of paracetamol, add 1 ml of hydrochloric acid R and heat to boiling for 3 min, add 10 ml of water and cool, no precipitate is produced. Add 0.05 ml of 0.5% potassium dichromate solution. A violet colour develops which does not change to red. Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the following solutions immediately before use and protect from light. Mobile phase: A mixture of 250 volumes of methanol R containing 4.6 g//l of 40% tetrabutyl ammonium hydroxide solution and 375 volumes of 0.05 M disodium hydrogen phosphate and 375 volumes of 0.05 M sodium dihydrogen phosphate. Test solution: Take 5 suppositories, cut into small pieces and dissolve in a minimum portion of ethanol 96% 729

VP V

PARACETAMOL TABLETS

R, warm up if necessary, dilute with water to obtain a solution having a concentration of paracetamol of about 0.5%, filter. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of the resulting solution to 10.0 ml with the mobile phase. Reference solution (2): A solution containing 0.0005% of 4-aminophenol R and 0.0005% of paracetamol RS in the mobile phase. Reference solution (3): Dilute a solution containing 0.005% of 4’-chloroacetamide R in methanol R with the mobile phase to obtain a 0.000005% solution of 4’-chloroacetanilide. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase B (5 μm). Zorbax Rx C8 is suitable. Column temperature: 35 °C. Detector: A spectrophotometer set at 245 nm. Flow rate: 1.5 ml/min. Volume of injection: 50 µl. Procedure: System suitability: Inject reference solution (2), the resolution factor between two peaks due to 4-aminophenol and paracetamol is not less than 4.0. Inject the test solution and record the chromatogram for 12 times the retention time of paracetamol. Limits: In the chromatogram obtained with the test solution, the area of the peak corresponding to 4-aminophenol is not greater than the area of the peak due to 4-aminophenol in the chromatogram obtained with reference solution (2) (0.1%). The area of the peak corresponding to 4’-chloroacetanilide is not greater than the area of the principal peak in the chromatogram obtained with reference solution (3) (10 ppm). The area of any other secondary peak is not greater than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%). The sum of the areas of other secondary peaks is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). Disregard any peak having the area less than 0.3 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.03%).

Assay For suppositories containing not less than 150 mg To 1 suppository add 30 ml of water and 100 ml of 1 M sulfuric acid R. Boil under a reflux condenser for 1 hour, cool and add 100 ml of water, 50 g of ice water, 50 ml of dilute hydrochloric acid R and 0.2 ml of ferroin sulfate solution R. Titrate with 0.2 M ammonium cerium sulfate VS until a yellow colour is obtained. Each ml of 0.2 M ammonium cerium sulfate VS is equivalent to 15.12 mg of paracetamol, C8H9NO2. Repeat the titration using 4 further suppositories and calculate the average content per suppository. 730

For suppositories containing more than 60 mg and less than 150 mg To 1 suppository add 10 ml of water and 30 ml of 1M sulfuric acid R. Boil under a reflux condenser for 1 hour, cool and add 40 ml of water, 40 g of ice water, 15 ml of dilute hydrochloric acid R and 0.1 ml of ferroin sulfate solution R. Titrate with 0.1 M ammonium cerium sulfate VS until a yellow colour is obtained. Each ml of 0.1 M ammonium cerium sulfate VS is equivalent to 7.56 mg of paracetamol, C8H9NO2. Repeat the titration using 4 further suppositories and calculate the average content per suppository. For suppositories containing 60 mg or less To 1 suppository add 10 ml of water and 30 ml of 1M sulfuric acid R. Boil under a reflux condenser for 1 hour, cool and add 40 ml of water, 40 g of ice water, 15 ml of dilute hydrochloric acid R and 0.1 ml of ferroin sulfate solution R. Titrate with 0.025 M ammonium cerium sulfate VS until a yellow colour is obtained. Each ml of 0.025 M ammonium cerium sulfate VS is equivalent to 1.89 mg of paracetamol, C8H9NO2. Repeat the titration using 4 further suppositories and calculate the average content per suppository.

Storage Store in a well-closed container, at a temperature from 4 to 8 °C. Action and use Analgesic and antipyretic. Usual strength 80 mg, 150 mg, 300 mg. PARACETAMOL TABLETS Tabellae Paracetamoli Paracetamol tablets contain paracetamol. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of paracetamol, C8H9NO2, 95.0% to 105.0% of the stated amount. Identification Shake a quantity of the powdered tablets containing 0.5 g of paracetamol with 20 ml of acetone R, filter, and evaporate the filtrate to dryness. Dry the residue at 105 °C. The infrared absorption spectrum of the dried residue (Appendix 4.2) is concordant with the reference spectrum of paracetamol. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of phosphate buffer pH 5.8 R.

VP V

Rotation speed: 50 rpm. Time: 45 min. Procedure: After the specified time, withdraw a sample of the medium, filter. Dilute the filtrate with 0.1 M sodium hydroxide R to obtain a solution containing about 7.5 µg of paracetamol per ml. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum wavelength at 257 nm, in a 1-cm cell, using 0.1 M sodium hydroxide R as a blank. Calculate the content of paracetamol dissolved taking 715 as the value of A (1%, 1 cm) at the maximum wavelength at 257 nm. Tolerance: Not less than 75% (Q) of the stated amount of paracetamol, C8H9NO2, is dissolved in 45 min.

Related substances Examined by liquid chromatography (Appendix 5.3). Prepare the following solutions immediately before use and protect from light. Mobile phase: A mixture of 250 volumes of methanol R containing 4.6 g/l of 40% solution of tetrabutyl ammonium hydroxide and 375 volumes of 0.05 M disodium hydrogen phosphate and 375 volumes of 0.05 M sodium dihydrogen phosphate. Test solution: Weigh accurately a quantity of the powdered tablets containing 0.2 g of paracetamol and transfer into a 10-ml volumetric flask. Add 8 ml of the mobile phase, sonicate, add sufficient mobile phase, mix and filter. Reference solution (1): Dilute 1.0 ml of the test solution to 20.0 ml with the mobile phase. Dilute 1.0 ml of the resulting solution to 20.0 ml with the mobile phase. Reference solution (2): A solution containing 0.002% of 4-aminophenol R and 0.002% of paracetamol RS in the mobile phase. Reference solution (3): Dilute a solution containing 0.02% of 4’-chloroacetamide R in methanol R with the mobile phase to obtain a 0.00002% solution of 4’-chloroacetanilide. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase B (5 μm). Zorbax Rx C8 is suitable. Column temperature: 35 °C. Detector: A spectrophotometer set at 245 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject reference solution (2), the resolution factor between two peaks due to 4-aminophenol and paracetamol is not less than 4.0. Inject the test solution with and record the chromatogram for 12 times the retention time of paracetamol. Limits: In the chromatogram obtained with the test solution, the area of the peak corresponding to 4-aminophenol is not greater than the area of the peak due to 4-aminophenol in the chromatogram obtained with reference solution (2) (0.1%). The area of the peak corresponding to 4’-chloroacetanilideis not greater than the area of the principal peak in the chromatogram obtained with reference solution (3) (10 ppm). The area

PARACETAMOL AND CAFFEINE TABLETS

of any other secondary peak is not greater than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.25%).

Assay Weigh 20 tablets, calculate the average mass and powder finely. Weigh accurately a quantity of the powdered tablets containing about 0.150 g of paracetamol, transfer into a 200-ml volumetric flask, add 50 ml of 0.1 M sodium hydroxide R and 100 ml of water, shake for 15 min. Dilute to volume with water and mix. Filter, discard the first 20 ml of the filtrate. Dilute 10.0 ml of the filtrate with water to 100.0 ml. Transfer 10.0 ml of this solution to a 100 ml volumetric flask, add 10 ml of 0.1 M sodium hydroxide R and dilute with water to volume. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum at about 257 nm, in a 1 cm cell, using 0.01 M sodium hydroxide R as the blank. Calculate the content of paracetamol, C8H9NO2, taking 715 as the value of A (1%, 1 cm) at the maximum at 257 nm. Storage Store in a well-closed container, in a cool place. Action and use Analgesic and antipyretic. Usual strength 300 mg; 500 mg. PARACETAMOL AND CAFFEINE TABLETS Tabellae Paracetamoli et Coffeini Paracetamol and caffeine tablets contain paracetamol and caffeine. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of paracetamol, C8H9NO2, and caffeine, C8H10N4O2, 90.0% to 110.0% of the stated amount. Identification In the Assay, the retention times of the principal peaks in the chromatogram of the test solution correspond to those of the reference solution, relative to the internal standard. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of water. Rotation speed: 100 rpm. Time: 60 min. Mobile phase, internal standard solution, solvent mixture, reference stock solution, chromatographic system and procedure: Proceed as directed in the Assay. Reference solution: Transfer 20.0 ml of the reference stock solution, 3.0 ml of the internal standard solution and 731

VP V

PARACETAMOL AND CHLORPHENIRAMINE TABLETS

20 ml of water to a 50 ml volumetric flask, mix and allow to stand for about 30 seconds. Dilute with the solvent mixture to volume and mix. Use this solution within 8 hours. Test solution: Withdraw a sample of the medium, filter, discard the first 20 ml of the filtrate. Measure accurately a portion of the filtrate to a 50 ml volumetric flask in order to obtain an expected concentration of about 0.1 mg of paracetamol and 0.1J mg of caffeine per ml, where J is defined for the reference stock solution. Add 3.0 ml of the internal standard solution and 20 ml of the solvent mixture, mix and allow to stand for about 30 seconds. Dilute with the solvent mixture to volume and mix. Calculate the content of paracetamol, C8H9NO2, and caffeine, C8H10N4O2, dissolved using the ratios of the peak areas of the corresponding analyte to the internal standard peak in the chromatograms obtained with the test solution and the reference solution and the declared content of paracetamol RS and caffeine RS. Tolerance: Not less than 75% (Q) of the stated amounts of paracetamol, C8H9NO2, and caffeine, C8H10N4O2, are dissolved in 60 minutes.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Water - methanol - glacial acetic acid (69 : 28 : 3). Make adjustments if necessary. Internal standard solution: Dissolve benzoic acid in methanol R to obtain a solution containing 6 mg per ml. Solvent mixture: Methanol - glacial acetic acid (95 : 5). Reference stock solution: Dissolve an accurately weighed quantity of paracetamol RS and caffeine RS in the solvent mixture to obtain a solution containing 0.25 mg of paracetamol and 0.25J mg of caffeine per ml, J being the ratio of the stated amount, in mg, of caffeine to the stated amount, in mg, of paracetamol per tablet. Reference solution: Transfer 20.0 ml of the reference stock solution, 3.0 ml of the internal standard solution to a 50 ml volumetric flask. Dilute with the solvent mixture to volume and mix. This solution contains about 0.1 mg of paracetamol and 0.1J mg of caffeine per ml. Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Transfer a quantity of the powdered tablets equivalent to about 250 mg of paracetamol, accurately weighed, to a 100 ml volumetric flask, add about 75 ml of the solvent mixture and shake on a mechanical shaker for 30 minutes. Dilute to volume with the solvent mixture. Transfer 2.0 ml of this solution and 3.0 ml of the internal standard solution to a 50 ml volumetric flask. Dilute with the solvent mixture to volume and mix. Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase C (5 µm). Column temperature: 45 ± 1 °C. Detector: A spectrophotometer set at 275 nm. 732

Flow rate: 2 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject the reference solution; the symmetry factor for each analyte peak is not more than 1.2; the resolution between the peaks corresponding to paracetamol, caffeine and internal standard peaks is not less than 1.4; and the relative standard deviation of the area responses for 6 replicate injections is not more than 2.0%. Inject alternately the test solution and the reference solution. The relative retention times are about 0.3 for paracetamol, 0.5 for caffeine, and 1.0 for benzoic acid. Calculate the content of paracetamol, C8H9NO2, and caffeine, C8H10N4O2, in the tablets using the ratios of the peak areas of the corresponding analyte to the internal standard peaks obtained from the test solution and the reference solution, respectively and the declared contents of paracetamol RS and caffeine RS.

Storage Store in a well-closed container, at 15 °C - 30 °C. Action and use Analgesic and antipyretic. Usual strength 200 mg of paracetamol and 50 mg of caffeine. 500 mg of paracetamol and 65 mg of caffeine. PARACETAMOL AND CHLORPHENIRAMINE TABLETS Tabellae Paracetamoli et Chlorpheniramini Paracetamol and chlorpheniramine tablets contain paracetamol and chlorpheniramine maleate. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of paracetamol, C8H9NO2, 90.0% to 110.0% of the stated amount. Content of chlorpheniramine maleate, C16H19ClN2,C4H4O4, 90.0% to 110.0% of the stated amount. Identification A. In the Assay for paracetamol, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that of paracetamol peak in the chromatogram obtained with the reference solution. B. In the Assay for chlorpheniramine maleate, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that of chlorpheniramine maleate peak in the chromatogram obtained with the reference solution.

VP V

Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the following solutions immediately before use and protect from light. Mobile phase: A mixture of 250 volumes of methanol R containing 4.6 g/l of 40% tetrabutyl ammonium hydroxide solution and 375 volumes of 0.05 M disodium hydrogen phosphate and 375 volumes of 0.05 M sodium dihydrogen phosphate. Test solution: Weigh accurately a quantity of the powdered tablets containing 0.2 g of paracetamol and transfer into a 10-ml volumetric flask. Add 8 ml of the mobile phase, sonicate, add sufficient mobile phase, mix and filter. Reference solution (1): Dilute 1.0 ml of the test solution to 20.0 ml with the mobile phase. Dilute 1.0 ml of the resulting solution to 20.0 ml with the mobile phase. Reference solution (2): A solution containing 0.002% of 4-aminophenol R and 0.002% of paracetamol RS in the mobile phase. Reference solution (3): Dilute a solution containing 0.02% of 4’-chloroacetanilide R in methanol R with the mobile phase to obtain a 0.00002% solution of 4’-chloroacetanilide. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase B (5 μm). Zorbax Rx C8 is suitable. Column temperature: 35 °C. Detector: A spectrophotometer set at 245 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject reference solution (2), the resolution factor between the peaks due to 4-aminophenol and paracetamol is not less than 4.0. Inject the test solution, record the chromatogram for 12 times the retention time of paracetamol. Limits: In the chromatogram obtained with the test solution, the area of the peak corresponding to 4-aminophenol is not greater than the area of the peak due to 4-aminophenol in the chromatogram obtained with reference solution (2) (0.1%). The area of the peak corresponding to 4’-chloroacetanilide is not greater than the area of the principal peak in the chromatogram obtained with reference solution (3) (10 ppm). The area of any other secondary peak is not greater than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.25%). Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of water. Rotation speed: 50 rpm. Time: 45 min. Test solution: After the specified time, withdraw a sample of the medium, filter, discard the first portion of the filtrate. Mix 9.0 ml of the filtrate with 1.0 ml of 1% phosphoric acid solution.

PARACETAMOL AND CHLORPHENIRAMINE TABLETS

Reference solution: Proceed as described in the Assay for paracetamol and assay for chlorpheniramine maleate. Dilute (if necessary) with 1% phosphoric acid solution to obtain a solution having the same concentration of paracetamol and chlorpheniramine maleate as that expected in the test solution. Determine the content of paracetamol, C8H9NO2 and chlorpheniramine maleate, C16H19ClN2,C4H4O4 by liquid chromatography as described in the assay for paracetamol and assay for chlorpheniramine maleate. Tolerance: Not less than 75% (Q) of the stated amounts of paracetamol, C8H9NO2, and chlorpheniramine maleate, C16H19ClN2,C4H4O4, are dissolved in 45 min.

Uniformity of content (Appendix 11.2) Examine by liquid chromatography (Appendix 5.3). Mobile phase, chromatographic system and procedure: Proceed as directed in the Assay for chlorpheniramine maleate. Test solution: Finely powder one tablet and transfer to a 250-ml volumetric flask, add 25 ml of methanol R, sonicate to disperse completely, add 1 ml of phosphoric acid R. Dilute to volume with water and mix. Reference solution: Proceed as directed in the Assay for chlorpheniramine maleate. Dilute (if necessary) to obtain a solution having the same concentration of chlorpheniramine maleate as that expected in the test solution. Calculate the content of chlorpheniramine maleate, C16H19ClN2,C4H4O4, in the tablets using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C16H19ClN2,C4H4O4 in chlorpheniramine maleate RS. Assay For paracetamol Examine by liquid chromatography (Appendix 5.3). Mobile phase: Water - methanol - glacial acetic acid (79 : 20 : 1). Make adjustments if necessary. Reference solution: Weigh accurately about 50 mg of paracetamol RS and transfer into a 100-ml volumetric flask, add 4 ml of methanol R, shake to dissolve completely, dilute to volume with 1% phosphoric acid solution R and mix. Test solution: Weigh 20 tablets, calculate the average weight and powder finely. Weigh accurately a quantity of the powdered tablets containing about 100 mg of paracetamol, transfer into a 50-ml volumetric flask, add about 7.5 ml of methanol R, sonicate to disperse completely, add 0.5 ml of phosphoric acid R. Dilute to volume with water and mix. Transfer 25.0 ml of this solution to a 100-ml volumetric flask. Dilute with water to volume and mix. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase B (5 to 10 µm). Detector: A spectrophotometer set at 280 nm. Flow rate: 1 ml/min. Volume of injection: 10 µl. 733

VP V

PARACETAMOL AND CODEINE TABLETS

Procedure: System suitability: Inject the reference solution, the symmetry factor for paracetamol peak is not more than 2.0; the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject alternately the test solution and the reference solution. Calculate the content of paracetamol, C8H9NO2, in the tablets using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C8H9NO2 in paracetamol RS. For chlorpheniramine maleate Examine by liquid chromatography (Appendix 5.3). Mobile phase: A mixture of methanol and water (60 : 40) containing 0.34 g of potassium dihydrogen phosphate R, 0.3 g of triethylamine hydrochloride R, 0.15 g of sodium lauryl sulfate R, and 0.1 ml of phosphoric acid R in each 100 ml of solution. Make adjustments if necessary. Reference solution: Weigh accurately a quantity of chlorpheniramine maleate RS and dissolve in water to obtain a solution containing about 0.8 mg per ml. Dilute a portion of this solution with 0.1% solution of phosphoric acid R to obtain a solution containing about 8 µg per ml. Test solution: Weigh 20 tablets, calculate the average weight and powder finely. Weigh accurately a quantity of the powdered tablets equivalent to about 2 mg of chlorpheniramine maleate, transfer into a 250-ml volumetric flask, add 25 ml of methanol R, sonicate to disperse completely, add 1 ml of phosphoric acid R. Dilute to volume with water and mix. Chromatographic system: A column (15 cm × 4.6 mm) packed with particles of silica the surface of which has been modified with chemicallybonded phenyl groups (5 to 10 µm). Detector: A spectrophotometer set at 214 nm. Flow rate: 2 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject the reference solution, the symmetry factor for chlorpheniramine maleate peak is not more than 2.0; the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject alternately the test solution and the reference solution. Calculate the content of chlorpheniramine maleate, C16H19ClN2,C4H4O4, in the tablets using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C16H19ClN2,C4H4O4 in chlorpheniramine maleate RS.

Storage Store in a well-closed container, at a temperature from 15 °C to 30 °C. Action and use Analgesic and antihistamine. 734

Usual strength 500 mg of paracetamol and either 4 mg or 2 mg of chlorpheniramine maleate. PARACETAMOL AND CODEINE TABLETS Tabellae Paracetamoli et Codeini Paracetamol and codeine tablets contain paracetamol and codeine phosphate. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of paracetamol, C8H9NO2, 90.0% to 110.0% of the stated amount. Content of codeine phosphate hemihydrate, C18H21NO3,H3PO4,½H2O, 90.0% to 110.0% of the stated amount. Identification A. In the Assay, the retention times of two principal peaks in the chromatogram obtained with the test solution correspond to those of the peaks due to paracetamol and codeine phosphate in the chromatogram obtained with the reference solution. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Methanol - ammonia (49 : 1). Test solution: Transfer a portion of powdered tablets, equivalent to about 12 mg of codeine phosphate, to a separating funnel, add 5 ml of water, 1 ml of ammonia R, and 5 ml of methylene chloride R, shake for 1 min, and allow the layers to separate. Use the clear, lower layer as the test solution. Reference solution: Dissolve paracetamol RS and codeine phosphate RS in methylene chloride R to obtain a solution having the same concentrations of paracetamol and codeine phosphate as in the test solution. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of about three fourths of the length of the plate. Remove the plate and allow the solvent to evaporate. Examine under the ultraviolet light at 254 nm. Two principal spots in the chromatogram obtained with the test solution correspond in position, colour and size to those in the chromatogram obtained with the reference solution. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of 0.01 M hydrochloric acid R. Rotation speed: 50 rpm. Time: 30 min. Procedure: After the specified time, withdraw a sample of the medium, filter, discard the first portion of the filtrate. Determine the contents of paracetamol, C8H9NO2, and

VP V

codeine phosphate hemihydrate, C18H21NO3,H3PO4,½H2O, dissolved by using the procedure described in the Assay, except use 0.01 M hydrochloric acid R to prepare the codeine phosphate reference stock solution, the reference solution and to make any other necessary volumetric adjustments. Tolerance: Not less than 75% (Q) of the stated amount of paracetamol, C8H9NO2, and codeine phosphate hemihydrate, C18H21NO3,H3PO4,½H2O, is dissolved in 30 minutes.

Assay Examine by liquid chromatography (Appendix 5.3). Buffer solution: Dissolve 2.04 g of potassium dihydrogen phosphate R in about 950 ml of water. Add 2 ml of triethylamine R, adjust to pH 2.35 with phosphoric acid R, dilute with water to 1000 ml and mix. Mobile phase: Buffer solution - methanol (98 : 2), filter and degas. Make adjustment if necessary. Codeine phosphate reference stock solution: Dissolve an accurately weighed quantity of codeine phosphate RS in the mobile phase to obtain a solution containing about 0.3 mg per ml. Reference solution: Transfer 30 mg of paracetamol RS, accurately weighed, and 100J ml of codeine phosphate reference stock solution, J being the ratio of the stated amount, in mg, of codeine phosphate hemihydrate to that, in mg, of paracetamol to a 100 ml volumetric flask. Dilute to volume with the mobile phase and mix. Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Transfer a quantity of the powdered tablets equivalent to about 300 mg of paracetamol, accurately weighed, to a 100 ml volumetric flask, add about 75 ml of the mobile phase, sonicate for 10 minutes. Dilute to volume with the mobile phase and mix. To 5.0 ml of this solution to a 50 ml volumetric flask. Dilute to volume with the mobile phase and mix. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 214 nm. Flow rate: 1.5 ml/min. Volume of injection: 30 µl. Procedure: System suitability: Inject the reference solution; the resolution factor between paracetamol and codeine phosphate peak is not less than 2.0; the relative standard deviation of the peak areas corresponding to paracetamol and codeine phosphate for replicate injections is not more than 2.0% and 3.0%, respectively. Inject alternately the test solution, the reference solution. Calculate the content of paracetamol, C8H9NO2, and codeine phosphate hemihydrate, C18H21NO3,H3PO4,½H2O, in the tablets using the peak areas in the chromatograms

PARACETAMOL AND IBUPROFEN TABLETS

obtained with the test solution, the reference solution and the declared content of C8H9NO2 in paracetamol RS and C18H21NO3,H3PO4,½H2O in codeine phosphate RS.

Uniformity of content of codeine phosphate Tablets with the content of codeine phosphate less than 60 mg comply with the requirements stated under “Uniformity of content” (Appendix 11.2). Examine by liquid chromatography (Appendix 5.3). Mobile phase, reference solution: Proceed as directed in the Assay. Test solution: Finely powder one tablet and transfer to a 100 ml volumetric flask, add about 75 ml of the mobile phase, sonicate for 10 minutes. Dilute to volume with the mobile phase, mix and filter. Dilute a portion of the filtrate with the mobile phase to obtain a solution having the same concentration of codeine phosphate as in the reference solution. Chromatographic system and procedure: Proceed as directed in the Assay. Calculate the content of codeine phosphate hemihydrate, C18H21NO3,H3PO4,½H2O, in the tablets using peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C18H21NO3,H3PO4,½H2O in codeine phosphate RS. Storage Store in a well-closed container, at 15 °C - 30 °C. Action and use Analgesic and antipyretic. Usual strength 300 mg of paracetamol, 60 mg of codeine phosphate. 600 mg of paracetamol, 60 mg of codeine phosphate. 500 mg of paracetamol, 30 mg of codeine phosphate. 500 mg of paracetamol, 15 mg of codeine phosphate. PARACETAMOL AND IBUPROFEN TABLETS Tabellae Paracetamoli et Ibuprofeni Paracetamol and ibuprofen tablets contain paracetamol and ibuprofen. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements:

Content of paracetamol, C8H9NO2, from 90.0% to 110.0% of the stated amount. Content of ibuprofen, C13H18O2, from 90.0% to 110.0% of the stated amount. Identification In the Assay, the retention times of two principal peaks in the chromatogram obtained with the test solution 735

VP V

LIQUID PARAFFIN

correspond to those of paracetamol peak and ibuprofen peak in the chromatograms obtained with paracetamol reference solution and ibuprofen reference solution.

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of phosphate buffer solution pH 7.2 R. Rotation speed: 50 rpm. Time: 45 min. Procedure: Test solution: After the specified time, withdraw a sample of the medium and filter, discard the first 20 ml of the filtrate. Paracetamol reference solution: Weigh accurately about 36 mg of paracetamol RS, transfer into a 100-ml volumetric flask, add 20 ml of methanol R, shake well, add the medium to volume, shake well. Ibuprofen Reference solution: Weigh accurately about 44 mg of ibuprofen RS into a 100-ml volumetric flask, add 20 ml of methanol R, shake well, add the medium to volume, shake well. If necessary dilute this solution with the medium to obtain a solution having a concentration of ibuprofen similar to that expected in the test solution. Determine the content of paracetamol and ibuprofen dissolved by liquid chromatography (Appendix 5.3) with the chromatographic conditions described in the Assay. Tolerance: Not less than 75% (Q) of the labeled amount of paracetamol, C8H9NO2, and not less than 75% (Q) of the labeled amount of ibuprofen, C13H18O2, are dissolved in 45 min. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - 0.01% solution of phosphoric acid (60 : 40), make adjustments if necessary. Paracetamol reference solution: Weigh accurately about 32.5 mg of paracetamol RS, transfer into a 100-ml volumetric flask, add about 80 ml of the mobile phase, sonicate for 15 min and dilute to volume with the same solvent, shake well. Ibuprofen reference solution: Weigh accurately about 40 mg of ibuprofen RS, transfer into 100-ml volumetric flask, add about 80 ml of the mobile phase, sonicate for 15 min and dilute to volume with the same solvent, shake well. If necessary, dilute the resulting solution with the mobile phase to obtain a solution having a concentration of ibuprofen similar to that expected in the test solution. Test solution: Weigh 20 tablets, calculate the average mass and finely powder. Weigh accurately a quantity of the powdered tablets containing about 325 mg of paracetamol and transfer into a 100-ml volumetric flask, add 80 ml of the mobile phase, sonicate for 15 min, add to volume with the mobile phase, shake well and filter. Dilute 10.0 ml of the filtrate to 100.0 ml with the same solvent. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (10 µm). 736

Detector: A spectrophotometer set at 220 nm. Flow rate: 2 ml/min. Volume of injection: 20 µl. Procedure: Inject alternately the reference solutions and the test solution, calculate the content of paracetamol, C8H9NO2 and of ibuprofen, C13H18O2 in the tablets using the area of paracetamol and ibuprofen peaks in the chromatogram obtained with the reference solutions, the test solution and the declared content of C8H9NO2 and C13H18O2 in paracetamol RS and of ibuprofen RS, respectively.

Storage Store in a cool place and protected from light. Action and use Analgesic; antipyretic. Usual strength 325 mg of paracetamol and 400 mg of ibuprofen. 325 mg of paracetamol and 200 mg of ibuprofen. 325 mg of paracetamol and 100 mg of ibuprofen. LIQUID PARAFFIN Paraffinum liquidum Liquid paraffin is a purified mixture of liquid saturated hydrocarbons obtained from petroleum.

Characters Colourless, transparent, oily liquid, free from fluorescence in daylight. Practically insoluble in water, slightly soluble in ethanol (96%), miscible with hydrocarbons. Identification Apply one of the two following identifications: First identification: A, C. Second identification: B, C. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of reference spectrum of liquid paraffin. B. In a test tube cautiously boil 1 ml with 1 ml of 0.1 M sodium hydroxide R, with continuous shaking, for about 30 s. On cooling to room temperature, 2 phases separate. To the aqueous phase add 0.1 ml of phenolphthalein solution R. The solution becomes red. C. It complies with the test for Viscosity. Acidity or alkalinity Add 20 ml of boiling water to 10 ml of the substance to be examined and shake vigorously for 1 min. Separate the aqueous layer and filter. To 10 ml of the filtrate, add 0.1 ml of phenolphthalein solution R. The solution is colourless. Not more than 0.1 ml of 0.1 N sodium hydroxide VS is required to change the colour of the indicator to pink.

PEFLOXACIN MESILATE

VP V

Relative density 0.827 to 0.890 (Appendix 6.5). Viscosity 110 mPa·s to 230 mPa·s (Appendix 6.3). Polycyclic aromatic hydrocarbons Use reagents for ultraviolet spectrophotometry. Test solution: Introduce 25.0 ml of the substance to be examined into a 125 ml separating funnel with unlubricated ground-glass parts (stopper, stopcock). Add 25 ml of hexane R which has been previously shaken twice with one-fifth its volume of dimethyl sulfoxide R. Mix and add 5.0 ml of dimethyl sulfoxide R. Shake vigorously for 1 min and allow to stand until 2 clear layers are formed. Transfer the lower layer to a 2nd separating funnel, add 2 ml of hexane R and shake the mixture vigorously. Allow to stand until 2 clear layers are formed. Separate the lower layer and measure its absorbance (Appendix 4.1) in the range 260 nm to 420 nm. Using blank solution: Vigorously shaking 5.0 ml of dimethyl sulfoxide R with 25 ml of hexane R for 1 min, use the clear lower layer. Reference solution: A 7.0 mg/l solution of naphthalene R in trimethylpentane R and measure the absorbance of the solution at the absorption maximum at 275 nm, using trimethylpentane R as blank solution. At no wavelength between 260 nm and 420 nm does the absorbance of the test solution exceed one-third that of the reference solution at 275 nm. Readily carbonisable substances Use a ground-glass-stoppered tube about 125 mm long and 18 mm in internal diameter, graduated at 5 ml and 10 ml; wash with hot water (temperature at least 60 °C), acetone R, heptane R and finally with acetone R, dry at 100 °C to 110 °C. Cool in a desiccator. Introduce 5 ml of the substance to be examined and add 5 ml of nitrogen-free sulfuric acid R1. Insert the stopper and shake as vigorously as possible, in the longitudinal direction of the tube, for 5 s. Loosen the stopper, immediately place the tube in a water-bath, avoiding contact of the tube with the bottom or side of the bath, and heat for 10 min. After 2 min, 4 min, 6 min and 8 min, remove the tube from the bath and shake as vigorously as possible, in the longitudinal direction of the tube for 5 s. At the end of 10 min of heating, remove the tube from the water-bath and allow to stand for 10 min. Centrifuge at 2000 g for 5 min. The lower layer is not more intensely coloured than a mixture of 0.5 ml of blue primary solution, 1.5 ml of red primary solution, 3.0 ml of yellow primary solution and 2 ml of 0,1 M hydrochloric acid R (Appendix 9.3). Solid paraffins Dry a suitable quantity of the substance to be examined by heating at 100 °C for 2 h and cool in a desiccator over sulfuric acid R. Place in a glass tube with an internal diameter of about 25 mm, close the tube and immerse in

a bath of iced water. After 4 h, the liquid is sufficiently clear for a black line, 0.5 mm wide, to be easily seen against a white background held vertically behind the tube.

Storage Protected from light. Action and use Faecal softener. Preparation Oral emulsion. PEFLOXACIN MESILATE Pefloxacini mesilas

C18H24FN3O6S,2H2O  

M. 465.5

Pefloxacin mesilate is 1-ethyl-6-fluoro-7-(4methylpiperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3carboxylic acid methanesulfonate dihydrate. It contains not less than 98.5% and not more than 101.5% of C18H24FN3O6S, calculated with reference to the anhydrous substance.

Characters Fine, white or almost white powder. Freely soluble in water, slightly soluble in ethanol (96%), very slightly soluble in methylene chloride. Identification A. Dissolve 0.1 g of the substance to be examined in 10 ml of water, add 5 ml of 1 M sodium hydroxide R. Adjust to pH 7.4 ± 0.1 with phosphoric acid R and shake with 2 quantities, each of 30 ml of methylene chloride R. Combine the organic layers and dry over anhydrous sodium sulfate R. Evaporate to dryness. Examine the infrared absorption spectrophotometry (Appendix 4.2) of the residue, prepare as a halide disc of potassium bromide R. The infrared absorption spectrum of the residue is concordant with the residue obtained when repeat the operations using pefloxacin mesilate dihydrate RS. B. Examine by thin-layer chromatography (Appendix 5.4). Plate: Silica gel G. Mobile phase: Water - ammonia - butanol - acetone (5 : 10 : 20 : 65). Test solution: Dissolve 40 mg of the substance to be examined in water and dilute to 1 ml with the same solvent. Reference solution: Dissolve 60 mg of methanesulfonic 737

VP V

PEFLOXACIN MESILATE

acid R in water and dilute to 10 ml with the same solvent. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm. Dry the plate in air and spray with a 0.04% solution of bromocresol purple R in ethanol (50%) R, adjusted to pH 10 using 1 M sodium hydroxide R. Examine in day light. The principal spot in the chromatogram obtained with the test solution is similar in position, size and colour to the principal spot in the chromatogram obtained with reference solution.

Appearance of solution Solution S: Dissolve 1.0 g in carbon dioxide-free water and dilute to 10.0 ml with the same solvent. Examin within 1 h after its preparation, solution S is not more opalescent than reference suspension II (Appendix 9.2) and not more intensely coloured than intensity 3 of the range of reference solutions of the most appropriate colour (Appendix 9.2, method 2). pH 3.5 to 4.5 (Appendix 6.2). Dilute 1 ml of solution S to 10 ml with carbon dioxide-free water. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - solution A - thiodiethylene glycol (30 : 70 : 0.2). Solution A: Dissolve 2.70 g of cetyltrimethylammonium bromide R and 6.18 g of boric acid R in 900 ml water, adjust to pH 8.3 with 1 M sodium hydroxide R, then dilute to 1000 ml with water. Test solution: Dissolve 20.0 mg of the substance to be examined in the mobile phase and dilute to 100.0 ml with the mobile phase. Reference solution (1): Dissolve 5.0 mg of pefloxacin impurity B RS in the mobile phase and dilute to 50.0 ml with the same solution. Dilute 1.0 ml of the solution to 100.0 ml with the mobile phase. Dissolve the contents of a vial of pefloxacin impurity C RS in 2.0 ml of this solution. Reference solution (2): Dissolve 10.0 mg of norfloxacin impurity A RS (impurity F) in the mobile phase and dilute to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 100.0 ml with the mobile phase. Chromatographic system: A column (15 cm × 6 mm) packed with octadecylsilyl vinyl polymer for chromatography R (5 µm). Detector: A spectrophotometer set at 258 nm and at 273 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. The run time is 4 times the retention time of pefloxacin (about 60 min). Procedure: Relative retentions and correction factors: 738

Approximate Relative retention

Correction factor

Impurity E

0.2

-

Impurity D

0.3

-

Impurity A

0.5

-

Impurity G

0.8

1.4

Pefloxacin

1

-

Impurity C

1.7

2.4

Impurity B

1.8

-

Impurity H

2.4

1.8

Impurity F

3.5

-

Procedure: System suitability: In the chromatogram obtained with reference solution (1) at 273 nm, the resolution between the peaks due to impurities C and B is at least 1.5. Inject the test solution and the reference solution (2). In the chromatogram obtained with the test solution at 258 nm, calculate the percentage content of impurities C, F, G and H using the area of the principal peak in the chromatogram obtained with reference solution (2) at 258 nm (external standardisation) taking into account the correction factors indicated in the table. In the chromatogram obtained with the test solution at 273 nm, calculate the percentage content of impurities A, B, D and E and of any other impurity from the areas of the peaks in the chromatogram obtained with the test solution by the normalisation procedure. Limits: Impurities A, B, D, E and any other impurity at 273 nm and impurities C, F, G, H at 258 nm: For each impurity, not more than 0.5% and not more than 3 impurities have a content between 0.2% and 0.5%. Total: Not more than 1.0%. Disregard any peak at 273 nm with an area less than 0.0005 times the area of the principal peak in the chromatogram obtained with the test solution (0.05%). Notes: Impurity A: 1-ethyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4dihydroquinoline-3-carboxylic acid (demethylated pefloxacin or norfloxacin). Impurity B: 6-chloro-1-ethyl-7-(4-methylpiperazin-1-yl)-4-oxo1,4-dihydroquinoline-3-carboxylic acid (chlorinated homologue of pefloxacin). Impurity C: 1-ethyl-6-fluoro-5-(4-methylpiperazin-1-yl)-4-oxo1,4-dihydroquinoline-3-carboxylic acid (isopefloxacin). Impurity D: 4-(3-carboxy-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinolin7-yl)-1-methylpiperazine 1-oxide (N-oxide of pefloxacin). Impurity E: 1-ethyl-6-fluoro-7-(4-methylpiperazin-1-yl)quinoline4(1H)-one (decarboxylated pefloxacin). Impurity F: 7-chloro-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline3-carboxylic acid (N-ethyl acid) (norfloxacin impurity A). Impurity G: ethyl 7-chloro-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline3-carboxylate (N- ethyl ester).

VP V Impurity H: 5-chloro-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline3-carboxylic acid (iso-N- ethyl acid).

Heavy metals Not more than 10 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 5. Prepare the standard using 10.0 ml of lead standard solution (1 ppm Pb) R. Water 7.0% to 8.5% (Appendix 10.3). Determined on 50.0 mg using a mixture of 10 volumes of methanol R and 50 volumes of methylene chloride R. Sulfated ash Not more than 0.1 % (Appendix 9.9). Determined on 1.0 g. Assay Dissolve 0.200 g of the substance to be examined in 15.0 ml of anhydrous acetic acid R and add 75.0 ml of acetic anhydride R. Titrate with 0.1 M perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 M perchloric acid VS is equivalent to 21.48 mg of C18H24FN3O6S. Storage In an airtight container, protected from light. Action and use Quinolone antibacterial. Preparations Tablets. PEFLOXACIN MESILATE TABLETS Tabellae Pefloxacini mesilati Pefloxacin mesilate tablets contain pefloxacin mesilate. They may be coated. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements:

Content of pefloxacin, C17H20FN3O3, 93.0% to 107.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Water - ammonia - butanol - aceton (5 : 10 : 20 : 65). Test solution: Shake a quantity of the powdered tablets containing 400 mg of pefloxacin with 10 ml of water, filter and use the filtrate.

PEFLOXACIN MESILATE TABLETS

Reference solution: Dissolve 60 mg of methanesulfonic acid R in water and dilute to 10 ml with the same solvent. Procedure: Apply separately to the plate 10 μl of each solution. Develop over a path of 15 cm with the mobile phase. Remove the plate, allow it to dry in air. Spray the plate with a 0.04% solution of bromocresol purple R in ethanol 50% R, adjusted to pH 10 with 1 M sodium hydroxide R. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to that in the chromatogram obtained with reference solution. B. In the assay, the retention time of the principal peak in the chromatogram obtained with the test solution coressponds to that of the principal peak in the chromatogram obtained with the reference solution.

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 1000 ml of 0.1 M hydrochloric acid R. Rotation speed: 50 rpm. Time: 45 min. Procedure: Test solution: After the specified time (45 min), withdraw a sample of the medium, and filter. Dilute the filtrate with the medium to obtain a solution containing 4 µg/ml of pefloxacin. Measure the absorbance (Appendix 4.1) of the resulting solution at 277 nm, using the medium as a blank, in comparison with a reference solution having the same concentration of pefloxacin in the medium. Tolerance: Not less than 75% (Q) of the stated amount of pefloxacin, C17H20FN3O3, is dissolved in 45 min. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase and chromatographic system: Prepare as directed in the Assay. Test solution: Weigh 20 tablets (coating layer removed if necessary), calculate the average mass and powder finely. Dissolve an appropriate quantity of the powdered tablets in the mobile phase to obtain a solution containing the equivalent of about 0.2 mg/ml of pefloxacin, mix and filter. Reference solution: Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Inject the reference solution, adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained is not less than 25% of the full scale of the recorder. Inject the test solution and continue the chromatography for 3 times the retention time of the principal peak. Limits: In the chromatogram obtained with the test solution, the area of any secondary peak is not more than 0.5 times the area of the principal peak in the chromatogram obtained with the reference solution (0.5%); the sum of areas of any secondary peaks is not greater than the area of the principal peak in the chromatogram obtained with the reference solution (1.0%). 739

VP V

PENICILLAMINE

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: a 0.55% solution of potassium hydrogen phosphate - a 1.61% solution of tetrabutylammonium bromide - acetonitrile (80 : 8 : 9). Adjust to pH 4.0 with phosphoric acid R. Reference solution: Dissolve an accurately weighed quantity of pefloxacin mesilate RS in the mobile phase to obtain a solution containing the equivalent of about 0.002% of pefloxacin. Test solution: Weigh 20 tablets (coating layer removed if necessary), calculate the average mass and powder finely. Transfer an accurately weighed quantity of the powdered tablets containing the equivalent of 100 mg of pefloxacin to a 200 ml volumetric flask, dissolve in water and dilute to volume with the same solvent, mix and filter. Dilute 2.0 ml of the filtrate to 50.0 ml with the mobile phase, mix. Resolution solution: A solution containing 0.002% of pefloxacin RS and 0.002% of norfloxacin RS in the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 μm), maintained at 40 °C. Detector: A spectrophotometer set at 273 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 μl. Procedure: System suitability: Inject the resolution solution, the order of elution is norfloxacin peak followed by pefloxacin. The resolution factor between pefloxacin peak and norfloxacin peak is at least 5.0. Inject the reference solution, the relative standard deviation of the peak areas due to pefloxacin for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of pefloxacin, C17H20FN3O3, in the tablets using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C17H20FN3O3 in pefloxacin mesilate RS. Storage Store in an airtight container, in a cool and dry place, protected from light. Action and use Quinolone antibacterial. Usual strength 400 mg of pefloxacin.

PENICILLAMINE Penicillaminum

C5H11NO2S 

M. 149.2 

Penicillamine is (2S)-2-amino-3-methyl-3-sulfanylbutanoic acid. It contains not less than 98.0% and not more than 101.0% of C5H11NO2S, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder. Freely soluble in water, slightly soluble in ethanol (96%). Identification Apply one of the two following identifications: First identification: A, B, D. Second identification: A, C, D. A. Dissolve 0.5 g of the substance to be examined in a mixture of 0.5 ml of hydrochloric acid R and 4 ml of warm acetone R, cool in iced water and initiate crystallisation by scratching the wall of the tube with a glass rod. A white precipitate is formed. Filter with the aid of vacuum, wash with acetone R and dry with suction. A 1% solution of the precipitate is dextrorotatory. B. In the chromatograms obtained in the test for Impurity A, the principal peak in the chromatogram obtained with the test solution is similar in retention time and area to the principal peak in the chromatogram obtained with reference solution (1). C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Glacial acetic acid - water - butanol (18 : 18 : 72). Test solution: Dissolve 10 mg of the substance to be examined in 4 ml of water. Reference solution: Dissolve 10 mg of penicillamine RS in 4 ml of water. Procedure: Apply separately to the plate 2 µl of each solution. Develop over a path of 10 cm. Heat the plate at 100 °C to 105 °C for 5 min to 10 min. Expose to iodine vapour for 5 min to 10 min. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. D. Dissolve 40 mg of the substance to be examined in 4 ml of water and add 2 ml of phosphotungstic acid solution R. Allow to stand for 5 min. A blue colour develops. Appearance of solution Solution S: Dissolve 2.5 g in carbon dioxide-free water R and dilute to 25 ml with the same solvent.

740

VP V

Solution S is clear (Appendix 9.2) and not more intensely coloured than intensity 6 of the range of reference solutions of the most appropriate colour (Appendix 9.3, method 2).

pH 4.5 to 5.5 (Appendix 6.2). Dilute 1 ml of solution S to 10 ml with carbon dioxide-free water R. Specific optical rotation -61.0° to -65.0°, calculated with reference to the dried substance (Appendix 6.4). Dissolve 0.500 g in 1 M sodium hydroxide R and dilute to 10.0 ml with the same solvent. Ultraviolet-absorbing substances Not more than 0.5% of penilloic acid. Dissolve 0.100 g in water and dilute to 50.0 ml with the same solvent. The absorbance of the solution at 268 nm is not more than 0.07. Impurity A Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use. Mobile phase: Solution containing 0.01% of sodium edetate R and 0.2% of methanesulfonic acid R in water. Test solution: Dissolve 40.0 mg of the substance to be examined in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (1): Dissolve 40 mg of penicillamine RS in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (2): Dissolve 20.0 mg of penicillamine disulfide RS (impurity A) in the mobile phase and dilute to 50.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase B (10 µm). Detector: A spectrophotometer set at 220 nm. Flow rate: 1.7 ml/min. Volume of injection: 20 µl. Procedure: Relative retention of impurity A with reference to penicillamine (retention time = about 6 min) is about 1.8. System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to penicillamine and impurity A is at least 4.0. Limit: In the chromatogram obtained with the test solution: The area of the peak due to impurity A is not more than the area of the corresponding peak in the chromatogram obtained with reference solution (2) (1%). Impurity B Not more than 0.1 ppm. Carry out all the operations in a penicillin-free atmosphere and with equipment reserved for this test. Sterilise the

PENICILLAMINE

equipment at 180 °C for 3 h and the buffer solutions at 121 °C for 20 min before use. Test solution (1): Dissolve 1.000 g of the substance to be examined in 8 ml of buffer solution pH 2.5 R and add 8 ml of ether R. Shake vigorously for 1 min. Repeat the extraction and combine the ether layers. Add 8 ml of buffer solution pH 2.5 R, shake for 1 min, allow to settle and quantitatively separate the upper layer, taking care to eliminate the aqueous phase completely (penicillin is unstable at pH 2.5; carry out operations at this pH within 6 min to 7 min). Add 8 ml of phosphate buffer solution pH 6.0 R2 to the ether phase, shake for 5 min, allow to settle, then separate the aqueous layer and check that the pH is 6.0. Test solution (2): Add 20 µl of penicillinase solution R to 2 ml of test solution (1) and incubate at 37 °C for 1 h. Reference solution (1): Dissolve 5 mg of benzylpenicillin sodium R in 500 ml of phosphate buffer solution pH 6.0 R2. Dilute 0.25 ml of the solution to 200.0 ml with buffer solution pH 2.5 R. Carry out the extraction using 8 ml of this solution as described for test solution (1). Reference solution (2): Add 20 µl of penicillinase solution R to 2 ml of reference solution (1) and incubate at 37 °C for 1 h. Blank solution: Prepare the solution as described for test solution (1) but omitting the substance to be examined. Liquefy a suitable nutrient medium such as that described below and inoculate it at a suitable temperature with a culture of Kocuria rhizophila (ATCC 9341), to give 5 × 104 micro-organisms per millilitre or a different quantity if necessary to obtain the required sensitivity and formation of clearly defined inhibition zones of suitable diameter. Immediately pour the inoculated medium into 5 Petri dishes 10 cm in diameter to give uniform layers 2 mm to 5 mm deep. The medium may alternatively consist of 2 layers, only the upper layer being inoculated. Store the dishes so that no appreciable growth or death of the micro-organisms occurs before use and so that the surface of the agar is dry at the time of use. In each dish, place 5 stainless steel hollow cylinders 6 mm in diameter on the surface of the agar evenly spaced on a circle with a radius of about 25 mm and concentric with the dish. For each dish, place in separate cylinders 0.15 ml of test solutions (1) and (2), reference solutions (1) and (2) and the blank solution. Maintain at 30 °C for at least 24 h. Measure the diameters of the inhibition zones with a precision of at least 0.1 mm. The test is valid if reference solution (1) gives a clear inhibition zone and if reference solution (2) and the blank solution give no inhibition zone. If test solution (1) gives an inhibition zone, this is caused by penicillin if test solution (2) gives no inhibition zone. If this is so, the average diameter of the inhibition zones given by test solution (1) for the 5 Petri dishes is less than the average diameter of the inhibition zones given by reference solution (1) measured in the same conditions. 741

PEPSIN Nutrient medium (pH 6.0) Peptone Yeast extract Meat extract Sodium chloride Agar Distilled water Note:

VP V

5.0 g 1.5 g 1.5 g 3.5 g 15.0 g 1000 ml

Impurity B: Penicillin.

Mercury Note more than 10 ppm. Examine by atomic absorption spectrometry (Appendix 4.4, method 1). Test solution: To 1.00 g of the substance to be examined add 10 ml of water and 0.15 ml of perchloric acid R and swirl until dissolution is complete. Add 1.0 ml of a 1% solution of  ammonium pyrrolidinedithiocarbamate R which has been washed immediately before use 3 times, each time with an equal volume of methyl isobutyl ketone R. Mix and add 2.0 ml of methyl isobutyl ketone R and shake for 1 min. Dilute to 25.0 ml with water and allow the 2 layers to separate; use the methyl isobutyl ketone layer. Reference solution: Dissolve a quantity of mercuric oxide R  equivalent to 0.108 g of HgO in the smallest necessary volume of dilute hydrochloric acid R and dilute to 1000.0 ml with water (100 ppm Hg). Prepare the reference solutions in the same manner as the test solution but using instead of the substance to be examined suitable volumes of the solution containing 100 ppm of Hg. Measure the absorbance at 254 nm using an mercury hollow-cathode lamp as a source of radiation and an airacetylene flame. Set the zero of the instrument using a methyl isobutyl ketone layer obtained as described for the test solution but omitting the substance to be examined. Heavy metals Not more than 20 ppm (Appendix 9.4.8). 12 ml of solution S complies with limit test for heavy metals, method 1. Prepare the standard using lead standard solution (2 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 60 °C; at a pressure not exceeding 0.67 kPa; diphosphorus pentoxide). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.1000 g of the substance to be examined in 30 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 742

1 ml of 0.1 N perchloric acid VS is equivalent to 14.92 mg of C5H11NO2S.

Storage Store in an airtight container, protected from light. Action and use Disease-modifying antirheumatic drug; chelating agent; heavy metal poisoning. Preparation Tablets. PEPSIN Pepsinum Pepsin is powder substance containing proteinases. It is prepared from the freshly gastric mucosa of pigs, cattle or sheep. It is active in acid medium (pH 1 to 5) and has an activity not less than 0.5 unit per mg, calculated with reference to the dried substance. The animals from which pepsin powder is derived must fulfil the requirements for the health of animals suitable for human consumption, to the satisfaction of the competent authority. It must have been shown to what extent the method of production allows inactivation or removal of any contamination by viruses or other infectious agents.

Characters A white or slightly yellow, crystalline or amorphous powder, hygroscopic. Soluble in water, practically insoluble in ethanol (96%). The solution in water may be slightly opalescent with a weak acidic reaction. Identification In a mortar, pound 30 mg of fibrin blue R. Suspend in 20 ml of dilute hydrochloric acid R. Filter the suspension on a filter paper and wash with dilute hydrochloric acid R until a colourless filtrate is obtained. Perforate the filter paper and wash the suspension of fibrin blue R through it into a conical flask using 20 ml of dilute hydrochloric acid R. Shake this mixture before use. Dissolve a quantity of the substance to be examined, equivalent to not less than 20 activity units, in 2 ml of dilute hydrochloric acid R and adjust to pH 1.6 ± 0.1 (test solution). Add 1 ml of this solution to a test-tube containing 4 ml of the fibrin blue suspension, mix and place in a water-bath at 25 °C with gentle shaking. Prepare a blank solution at the same time and in the same manner using 1 ml of water. After 15 min of incubation the blank solution is colourless and the test solution is blue. Loss on drying Not more than 5.0% (Appendix 9.6).

PEPSIN

VP V

(0.500 g; phosphorus pentoxide; at a pressure not exceeding 0.7 kPa; 60 °C; 4 h)

Microbial contamination Total aerobic microbial count: Not more than 103 CFU/g. 1.0 g of the substance to be examined is free from Escherichia coli and 10 g of the substance to be examined is free from Salmonella (Appendix 13.6). Assay The activity of pepsin powder is determined by comparing the quantity of peptides, non-precipitable by trichloroacetic acid solution R and assayed using the phosphomolybdotungstic reagent R, which are released per minute from a substrate of haemoglobin solution R, with the quantity of such peptides released by pepsin powder RS from the same substrate in the same conditions. Avoid shaking and foaming during preparation of the test and reference solutions. Test solution: Immediately before use, prepare a solution of the substance to be examined expected to contain 0.5 activity units/ml in dilute hydrochloric acid R; before dilution to volume, adjust to pH 1.6 ± 0.1, if necessary, using 1 M hydrochloric acid R. Reference solution: Prepare a solution of pepsin powder RS containing 0.5 activity units/ml in dilute hydrochloric acid R; before dilution to volume, adjust to pH 1.6 ± 0.1, if necessary, using 1 M hydrochloric acid R. Reference solution is prepare within 15 min before use. Designate tubes in duplicate T, Tb, S1, S1b, S2, S2b, S3, S3b; designate a tube B. Add dilute hydrochloric acid R to the tubes as follows: B: 1.0 ml S1 and S1b: 0.5 ml

S2, S2b, T and Tb: 0.25 ml Add the reference solution to the tubes as follows: S1 and S1b: 0.5 ml S2 and S2b: 0.75 ml S3 and S3b: 1.0 ml Add 0.75 ml of the test solution to tubes T and Tb. Add 10.0 ml of trichloroacetic acid solution R to tubes S1b, S2b, S3b, Tb and B. Mix by shaking. Place the tubes and haemoglobin solution R in a water bath at 25 ± 0.1°C. When temperature equilibrium is reached, add 5.0 ml of haemoglobin solution R to tubes B, S1b, S2b, S3b and Tb. Mix. At time zero add 5.0 ml of haemoglobin solution R successively and at intervals of 30 s to tubes S1, S2, S3 and T. Mix immediately after each addition. Exactly 10 min after adding the haemoglobin solution R, stop the reaction by adding 10.0 ml of trichloroacetic acid solution R to tubes S1, S2, S3 and T, at intervals of 30 s, mix. Filter the contents of each tube (samples and blanks) twice through the same suitable filter paper previously washed with a 5% solution of trichloroacetic acid R, then with water and dried. Discard the first 5 ml of filtrate. Place 3.0 ml of each filtrate separately in a tube containing 20 ml of water. Mix. (A suitable filter paper complies with the following test: filter 5 ml of a 5% solution of trichloroacetic acid R through a 7 cm disc of white filter paper: the absorbance (Appendix 4.1) of the filtrate, measured at 275 nm using unfiltered 5% solution of trichloroacetic acid R as the compensation liquid, is less than 0.04). A schematic presentation of the above operations is shown in following Tablet A. Add to each tube 1.0 ml of 20% sodium hydroxide solution R and 1.0 ml of phosphomolybdotungstic reagent R,

Table A: Tubes S2

S1

S1b

S2b

Dilute hydrochloric acid R (ml)

0.5

0.5

0.25

0.25

Reference slution (ml)

0.5

0.5

0.75

0.75

S3 1.0

S3b

Mix Water bath at 25 °C

+

Hemoglobin solution R (ml) Mix

Tb

B

0.25

0.25

1.0

0.75

0.75

1.0

Test solution (ml) Tricloroacetic acid solution R (ml)

T

10.0

10.0

10.0

10.0

10.0

+

+

+

+

+

+

+

+

+

+

+

+

+

5.0

5.0

5.0

5.0

5.0

+

+

+

+

+

+

+

+

+

5.0

5.0

5.0

5.0

Mix

+

+

+

+

Water bath at 25 °C, 10 min

+

Hemoglobin solution R (ml)

+

+

+

+

+

+

10.0

10.0

10.0

10.0

Mix

+

+

+

+

Filter

+

Tricloroacetic acid solution R (ml)

+

+

+

+

+

+

743

VP V

PERINDOPRIL tert-BUTYLAMINE

beginning with the blanks and then the samples of each set, in a known order. After 15 min, measure the absorbance (Appendix 4.1) of solutions S1, S2, S3, S1b, S2b, S3b, T and Tb at 540 nm using the filtrate obtained from tube B as the compensation liquid. Correct the average absorbance values for the filtrates obtained from tubes S1, S2 and S3 by subtracting the average values obtained for the filtrates from tubes S1b, S2b, S3b respectively. Draw a calibration curve of the corrected values against volume of reference solution used. Determine the activity of the substance to be examined using the corrected absorbance for the test solution (T - Tb) together with the calibration curve and taking into account the dilution factors. Storage Store in an airtight container, protected from light, at a temperature of 2 - 8 °C. PERINDOPRIL tert-BUTYLAMINE Tert-butylamini perindoprilum Perindopril Erbumine

C19H32N2O5,C4H11N  

M: 441.6

Perindopril tert-butylamine is 2-methylpropan-2-amine (2S,3aS,7aS)-1-[(2S)-2-[[(1S)-1-(ethoxycarbonyl)butyl] amino]propanoyl]octahydro-1H-indole-2-carboxylate. It contains not less than 99.0% and not more than 101.0% of C19H32N2O5,C4H11N, calculated with reference to the anhydrous substance.

Characters White or almost white, slightly hygroscopic, crystalline powder, it shows polymorphism. Freely soluble in water and in ethanol (96%), soluble or sparingly soluble in dichloromethane. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of perindopril tert-butylamine RS. If the spectra obtained show differences, dissolve the substance to be examined and the reference substance separately in dichloromethane R, evaporate to dryness and record new spectra using the residues. B. In the test for Impurity A, in the chromatogram obtained with the test solution a spot is observed with the same Rf as the spot with the higher Rf in the chromatogram obtained with reference solution (3) (tert-butylamine). 744

C. Specific optical rotation: -66° to -69°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.250 g of the substance to be examined in ethanol (96%) R and dilute to 25.0 ml with the same solvent.

Impurity A Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Glacial acetic acid R - toluene R - methanol R (1 : 40 : 60). Test solution: Dissolve 0.20 g of the substance to be examined in methanol R and dilute to 10.0 ml with the same solvent. Reference solution (1): Dissolve 5 mg of perindopril impurity A RS in methanol R and dilute to 25.0 ml with the same solvent. Reference solution (2): Dilute 5.0 ml of reference solution (1) to 20.0 ml with methanol R. Reference solution (3): To 5 ml of reference solution (1) add 5 ml of a 2% solution of 1,1-dimethylethylamine R in methanol R. Procedure: Apply separately to the plate 10 µl of each test solution and reference solutions (2), (3). Develop over a path of 2/3 of the plate. Dry the plate in a current of warm air and expose to iodine vapour for at least 20 h. In the chromatogram obtained with the test solution, any spot due to impurity A is not more intense than the spot in the chromatogram obtained with reference solution (2) (0.25%). The test is not valid unless the chromatogram obtained with reference solution (3) shows two clearly separated spots. Stereochemical purity Examine by liquid chromatography (Appendix 5.3). Mobile phase: Mix, in the following order: Acetonitrile R pentanol R - a 0.15% solution of sodium heptanesulfonate R previously adjusted to pH 2.0 with a mixture of equal volumes of perchloric acid R and water (21.7 : 0.3 : 78). Test solution: Dissolve 20 mg of the substance to be examined in ethanol (96%) R and dilute to 10.0 ml with the same solvent. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with ethanol (96%) R. Dilute 1.0 ml of this solution to 10.0 ml with the same solvent. Reference solution (2): Dissolve 10 mg of perindopril for stereochemical purity RS (containing impurity I) in ethanol (96%) R and dilute to 5.0 ml with the same solvent. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase spherical octadecylsilyl silica gel for chromatography R (5 µm). Column temperature and at least 30 cm of the tubing preceding the column: 50 °C. Detector: A spectrophotometer set at 215 nm. Flow rate: 0.8 ml/min. Equilibration time: Minimum 4 h. Volume of injection: 10 µl.

VP V

Procedure: Inject the test solution and the reference solutions. The run time is 1.5 times the retention time of perindopril. Identification of impurities: Use the chromatogram supplied with perindopril for stereochemical purity RS and the chromatogram obtained with reference solution (2) to identify the peak due to impurity I. Relative retention with reference to perindopril (retention time = about 100 min): Impurity I = about 0.9. System suitability: The chromatogram obtained with reference solution (2) is similar to the chromatogram supplied with perindopril for stereochemical purity RS. The signal-to-noise ratio is at least 3 for the principal peak in the chromatogram obtained with reference solution (1). Peak-to-valley ratio (Hp/Hv) is at least 3, where Hp = height above the baseline of the peak due to impurity I and Hv = height above the baseline of the lowest point of the curve separating this peak from the peak due to perindopril in the chromatogram obtained with reference solution (2). Limits: In the chromatogram obtained with the test solution: The area of the peak due to impurity I is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%). Disregard any peak with a relative retention with reference to perindopril of less than 0.6 or more than 1.4. Note: Impurity I. (2RS,3aRS,7aRS)-1-[(2RS)-2-[[(1SR)-1(ethoxycarbonyl)butyl]amino]propanoyl]octahydro-1H-indole2-carboxylic acid ((±)-1’’-epi-perindopril).

Related substances

Examine by liquid chromatography (Appendix 5.3). Mobile phase A: water adjusted to pH 2.5 with a mixture of equal volumes of perchloric acid R and water. Mobile phase B: A 0.03% solution of perchloric acid R in acetonitrile R1. Prepare the following solutions immediately before use or maintain them at a temperature below 10 °C. Test solution: Dissolve 60 mg of the substance to be examined in mobile phase A and dilute to 20.0 ml with the same solvent. Reference solution (1): Dissolve 3 mg of perindopril for peak identification RS (containing impurities B, E, F, H and K) in 1 ml of mobile phase A. Reference solution (2): Dilute 1.0 ml of the test solution to 200.0 ml with mobile phase A. Reference solution (3): Dilute 1.0 ml of reference solution (2) to 10.0 ml with mobile phase A. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase spherical end-capped octylsilyl silica gel for chromatography R (5 µm) with a pore size of 15 nm.

PERINDOPRIL tert-BUTYLAMINE

Temperature for the column and the tubing preceding the column: 60 °C. Detector: A spectrophotometer set at 215 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 – (5 - t)

95

5

(5 - t) – (60 - t)

95 → 40

5 → 60

(60 - t) – (65 - t)

40 → 95

60 → 5

The isocratic step is described for a chromatographic system with a dwell volume (D) of 2 ml. If D is different from 2 ml, correct the gradient times with the value t, calculated using the following expression: D−2 Flow rate Inject the test solution and the reference solutions. Identification of impurities: Use the chromatogram supplied with perindopril for peak identification RS and the chromatogram obtained with reference solution (1) to identify the peaks due to impurities B, E, F, H and K. Relative retention with reference to perindopril (retention time = about 25 min): Impurity B = about 0.68; impurity K = about 0.72; impurity E = about 1.2; impurity F = about 1.6; impurity H = about 1.8 (impurity H may be eluted as 1 or 2 peaks). System suitability: In the chromatogramobtained with reference solution (1), peak-to-valley ratio (Hp/Hv) is at least 3, where Hp = height above the baseline of the peak due to impurity B and Hv = height above the baseline of the lowest point of the curve separating this peak from the peak due to impurity K. Limits: In the chromatogram obtained with the test solution: Impurity E: The area of the peak due to impurity E is not more than 0.8 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.4%). Impurity B: The area of the peak due to impurity B is not more than 0.6 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.3%). Impurities F, H: For each impurity, the area is not more than 0.4 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.2%). Any other impurity: For each impurity, the area is not more than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.1%). The sum of the peak areas of all impurities is not more than 2 times the area of the principal peak in the chromatogram obtained with reference solution (2) (1.0%). Disregard any peak with an area less than the area of the principal peak in the chromatogram obtained with reference solution (3) (0.05%). 745

PERINDOPRIL tert-BUTYLAMINE TABLETS Note: Impurity B: (2S,3aS,7aS)-1-[(2S)-2-[[(1S)-1- carboxybutyl]amino] propanoyl]octahydro-1H-indole-2-carboxylic acid (perindoprilat). Impurity E: (2S,3aS,7aS)-1-[(2S)-2-[[(1S)-1-[(1- methylethoxy) carbonyl]butyl]amino]propanoyl]octahydro-1H-indole-2carboxylic acid. Impurity F: ethyl (2S)-2-[(3S,5aS,9aS,10aS)-3-methyl-1,4dioxodecahydropyrazino[1,2-a]indol-2(1H)-yl] pentanoate. Impurity H: (2S,3aS,7aS)-1-[(2S)-2-[(5RS)-3-cyclohexyl-2(cyclohexylimino)-4-oxo-5- propylimidazolidin-1-yl]propanoyl] octahydro-1H-indole-2-carboxylic acid. Impurity K: (3S,5aS,9aS,10aS)-3-methyldecahydropyrazino[1,2-a] indole-1,4-dione.

Water Not more than 1.0% (Appendix 10.3). Determined on 0.50 g. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.160 g of the substance to be examined in 50 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 22.08 mg of C23H43N3O5. Storage In an airtight container. Action and use Angiotensin converting enzyme inhibitor. Preparation Tablets. PERINDOPRIL tert-BUTYLAMINE TABLETS Tabellae tert-butylamini Perindoprilum Perindopril tert-butylamine tablets contain perindopril tert-butylamine. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of perindopril tert-butylamine, C19H32N2O5, C4H11N, 90.0% to 110.0% of the stated amount. Identification A. To a quantity of the powdered tablets containing the equivalent of 50 mg of perindopril tert-butylamine, add 10 ml of dichloromethane R, sonicate for 2 min to dissolve and centrifuge for 5 min. Decant the supernatant liquid and filter, extract the filtrate with 10 ml of water. Allow 746

VP V

to stand, decant the water layer which is the upper layer and wash with 2 quantities, each of 10 ml, of hexane R. Evaporate the aqueous extracts on a water bath and dry at 60 °C at a pressure not exceeding 0.7 kPa, note: keep temperature at evaporating and drying stages not too high. The infrared absorption spectrum (Appendix 4.2) of the residue is concordant with the reference spectrum of perindopril tert-butylamine RS. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that of the perindopril tert-butylamine peak in the chromatogram obtained with the reference solution.

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 500 ml of 0.05 M hydrochloric acid R. Rotation speed: 50 rpm. Time: 45 min. Procedure: Examine by liquid chromatography (Appendix 5.3). Chromatographic system: Proceed as dirrected in the Assay. Volume of injection: 50 μl. Test solution: After the specified time, withdraw a sample of the medium and filter. Dilute the filtrate, if necessary, with 0.05 M hydrochloric acid R to obtain a solution containing 0.004 mg of perindopril tert-butylamine per ml. Reference solution: Transfer an accurately weighed quantity of about 20.0 mg of perindopril tert-butylamine RS to a 250 ml volumetric flask. Add about 150 ml of 0.05 M hydrochloric acid R, sonicate to dissolve and dilute to volume with the same solvent, mix. Dilute 5.0 ml of the resulting solution to 100.0 ml with 0.05 M hydrochloric acid R. Tolerance: Not less than 75% (Q) of the stated amount of perindopril tert-butylamine, C19H32N2O5,C4H11N, is dissolved in 45 min. Uniformity of content (Appendix 11.2) Examine by liquid chromatography (Appendix 5.3). Mobile phase, chromatographic system, reference solution: Prepare as dirrected in the Assay. Test solution: Finely powder one tablet and transfer to a 50 ml volumetric flask, add 40 ml of the mobile phase, sonicate to dissolve. Dilute to volume with the mobile phase, mix and filter. Dilute a portion of the filtrate with the mobile phase to obtain a solution containing 0.04 mg of perindopril tert-butylamine per ml. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - water previously adjusted to pH 2.0 with a mixture of equal volumes of perchloric acid and water (34 : 66). Reference solution: Transfer an accurately weighed quantity of about 20.0 mg of perindopril tert-butylamine RS to a 100 ml volumetric flask. Add about 70 ml of the mobile phase, sonicate to dissolve. Allow to cool and

VP V

dilute to volume with the same solvent, mix. Dilute 10.0 ml of this solution to 50.0 ml with the mobile phase. Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Transfer an accurately weighed quantity of the powder containing the equivalent of 20 mg of perindopril tert-butylamine to a 100 ml volumetric flask, add 70 ml of the mobile phase, sonicate for 20 min. Allow to cool and dilute to volume with the mobile phase, mix and filter. Dilute 10.0 ml of the filtrate to 50.0 ml the mobile phase. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 μm). Column temperature: 40 °C. Detector: A spectrophotometer set at 215 nm. Flow rate: 2.0 ml/min. Volume of injection: 20 μl. Procedure: System suitability: Inject the reference solution for six times, the relative standard deviation of the peak areas is not more than 2.0%. The symmetry factor is not more than 2.0. Inject alternately the reference solution and the test solution. Calculate the content of perindopril tert-butylamine, C19H32N2O5.C4H11N, in the tablets using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C19H32N2O5. C4H11N in perindopril tert-butylamine RS.

Storage Store in an airtight container, in a cool and dry place, at a temparature not exceeding 30 °C, protected from light. Action and use Treatment of hypertension. Usual strength 2 mg, 4 mg and 8 mg. PETHIDINE HYDROCHLORIDE Pethidini hydrochloridum

C15H21NO2,HCl M.283.8 Pethidine hydrochloride is ethyl 1-methyl-4-phenylpiperidine-4-carboxylate hydrochloride. It contains not less than 99.0% and not more than 101.0% of C15H21NO2, HCl, calculated with reference to the dried substance. Production: If intended for use in the manufacture of parenteral preparations, the manufacturing process is

PETHIDINE HYDROCHLORIDE

validated to show that the content of impurity B is not more than 0.1 ppm.

Characters White, crystalline powder. Very soluble in water, freely soluble in ethanol (96%), practically insoluble in ether. Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of pethidine hydrochloride RS. B. Melting point: 187 °C to 190 °C (Appendix 6.7). C. Dissolve 0.1 g of the substance to be examined in 10 ml of ethanol R and add 10 ml of a 1% solution of picric acid R. A crystalline precipitate is formed which, when washed with water and dried at 100 - 105 °C, melts at 186 to 193 °C. Mix equal quantities of the precipitate and the substance to be examined and determine the melting point of the mixture. The melting point is at least 20 °C lower than that of the precipitate (Appendix 6.7). D. To 5 ml of solution S add 5 ml of water. The solution gives reaction A of chlorides (Appendix 8.1). Appearance of solution Solution S: Dissolve 0.5 g of the substance to be examined in carbon dioxide-free water R and dilute to 25 ml with the same solvent, and mix. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). Acidity or alkalinity To 10 ml of solution S add 0.2 ml of methyl red solution R and 0.2 ml of 0.01 N sodium hydroxide VS. The solution is yellow. Add 0.3 ml of 0.01 N hydrochloric acid VS. The solution is red. Impurity B Not more than 10 ppm if intended for non-parenteral use. Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Mix equal volumes of a 4.2% solution of sodium perchlorate R and of a 1.16% solution of phosphoric acid R, adjust to pH 2.0 with triethylamine, and mix. Mobile phase B: Acetonitrile R. Test solution (1): Dissolve 0.100 g of the substance to be examined in a mixture of acetonitrile R and water (20 : 80) and dilute to 25.0 ml with the same mixture of solvents, and mix. Test solution (2): Dissolve 0.125 g of the substance to be examined in a mixture of acetonitrile R and water (20 : 80) and dilute to 10.0 ml with the same mixture of solvents, and mix. Reference solution (1): Dilute 0.5 ml of test solution (1) to 100.0 ml with a mixture of acetonitrile R and water (20 : 80), and mix. 747

VP V

PHENOBARBITAL

Reference solution (2): Dissolve 10.0 mg of impurity A (1-methyl-4-phenylpiperidine) RS in a mixture of acetonitrile R and water (20 : 80) and dilute to 100.0 ml with the same mixture of solvents, and mix. Reference solution (3): Dissolve 12.5 mg of impurity B (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) RS in a mixture of acetonitrile R and water (20 : 80) and dilute to 10.0 ml with the same mixture of solvents, and mix. Dilute 1.0 ml of the solution to 100.0 ml with a mixture of acetonitrile R and water (20 : 80), and mix. Reference solution (4): Dilute 5.0 ml of reference solution (2) and 1.0 ml of reference solution (3) to 100.0 ml with a mixture of acetonitrile R and water (20 : 80), and mix. Chromatographic system: A column (25 cm × 4.0 mm) packed with spherical endcapped octadecylsilyl silica gel for chromatography (5 µm) with a specific surface area of 340 m2/g, a pore size of 10 nm and a carbon loading of 19%. Detector: A spectrophotometer set at 210 nm. Flow rate: 1.0 ml/min. Volume of injection: 50 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 15

80 → 75

20 → 25

15 - 31

75 → 55

25 → 45

31 - 40

55

45

40 - 41

55 → 80

45 → 20

41 - 50

80

20

Inject test solution (2) and reference solution (4). Relative retention: With reference to pethidine (retention time = about 24 min): impurity B = about 0.66; impurity A = about 0.68. System suitability: The test is not valid unless, in the chromatogram obtained with reference solution (4), the signal-to-noise ratio is minimum 10 for the first peak and the peak-to-valley ratio is minimum 4, where Hp = height above the baseline of the peak due to impurity B, and Hv = height above the baseline of the lowest point of the curve separating this peak from the peak due to impurity A. Limits: In the chromatogram obtained with test solution (2), the area of the impurity B peak is not greater than the area of the corresponding peak in the chromatogram obtained with reference solution (4).

Related substances Examine by liquid chromatography (Appendix 5.3) as described in the test for impurity B with the following modifications: Volume of injection: 20 µl. Inject test solution (1) and reference solution (1). 748

Limits: In the chromatogram obtained with test solution (1): the area of any secondary peak is not greater than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). The sum of the areas of all the secondary peaks is not greater than 2 times the area of the principal peak in the chromatogram obtained with reference solution (1) (1.0%). Disregard any peak with an area less than 0.1 times that of the principal peak in the chromatogram obtained with reference solution (1) (0.05%).

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 100 °C - 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.220 g of the substance to be examined in 50 ml of ethanol (96%) R. Add 5.0 ml of 0.01 N hydrochloric acid VS. Titrate with 0.1 N sodium hydroxide VS, determining the end-point potentiometrically (Appendix 10.2). Read the volume added between the 2 points of inflexion. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 28.38 mg of C15H21NO2. HCl. Storage Store in an airtight container, protected from light. Action and use Opoid analgesic. PHENOBARBITAL Phenobarbitalum H 3C

O

H N

O NH

O

C12H12N2O3  

M. 232.2

Phenobarbitalis5-ethyl-5-phenylpyrimidine-2,4,6(1H,3H,5H)trione. It contains not less than 99.0% and not more than 101.0% of C12H12N2O3, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder or colourless crystals. Very slightly soluble in water, freely soluble in ethanol (96%). It forms water-soluble compounds with alkali hydroxides, carbonates and ammonia.

PHENOBARBITAL

VP V

Identification Apply one of the two following identifications: First identification: A, B. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of phenobarbital RS. B. Determine the melting point (Appendix 4.2) of the substance to be examined and a mix equal parts of the substance to be examined and phenobarbital RS. The difference between the melting points (which are about 176 °C) is not greater than 2 °C. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Ammonia - ethanol (96%) - methylene chloride (5 : 15 : 80); use the lower layer. Test solution: Dissolve 10 mg of the substance to be examined in ethanol (96%) R and dilute to 10.0 ml with the same solvent. Reference solution: Dissolve 10 mg of phenobarbital RS in ethanol (96%) R and dilute to 10.0 ml with the same solvent. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of two thirds of the plate. Examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. D. It gives the reaction of non-nitrogen substituted barbiturates (Appendix 8.1). Appearance of solution Dissolve 1.0 g in a mixture of 4 ml of 2 M sodium hydroxide R and 6 ml of water. The solution is clear (Appendix 9.2) and not more intensely coloured than reference solution Y6 (Appendix 9.3, method 2). Acidity Boil 1.0 g of the substance to be examined with 50 ml of water for 2 min, allow to cool and filter. Add 0.15 ml of methyl red solution R to 10 ml of the filtrate. The solution is orange-yellow. Not more than 0.1 ml of 0.1 N sodium hydroxide VS is required to produce a pure yellow colour. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 6.60 g of sodium acetate R in 900 ml of water, add 3 ml of glacial acetic acid R, adjust to pH 4.5 with glacial acetic acid R and dilute to 1000 ml with water R. Mix 60 volumes of this solution with 40 volumes of methanol R. Test solution: Dissolve 0.125 g of the substance to be examined in 5.0 ml of methanol R and dilute to 25.0 ml with the mobile phase. Reference solution (1): Mix 1.0 ml of the test solution and 20.0 ml of methanol R and dilute to 100.0 ml with the

mobile phase. Mix 1.0 ml of this solution with 2.0 ml of methanol R and dilute to 10.0 ml with the mobile phase. Reference solution (2): Dissolve 5.0 mg of phenobarbital impurity A RS and 5.0 mg of phenobarbital impurity B RS in 2.0 ml of methanol R and dilute to 10.0 ml with the mobile phase. Mix 1.0 ml of this solution with 20.0 ml of methanol R and dilute to 100.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: The run time is 2.1 times the retention time of phenobarbital. Identification of impurities: Use the chromatogram obtained with reference solution (2) to identify the peaks due to impurities A and B. Relative retention with reference to phenobarbital (retention time = about 14 min): impurity A = about 0.2; impurity B = about 0.3. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurities A and B is at least 1.5. Limits: Impurity A: The area of the peak due to impurity A is not more than 1.5 times the area of the corresponding peak in the chromatogram obtained with reference solution (2) (0.15%). Impurity B: The area of the peak due to impurity B is not more than 1.5 times the area of the corresponding peak in the chromatogram obtained with reference solution (2) (0.15%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). The sum of the peak areas of all impurities is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: ImpurityA: (5RS)-5-ethyl-2,6-diimino-5-phenyltetrahydropyrimidin4(1H)-one. Impurity B: (5RS)-5-ethyl-6-imino-5-phenyldihydropyrimidine2,4(1H,3H)-dione. Impurity C: 5-methyl-5-phenylpyrimidine-2,4,6(1H,3H,5H)-trione.

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. 749

VP V

PHENOBARBITAL TABLETS

Assay Dissolve 0.200 g of the substance to be examined in 40 ml of ethanol (96%) R and add 20 ml of water. Titrate with 0.1 N sodium hydroxide VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N sodium hydroxide VS is equivalent to 23.22 mg of C12H12N2O3. Storage Store in an airtight container. Action and use Barbiturate. Preparations Elixir, tablets. PHENOBARBITAL TABLETS Tabellae Phenobarbitali Gardenal, Luminal tablets Phenobarbital tablets contain phenobarbital. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of phenobarbital, C12H12N2O3, 90.0% to 110.0% of the stated amount. Identification A. Shake a quantity of the powdered tablets containing the equivalent of 60 mg of phenobarbital with 50 ml of chloroform R and filter. Evaporate the filtrate to dryness and dry at 105 °C for 2 hours. The infrared absorption spectrum (Appendix 4.2) of the residue is concordant with the spectrum of phenobarbital RS. B. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the peak due to phenobarbital in the chromatogram obtained with the reference solution. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of water. Rotation speed: 50 rpm. Time: 45 min. Procedure: After the specified time, withdraw a sample of the medium, filter and discard the first 20 ml of the filtrate. Dilute the filtrate with borate buffer solution pH 9.6 R to obtain a suitable concentration. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum at about 240 nm, in comparison with the reference solution of phenobarbital RS having the same concentration in the same solvent. Calculate the content of phenobarbital dissolved using the absorbances obtained and the declared content of 750

C12H12N2O3 in phenobarbital RS. Tolerance: Not less than 75% (Q) of the stated amount of phenobarbital, C12H12N2O3, is dissolved in 45 min.

Assay Examine by liquid chromatography (Appendix 5.3). Buffer solution pH 4.5: Dissolve 6.6 g of sodium acetate R and 3.0 ml glacial acid acetic R in 1000 ml of water, adjust to pH 4.5 ± 0.1 with glacial acid acetic R. Mobile phase: Buffer solution pH 4.5 - methanol (3 : 2). Internal standard solution: Dissolve caffeine RS in a mixture of equal volumes of methanol R and buffer solution pH 4.5 to obtain a solution containing about 125 µg per ml. Reference solution: Dissolve about 20 mg of phenobarbital RS, accurately weighed, in 15.0 ml of the internal standard solution. Sonicate if necessary, filter. Test solution: Weigh 20 tablets, calculate the average mass and powder finely. To an accurately weighed quantity of the powdered tablets equivalent to about 20 mg of phenobarbital, add 15.0 ml of the internal standard solution. Sonicate for 15 minutes and filter. Chromatographic system: A column (25 cm × 4 mm) packed with stationary phase C (3 to 10 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 2.0 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject the reference solution; the resolution factor between phenobarbital and internal standard peak is not less than 1.2; the symmetry factors of phenobarbital and internal standard peak are not more than 2.0; the relative standard deviation for replicate injections is not more than 2.0%; the relative retention times are about 0.6 for caffeine and 1.0 for phenobarbital; adjust the ratio of the components of the mobile phase if necessary. Inject alternately the reference solution and the test solution. Calculate the content of phenobarbital, C12H12N2O3, in the tablets using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C12H12N2O3 in phenobarbital RS. Storage Store in a well-closed container, protected from light. Action and use Sedative and anticonvulsant. Usual strength 10 mg, 50 mg, 100 mg.

PHENOXYMETHYLPENICILLIN

VP V

PHENOL Phenolum OH

C6H6O M.94.1 Phenol contains not less than 99.0% and not more than 100.5% of C6H6O.

Characters Colourless or faintly pink or faintly yellowish crystals or crystalline masses, characteristic odour, deliquescent. Soluble in water, very soluble in dichloromethane, in ethanol (96%) and in glycerol. Identification A. Dissolve 0.5 g of the substance to be examined in 2 ml of 13.5 M ammonia R. The substance dissolves completely. Dilute to about 100 ml with water, and mix. To 2 ml of the dilute solution add 0.05 ml of sodium hypochlorite solution (3% Cl) R. A blue colour develops and becomes progressively more intense. B. To 1 ml of solution S add 10 ml of water and 0.1 ml of 10.5% solution of ferric chloride R. A violet colour is produced which disappears on addition of 5 ml of 2-propanol R. C. To 1 ml of solution S add 10 ml of water and 1 ml of bromine water R. A pale-yellow precipitate is formed. Appearance of solution Solution S: Dissolve 1.0 g of the substance to be examined in water and dilute to 15 ml with the same solvent, and mix. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution B6 (Appendix 9.3, method 2). Acidity To 2 ml of solution S add 0.05 ml of methyl orange solution R. The solution is yellow. Freezing point Not less than 39.5 °C (Appendix 6.6). Residue on evaporation Not more than 0.05%. Evaporate 5.000 g of the substance to be examined to dryness on a water bath and dry the residue at 100 °C to 105 °C for 1 hour. Assay Dissolve 2.000 g of the substance to be examined in water and dilute to 1000.0 ml with the same solvent, and mix. Transfer 25.0 ml of the solution to a glass-stoppered flask, add 50.0 ml of 0.1 N bromine VS and 5 ml of hydrochloric acid R, close the flask, allow to stand with occasional

swirling for 30 minutes and then allow to stand for a further 15 minutes. Add 5 ml of a 20% solution of potassium iodide R, shake and titrate with 0.1 N sodium thiosulphate until a faint yellow colour remains. Add 0.5 ml of starch solution R and 10 ml of chloroform R and continue the titration with vigorous shaking. Carry out a blank titration. 1 ml of 0.1 N bromine VS is equivalent to 1.569 mg of C6H6O.

Storage Store in an airtight container, protected from light. Action and use Antiseptic, antimicrobial preservative, antipruritic. PHENOXYMETHYLPENICILLIN Phenoxymethylpenicillinum

C16H18N2O5S M.350.4 Phenoxymethylpenicillin is (2S,5R,6R)-3,3-dimethyl-7oxo-6-[(phenoxyacetyl)amino]-4-thia-1-azabicyclo[3.2.0] heptane-2-carboxylic acid, a substance produced by the growth of certain strains of Penicillium notatum or related organisms on a culture medium containing an appropriate precursor, or obtained by any other means. The sum of the percentage contents of phenoxymethylpenicillin and 4-hydroxyphenoxymethylpenicillin is not less than 95.0% and not more than 100.5%, calculated with reference to the anhydrous substance.

Characters A white or almost white, crystalline powder, slightly hygroscopic. Very slightly soluble in water, soluble in ethanol (96%). Identification Apply one of the two following identifications: First identification: A. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of phenoxymethylpenicillin RS. B. It complies with the test for pH. C. Examine by thin-layer chromatography as described under Identification for penicillins (Appendix 8.2). D. It gives reaction B described under Colour reactions for penicillin and cephalosporins (Appendix 8.3). pH Suspend 50 mg in 10 ml of carbon dioxide-free water R. The pH of the suspension is 2.4 to 4.0. (Appendix 6.2). 751

VP V

PHENOXYMETHYLPENICILLIN

Specific optical rotation +186° to +200°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.250 g of the substance to be examined in butanol R and dilute to 25.0 ml with the same solvent, and mix. Related substances Not more than 1.0%. Examine by liquid chromatography (Appendix 5.3). Chromatographic system, solvent mixture, reference solutions: Prepare and proceed as described under Assay. Test solution: Dissolve 80.0 mg of the substance to be examined in the solvent mixture and dilute to 20.0 ml with the same mixture. Prepare immediately before use. Procedure: Inject reference solution (4) and elute isocratically with the chosen mobile phase until elution of the phenoxymethylpenicillin peak. Adjust the sensitivity of the system to obtain a peak with a signal-to-noise ratio of at least 3. Inject reference solution (5). Inject the test solution and start the elution isocratically. Immediately after elution of the phenoxymethylpenicillin peak start the following linear gradient. Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

Comment

0 → 20

60 → 0

40 → 100

Linear gradient

100

Isocratic

20 → 35

0

35 → 50

0 → 60

100 → 40 Re-equilibration

Inject the solvent mixture and use the same elution pattern to obtain a blank. Limits: In the chromatogram obtained with the test solution, the area of any peak, apart from the principal peak and any peak corresponding to 4-hydroxyphenoxymethylpenicillin, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (5).

4-Hydroxyphenoxymethylpenicillin Not more than 4.0%, calculated with reference to the anhydrous substance. Examine by liquid chromatography (Appendix 5.3), as described under Assay. Water Not more than 0.5% (Appendix 10.3). Determined on 1.000 g of the substance to be examined. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Phosphate buffer solution pH 3.5 methanol - water (10 : 30 : 60). Mobile phase B: Phosphate buffer solution pH 3.5 - water - methanol (10 : 35 : 55). Solvent mixture: To 250 ml of 0.2 M potassium dihydrogen phosphate R add 500 ml of water and adjust to pH 6.5 with 752

a 0.84% solution of sodium hydroxide R. Dilute to 1000 ml with water, and mix. Test solution: Dissolve 50.0 mg of the substance to be examined in the solvent mixture and dilute to 50.0 ml with the same mixture, and mix. Reference solution (1): Dissolve 55.0 mg of phenoxymethylpenicillin potassium RS in the solvent mixture and dilute to 50.0 ml with the same mixture, and mix. Reference solution (2): Dissolve 4.0 mg of 4-hydroxyphenoxymethylpenicillin RS in the solvent mixture and dilute to 10.0 ml with the same mixture, and mix. Dilute 5.0 ml of the solution to 100.0 ml with the solvent mixture, and mix. Reference solution (3): Dissolve 10 mg of phenoxymethylpenicillin potassium RS and 10 mg of benzylpenicillin sodium RS in the solvent mixture and dilute to 50 ml with the same mixture, and mix. Reference solution (4): Dilute 1.0 ml of reference solution (1) to 20 ml with the solvent mixture, and mix. Dilute 1.0 ml of the solution to 50 ml with the solvent mixture, and mix. Reference solution (5): Dilute 1.0 ml of reference solution (1) to 25.0 ml with the solvent mixture. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Equilibrate the column with a mobile phase ratio A : B of 60 : 40. Inject reference solution (3). The test is not valid unless, in the chromatogram obtained, the resolution between the 2 principal peaks is at least 6.0 (if necessary, adjust the ratio A : B of the mobile phase) and the mass distribution ratio (Dm) for the second peak (phenoxymethylpenicillin) is 5.0 to 7.0. Inject reference solution (1) 6 times. The test is not valid unless the relative standard deviation for the area of the principal peak is not more than 1.0%. Inject alternately the test solution and reference solutions (1) and (2). Calculate the percentage content of phenoxymethylpenicillin by multiplying the percentage content of phenoxymethylpenicillin potassium by 0.902. Calculate the percentage content of 4-hydroxyphenoxy-methylpenicillin by multiplying, if necessary, by the correction factor supplied with the reference subtance.

Storage Store in an airtight container. Action and use Penicillin antibacterial. Preparation Tablets.

VP V

PENICILLIN V TABLETS Tabellae Phenoxymethylpenicillini Penicillin V tablets contain phenoxymethylpenicillin. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of phenoxymethylpenicillin, C16H18N2O5S, 90.0% to 120.0% of the stated amount. Identification A. Transfer a quantity of the powdered tablets containing the equivalent of 80 mg of phenoxymethylpenicillin to a 250 ml volumetric flask, add 5 ml of methanol R, shake well to dissolve and dilute to volume with water, mix and filter. The ultraviolet absorption spectrum (Appendix 4.1) of the filtrate exhibits absorption maxima at 268 nm and 274 nm and a minimum at 272 nm. B. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the peak due to phenoxymethylpenicillin in the chromatogram obtained with the reference solution. Water Not more than 3.0% (Appendix 10.3). Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of water. Rotation speed: 50 rpm. Time: 45 min. Procedure: After the specified time, withdraw a sample of the medium and filter, discard the first 20 ml of the filtrate. Dilute a portion of the filtrate with the medium to obtain a solution containing a suitable concentration (if necessary). Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum at 268 nm, in a 1 cm cell, using the medium as a blank, in comparison with the reference solution of phenoxymethylpenicillin potassium RS having the same concentration in the same medium. Calculate the content of phenoxymethylpenicillin, C16H18N2O5S, dissolved using the absorbances obtained and the declared content of C16H18N2O5S in phenoxymethylpenicillin potassium RS. Tolerance: Not less than 75% (Q) of the stated amount of phenoxymethylpenicillin, C16H18N2O5S, is dissolved in 45 min. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Water - acetonitrile - glacial acetic acid (650 : 350 : 5.75). Resolution solution: A solution in the mobile phase contains 2.5 mg of benzylpenicillin potassium and 2.5 mg of phenoxymethylpenicillin potassium per ml.

PHENOXYMETHYLPENICILLIN POTASSIUM

Reference solution: A solution in the mobile phase contains 2.5 mg of phenoxymethylpenicillin potassium RS per ml. Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Dissolve a quantity of the powdered tablets equivalent to about 400 000 IU of phenoxymethylpenicillin, accurately weighed, in sufficient mobile phase to produce 100.0 ml. Shake for 5 minutes and filter. Chromatographic system: A column (30 cm × 4 mm) packed with stationary phase C (3 to 10 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: Inject the resolution solution; the relative retention times are about 0.8 for benzylpenicillin and 1.0 for phenoxymethylpenicillin; the column efficiency determined on the phenoxymethylpenicillin peak is not less than 1800 theoretical plates; the resolution factor between benzylpenicillin and phenoxymethylpenicillin peak is not less than 3.0. Inject the reference solution, the relative standard deviation of phenoxymethylpenicillin peak areas for 6 replicate injections is not more than 1.0%. Adjust the ratio of the components of the mobile phase if necessary. Inject alternately the reference solution and the test solution. Calculate the content of phenoxymethylpenicillin, C16H18N2O5S, in the tablets using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C16H18N2O5S in phenoxymethylpenicillin potassium RS.

Storage Store protected from humidity and light, at a temperature not exceeding 30 °C. Action and use Penicillin antibiotic. Usual strength 200 000 IU; 400 000 IU. PHENOXYMETHYLPENICILLIN POTASSIUM Phenoxymethylpenicillinum kalicum

C16H17KN2O5S M.388.5

753

VP V

PHENOXYMETHYLPENICILLIN POTASSIUM

Phenoxymethylpenicillin potassium is the potassium salt of (2S,5R,6R)-3,3-dimethyl-7-oxo-6-[(phenoxyacetyl) amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid, a substance produced by the growth of certain strains of Penicillium notatum or related organisms on a culture medium containing an appropriate precursor, or obtained by any other means. The sum of the percentage contents of phenoxymethylpenicillin potassium and 4-hydroxyphenoxymethylpenicillin potassium is not less than 95.0% and not more than 100.5%, calculated with reference to the anhydrous substance.

Characters A white or almost white, crystalline powder. Freely soluble in water, practically insoluble in ethanol (96%). Identification Apply one of the two following identifications: First identification: A, D Second identification: B, C, D A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of phenoxymethylpenicillin potassium RS. B. Examine by thin-layer chromatography as described under Identification for penicillins (Appendix 8.2). C. It gives reaction B described under Colour reactions for penicillin and cephalosporins (Appendix 8.3). D. It gives reaction A of potassium (Appendix 8.1). pH 5.5 to 7.5 (Appendix 6.2). Dissolve 100 mg of the substance to be examined in carbon dioxide-free water R and dilute to 20 ml with the same solvent, and mix. Specific optical rotation +215° to +230°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.250 g of the substance to be examined in carbon dioxide-free water R and dilute to 25.0 ml with the same solvent, and mix. Related substances Not more than 1.0%. Examine by liquid chromatography (Appendix 5.3), Chromatography system, solvent mixture and reference solutions: Prepare and proceed as described under Assay. Test solution: Dissolve 80.0 mg of the substance to be examined in the solvent mixture and dilute to 20.0 ml with the same mixture. Prepare immediately before use. Procedure: Inject reference solution (4) and elute isocratically with the chosen mobile phase until elution of the phenoxymethylpenicillin peak. Adjust the sensitivity of the system to obtain a peak with a signal-to-noise ratio of at least 3. Inject reference solution (5). Inject the test solution and start the elution isocratically. Immediately 754

after elution of the phenoxymethylpenicillin peak, start the following linear gradient: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

Comment

0 → 20

60 → 0

40 → 100

Linear gradient

20 → 35

0

100

Isocratic

35 → 50

0 → 60

100 → 40

Re-equilibration

Inject the solvent mixture and use the same elution pattern to obtain a blank. Limits: In the chromatogram obtained with the test solution, the area of any peak, apart from the principal peak and any peak corresponding to 4-hydroxyphenoxymethylpenicillin, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (5).

4-Hydroxyphenoxymethylpenicillin potassium Not more than 4.0%, calculated with reference to the anhydrous substance. Examine by liquid chromatography (Appendix 5.3), as described under Assay. Water Not more than 1.0% (Appendix 10.3). Determined on 1.000 g of the substance to be examined. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Phosphate buffer solution pH 3.5 methanol - water (10 : 30 : 60). Mobile phase B: Phosphate buffer solution pH 3.5 - water - methanol (10 : 35 : 55). Solvent mixture: To 250 ml of 0.2 M potassium dihydrogen phosphate R add 500 ml of water and adjust to pH 6.5 with a 0.84% solution of sodium hydroxide R. Dilute to 1000 ml with water, and mix. Test solution: Dissolve 50.0 mg of the substance to be examined in the solvent mixture and dilute to 50.0 ml with the same mixture, and mix. Reference solution (1): Dissolve 50.0 mg of phenoxymethylpenicillin potassium RS in the solvent mixture and dilute to 50.0 ml with the same mixture, and mix. Reference solution (2): Dissolve 4.0 mg of 4-hydroxyphenoxymethylpenicillin RS in the solvent mixture and dilute to 10.0 ml with the same mixture, and mix. Dilute 5.0 ml of the solution to 100.0 ml with the solvent mixture, and mix. Reference solution (3): Dissolve 10 mg of phenoxymethylpenicillin potassium RS and 10 mg of benzylpenicillin sodium RS in the solvent mixture and dilute to 50 ml with the same mixture, and mix. Reference solution (4): Dilute 1.0 ml of reference solution (1) to 20 ml with the solvent mixture, and mix. Dilute 1.0 ml of the solution to 50 ml with the solvent mixture, and mix.

VP V

Reference solution (5): Dilute 1.0 ml of reference solution (1) to 25.0 ml with the solvent mixture. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Equilibrate the column with a mobile phase ratio A : B of 60 : 40. System suitability: Inject reference solution (3). The test is not valid unless, in the chromatogram obtained, the resolution between the 2 principal peaks is at least 6.0 (if necessary, adjust the ratio A : B of the mobile phase) and the mass distribution ratio (Dm) for the second peak (phenoxymethylpenicillin) is 5.0 to 7.0. Inject reference solution (1) 6 times. The test is not valid unless the relative standard deviation for the area of the principal peak is not more than 1.0%. Inject alternately the test solution and reference solutions (1) and (2). Calculate the percentage content of phenoxymethylpenicillin potassium. Calculate the percentage content of 4-hydroxyphenoxymethylpenicillin potassium by multiplying if necessary, by the correction factor supplied with the reference substance.

Storage Store in an airtight container. Action and use Penicillin antibacterial. Preparations Tablets, oral powder for suspensions. PENICILLIN V POTASSIUM TABLETS Tabellae Phenoxymethylpenicillini Kalii Penicillin V potassium tablets contain phenoxymethylpenicillin potassium. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of phenoxymethylpenicillin, C16H18N2O5S, 90.0% to 120.0% of the stated amount. Identification A. Transfer a quantity of the powdered tablets containing the equivalent of 80 mg of phenoxymethylpenicillin to a 250 ml volumetric flask, add about 200 ml of water, shake and dilute to volume with water and filter. The ultraviolet absorption spectrum (Appendix 4.1) of the resulting

PENICILLIN V POTASSIUM TABLETS

solution exhibits absorption maxima at 268 nm and 274 nm and a minimum at 272 nm. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution conrresponds to that of phenoxymethylpenicillin peak in the chromatogram obtained with the reference solution. C. Ignite 0.5 g of the powdered tablets until an almost white residue is obtained. Allow to cool and add 5 ml of 2 M acetic acid R, boil, cool and filter. The filtrate yields reaction B characteristic of potassium (Appendix 8.1).

Loss on drying Not more than 1.5% (Appendix 9.6). Determined on 0.5 g of the powder finely by drying under vacuum 60 °C for 3 hours. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of phosphate buffer pH 6.0 R. Rotation speed: 50 rpm. Time: 45 min. Procedure: After the specified time, withdraw a sample of the medium, filter, discard the first 20 ml of the filtrate. Dilute the filtrate with the medium to obtain a solution containing a suitable concentration (if necessary). Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum at 268 nm, in a 1 cm cell, using the medium as a blank, in comparison with a reference solution of phenoxymethylpenicillin potassium RS having the same concentration in the same medium. Calculate the content of phenoxymethylpenicillin, C16H18N2O5S, dissolved using the absorbances obtained and the declared content of C16H18N2O5S in phenoxymethylpenicillin potassium RS. Tolerance: Not less than 75% (Q) of the stated amount of phenoxymethylpenicillin, C16H18N2O5S, is dissolved in 45 minutes. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Water - acetonitrile - glacial acetic acid (650 : 350 : 5.75). Resolution solution: A solution in the mobile phase contains 2.5 mg of benzylpenicillin potassium and 2.5 mg of phenoxymethylpenicillin potassium per ml. Reference solution: A solution in the mobile phase contains 2.5 mg of phenoxymethylpenicillin potassium RS per ml. Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Dissolve a quantity of the powdered tablets equivalent to about 400 000 IU of phenoxymethylpenicillin, accurately weighed, in sufficient mobile phase to produce 100.0 ml. Shake for 5 min and filter. Chromatographic system: A column (30 cm × 4 mm) packed with stationary phase C (3 to 10 µm). Detector: A spectrophotometer set at 254 nm. 755

PHENYLPROPANOLAMINE HYDROCHLORIDE

Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: Inject the resolution solution; the relative retention times are about 0.8 for benzylpenicillin and 1.0 for phenoxymethylpenicillin; the column efficiency determined on the phenoxymethylpenicillin peak is not less than 1800 theoretical plates; the resolution factor between benzylpenicillin and phenoxymethylpenicillin peak is not less than 3.0. Adjust the ratio of the components of the mobile phase if necessary. Inject the reference solution, the relative standard deviation of phenoxymethylpenicillin peak areas for 6 replicate injections is not more than 1.0%. Inject alternately the reference solution and the test solution. Calculate the content of phenoxymethylpenicillin, C16H18N2O5S, in the tablets using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C16H18N2O5S in phenoxymethylpenicillin potassium RS.

Storage Store protected from humidity and light, at a temperature not exceeding 30 °C. Action and use Penicillin antibacterial. Usual strength 200 000 IU; 400 000 IU. PHENYLPROPANOLAMINE HYDROCHLORIDE Phenylpropanolamini hydrochloridum

and enantiomer , HCl

C9H13NO,HCl M.187.7 Phenylpropanolamine hydrochloride is (1RS,2SR)-2amino-1-phenylpropan-1-ol hydrochloride. It contains not less than 99.0% and not more than 101.5% of C9H13NO, HCl, calculated with reference to the dried substance.

Characters A white or almost white, crystalline powder. Freely soluble in water and in ethanol (96%), practically insoluble in dichloromethane. Identification Apply one of the two following identifications: First identification: A, E. Second identification: B, C, D, E. 756

VP V

A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of phenylpropanolamine hydrochloride RS. Examine the substances prepared as disc without recrystallisation. B. Melting point: 194 °C to 197 °C (Appendix 6.7). C. Examine the chromatograms obtained in the test for related substances. The principal spot in the chromatogram obtained with test solution (2) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). D. Dissolve 50 mg of the substance to be examined in 5 ml of water, add 0.2 ml of a 12.5% solution of copper sulphate R and 0.3 ml of dilute sodium hydroxide solution R. A violet colour develops. Add 2 ml of ether R and shake. A violet precipitate is formed between the two layers. E. It gives reaction A of chlorides (Appendix 8.1).

Appearance of solution Solution S: Dissolve 1.25 g of the substance to be examined in water and dilute to 25 ml with the same solvent, and mix. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). Acidity or alkalinity To 10 ml of solution S add 0.1 ml of methyl red solution R and 0.2 ml of 0.01 N sodium hydroxide VS. The solution is yellow. Add 0.4 ml of 0.01 N hydrochloric acid VS. The solution is red. Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel H. Mobile phase: Ammonia - ethanol (96%) - butanol (6 : 24 : 70). Test solution (1): Dissolve 0.20 g of the substance to be examined in ethanol (96%) R and dilute to 10 ml with the same solvent, and mix. Test solution (2): Dilute 1 ml of test solution (1) to 10 ml with ethanol (96%) R, and mix. Reference solution (1): Dissolve 20 mg of phenylpropanolamine hydrochloride RS in ethanol (96%) R and dilute to 10 ml with the same solvent, and mix. Reference solution (2): Dilute 1 ml of reference solution (1) to 10 ml with ethanol (96%) R, and mix. Reference solution (3): Dissolve 20 mg of norpseudoephedrine hydrochloride RS in ethanol (96%) R, add 1 ml of test solution (1) and dilute to 10 ml with ethanol (96%) R, and mix. Reference solution (4): Dissolve 60 mg of ammonium chloride R in methanol R and dilute to 10 ml with the same solvent, and mix. Procedure: Before applying the solutions, spray the plate with a 2% solution of disodium tetraborate R, using 8 ml for a plate 100 mm by 200 mm and dry in a stream of cold air for 30 minutes. Apply separately to the plate as bands about 10 mm by 3 mm 10 µl of each solution. Develop over a path of 10 cm. Dry the plate in a current of warm air until

PHENYTOIN

VP V

the solvents have evaporated, allow to cool, spray with a 0.2% solution of ninhydrin R in ethanol (96%) R and heat at 110°C for 15 minutes. In the chromatogram obtained with test solution (1), any spot, apart from the principal spot and the spot corresponding to ammonium chloride, is not more intense than the spot in the chromatogram obtained with reference solution (2) (1.0%). The test is not valid unless the chromatogram obtained with reference solution (3) shows two clearly separated spots.

Phenylpropanolamine Dissolve 1.0 g of the substance to be examined in 0.01 N hydrochloric acid R and dilute to 50.0 ml with the same acid, and mix. The absorbance (Appendix 4.1) of the solution measured at 283 nm is not greater than 0.10. Heavy metals Not more than 20 ppm (Appendix 9.4). 12 ml of solution S complies with the limit test for heavy metals, method 1. Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 100 °C - 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.1500 g of the substance to be examined in a mixture of 5 ml of 0.01 N hydrochloric acid VS and 50 ml of ethanol (96%) R. Carry out a potentiometric titration (Appendix 10.2), using 0.1 N sodium hydroxide VS. Read the volume added between the two points of inflexion. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 18.77 mg of C9H13NO, HCl. Storage Store in an airtight container, protected from light. Action and use Sympathomimetic. Preparations Tablets or capsules (in combination with other active ingredients).

PHENYTOIN Phenytoinum

H N

O NH

O C15H12N2O2  

M. 252.3  

Phenytoin is 5,5-diphenylimidazolidine-2,4-dione. It contains not less than 99.0% and not more than 101.0% of C15H12N2O2, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder. Practically insoluble in water, sparingly soluble in ethanol (96%), very slightly soluble in methylene chloride. It dissolves in dilute solutions of alkali hydroxides. Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of phenytoin RS. Appearance of solution Dissolve 1.0 g of the substance to be examined in a mixture of 5 ml of 1 M sodium hydroxide R and 20 ml of water. Solution is clear (Appendix 9.2) and not more intensely coloured than reference solution BY6 (Appendix 9.3, method 2). Acidity or alkalinity Add 45 ml of water to 1.0 g of the substance to be examined and boil for 2 min. Allow to cool and filter. Wash the filter with carbon dioxide-free water R and dilute the combined filtrate and washings to 50 ml with carbon dioxide-free water R. To 10 ml of the solution add 0.15 ml of methyl red solution R. Not more than 0.5 ml of 0.01 N hydrochloric acid VS is required to change the colour of the indicator to red. To 10 ml of the solution add 0.15 ml of bromothymol blue solution R. Not more than 0.5 ml of 0.01 N sodium hydroxide VS is required to change the colour of the indicator to blue. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol R2 - acetonitrile R1 - a 0.575% solution of ammonium dihydrogen phosphate R adjusted to pH 2.5 with phosphoric acid R (20 : 35 : 45). Test solution: Dissolve 50 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. 757

VP V

PHENYTOIN TABLETS

Reference solution (2): Dissolve 2 mg of 2,2-diphenylglycine R (impurity C) in 100.0 ml of the mobile phase. Reference solution (3): Dissolve 10 mg of phenytoin for system suitability RS (containing impurities D and E) in the mobile phase, add 1.0 ml of reference solution (2) and dilute to 10.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 220 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: Inject the test solution, the reference solutions (1) and (3). The run time is 4 times the retention time of phenytoin. Identification of impurities: Use the chromatogram supplied with phenytoin for system suitability RS and the chromatogram obtained with reference solution (3) to identify the peaks due to impurities C, D and E. Relative retention with reference to phenytoin (retention time = about 4 min): impurity C = about 0.5; impurity D = about 0.6; impurity E = about 0.8. System suitability: In the chromatogram obtained with reference solution (3), the resolution between the peaks due to impurities D and E is at least 3.5. Limits: Correction factors: for the calculation of content, multiply the peaks areas of the following impurities by the corresponding correction factor: impurity D = 1.7; impurity E = 1.4; Impurity E: The corrected area of the peak due to impurity E is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.3%). Impurity C: The area of the peak due to impurity C is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%). Impurity D: The corrected area of the peak due to impurity D is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). The sum of the peak areas of all impurities is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A: diphenylmethanone (benzophenone). Impurity B: diphenylethanedione (benzil). Impurity C: amino(diphenyl)acetic acid (2,2-diphenylglycine). Impurity D: 3a,6a-diphenyltetrahydroimidazo[4,5-d]imidazole2,5(1H,3H)-dione.

758

Impurity E: (carbamoylamino)(diphenyl)acetic acid. Impurity F: 5-(4-methylphenyl)-5-phenylimidazolidine-2,4-dione.

Heavy metals Not more than 10 ppm (Appendix 9.4.8). 2.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.200 g of the substance to be examined in 50 ml of dimethylformamide R. Titrate with 0.1 M sodium methoxide VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 M sodium methoxide VS is equivalent to 25.23 mg of C15H12N2O2. Storage Store in an airtight container. Action and use Antiepileptic. Preparation Cream, ointment. PHENYTOIN TABLETS Tabellae Phenytoini Phenytoin tablets contain phenytoin sodium. They are tablets or film-coated tablets. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of phenytoin sodium, C15H11N2NaO2, 90.0% to 110.0% of the stated amount. Identification A. To a quantity of the powdered tablets containing about 0.5 g of phenytoin sodium add 10 ml of water R, shake for 10 minutes, filter (solution A). To 2 ml of solution A, add a few drops of a 5% solution of mercury (II) chloride R, a white precipitate is produced and not dissolved in a 10% solution of ammonia R. B. Evaporate 0.5 ml of solution A to dryness. The residue gives the flame reaction of sodium (Appendix 8.1). C. In the Related substances, the principal spot in the chromatogram obtained with test solution (2) is similar

PHTHALYLSULFATHIAZOLE

VP V

in position, colour and size to the principal spot in the chromatogram obtained with reference solution (3).

Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Propan-2-ol - chloroform - 13.5 M ammonia (45 : 45 : 10). Test solution (1): Shake well a quantity of the powdered tablets containing 0.2 g of phenytoin sodium with 5 ml of methanol R, warm on a water bath with shaking, filter and use the filtrate. Test solution (2): Dilute 1 ml of test solution (1) to 10 ml with methanol R. Reference solution (1): Dilute 1 ml of test solution (2) to 20 ml with methanol R. Reference solution (2): A solution contains 0.020% of benzophenone RS in methanol R. Reference solution (3): A solution contains 0.4% of phenytoin sodium RS in methanol R. Procedure: Apply separately to the plate 10 µl of each of the above solutions. Allow the plate to dry in a current of cold air for 2 minutes. Develop over a path of 15 cm. After removal of the plate, allow it to dry at 80 °C for 5 minutes and examine under ultraviolet light at 254 nm. In the chromatogram obtained with test solution (1) any secondary spot corresponding to benzophenone is not more intense than the spot in the chromatogram obtained with reference solution (2) (0.5%) and any other secondary spot is not more intense than the spot in the chromatogram obtained with reference solution (1) (0.5%). Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 500 ml of water. Rotation speed: 100 rpm. Time: 45 min. Procedure: After the specified time, withdraw a sample of the medium, filter and discard the first 20 ml of the filtrate. Measure the absorbance (Appendix 4.1) of the filtrate at the maximum at 258 nm, in a 1 cm cell, using water as a blank, in comparison with the reference solution of phenytoin sodium RS containing accurately about 0.2 mg/ml. Calculate the content of phenytoin sodium, C15H11N2NaO2, dissolved using the absorbances obtained with the reference and test solution and the declared content of phenytoin sodium RS. Tolerance: Not less than 75% (Q) of the stated amount of phenytoin sodium is dissolved in 45 minutes. Assay Weigh 20 tablets (remove the coating in case of coated tablets), calculate the average mass and powder finely. Transfer an accurately weighed quantity of the powdered tablets containing about 0.3 g of phenytoin sodium to a separating funnel, add 25 ml of water and shake for

10 minutes. Add 50 ml of ether R and 10 drops of bromophenol blue solution R. Titrate with 0.1 N hydrochloric acid VS, shake vigorously, until the colour of the aqueous layer turns to bluish-grey. Transfer the aqueous layer to a ground-glass stoppered conical flask. Wash the ether layer with 5 ml of water and combine the washing with the aqueous layer in the conical flask. Add 20 ml of ether R and continue the titration with 0.1 N hydrochloric acid VS, shaking vigorously, until the colour of the aqueous layer turns to pale green. Each ml of 0.1 N hydrochloric acid VS is equivalent to 27.43 mg of C15H11N2NaO2.

Storage Store in a cool and dry place, protected from light. Action and use Antiepileptic. Usual strength 50 mg, 100 mg. PHTHALYLSULFATHIAZOLE Phthalylsulfathiazolum

C17H13N3O5S2

M. 403.4

Phthalylsulfathiazole is 2-[[4-(thiazol-2-ylsulfamoyl) phenyl]carbamoyl]benzoic acid. It contains not less than 98.5% and not more than 101.5% of C17H13N3O5S2, calculated with reference to the dried substance.

Characters A white or yellowish-white, crystalline powder. Practically insoluble in water, freely soluble in dimethylformamide, slightly soluble in acetone and in ethanol (96%). Identification Apply one of the two following identifications: First identification: A, B, E. Second identification: B, C, D, E. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of phthalylsulfathiazole RS. B. To 1 g of the substance to be examined add 8.5 ml of 2 M sodium hydroxide R and boil under a reflux condenser for 30 minutes. Cool and add 17.5 ml of dilute hydrochloric acid R. Shake vigorously and filter. Neutralise the filtrate with 2 M sodium hydroxide R. Filter, wash the precipitate 759

VP V

PHTHALYLSULFATHIAZOLE TABLETS

with water, recrystallise from water and dry the crystals at 100 °C to 105 °C. The crystals melt at 200 °C to 203 °C (Appendix 6.7). C. To 0.1 g of the substance to be examined in a test-tube add 3 ml of 1 M sulfuric acid R and 0.5 g of zinc powder R. Fumes are evolved which produce a black stain on lead acetate paper R. D. To 0.1 g of the substance to be examined add 0.5 g of resorcinol R and 0.3 ml of sulphuric acid R and heat on a water bath until a homogeneous mixture is obtained. Allow to cool. Add 5 ml of 2 M sodium hydroxide R, shake well. Dilute 0.1 ml of this brownish-red mixture to 25 ml with water. An intense green fluorescence appears which disappears on acidification. E. Dissolve about 10 mg of the crystals obtained in identification test B in 200 ml of 0.1 M hydrochloric acid R. 2 ml of the solution gives the reaction of primary aromatic amines (Appendix 8.1) with formation of an orange precipitate.

Appearance of solution Dissolve 1.0 g of the substance to be examined in 20 ml of 1 M sodium hydroxide R. The solution is clear (Appendix 9.2) and and not more intensely coloured than reference solution BY5 (Appendix 9.3, method 2). Acidity To 2.0 g of the substance to be examined add 20 ml of water, shake for 30 minutes and filter. To 10 ml of the filtrate add 0.1 ml of phenolphthalein solution R. Not more than 0.2 ml of 0.1 N sodium hydroxide VS is required to change the colour of the indicator. Sulfathiazole and other primary aromatic amines Dissolve 5 mg of the substance to be examined in a mixture of 3.5 ml of water, 6 ml of dilute hydrochloric acid R and 25 ml of ethanol (96%) R, previously cooled to 15 °C. Place immediately in iced water and add 1 ml of a 0.25% solution of sodium nitrite R. Allow to stand for 3 minutes, add 2.5 ml of a 4% solution of sulphamic acid R and allow to stand for 5 minutes. Add 1 ml of a 0.4% solution of naphthylethylenediamine dihydrochloride R and dilute to 50 ml with water, and mix. Measured at 550 nm the absorbance (Appendix 4.1) is not greater than that of a standard prepared at the same time and in the same manner using a mixture of 1 ml of a solution containing 10 mg of sulfathiazole RS and 0.5 ml of hydrochloric acid R in 100 ml, 2.5 ml of water, 6 ml of dilute hydrochloric acid R and 25 ml of ethanol (96%) R. Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g of the substance to be examined complies with the limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R.

760

Loss on drying Not more than 2.0% (Appendix 9.6). (1.00 g; 100 °C - 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.300 g of the substance to be examined in 40 ml of dimethylformamide R. Titrate with 0.1 N sodium hydroxide VS until the colour becomes blue, using 0.2 ml of thymolphthalein solution R as indicator. Carry out a blank titration. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 20.17 mg of C17H13N3O5S2. Storage Protected from light. Action and use Antibacterial. Preparation Tablets. PHTHALYLSULFATHIAZOLE TABLETS Tabellae Phthalylsulfathiazoli Phthalylsulfathiazole tablets contain phthalylsulfathiazole. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of phthalylsulfathiazole, C17H13N3O5S2, 95.0% to 105.0% of the stated amount. Identification Shake a quantity of the powdered tablets containing 1.5 g of phthalylsulfathiazole with 60 ml of hot acetone R, filter and evaporate the filtrate to dryness. Dry the residue at 105 °C and perform the following reactions: A. The residue yields reaction D described in the Identification under “Phthalylsulfathiazole”. B. To 1 g of the residue add 8.5 ml of 2 M sodium hydroxide R and boil under a reflux condenser for 30 minutes. Cool and add 17.5 ml of dilute hydrochloric acid R. Shake well and filter. Neutralise the filtrate with 2 M sodium hydroxide R. Filter, wash the precipitate with water, recrystallise from water and dry the crystals at 100 °C to 105 °C. Dissolve about 10 mg of the crystals in 200 ml of 0.1 M hydrochloric acid R, 2 ml of this solution gives the reaction of primary aromatic amines (Appendix 8.1) with formation of an orange precipitate. C. To 10 mg of the residue add 20 mg of phenol R and 3 drops of concentrated sulphuric acid R, heat until the mixture turns to brown. Allow to cool, add 20 ml of water and

PHYTOMENADIONE

VP V

make alkaline with 2 M sodium hydroxide R, a pink colour is produced (to distinguish from sucinylsulfathiazole).

Sulfathiazole Shake a quantity of the powdered tablets containing 7.5 mg of phthalylsulfathiazole with a mixture of 25 ml of ethanol (96%) R, 6 ml of dilute hydrochloric acid R and 3.5 ml of water, previously cooled to 15 °C. Filter and place immediately the filtrate in iced water and add 1 ml of a 0.25% solution of sodium nitrite R and mix. Allow to stand for 3 minutes, add 2.5 ml of a 4% solution of sulfamic acid R and allow to stand for 5 minutes. Add 1 ml of a 0.4% solution of N-(1-naphthyl)ethylenediamine dihydrochloride R and dilute to 50 ml with water R. The absorbance (Appendix 4.1) of the resulting solution at 550 nm is not greater than that of a standard prepared at the same time and in the same manner: Using a mixture of 25 ml of ethanol (96%) R, 2 ml of water, 6 ml of dilute hydrochloric acid R and 1.5 ml of a solution prepared by dissolving 10 mg of sulfathiazole RS and 0.5 ml of concentrated hydrochloric acid R in water to produce 100 ml. Place immediately the mixture in iced water and add 1 ml of a 0.25% solution of sodium nitrite R, mix and allow to stand for 3 minutes. Proceed as directed above, beginning at the words “add 2.5 ml of a 4% solution of sulfamic acid R …” Assay Weigh 20 tablets, calculate the average mass and powder finely. Transfer an accurately weighed quantity of the powdered tablets containing about 0.5 g of phthalylsulfathiazole to a round-bottomed flask, add 20 ml of concentrated hydrochloric acid R and 10 ml of water. Boil under a reflux condenser for 1 hour. Transfer this solution to a 200 ml beaker by using 20 ml of dilute hydrochloric acid R. Titrate with 0.1 M sodium nitrite VS, using the method under nitrite titration (Appendix 10.4). Each ml of 0.1 M sodium nitrite VS is equivalent to 40.34 mg of C17H13N3O5S2. Storage Store in a well-closed container, in a cool and dry place, protected from light. Action and use Antibacterial. Usual strength 500 mg.

PHYTOMENADIONE Phytomenadionum Vitamin K1 O CH3

H 3C H

O

CH3

CH3

CH3

C31H46O2

N

CH3

M. 450.7  

Phytomenadione is a mixture of 2-methyl-3-[(2E)-(7R,11R)3,7,11,15-tetramethylhexadec-2-enyl]naphthalene-1,4dione (trans-phytomenadione), 2-methyl-3-[(2Z)-(7R,11R)3,7,11,15-tetramethylhexadec-2-enyl]naphthalene-1,4dione(cis-phytomenadione) and 2,3-epoxy-2-methyl3-[(2E)-(7R,11R)-3,7,11,15-tetramethylhexadec2-enyl]-2,3-dihydronaphthalene-1,4-dione(transepoxyphytomenadione). It contains not more than 4.0% of trans-epoxyphytomenadione and not less than 75.0% of trans-phytomenadione. The total of the three components is not less than 97.0% and not more than 103.0%.

Characters A clear, yellow or orange viscous liquid, odourless. Freely soluble in ether, in iso-octane, in chloroform and in fatty oils. Sparingly soluble in ethanol (96%) and in methanol, practically insoluble in water. It decomposes on exposure to actinic light. The refractive index is about 1.526. Identification Carry out all operations as rapidly as possible avoiding exposure to actinic light. A. Dissolve 10.0 mg of the subtance to be examined in trimethylpentane R and dilute to 100.0 ml with the same solvent. The ultraviolet absorption spectrum (Appendix 4.1) of the solution in the range of 275 nm to 340 nm exhibits an absorption maximum at 327 nm and an absorption minimum at 285 nm. The specific absorbance at the absorption maximum is 67 to 73. Dilute 10.0 ml of the solution to 50.0 ml with trimethylpentane R. The ultraviolet absorption spectrum (Appendix 4.1) of the solution in the range of 230 nm to 280 nm exhibits four absorption maxima, at 243 nm, 249 nm, 261 nm and 270 nm. B. Examine the chromatograms obtained in the test for Menadione and other related substances. The principal spot in the chromatogram obtained with test solution (2) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). C. Dissolve 50 mg in 10 ml of methanol R and add 1 ml of a 20% solution of potassium hydroxide R in methanol R. A green colour is produced which becomes violet-red on heating in a water-bath at 40 °C and then reddish-brown on standing. 761

VP V

PHYTOMENADIONE

Clarity of solution Dissolve 2.5 g in trimethylpentane R and dilute to 25 ml with the same solvent. The solution is clear (Appendix 9.2). Acid value Not more than 2.0 (Appendix 7.2). Determined on 2.00 g. Menadione and other related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Using silica gel GF254. Mobile phase: Cyclohexane - toluene (20 : 80). Test solution (1): Dissolve 0.40 g of the substance to be examined in cyclohexane R and dilute to 10 ml with the same solvent. Test solution (2): Dilute 1 ml of test solution (1) to 10 ml with cyclohexane R. Reference solution (1): Dissolve 40 mg of phytomenadione RS in cyclohexane R and dilute to 10 ml with the same solvent. Reference solution (2): Dilute 1 ml of test solution (2) to 20 ml with cyclohexane R. Reference solution (3): Dissolve 4.0 mg of menadione R in cyclohexane R and dilute to 50 ml with the same solvent. Procedure: Apply to the plate 10 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in air for 5 min. Examine in ultraviolet light at 254 nm and spray with a 10% solution of phosphomolybdic acid R in ethanol R. Heat at 120 °C for 5 min. Examine in daylight. In the chromatogram obtained with test solution (1): any spot corresponding to menadione is not more intense than the spot in the chromatogram obtained with reference solution (3) (0.2%); any spot, apart from the principal spot and any spot corresponding to menadione, is not more intense than the spot in the chromatogram obtained with reference solution (2) (0.5%). Disregard any spot below the principal spot, which may not be completely separated from the principal spot. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by liquid chromatography (Appendix 5.3) Mobile phase: Octanol - di-isopropyl ether - heptane (0.67 : 3.3 : 1000). Test solution: Dissolve 15.0 mg of the substance to be examined in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (1): Dissolve 15.0 mg of phytomenadione RS in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (2): Dissolve 15.0 mg of phytomenadione RS and 4.0 mg of trans-epoxyphytomenadione RS in the mobile phase and dilute to 10.0 ml with the mobile phase. 762

Chromatographic system: A stainless steel column (25 cm × 4.6 mm) packed with spherical silica gel for chromatography R (5 µm) with a porosity of 8 nm. Flow rate: 0.4 ml/min. Detector: A spectrophotometer set at 254 nm. Volume of injection: 20 µl. Inject reference solution (2). When using a recorder, adjust the sensitivity of the system so that the height of the principal peak is at least 50% of the full scale of the recorder. The test is not valid unless the order of elution of the peaks is: trans-epoxyphytomenadione, cis-phytomenadione and trans-phytomenadione. Carry out six replicate injections of reference solution (1). The assay is not valid unless the relative standard deviation of the peak area of the transisomer is less than 1.0% and the resolution between the peaks corresponding to trans-phytomenadione and cisphytomenadione is at least 2.5. Inject the test solution and reference solution (1) and calculate the percentage contents of trans-phytomenadione, cis-phytomenadione and transepoxyphytomenadione using the following expressions: Trans-phytomenadione = Cis-phytomenadione =

m'× A'trans ×S trans m × S 'trans

m'× A' cis ×S cis m × S ' cis

Trans-epoxyphytomenadione =

m'× A' epoxy ×S epoxy m × S ' epoxy

m’ = Mass of the reference substance in reference solution (1), in milligrams; m = Mass of the substance to be examined in the test solution, in milligrams; A’trans = Percentage content of trans-phytomenadione in phytomenadione RS; A’cis = Percentage content of cis-phytomenadione in phytomenadione RS; A’epoxy = Percentage content of trans-epoxyphytomenadione in phytomenadione RS; Strans = Area of the peak corresponding to the trans-isomer in the chromatogram obtained with the test solution; Scis = Area of the peak corresponding to the cis-isomer in the chromatogram obtained with the test solution; Sepoxy = Area of the peak corresponding to transepoxyphytomenadione in the chromatogram obtained with the test solution; S’trans = Area of the peak corresponding to the trans-isomer in the chromatogram obtained with reference solution (1), S’cis = Area of the peak corresponding to the cis-isomer in the chromatogram obtained with reference solution (1), S’epoxy = Area of the peak corresponding to transepoxyphytomenadione in the chromatogram obtained with reference solution (1).

PILOCARPINE NITRATE

VP V

Sorage Store in an airtight container, protected from light. Action and use Vitamin K analogue. Preparations Injection, tablets. PHYTOMENADIONE TABLETS Tabellae Phytomenadioni Phytomenadione tablets contain phytomenadione. They are sublingual or chewing tablets. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of phytomenadione, C31H46O2, 90.0% to 110.0% of the stated amount. Identification Shake a quantity of the powdered tablets containing 50 mg of phytomenadione with 50 ml of ethanol R for about 1 hour, allow to stand and use the supernatant liquid. Dilute 5 ml of the supernatant liquid to 50.0 ml with ethanol R (solution A). The absorbance (Appendix 4.1) of solution A in the range 230 to 350 nm exhibits a maximum at 328 nm and a minimum at 292 nm. Dilute a suitable volume of solution A with sufficient ethanol R to produce a solution containing 0.001% of phytomenadione. The absorption (Appendix 4.1) of the resulting solution, in the range 230 to 350 nm, exhibits maxima at 245, 249, 263 and 271 nm and minima at 256 nm and 266 nm. Disintegration The requirement for Disintegration does not apply to phytomenadione tablets. Menadione Not more than 1.0%. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Methanol - ether - cyclohexane (1 : 20 : 80). Test solution: Disperse a quantity of the powdered tablets containing 50 mg of phytomenadione in 5 ml of ethanol R with the aid of ultrasound for 5 min, add 15 ml of 2,2,4-trimethylpentane R, shake for 1 min, centrifuge and use the supernatant liquid. Reference solution: The solution contains 0.0025% of menadione in 2,2,4-trimethylpentane R. Procedure: Apply separately to the plate 50 µl of each of the above solutions. Develop over a path of 15 cm. After removal of the plate, allow to dry in air. Examine under ultraviolet light at 254 nm.

Any secondary spot corresponding to menadione in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Water - ethanol (96%) (5 : 95). Reference solution: The solution contains 0.01% of phytomenadione RS in the mobile phase. Test solution: Weigh 20 tablets, remove the coating layer, if neccesary, calculate the average mass and powder finely. Add 5 ml of 0.5 M ammonia R to an accurately weighed quantity of the powdered tablets containing about 10 mg of phytomenadione. Sonicate for 5 min. Add 90 ml of ethanol (96%) R and sonicate for 10 min. Shake mechanically for 10 min, add sufficient ethanol (96%) R to produce 100.0 ml, centrifuge and use the clear supernatant layer. Chromatographic system: A steel column (20 cm × 4 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution, the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject alternately the test solution and the reference solution. Calculate the content of phytomenadione, C31H46O2, in the tablets using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C31H46O2 in phytomenadione RS. Storage Store in a well-closed container, protected from light. Action and use Vitamin K analogue. Usual strength 2 mg, 5 mg, 10 mg. PILOCARPINE NITRATE Pilocarpini nitras

C11H16N2O2,HNO3  

M. 271.3

763

PILOCARPINE NITRATE

Pilocarpine nitrate is (3S,4R)-3-ethyl-4-[(1-methyl-1Himidazol-5-yl)methyl]dihydrofuran-2(3H)-one nitrate. It contains not less than 98.5% and not more than 101.0% of C11H16N2O2,HNO3, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder or colourless crystals, sensitive to light. Freely soluble in water, sparingly soluble in ethanol (96%). Melting point: About 174 °C, with decomposition. Identification Apply one of the two following identifications: First identification: A, C, D. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of pilocarpine nitrate RS. A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: silica gel G. Mobile phase: Concentrated ammonia - methanol methylene chloride (1 : 14 : 85). Test solution: Dissolve 10 mg of the substance to be examined in water and dilute to 10 ml with the same solvent. Reference solution: Dissolve 10 mg of pilocarpine nitrate RS in water and dilute to 10 ml with the same solvent. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm. Dry the plate at 100 °C to 105 °C for 10 min and allow to cool. Spray with Dragendorff reagent R. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. B. It complies with the test for Specific optical rotation. D. It gives the reaction of nitrates (Appendix 8.1). Appearance of solution Solution S: Dissolve 2.50 g of the substance to be examined in carbon dioxide-free water R and dilute to 50.0 ml with the same solvent. Prepare immediately before use. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution Y6 (Appendix 9.3, method 2). pH 3.5 to 4.5 (Appendix 6.2). Determined on solution S. Specific optical rotation +80° to +83°, calculated with reference to the dried substance (Appendix 6.4). Determined on solution S. Related substances Examine by liquid chromatography (Appendix 5.3). 764

VP V

Mobile phase: Methanol - acetonitrile - a 0.679 g/L solution of tetrabutylammonium dihydrogen phosphate R previously adjusted to pH 7.7 with 2 M ammonia R (55 : 60 : 885). Test solution: Dissolve 0.100 g of the substance to be examined in water and dilute to 100.0 ml with the same solvent. Reference solution (1): Dilute 5.0 ml of the test solution to 100.0 ml with water. Dilute 2.0 ml of this solution to 20.0 ml with water. Reference solution (2): Dissolve 5.0 mg of pilocarpine nitrate for system suitability RS (containing impurity A) in water R and dilute to 50.0 ml with the same solvent. Reference solution (3): To 5 ml of the test solution, add 0.1 ml of ammonia R and heat the solution on a water-bath for 30 min, cool and dilute to 25 ml with water. Dilute 3 ml of this solution to 25 ml with water. Mainly pilocarpic acid (impurity B) is formed. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 µm) with a pore size of 10 nm and a carbon loading of 19%. Detector: A spectrophotometer set at 220 nm. Flow rate: 1.2 ml/min. Volume of injection: 20 µl. Procedure: The run time is twice the retention time of pilocarpine. Elution order: Impurity B, impurity C, impurity A, pilocarpine. Retention time: Pilocarpine = about 20 min. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurity A and pilocarpine is at least 1.6. Limits: Impurity A: The area of the peak due to impurity A is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (1) (1%). Sum of impurities A and B is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (1) (1.5%). Sum of impurities other than A and B is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). Disregard any peak with an area less than 0.4 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%); disregard any peak due to the nitrate ion with a relative retention with reference to pilocarpine of about 0.3. Note: Impurity A: (3R,4R)-3-ethyl-4-[(1-methyl-1H-imidazol-5-yl) methyl]dihydrofuran-2(3H)-one (isopilocarpine). Impurity B: (2S,3R)-2-ethyl-3-(hydroxymethyl)-4-(1-methyl1H-imidazol-5-yl)butanoic acid (pilocarpic acid). Impurity C: (2R,3R)-2-ethyl-3-(hydroxymethyl)-4-(1-methyl1H-imidazol-5-yl)butanoic acid (isopilocarpic acid).

PIPERACILLIN SODIUM

VP V

Chlorides Not more than 70 ppm (Appendix 9.4.5). Determined on 15 ml of solution S. Iron Not more than 10 ppm (Appendix 9.4.13). Determined on 10 ml of solution S. Prepare the standard using 5 ml of iron standard solution (1 ppm Fe) R and 5 ml of water. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.250 g of the substance to be examined in 30 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 27.13 mg of C11H16N2O2, HNO3 Storage Store in an airtight container, protected from light Action and use Cholinoceptor agonist; treatment of glaucoma. Preparation Eye drops. PIPERACILLIN SODIUM Piperacillinum natricum

C23H26N5NaO7S

M. 539.5

Piperacinllin sodium is sodium (2S,5R,6R)-6-[[(2R)-2[[(4-ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]2-phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1azabicyclo[3.2.0]heptane-2- carboxylate. It contains not less than 95.0% and not more than 102.0% of C23H26N5NaO7S, calculated with reference to the anhydrous substance. Semi-synthetic product derived from a fermentation product.

Characters White or almost white, hygroscopic powder. Freely soluble in water and in methanol, practically insoluble in ethyl acetate. Identification A. Dissolve 0.250 g of the substance to be examined in water, add 0.5 ml of dilute hydrochloric acid R and 5 ml of ethyl acetate R; stir and allow to stand for 10 min in iced water. Filter through a small sintered-glass filter (40), applying suction. Wash the crystals with 5 ml of water and 5 ml of ethyl acetate R, then dry in an oven at 60 °C for 60 min. The infrared absorption spectrum (Appendix 4.2) of crystals is concordant with the spectrum of piperacillin RS. B. It gives reaction (A) of sodium (Appendix 8.1) Appearance of solution Solution S: Dissolve 2.50 g in carbon dioxide-free water R and dilute to 25 ml with the same solvent. Solution S is clear (Appendix 9.2) and its absorbance (Appendix 4.1) at 430 nm is not greater than 0.10. pH 5.0 to 7.0 (Appendix 6.2). Determined on solution S. Specific optical rotation +175° to +190°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.250 g in water and dilute to 25.0 ml with the same solvent. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Mix 576 ml of water, 200 ml of a 3.12% solution of sodium dihydrogen phosphate R and 24 ml of a 8% solution of tetrabutylammonium hydroxide R; if necessary, adjust to pH 5.5 with dilute phosphoric acid R or dilute sodium hydroxide solution R; add 200 ml of acetonitrile R. Mobile phase B: Mix 126 ml of water, 200 ml of a 3.12% solution of sodium dihydrogen phosphate R and 24 ml of a 8% solution of tetrabutylammonium hydroxide R; if necessary, adjust to pH 5.5 with dilute phosphoric acid R or dilute sodium hydroxide solution R; add 650 ml of acetonitrile R. Solvent mixture: Acetonitrile R - a 3.12% solution of sodium dihydrogen phosphate R (25 : 75). Test solution: Prepare the solution immediately before use. Dissolve 40.0 mg of the substance to be examined in the solvent mixture and dilute to 20.0 ml with the solvent mixture. Reference solution (1): Dissolve 25.0 mg of piperacillin RS in the solvent mixture and dilute to 50.0 ml with the solvent mixture. Reference solution (2): Dilute 1.0 ml of reference solution (1) to 25.0 ml with the solvent mixture. 765

VP V

PIPERAZINE ADIPATE

Reference solution (3): Dilute 1.0 ml of reference solution (1) to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 50.0 ml with the solvent mixture. Resolution solution: Dissolve 10.0 mg of piperacillin RS and 10.0 mg of anhydrous ampicillin RS (impurity A) in the solvent mixture and dilute to 50.0 ml with the solvent mixture. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 220 nm. Flow rate: 1,0 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A Mobile phase B (% v/v) (% v/v)

0 - tR

88

12

tR - (tR + 30)

80 → 0

12 → 100

(tR + 30) - (tR + 45)

0 → 88

100 → 12

tR is the retention time of piperacillin in the chromatogram obtained with reference solution (2)

If the mobile phase composition has been adjusted to achieve the required resolution, the adjusted composition will apply at time zero in the gradient. Inject reference solutions (2), (3) and the resolution solution with isocratic elution at the initial mobile phase composition and inject the test solution according to the elution gradient described in the table. System suitability: In the chromatogram obtained with resolution solution, resolution between the peaks due to impurity A and piperacillin is at least 10. If necessary, adjust the ratio mobile phase A : mobile phase B. The mass distribution ratio for the peak due to piperacillin is 2.0 to 3.0. In the chromatogram obtained with reference solution (3), the signal-to-noise ratio of the principal peak is not less than 3. Limit: Any impurity: For each impurity, not more than twice the area of the principal peak in the chromatogram obtained with reference solution (2) (2%).

N,N-Dimethylaniline Not more than 20 ppm (Appendix 10.16, method 1). Heavy metals Not more than 20 ppm (Appendix 9.4.8) 1.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Water Not more than 2.0% (Appendix 10.3). Determined on 0.500 g. 766

Bacterial endotoxins Less than 0.07 EU/mg (Appendix 13.2). If intended for use in the manufacture of parenteral preparations without a further appropriate procedure for the removal of bacterial endotoxins. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phases A, mobile phases B, solvent mixture and chromatographic system as described in the test for Related substances. Mobile phase: Mobile phases A - mobile phases B (88 : 12), adjusted if necessary. Test solution: Dissolve 25.0 mg of the substance to be examined in the solvent mixture and dilute to 50.0 ml with the solvent mixture. Standard solution: Use reference solution (1) in the test for Related substances. Procedure: System suitability: In the chromatogram obtained with the standard solution, the relative standard deviation of the peak areas for 6 replicate injections is not more than 1.0%. Inject the test solution and the standard solution. Calculate the content of piperacillin sodium, C23H26N5NaO7S, using the peak areas in the chromatograms obtained with the test solution, standard solution and the declared content of C23H26N5NaO7S in piperacillin RS, the correction factor from piperacillin to piperacillin sodium is 1.042. Storage Store in an airtight container. If the substance is sterile, store in a sterile, airtight container. Action and use Penicillin antibacterial. Preparations Injection powder. PIPERAZINE ADIPATE Piperazini adipas NH HN

, HO2C

C4H10N2,C6H10O4

CO2H

M. 232.3

Piperazine adipate contains not less than 98.0% and not more than 101.0% of C4H10N2.C6H10O4, calculated with reference to the anhydrous substance.

Characters A white crystalline powder, it melts at about 250 °C with decomposition. Soluble in water, practically insoluble in ethanol (96%).

PIPERAZINE CITRATE

VP V

Identification Apply one of the two following identifications: First identification: A. Second identification: B, C. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of piperazine adipate RS. B. In the test for Related substances, after spraying with the ninhydrin solutions, the principal spot in the chromatogram obtained with test solution (2) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). C. To 10 ml of solution S (see Appearance of solution) add 5 ml of hydrochloric acid R and shake with three quantities, each of 10 ml of ether R. Evaporate the combined ether layers to dryness. Wash the residue with 5 ml of water and dry it at 100 - 105 °C. The melting point (Appendix 6.7) of the residue is 150 - 154 °C. Appearance of solution Solution S: Dissolve 2.5 g of the substance to be examined in water and dilute to 50 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution B8 (Appendix 9.3, method 2). Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Concentrated ammonia - acetone (20 : 80), freshly prepared. Solvent mixture: Ethanol - concentrated ammonia (2 : 3). Test solution (1): Dissolve 1.0 g of the substance to be examined in 6 ml of concentrated ammonia R and dilute to 10 ml with ethanol R. Test solution (2): Dilute 1 ml of test solution (1) to 10 ml with the solvent mixture. Reference solution (1): Dissolve 0.1 g of piperazine adipate RS in the solvent mixture and dilute to 10 ml with the same solvent. Reference solution (2): Dissolve 25 mg of ethylenediamine R in the solvent mixture and dilute to 100 ml with the same solvent. Reference solution (3): Dissolve 25 mg of triethylenediamine R in the solvent mixture and dilute to 100 ml with the same solvent. Reference solution (4): Dissolve 12.5 mg of triethylenediamine R in 5.0 ml of test solution (1) and dilute to 50 ml with the solvent mixture. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm. Dry the plate at 105 °C and spray successively with a 0.3% solution of ninhydrin R in a mixture of anhydrous acetic acid - butanol (3 : 100), and a 0.15% solution of ninhydrin R in ethanol R. Dry the plate at 105 °C for 10 min. Any spot in the chromatogram obtained with test solution (1), apart from the principal spot, is not more intense than

the spot in the chromatogram obtained with reference solution (2) (0.25%). Spray the plate with 0.1 N iodine solution R and allow to stand for about 10 minutes. Any spot corresponding to triethylenediamine in the chromatogram obtained with test solution (1) is not more intense than the spot in the chromatogram obtained with reference solution (3) (0.25%). The test is not valid unless the chromatogram obtained with reference solution (4) shows two clearly separated spots. Disregard any spot remaining on the starting line.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 12 ml of solution S complies with limit test for heavy metals (method 1). Prepare the standard using lead standard solution(1 ppm Pb) R. Water Not more than 0.5% (Appendix 10.3). Determined on 1.00 g. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.100 g of the substance to be examined in 10 ml of anhydrous acetic acid R with gentle heating and dilute to 70 ml with the same solvent. Titrate with 0.1 N perchloric acid VS using 0.25 ml of 1-naphtholbenzein solution R as indicator until the colour changes from brownish-yellow to green. 1 ml of 0.1 N perchloric acid VS is equivalent to 11.61 mg of C4H10N2,C6H10O4. Storage Store in a well-closed container. Action and use Anthelmintic. PIPERAZINE CITRATE Piperazini citras

(C4H10N2)3,2C6H8O7 (anhydrous)

M. 643.0

Piperazine citrate contains not less than 98.0% and not more than 101.0% of (C4H10N2)3,2C6H8O7, calculated with reference to the anhydrous substance.

767

VP V

PIPERAZINE HYDRATE

Characters A white granular powder; after drying at 100 - 105 °C, it melts at about 190 °C. Freely soluble in water, practically insoluble in ethanol (96%) and in ether. Identification Apply one of the two following identifications: First identification: A. Second identification: B, C. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of piperazine citrate RS. Dry the substance to be examined and the reference substance at 120 °C for 5 hours, avoiding moisture, record the spectra without delay. B. In the test for Related substances, after spraying with the ninhydrin solutions, the principal spot in the chromatogram obtained with test solution (2) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). C. Dissolve 0.5 g of the substance to be examined in 5 ml of water, the solution gives the reaction of citrates (Appendix 8.1). Appearance of solution Solution S: Dissolve 1.25 g of the substance to be examined in water and dilute to 25 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution B8 (Appendix 9.3, method 2). Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Concentrated ammonia - acetone (20 : 80), freshly prepared. Solvent mixture: Ethanol - concentrated ammonia (2 : 3). Test solution (1): Dissolve 1.0 g of the substance to be examined in 6 ml of concentrated ammonia R and dilute to 10 ml with ethanol R. Test solution (2): Dilute 1 ml of test solution (1) to 10 ml with the solvent mixture. Reference solution (1): Dissolve 0.1 g of piperazine citrate RS in the solvent mixture and dilute to 10 ml with the same solvent. Reference solution (2): Dissolve 25 mg of ethylenediamine R in the solvent mixture and dilute to 100 ml with the same solvent. Reference solution (3): Dissolve 25 mg of triethylenediamine R in the solvent mixture and dilute to 100 ml with the same solvent. Reference solution (4): Dissolve 12.5 mg of triethylenediamine R in 5.0 ml of test solution (1) and dilute to 50 ml with the solvent mixture. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm. Dry the plate at 105 °C and spray successively with a 0.3% solution of 768

ninhydrin R in a mixture of anhydrous acetic acid - butanol (3 : 100), and a 0.15% solution of ninhydrin R in ethanol R. Dry the plate at 105 °C for 10 min. Any spot in the chromatogram obtained with test solution (1), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (2) (0.25%). Spray the plate with 0.1 N iodine solution R and allow to stand for about 10 minutes. Any spot corresponding to triethylenediamine in the chromatogram obtained with test solution (1) is not more intense than the spot in the chromatogram obtained with reference solution (3). The test is not valid unless the chromatogram obtained with reference solution (4) shows two clearly separated spots.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 12 ml of solution S complies with the limit test for heavy metals, method 1. Prepare the standard using lead standard solution(1 ppm Pb) R. Water 10.0 to 14.0% (Appendix 10.3). Determined on 0.300 g. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.100 g of the substance to be examined in 10 ml of anhydrous acetic acid R with gentle heating and dilute to 70 ml with the same solvent. Titrate with 0.1 N perchloric acid VS using 0.25 ml of 1-naphtholbenzein solution R as indicator until the colour changes from brownish-yellow to green. 1 ml of 0.1 N perchloric acid VS is equivalent to 10.71 mg of (C4H10N2)3,2C6H8O7. Storage Store in a well-closed container. Action and use Anthelmintic. PIPERAZINE HYDRATE Piperazini hydras NH HN

, 6 H 2O

C4H10N2,6H2O

M. 194.2

Piperazine hydrate contains not less than 98.0% and not more than 101.0% of C4H10N2,6H2O.

PIPERAZINE HYDRATE

VP V

Characters Colourless, deliquescent crystals. Freely soluble in water and in ethanol (96%), very slightly soluble in ether. It melts at about 43 °C. Identification Apply one of the two following identifications: First identification: A. Second identification: B, C. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of piperazine hydrate RS. Dry the substance to be examined and the reference substance over phosphorus pentoxide R in vacuo for 48 hours. Powder the substances avoiding uptake of water, prepare discs and record the spectra without delay. B. Examine the chromatograms obtained in the test for Related substances, after spraying with the ninhydrin solutions. The principal spot in the chromatogram obtained with test solution (2) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). C. Dissolve 0.5 g of the substance to be examined in 5 ml of dilute sodium hydroxide solution R. Add 0.2 ml of benzoyl chloride R and mix. Continue to add benzoyl chloride R in portions of 0.2 ml until no further precipitate is formed. Filter and wash the precipitate with a total of 10 ml of water added in small portions. Dissolve the precipitate in 2 ml of hot ethanol (96%) R and pour the solution into 5 ml of water. Allow to stand for 4 hours, filter, wash the crystals with water and dry at 100 °C to 105 °C. The crystals melt at 191 to 196 °C (Appendix 6.7). Appearance of solution Solution S: Dissolve 1.0 g of the substance to be examined in carbon dioxide-free water R to produce 20 ml. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution B8 (Appendix 9.3, method 2). pH The pH of solution S is 10.5 to 12.0 (Appendix 6.2). Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Concentrated ammonia - acetone (20 : 80), freshly prepared. Solvent mixture: Ethanol - concentrated ammonia (2 : 3). Test solution (1): Dissolve 1.0 g of the substance to be examined in 6 ml of concentrated ammonia R and dilute to 10 ml with ethanol R. Test solution (2): Dilute 1 ml of test solution (1) to 10 ml with the solvent mixture. Reference solution (1): Dissolve 0.1 g of piperazine hydrate RS in the solvent mixture and dilute to 10 ml with the same solvent.

Reference solution (2): Dissolve 25 mg of ethylenediamine R in the solvent mixture and dilute to 100 ml with the same solvent. Reference solution (3): Dissolve 25 mg of triethylenediamine R in the solvent mixture and dilute to 100 ml with the same solvent. Reference solution (4): Dissolve 12.5 mg of triethylenediamine R in 5.0 ml of test solution (1) and dilute to 50 ml with the solvent mixture. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm. Dry the plate at 105 °C and spray successively with a 0.3% solution of ninhydrin R in a mixture of anhydrous acetic acid - butanol (3 : 100), and a 0.15% solution of ninhydrin R in ethanol R. Dry the plate at 105 °C for 10 min. Any spot in the chromatogram obtained with test solution (1), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (2) (0.25%). Spray the plate with 0.1 N iodine solution R and allow to stand for about 10 minutes. Any spot corresponding to triethylenediamine in the chromatogram obtained with test solution (1) is not more intense than the spot in the chromatogram obtained with reference solution (3). The test is not valid unless the chromatogram obtained with reference solution (4) shows two clearly separated spots.

Heavy metals Not more 20 ppm (Appendix 9.4.8). 12 ml of solution S complies with the limit test for heavy metals, method 1. Prepare the standard using lead standard solution (1 ppm Pb) R. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 80.0 mg of the substance to be examined in 10 ml of anhydrous acetic acid R with gentle heating and dilute to 70 ml with the same solvent. Titrate with 0.1 N perchloric acid VS using 0.25 ml of 1-naphtholbenzein solution R as indicator until the colour changes from brownish-yellow to green. 1 ml of 0.1 N perchloric acid VS is equivalent to 9.705 mg of C4H10N2,6H2O. Storage Store in an airtight container, protected from light. Action and use Anthelmintic.

769

VP V

PIPERAZINE PHOSPHATE

Storage Store in a well-closed container.

PIPERAZINE PHOSPHATE Piperazini phosphas

HN

Action and use Anthelmintic.

NH ., H3PO4 ., H2O

C4H10N2,H3PO4,H2O

M. 202.1

Piperazine phosphate contains not less than 98.5% and not more than 100.5% of C4H10N2,H3PO4, calculated with reference to the anhydrous substance.

Characters A white, crystalline powder; odourless or almost odourless. Sparingly soluble in water, practically insoluble in ethanol (96%). Identification A. Dissolve 0.1 g of the substance to be examined in 5 ml of water, add 0.5 g of sodium hydrogen carbonate R, 0.5 ml of a 5% solution of potassium ferricyanide R and 0.1 ml of mercury R. Shake vigorously for 1 min and allow to stand for 20 min. A reddish colour is produced slowly. B. Dissolve 0.2 g of the substance to be examined in 5 ml of 2 M hydrochloric acid R, add with stirring 1 ml of a 50% solution of sodium nitrite R and cool in iced water for 15 min, stirring if necessary to induce crystallisation. The melting point (Appendix 6.7) of the crystals (after washing with 10 ml of iced water and drying at 105 °C) is about 159 °C. C. A solution of the substance to be examined gives the reactions characteristic of phosphates (Appendix 8.1). pH The pH of a 1% solution of the substance to be examined is 6.0 to 6.5 (Appendix 6.2). Heavy metals Not more than 20 ppm (Appendix 9.4.8). Dissolve 2.0 g of the substance to be examined in 20 ml of 2 M acetic acid R. 12 ml of the resulting solution complies with the limit test for heavy metals, method 1. Prepare the standard using lead standard solution (2 ppm Pb) R. Water 8.0 to 9.5% (Appendix 10.3). Determined on 0.250 g. Assay Dissolve 0.200 g of the substance to be examined in a mixture of 3.5 ml of 0.5 M sulphuric acid R and 10 ml of water. Add 100 ml of picric acid solution R, heat on a water-bath for 15 min and allow to stand for 1 hour. Filter through a sintered-glass filter G4 and wash the residue with successive 10 ml quantities of a mixture of equal volumes of a saturated solution of picric acid R and water until the washings are free from sulphate. Wash the residue with five 10 ml quantities of ethanol R and dry to constant mass at 100 °C to 105 °C. 1 g of residue is equivalent to 338.2 mg of C4H10N2,H3PO4. 770

Preperations Tablets. PIPERAZINE PHOSPHATE TABLETS Tabellae Piperazini phosphatis Piperazine phosphate tablets contain piperazine phosphate. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of piperazine phosphate, C4H10N2,H3P O4,H2O, 92.5% to 107.5% of the stated amount. Identification Extract a quantity of the powdered tablets containing 1 g of piperazine phosphate with 20 ml of water and filter. The filtrate complies with the following tests. A. To 4 ml of the filtrate add 1 ml of hydrochloric acid R with stirring. Continue to add 1 ml of a 50% solution of sodium nitrite R and cool in ice for 15 min, stirring if necessary to induce crystallization. The melting point (Appendix 6.7) of the crystals (after washing with 10 ml of cold water and drying at 105 °C), is about 159 °C. B. The filtrate yields the reactions characteristic of phosphates (Appendix 8.1). Disintegration The requirement for Disintegration does not apply to piperazine phosphate tablets intended to be chewed before swallowing. Assay Weigh 20 tablets and powder finely. Shake a quantity of the powder containing the equivalent of about 0.15 g of piperazine phosphate with 10 ml of water for 1 hour and filter. Wash the residue with two 10-ml quantities of water. To the combined extracts and washings add 5 ml of 1 M sulfuric acid R and 50 ml of picric acid solution, bring to the boil, allow to stand for several hours, filter through a sintered-glass filter No. 4 and wash the residue with successive 10-ml quantities of a mixture of equal volumes of a saturated solution of picric acid R and water until the washings are free from sulphate. Wash the residue with five 10-ml quantities of ethanol R and dry to constant weight at 100 °C to 105 °C. Each g of residue is equivalent to 0.3714 g of C4H10N2, H3PO4,H2O.

PIRACETAM

VP V

Storage Store in a dry place, in a well-closed container. Action and use Anthelmintic. Usual strength 300 mg of piperazine. PIRACETAM Piracetamum O N

NH2

O

C6H10N2O2  

M. 142.2

Piracetam is 2-(2-oxopyrrolidin-1-yl)acetamide. It contains not less than 98.0% and not more than 102.0% of C6H10N2O2, calculated with reference to the dried substance.

Characters White or almost white, powder, it shows polymorphism. Freely soluble in water, soluble in ethanol (96%). Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of piracetam RS. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in ethanol (96%) R, evaporate to dryness on a water-bath and record new spectra using the residues. Appearance of solution Dissolve 2.0 g of the substance to be examined in water and dilute to 10 ml with the same solvent. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile R1 - a 0.1% solution of dipotassium hydrogen phosphate R (10 : 90). Adjust to pH 6.0 with 2 M phosphoric acid R. Solvent mixture: Acetonitrile R1 - water (10 : 90). Test solution (1): Dissolve 50.0 mg of the substance to be examined in solvent mixture and dilute to 100.0 ml with the same solvent. Test solution (2): Dilute 10.0 ml of test solution (1) to 50.0 ml with solvent mixture. Reference solution (1): Dissolve 5 mg of the substance to be examined and 10 µl of 2-pyrrolidone R in solvent mixture and dilute to 100.0 ml with the same solvent.

Reference solution (2): Dilute 1.0 ml of test solution (1) to 100.0 ml with solvent mixture. Dilute 5.0 ml of this solution to 50.0 ml with solvent mixture. Reference solution (3): Dissolve 50.0 mg of piracetam RS in solvent mixture and dilute to 100.0 ml with the same solvent. Dilute 10.0 ml of this solution to 50.0 ml with solvent mixture. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 205 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Inject the test solution (1) and reference solutions (1) and (2). The run time is 8 times the retention time of piracetam. Relative retention with reference to piracetam (retention time = about 4 min): impurity D = about 0.8; impurity A = about 1.15; impurity B = about 2.8; impurity C = about 6.3. System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to piracetam and impurity A is at least 3.0; symmetry factor is not more than 2.0 for the peak due to piracetam. Limits: Impurities A, B, C, D: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.1%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.1%). The sum of the peak areas of all impurities is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.3%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Note: Impurity A: Pyrrolidin-2-one (2-pyrrolidone). Impurity B: Methyl (2-oxopyrrolidin-1-yl)acetate. Impurity C: Ethyl (2-oxopyrrolidin-1-yl)acetate. Impurity D: (2-oxopyrrolidin-1-yl)acetic acid.

Heavy metals Not more than 10 ppm (Appendix 9.4.8). Dissolve 2.0 g of the substance to be examined in 20 ml of water. 12 ml of this solution complies with limit test for heavy metals, method 1. Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on drying Not more than 1.0% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. 771

VP V

PIRACETAM CAPSULES

Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. Inject test solution (2) and reference solution (3). Calculate the content of C6H10N2O2 using the peak areas in the chromatograms obtained with test solution (2), reference solution (3) and the declared content of C6H10N2O2 in piracetam RS. Storage Protected from light. Action and use Nootropic; cortical myoclonus. Preparations Capsules, injection. PIRACETAM CAPSULES Capsulae Piracetami Piracetam capsules contain piracetam. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of piracetam, C6H10N2O2, 93.0% to 107.0% of the stated amount. Identification A. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that of the peak due to piracetam in the chromatogram obtained with the reference solution. B. Shake well a quantity of the contents of the capsules containing 0.5 g of piracetam with 10 ml of water and filter. To 2 ml of the filtrate add one drop of a 5.0% solution of potassium permanganate R and a 10% solution of sodium hydroxide R. A violet colour is produced and turns to blue then green. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol - water (10 : 90). Test solution: Weigh 20 capsules, calculate the average weight of the capsule contents, mix well, and powder finely. Transfer a quantity of the contents of the capsules containing 0.1 g of piracetam to a 100 ml volumetric flask. Add about 60 ml of the mobile phase, sonicate to dissolve. Add the mobile phase to volume and mix. Filter and dilute 5.0 ml of the filtrate to 50.0 ml with the mobile phase and mix. Reference solution: Transfer about 100 mg of piracetam RS, accurately weighed, to a 100 ml volumetric flask. Add about 60 ml of the mobile phase, sonicate to dissolve. Add the mobile phase to volume and mix. Dilute 5.0 ml of this solution to 50.0 ml with the mobile phase and mix. 772

Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 210 nm. Flow rate: 1.5 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject the reference solution, the column efficiency, determined on the peak due to piracetam, is not less than 2000 theoretical plates. Inject alternately the test solution and the reference solution. Calculate the content of piracetam, C6H10N2O2, in the capsules using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C6H10N2O2 in piracetam RS.

Storage Store in a well-closed container, protected from light. Action and use Nootropic, cortical myoclonus. Usual strength 200 mg, 400 mg. PIRACETAM INJECTION Injectio Piracetami Piracetam injection is a sterile solution of piracetam in water for injection. The injection complies with the requirements stated under “Injection, infusions” (Appendix 1.19) and with the following requirements:

Content of piracetam, C6H10N2O2, 95.0% to 105.0% of the stated amount. Characters A clear and colourless solution. Identification A. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that of the piracetam peak in the chromatogram obtained with the reference solution. B. To a volume of the injection containing about 0.1 g of piracetam, add 1 drop of a 5% solution of potassium permanganate R. Further add 1 drop of a 10% solution of sodium hydroxide R, mix well, a violet colour will change to blue and then to green. pH 4.0 to 7.0 (Appendix 6.2). Related substances Examine by liquid chromatography (Appendix 5.3).

PIROXICAM

VP V

Mobile phase and chromatographic system are described in the Assay. Test solution: Dilute a volume of the injection with the mobile phase to obtain a solution having a concentration of piracetam of about 0.5 mg per ml. Reference solution: Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Procedure: Inject the reference solution, adjust the sensitivity of the system so that the height of the principal peak is about 10% of the full scale of the recorder. Inject the reference solution and the test solution, record chromatograms for 3 times the retention time of the principal peak. In the chromatogram obtained with the test solution, the sum of areas of all secondary peaks is not more than the area of the principal peak in the chromatogram obtained with the reference solution (1.0%).

Bacterial endotoxins (Appendix 13.2) Not more than 0.04 EU per 1 mg of piracetam. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol - water (10 : 90). Test solution: Dilute accurately a volume of the injection with the mobile phase to obtain a solution having a concentration of about 0.1 mg of piracetam per ml. Reference solution: Weigh accurately about 100 mg of piracetam RS, transfer into a 100-ml volumetric flask, add 60 ml of the mobile phase, sonicate to dissolve, dilute to volume with the mobile phase. Dilute 5.0 ml of the solution to 50.0 ml with the mobile phase, shake well. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 210 nm. Flow rate: 1.5 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject the reference solution. The column efficiency determined on the piracetam peak is not less than 2000 theoretical plates. The relative standard deviation of the areas of piracetam peaks for 6 replicate injections of the reference solution is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of piracetam, C6H10N2O2, in the injection using the peak areas in the chromatograms obtained with the reference solution, the test solution and the declared content of C6H10N2O2 in piracetam RS. Storage Store in a tight container, in a cool place, protected from light.

Action and use Nootropic; cortical myoclonus. Usual strength Ampoule of 1g/5 ml. PIROXICAM Piroxicamum

C15H13N3O4S 

M. 331.4

Piroxicam is 4-hydroxy-2-methyl-N-(pyridin-2-yl)-2H-1,2benzothiazine-3-carboxamide 1,1- dioxide. It contains not less than 98.5% and not more than 101.0% of C15H13N3O4S, calculated with reference to the dried substance.

Characters White or slightly yellow, crystalline powder, it shows polymorphism. Practically insoluble in water, soluble in methylene chloride, slightly soluble in anhydrous ethanol. Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of piroxicam RS. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in the minimum volume of methylene chloride R, evaporate to dryness on a water-bath and record new spectra using the residues. Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use. Mobile phase: Acetonitrile R1 - a 0.681% solution of potassium dihydrogen phosphate R previously adjusted to pH 3.0 with phosphoric acid R (30 : 70). Test solution: Dissolve 75 mg of the substance to be examined in acetonitrile R1, warming slightly if necessary, and dilute to 50.0 ml with the same solvent. Reference solution (1): Dissolve 7 mg of piroxicam for system suitability RS (containing impurities A, B, D, G and J) in acetonitrile R1 and dilute to 5.0 ml with the same solvent. Reference solution (2): Dilute 1.0 ml of the test solution to 10.0 ml with acetonitrile R1. Dilute 1.0 ml of this solution to 50.0 ml with acetonitrile R1. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase base-deactivated end-capped octadecylsilyl silica gel for 773

VP V

PIROXICAM CAPSULES

chromatography R (5 µm). Column temperature: 50 °C. Detector: A spectrophotometer set at 230 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Inject the blank, the test solution and the reference solutions. The run time is 5 times the retention time of piroxicam. Identification of impurities: Use the chromatogram supplied with piroxicam for system suitability RS and the chromatogram obtained with reference solution (1) to identify the peaks due to impurities A, B, D, G and J. Relative retention with reference to piroxicam (retention time = about 16 min): impurity A = about 0.1; impurity D = about 0.6; impurity G = about 0.7; impurity B = about 0.8; impurity J = about 1.8. System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to impurities G and B is at least 1.5. Limits: Correction factor: for the calculation of content, multiply the peak area of impurity A by 0.6. Impurities A, B, D, G, J: For each impurity, the area corrected if necessary, is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.2%). Any other impurity: For each impurity, the area is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.10%). The sum of the peak areas of all impurities is not more than twice times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.4%). Disregard any peak with an area less than 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Note: Impurity A: Pyridin-2-amine. Impurity B: 4-hydroxy-N-(pyridin-2-yl)-2H-1,2-benzothiazine3-carboxamide 1,1-dioxide. Impurity C: 4-hydroxy-2-methyl-2H-1,2-benzothiazine-3-carboxamide 1,1-dioxide. Impurity D: Methyl (1,1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)yl)acetate. Impurity E: Ethyl (1,1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)yl)acetate. Impurity F: 1-methylethyl (1,1-dioxido-3-oxo-1,2-benzisothiazol2(3H)-yl)acetate. Impurity G: Methyl 4-hydroxy-2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide. Impurity H: Ethyl 4-hydroxy-2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide. Impurity I: 1-methylethyl 4-hydroxy-2H-1,2-benzothiazine-3carboxylate 1,1-dioxide. Impurity J: Methyl 4-hydroxy-2-methyl-2H-1,2-benzothiazine3-carboxylate 1,1-dioxide.

774

Impurity K: Ethyl 4-hydroxy-2-methyl-2H-1,2-benzothiazine3-carboxylate 1,1-dioxide. Impurity L: 1-methylethyl 4-hydroxy-2-methyl-2H-1,2-benzothiazine3-carboxylate 1,1- dioxide.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C; in vacuo; 4 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.250 g of the substance to be examined in 60 ml of a mixture of equal volumes of acetic anhydride R and anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appenidx 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 33.14 mg of C15H13N3O4S. Storage Store in an airtight container, protected from light Action and use Non-steroid anti-inflammatory, cyclo-oxy genase inhibitor Preparations Capsules, tablets, injection, suppository. PIROXICAM CAPSULES Capsulae Piroxicami Piroxicam capsules contain piroxicam. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of piroxicam, C15H13N3O4S, 92.5% to 107.5% of the stated amount. Identification A. In the Related substances, the principal spot in the chromatogram obtained with test solution (2) is similar in position, colour and size to that in the chromatogram obtained with reference solution (1). B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that of the peak due to piroxicam in the chromatogram obtained with the reference solution.

PIROXICAM CAPSULES

VP V

Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 900 ml of 0.1 M hydrochloric acid R. Rotation speed: 100 rpm. Time: 45 min. Procedure: Test solution: After the specified time, withdraw a sample of the medium, filter and discard the first portion of the filtrate. Dilute the filtrate with 0.1 M hydrochloric acid R if necessary, to obtain a solution containing about 10 µg of piroxicam per ml. Reference solution: Transfer about 10 mg of piroxicam RS, accurately weighed, to a 100 ml volumetric flask, add 20 ml of methanol R to dissolve. Dilute to volume with water and mix. Transfer 10 ml of the resulting solution, accurately measured, to an another 100 ml volumetric flask, dilute to volume with 0.1 M hydrochloric acid R and mix. Measure the absorbances (Appendix 4.1) of the test solution and the reference solution at the maximum at 242 nm in a 1-cm cell, using 0.1 M hydrochloric acid R as a blank. Calculate the content of piroxicam dissolved using the absorbances obtained with the reference and test solution and the declared content of C15H13N3O4S in piroxicam RS. Or another way, taking 352 as the value of A (1%, 1 cm) at the maximum at 242 nm to calculate the content of piroxicam dissolved. Tolerance: Not less than 70% (Q) of the stated amount of piroxicam, C15H13N3O4S, is dissolved in 45 min. Note: If the capsule shells interfere the analytical results, take 6 empty capsules and dissolve with a prescribed volume of the medium. Proceed as directed above and calculate the correction factor. The correction factor should not be more than 25% of the stated amount.

2-Pyridylamine Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Diethylamine - dichloromethane (1 : 8). Test solution: Shake a quantity of the contents of the capsules containing 80 mg of piroxicam with 25 ml of dichloromethane R, filter, evaporate the filtrate to dryness using a rotary evaporator and dissolve the residue in 2 ml of dichloromethane R. Reference solution: A solution contains 0.010% of 2-pyridylamine in dichloromethane R. Procedure: Apply separately to the plate 20 µl of each solution. Develop over a path of 15 cm. Allow to dry in air. Examine under ultraviolet light at 254 nm. Any spot corresponding to 2-pyridylamine in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution (0.25%). Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Toluene - acetic acid (90 : 10).

Test solution (1): Shake a quantity of the contents of the capsules containing 80 mg of piroxicam with 25 ml of dichloromethane R, filter, evaporate the filtrate to dryness using a rotary evaporator and dissolve the residue in 2 ml of dichloromethane R. Test solution (2): Dilute 1 ml of test solution (1) to 20 ml with dichloromethane R. Reference solution (1): A solution contains 0.20% of piroxicam RS in dichloromethane R. Reference solution (2): Dilute 2 ml of test solution (2) to 50 ml with dichloromethane R. Procedure: Apply separately to the plate 7.5 µl of each solution. After removal of the plate, allow to dry in air. Examine under ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with test solution (1) is not more intense than the spot in the chromatogram obtained with reference solution (2) (0.2%). Disregard any spot remaining on the line of application.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol - buffer solution (60 : 40). Buffer solution: Prepared by adding a solution containing 5.35 g of disodium hydrogen phosphate R in 100 ml of water to a solution containing 7.72 g of citric acid R in 400 ml of water and diluting to 1000 ml with water. Test solution: Weigh 20 capsules, calculate the average weight of the capsule contents and powder finely. Transfer an accurately weighed quantity of the contents of the capsules containing about 10 mg of piroxicam to a 200 ml volumetric flask. Add 150 ml of 0.01 M methanolic hydrochloric acid R, sonicate for 30 minutes, allow to cool and dilute to volume with the same solvent. Mix and filter. Reference solution: The solution contains 0.005% of piroxicam RS in 0.01 M methanolic hydrochloric acid R. Chromatographic system: A column (30 cm × 3.9 mm) packed with stationary phase C (10 µm). Detector: A spectrophotometer set at 242 nm. Flow rate: 2 ml/min. Volume of injection: 20 µl. Procedure: Inject alternately the test solution and the reference solution. Calculate the content of piroxicam, C15H13N3O4S, in the capsules using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C15H13N3O4S in piroxicam RS. Storage Store in a well-closed container, in a cool place, protected from light. Action and use Non-steroidal anti-inflammatory drug. Usual strength 10 mg, 20 mg. 775

PIROXICAM TABLETS

PIROXICAM TABLETS Tabellae Piroxicami Piroxicam tablets contain piroxicam. They may be filmcoated or sugar-coated tablets. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of piroxicam, C15H13N3O4S, 92.5% to 107.5% of the stated amount. Identification A. In the Related substances, the principal spot in the chromatogram obtained with the test solution (2) is similar in position, colour and size to that in the chromatogram obtained with reference solution (1). B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that of the peak due to piroxicam in the chromatogram obtained with the reference solution. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of 0.1 M hydrochloric acid R. Rotation speed: 100 rpm. Time: 45 min. Procedure: After the specified time, withdraw a sample of the medium, filter. Dilute a portion of the filtrate with 0.1 M hydrochloric acid R to obtain a solution containing about 10 µg of piroxicam per ml. Measure the absorbances (Appendix 4.1) of the resulting solution at the maximum at 242 nm in a 1 cm cell, using 0.1 M hydrochloric acid R as a blank, taking 352 as the value of A (1%, 1 cm) at the maximum at 242 nm. Tolerance: Not less than 70% (Q) of the stated amount of piroxicam, C15H13N3O4S, is dissolved in 45 min. 2-Pyridylamine Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Diethylamine - dichloromethane (1 : 8). Test solution: Shake a quantity of powdered tablets containing 80 mg of piroxicam with 25 ml of dichloromethane R, filter, evaporate the filtrate to dryness using a rotary evaporator and dissolve the residue in 2 ml of dichloromethane R. Reference solution: A solution contains 0.010% of 2-pyridylamine in dichloromethane R. Procedure: Apply separately to the plate 20 µl of each solution. Develop over a path of 15 cm. Allow to dry in air. Examine under ultraviolet light at 254 nm. Any spot corresponding to 2-pyridylamine in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution (0.25%). 776

VP V

Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Toluene - acetic acid (90 : 10). Test solution (1): Shake a quantity of powdered tablets containing 80 mg of piroxicam with 25 ml of dichloromethane R, filter, evaporate the filtrate to dryness using a rotary evaporator and dissolve the residue in 2 ml of dichloromethane R. Test solution (2): Dilute 1 ml of test solution (1) to 20 ml with dichloromethane R. Reference solution (1): A solution contains 0.20% of piroxicam RS in dichloromethane R. Reference solution (2): Dilute 2 ml of test solution (2) to 50 ml with dichloromethane R. Procedure: Apply separately to the plate 7.5 µl of each of the above solutions. After removal of the plate, allow to dry in air. Examine under ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with test solution (1) is not more intense than the spot in the chromatogram obtained with reference solution (2) (0.2%). Disregard any spot remaining on the line of application. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol - buffer solution (60 : 40). Buffer solution: Prepared by adding a solution containing 5.35 g of disodium hydrogen phosphate R in 100 ml of water to a solution containing 7.72 g of citric acid R in 400 ml of water and diluting to 1000 ml with water. Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Transfer an accurately weighed quantity of powdered tablets containing about 10 mg of piroxicam to a 200 ml volumetric flask. Add 150 ml of 0.01 M methanolic hydrochloric acid R, sonicate for 30 minutes, allow to cool and dilute to volume with the same solvent. Mix and filter. Reference solution: A solution containing 0.005% of piroxicam RS in 0.01 M methanolic hydrochloric acid R. Chromatographic system: A column (30 cm × 3.9 mm) packed with stationary phase C (10 µm). Detector: A spectrophotometer set at 242 nm. Flow rate: 2 ml/min. Volume of injection: 20 µl. Procedure: Inject alternately the test solution and the reference solution. Calculate the content of piroxicam, C15H13N3O4S, in the capsules using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C15H13N3O4S in piroxicam RS. Storage Store in a well-closed container, store in a cool place, protected from light.

POLYMYCIN B SULFATE

VP V

Action and use Non-steroidal anti-inflammatory drug. Usual strength 10 mg, 20 mg. POLYMYCIN B SULFATE Polymyxini B Sulfas

Polymyxin B1

R

B2

H

B3

CH3

B1-I

R’

X

Molecular formula

Mr

C56H98N16O13

1204

C55H96N16O13

1190

L-Leu

C55H96N16O13

1190

L-Ile

C56H98N16O13

1204

CH3 CH3 L-Leu CH3 L-Leu H

CH3 CH3

Polymyxin B is a mixture of the sulfates of polypeptides produced by the growth of certain strains of Paenibacillus polymyxa, or obtained by any other means, the main component being polymyxin B1.

Content Sum of polymyxins B1, B2, B3 and B1-I: Not less than 80.0%, calculated with reference to the dried substance. Polymyxin B3: Not more than 6.0%, calculated with reference to the dried substance. Polymyxin B1-I: Not more than 15.0%, calculated with reference to the dried substance. Characters White or almost white, hygroscopic powder. Soluble in water, slightly soluble in ethanol (96%). Identification Apply one of the two following identifications: First identification: B, D. Second identification: A, C, D. A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Water - phenol (25 : 75). Test solution: Dissolve 5 mg of the substance to be examined in 1 ml of a mixture of equal volumes of hydrochloric acid R and water. Heat at 135 °C in a sealed tube for 5 h. Evaporate to dryness on a water-bath and continue the heating until the hydrochloric acid has evaporated. Dissolve the residue in 0.5 ml of water. Reference solution (1): Dissolve 20 mg of leucine RS in water and dilute to 10 ml with the same solvent.

Reference solution (2): Dissolve 20 mg of threonine RS in water and dilute to 10 ml with the same solvent. Reference solution (3): Dissolve 20 mg of phenylalanine RS in water and dilute to 10 ml with the same solvent. Reference solution (4): Dissolve 20 mg of serine RS in water and dilute to 10 ml with the same solvent. Procedure: Carry out the following procedures protected from light. Apply separately to the plate 5 µl of each solution as bands of 10 mm, then place the plate in the chromatographic tank so that it is not in contact with the mobile phase, and allow it to become impregnated with the vapour of the mobile phase for at least 12 h. Develop over a path of 12 cm. Dry the plate at 100° C to 105 °C. Allow to cool, then spray with ninhydrin solution R1 and heat at 110 °C for 5 min. Results: The chromatogram obtained with the test solution shows zones corresponding to those in the chromatograms obtained with reference solutions (1), (2) and (3), but shows no zone corresponding to that in the chromatogram obtained with reference solution (4); the chromatogram obtained with the test solution also shows a zone with a very low Rf value (2,4-diaminobutyric acid). B. In the assay, the peaks due to polymyxins B1, B2, B3 and B1-I in the chromatogram obtained with the test solution are similar in retention time to the corresponding peaks in the chromatogram obtained with reference solution (1). C. Dissolve about 2 mg of the substance to be examined in 5 ml of water and add 5 ml of a 10% solution of sodium hydroxide R. Shake and add dropwise 0.25 ml of a 1% solution of copper sulfate R, shaking after each addition. A reddish-violet colour develops. D. It gives reaction (A) of sulfates (Appendix 8.1).

pH 5.0 to 7.0 (Appendix 6.2). Dissolve 0.2 g in carbon dioxide-free water R and dilute to 10 ml with the same solvent. Specific optical rotation -78° to -90°, calculated with reference to the dried substance (Appendix 6.4). Dissolve 0.50 g in water and dilute to 25.0 ml with the same solvent. Related substances Examine by liquid chromatography (Appendix 5.3). Solution A: Dissolve 4.46 g of anhydrous sodium sulfate R in 900 ml of water, adjust to pH 2.3 with 2 M phosphoric acid R and dilute to 1000 ml with water. Mobile phase: Acetonitrile - solution A (20 : 80). Solvent mixture: Acetonitrile - water (20 : 80). Test solution: Dissolve 50.0 mg of the substance to be examined in solvent mixture and dilute to 100.0 ml with the same solvent. Reference solution (1): Dissolve 50.0 mg of polymyxin B sulfate RS in solvent mixture and dilute to 100.0 ml with the same mixture of solvent. 777

VP V

POLYSORBATE 20

Reference solution (2): Dilute 1.0 ml of reference solution (1) to 100.0 ml with solvent mixture. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase base-deactivated end-capped octadecylsilyl silica gel for chromatography R (5 µm). Column temperature: 30 °C. Detector: A spectrophotometer set at 215 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: The run time is 1.4 times the retention time of polymyxin B1. Relative retention with reference to polymyxin B1 (retention time = about 35 min): polymyxin B2 = about 0.5; polymyxin B3 = about 0.6; polymyxin B1-I = about 0.8. System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to polymyxin B2 and polymyxin B3 is at least 3.0. Limits: Any impurity: For each impurity, not more than 3.0%. Total: Not more than 17.0 %. Disregard any peak with an area less than 0.7 times the area of the principal peak in the chromatogram obtained with reference solution (2).

Sulfate 15.5% to 17.5%, calculated with reference to the dried substance. Dissolve 0.250 g of the substance to be examined in 100 ml of water and adjust the solution to pH 11 with ammonia R. Add 10.0 ml of 0.1 M barium chloride VS and about 0.5 mg of phthalein purple R. Titrate with 0.1 M sodium edetate VS, adding 50 ml of ethanol (96%) R when the colour of the solution begins to change and continuing the titration until the violet-blue colour disappears. 1 ml of 0.1 M barium chloride VS is equivalent to 9.606 mg of SO4. Loss on drying Not more than 6.0% (Appendix 9.6). (1.000 g; 60 °C; phosphorus pentoxide, at a pressure not exceeding 670 Pa; 3 h). Sulfated ash Not more than 0.75% (Appendix 9.9, method 2). Determined on 1.0 g. Pyrogens If intended for use in the manufacture of parenteral preparations without a further appropriate procedure for the removal of pyrogens, it complies with the test for pyrogens (Appendix 13.4). Inject, per kilogram of the rabbit's mass, 1 ml of a solution containing 1.5 mg of the substance to be examined in 1 ml water for injections R.

778

Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances with the following modification. Inject the test solution and reference solution (1). Calculate the percentage content of polymyxin B3, of polymyxin B1-I, and of the sum of polymyxins B1, B2, B3 and B1-I, using the following expression: CBi

=

ABi × m2 × DBi m1 × BBi

Where: CBi: Percentage content of polymyxin Bi. ABi: Area of the peak due to polymyxin Bi in the chromatogram obtained with the test solution (mg). m1: Mass of the substance to be examined (dried substance) in the test solution (mg). BBi: Area of the peak due to polymyxin Bi in the chromatogram obtained with reference solution (1). m2: Mass of polymyxin B sulfate RS in reference solution (1) (mg). DBi: Declared percentage content for polymyxin Bi in polymyxin B sulfate RS.

Storage In an airtight container, protected from light. If the substance is sterile, store in a sterile, airtight, tamper-proof container. Action and use Antibacterial. Preparations Polymyxin and Bacitracin Eye Ointment. POLYSORBATE 20 Polysorbatum 20 Polyoxyethylene Polysorbate 20 is a mixture of partial esters of fatty acids, mainly lauric (dodecanoic) acid, with sorbitol and its anhydrides ethoxylated with approximately 20 moles of ethylene oxide for each mole of sorbitol and sorbitol anhydrides.

Characters Oily, yellow or brownish-yellow, clear or slightly opalescent liquid. Soluble in water, in anhydrous ethanol, in ethyl acetate and in methanol, practically insoluble in fatty oils and in liquid paraffin. Relative density: About 1.10. Viscosity: About 400 mPa·s at 25 °C. Identification Apply one of the two following identifications: First identification: A, D.

POLYSORBATE 60

VP V

Second identification: B, C, D, E. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the reference spectrum of polysorbate 20. B. It complies with the test for Hydroxyl value. C. It complies with the test for Saponification value. D. It complies with the test for Composition of fatty acids. E. Dissolve 0.1 g of the substance to be examined in 5 ml of methylene chloride R. Add 0.1 g of potassium thiocyanate R and 0.1 g of cobalt nitrate R. Stir with a glass rod. The solution becomes blue.

is coated with macrogol 20 000 R (film thickness 0.5 µm). Carrier gas: Helium for chromatography R. Flow rate: 50 cm/s. Temperature programme:

Acid value Not more than 2.0 (Appendix 7.2). Dissolve 5.0 g of the substance to be examined in 50 ml of the prescribed solvent mixture.

Detector: A flame-ionisation detector. Volume of injection: 1 µl. Limits: Composition of the fatty-acid fraction of the substance: Caproic acid: Not more than 1.0%. Caprylic acid: Not more than 10.0%. Capric acid: Not more than 10.0%. Lauric acid: 40.0% to 60.0%. Myristic acid: 14.0% to 25.0%. Palmitic acid: 7.0% to 15.0%. Stearic acid: Not more than 7.0%. Oleic acid: Not more than 11.0%. Linoleic acid: Not more than 3.0%.

Hydroxyl value 96 to 108 (Appendix 7.4, method A). Saponification value 40 to 50 (Appendix 7.7). Determined on 4.0 g. Use 15.0 ml of 0.5 M potassium hydroxide in ethanol VS and dilute with 50 ml of ethanol (96%) R before carrying out the titration. Heat under reflux for 60 min. Peroxide value Not more than 10.0. Introduce 10.0 g of the substance to be examined into a 100 ml beaker and dissolve with 20 ml of glacial acetic acid R. Add 1 ml of saturated potassium iodide solution R, mix and allow to stand for 1 min. Add 50 ml of carbon dioxide-free water R and a magnetic stirring bar. Titrate with 0.01 N sodium thiosulfate VS, determining the endpoint potentiometrically (Appendix 10.2). Carry out a blank titration. If the result of the blank determination exceeds 0.1 ml of 0.01 N sodium thiosulfate VS, replace the reagents and repeat the determination. Determine the peroxide value using the following expression: (n1 − n2) × M × 1000 m Where: n1: volume of 0.01 N sodium thiosulfate VS required for the substance to be examined, in millilitres. n2: volume of 0.01 N sodium thiosulfate VS required for the blank, in millilitres. M: molarity of the sodium thiosulfate solution, in moles per litre. M: mass of substance to be examined, in grams. Composition of fatty acids (Appendix 12.9, method C) Use a reference mixture as described in Table 12.9.2 to prepare a reference solution (a). Chromatographic system: A fused-silica capillary column (30 m × 0.32 mm), the wall

Column

Time (min)

Temperature (°C)

0 - 14 14 - 54

80 → 220 220

Injection port

250

Detector

250

Ethylene oxide and dioxan (Appendix 10.15, method 1). Ethylene oxide: Not more than 1 ppm. Dioxan: Not more than10 ppm. Heavy metals Not more than 10 ppm (Appendix 9.4.8). 2.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Water Not more than 3.0% (Appendix 10.3). Determined on 1.00 g. Total ash Not more than 0.25% (Appendix 9.8, method 2). Determined on 2.0 g. Storage Store in an airtight container, protected from light. Action and use Non-ionic surfactants. POLYSORBATE 60 Polysorbatum 60 Polysorbate 60 is a mixture of partial esters of fatty acids, mainly stearic acid 50, with sorbitol and its anhydrides ethoxylated with approximately 20 moles of ethylene oxide for each mole of sorbitol and sorbitol anhydrides. 779

VP V

POLYSORBATE 60

Characters Yellowish-brown gelatinous mass which becomes a clear liquid at temperatures above 25 °C. Soluble in water, in anhydrous ethanol, in ethyl acetate and in methanol, practically insoluble in fatty oils and in liquid paraffin. Relative density: About 1.10. Viscosity: About 400 mPa·s at 30 °C. Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D, E. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the reference spectrum of polysorbate 60. B. It complies with the test for Hydroxyl value. C. It complies with the test for Saponification value. D. It complies with the test for Composition of fatty acids. E. Dissolve 0.1 g of the substance to be examined in 5 ml of methylene chloride R. Add 0.1 g of potassium thiocyanate R and 0.1 g of cobalt nitrate R. Stir with a glass rod. The solution becomes blue. Acid value Not more than 2.0 (Appendix 7.2). Dissolve 5.0 g of the substance to be examined in 50 ml of the prescribed solvent mixture. Hydroxyl value 81 to 96 (Appendix 7.4, method A). Saponification value 45 to 55 (Appendix 7.7). Determined on 4.0 g. Use 15.0 ml of 0.5 M potassium hydroxide in ethanol and dilute with 50 ml of ethanol (96%) R before carrying out the titration. Heat under reflux for 60 min. Peroxide value Not more than 10.0. Introduce 10.0 g of the substance to be examined into a 100 ml beaker and dissolve with 20 ml of glacial acetic acid R. Add 1 ml of saturated potassium iodide solution R, mix and allow to stand for 1 min. Add 50 ml of carbon dioxide-free water R and a magnetic stirring bar. Titrate with 0.01 N sodium thiosulfate VS, determining the endpoint potentiometrically (Appendix 10.2). Carry out a blank titration. If the result of the blank determination exceeds 0.1 ml of 0.01 N sodium thiosulfate VS, replace the reagents and repeat the determination. Determine the peroxide value using the following expression:

(n1 − n2) × M × 1000 m

Where: n1: volume of 0.01 N sodium thiosulfate VS required for the substance to be examined, in millilitres. 780

n2: volume of 0.01 N sodium thiosulfate VS required for the blank, in millilitres. M: molarity of the sodium thiosulfate solution, in moles per litre. M: mass of substance to be examined, in grams.

Composition of fatty acids (Appendix 12.9, method C) Use a reference mixture as described in Table 12.9.1 to prepare a reference solution (a). Chromatographic system: A fused-silica capillary column (30 m × 0.32 mm), the wall is coated with macrogol 20 000 R (film thickness 0.5 µm). Carrier gas: Helium for chromatography R. Flow rate: 50 cm/s. Temperature programme:

Column

Time (min)

Temperature (°C)

0 - 14 14 - 54

80 → 220 220

Injection port

250

Detector

250

Detector: A flame-ionisation detector. Volume of injection: 1 µl. Limits: Composition of the fatty-acid fraction of the substance: Stearic acid : 40.0% to 60.0%. Sum of the contents of palmitic and stearic acids: Not less than 90.0%.

Ethylene oxide and dioxan (Appendix 10.15, method 1). Ethylene oxide: Not more than 1 ppm. Dioxan: Not more than10 ppm. Heavy metals Not more than 10 ppm (Appendix 9.4.8). 2.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Water Not more than 3.0% (Appendix 10.3). Determined on 1.00 g. Total ash Not more than 0.25% (Appendix 9.8, method 2). Determined on 2.0 g. Storage Store in an airtight container, protected from light. Action and use Non-ionic surfactants.

POLYSORBATE 80

VP V

POLYSORBATE 80 Polysorbatum 80 Polysorbate 80 is a mixture of partial esters of fatty acids, mainly oleic acid, with sorbitol and its anhydrides ethoxylated with approximately 20 moles of ethylene oxide for each mole of sorbitol and sorbitol anhydrides.

Characters Oily, yellow or brownish-yellow, clear or slightly opalescent liquid. Dispersible in water, in anhydrous ethanol, in ethyl acetate and in methanol, practically insoluble in fatty oils and in liquid paraffin. Relative density: About 1.10. Viscosity: About 400 mPa·s at 25 °C. Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D, E. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the reference spectrum of polysorbate 80. B. It complies with the test for Hydroxyl value. C. It complies with the test for Saponification value. D. It complies with the test for Composition of fatty acids. E. Dissolve 0.1 g of the substance to be examined in 5 ml of methylene chloride R. Add 0.1 g of potassium thiocyanate R and 0.1 g of cobalt nitrate R. Stir with a glass rod. The solution becomes blue. Acid value Not more than 2.0 (Appendix 7.2). Dissolve 5.0 g of the substance to be examined in 50 ml of the prescribed solvent mixture. Hydroxyl value 65 to 80 (Appendix 7.4, method A). Saponification value 45 to 55 (Appendix 7.7). To 4.0 g add 30.0 ml of 0.5 M potassium hydroxide in ethanol, heat under reflux for 60 min and dilute with 50 ml of ethanol (96%) R before carrying out the titration. Peroxide value Not more than 10.0. Introduce 10.0 g of the substance to be examined into a 100 ml beaker and dissolve with 20 ml of glacial acetic acid R. Add 1 ml of saturated potassium iodide solution R, mix and allow to stand for 1 min. Add 50 ml of carbon dioxide-free water R and a magnetic stirring bar. Titrate with 0.01 N sodium thiosulfate VS, determining the endpoint potentiometrically (Appendix 10.2). Carry out a blank titration. Determine the peroxide value using the following expression:

(n1 − n2) × M × 1000 m

Where: n1: volume of 0.01 N sodium thiosulfate VS required for the substance to be examined, in millilitres. n2: volume of 0.01 N sodium thiosulfate VS required for the blank, in millilitres. M: molarity of the sodium thiosulfate solution, in moles per litre. M: mass of substance to be examined, in grams.

Composition of fatty acids (Appendix 12.9, method C) Use a reference mixture as described in Table 12.9.3 to prepare a reference solution (a). Chromatographic system: A fused-silica capillary column (30 m × 0.32 mm) coated with a film of macrogol 20 000 R (film thickness 0.5 µm). Carrier gas: Helium for chromatography R. Flow rate: 50 cm/s. Temperature programme:

Column

Time (min)

Temperature (°C)

0 - 14 14 - 54

80 → 220 220

Injection port

250

Detector

250

Detector: A flame-ionisation detector. Volume of injection: 1 µl. Limits: Composition of the fatty-acid fraction of the substance: Myristic acid : Not more than 5.0%. Palmitic acid : Not more than 16.0%. Palmitoleic acid : Not more than 8.0%. Stearic acid : Not more than 6.0%. Oleic acid : Not less than 58.0%. Linoleic acid : Not more than 18.0%. Linolenic acid : Not more than 4.0%.

Ethylene oxide and dioxan Not more than 1 ppm of ethylene oxide and not more than 10 ppm of dioxan. Examine by gas chromatography (Appendix 5.2). Ethylene oxide stock solution: Dilute 0.5 ml of a commercially available solution of ethylene oxide in methylene chloride (50 mg/ml) to 50.0 ml with water. [NOTE: the solution is stable for 3 months, if stored in vials with polytetrafluoroethylene coated silicone membrane crimped caps at - 20 °C]. Allow to cool at room temperature. Dilute 1.0 ml of this solution to 250.0 ml with water. Dioxan stock solution: Dilute 1.0 ml of dioxan R to 200.0 ml with water. Dilute 1.0 ml of this solution to 100.0 ml with water. Acetaldehyde stock solution: Weigh about 0.100 g of acetaldehyde R into a 100 ml volumetric flask and dilute 781

VP V

POTASSIUM BROMIDE

to 100.0 ml with water. Dilute 1.0 ml of this solution to 100.0 ml with water. Standard solution: To 6.0 ml of ethylene oxide stock solution add 2.5 ml of dioxan stock solution and dilute to 25.0 ml with water. Test solution (1): Weigh 1.00 g of the substance to be examined into a 10 ml head-space vial. Add 2.0 ml of water R, seal the vial immediately with a polytetrafluoroethylene coated silicon membrane and an aluminum cap. Mix carefully. Test solution (2): Weigh 1.00 g of the substance to be examined into a 10 ml head-space vial. Add 2.0 ml of standard solution, seal the vial immediately with a polytetrafluoroethylene coated silicon membrane and an aluminum cap. Mix carefully. Reference solution  Introduce 2.0 ml of acetaldehyde stock solution and 2.0 ml of ethylene oxide stock solution into a 10 ml head-space vial and seal the vial immediately with a polytetrafluoroethylene coated silicon membrane and an aluminum cap. Mix carefully. Chromatographic system: A fused-silica column (50 m × 0.53 mm) the wall is coated with poly(dimethyl)(diphenyl)siloxane R (film thickness 0,5 µm). Carrier gas: Helium for chromatography R. Flow rate: 4.0 ml/min. Split ratio: 1 : 3 : 5. Static head-space conditions: equilibration temperature: 80 °C; equilibration time: 30 min Temperature program:

Column

Time (min)

Temperature (°C)

0 - 18 18 - 23

70 → 250 250

Injection port

85

Detector

250

Detector: A flame-ionisation detector. Volume of injection: 1.0 ml. Procedure: Inject the test solutions (1) and (2) and of the reference solution. Relative retention with reference to ethylene oxide (retention time = about 6.5 min): acetaldehyde = about 0.9; dioxan = about 1.9. System suitability: In the chromatogram obtained with reference solution, the resolution between the peaks due to acetaldehyde and ethylene oxide is at least 2.0. Calculate the content of ethylene oxide using the following expression: 2CEO × Aa Ab − Aa Where: CEO: concentration of ethylene oxide in test solution (2), in micrograms per millilitre. 782

Aa: peak area of ethylene oxide in the chromatogram obtained with test solution (1). Ab: peak area of ethylene oxide in the chromatogram obtained with test solution (2). Calculate the content of dioxan using the following expression: 2 × 1.03 × CD × Aa' Ab' − Aa' Where: CD: concentration of dioxan in test solution (2), in microlitres per millilitre. 1.03: density of dioxan, in grams per millilitre. Aa’: peak area of dioxan in the chromatogram obtained with test solution (1). Ab’: peak area of dioxan in the chromatogram obtained with test solution (2).

Heavy metals Not more than 10 ppm (Appendix 9.4.8). 2.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Water Not more than 3.0% (Appendix 10.3). Determined on 1.00 g. Total ash Not more than 0.25% (Appendix 9.8, method 2). Determined on 2.0 g. Storage Store in an airtight container, protected from light. Action and use Non-ionic surfactants. POTASSIUM BROMIDE Kalii bromidum KBr   M. 119.0 Potassium bromide contains not less than 98.5% and not more than 101.0% of KBr, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder or colourless crystals. Freely soluble in water and in glycerol, slightly soluble in ethanol (96%). Identification A. It gives reaction (A) of bromides (Appendix 8.1). B. Solution S: Dissolve 10.0 g in carbon dioxide-free water R and dilute to 100 ml with the same solvent. Solution S gives the reactions of potassium (Appendix 8.1).

VP V

Appearance of solution Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). Acidity or alkalinity To 10 ml of solution S, add 0.1 ml of bromothymol blue solution R. Not more than 0.5 ml of 0.01 N hydrochloric acid VS or 0.01 N sodium hydroxide VS is required to change the colour of the indicator. Chlorides and sulfates Examine by liquid chromatography (Appendix 5.3) Mobile phase: Dissolve 0.600 g of potassium hydroxide R in water for chromatography R and dilute to 1000.0 ml with the same solvent. Test solution (1): Dissolve 0.400 g of the substance to be examined in 50 ml of water for chromatography R and dilute to 100.0 ml with the same solvent. Test solution (2): Dilute 25.0 ml of test solution (1) to 50.0 ml with water for chromatography R. Reference solution (1): To 25.0 ml of test solution (1) add 1.0 ml of sulfate standard solution (10 ppm SO4) R and 12.0 ml of chloride standard solution (50 ppm Cl) R and dilute to 50.0 ml with water for chromatography R. Reference solution (2): Dilute 10.0 ml of test solution (1) to 100.0 ml with water for chromatography R. To 2.0 ml of this solution add 8.0 ml of chloride standard solution (50 ppm Cl) R and dilute to 20.0 ml with water for chromatography R. Blank solution: water for chromatography R. Chromatographic system: A column (25 cm × 2.06 mm) packed with stationary phase strongly basic anion-exchange resin for chromatography R (13 µm). Detector: Conductivity detector equipped with a suitable ion suppressor. Flow rate: 0.4 ml/min. Volume of injection: 50 µl. Procedure: Inject the blank, the test solution (2) and the reference solution (1), (2). The run time is 2.5 times the retention time of bromide. Retention time: Chloride = about 5 min; bromide = about 8 min; sulfate = about 16 min. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to chloride and bromide is at least 8.0. Limits: Correct the areas of the peaks obtained with test solution (2) and reference solution (1) using the areas of the peaks obtained with the blank solution: Chlorides: the area of the peak due to chloride in test solution (2) is not more than the difference between the areas of the peaks due to chloride in the chromatograms obtained with test solution (2) and reference solution (1) (0.6%).

POTASSIUM BROMIDE

Sulfates: the area of the peak due to sulfate in test solution (2) is not more than the difference between the areas of the peaks due to sulfate in the chromatograms obtained with test solution (2) and reference solution (1) (0,01%).

Bromates To 10 ml of solution S add 1 ml of starch solution R, 0.1 ml of a 10% solution of potassium iodide R and 0.25 ml of 0.5 M sulfuric acid R and allow to stand protected from light for 5 min. No blue or violet colour develops. Iodides To 5 ml of solution S add 0.15 ml of a 10.5% solution of ferric (III) chloride R and 2 ml of methylene chloride R. Shake and allow to separate. The lower layer is colourless (Appendix 9.3, method 1). Iron Not more than 20 ppm (Appendix 9.4.13). Dilute 5 ml of solution S to 10 ml with water. Magnesium and alkaline-earth metals Not more than 0.02%, calculated as Ca (Appendix 9.4.16). 10.0 g complies with the test for magnesium and alkalineearth metals. The volume of 0.01 M sodium edetate VS used does not exceed 5.0 ml. Heavy metals Not more than 10 ppm (Appendix 9.4.8). 12 ml of solution S complies with limit test for heavy metals, method 1. Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on drying Not more than 1.0% (Appendix 9.6). (1.000 g; 105 °C; 3 h). Assay Dissolve 100.0 mg of the substance to be examined in water, add 5 ml of 2 M nitric acid R and dilute to 50 ml with water. Titrate with 0.1 N silver nitrate VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 M silver nitrate is equivalent to 11.90 mg of KBr. Calculate the percentage content of KBr using the following expression: a – 3.357 × b Where: a: percentage content of KBr and KCl obtained in the assay and calculated as KBr. b: percentage content of Cl obtained in the test for Chlorides and sulfates.

783

VP V

POTASSIUM CHLORIDE

POTASSIUM CHLORIDE Kalii chloridum KCl



the solution is not more intense than that in a mixture of 5.0 ml of solution S and 6.0 ml of water. M. 74.6

Potassium chloride contains not less than 99.0% and not more than 100.5% of KCl, calculated with reference to the dried substance.

Heavy metals Not more than 10 ppm (Appendix 9.4.8). Take 12.0 ml of solution S and carry out method 1. Prepare the standard using lead standard solution(1 ppm Pb) R.

Characters White, crystalline powder or colourless crystals, odourless. Freely soluble in water, practically insoluble in ethanol.

Iron Not more than 20 ppm (Appendix 9.4.13). Take 5.0 ml of solution S, diluted to 10 ml with water and carry out the limit test for iron.

Identification Solution S: Dissolve 10.0 g in carbon dioxide-free water R, dilute to 100 ml with the same solvent. Solution S gives reactions of chlorides and potassium (Appendix 8.1).

Magnesium and alkaline-earth metals Not more than 0.02%, calculated as Ca (Appendix 9.4.16). 10.0 g complies with the limit test for magnesium and alkaline-earth metals. The volume of 0.01 M sodium edetate VS used does not exceed 5.0 ml.

Appearance of solution Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2).

Loss on drying Not more than 1.0% (Appendix 9.6). (1.000 g; 100 °C - 105 °C, 3 h).

Acidity or alkalinity Take 50.0 ml of solution S, add 0.1 ml of bromothymol blue solution R. The colour of solution is changed when adding not more than 0.5 ml of 0.01 N hydrochloric acid VS or 0.01 N sodium hydroxide VS.

Sodium Not more than 0.1% of Na, if intended for use in the manufacture of parenteral preparations or haemodialysis solutions. Determined by atomic emission spectrometry (Appendix 4.4, method 1). Test solution: Dissolve 1.00 g of the substance to be examined in water and dilute to 100.0 ml with the same solvent. Reference solutions: Dissolve in water 0.5084 g of sodium chloride R, previously dried at 100 °C to 105 °C for 3 h, and dilute to 1000.0 ml with the same solvent (200 µg of Na per ml). Dilute as required. Measure the emission intensity at 589 nm.

Sulfates Not more than 0.03% (Appendix 9.4.14). Take 5 ml of solution S, dilute to 15 ml with water and carry out the limit test for sulphates. Iodides Moisten 5.0 g by the dropwise addition of a freshly prepared mixture of 25 ml of iodide-free starch solution R, 2 ml of 0.5 M sulphuric acid R, 0.15 ml of a 10% sodium nitrite solution R and 25 ml of water. After 5 min, examine in daylight. The mixture shows no blue colour spot or particle. Bromides Not more than 0.1%. Dilute 1.0 ml of solution S to 50 ml with water. To 5.0 ml of the solution add 2.0 ml of phenol red solution R2 and 1.0 ml of 0.02% solution of chloramines T R and mix immediately. After exactly 2 min add 0.15 ml of 0.1 M sodium thiosulfate R, mix and dilute to 10.0 ml with water. The absorbance (Appendix 4.1) of the solution measured at 590 nm, using water as the blank, is not greater than that of a standard prepared at the same time and in the same manner using 5.0 ml of a 3.0 mg/l solution of potassium bromide R. Barium Take 5.0 ml of solution S, add 5.0 ml of water and 1.0 ml of 1 M sulfuric acid R. After 15 min, any opalescence in 784

Aluminium Not more than 1 ppm (Appendix 9.4.9), if intended for use in the manufacture of haemodialysis solutions. Test solution: Dissolve 4.0 g of the substance to be examined in 100 ml of water and add 10 ml of acetate buffer solution pH 6.0 R. Reference solution: Mix 2.0 ml of aluminium standard solution (2 ppm Al) R, 10 ml of acetate buffer solution pH 6.0 R and 98 ml of water. Blank solution: Mix 10 ml of acetate buffer solution pH 6.0 R and 100 ml of water. Assay Dissolve 1.300 g in water and dilute to 100.0 ml with the same solvent. Take 10.0 ml of the solution into a conical flask, add 50 ml of water, 5 ml of 12.5% solution of nitric acid R, 25.0 ml of 0.1 N silver nitrate VS and 2 ml of dibutyl phthalate R (or nitrobenzene). Shake. Titrate with 0.1 N ammonium thiocyanate VS , using 2 ml of 10% solution of ferric ammonium sulfate R as indicator.

POTASSIUM CHLORIDE TABLETS

VP V

1 ml of 0.1 N silver nitrate VS is equivalent to 7.46 mg of KCl.

valid dilution of solution A calculated from the declared sensitivity of the lysate used in the test.

Storage Store in a well-closed container, in a dry place and protected from light.

Assay To an accurately measured volume of the injection, equivalent to about 0.15 g of potassium chloride add 30 ml of water, and mix. Titrate with 0.1 N silver nitrate VS using a 5% solution of potassium chromate R as indicator. 1 ml of 0.1 N silver nitrate VS is equivalent to 7.46 mg of KCl.

Action and use Treatment of electrolyte deficiency. Preparations Potassium chloride injection; potassium chloride, sodium chloride and glucose intravenous infusion; potassium chloride and glucose intravenous infusion; oral rehydration salts; prolonged-release potassium chloride tablets.

Storage Store in a dry and cool place, protected from light.

Labelling The label states, where applicable, that the substance is suitable for use in the manufacture of parenteral preparations or haemodialysis solutions.

Usual strength 10%.

POTASSIUM CHLORIDE INJECTION CONCENTRATE Injectio Kalii chloridi concentrata Potassium chloride injection concentrate is a sterile solution of potassium chloride in water for injections. The injection complies with the requirements stated under "Injections, infusions" (Appendix 1.19) and with the following requirements.

Content of potassium chloride, KCl, 95.0% to 105.0% of the stated amount. Characters A clear, colourless solution. Identification It gives the reactions characteristic of potassium and chlorides (Appendix 8.1). Acidity or alkalinity Dilute, if necessary, the injection with carbon dioxide-free water R to contain 10.0% of potassium chloride. To 50 ml of the solution add 0.1 ml of bromothymol blue solution R. Not more than 0.5 ml of 0.01 M hydrochloric acid VS or 0.01 M sodium hydroxide VS is required to change the colour of the indicator. Bacterial endotoxins Carry out the test for bacterial endotoxins (Appendix 13.2). Dilute the injection with water BET to obtain a 0.5% solution of potassium chloride and adjust the pH, if necessary, to 7 (solution A). The endotoxin limit concentration of solution A is 3.0 EU of endotoxin per ml. Carry out the test using a lysate with a declared sensitivity not less sensitive than 0.0625 EU of endotoxin per ml, and using the maximum

Action and use Treatment of electrolyte deficiency.

POTASSIUM CHLORIDE TABLETS Tabellae Kalii chloridi Potassium chloride tablets contain potassium chloride and are formulated so as to release the medicament over a period of several hours. They are coated. The tablets comply with the requirements stated for “Coated tablets” under “Tablets” (Appendix 1.20) and with the following requirements.

Content of potassium chloride, KCl, 95.0% to 105.0% of the stated amount. Identification To a quantity of the coating-removed and powdered tablets, equivalent to about 1 g of potassium chloride, add 20 ml of water, sonicate for 20 minutes, and filter. The filtrate yields the reactions characteristic of potassium and the reactions characteristic of chlorides (Appendix 8.1). Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of water. Rotation speed: 50 rpm. Time: 1 h, 2 h and 6 h. Procedure: Withdraw a sample of 10.0 ml of the medium after 1 h, 2 h and 6 h and treat each sample in the following manner. Add 25 ml of water, 5 ml of a 25% v/v solution of glacial acetic acid R and 0.1 ml of a saturated solution of potassium sulfate R and titrate with 0.01 N silver nitrate VS determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.01 N silver nitrate VS is equivalent to 0.746 mg of KCl. Tolerance: The amount of potassium chloride, KCl, released after 1 hour is not more than 50%, after 2 hours is not less than 25% and not more than 75%, and after 785

VP V

POTASSIUM CLAVULANATE

6 hours is not less than 75%, calculated with reference to the declared content of potassium chloride.

Assay Test solution: Shake 10 whole tablets with 400 ml of water for 30 minutes, then heat on a water bath for 45 min. Cool, add sufficient water to produce 500.0 ml and allow to stand for 24 h. Filter, discarding the first 20 ml of filtrate, and dilute with water to give a suitable concentration. Reference solution: Dilute an accurately measured volume of potassium standard solution (600 ppm K) with water to give a suitable concentration. Carry out the method for atomic emission spectrophotometry (Appendix 4.4), measuring at 766.5 nm. 1 mg of potassium, K, is equivalent to 1.908 mg of KCl. Storage Store in an airtight container in a dry and cool place, protected from light. Action and use Treatment of electrolyte deficiency. Usual strength 600 mg. POTASSIUM CLAVULANATE Kalii clavulanas

C8H8KNO5

M. 237.3

Potassium clavulanate is potassium (2R,3Z,5R)-3-(2hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo[3.2.0] heptane-2-carboxylate, the potassium salt of a substance produced by the growth of certain strains of Streptomyces clavuligerus or obtained by any other means. It contains not less than 96.5% and not more than 102.0% of C8H8KNO5, calculated with reference to the anhydrous substance.

Characters White or almost white, crystalline powder, hygroscopic. Freely soluble in water, slightly soluble in ethanol (96%), very slightly soluble in acetone. Production The method of production, extraction and purification are such that clavam-2-carboxylate is eliminated or present at a level not exceeding 0.01%. Identification A. The infrared absorption spectrum (Appendix 4.2) of the 786

substance to be examined is concordant with the spectrum obtained with potassium clavulanate RS. B. It gives reaction (B) of potassium (Appendix 8.1).

pH 5.5 to 8.0 (Appendix 6.2). Solution S: Dissolve 0.400 g in carbon dioxide-free water R and dilute to 20.0 ml with the same solvent. Dilute 5 ml of solution S to 10 ml with carbon dioxide-free water R. Specific optical rotation +53° to +63°, calculated with reference to the anhydrous substance (Appendix 6.4). Determine on solution S. Absorbance Not more than 0.40 at 278 nm (Appendix 4.1). Dissolve 50.0 mg in 0.1 M phosphate buffer solution pH 7.0 and dilute to 50.0 ml with the same solution. Measure immediately the absorbance of this solution. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: A 0.78% solution of sodium dihydrogen phosphate adjusted to pH 4.0 with phosphoric acid R and filtered through a 0.5 µm filter. Mobile phase B: Mobile phase A - methanol (1 : 1). Prepare the solutions immediately before use. Test solution: Dissolve 0.250 g of the substance to be examined in mobile phase A and dilute to 25.0 ml with mobile phase A. Reference solution: Dilute 1.0 ml of the test solution to 100.0 ml with mobile phase A. Resolution solution: Dissolve 10 mg of lithium clavulanate RS and 10 mg of amoxicillin trihydrate RS in mobile phase A and dilute to 100 ml with mobile phase A. Chromatographic system: A stainless steel column (10 cm × 4.6 mm) packed with stationary phase C (5 µm), maintaining the temperature of the column at 40 °C. Detector: A spectrophotometer set at 230 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0-4

100

0

4 - 15

100 → 50

0 → 50

15 - 18

50

50

18 - 24

50 → 100

50 → 0

24 - 39

100

0

POTASSIUM CLAVULANATE

VP V

System suitability: In the chromatogram obtained with the resolution solution, the resolution between the first peak (clavulanate) and the second peak (amoxicillin) is at least 13. Limits: In the chromatogram obtained with the test solution: The area of any impurity peak is not more than the area of the principal peak in the chromatogram obtained with the reference solution (1.0%). The sum of the areas of all peaks, apart from the principal peak is not more than twice the area of the principal peak in the chromatogram obtained with the reference solution (2.0%). Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with the reference solution (0.05%).

Aliphatic amines Not more than 0.2%. Examine by gas chromatography (Appendix 5.2). The method shown below can be used to determine the following aliphatic amines: 1,1-dimethylethylamine; diethylamine; N,N,N’,N’-tetramethylethylenediamine; 1,1,3,3 -tetramethylbutylamine; N,N’-diisopropylethylenediamine; 2,2’-oxydi(N,N) dimethylethylamine. Internal standard solution: Dissolve 50 µl of 3-methylpentan2-one in water and dilute to 100.0 ml with the same solvent. Test solution: Weigh 1.00 g of the substance to be examined into a centrifuge tube. Add 5.0 ml of the internal standard solution, 5.0 ml of dilute sodium hydroxide solution R, 10.0 ml of water, 5.0 ml of 2-methylpropanol R and 5 g of sodium chloride R. Shake vigorously for 1 minute. Centrifuge to separate the layers. Use the upper layer. Reference solution: Dissolve 80.0 mg of each of the following amines: 1,1-dimethylethylamine (Impurity H); diethylamine (Impurity I); N,N,N’,N’-tetramethylethylenediamine (Impurity J); 1,1,3,3-tetramethylbutylamine (Impurity K); N,N’-diisopropylethylenediamine (Impurity L) and 2,2’-oxybis(N,N-dimethylethylamine) (Impurity M) in dilute hydrochloric acid R and dilute to 200.0 ml with the same solvent. Introduce 5.0 ml of this solution into a centrifuge tube. Add 5.0 ml of the internal standard solution, 10.0 ml of dilute sodium hydroxide solution R, 5.0 ml of 2-methylpropanol R and 5 g of sodium chloride R. Shake vigorously for 1 minute. Centrifuge to separate the layers. Use the upper layer. Chromatographic system: A fused silica column (50 m × 0.53 mm), the wall is coated with stationary phase poly(dimethyl)(diphenyl) siloxane R (film thickness 5 µm). Carrier gas: Helium for chromatography R. Flow rate: 8 ml/min. Split ratio: 1:10. Temperature:

Column

Time (minutes)

Temperature (°C)

0→7

35

7 → 10.8

35 → 150

10.8 → 25.8

150

Injection port

200

Detector

250

Detector: A flame-ionisation detector. Volume of injection: 1 µl. Procedure: Relative retention times with reference to 3-methylpentan2-one (retention time = about 11.4 minutes): impurity H = about 0.55; impurity I = about 0.76; impurity J = about 1.07; impurity K = about 1.13; impurity L = about 1.33; impurity M = about 1.57. Limits: In the chromatogram obtained with the test solution: The area of any impurity peaks is not more than that of the corresponding peaks in the chromatogram obtained with the reference solution.

2-Ethylhexanoic acid Not more than 0.8% (Appendix 10.17). Water Not more than 0.5% (Appendix 10.3). Determine on 1.0 g. Sterility If intended for use in the manufacture of parenteral preparations without a further appropriate sterilisation procedure, it complies with the test for sterility (Appendix 13.7). Bacterial endotoxins Less than 0.03 EU/mg if intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins (Appendix 13.2). Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol - phosphate buffer solution pH 4.0 (dissolve 15 g of sodium dihydrogen phosphate R in 1000 ml of water, adjusted to pH 4.0 with dilute phosphoric acid R) (5 : 95). Prepare the solutions immediately before use. Test solution: Dissolve 50.0 mg of the substance to be examined in a 0.41% solution of sodium acetate previously adjusted to pH 6.0 with glacial acetic acid R, and dilute to 50.0 ml with the same solvent. Reference solution: Dissolve 50.0 mg of lithium clavulanate RS in a 0.41% solution of sodium acetate previously adjusted to pH 6.0 with glacial acetic acid R, and dilute to 50.0 ml with the same solvent. Resolution solution: Dissolve 50.0 mg of lithium clavulanate RS and 50.0 mg of amoxicillin trihydrate RS 787

VP V

POTASSIUM IODIDE

in a 0.41% solution of sodium acetate previously adjusted to pH 6.0 with glacial acetic acid R and dilute to 50.0 ml with the same solvent. Chromatographic system: A column (30 cm × 4.6 mm) packed with octadecylsilyl silica gel for chromatography (5 µm). Detector: A spectrophotometer set at 230 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Procedure: System suitability: In the chromatogram obtained with the resolution solution, the resolution between the first peak (clavulanate) and the second peak (amoxicillin) is at least 3.5. Inject the reference solution and the test solution. 1 mg of clavulanate (C8H9NO5) is equivalent to 1.191 mg of C8H8KNO5. Calculate the content of clavulanate (C8H9NO5), using the areas of the clavulanate peaks in the chromatograms obtained with the test solution and the reference solution, and the declared content of C8H9NO5 in lithium clavulanate RS.

Storage Store in an airtight container, at a temperature of 2 °C to 8 °C. If the substance is sterile, store in a sterile, airtight, tamper-proof container. Labelling The label states, where applicable, that the substance is sterile and free from bacterial endotoxins.

Appearance of solution Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). Alkalinity Take 10 ml of solution S, add 0.1 ml of 0.05 M sulfuric acid R and 1 drop of phenolphthalein solution R. Solution is colourless. Iodates Take 10 ml of solution S, add 0.25 ml of iodide-free starch solution R and 0.2 ml of 10% solution of sulfuric acid R and allow to stand protected from light for 2 min. No blue colour develops. Sulfates Not more than 0.015% (Appendix 9.4.14). Dilute 10 ml of solution S to 15 ml with water and carry out the limit test for sulfates. Thiosulfates To 10 ml of solution S add 0.1 ml of starch solution R and 0.1 ml of 0.005 M iodine. A blue colour is produced. Heavy metals Not more than 10 ppm (Appendix 9.4.8). Take 12 ml of solution S and carry out method 1. Prepare the standard using lead standard solution (1 ppm Pb) R.

Action and use Beta-lactamase inhibitor.

Iron Not more than 20 ppm (Appendix 9.4.13). Take 5 ml of solution S, diluted to 10 ml with water and carry out the limit test for iron.

Preparations Tablets, powder for suspension, injection (in combination with amoxicillin).

Loss on drying Not more than 1.0% (Appendix 9.6). (1.000 g; 100 °C - 105 °C, 3 h).

POTASSIUM IODIDE Kalii iodidum KI



M. 166.0

Potassium iodide contains not less than 99.0% and not more than 100.5% of KI, calculated with reference to the dried substance.

Characters White, crystalline powder or colourless crystals, odourless. Liquefied on contact with moisture. Very soluble in water, freely soluble in glycerin, soluble in ethanol 96%. Identification Solution S: Dissolve 10.0 g in carbon dioxide-free water R, dilute to 100 ml with the same solvent. Solution S gives reaction characteristic of iodides and reaction characteristic of potassium (Appendix 8.1). 788

Assay Dissolve 1.500 g in water and dilute to 100.0 ml with the same solvent. To 20.0 ml of the solution add 40 ml of hydrochloric acid R and titrate with 0.05 M potassium iodate VS until the colour changes from red to yellow. Add 5 ml of chloroform R and continue the titration, shaking vigorously, until the chloroform layer is decolourised. 1 ml of 0.05 M potassium iodate VS is equivalent to 16.60 mg of KI. Storage Store in a well-closed container, protected from light. Action and use Antithyroid. Preparations Oral solution.

POVIDONE

VP V

POTASSIUM PERMANGANATE Kalii permanganatum KMnO4 M.158.0 Potassium permanganate contains not less than 99.0% and not more than 100.5% of KMnO4.

Characters A dark purple or brownish-black, granular powder or dark purple or almost black crystals, usually having a metallic lustre, odourless. It decomposes and explodes on contact with certain organic and oxidizing substances. Soluble in cold water, freely soluble in boiling water. Caution should be taken when working with potassium permanganate, because on direct contact with certain organic substances and oxidizing substances can cause the explosion even in both liquid and solid state. Identification A. Dissolve about 50 mg in 5 ml of water and add 1 ml of ethanol (96%) R and 0.3 ml of dilute sodium hydroxide solution R. A green colour develops. Heat to boiling. A dark brown precipitate is formed. B. Filter the mixture obtained in Identification test A. The filtrate gives reactions of potassium (Appendix 8.1). Colour of solution Solution S: Dissolve 0.75 g in 25 ml of water, add 3 ml of ethanol (96%) R and heat to boiling for 2 - 3 minutes. Cool, dilute to 30 ml with water and filter. Solution S is colourless (Appendix 9.3, method 2). Chlorides Not more than 0.02% (Appendix 9.4.5). Dilute 10 ml of solution S to 15 ml with water and carry out the test for chlorides. Sulfates Not more than 0.05% (Appendix 9.4.14). Dilute 12 ml of solution S to 15 ml with water and carry out the limit test for sulfates. Substances insoluble in water Not more than 1.0%. Dissolve 0.5 g in 50 ml of water and heat to boiling. Filter through a tared sintered-glass filter (16). Wash the filter with water until the filtrate is colourless and collect the residue on the filter. The residue, dried in an oven at 100 °C to 105 °C to constant weight, weighs not more than 5 mg. Assay Dissolve 0.300 g in water and dilute to 100.0 ml with the same solvent. To 20.0 ml of the solution in a conical flask with a ground-glass stopper add 20 ml of water, 1 g of potassium iodide R and 10 ml of dilute hydrochloric acid R. Titrate the liberated iodine with

0.1 N sodium thiosulfate VS. Using 1 ml of starch solution R as indicator and add the indicator when solution become pale colour. 1 ml of 0.1 N sodium thiosulfate VS is equivalent to 3.16 mg of KMnO4.

Storage Store in an airtight bottle, protected from light. Action and use Antiseptic. POVIDONE Povidonum

C6nH9n+2NnOn Povidone is α-hydro-ω-hydropoly[1-(2-oxopyrrolidin1-yl)ethylene]. It consists of linear polymers of 1-ethenylpyrrolidin-2-one. It contains 11.5% to 12.8% of nitrogen (N; Ar 14.01), calculated with reference to the anhydrous substance. The different types of povidone are characterised by their viscosity in solution expressed as a K-value.

Characters White or yellowish-white, hygroscopic powder or flakes. Freely soluble in water, in ethanol (96%) and in methanol, very slightly soluble in acetone. Identification Apply one of the two following identifications: First identification: A, E. Second identification: B, C, D, E. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of povidone RS. Dry the substances beforehand at 105 °C for 6 h; record the spectra using 4 mg of substance. B. To 0.4 ml of solution S1 (see Appearance of solution), add 10 ml of water 5 ml of dilute hydrochloric acid R and 2 ml of a 10.6% solution of potassium dichromate R. An orange-yellow precipitate is formed. C. Add 0.2 ml of dimethylaminobenzaldehyde solution R1 and 0.1 ml of sulfuric acid R to 1 ml of solution S1 a pink colour is produced. D. Add 5 ml of water and 0.2 ml of 0.05 M iodine R to 0.1 ml of solution S1. A red colour is produced. E. Add 10 ml of water to 0.5 g of the substance to be examined and shake. The substance dissolves. 789

POVIDONE

Appearance of solution Solution S1: Dissolve 2.5 g of the substance to be examined in carbon dioxide-free water R and dilute to 25 ml with the same solvent. Add the substance to be examined to the water in small portions, stirring using a magnetic stirrer. Solution S: Dissolve 1.0 g of the substance to be examined in carbon dioxide-free water R and dilute to 20.0 ml with the same solvent. Add the substance to be examined to the water in small portions, stirring using a magnetic stirrer. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution B6, BY6 or R6 (Appendix 9.3, method 2). pH (Appendix 6.2). 3.0 to 5.0 for solution S, for povidone having a stated K-value of not more than 30. 4.0 to 7.0 for solution S, for povidone having a stated K-value of more than 30. Viscosity, expressed as K-value For povidone having a stated value of 18 or less, use a 50 g/L solution. For povidone having a stated value of more than 18 and not more than 95, use a 10 g/L solution. For povidone having a stated value of more than 95, use a 1.0 g/L solution. Allow to stand for 1 h and determine the viscosity (Appendix 6.3, method 1) of the solution at 25 °C, using a size no. 1 viscometer with a minimum flow time of 100 s. Calculate the K-value using the following expression:

1,5 log η -1 + 300c log η +(c + 1,5 c log η)2 0,15 + 0,003 c 0,15 c + 0,003 c 2 Where: c: concentration of the substance to be examined, calculated with reference to the anhydrous substance, in grams per 100 ml. η: kinematic viscosity of the solution relative to that of water. The K-value of povidone having a stated K-value of not 15 or less is 85.0% to 115.0% of the stated value. The K-value of povidone having a stated K-value or a stated K-value range with an average of more than 15 is 90.0% to 108.0% of the stated value or of the average of the stated range.

Aldehydes Not more than 0.05%, expressed as acetaldehyde. Test solution: Dissolve 1.0 g of the substance to be examined in phosphate buffer solution pH 9.0 R and dilute to 100.0 ml with the same solvent. Stopper the flask tightly and heat at 60 °C for 1 h. Allow to cool to room temperature. Reference solution: Dissolve 0.140 g of acetaldehyde ammonia trimer trihydrate R in water and dilute to 200.0 ml with the same solvent. Dilute 1.0 ml of the solution to 100.0 ml with 790

VP V

phosphate buffer solution pH 9.0 R. Procedure: Into 3 identical spectrophotometric cells with a path length of 1 cm, introduce separately 0.5 ml of the test solution, 0.5 ml of the reference solution and 0.5 ml of water (blank). To each cell add 2.5 ml of phosphate buffer solution pH 9.0 R and 0.2 ml of nicotinamide-adenine dinucleotide solution R. Mix and stopper tightly. Allow to stand at 22 °C ± 2 °C for 2 min to 3 min and measure the absorbance (Appendix 4.1) of each solution at 340 nm, using water as the compensation liquid. To each cell add 0.05 ml of aldehyde dehydrogenase solution R, mix and stopper tightly. Allow to stand at 22 °C ± 2 °C for 5 min. Measure the absorbance of each solution at 340 nm using water as the compensation liquid. Calculate the content of aldehydes using the following expression: (At2 − At1) − (Ab2 − Ab1) 100 000 × C × (As2 − As1) − (Ab2 − Ab1) m Where: At1: absorbance of the test solution before the addition of aldehyde dehydrogenase. At2: absorbance of the test solution after the addition of aldehyde dehydrogenase. As1: absorbance of the reference solution before the addition of aldehyde dehydrogenase. As2: absorbance of the reference solution after the addition of aldehyde dehydrogenase. Ab1: absorbance of the blank before the addition of aldehyde dehydrogenase. Ab2: absorbance of the blank after the addition of aldehyde dehydrogenase. m: mass of povidone calculated with reference to the anhydrous substance, in grams. C: concentration of acetaldehyde in the reference solution, calculated from the weight of the acetaldehyde ammonia trimer trihydrate with the factor 0.72, in milligrams per millilitre.

Peroxides Not more than 0.04%, expressed as H2O2. Stock solution: Dissolve a quantity of the substance to be examined equivalent to 4.0 g of the anhydrous substance in water and dilute to 100.0 ml with the same solvent. Test solution: Add 2.0 ml of titanium trichloride-sulfuric acid reagent R to 25.0 ml of the stock solution. Allow to stand for 30 min. Blank solution: Add 2.0 ml of a 13% (v/v) solution of sulfuric acid R to 25.0 ml of the stock solution. The absorbance (Appendix 4.1) of the test solution, measured at 405 nm is not greater than 0.35. Hydrazine Not more than 1 ppm. Examine by thin-layer chromatography (Appendix 5.4). Use freshly prepared solutions.

VP V

Coating substance: Silanised silica gel F254. Mobile phase: Water - methanol R (1 : 2). Test solution: Dissolve a quantity of the substance to be examined equivalent to 2.5 g of the anhydrous substance in 25 ml of water. Add 0.5 ml of a 5% solution of salicylaldehyde R in methanol R, mix and heat in a waterbath at 60 °C for 15 min. Allow to cool, add 2.0 ml of toluene R, shake for 2 min and centrifuge. Use the upper layer of the mixture. Reference solution: Dissolve 90 mg of salicylaldehyde azine R in toluene R and dilute to 100 ml with the same solvent. Dilute 1 ml of the solution to 100 ml with toluene R. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of three quarters of the plate. Allow the plate to dry in air and examine in ultraviolet light at 365 nm. Retardation factor of salicylaldehyde azine is about 0.3. Any spot due to salicylaldehyde azine is not more intense than the spot in the chromatogram obtained with the reference solution.

Formic acid Not more than 0.5%. Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dilute 5 ml of perchloric acid R to 1000 ml with water. Test solution: Dissolve a quantity of the substance to be examined equivalent to 2.0 g of the anhydrous substance in water and dilute to 100.0 ml with the same solvent (test stock solution). Transfer a suspension of strongly acidic ion-exchange resin R for column chromatography in water to a column of about 0.8 cm in internal diameter to give a packing of about 20 mm in length and keep the strongly acidic ion-exchange resin layer constantly immersed in water. Pour 5 ml of water and adjust the flow rate so that the water drops at a rate of about 20 drops per minute. When the level of the water comes down to near the top of the strongly acidic ion-exchange resin layer, put the test stock solution into the column. After dropping 2 ml of the solution, collect 1.5 ml of the solution and use this solution as the test solution. Reference solution: Dissolve 0.100 g of anhydrous formic acid R in water and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of the solution to 100.0 ml with water. Chromatographic system: A column (25 cm - 30 cm), 4 mm - 8 mm in internal diameter, packed with stationary phase strongly acidic ionexchange resin R for column chromatography (5 - 10 µm). Column temperature: 30 °C. Detector: A spectrophotometer set at 210 nm. Flow rate: Adjusted so that the retention time of formic acid is about 11 min. Volume of injection: 50 µl. Procedure: System suitability: In the chromatogram obtained with standard solution, the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%.

POVIDONE

Limit: Formic acid: not more than 10 times the area of the principal peak in the chromatogram obtained with the reference solution (0.5%).

Impurity A Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - water (10 : 90). Test solution: Dissolve a quantity of the substance to be examined equivalent to 0.250 g of the anhydrous substance in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (1): Dissolve 50.0 mg of 1-vinylpyrrolidin-2-one R (impurity A) in methanol R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of the solution to 100.0 ml with methanol R. Dilute 5.0 ml of this solution to 100.0 ml with the mobile phase. Reference solution (2): Dissolve 10 mg of 1-vinylpyrrolidin2-one R and 0.5 g of vinyl acetate R in methanol R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of the solution to 100.0 ml with the mobile phase. Chromatographic system: Precolumn (2,5 cm × 4 mm) packed with stationary phase C (5 µm). A column (25 cm × 4 mm) packed with stationary phase C (5 µm). Column temperature: 40 °C. Detector: A spectrophotometer set at 235 nm. Flow rate: Adjusted so that the retention time of impurity A is about 10 min. Volume of injection: 50 µl. Procedure: System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurity A and vinyl acetate is at least 2.0. The relative standard deviation for 6 replicate injections of the reference solution (1) is not more than 2.0%. After injection of the test solution, wait for about 2 min and wash the precolumn by passing the mobile phase through the column backwards for 30 min at the same flow rate as applied in the test. Limit: In the chromatogram obtained with the test solution: The area of the peak due to impurity A is not more than the area of the principal peak in the chromatogram obtained with reference solution (1). Note: Impurity A: 1-ethenylpyrrolidin-2-one (1-vinylpyrrolidin-2-one).

Impurity B Examine by liquid chromatography (Appendix 5.3). Mobile phase: Water adjusted to pH 2.4 with phosphoric acid R. Test solution: Dissolve a quantity of the substance to be examined equivalent to 0.100 g of the anhydrous substance in water and dilute to 50.0 ml with the same solvent. 791

VP V

IODINATED POVIDONE

Reference solution: Dissolve 0.100 g of 2-pyrrolidone R (impurity B) in water and dilute to 100.0 ml with the same solvent. Dilute 3.0 ml of the solution to 50.0 ml with water. Chromatographic system: Precolumn (2,5 cm × 3 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (5 µm). A column (25 cm × 3 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (5 µm). Column temperature: 30 °C. Detector: A spectrophotometer set at 205 nm. Flow rate: Adjusted so that the retention time of impurity B is about 11 min. Volume of injection: 50 µl. Procedure: System suitability: The relative standard deviation of the peak areas for 6 replicate injections of the reference solution is not more than 2.0%. After each injection of the test solution, wash away the polymeric material of povidone from the precolumn by passing the mobile phase through the column backwards for about 30 min at the same flow rate as applied in the test. Limit: In the chromatogram obtained with test solution: The area of the peak due to impurity B is not more than the area of the principal peak in the chromatogram obtained with the reference solution (3.0%). Note: Impurity B: pyrrolidin-2-one (2-pyrrolidone).

Heavy metals Not more than 10 ppm (Appendix 9.4.8). 2.0 g complies with limit test for heavy metals, method 4. Prepare the standard using 2.0 ml of lead standard solution (10 ppm Pb) R. Water Not more than 5.0% (Appendix 10.3). Determined on 0.500 g. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Place 0.100 g of the substance to be examined (m mg) in a combustion flask and add 5 g of a mixture of 1 g of copper sulfate R, 1 g of titanium dioxide R and 33 g of dipotassium sulfate R, and 3 glass beads. Wash any adhering particles from the neck into the flask with a small quantity of water R. Add 7 ml of sulfuric acid R, allowing it to run down the insides of the flask. Heat the flask gradually until the solution has a clear, yellowish-green colour, and the inside wall of the flask is free from any carbonised material, and then heat for a further 45 min. After cooling, add cautiously 792

20 ml of water R, and connect the flask to the distillation apparatus, which has been previously washed by passing steam through it. To the absorption flask add 30 ml of a 4% solution of boric acid R, 3 drops of bromocresol greenmethyl red solution R and sufficient water to immerse the lower end of the condenser tube. Add 30 ml of strong sodium hydroxide solution R through the funnel, rinse the funnel cautiously with 10 ml of water, immediately close the clamp on the rubber tube, then start distillation with steam to obtain 80-100 ml of distillate. Remove the absorption flask from the lower end of the condenser tube, rinsing the end part with a small quantity of water. Titrate the distillate with 0.025 M sulfuric acid VS until the colour of the solution changes from green through pale greyish blue to pale greyish reddish-purple. Carry out a blank determination. 1 ml of 0.025 M sulfuric acid VS is equivalent to 0.7004 mg of N.

Storage In an airtight container. FUNCTIONALITY-RELATED CHARACTERISTICS The following characteristics may be relevant for povidone used as solubiliser and stabiliser in liquid dosage forms. Viscosity Determine the dynamic viscosity using a capillary viscometer on a 10% solution (dried substance) at 25 °C. Typical values are shown in Table 1. Molecular mass (see Viscosity, expressed as K-value) Typical values are shown in Table 1. The following characteristic may be relevant for povidone used as binder in tablets and granules. Table 1 - Molecular mass (see Viscosity, expressed as K-value) Viscosity range (mPa·s)

Molecular mass: Viscosity, expressed as K-value

Povidone K 12

1.3 - 2.3

11 - 14

Povidone K 17

1.5 - 3.5

16 - 18

Povidone K 25

3.5 - 5.5

24 - 27

Povidone K 30

5.5 - 8.5

28 - 32

Povidone K 90

300 - 700

85 - 95

IODINATED POVIDONE Povidonum iodinatum Iodinated povidone is complex of iodine and povidone. It contains 9.0% to 12.0% of available iodine, calculated with reference to the dried substance.

POVIDONE-IODINE SOLUTION

VP V

Production It is produced using povidone that complies with the monograph on Povidone, except that the povidone used may contain not more than 2.0% of formic acid and not more than 8.0% of water. Characters Yellowish-brown or reddish-brown, amorphous powder. Soluble in water and in ethanol (96%), practically insoluble in acetone. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of iodinated povidone RS. B. Dissolve 10 mg of the substance to be examined in 10 ml of water and add 1 ml of starch solution R. An intense blue colour is produced. pH 1.5 to 5.0 (Appendix 6.2). Dissolve 1.0 g of the substance to be examined in 10 ml of carbon dioxide-free water R. Iodide Not more than 6.0% calculated with reference to the dried substance. Dissolve 0.500 g of the substance to be examined in 100 ml of water R. Add sodium metabisulfite R until the colour of the iodine has disappeared. Add 25.0 ml of 0.1 N silver nitrate VS, 10 ml of nitric acid R and 5 ml of a 10% solution of ferric ammonium sulfate R. Titrate with 0.1 N ammonium thiocyanate. Carry out a blank titration. 1 ml of 0.1 N silver nitrate VS is equivalent to 12.69 mg of total iodine. From the percentage of total iodine, calculated with reference to the dried substance, subtract the percentage of available iodine as determined in the test for Assay to obtain the percentage of iodide. Loss on drying Not more than 8.0% (Appendix 9.6). (0.500 g; 105 °C; 3 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Transfer 1.000 g of the substance to be examined into a ground-glass-stoppered flask containing 150 ml of water and stir for 1 h. Add 0.1 ml of dilute acetic acid R and titrate with 0.1 N sodium thiosulfate VS using starch solution R as indicator. 1 ml of 0.1 N sodium thiosulfate VS is equivalent to 12.69 mg of available iodine. Storage Protected from light.

Action and use Antiseptic. Preparations Eye drops, mouthwash, solution. POVIDONE-IODINE SOLUTION Solutio Povidoni Iodini Povidone-iodine solution is a solution for cutaneous application of iodinated povidone in water. The preparation complies with the requirements stated under “Solutions” (Appendix 1.3) and with the following requirements.

Content of iodine, I, 0.85 to 1.20% (w/v) of the stated amount. Characters A deep brown liquid. Identification A. Dilute 1 ml of the solution being examined to 20 ml with water and to 1 ml of the resulting solution add a mixture of 1 ml of starch mucilage R and 9 ml of water. A deep blue colour is produced. B. Transfer 10 ml of the solution being examined to a 50 ml conical flask and cover the mouth of the flask with filter paper moistened with 0.05 ml of starch mucilage R. No blue colour is produced on the paper within 60 seconds. C. Dilute 20 ml of the solution being examined to 100 ml with water. To 10 ml of this solution add drop wise 0.1 M sodium thiosulphate R until the colour of iodine is just discharged. Reserve 5 ml of the resulting solution for test D. To 5 ml of the resulting solution add 10 ml of 1 M hydrochloric acid R and 5 ml of a 7.0% solution of potassium dichromate R. A red precipitate is produced. D. To 5 ml of the solution reserved in test C add 2 ml of ammonium cobaltthiocyanate solution R (prepared by dissolving 3.75 g of cobalt nitrate R and 15 g of ammonium thiocyanate R in water and add sufficient water to produce 100 ml, use within 1 day of preparation) previously acidified with 5 M hydrochloric acid R. A blue precipitate is produced. pH 1.5 to 6.5 (Appendix 6.2). Iodide Not more than 0.6% when determined by the following method. Dilute 5 ml of the solution being examined to 100 ml with water and add sodium metabisulphite R until the colour of iodine has disappeared. Add 25 ml of 0.1 N silver nitrate VS, 10 ml of nitric acid R and 5 ml of a 10% solution of ferric ammonium sulfate solution R. Titrate with 0.1 N 793

VP V

PRAZIQUANTEL

ammonium thiocyanate VS. Repeat the procedure without the solution being examined. Each ml of 0.1 N silver nitrate VS is equivalent to 12.69 mg of total iodine. Calculate the percentage content of total iodine and subtract the percentage content of available iodine determined in the Assay to obtain the percentage content of iodide.

Assay To 10.0 ml of the solution being examined add 10 ml of 0.1 M hydrochloric acid R and sufficient water to produce 150 ml. Titrate with 0.02 N sodium thiosulphate VS determining the end-point potentiometrically (Appendix 10.2). Each ml of 0.02 N sodium thiosulphate VS is equivalent to 2.538 mg of iodine, I. Storage Store in a well-closed container, in a cool place, protected from light. Action and use Antiseptic. Usual strength 10% solution. PRAZIQUANTEL Praziquantelum

C19H24N2O2 

M. 312.4

Praziquantel is (11bRS)-2-(cyclohexylcarbonyl)-1,2,3,6,7,11bhexahydro-4H-pyrazino[2,1-a]isoquinolin-4-one. It contains not less than 97.5% and not more than 102.0% of C19H24N2O2, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder, it shows polymorphism. Very slightly soluble in water, freely soluble in ethanol (96%) and in methylene chloride. Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of praziquantel RS. If the spectra obtained show differences, dissolve 50 mg of the substance to be examined and 50 mg of the reference substance separately in 2 ml of methanol R. Evaporate and dry the residue at 60 °C at a pressure not exceeding 0.7 kPa. Record new spectra using the residues. 794

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile R1 - water for chromatography R (45 : 55). Test solution (1): Dissolve 40.0 mg of the substance to be examined in the mobile phase and dilute to 10.0 ml with the mobile phase. Test solution (2): Dilute 5.0 ml of test solution (1) to 100.0 ml with the mobile phase. Reference solution (1): Dissolve 40.0 mg of praziquantel RS in the mobile phase and dilute to 10.0 ml with the mobile phase. Dilute 5.0 ml of this solution to 100.0 ml with the mobile phase. Reference solution (2): Dissolve 2 mg of praziquantel for system suitability RS (containing impurities A and B) in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (3): Dilute 1.0 ml of test solution (1) to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.0 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 210 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Inject the test solution (1) and reference solutions (2) and (3). The run time is 4 times the retention time of praziquantel. Identification of impurities: Use the chromatogram supplied with praziquantel for system suitability RS and the chromatogram obtained with reference solution (2) to identify the peaks due to impurities A and B. Relative retention with reference to praziquantel (retention time = about 10 min): impurity A = about 0.6; impurity B = about 2.2. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurity A and praziquantel is at least 3.0. Limits: Correction factor: for the calculation of content, multiply the peak area of impurity B by 1.4; Impurities A, B: For each impurity, the area corrected if necessary, is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (3) (0.2%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (3) (0.10%). The sum of the peak areas of all impurities is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (3) (0.5%).

VP V

Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (3) (0.05%). Note: Impurity A: (11bRS)-2-benzoyl-1,2,3,6,7,11b-hexahydro-4Hpyrazino[2,1-a]isoquinolin-4-one. Impurity B: 2-(cyclohexylcarbonyl)-2,3,6,7-tetrahydro-4Hpyrazino[2,1-a]isoquinolin-4-one. Impurity C: N-formyl-N-[2-oxo-2-(1-oxo-3,4-dihydroisoquinolin2(1H)- yl)ethyl]cyclohexanecarboxamide.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 50 °C; over diphosphorus pentoxide R at a pressure not exceeding 0.7 kPa; 2 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for related substances. Inject the test solution (2) and reference solution (1). Calculate the content of C19H24N2O2, using the areas of the praziquantel peaks in the chromatograms obtained with the test solution (2), reference solution (1) and the declared content of C19H24N2O2 in praziquantel RS. Storage Protected from light. Action and use Anthelminthic. Preparation Tablets. PRAZIQUANTEL TABLETS Tabellae Praziquanteli Praziquantel tablets contain praziquantel. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of praziquantel, C19H24N2O2, 90.0% to 110.0% of the stated amount. Identification A. In the Dissolution, the ultraviolet absorption spectrum

PRAZIQUANTEL TABLETS

(Appendix 4.1) of the test solution is concordant with that of the reference solution and exhibits an absorption maximum at 263 ± 1 nm. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that of the principal peak in the chromatogram obtained with the reference solution.

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of 0.1 M hydrochloric acid containing 2 mg of sodium lauryl sulfate R per ml. Rotation speed: 50 rpm. Time: 60 min. Procedure: After the specified time, withdraw a sample of the medium, filter and discard the first 20 ml of the filtrate. Dilute a portion of the filtrate with the medium, if necessary. Measure the absorbances (Appendix 4.1) of the resulting solution at the maximum at 263 ± 1 nm in a 1 cm cell, in comparison with a 0.06% solution of praziquantel RS in the medium using the medium as a blank. Calculate the content of praziquantel, C19H24N2O2, dissolved using the absorbances obtained and the declared content of C19H24N2O2 in praziquantel RS. Tolerance: Not less than 75% (Q) of the stated amount of praziquantel, C19H24N2O2, is dissolved in 60 minutes. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - water (60 : 40), make adjustments if necessary. Reference solution: Dissolve an accurately weighed quantity of praziquantel RS in the mobile phase to obtain a solution containing 0.18 mg per ml. Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Transfer a quantity of the powdered tablets containing 0.15 g of praziquantel, accurately weighed, to a 100 ml volumetric flask, add 70 ml of the mobile phase, sonicate for 5 minutes. Dilute to volume with the mobile phase and mix. Filter and dilute 3.0 ml of the filtrate to 25.0 ml with the mobile phase and mix. Chromatographic system: A column (25 cm × 4 mm) packed with stationary phase C (10 µm). Detector: A spectrophotometer set at 210 nm. Flow rate: 1.5 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject the reference solution, the symmetry factor of praziquantel peak is not more than 1.5 and the relative standard deviation of the peak areas for replicate injections is not more than 1.0%. Inject alternately the test solution and the reference solution. Calculate the content of praziquantel, C19H24N2O2, in the tablets using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C19H24N2O2 in praziquantel RS. 795

VP V

PREDNISOLONE

Storage Store in a well-closed container, at a temperature not exceeding 30 °C. Action and use Anthelmintic. Usual strength 600 mg. PREDNISOLONE Prednisolonum

C21H28O5  

M. 360.4

Prednisolone is 11β,17,21-trihydroxypregna-1,4-diene3,20-dione. It contains not less than 96.5% and not more than 102.0% of C21H28O5, calculated with reference to the dried substance.

Characters White or almost white, crystalline, hygroscopic powder, it shows polymorphism. Very slightly soluble in water, soluble in ethanol (96%) and in methanol, sparingly soluble in acetone, slightly soluble in methylene chloride. Dentification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of prednisolone RS. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in the minimum volume of acetone R, evaporate to dryness on a water-bath and record new spectra using the residues. B. In the test for Assay, the principal peak in the chromatogram obtained with test solution (2) is similar in retention time and size to the principal peak in the chromatogram obtained with reference solution (4). Specific optical rotation +113° to +119°, calculated with reference to the dried substance (Appendix 6.4). Dissolve 0.250 g of the substance to be examined in ethanol (96%) R and dilute to 25.0 ml with the same solvent. Related substances Examine by liquid chromatography (Appendix 5.3). Carry out the test protected from light. 796

Mobile phase A: Water. Mobile phase B: Acetonitrile - methanol (50 : 50). Solvent mixture: Acetonitrile - water (40 : 60). Test solution (1): Dissolve 10 mg of the substance to be examined in the solvent mixture and dilute to 20.0 ml with the solvent mixture. Test solution (2): Dissolve 25.0 mg of the substance to be examined in the solvent mixture and dilute to 20.0 ml with the solvent mixture. Dilute 1.0 ml of the solution to 10.0 ml with the solvent mixture. Reference solution (1): Dissolve 5 mg of prednisolone for system suitability RS (containing impurities A, B and C) in the solvent mixture and dilute to 10.0 ml with the solvent mixture. Reference solution (2): Dissolve 5 mg of prednisolone for peak identification RS (containing impurities F and J) in the solvent mixture and dilute to 10.0 ml with the solvent mixture. Reference solution (3): Dilute 1.0 ml of test solution (1) to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 10.0 ml with the solvent mixture. Reference solution (4): Dissolve 25.0 mg of prednisolone RS in the solvent mixture and dilute to 20.0 ml with the solvent mixture. Dilute 1.0 ml of the solution to 10.0 ml with the solvent mixture. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (3 µm). Column temperature: 40 °C. Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 14

60

40

14 - 20

60 → 20

40 → 80

20 - 25

20

80

Inject the test solution (1) and reference solutions (1), (2) and (3). Identification of impurities: Use the chromatogram supplied with prednisolone for system suitability RS and the chromatogram obtained with reference solution (1) to identify the peaks due to impurities A, B and C; use the chromatogram supplied with prednisolone for peak identification RS and the chromatogram obtained with reference solution (2) to identify the peaks due to impurities F and J. Relative retention with reference to prednisolone (retention time = about 12 min): impurity F = about 0.7; impurity B = about 0.9; impurity A = about 1.05; impurity J = about 1.5; impurity C = about 1.7.

PREDNISOLONE TABLETS

VP V

System suitability: In the chromatogram obtained with reference solution (1), peak-to-valley ratio (Hp/Hv) is at least 3; where Hp = height above the baseline of the peak due to impurity A and Hv = height above the baseline of the lowest point of the curve separating this peak from the peak due to prednisolone. Limits: Impurity A: The area of the peak due to impurity A is not more than 10 times the area of the principal peak in the chromatogram obtained with reference solution (3) (1.0%). Impurity F: The area of the peak due to impurity F is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (3) (0.5%). Impurities B, C, J: For each impurity, the area is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (3) (0.3%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (3) (0.10%). The sum of the peak areas of all impurities is not more than 15 times the area of the principal peak in the chromatogram obtained with reference solution (3) (1.5%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (3) (0.05%). Note: Impurity A: 11β,17,21-trihydroxypregn-4-ene-3,20-dione (hydrocortisone). Impurity B: 17,21-dihydroxypregna-1,4-diene-3,11,20-trione (prednisone). Impurity C: 11β,17-dihydroxy-3,20-dioxopregna-1,4-dien-21-yl acetate (prednisolone acetate). Impurity D: 6β,11β,17,21-tetrahydroxypregna-1,4-diene-3,20dione (6β- hydroxyprednisolone). Impurity E: 11β,14α,17,21-tetrahydroxypregna-1,4-diene-3,20dione (14α- hydroxyprednisolone). Impurity F: 11α,17,21-trihydroxypregna-1,4-diene-3,20-dione (11-epi-prednisolone). Impurity G: 11β,17,20β,21-tetrahydroxypregna-1,4-dien-3-one (20β-hydroxyprednisolone). Impurity H: 11β,17,21-trihydroxypregna-1,4,6-triene-3,20-dione (∆6-prednisolone). Impurity I: 11β,21-dihydroxypregna-1,4-diene-3,20-dione (17-deoxyprednisolone). Impurity J: 17,21-dihydroxypregna-1,4-diene-3,20-dione (11-deoxyprednisolone).

Loss on drying Not more than 1.0% (Appendix 9.6). (0.500 g; 105 °C). Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. Inject test solution (2) and reference solution (4). Calculate the content of C21H28O5, using the peak areas in the chromatograms obtained with test solution (2),

reference solution (4) and the declared content of C21H28O5 in prednisolone RS.

Storage In an airtight container, protected from light. Action and use Glucocorticoid. Preparation Tablets. PREDNISOLONE TABLETS Tabellae Prednisoloni Prednisolone tablets contain prednisolone. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of prednisolone, C21H28O5, 90.0% to 110.0% of the stated amount. Identification A. Extract a quantity of the powdered tablets with acetone R, filter and evaporate the filtrate to dryness. The infrared absorption spectrum (Appendix 4.2) of the residue is concordant with the reference spectrum of prednisolone. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Kieselguhr G plate prepared by impregnating with a mixture of formamide-acetone (1 : 9). Allow the solvent to ascend to the top, remove the plate and allow the solvent to evaporate completely. Use the plate within 2 h. Mobile phase: Chloroform. Diluent: Chloroform - methanol (9 : 1). Test solution: Dissolve 25 mg of the residue obtained in the test A in 10 ml of the diluent. Reference solution (1): Dissolve 25 mg of prednisolone RS in 10 ml of the diluent. Reference solution (2): A mixture of equal volume of the test solution and reference solution (1). Procedure: Apply separately to the plate 2 µl of each solution. Develop over a path of 15 cm. Remove the plate, allow to dry in air, dry the plate at 120 °C for 15 min, spray with sulfuric acid solution in ethanol R. Heat the plate in the oven at 120 °C for 10 min. Allow to cool. Examine in daylight and under ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, colour under daylight, in fluorescent at 365 nm, and in size to the principal spot in the chromatogram obtained with reference solution (1). The test is not valid unless the principal spot in the chromatogram obtained with reference solution (2) appears a single spot. 797

VP V

PREDNISOLONE TABLETS

Related substances Examined by liquid chromatography (Appendix 5.3). Prepare the following solutions immediately before use and protect from light. Mobile phase: Mix 220 ml of tetrahydrofuran R with 700 ml of water, mix carefully and allow it to stand. Dilute to 1000.0 ml with water, mix. Test solution: Shake a quantity of the powdered tablets equivalent to 10 mg of prednisolone with 25 ml of methanol R for 10 min, sonicate for 2 min. Filter the extract and wash the filter with 2 portions of 10 ml of methanol R. Combine the filtrate and the washings, evaporate to dryness using a rotary evaporator and a warm water - bath. Dissolve the obtained residue in 10 ml of tetrahydrofuran R and dilute to 20.0 with water. Reference solution: Dilute 1.0 ml of the test solution to 100.0 ml with a 50% v/v solution of tetrahydrofuran R. Resolution solution: Dissolve 2 mg of prednisolone RS and 2 mg of hydrocortisone RS in the mobile phase to obtain 100.0 ml. Blank: A 50% v/v solution of tetrahydrofuran R. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 μm). Alltima C18 is suitable. Column temperature: 45 °C. Detector: A spectrophotometer set at 254 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Procedure: Equilibrate the column with the mobile phase for 30 min. System suitability: With the above conditions, the retention time of prednisolone is about 14 min and of hydrocortisone is about 15.5 min. The test is not valid unless the resolution factor between the peaks due to prednisolone and hydrocortisone is at least 2.2. If necessary, adjust the concentration of tetrahydrofuran in the mobile phase. Inject alternately the blank, reference solution, test solution. Record the chromatogram for 4.5 times the retention time of the principal peak. Limits: In the chromatogram obtained with the test solution, the area of any secondary peak is not greater than the area of the peak in the chromatogram obtained with the reference solution (1%). The sum of the areas of all secondary peaks is not greater than 3 times the area of the principal peak in the chromatogram obtained with the reference solution (3%). Disregard any secondary peak having the area less than 0.05 times the area of the principal peak in the chromatogram obtained with the reference solution and any peak with the retention time of 3 min or less. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of water. Rotation speed: 50 rpm. Time: 45 min. 798

Procedure: After the specified time, withdraw a sample of the medium, filter. Determine the content of prednisolone dissolved by liquid chromatography (Appendix 5.3). Mobile phase and chromatographic system: Proceed as described in the Assay. Use the reference solution of prednisolone RS in water having the same concentration as that expected in the test solution. An amount of methanol R not to exceed 5% of the total volume of the reference solution may be used to dissolve prednisolone RS before diluting with water. Tolerance: Not less than 75% (Q) of the stated amount of prednisolone, C21H28O5, is dissolved in 45 min.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol - water (58 : 42). Reference solution: A solution containing 0.005% of prednisolone RS and 0.0075% of dexamethasone RS (internal standard) in a mixture of methanol and water (58 : 42). Test solution (1): Weigh 20 tablets, calculate the average mass and powder finely. Weigh accurately a quantity of the powdered tablets containing 5 mg of prednisolone, transfer into a 100-ml volumetric flask, add 58 ml of methanol R, shake for 10 min. Dilute to volume with water, mix and filter. Test solution (2): Prepare in the same manner as test solution (1) but add 10 ml of a 0.075% solution of dexamethasone in methanol R (internal standard) and 48 ml of methanol R. Chromatographic system: A column (20 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution, the resolution factor between the prednisolone and dexamethasone peaks is greater than 2.5 and the column efficiency, determined on prednisolone peak, is greater than 15 000 theoretical plates per meter. Inject alternately the test solutions and the reference solution. Calculate the content of prednisolone, C21H28O5, in the tablets using the ratios of the peak areas of prednisolone to those of dexamethasone in the chromatograms obtained with the test solution, the reference solution and the declared content of C21H28O5 in prednisolone RS. Storage Store in a well-closed container, protected from light. Action and use Corticosteroid. Usual strength 5 mg.

PREDNISONE

VP V

PREDNISONE Prednisonum

O CH3

O CH3

H O

H

OH OH

H

C21H26O5

M. 358.4

Prednisone is 17,21-dihydroxypregna-1,4-diene-3,11,20trione, contains not less than 97.0% and not more than 103.0% of C21H26O5, calculated with reference to the dried substance.

Characters A white or almost white, polymorphic crystalline powder. Practically insoluble in water, slightly soluble in ethanol (96%) and in methylene chloride. Identification Apply one of the two following identifications: First identification: A, B. Second identification: C, D. A. The infrared absorption spectrophotometry (Appendix 4.2) of the substance to be examined is concordant with the spectrum of prednisone RS. If the spectra show differences, dissolve the substance to be examined and the reference substance separately in the minimum volume of acetone R, evaporate to dryness on a water-bath and record new spectra using the residues. B. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel GF254. Mobile phase: Water - methanol - ether - methylene chloride (1.2 : 8 : 15 : 77). Test solution: Dissolve 10 mg of the substance to be examined in a mixture of methanol R and methylene chloride R (1 : 9) and dilute to 10 ml with the same mixture of solvents. Reference solution (1): Dissolve 20 mg of prednisone RS in a mixture of methanol R and methylene chloride R (1 : 9) and dilute to 20 ml with the same mixture of solvents. Reference solution (2): Dissolve 10 mg of betamethasone RS in reference solution (1) and dilute to 10 ml with the same solvent. Procedure: Apply to the plate 5 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (1). Spray with alcoholic solution of sulfuric acid R. Heat at 120 °C for 10 min or until the spots appear. Allow to cool. Examine the plate in daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and

size to the principal spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) shows two clearly separated spots. C. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel GF254. Mobile phase: Water - methanol - ether - methylene chloride (1.2 : 8 : 15 : 77). Test solution (1): Dissolve 25 mg of the substance to be examined in methanol R and dilute to 5 ml with the same solvent (solution A). Dilute 2 ml of solution A to 10 ml with methylene chloride R. Test solution (2): Transfer 0.4 ml of solution A to a glass tube (100 mm × 20 mm) and fitted with a ground-glass stopper or a polytetrafluoroethylene cap and evaporate the solvent with gentle heating under a stream of nitrogen R. Add 2 ml of a 15% (v/v) solution of acetic acid R and 50 mg of sodium bismuthate R. Stopper the tube and shake the suspension in a mechanical shaker, protected from light, for 1 h. Add 2 ml of a 15% (v/v) solution of acetic acid R and filter into a 50 ml separating funnel, washing the filter with two quantities, each of 5 ml, of water. Shake the clear filtrate with 10 ml of methylene chloride R. Wash the organic layer with 5 ml of 1M sodium hydroxide R and then wash with two quantities, each of 5 ml, of water. Dry over anhydrous sodium sulfate R. Reference solution (1): Dissolve 25 mg of prednisone RS in methanol R and dilute to 5 ml with the same solvent (solution B). Dilute 2 ml of solution B to 10 ml with methylene chloride R. Reference solution (2): Carry out the same procedure as described in Test solution (2) using 0.4 ml of solution B instead of solution A. Procedure: Apply to the plate 5 µl of test solution (1) and reference solution (1); 50 µl of test solution (2) and reference solution (2), applying the latter two solution in small quantities in order to obtain small spots. Develop over a path of 15 cm. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in each of the chromatograms obtained with the test solutions is similar in position and size to the principal spot in the chromatogram obtained with the corresponding reference solution. Spray with alcoholic solution of sulphuric acid R. Heat at 120 °C for 10 min or until the spots appear. Allow to cool. Examine the chromatograms in daylight and in ultraviolet light at 365 nm. The principal spot in each of the chromatograms obtained with the test solutions is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with the corresponding reference solution. The principal spots in the chromatograms obtained with test solution (2) and reference solution (2) have an Rf value distinctly higher than that of the principal spots in the chromatograms obtained with test solution (1) and reference solution (1). D. Add about 2 mg of the substance to be examined to 799

VP V

PRIMAQUINE DIPHOSPHATE

2 ml of sulphuric acid R and shake to dissolve. Within 5 min, a yellow colour develops with a blue fluorescence in ultraviolet light at 365 nm. Add the solution to 10 ml of water and mix. The colour fades but the blue fluorescence in ultraviolet light does not disappear.

Specific optical rotation +167° to +175°, calculated with reference to the dried substance (Appendix 6.4). Dissolve 0.125 g of the substance to be examined in dioxan R and dilute to 25.0 ml with the same solvent. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: In a 1000 ml volumetric flask mix 100 ml of acetonitrile R with 200 ml of methanol R and 650 ml of water, allow to equilibrate; make up to the volume with water and mix again. Mobile phase B: Acetonitrile R. Test solution: Dissolve 25.0 mg of the substance to be examined in methanol R and dilute to 10.0 ml with the same solvent. Reference solution: Dilute 1.0 ml of the test solution to 100.0 ml with methanol R. Resolution solution: Dissolve 2 mg of prednisone RS and 2 mg of prednisolone RS in methanol R and dilute to 100.0 ml with the same solvent. Chromatographic system: A stainless steel column (0.25 m × 4.6 mm) packed with octadecylsilyl silica gel for chromatography R (5 µm). Column temperature: 45 °C. Detector: A spectrophotometer set at 254 nm, Flow rate: 2.5 ml/min. Volume of injection: 20 µl Procedure: A linear gradient program using the following conditions: Time (min)

Mobile phase A (% V/V)

Mobile phase B (% V/V)

Comment

0

100

0

Isocratic

25

100

0

Begin linear gradient

40

40

60

End chromatogram, change to 100 B

41

0

100

Begin treatment with B

46

0

100

End treatment, return to 100 A

47

100

0

Begin equilibration with A

52 = 0

100

0

End equilibration, begin next chromatogram

Equilibrate the column for at least 30 min with mobile phase B and then with mobile phase A for 5 minutes. For subsequent chromatograms, use the conditions described from 40.0 to 52.0 min. Adjust the sensitivity of the system 800

so that the height of the principal peak in the chromatogram obtained with the reference solution is not less than 50% of the full scale of the recorder. System suitability: Inject resolution solution. When the chromatograms are recorded in the prescribed conditions, the retention times are: prednisone = about 19 minutes and prednisolone = about 23 minutes. The test is not valid unless the resolution between the peaks corresponding to prednisone and prednisolone is at least 2.7; if necessary, adjust the concentration of acetonitrile in mobile phase A. Inject separately methanol R as a blank, the test solution and reference solution. Limits: In the chromatogram obtained with the test solution: the area of any peak apart from the principal peak is not greater than 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (0.25%); the sum of the areas of all the peaks, apart from the principal peak, is not greater than 0.75 times the area of the principal peak in the chromatogram obtained with reference solution (0.75%). Disregard any peak due to the blank and any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution.

Loss on drying Not more than 1.0% (Appendix 9.6) (0.500 g; 100 °C - 105 °C). Assay Dissolve 0.100 g of the substance to be examined in ethanol (96%) R and dilute to 100.0 ml with the same solvent. Dilute 2.0 ml of the solution to 100.0 ml with ethanol (96%) R. Measure the absorbance (Appendix 4.1) at the maximum at 238 nm. Calculate the content of C21H26O5 taking 425 as the value of A (1%, 1 cm) at the maximum at 238 nm Storage Protected from light. Action and use Corticosteroid. Preparation Tablets. PRIMAQUINE DIPHOSPHATE Primaquini Diphosphas

C15H21N3O,2H3PO4  

M. 455.3

VP V

Primaquine diphosphate is (4RS)-N4-(6-methoxyquinolin8-yl)pentane-1,4-diamine bisphosphate. It contains not less than 98.5% and not more than 101.5% of C15H21N3O,2H3PO4, calculated with reference to the dried substance.

Characters Orange crystalline powder. Soluble in water, practically insoluble in ethanol (96%). Melting point: About 200 °C with decomposition. Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of primaquine diphosphate RS. Examine the substances prepared as discs. Dissolve separately 0.1 g of the substance to be examined and 0.1 g of the reference substance in 5 ml of water R, add 2 ml of 2 M ammonia R and 5 ml of methylene chloride R, then shake. Dry the methylene chloride layer over 0.5 g of anhydrous sodium sulfate R. Prepare a blank disc using about 0.3 g of potassium bromide R. Apply dropwise to the disc 0.1 ml of the methylene chloride layer, allowing the methylene chloride to evaporate between applications. Dry the disc at 50 °C for 2 min. B. Ultraviolet and visible absorption spectrophotometry (Appendix 4.1). Test solution (1): Dissolve 15 mg of the substance to be examined in 0.01 M hydrochloric acid R and dilute to 100.0 ml with the same acid. Test solution (2): Dilute 5.0 ml of test solution (1) to 50.0 ml with 0.01 M hydrochloric acid R. The ultraviolet absorption spectrum of test solution (1) in the range 310 nm to 450 nm, exhibits two absorption maxima at 332 mn and 415 nm. The specific absorbance at this maxima are 45 to 52, and 27 to 35 respectively. The ultraviolet absorption spectrum of test solution (2) in the range 215 nm to 310 nm, exhibits three absorption maxima at 225 mn, 265 nm and 282 nm. The specific absorbance at this maxima are 495 to 515; 335 to 350 and 330 to 345, respectively. C. Examine by thin-layer chromatography (Appendix 5.4). Carry out all operations as rapidly as possible, protected from light. Prepare the solutions immediately before use. Coating substance: Silica gel GF254. Mobile phase: Concentrated ammonia - methanol methylene chloride R (1 : 40 : 60). Test solution: Dissolve 0.20 g of the substance to be examined in 5 ml of water and dilute to 10 ml with methanol R. Dilute 1 ml of this solution to 10 ml with a mixture of equal volumes of methanol R and water. Reference solution: Dissolve 20 mg of primaquine diphosphate RS in 5 ml of water and dilute to 10 ml with methanol R.

PRIMAQUINE DIPHOSPHATE

Procedure: Wash the plate with the mobile phase and allow to dry in air. Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. D. Dissolve 50 mg of the substance to be examined in 5 ml of water. Add 2 ml of dilute sodium hydroxide solution R and shake with 2 quantities, each of 5 ml, of methylene chloride R. The aqueous layer, acidified by addition of nitric acid R, gives reaction (B) of phosphates (Appendix 8.1).

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Concentrated ammonia - methanol - hexane - methylene chloride (0.1 : 10 : 45 : 45). Test solution: Dissolve 50 mg of the substance to be examined in water and dilute to 5.0 ml with the same solvent. To 1.0 ml of this solution add 0.2 ml of concentrated ammonia R and shake with 10.0 ml of the mobile phase. Use the clear lower layer. Reference solution (1): Dissolve 50 mg of primaquine diphosphate RS in water and dilute to 5.0 ml with the same solvent. To 1.0 ml of this solution add 0.2 ml of concentrated ammonia R and shake with 10.0 ml of the mobile phase. Use the clear lower layer. Reference solution (2): Dilute 3.0 ml of the test solution to 100.0 ml with the mobile phase. Reference solution (3): Dilute 1.0 ml of the test solution to 10.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 50.0 ml with the mobile phase. Chromatographic system: A column (20 cm × 4.6 mm) packed with stationary phase silica gel for chromatography R (10 µm). Detector: A spectrophotometer set at 261 nm. Flow rate: 3.0 ml/min. Volume of injection: 20 µl. Procedure: Inject the blank, the test solution and the reference solutions. The run time is at least twice the retention time of primaquine. System suitability: In the chromatogram obtained with reference solution (1), it shows just before the principal peak a peak whose area is about 6% of that of the principal peak; the resolution between the peak just before the principal peak and the principal peak is at least 2.0. In the chromatogram obtained with reference solution (3), the signal-to-noise ratio of the principal peak is not less than 5. Limits: The sum of the peak areas of all impurities is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (3.0 %). Disregard any peak with an area less than the area of the principal peak in the chromatogram obtained with reference solution (3) (0.2%). 801

VP V

PRIMAQUINE DIPHOSPHATE TABLETS

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Assay Dissolve 0.2000 g of the substance to be examined in 40 ml of anhydrous acetic acid R, heating gently. Allow to cool and titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 22.77 mg of C15H21N3O,2H3PO4 Storage Protected from light. Action and use Antiprotozoal (malaria). Preparations Tablets. PRIMAQUINE DIPHOSPHATE TABLETS Tabellae Primaquini diphosphas Primaquine diphosphate tablets contain primaquine diphosphate. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of primaquine diphosphate, C15H21N3O,2H3PO4, 90.0% to 110.0% of the stated amount. Identification A. Shake a quantity of the powdered tablets containing 10 mg of primaquine diphosphate with 5 ml of water and filter. To the filtrate add 1 ml of a 5% solution of ammonium cerium(IV)sulphate in dilute nitric acid R. A dark violet is produced. B. Shake a quantity of the powdered tablets containing 50 mg of primaquine diphosphate with 5 ml of water and filter. To the filtrate add 2 ml of 1 M sodium hydroxyde R and filter. Neutralise the filtrate with dilute nitric acid R, the resulting solution yields reaction characteristic of phosphate (Appendix 8.1). C. Shake a quantity of the powdered tablets with 0.01 M hydrochloric acid R to obtain a solution containing 15 µg of primaquine diphosphate per ml and filter. The ultraviolet absorption spectrum (Appendix 4.1) of the resulting solution exhibits absorption maxima at 265 nm and 282 nm. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of 0.01 M hydrochloric acid R. Rotation speed: 50 rpm. Time: 60 min. 802

Procedure: Test solution: After the specified time, withdraw a sample of the medium, filter and discard the first 20 ml of the filtrate. Dilute a portion of the filtrate with the medium, if necessary. Determine the content of primaquine diphosphate dissolved by liquid chromatography (Appendix 5.3) in comparison with a reference solution of primaquine diphosphate RS having the same concentration in the same medium. Mobile phase: Methanol - sodium pentanesulfonate solution (60 : 40). Make adjustments if necessary. Sodium pentanesulphonate solution: Dissolve 961 mg of sodium pentanesulfonate R and 1 ml of glacial acetic acid R in 400 ml of water, mix and filter. Chromatographic system: A column (30 cm × 3.9 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 2 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution, the relative standard deviation of the peak areas for 6 replicate injections is not more than 3.0%. Inject alternately the test solution and the reference solution. Calculate the content of primaquine diphosphate, C15H21N3O,2H3PO4, dissolved using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C15H21N3O,2H3PO4 in primaquine diphosphate RS. Tolerance: Not less than 75% (Q) of the stated amount of primaquine diphosphate, C15H21N3O,2H3PO4, is dissolved in 60 min.

Assay Weigh 20 tablets, calculate the average mass and powder finely. Transfer a quantity of the powdered tablets containing 0.2 g of primaquine diphosphate, accurately weighed, to a beaker, add 50 ml of water and about 5 ml of hydrochloric acid R and titrate with 0.05 M sodium nitrite VS, using the method under nitrite titration (Appendix 10.4). Each ml of 0.05 M sodium nitrite VS is equivalent to 22.77 mg of C15H21N3O,2H3PO4. Storage Stored in a well-closed container, in a cool and dry place, protected from light. Action and use Antimalarial. Usual strength 13.2 mg of primaquine diphosphate (equivalent to 7.5 mg of primaquine).

PROCAINE HYDROCHLORIDE

VP V

PROCAINAMIDE HYDROCHLORIDE Procainamidi hydrochloridum

C13H21N3O,HCl

M. 271.8

Procainamide hydrochloride is 4-amino-N-[2-(diethylamino) ethyl]benzamide hydrochloride. It contains not less than 98.0% and not more than 101.0% of C13H21N3O,HCl, calculated with reference to the dried substance.

Characters A white or very slightly yellow, crystalline powder, hygroscopic. Very soluble in water, freely soluble in ethanol (96%), slightly soluble in acetone, practically insoluble in ether. Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D, E. A. The infrared absorption spectrophotometry (Appendix 4.2) of the substance to be examined is concordant with the spectrum procainamide hydrochloride RS. B. Dissolve 10.0 mg of the substance to be examined in 0.1M sodium hydroxide R and dilute to 100.0 ml with the same solvent. Dilute 10.0 ml of the solution to 100.0 ml with 0.1M sodium hydroxide R. The ultraviolet absorption spectrum (Appendix 4.1) of the solution in the range 220 nm to 350 nm exhibits an absorption maximum at 273 nm. The value of A (1%, 1 cm) at the maximum is 580 to 610. C. Melting point: 166 °C to 170 °C (Appendix 6.7). D. Dilute 1 ml of solution S to 5 ml with water. The solution gives reaction A of chlorides (Appendix 8.1). E. Dilute 1 ml of solution S to 2 ml with water. 1 ml of this solution gives the reaction of primary aromatic amines (Appendix 8.1). Appearance of solution Solution S: Dissolve 2.5 g of the substance to be examined in carbon dioxide-free water R and dilute to 25.0 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution B6 (Appendix 9.3, method 2).

Mobile phase: Glacial acetic acid - water - butanol (15 : 30 : 60). Test solution: Dissolve 0.10 g of the substance to be examined in ethanol (96%) R and dilute to 10 ml with the same solvent. Reference solution: Dilute 1 ml of the test solution to 200 ml with ethanol (96%) R. Procedure: Apply to the plate 5 µl of each solution. Develop over a path of 12 cm. Place the plate in a stream of cold air until the plate appears dry. Examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with the reference solution.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). Take 1.0 g of the substance to be examined and carry out method 3. Prepare the reference solution using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 100 °C - 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.250 g of the substance to be examined in 50 ml of 2 M hydrochloric acid R, add 3 g of potassium bromide R. Cool in ice-water and titrate slowly with 0.1 M sodium nitrite VS. Determine the end-point amperometrically (Appendix 10.1). 1 ml of 0.1 M sodium nitrite VS is equivalent to 27.18 mg of C13H21N3O, HCl. Storage Store in an airtight container, protected from light. Action and use Anti arrhythmic. Preparations Injection, tablets. PROCAINE HYDROCHLORIDE Procaini hydrochloridum

pH The pH of solution S is 5.6 to 6.3 (Appendix 6.2). Related substances Not more than 0.5%. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254.

C13H20N2O2,HCl  

M. 272.8 803

VP V

PROCAINE HYDROCHLORIDE INJECTION

Procaine hydrochloride is 2-(diethylamino)ethyl 4-aminobenzoate hydrochloride. It contains not less than 99.0% and not more than 101.0% of C13H20N2O2,HCl, calculated with reference to the dried substance.

Characters A white or almost white, crystalline powder or colourless crystals, very soluble in water, soluble in ethanol (96%). Identification Apply one of the two following identifications: First identification: A, B, E. Second identification: B, C, D, E, F. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of procaine hydrochloride RS. B. Melting point: 154 °C to 158 °C (Appendix 6.7). C. Add 0.5 ml of fuming nitric acid R to about 5 mg of the substance to be examined. Evaporate to dryness on a water-bath, allow to cool and dissolve the residue in 5 ml of acetone R. Add 1 ml of 0.1 M potassium hydroxide in ethanol R. Only a brownish-red colour develops. D. Add 2 ml of water and 0.5 ml of 1 M sulfuric acid R to 0.2 ml of solution S (see Appearance of solution) and shake. Add 1 ml of a 0.1% solution of potassium permanganate R. The colour is immediately discharged. E. It gives reaction (A) of chlorides (Appendix 8.1). F. Dilute 1 ml of solution S to 100 ml with water. 2 ml of this solution gives the reaction of primary aromatic amines (Appendix 8.1). Appearance of solution Solution S: Dissolve 2.5 g in carbon dioxide-free water R and dilute to 50 ml with the same solvent. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). pH 5.0 to 6.5 (Appendix 6.2). Dilute 4 ml of solution S to 10 ml with carbon dioxide-free water R. Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Glacial acetic acid - hexane - dibutyl ether (4 : 16 : 80). Test solution: Dissolve 1.0 g of the substance to be examined in water and dilute to 10 ml with the same solvent. Reference solution: Dissolve 50 mg of 4-aminobenzoic acid R in water and dilute to 100 ml with the same solvent. Dilute 1 ml of the solution to 10 ml with water. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 10 cm. Dry the plate at 100 °C to 105 °C for 10 min and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained 804

with the reference solution (0.05%). The principal spot in the chromatogram obtained with the test solution remains on the point of application.

Heavy metals Not more than 5 ppm (Appendix 9.4.8). Dissolve 1.0 g of the substance to be examined in water and dilute to 25.0 ml with the same solvent. Carry out the prefiltration. 10 ml of the prefiltrate complies with limit test for heavy metals, method 5. Prepare the standard using 5 ml of lead standard solution (1 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.00 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.400 g of the substance to be examined in 50 ml of 2 M hydrochloric acid R, add 3 g of potassium bromide R. Cool in ice-water and titrate by slowly adding 0.1 M sodium nitrite VS. Determine the end-point by amperometric titration (Appendix 10.1). 1 ml of 0.1 M sodium nitrite is equivalent to 27.28 mg of C13H20N2O2,HCl. Storage Store protected from light. Action and use Local anaesthetic. Preparation Injection. PROCAINE HYDROCHLORIDE INJECTION Injectio Procaini hydrochloridi Procaine hydrochloride injection is a sterile solution of procaine hydrochloride in water for injections. The preparation complies with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Content of procaine hydrochloride, C13H20N2O2,HCl, 95.0% to 105.0% of the stated amount. Characters A clear and colourless solution. Identification A. Evaporate a portion of the injection containing 30 mg of procaine hydrochloride on a water bath to dryness, and

PROGESTERONE

VP V

dry over silica gel for 18 hours. The infrared absorption spectrum (Appendix 4.2) of the residue is concordant with the spectrum of procaine hydrochloride RS. B. Dissolve 10 mg of the residue in test A in 1 ml of water, add 1 drop of hydrochloric acid R and 1 drop of a 10% solution of sodium nitrite R. After 1 min to 2 min, add 1 ml of alkaline 2-naphtol solution R and shake well. A red precipitate is produced. C. It gives reaction characteristic of chlorides (Appendix 8.1).

pH 3.0 to 5.5 (Appendix 6.2). 4-Aminobenzoic acid Not more than 1.2%. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel H and sodium carboxymethylcellulose. Mobile phase: Benzene - glacial acetic acid - acetone methanol (14 : 1 : 1 : 4). Test solution: Dilute a portion of the injection with ethanol R to obtain a solution containing 2.5 mg of procaine hydrochloride per ml. Reference solution: A solution of 4-aminobenzoic acid R in ethanol R containing 30 µg per ml. Procedure: Apply separately to the plate 10 µl of each of the above solutions. After developing and removal of the plate, allow to dry in air and spray with p-dimethylaminobenzaldehyde solution (prepared by mixing 100 ml of a 2% solution of p-dimethylaminobenzaldehyde in ethanol R and 5 ml of glacial acetic acid R). The spot corresponding to 4-aminobenzoic acid in the chromatogram obtained with the test solution is not more intense than the principal spot in the chromatogram obtained with the reference solution. Assay To an accurately measured volume of the injection containing 0.2 g of procaine hydrochloride add 50 ml of dilute hydrochloric acid R and 3 g of potassium bromide R. Cool in iced-water to a temperature about 10 °C to 15 °C and titrate with 0.05 M sodium nitrite VS (Appendix 10.4). Determine the end-point potentiometrically (Appendix 10.2). Each ml of 0.05 M sodium nitrite VS is equivalent to 13.64 mg of C13H20N2O2,HCl. Storage Store in a well-close container, protected from light. Action and use Local anaesthetic, spinal anaesthesia. Usual strength 1%, 2%, 5%.

PROGESTERONE

Progesteronum

O CH3 CH3 H

H

CH3 H

H

O

C21H30O2  

M. 314.5

Progesterone is pregn-4-ene-3,20-dione. It contains not less than 97.0% and not more than 103.0% of C21H30O2, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder or colourless crystals, it shows polymorphism. Practically insoluble in water, freely soluble in ethanol, sparingly soluble in acetone and in fatty oils. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of progesterone RS. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in ethanol R, evaporate to dryness and record new spectra using the residues. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel F254. Mobile phase: Ethyl acetate - methylene chloride (33 : 66). Solvent mixture: Methylene chloride - methanol (9 : 1). Test solution: Dissolve 10 mg of the substance to be examined in solvent mixture and dilute to 10 ml with the same of solvent. Reference solution: Dissolve 10 mg of progesterone RS in solvent mixture and dilute to 10 ml with the same of solvent. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of three quarters of the plate. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. Spray with alcoholic solution of sulfuric acid R, heat at 120 °C for 15 min and allow to cool. Examine in daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, fluorescence in ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with the reference solution. Specific optical rotation +186° to +194°, calculated with reference to the dried substance (Appendix 6.4). 805

VP V

PROGESTERONE INJECTION

Dissolve 0.250 g in ethanol R and dilute to 25.0 ml with the same solvent.

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Water. Mobile phase B: Acetonitrile. Test solution: Dissolve 20.0 mg of the substance to be examined in methanol R and dilute to 50.0 ml with the same solvent. Reference solution (1): Dissolve 2.0 mg of progesterone RS and 2.0 mg of progesterone impurity C RS in methanol R and dilute to 50.0 ml with the same solvent. Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with methanol R. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase spherical end-capped octadecylsilyl silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 241 nm. Flow rate: 0.8 ml/min. Volume of injection: 10 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 20

50

50

20 - 27

50 → 20

50 → 80

27 - 45

20

80

45 - 50

50

50

System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to impurity C and to progesterone is at least 1.5. Limits: Any other impurity: For each impurity, the area is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.5%). The sum of the peak areas of all impurities is not more than 0.8 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.8%). Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Note: Impurity A: pregna-4,14-diene-3,20-dione. Impurity B: (20S)-20-hydroxypregn-4-en-3-one. Impurity C: (20R)-20-hydroxypregn-4-en-3-one. Impurity D: (20S)-3-oxopregn-4-en-20-yl acetate. Impurity E: (20R)-3-oxopregn-4-en-20-yl acetate. Impurity F: 21-(cyclohex-1-enyl)pregn-4-ene-3,20-dione. Impurity G: 21-(cyclohexylidene)pregn-4-ene-3,20-dione.

806

Loss on drying Not more than 0.5% (Appendix 9.6). (0.500 g; 105 °C; 2 h). Assay Dissolve 25.0 mg of the substance to be examined in ethanol (96%) R and dilute to 250.0 ml with the same solvent. Dilute 5.0 ml of the solution to 50.0 ml with ethanol (96%) R. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum at 241 nm. Calculate the content of C21H30O2 taking 535 as the value of A (1%, 1 cm) at 241 nm. Storage Store in an airtight container, protected from light. Action and use Progestogen. Preparations Oil injection, soft capsules vaginal gel. PROGESTERONE INJECTION Injectio Progesteroni Progesterone injection is a sterile solution of progesterone in the suitable solvent. The injection complies with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Content of progesterone, C21H30O2, 90.0% to 110.0% of the stated amount. Characters A clear and colourless solution. Identification Insert a piece of fine glass wool into the base of a chromatographic column with 20 cm long and 2.5 cm in diameter. Mix 8.0 ml of nitromethane R with 7.0 g of silica gel for column chromatography R in a 150 ml beaker to homogenize the mixture, and transfer to the chromatographic column, tapping lightly with a suitable tamping rod. Pack a piece of glass wool on the top of the column. Dilute a portion of the injection with n-heptane R to obtain a solution containing about 1 mg of progesterone per ml. Transfer 4 ml of this solution to the prepared column. Pass gradually 300 ml of n-heptane R through the column, discarding the first 120 ml of the eluate. Collect the subsequent eluate in a 250 ml beaker. Evaporate the solution under a stream of nitrogen on a water bath to about 50 ml, transfer to a 100 ml beaker, and evaporate to dryness. Remove the last traces of n-heptane by adding 1 ml of methanol R and evaporate to dryness. Dry the residue over silica gel in the desiccator for 4 hours. The infrared absorption spectrum (Appendix 4.2) of the residue

VP V

PROGESTERONE SOFT CAPSULES

is concordant with the spectrum of a similar preparation of progesterone RS.

Content of progesterone, C21H30O2, 90.0% to 110.0% of the stated amount.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Ethanol (96%) - water (11 : 9). Test solution: Transfer an accurately measured volume of the injection containing 100 mg of progesterone to a 100 ml volumetric flask. Add about 20 ml of tetrahydrofuran R to dissolve, and dilute with ethanol (96%) R to volume. Transfer 8.0 ml of this solution to a 100 ml volumetric flask, dilute with ethanol (96%) R to volume, and mix. Reference solution: Transfer about 50 mg of progesterone RS, accurately weighed, to a 50 ml volumetric flask. Add about 10 ml of tetrahydrofuran R to dissolve, and dilute with ethanol (96%) R to volume. Transfer 8.0 ml of this solution to a 100 ml volumetric flask, dilute with ethanol (96%) R to volume, and mix. Chromatographic system: A column (25 cm × 4 mm) packed with stationary phase C (5 µm). Column temperature: 40 °C. Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject a sample of dimethyl sulfoxide, and identify the retention time, to, of this nonretarded compound to calculate the capacity factor. Inject the reference solution, and record the peak responses. The capacity factor, k′, for progesterone is not less than 2.0; the symmetry factor is not more than 2.0; and the relative standard deviation of the peak areas for replicate injections is not more than 2.0%. Inject alternately the test solution and the reference solution. Calculate the content of progesterone, C21H30O2, in the injection using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C21H30O2 in progesterone RS.

Identification Insert a piece of fine glass wool into the base of a chromatographic column with 20 cm long and 2.5 cm in diameter. Mix 8.0 ml of nitromethane R with 7.0 g of silica gel for column chromatography R in a 150 ml beaker to homogenize the mixture, and transfer to the chromatographic column, tapping lightly with a suitable tamping rod. Pack a piece of glass wool on the top of the column. Dilute a portion of the contents of the capsules with n-heptane R to obtain a solution containing about 1 mg of progesterone per ml. Transfer 4 ml of this solution to the prepared column. Pass gradually 300 ml of n-heptane R through the column, discarding the first 120 ml of the eluate. Collect the subsequent eluate in a 250 ml beaker. Evaporate the solution under a stream of nitrogen on a water bath to about 50 ml, transfer to a 100 ml beaker, and evaporate to dryness. Remove the last traces of n-heptane by adding 1 ml of methanol R and evaporate to dryness. Dry the residue over silica gel in the desiccator for 4 hours. The infrared absorption spectrum (Appendix 4.2) of the residue is concordant with the spectrum of a similar preparation of progesterone RS.

Storage Protected from light. Action and use Progestin hormone. Usual strength 50 mg/ml. PROGESTERONE SOFT CAPSULES Molles capsulae Progesteroni Progesterone soft capsules contain progesterone. The soft capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Ethanol (96%) - water (11 : 9). Test solution: Weigh 20 soft capsules, calculate the average weight of the capsule contents and mix. Transfer an accurately weighed quantity of the contents of the capsules containing about 100 mg of progesterone to a 100 ml volumetric flask. Add about 20 ml of tetrahydrofuran R to dissolve, and dilute with ethanol (96%) R to volume. Transfer 8.0 ml of this solution to a 100 ml volumetric flask, dilute with ethanol (96%) R to volume, and mix. Reference solution: Transfer about 50 mg of progesterone RS, accurately weighed, to a 50 ml volumetric flask. Add 10 ml of tetrahydrofuran R to dissolve, and dilute with ethanol (96%) R to volume. Transfer 8.0 ml of this solution to a 100 ml volumetric flask, dilute with ethanol (96%) R to volume, and mix. Chromatographic system: A column (25 cm × 4 mm) packed with stationary phase C (5 µm). Column temperature: 40 °C. Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject a sample of dimethyl sulfoxide, and identify the retention time, to, of this nonretarded compound to calculate the capacity factor. Inject the reference solution, and record the peak responses. The capacity factor, k ′, for progesterone is not less than 2.0; 807

VP V

PROMETHAZINE HYDROCHLORIDE

the symmetry factor is not more than 2.0; and the relative standard deviation of the peak areas for replicate injections is not more than 2.0%. Inject alternately the test solution and the reference solution. Calculate the content of progesterone, C21H30O2, in the soft capsules using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C21H30O2 in progesterone RS.

Promethazine hydrochloride is (2RS)-N,N-dimethyl-1(10H-phenothiazin-10-yl)propan-2-amine hydrochloride. It contains not less than 99.0% and not more than 101.0% of C17H20N2S,HCl, calculated with reference to the dried substance.

the impregnation mixture has risen at least 17 cm from the lower edge of the plate, remove the plate and use immediately for chromatography. Mobile phase: A mixture of 50 ml of petroleum ether (50 °C to 70 °C) R and 1 ml of diethylamine R saturated with phenoxyethanol R (i.e. add about 3 ml to 4 ml of phenoxyethanol R to the above mixture of solvents to give a persistent cloudiness on shaking, decant, and use the supernatant liquid, even if it is cloudy). Test solution: Dissolve 20 mg of the substance to be examined in chloroform R and dilute to 10 ml with the same solvent. Reference solution: Dissolve 20 mg of promethazinehydrochloide RS in chloroform R and dilute to 10 ml with the same solvent. Procedure: Apply separately to the plate 2 µl of each solution. Develop over a path of 15 cm. After development place the plate under ultraviolet light at 365 nm and examine after a few minutes. The spot in the chromatogram obtained with the test solution is similar in position, fluorescence and size to the spot in the chromatogram obtained with the reference solution. Spray with a 10% v/v solution of sulfuric acid R in ethanol (96%) R. The spot in the chromatogram obtained with the test solution is of the same colour as that in the chromatogram obtained with the reference solution and has similar stability over a period of at least 20 min. C. Dissolve 0.1 g of the substance to be examined in 3 ml of water. Add dropwise 1 ml of nitric acid R. A precipitate is formed which rapidly dissolves to give a red solution, becoming orange and then yellow. Heat to boiling. The solution becomes orange and an orange-red precipitate is formed. D. It gives reaction (B) of chlorides (Appendix 8.1).

Characters White or faintly yellowish, crystalline powder. Very soluble in water, freely soluble in ethanol (96%) and in methylene chloride. Melting point: About 222 °C, with decomposition.

pH 4.0 to 5.0 (Appendix 6.2). Dissolve 1.0 g of the substance to be examined in carbon dioxide-free water R and dilute to 10 ml with the same solvent, measured immediately after preparation.

Identification Apply one of the two following identifications: First identification: A, B, D. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of promethazine hydrochloride RS. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Kieselguhr G R. Impregnate the plate by placing it in a closed tank containing the necessary quantity of the impregnation mixture composed of a solution containing 10% v/v of phenoxyethanol R and 50 g/L of macrogol 300 R in acetone R so that the plate dips about 5 mm beneath the surface of the liquid. When

Related substances Examine by liquid chromatography (Appendix 5.3). Carry out the test protected from light and use freshly prepared solutions. Mobile phase: Methanol - acetonitrile - buffer solution pH 7.0 (20 : 30 : 50). Buffer solution pH 7.0: A 3.4 g/L solution of potassium dihydrogen phosphate R previously adjusted to pH 7.0 with 2 M potassium hydroxide R. Solvent mixture: Triethylamine - methanol (1 : 1000). Test solution: Dissolve 25.0 mg of the substance to be examined in the solvent mixture and dilute to 50.0 ml with the solvent mixture. Reference solution (1): Dissolve 2.5 mg of promethazine for peak identification RS (containing impurities A, B

Storage Protected from light. Action and use Progestin hormone. Usual strength 100 mg, 200 mg. PROMETHAZINE HYDROCHLORIDE Promethazini hydrochloridum

C17H20N2S,HCl

808

M. 320.9

VP V

and C) in the solvent mixture and dilute to 5 ml with the solvent mixture. Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 10.0 ml with the solvent mixture. Reference solution (3): Dissolve 5.0 mg of promethazine impurity D RS in the solvent mixture and dilute to 100.0 ml with the solvent mixture. Dilute 1 ml of this solution to 100 ml with the solvent mixture. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase end-capped octylsilyl silica gel for chromatography with polar incorporated groups R (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: The run time is 2.5 times the retention time of promethazine. Identification of impurities: Use the chromatogram supplied with promethazine for peak identification RS and the chromatogram obtained with reference solution (1) to identify the peaks due to impurities A, B and C; use the chromatogram obtained with reference solution (3) to identify the peak due to impurity D. Relative retention with reference to promethazine (retention time = about 18 min): impurity D = about 0.2; impurity C = about 0.5; impurity B = about 1.4; impurity A = about 1.8. System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to impurities B and A is at least 2.0. The chromatogram obtained with reference solution (1) is similar to the chromatogram supplied with promethazine for peak identification RS. Limits: Correction factor: for the calculation of content, multiply the peak area of impurity A by 0.5. Impurity B: The area of the peak due to impurity B is not more than 8 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.8%). Impurity C: The area of the peak due to impurity C is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (2) (0.2%). Impurity A: The corrected area of the peak due to impurity A is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.1%). Impurity D: The area of the peak due to impurity D is not more than the area of the corresponding peak in the chromatogram obtained with reference solution (3) (0.1%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.10%). The sum of the peak areas of all impurities is not more than 12 times the area of the principal peak in the chromatogram obtained with reference solution (2) (1.2%).

PROMETHAZINE HYDROCHLORIDE CREAM

Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Note: Impurity A: phenothiazine. Impurity B: (2RS)-N,N-dimethyl-2-(10H-phenothiazin-10-yl) propan-1-amine (isopromethazine). Impurity C: (2RS)-N-methyl-1-(10H-phenothiazin-10-yl)propan2-amine. Impurity D: (2RS)-N,N-dimethyl-1-(10H-phenothiazin-10-yl) propan-2- amine S-oxide.

Heavy metals Not more than 10 ppm (Appendix 9.4.8). Dissolve 1.0 g of the substance to be examined in 5 ml of water, then add 5 ml of acetone R and 5 ml of buffer solution pH 3.5 R. Carry out the prefiltration. The prefiltrate complies with limit test for heavy metals, method 5. Prepare the standard using 5 ml of lead standard solution (2 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.250 g of the substance to be examined in a mixture of 5.0 ml of 0.01 N hydrochloric acid VS and 50 ml of ethanol (96%) R. Carry out a potentiometric titration (Appendix 10.2), using 0.1 N sodium hydroxide VS. Read the volume added between the 2 points of inflexion. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 32.09 mg of C17H21ClN2S. Storage Store in an airtight container, protected from light. Action and use Histamine H1 receptor antagonist; antihistamine. Preparations Tablets, injection, syrups, cream. PROMETHAZINE HYDROCHLORIDE CREAM Cremoris Promethazini hydrochloridi Promethazine hydrochloride cream is a topical cream containing promethazine hydrochloride. Promethazine hydrochloride cream complies to the requirements stated under “Semi-solid preparation for cutaneous and muscosal application” (Appendix 1.12) and with the following requirements. 809

VP V

PROMETHAZINE HYDROCHLORIDE SYRUP

Content of promethazine hydrochloride, C17H20N2S,HCl, 90.0% to 110.0% of the stated amount. Characters A off-white, smooth, soft and homogenous cream. Identification A. Transfer a quantity of the cream containing about 50 mg of promethazine hydrochloride into a 100 ml beaker. Add 25 ml of 0.01 M hydrochloric acid R, heat in a water-bath at 60 °C, sonicate for 5 min. Cold in ice water for 30 min, filter, transfer the filtrate into a 100-ml separating funnel. Add 2 ml of 1 M sodium hydroxide R and 4 ml of carbon disulfide R, shake for 2 min. Use the carbon disulfide layer, centrifuge (if necessary) and filter through a dry membrane filter to obtain a clear solution. The infrared spectrum (Appendix 4.2) of the obtained solution is concordant with that of the reference solution prepared by dissolving 50 mg of promethazine hydrochloride RS in 25 ml 0.01 M hydrochloric acid R and transfer into a 100 ml separating funnel. Add 2 ml of 1 M sodium hydroxyde R and 4 ml of carbon disulfide R, shake for 2 min. Use the carbon disulfide layer, centrifuge (if necessary) and filter through a dry membrane filter to obtain a clear solution. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of the promethazine hydrochloride peak in the chromatogram obtained with the reference solution. Assay Examine by liquid chromatography (Appendix 5.3), protected from light. Mobile phase: Water adjusted to pH 2.3 with glacial acetic acid - methanol (55 : 45). Make adjustments if necessary. Test solution: Weigh accurately a quantity of the cream containing about 10 mg of promethazine hydrochloride, transfer into a 100-ml beaker. Add 30 ml of 0.01 M hydrochloric acid R, heat in a water-bath at 60 °C and sonicate for 3 min. Repeat the procedure with two 10 ml quantities of 0.01 M hydrochloric acid R. Transfer the solution into a 50-ml volumetric flask, rinse the beaker with 0.01 M hydrochloric acid R and transfer into the volumetric flask. Allow to cool and add 0.01 M hydrochloric acid R to volume, mix. Allow to cold in icewater for about 30 min. Filter through a membrane filter, discard the portion of the filtrate and allow the filtrate to cool to the room temperature. Transfer 5.0 ml of the filtrate into a 50-ml volumetric flask and dilute to volume with 0.01 M hydrochloric acid R, mix well. Reference solution: A 0.002% solution of promethazine hydrochloride RS in 0.01 M hydrochloric acid R. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary C (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. 810

Procedure: System suitability: Inject the reference solution, the test is not valid unless: the number of theoretical plates of the column determined on the promethazine hydrochloride peak is not less than 3000, the resolution factor between the promethazine hydrochloride peak and the impurity peak (the relative retention time are 1.1 and 1.2, respectively) is not less than 2.0. The relative standard deviation of the peak areas for six replicate injections is not more than 2.0%. Inject alternately the reference solution, the test solution. Calculate the content of promethazine hydrochloride, C17H20N2S,HCl, in the cream using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C17H20N2S,HCl in promethazine hydrochloride RS.

Storage Store in a cool place, in a tight container and protect from light. Action and use Antihistamine, anti-allergic. Usual strength 2%. PROMETHAZINE HYDROCHLORIDE SYRUP Sirupi Promethazini hydrochloridi Promethazine hydrochloride syrup contains promethazine hydrochloride. The syrup complies with the requirements stated under “Syrups” (Appendix 1.4) and with the following requirements.

Content of promethazine hydrochloride, C17H20N2S,HCl, 90.0% to 110.0% of the stated amount. Identification A. Transfer a quantity of the syrup containing about 25 mg of promethazine hydrochloride into a 250-ml separating funnel. Add 10 ml of ammonia R and extract with two 40-ml quantities of chloroform R. Combine the chloroform extracts, rinse with 25 ml of water and eliminate water by anhydrous sodium sulfate R. Evaporate the chloroform extract on a water bath to dryness. The infrared absorption spectrum of the obtained residue (Appendix 4.2) is concordant with the reference spectrum of promethazine. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of the promethazine peak in the chromatogram obtained with the reference solution. Assay Examine by liquid chromatography (Appendix 5.3), protected from light.

VP V

Mobile phase: Buffer solution pH 2.3 - methanol (40 : 60). Make adjustments if necessary. Buffer solution pH 2.3: Add 2 ml of triethylamine R in 1000 ml water, adjust the pH to 2.3 with glacial acetic acid R. Test solution: Weigh accurately a quantity of the syrup containing about 10 mg of promethazine hydrochloride, transfer into a 50-ml volumetric flask, dilute to volume with 0.1 M hydrochloric acid R, mix well. Transfer accurately 5.0 ml of this solution into a 50-ml volumetric flask and dilute to volume with 0.1 M hydrochloric acid R, mix. Filter through a 0.45 µm membrane filter. Reference solution: A 0.002 % solution of promethazine hydrochloride RS in 0.1 M hydrochloric acid R. Filter through a 0.45 µm membrane filter. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution, the test is not valid unless the number of theoretical plates determined on promethazine hydrochloride peak is not less than 3000, the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of promethazine hydrochloride, C17H20N2S,HCl, in the syrup using the peak areas in the chromatograms obtained with the reference solution, the test solution, the density of the syrup and the declared content of C17H20N2S,HCl in promethazine hydrochloride RS.

Storage Store in a tight container, protected from light. Action and use Antihistamine. Usual strength 6.5 mg/5 ml and 25 mg/5 ml. PROMETHAZINE HYDROCHLORIDE TABLETS Tabellae Promethazini hydrochloridi Promethazine hydrochloride tablets contain promethazine hydrochloride. They are coated tablets. The tablets comply with the requirements stated under “Tablets” in “Coated tablets” section (Appendix 1.20) and with the following requirements.

Content of promethazine hydrochloride, C17H20N2S,HCl, 92.5% to 107.5% of the stated amount.

PROMETHAZINE HYDROCHLORIDE TABLETS

Identification A. To a quantity of the powdered tablets containing 40 mg of promethazine hydrochloride add 10 ml of water and 2 ml of 1 M sodium hydroxide R, shake and extract with 15 ml of ether R. Wash the ether layer with 5 ml of water, filter through filter paper containing anhydrous sodium sulfate R, evaporate to dryness and dissolve the residue in 0.4 ml of chloroform R. The infrared absorption spectrum (Appendix 4.2) of the resulting solution is concordant with the reference spectrum of promethazine. B. To a quantity of the powdered tablets containing about 5 mg of promethazine hydrochloride add 5 ml of sulfuric acid R and allow to stand for 5 minutes. A red colour is produced. C. Dissolve a quantity of the powdered tablets containing 0.2 g of promethazine hydrochloride in 2 ml of water, filter, saturate with potassium carbonate R, extract with two 10 ml quantities of ether R and evaporate the combined extracts to dryness. Dissolve the residue in 2 ml of methanol R and pour into a solution of 0.4 g of picric acid R in 10 ml of methanol R at about 50 °C. Cool, scratch with a glass rod the wall of the tube to induce crystallisation, allow to stand for 3 to 4 hours and filter. The melting point (Appendix 6.7) of the crystals, after washing with methanol R and drying, is about 160 °C. Dissolution (Appendix 11.4) Carry out the following procedure protected from light. Apparatus: Basket. Medium: 900 ml of 0.01 M hydrochloric acid R. Rotation speed: 100 rpm. Time: 45 min. Procedure: After the specified time, withdraw a sample of the medium, filter and discard the first 20 ml of the filtrate. Dilute the filtrate with 0.01 M hydrochloric acid R to obtain a solution containing a suitable concentration. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum at 249 nm in a 1 cm cell, using 0.01 M hydrochloric acid R as a blank. Calculate the content of promethazine hydrochloride, C17H20N2S,HCl, dissolved taking 910 as the value of A (1%, 1 cm) at the maximum at 249 nm. Tolerance: Not less than 80% (Q) of the stated amount of promethazine hydrochloride, C17H20N2S,HCl, is dissolved in 45 minutes. Assay Carry out the following procedure protected from light. Weigh 20 tablets, remove the coating if necessary. Calculate the average mass and powder finely. Transfer a quantity of the powdered tablets equivalent to about 25 mg of promethazine hydrochloride, accurately weighed, to a mortar. Triturate with 5 ml of 2 M hydrochloric acid R and transfer the mixture to a 250 ml volumetric flask by 100 ml of water. Shake for 15 minutes, add water to volume and mix. Filter, discard the first portion of filtrate. To 5.0 ml of the filtrate add 10 ml of 0.1 M hydrochloric 811

PROPRANOLOL HYDROCHLORIDE

acid R and sufficient water to produce 100.0 ml. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum at 249 ± 1 nm, using 0.01 M hydrochloric acid R as a blank. Calculate the content of promethazine hydrochloride, C17H20N2S,HCl, taking 910 as the value of A(1%, 1 cm) at the maximum at 249 nm.

Storage Store in a well-closed container, in a cool and dry place. Action and use Histamine H1-receptor antagonist. Usual strength 15 mg, 25 mg. PROPRANOLOL HYDROCHLORIDE Propranololi hydrochloridum

VP V

The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. D. It gives reaction (A) of chlorides (Appendix 8.1).

Appearance of solution Dissolve 2.0 g in methanol R and dilute to 20 ml with the same solvent. The solution is clear (Appendix 9.2) and not more intensely coloured than intensity 6 of the range of reference solutions of the most appropriate colour (Appendix 9.3, method 2). Acidity or alkalinity Dissolve 0.20 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent. Add 0.2 ml of methyl red solution R and 0.2 ml of 0.01 N hydrochloric acid VS; the solution is red. Add 0.4 ml of 0.01 N sodium hydroxide VS; the solution is yellow.

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Mix 1.6 g of sodium laurylsulfate R and 0.31 g of tetrabutylammonium dihydrogen phosphate R in a mixture of 1 ml of sulfuric acid R, 450 ml of water C16H21NO2,HCl   M. 295.8  and 550 ml of acetonitrile R; adjust to pH 3.3 using dilute sodium hydroxide solution R. Propranolol hydrochloride is (2RS)-1-[(1-methylethyl) Test solution: Dissolve 20.0 mg of the substance to be amino]-3-(naphthalen-1-yloxy)propan-2-ol hydrochloride. examined in the mobile phase and dilute to 10.0 ml with It contains not less than 99.0% and not more than 101.0% the mobile phase. of C16H21NO2,HCl, calculated with reference to the dried Reference solution (1): Dissolve 10.0 mg of propranolol substance. hydrochloride for performance test RS in the mobile phase and dilute to 10.0 ml with the mobile phase. Characters White or almost white powder. Soluble in water and in Reference solution (2): Dilute 2.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of this ethanol (96%). solution to 10.0 ml with the mobile phase. Identification Chromatographic system: Apply one of the two following identifications: A column (25 cm × 4.6 mm) packed with stationary phase First identification: A, D. C (5 µm). Second identification: B, C, D. Detector: A spectrophotometer set at 292 nm. A. The infrared absorption spectrum (Appendix 4.2) of the Equilibration: For at least 30 min. substance to be examined is concordant with the spectrum Flow rate: 1.8 ml/min. of propranolol hydrochloride RS. Volume of injection: 20 µl. B. Melting point (Appendix 6.7): 163 °C to 166 °C. Procedure: C. Thin-layer chromatography (Appendix 5.4). The run time is 7 times the retention time of propranolol. Coating substance: Silica gel G. Identification of impurities: Use the chromatogram Mobile phase: Ammonia 18 M - methanol R (1 : 99). supplied with propranolol hydrochloride for performance Test solution: Dissolve 10 mg of the substance to be test RS to identify the peak due to impurity A. examined in 1 ml of methanol R. System suitability: In the chromatogram obtained with Reference solution: Dissolve 10 mg of propranolol reference solution (1), baseline separation is obtained hydrochloride RS in 1 ml of methanol R. between the peaks due to impurity A and propranolol. Procedure: Apply separately to the plate 10 µl of each Limits: solution. Develop over a path of 15 cm. Dry the plate at Any other impurity: For each impurity, the area is not 100 °C to 105 °C. Spray with anisaldehyde solution R more than 0.5 times the area of the principal peak in the and heat at 100 °C to 105 °C until the colour of the spots chromatogram obtained with reference solution (2) (0.1%). reaches maximum intensity (10 min to 15 min). The sum of the peak areas of all impurities is not more than 812

VP V

twice the area of the principal peak in the chromatogram obtained with reference solution (2) (0.4%). Note: Impurity A: (2RS)-3-(naphthalen-1-yloxy)propane-1,2-diol (diol derivative). Impurity B: 1,1’-[(1-methylethyl)imino]bis[3-(naphthalen-1yloxy)propan-2-ol] (tertiary amine derivative). Impurity C: 1,3-bis(naphthalen-1-yloxy)propan-2-ol (bis-ether derivative).

Heavy metals Not more than 20 ppm (Appendix 9.4.8). Dissolve 1.0 g of the substance to be examined in a mixture of water - methanol (15 : 85) and dilute to 20 ml with the same mixture of solvents. 12 ml of the solution complies with limit test for heavy metals, method 2. Prepare the reference solution using lead standard solution (1 ppm Pb) prepared by diluting lead standard solution (100 ppm Pb) R with a mixture of water - methanol (15 : 85). Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.250 g of the substance to be examined in 25 ml of ethanol (96%) R. Titrate with 0.1 N sodium hydroxide VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N sodium hydroxide VS is equivalent to 29.58 mg of C16H22ClNO2. Storage Store in an airtight container. Action and use Beta-adrenoceptor antagonist. Preparations Injection, tablets. PROPRANOLOL TABLETS Tabellae Propranololi Propranolol tablets contain propranolol hydrochloride. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of propranolol hydrochloride, C16H21NO2,HCl, 92.5% to 107.5% of the stated amount.

PROPRANOLOL TABLETS

Identification A. Shake a quantity of the powdered tablets containing 0.1 g of propranolol hydrochloride with 20 ml of water, filter. Make the filtrate alkaline with 1 M sodium hydroxide R and extract with 3 portions, each of 10 ml, of ether R. Wash the ether layer with water until washings are free from alkali. Filter the extract through anhydrous sodium sulfate R, evaporate the extract to dryness and dry at 50 °C, at pressure of 2 kPa for 1 h. The infrared absorption spectrum (Appendix 4.2) of the residue is concordant with the reference spectrum of propranolol. B. In the Assay, the ultraviolet absorption spectrum (Appendix 4.1) of the test solution in the range 230 nm to 350 nm exhibits absorption maxima at 290 nm and 319 nm and a shoulder at 306 nm. Related substances Examined by liquid chromatography (Appendix 5.3). Mobile phase: A mixture of 1.15 g of sodium dodecyl sulfate R, 10 ml of a mixture of sulfuric acid - water (1 : 9), 20 ml of 1.7% tetrabutyl ammoni dihydrogen phosphate solution, 370 ml of water and 600 ml of acetonitrile R, adjust the pH to 3.3 with 2 M sodium hydroxide R. Test solution: Shake a quantity of the powdered tablets containing 100 mg of propranolol hydrochloride with 100 ml of methanol R and filter. Reference solution: Dilute 1.0 ml of the test solution to 500.0 ml with the mobile phase. Chromatographic system: A column (20 cm × 5 mm) packed with end-capped octadecylsilyl silica gel for chromatography R (5 μm). Hypersil ODS column is suitable. Detector: A spectrophotometer set at 292 nm. Flow rate: 1.8 ml/min. Volume of injection: 10 µl. Procedure: Equilibrate the column with the mobile phase for 30 min. Inject the reference solution and the test solution. Record the chromatogram for 5 times the retention time of propranolol. In the chromatogram obtained with the test solution, the area of any secondary peak is not greater than the area of the principal peak in the chromatogram obtained with the reference solution (0.2%).The sum of all secondary peaks is not greater than 4 times the area of the principal peak in the chromatogram obtained with the reference solution (0.8%). Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 1000 ml of 0.1 M hydrochloric acid R. Rotation speed: 100 rpm. Time: 30 min. Procedure: After the specified time, withdraw a sample of the medium, filter and discard the first portion of the filtrate. Dilute the filtrate with the medium, if necessary, to obtain a solution containing 10 μg to 30 µg of propranolol hydrochloride per ml. Measure the absorbance (Appendix 813

VP V

PROPYL PARAHYDROXYBENZOATE

4.1) at the maximum wavelength at about 290 nm in a 1-cm cell, using the medium as the blank. Calculate the content of propranolol hydrochloride, C16H21NO2,HCl, dissolved taking 206 as the value of A (1%, 1 cm) at the maximum at 290 nm. Tolerance: Not less than 75% (Q) of the stated amount of propranolol hydrochloride, C16H21NO2,HCl, is dissolved in 30 min.

Propyl parahydroxybenzoate is propyl 4-hydroxybenzoate. It contains not less than 98.0% and not more than 102.0% of C10H12O3.

Uniformity of content (Appendix 11.2) Transfer 1 tablet into a 50-ml volumetric flask, add 1 ml of water and shake to disintegrate completely. Add 30 ml of methanol R, shake for 10 min and dilute to volume with methanol R. Mix, filter and discard the first portion of the filtrate and dilute a portion of the filtrate with methanol R to obtain a solution containing 20 µg of propranolol hydrochloride per ml. Perform the procedure as described under the Assay, beginning at the words “Measure the absorbance…”

Identification Apply one of the two following identifications: First identification: A, B. Second identification: B, C. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of propyl parahydroxybenzoate RS. B. Melting point: 96 °C to 99 °C (Appendix 6.7). A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Octadecylsilyl silica gel F254. Mobile phase: Glacial acetic acid - water - methanol (1 : 30 : 70). Test solution (1): Dissolve 0.10 g of the substance to be examined in acetone R and dilute to 10 ml with the same solvent. Test solution (2): Dilute 1 ml of test solution (1) to 10 ml with acetone R. Reference solution (1): Dissolve 10 mg of propyl parahydroxybenzoate RS in acetone R and dilute to 10 ml with the same solvent. Reference solution (2): Dissolve 10 mg of ethyl parahydroxybenzoate RS in 1 ml of test solution (1) and dilute to 10 ml with acetone R. Procedure: Apply separately to the plate 2 µl of test solution (2), reference solutions (1) and (2). Develop over 2/3 of the plate. Allow to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with test solution (2) is similar in position and size to the principal spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) shows 2 clearly separated principal spots.

Assay Weigh 20 tablets, calculate the average weight and powder finely. Weigh accurately a quantity of the powdered tablets containing about 20 mg of propranolol hydrochloride, transfer into a 100-ml volumetric flask. Add 2 ml of water and shake for 5 min. Add methanol R to volume and mix. Filter, discard the first portion of the filtrate. Dilute 5.0 ml of the filtrate to 50.0 ml with methanol R. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum wavelength at 290 nm in 1-cm cell, using methanol R as the blank. Calculate the content of propranolol hydrochloride, C16H21NO2,HCl, in the tablets taking 206 as the value of A(1%, 1 cm) at the maximum at 290 nm. Storage Stored in a cool and dry place, protected from light. Action and use Beta-adrenoceptor antagonist. Usual strength 10 mg; 20 mg; 40 mg.

Appearance of solution Solution S: Dissolve 1.0 g in ethanol (96%) R and dilute to 10 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution BY6 (Appendix 9.3, method 2).

PROPYL PARAHYDROXYBENZOATE Propylis parahydroxybenzoas Propylparaben, Nipasol M O O

CH3

HO

C10H12O3  

814

Characters White or almost white, crystalline powder. Very slightly soluble in water, freely soluble in ethanol (96%) and in methanol.

M. 180.2

Acidity To 2 ml of solution S add 3 ml of ethanol (96%) R, 5 ml of carbon dioxide- free water R and 0.1 ml of bromocresol green solution R. Not more than 0.1 ml of 0.1 N sodium hydroxide VS is required to change the colour of the indicator to blue.

PROPYLENE GLYCOL

VP V

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: A 0.68% solution of potassium dihydrogen phosphate - methanol (35 : 65). Test solution: Dissolve 50.0 mg of the substance to be examined in 2.5 ml of methanol R and dilute to 50.0 ml with the mobile phase. Dilute 10.0 ml of this solution to 100.0 ml with the mobile phase. Reference solution (1): Dissolve 5 mg of 4-hydroxybenzoic acid R (impurity A), 5 mg of ethyl parahydroxybenzoate R (impurity C) and 5 mg of the substance to be examined in the mobile phase and dilute to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (2): Dissolve 50.0 mg of propyl parahydroxybenzoate RS in 2.5 ml of methanol R and dilute to 50.0 ml with the mobile phase. Dilute 10.0 ml of this solution to 100.0 ml with the mobile phase. Reference solution (3): Dilute 1.0 ml of the test solution to 20.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 272 nm. Flow rate: 1.3 ml/min. Volume of injection: 10 µl. Procedure: Inject the test solution and reference solutions (1) and (3). Run time is 2.5 times the retention time of propyl parahydroxybenzoate. Relative retention with reference to propyl parahydroxybenzoate (retention time = about 4.5 min): impurity A = about 0.3; impurity C = about 0.7. System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to impurity C and propyl parahydroxybenzoate is at least 3.0. Limits: Correction factor: For the calculation of content, multiply the peak area of impurity A by 1.4. Impurity A: The area of the peak due to impurity A is not more than the area of the principal peak in the chromatogram obtained with reference solution (3) (0.5%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (3) (0.5%). The sum of the peak areas of all impurities is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (3) (1.0%). Disregard any peak with an area less than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (3) (0.1%). Note: Impurity A. 4-hydroxybenzoic acid. Impurity B. Methyl 4-hydroxybenzoate (methyl parahydroxybenzoate).

Impurity C. Ethyl 4-hydroxybenzoate (ethyl parahydroxybenzoate). Impurity D. Butyl 4-hydroxybenzoate (butyl parahydroxybenzoate).

Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. Inject the test solution and reference solution (2). Calculate the percentage content of C10H12O3 using the peak areas in the chromatograms obtained with the test solution, reference solution (2) and the declared content of C10H12O3 in propyl parahydroxybenzoate RS. Storage Store in an airtight container. Action and use Antimicrobial preservative. PROPYLENE GLYCOL

Propylenglycolum

C3H8O2   M. 76.1 Propylene glycol is (RS)-propane-1,2-diol.

Characters A viscous, clear, colourless, hygroscopic liquid, miscible with water and with ethanol (96%). Identification B. It complies with the test for Relative density. B. It complies with the test for Refractive index. C. Boiling point: 184 °C to 189 °C (Appendix 6.8). D. Add 5 ml of pyridine R and 2 g of finely ground nitrobenzoyl chloride R to 0,5 ml of the substance to be examined. Boil for 1 min and pour into 15 ml of cold water with shaking. Filter, wash the precipitate with 20 ml of a saturated solution of sodium hydrogen carbonate R and then with water and dry. Dissolve in boiling ethanol (80%) R and filter the hot solution. On cooling, crystals are formed. After drying at 100 °C to 105 °C, the crystals melt at 121 °C to 128 °C (Appendix 6.7). Appearance of solution It is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2).

815

VP V

PROPYLTHIOURACIL

Relative density 1.035 to 1.040 (Appendix 6.5).

PROPYLTHIOURACIL Propylthiouracillum

Refractive index 1.431 to 1.433 (Appendix 6.1). Acidity Add 40 ml of water and 0.1 ml of bromothymol blue solution R to 10 ml of the substance to be examined. The solution is greenish-yellow. Not more than 0.05 ml of 0.1 M sodium hydroxide is required to change the colour of the indicator to blue. Oxidising substances Add 5 ml of water, 2 ml of potassium iodide solution R and 2 ml of dilute sulfuric acid R to 10 ml of the substance to be examined in a ground-glass-stoppered flask and allow to stand, protected from light for 15 min. Titrate with 0.05 M sodium thiosulfate VS, using 1 ml of starch solution R as indicator. Not more than 0.2 ml of 0.05 M sodium thiosulfate VS is required. Reducing substances Add 1 ml of dilute ammonia solution R to 1 ml of the substance to be examined and heat in a water-bath at 60 °C for 5 min. The solution is not yellow. Immediately add 0.15 ml of 0.1 M silver nitrate R and allow to stand for 5 min. The solution does not change its appearance. Heavy metals Not more than 5 ppm (m/v) (Appendix 9.4.8). Mix 4 ml of the substance to be examined with 16 ml of water. 12 ml of the solution complies with limit test for heavy metals, method 1. Prepare the standard using lead standard solution (1 ppm Pb) R. Water Not more than 0.2% (Appendix 10.3). Determined on 5.00 g. Sulfated ash Not more than 0.01% (Appendix 9.9, method 2). Heat 50 g of the substance to be examined until it burns and ignite. Allow to cool. Moisten the residue with sulfuric acid R and ignite; repeat the operations to constant weight. The residue weighs not more than 5 mg. Action and use Excipients. Storage Store in an airtight container.

816

C7H10N2OS

M. 170.2

Propylthiouracil is 2,3-dihydro-6-propyl-2-thioxopyrimidin4(1H)-one. It contains not less than 98.0% and not more than 100.5% of C7H10N2OS, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder or crystals. Very slightly soluble in water and in ether, sparingly soluble in ethanol (96 %). It dissolves in solutions of alkali hydroxides. Identification Apply one of the two following indentifications: First identification: A, B. Second identification: B, C, D. A. The infrared absorption spectrophotometry (Appendix 4.2) of the substance to be examined is concordant with the spectrum of propylthiouracil RS. Examine as discs prepared using 1 mg of substance and 0.3 g of potassium bromide R. B. Melting point: 217 °C to 221 °C (Appendix 6.7). C. Examine the chromatograms obtained in the test for thiourea and related substances in ultraviolet light at 254 nm before exposure of the plate to iodine vapour. The principal spot in the chromatogram obtained with test solution (2) is similar in position and size to the principal spot in the chromatogram obtained with reference solution (1). D. To about 20 mg of the substance to be examined add 8 ml of bromine water R and shake for a few minutes. Boil until the mixture is decolourised, allow to cool and filter. To the filtrate add 2 ml of 6.1% barium chloride solution R. A white precipitate is formed whose colour does not become violet on the addition of 5 ml of dilute sodium hydroxide solution R. Thiourea and related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Glacial acetic acid - isopropanol chloroform (0.1 : 6 : 50). Test solution (1): Dissolve 0.1 g of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Test solution (2): Dilute 1 ml of test solution (1) to 10 ml with methanol R. Reference solution (1): Dissolve 10 mg of propylthiouracil RS in methanol R and dilute to 10 ml with the same solvent.

VP V

Reference solution (2): Dissolve 50 mg of thiourea R in methanol R and dilute to 100 ml with the same solvent. Dilute 1 ml of this solution to 100 ml with methanol R. Reference solution (3): Dilute 1 ml of test solution (1) to 100 ml with methanol R. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in air. Examine in ultraviolet light at 254 nm. Expose the plate to iodine vapour for 10 minutes. In the chromatogram obtained with test solution (1), any spot corresponding to thiourea is not more intense than the spot in the chromatogram obtained with reference solution (2) (0.05%) and any spot apart from the principal spot and any spot corresponding to thiourea is not more intense than the spot in the chromatogram obtained with reference solution (3) (1.0%).

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 6. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 100 °C - 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Weigh accurately about 0.300 g of the substance to be examined, add 30 ml of water and 30.0 ml of 0.1 N sodium hydroxide VS. Boil and shake until dissolution is complete. Add 50 ml of 0.1 N silver nitrate VS while stirring, boil gently for 5 min and cool. Titrate with 0.1 N sodium hydroxide VS, determining the end-point potentiometrically (Appendix 10.2). The volume of 0.1 N sodium hydroxide VS used is equal to the sum of the volume (30.0 ml) added initially and the volume used in the final titration. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 8.511 mg of C7H10N2OS. Storage Store protected from light. Action and use Antithyroid. Preparation Tablets.

PROPYLTHIOURACIL TABLETS

PROPYLTHIOURACIL TABLETS Tabellae Propylthiouracili Propylthiouracil tablets contain propylthiouracil. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of propylthiouracil, C7H10N2OS, 92.5% to 107.5% of the stated amount. Identification A. Shake a quantity of the powdered tablets containing 50 mg of propylthiouracil with 20 ml of methanol R, filter and evaporate on a water bath to dryness. The infrared absorption spectrum (Appendix 4.2) of the residue is concordant with the spectrum of propylthiouracil RS. B. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the principal peak in the chromatogram obtained with the reference solution Dissolution (Appendix 11.4) Apparatus: Basket Medium: 900 ml of water. Rotation speed: 100 rpm. Time: 30 min. Procedure: After the specified time, withdraw a sample of the medium, filter and discard the first portion of the filtrate. Dilute a portion of the filtrate with water, if necessary, to obtain a solution containing 5 µg of propylthiouracil per ml. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum at about 274 nm in a 1 cm cell, using water as a blank in comparison with the reference solution of propylthiouracil RS having the same concentration in water. Calculate the content of propylthiouracil, C7H10N2OS, dissolved using the absorbances of the test solution and the reference solution and the declared content of C7H10N2OS in propylthiouracil RS. Tolerance: Not less than 85% (Q) of the stated amount of propylthiouracil, C7H10N2OS, is dissolved in 30 min. Assay Examine by liquid chromatography (Appendix 5.3). 0.025 M phosphate buffer: Transfer an accurately weighed quantity of 3.40 g of potassium dihydrogen phosphate R to a 1000 ml beaker. Add 500 ml of water, and stir until dissolved. Adjust the resulting solution with phosphoric acid R or 0.1 M sodium hydroxide to pH 4.6. Add sufficient water to produce 1000.0 ml, mix and filter. Mobile phase: 0.025 M phosphate buffer - acetonitrile (80 : 20). Reference solution: Transfer an accurately weighed quantity of about 25 mg of propylthiouracil RS to a 50 ml volumetric flask, add 5 ml of methanol R, and sonicate for 5 minutes. Add 25 ml of water and shake for 15 minutes. 817

VP V

PYRANTEL PAMOATE

Dilute with water to volume and mix. Dilute 10.0 ml of this solution to 100.0 ml with water and shake. Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Weigh accurately a quantity of the powdered tablets containing about 50 mg of propylthiouracil to a 100 ml volumetric flask, add 10 ml of methanol R and sonicate for 5 minutes. Add 50 ml of water and shake for 20 minutes. Dilute with water to volume, mix and filter. Dilute 10.0 ml of the filtrate to 100.0 ml with water and shake. Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 272 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution, the column efficiency, determined from propylthiouracil peak, is not less than 3500 theoretical plates; the symmetry factor for propylthiouracil peak is not more than 2.0, and the relative standard deviation of the peak areas for replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of propylthiouracil, C7H10N2OS, in the tablets using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C7H10N2OS in propylthiouracil RS.

Storage Protected from light. Action and use Hyperthyrodism. Usual strength 25 mg, 50 mg. PYRANTEL PAMOATE Pyranteli pamoatum Pyrantel embonate

C34H30N2O6S  

M. 594.7

Pyrantel pamoate is 1-methyl-2-[(E)-2-(thiophen-2-yl) ethenyl]-1,4,5,6-tetrahydropyrimidine hydrogen 4,4’ -methylenebis(3-hydroxynaphthalene-2-carboxylate). It contains not less than 98.0% and not more than 102.0% of C34H30N2O6S, calculated with reference to the dried substance. 818

Characters Pale yellow or yellow powder. Soluble in dimethyl sulfoxide, pratically insoluble in methanol and in water. Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of pyrantel pamoate RS. Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use and strictly protect from light at all stages. Solvent mixture: Mix 5 volumes of glacial acetic R with 5 volumes of water and add 2 volumes of diethylamine R with cooling. Mobile phase: Solvent mixture - acetonitrile for chromatography R (72 : 928). Test solution: Dissolve 80 mg of the substance to be examined in 7 ml of the solvent mixture and dilute to 100.0 ml with acetonitrile R. Reference solution (1): Dissolve 10.0 mg of pyrantel impurity A RS in the solvent mixture, add 2.5 ml of the test solution and dilute to 50.0 ml with the solvent mixture. Dilute 2.0 ml of this solution to 100.0 ml with the solvent mixture. Reference solution (2): Dilute 1.0 ml of the test solution to 200.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase A (5 µm). Detector: A spectrophotometer set at 288 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Procedure: The run time of the test solution is 4 times the retention time of pyrantel. Relative retention with reference to pyrantel (retention time = about 11 min): embonic acid = about 0.5; impurity A = about 1.3; impurity B = about 1.8 (impurity A also gives rise to an embonate peak). System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to pyrantel and impurity A is at least 4.0. Limits: Correction factor: For the calculation of content, multiply the peak area of impurity B by 0.4. Impurity A: The area of the peak due to impurity A is not more than the area of the corresponding peak in the chromatogram obtained with reference solution (1) (0.5%). Impurity B: The corrected area of the peak due to impurity B is not more than 0.4 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.2%). Any other impurity: For each impurity, the area is not more than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.10%).

PYRANTEL PAMOATE TABLETS

VP V

The sum of the peak areas of all impurities other than A and B is not more than 0.6 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.3%). Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Note: Impurity A: 1-methyl-2-[(Z)-2-(thiophen-2-yl)ethenyl]-1,4,5,6tetrahydropyrimidine. Impurity B: (E)-N-[3-(methylamino)propyl]-3-(thiophen-2-yl) prop-2-enamide.

Chlorides Not more than 360 ppm (Appendix 9.4.5). Add 10 ml of 2 M nitric acid R and 30 ml of water to 0.46 g of the substance to be examined. Heat on a water- bath for 5 min. Cool, dilute to 50 ml with water, mix well and filter. Use 15 ml of filtrate. Sulfates Not more than 0.1% (Appendix 9.4.14). Add 2.5 ml of dilute nitric acid R to 0.50 g of the substance to be examined and dilute to 50 ml with distilled water R. Heat on a water-bath for 5 min, shake for 2 min, cool and filter. Iron Not more than 75 ppm (Appendix 9.4.13). Ignite 0.66 g of the substance to be examined at 800 °C ± 50 °C for 2 h. Dissolve the residue in 2.5 ml of dilute hydrochloric acid R with gentle heating for 10 min. Cool and dilute to 50 ml with water. Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 4. Prepare the standard using 2.0 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 1.0% (Appendix 9.6). (1.000 g; 60 °C; in vacuo; 3 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Add 10 ml of acetic anhydride R and 50 ml glacial acetic acid R to 0.450 g of the substance to be examined, heat at 50 °C and stir for 10 min. Allow to cool (a clear solution is not obtained). Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). Carry out a blank titration. 1 ml of 0.1 N perchloric acid VS is equivalent to 59.47 mg of C34H30N2O6S.

Storage Store in an airtight container, protected from light. Action and use Anthelminthic. Preparation Tablets. PYRANTEL PAMOATE TABLETS Tabellae Pyranteli pamoati Pyrantel pamoate tablets contain pyrantel pamoate. They are coated tablets. The tablets comply with the requirements stated under “Tablets” in “Coated tablets” section (Appendix 1.20) and with the following requirements.

Content of pyrantel, C11H14N2S, 93.0% to 107.0% of the stated amount. Identification A. Shake a quantity of the powdered tablets containing 40 mg of pyrantel pamoate with 20 ml of a mixture of dioxane - a 0.1% solution of ammonia (1 : 1), filter. Use the filtrate to perform the following tests: To 5 ml of the filtrate add 2 ml of dilute hydrochloric acid R. A yellow precipitate is produced. Evaporate 10 ml of the filtrate to dryness. To the residue add 1 ml of sulfuric acid R and shake. A red colour is produced. B. In the Assay, the retention times of two principal peaks in the chromatogram obtained with the test solution correspond to those of two principal peaks in the chromatogram obtained with the reference solution. Assay Examine by liquid chromatography (Appendix 5.3) and carry out the following procedure protected from light. Mobile phase: Acetonitrile - water - glacial acetic acid diethylamine (94 : 2.5 : 2.5 : 1). Make adjustments if necessary. Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Dissolve an accurately weighed quantity of the powdered tablets in the mobile phase to obtain a solution containing about 80 µg of pyrantel pamoate per ml and filter. Reference solution: Dissolve an accurately weighed quantity of pyrantel pamoate RS in the mobile phase to obtain a solution containing the same concentration as the test solution. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase A (5 µm to 10 µm). Detector: A spectrophotometer set at 288 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. 819

VP V

PYRAZINAMIDE

Procedure: System suitability: Inject the reference solution, record the chromatograms not less than 2.5 times the retention time of pyrantel peak; the column efficiency, determined from pyrantel peak, is not less than 8000 theoretical plates; the relative retention times for pamoic acid and pyrantel are about 0.6 and 1.0, respectively; the resolution, R, between pyrantel and pamoic acid peak is not less than 10; the symmetry factor for the pyrantel peak is not greater than 1.3; and the relative deviation of the peak areas for replicate injections is not more than 1.0%. Inject alternately the reference solution and the test solution. Calculate the content of pyrantel, C11H14N2S, in the tablets using the areas of pyrantel peaks in the chromatograms obtained with the test solution, the reference solution and the declared content of C11H14N2S in pyrantel pamoate RS. Each mg of pyrantel pamoate is equivalent to 0.347 mg of pyrantel base.

Storage Store in a well-closed container, protected from light. Action and use Anthelmintic. Usual strength 300 mg.

O NH2

N

C5H5N3O  

M. 123.1

Pyrazinamide is pyrazine-2-carboxamide. It contains not less than 99.0% and not more than 101.0% of C5H5N3O, calculated with reference to the anhydrous substance.

Characters White or almost white, crystalline powder, it shows polymorphism. Sparingly soluble in water, slightly soluble in ethanol (96%) and in methylene chloride. Identification Apply one of the two following identifications: First identification: A. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of pyrazinamide RS. If the spectra obtained show differences, dissolve the substance to be examined and the reference 820

Appearance of solution Solution S: Dissolve 0.5 g in carbon dioxide-free water R and dilute to 50 ml with the same solvent. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). Acidity or alkalinity Add 0.05 ml of phenolphthalein solution R1 and 0.2 ml of 0.01 N sodium hydroxide VS to 25 ml of solution S. The solution is red. Add 1.0 ml of 0.01 N hydrochloric acid VS. The solution is colourless. Add 0.15 ml of methyl red solution R. The solution is red.

PYRAZINAMIDE Pyrazinamidum N

substance separately in ethanol (96%) R, evaporate to dryness and record new spectra using the residues. B. Melting point: 188 °C to 191 °C (Appendix 6.7). C. Dissolve 50.0 mg of the substance to be examined in water and dilute to 100.0 ml with the same solvent (Solution A). Dilute 1.0 ml of the solution A to 10.0 ml with water. The ultraviolet absorption spectrum (Appendix 4.1) of the resulting solution in the range 290 nm to 350 nm exhibits an absorption maximum at 310 nm. Dilute 2.0 ml of the solution A to 100.0 ml with water. The ultraviolet absorption spectrum (Appendix 4.1) of the resulting solution in the range 230 nm to 290 nm exhibits an absorption maximum at 268 nm. The specific absorbance at this maximum is 640 to 680. D. Dissolve 0.1 g in 5 ml of water. Add 1 ml of ferrous sulfate solution R2, the solution becomes orange. Add 1 ml of dilute sodium hydroxide solution R, the solution becomes dark blue.

Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use. Mobile phase: Dissolve 6.80 g of potassium dihydrogen phosphate R in 800 ml of water, add 1.84 g of sodium hydroxide R, adjust to pH 3.0 with dilute phosphoric acid R and dilute to 1000 ml with water; add 10.0 ml of acetonitrile R and 1.0 ml of tetrahydrofuran R. Test solution: Dissolve 50 mg of the substance to be examined in water and dilute to 25.0 ml with the same solvent. Dilute 5.0 ml of the solution to 25.0 ml with water. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with water. Dilute 1.0 ml of this solution to 10.0 ml with water. Reference solution (2): Dissolve 10 mg of pyrazine2-carbonitrile R (impurity B) in water and dilute to 50.0 ml with the same solvent. Dilute 5.0 ml of the solution to 50.0 ml with water. To 5.0 ml of this solution add 5.0 ml of the test solution and dilute to 25.0 ml with water. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (5 µm).

PYRAZINAMIDE TABLETS

VP V

Column temperature: 30 °C. Detector: A spectrophotometer set at 270 nm. Flow rate: 2.0 ml/min. Volume of injection: 40 µl. Procedure: The run time is 4 times the retention time of pyrazinamide. Identification of impurities: Use the chromatogram obtained with reference solution (2) to identify the peak due to impurity B. Relative retention with reference to pyrazinamide (retention time = about 5 min): impurity B = about 1.6. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to pyrazinamide and impurity B is at least 4.0. Limits: Impurity B: The area of the peak due to impurity B is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). Any other impurity: For each impurity, the area is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Total peak area of all impurities is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%). Disregard any peak with an area less than 0.3 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.03%). Note: Impurity A: Pyrazine-2-carboxylic acid. Impurity B: Pyrazine-2-carbonitrile.

Heavy metals Not more than 10 ppm (Appendix 9.4.8). Solvent mixture: water - ethanol (96%) (50 : 50). 0.25 g complies with limit test for heavy metals, method 8. Prepare the standard using 0.25 ml of lead standard solution (10 ppm Pb) R. Water Not more than 0.5% (Appendix 10.3). Determined on 2.00 g. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.100 g of the substance to be examined in 50 ml of acetic anhydride R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 12.31 mg of C5H5N3O. Storage Store in an airtight container, protected from light.

Action and use Antituberculosis drug. Preparation Tablets. PYRAZINAMIDE TABLETS Tabellae Pyrazinamidi Pyrazinamide tablets contain pyrazinamide. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of pyrazinamide, C5H5N3O, 95.0% to 105.0% of the stated amount. Identification A. Shake a quantity of the powdered tablets containing 0.25 g of pyrazinamide with 20 ml of ethanol R, filter and evaporate the filtrate to dryness and dry the residue at 105 °C for 30 min. The infrared absorption spectrum (Appendix 4.2) of the residue is concordant with the reference spectrum of pyrazinamide. B. Shake a quantity of the powdered tablets containing 50 mg of pyrazinamide with 50 ml of water, filter. Dilute 1 ml of the filtrate to 100 ml with water. The ultraviolet absorption spectrum (Appendix 4.1) of the resulting solution in the range 230 nm to 350 nm exhibits two absorption maxima at 268 nm and 310 nm. C. To a quantity of the powdered tablets containing 20 mg of pyrazinamide add 5 ml of 5 M sodium hydroxide R. Boil this mixture, an odour of ammonia is produced. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of water. Rotation speed: 50 rpm. Time: 45 min. Procedure: After the specified time, withdraw a sample of the medium, filter. Dilute the filtrate with water to obtain a solution containing about 10 µg of pyrazinamide per ml. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum at 268 nm, using water as a blank, in comparison with the reference solution of pyrazinamide RS having the same concentration in water. Calculate the content of pyrazinamide, C5H5N3O, dissolved using the absorbances of the test solution and the reference solution and the declared content of C5H5N3O in pyrazinamide RS. Tolerance: Not less than 75% (Q) of the stated amount of pyrazinamide, C5H5N3O, is dissolved in 45 min. Related substances Not more than 0.2%. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. 821

PYRIDOXINE HYDROCHLORIDE

VP V

Mobile phase: Glacial acetic acid - water - n-butanol (20 : 20 : 60). Test solution: Shake a quantity of the powdered tablets containing 0.1 g of pyrazinamide with 50 ml of a mixture of chloroform - methanol (9 : 1), filter, evaporate on a water bath to dryness and dissolve the residue in the same solvent mixture to produce 10 ml. Reference solution: Dilute 1 volume of the test solution to 500 volumes with a mixture of chloroform - methanol (9 : 1). Procedure: Apply separately to the plate 20 µl of each solution. Develop over a path of 15 cm. After removal of the plate, allow it to dry in air. Examine immediately under ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution.

Assay Weigh 20 tablets, calculate the average mass and powder finely. Transfer a quantity of the powdered tablets equivalent to about 0.1 g of pyrazinamide, accurately weighed, to a 500 ml volumetric flask, add 200 ml of water, allow to stand for 10 min with occasionally shaking. Sonicate for 10 min and add water to volume, mix and filter, discard the first 20 ml of the filtrate. Dilute 5.0 ml of the filtrate to 100.0 ml with water, mix. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum at 268 nm, using water as a blank in comparison with the reference solution of pyrazinamide RS having the same concentration in water. Calculate the content of pyrazinamide, C5H5N3O, in the tablets using the absorbances of the test solution and the reference solution and the declared content of C5H5N3O in pyrazinamide RS. Storage Store in a well-closed container, at a temperature not exceeding 30 °C. Action and use Antituberculosis. Usual strength 500 mg.

PYRIDOXINE HYDROCHLORIDE Pyridoxini hydrochloridum Vitamin B6 H 3C

N OH

HO

, HCl

OH

C8H11NO3,HCl  

822

M. 205.6

Pyridoxine hydrochloride is (5-hydroxy-6-methylpyridine3,4-diyl)dimethanol hydrochloride. It contains not less than 99.0% and not more than 101.0% of C8H11NO3,HCl, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder. Freely soluble in water, slightly soluble in ethanol (96%). Melting point: About 205 °C, with decomposition. Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of pyridoxine hydrochloride RS. B. Solution A: Dilute 1.0 ml of solution S (see Appearance of solution) to 50.0 ml with 0.1 M hydrochloric acid R. Solution B: Dilute 1.0 ml of solution A to 100.0 ml with 0.1 M hydrochloric acid R. Solution C: Dilute 1.0 ml of solution A to 100.0 ml with the 0.025 M phosphate buffer R. The ultraviolet absorption spectrum (Appendix 4.1) of solution B in the range 250 nm to 350 nm, exhibits an absorption maximum at 288 nm to 296 nm. The specific absorbance at this maximum is 425 to 445. The ultraviolet absorption spectrum (Appendix 4.1) of solution C in the range 220 nm to 350 nm exhibits two absorption maxima at 248 nm to 256 nm and 320 nm to 327 nm. The specific absorbance at this maxima is 175 to 195 and 345 to 365, respectively. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Ammonia - methylene chloride tetrahydrofuran - acetone (9 : 13 : 13 : 65). Test solution: Dissolve 1.0 g of the substance to be examined in water and dilute to 10 ml with the same solvent. Dilute 1 ml of this solution to 10 ml with water. Reference solution: Dissolve 0.10 g of pyridoxine hydrochloride RS in water and dilute to 10 ml with the same solvent. Procedure: Apply separately to the plate 2 µl of each solution. Develop in an unsaturated tank to over a path of 15 cm. Allow the plate to dry in air. Spray with a 5% solution of sodium carbonate R in a mixture of ethanol (96%) - water (30 : 70). Allow to dry in air, spray with a 0.1% solution of dichloroquinonechlorimide R in ethanol (96%) R and examine immediately. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. D. Solution S gives reaction (A) of chlorides (Appendix 8.1).

VP V

Appearance of solution Solution S: Dissolve 2.50 g in carbon dioxide-free water R and dilute to 50.0 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution Y7 (Appendix 9.3, method 2). pH 2.4 to 3.0 (Appendix 6.2). Determined on solution S. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 2.72 g of potassium dihydrogen phosphate R in 900 ml of water, adjust to pH 3.0 with dilute phosphoric acid R and dilute to 1000 ml with water. Test solution: Dissolve 25 mg of the substance to be examined in water and dilute to 10.0 ml with the same solvent. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with water. Dilute 1.0 ml of this solution to 10.0 ml with water. Reference solution (2): Dissolve 2.5 mg of pyridoxine impurity A RS and 2.5 mg of 4-deoxypyridoxine hydrochloride R (impurity B) in water and dilute to 10.0 ml with the same solvent. Dilute 2.0 ml of this solution to 10.0 ml with water. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase base-deactivated end-capped octadecylsilyl silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 210 nm. Flow rate: 1.0 ml/min. Volume of injection: 5 µl. Procedure: The run time is 2.5 times the retention time of pyridoxine. Identification of impurities: Use the chromatogram obtained with reference solution (2) to identify the peaks due to impurities A and B. Relative retention with reference to pyridoxine (retention time = about 12 min): impurity A = about 1.7; impurity B = about 1.9. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurities A and B is at least 1.5. Limits: Correction factor: For the calculation of content, multiply the peak area of impurity B by 1.5. Impurity B: The corrected area of the peak due to impurity B is not more 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.15%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%).

PYRIDOXINE HYDROCHLORIDE INJECTION

The sum of the peak areas of all impurities is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A: 6-methyl-1,3-dihydrofuro[3,4-c]pyridin-7-ol. Impurity B: 5-(hydroxymethyl)-2,4-dimethylpyridin-3-ol.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 12 ml of solution S complies with limit test for heavy metals, method 1. Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay In order to avoid overheating in the reaction medium, mix thoroughly throughout and stop the titration immediately after the end-point has been reached. Dissolve 0.150 g of the substance to be examined in 5 ml of anhydrous formic acid R, add 50 ml of acetic anhydride R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). Carry out a blank titration. 1 ml of 0.1 N perchloric acid VS is equivalent to 20.56 mg of C8H11NO3,HCl. Storage Store in an airtight container, protected from light. Action and use Vitamin B group. Preparations Tablets, capsules, injection. PYRIDOXINE HYDROCHLORIDE INJECTION Injectio Pyridoxini hydrochloridi Vitamin B6 injection Pyridoxine hydrochloride injection is a sterile solution of pyridoxine hydrochloride in water for injections. The preparation complies with the general requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements.

823

VP V

PYRIDOXINE HYDROCHLORIDE TABLETS

Content of pyridoxine hydrochloride, C8H11NO3, HCl, 95.0% to 110.0% of the stated amount. Characters A clear and colourless solution. Identification Solution A: Dilute a portion of the injection containing 100 mg of pyridoxine hydrochloride with water to 100 ml. A. In the Assay, the ultraviolet absorption spectrum (Appendix 4.1) of the test solution exhibits an absorption maximum at about 290 nm. B. Dilute 1 ml of solution A to 10 ml with water. To 1 ml of the resulting solution add 2 ml of a 20% solution of sodium acetate R, 1 ml of water and 1 ml of a 0.5% solution of 2,6-dichloroquinone chlorimide in ethanol and shake. A blue colour is produced but discolours and turns red after. Repeat the test but using 1 ml of a 4% solution of boric acid in place of 1 ml of water, no blue colour is produced. C. To 1 ml of solution A add 2 drops of a 5% solution of ferric chloride R, a red colour is produced. Add a 10% solution of sulfuric acid dropwise, the red colour is fade slowly. pH 2.5 to 4.0 (Appendix 6.2). Bacterial endotoxins Not more than 0.4 EU/mg Pyridoxine hydrochloride. Carry out the test for bacterial endotoxins (Appendix 13.2). Assay Dilute an accurately measured volume of the injection containing 0.1 g of pyridoxine hydrochloride to 500.0 ml with 0.1 M hydrochloric acid R and mix. Dilute 5.0 ml of this solution to 100.0 ml with 0.1 M hydrochloric acid R and mix. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum at about 290 nm in a 1 cm cell, using 0.1 M hydrochloric acid R as a blank. Calculate the content of pyridoxine hydrochloride, C8H11NO3,HCl, in the injection taking 430 as the value of A (1%, 1 cm) at the maximum at 290 nm. Storage Protected from light. Action and use Vitamin B group. Usual strength 2.5%; 5.0%; 10.0%.

PYRIDOXINE HYDROCHLORIDE TABLETS Tabellae Pyridoxini hydrochloridi Vitamin B6 tablets Pyridoxine hydrochloride tablets contain pyridoxine hydrochloride. The tablets comply with the general requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of pyridoxine hydrochloride, C8H11NO3,HCl, 90.0% to 110.0% of the stated amount. Identification Solution A: To a quantity of the powder tablets containing about 50 mg of pyridoxine hydrochloride add 50 ml of water, mix and filter. A. In the Assay, the ultraviolet absorption spectrum (Appendix 4.1) of the test solution exhibits an absorption maximum at about 290 nm. B. Dilute 1 ml of solution A to 10 ml with water. To 1 ml of the resulting solution add 2 ml of a 20% solution of sodium acetate R, 1 ml of water and 1 ml of a 0.5% solution of 2,6-dichloroquinone chlorimide in ethanol and shake. A blue colour is produced but discolours and turns red after. Repeat the test but using 1 ml of a 4% solution of boric acid in place of 1 ml of water, no blue colour is produced. C. To 1 ml of solution A add 2 drops of a 5% solution of ferric chloride R, a red colour is produced. Add a 10% solution of sulfuric acid dropwise, the red colour is fade slowly. Assay Weigh 20 tablets, powder finely. Weigh accurately a quantity of the powdered tablets containing 25 mg of pyridoxine hydrochloride, add 50 ml of 0.1 M hydrochloric acid R and heat on a water bath for 15 minutes, with occasionally shaking. Cool, dilute to 100.0 ml with 0.1 M hydrochloric acid R and mix. Filter, discard the first portion of the filtrate. Dilute 5.0 ml of the filtrate to 100.0 ml with 0.1 M hydrochloric acid R. Measure the absorbance (Appendix 4.1) of the resulting solution at the maximum at about 290 nm in a 1 cm cell, using 0.1 M hydrochloric acid R as a blank. Calculate the content of pyridoxine hydrochloride, C8H11NO3,HCl, in the tablets taking 430 as the value of A (1%, 1 cm) at the maximum at 290 nm. Storage Store in a cool and dry place and protected from light. Action and use Vitamin B group. Usual strength 25 mg, 50 mg.

824

PYRIMETHAMINE

VP V

PYRIMETHAMINE Pyrimethaminum

C12H13ClN4 M.248.7 Pyrimethamine is 5-(4-chlorophenyl)-6-ethylpyrimidine -2,4-diamine. It contains not less than 99.0% and not more than 101.0% of C12H13ClN4, calculated with reference to the dried substance.

Characters An almost white, crystalline powder or colourless crystals. Practically insoluble in water, slightly soluble in ethanol (96 %). Identification Apply one of the two following identifications: First identification: A. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of pyrimethamine RS. B. Melting point: 239 °C to 243 °C (Appendix 6.7). C. Dissolve 0.14 g of the substance to be examined in ethanol R, dilute to 100.0 ml with the same solvent, and mix. Dilute 10.0 ml of this solution to 100.0 ml with 0.1 M hydrochloric acid R, and mix. Dilute 10.0 ml of this solution to 100.0 ml with 0.1 M hydrochloric acid R, and mix. The ultraviolet absorptiom spectrum (Appendix 4.1) of the solution, in the range between 250 nm and 300 nm, exhibits an absorption maximum at 272 nm and an absorption minimum at 261 nm. The specific absorbance at the maximum is 310 to 330. D. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (2) is similar in position and size to the principal spot in the chromatogram obtained with reference solution (1). Appearance of solution Prepare the solution immediately before use. Dissolve 0.25 g of the substance to be examined in a mixture of methanol - methylene chloride (1 : 3) and dilute to 10 ml with the same mixture of solvents. The solution is clear (Appendix 9.2) and not more intensely coloured than reference solution BY6 (Appendix 9.3, method 2). Acidity or alkalinity Solution S: Shake 1.0 g of the substance to be examined with 50 ml of water for 2 min and filter. To 10 ml of solution S add 0.05 ml of phenolphthalein solution R. The solution is colourless. Not more than

0.2 ml of 0.01 N sodium hydroxide VS is required to change the colour of the indicator to pink. Add 0.4 ml of 0.01 N hydrochloric acid VS and 0.05 ml of methyl red solution R. The solution is red or orange.

Related substances Not more than 0.25%. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel GF254. Mobile phase: Chloroform - propanol - glacial acetic acid - toluene (4 : 8 : 12 : 76). Prepare the solutions immediately before use. Test solution (1): Dissolve 0.25 g of the substance to be examined in a mixture of methanol - chloroform (1 : 9), dilute to 25 ml with the same mixture of solvents, and mix. Test solution (2): Dilute 1.0 ml of test solution (1) to 10 ml with a mixture of methanol - chloroform (1 : 9), and mix. Reference solution (1): Dissolve 0.10 g of pyrimethamine RS in a mixture of methanol - chloroform (1 : 9), dilute to 100 ml with the same mixture of solvents, and mix. Reference solution (2): Dilute 5.0 ml of test solution (1) to 200.0 ml with a mixture of methanol - chloroform (1 : 9), and mix. Dilute 1.0 ml of the solution to 10.0 ml with the same mixture of solvents. Procedure: Apply separately to the plate 20 µl of each solution. Develop over a path of about 10 cm. Remove the plate, allow it to dry in air. Examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with test solution (1), apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (2). Sulphates Not more than 80 ppm (Appendix 9.4.14). 15 ml of solution S complies with the limit test for sulphates. Prepare the reference using a mixture of 2.5 ml of sulfate standard solution (10 ppm SO4) R and 12.5 ml of water. Loss on drying Not more than 0.5% (Appendix 9.6). (0.50 g; 100 °C - 105 °C; 4 hours). Sulphated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.200 g, accurately weighed, of the substance to be examined in 25 ml of anhydrous acetic acid R, heating gently. Cool, and titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 24.87 mg of C12H13ClN4. Storage Protected from light. 825

VP V

QUINAPRIL HYDROCHLORIDE

Action and use Antimalarial. Preparation Tablets. QUINAPRIL HYDROCHLORIDE Quinaprili hydrochloridum

C25H31ClN2O5

M. 475.0

Quinapril hydrochloride is (3S)-2-[(2S)-2-[[(1S)-1(ethoxycarbonyl)-3-phenylpropyl]amino]propanoyl]-1,2,3,4tetrahydroisoquinoline-3-carboxylic acid hydrochloride. It contains not less than 98.5% and not more than 101.5% of C25H31ClN2O5, calculated with reference to the anhydrous substance.

Characters White or almost white or slightly pink, hygroscopic powder. Freely soluble in water and in ethanol (96%), very slightly soluble in acetone. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of quinapril hydrochloride RS. B. It complies with the test for Specific optical rotation. C. It gives reaction (A) of chlorides (Appendix 8.1). Specific optical rotation +14.4° to +16.6°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.500 g in methanol R and dilute to 25.0 ml with the same solvent. Diastereoisomers Examine by liquid chromatography (Appendix 5.3). Prepare the solution immediately before use. Mobile phase: Mix 260 ml of tetrahydrofuran R with 740 ml of a freshly prepared solution containing 0.80 g of  sodium octanesulfonate R and 2.13 g of ammonium dihydrogen phosphate R, previously adjusted to pH 4.5 with phosphoric acid R. Solvent mixture: Adjust 500 ml of the mobile phase to pH 6.5 with ammonia R. Test solution: Dissolve 100 mg of the substance to be examined in the solvent mixture and dilute to 50.0 ml with the solvent mixture. 826

Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 20.0 ml with the solvent mixture. Reference solution (2): Dissolve the contents of a vial of  quinapril for peak identification RS (containing impurities G, H and I) in the solvent mixture and dilute to 5.0 ml with the solvent mixture. Chromatographic system: A column (25 cm × 4.0 mm) packed with stationary phase C (5 µm). Column temperature: 25 °C. Detector: A spectrophotometer set at 220 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: The run time is 3.5 times the retention time of quinapril. Identification of impurities: Use the chromatogram supplied with quinapril for peak identification RS and the chromatogram obtained with reference solution (2) to identify the peaks due to impurities G, H and I. Relative retention with reference to quinapril (retention time = about 18 min): Impurity G = about 0.9; impurity H = about 1.2; impurity I = about 1.3. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurity G and quinapril is at least 1.5. Peak-tovalley ratio (Hp/Hv) is at least 2.0, where Hp =height above the baseline of the peak due to impurity H and Hv = height above the baseline of the lowest point of the curve separating this peak from the peak due to quinapril. Limits: Impurities G, H, I: For each impurity, the area is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.15%). Note: Impurity G:  (3R)-2-[(2S)-2-[[(1S)-1-(ethoxycarbonyl)-3-phenylpropyl] amino]propanoyl]-1,2,3,4-tetrahydro isoquinoline-3-carboxylic acid. Impurity H:  (3R)-2-[(2S)-2-[[(1R)-1-(ethoxycarbonyl)-3-phenylpropyl] amino]propanoyl]-1,2,3,4-tetrahydro isoquinoline-3-carboxylic acid. Impurity I:  (3S)-2-[(2S)-2-[[(1R)-1-(ethoxycarbonyl)-3-phenylpropyl] amino]propanoyl]-1,2,3,4-tetrahydro isoquinoline-3-carboxylic acid.

Related substances Examine by liquid chromatography (Appendix 5.3).  Mobile phase: Acetonitrile R1 - a 5.77 g/L solution of  sodium dodecyl sulfate R adjusted to pH 2.2 with phosphoric acid R (48 : 52). Solvent mixture: Acetonitrile R1 - a 2.88 g/L solution of ammonium dihydrogen phosphate R previously adjusted to pH 6.5 with dilute ammonia R1 (40 : 60). Test solution: Dissolve 50 mg of the substance to be examined in the solvent mixture and dilute to 25.0 ml with the solvent mixture. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 10.0 ml with the solvent mixture.

QUINAPRIL HYDROCHLORIDE

VP V

Reference solution (2): Dissolve the contents of a vial of quinapril for system suitability RS (containing impurities A, C, D, E and G) in the solvent mixture and dilute to 5.0 ml with the solvent mixture. Reference solution (3): In order to prepare impurity M in situ, dissolve 250 mg of the substance to be examined in methylene chloride R and dilute to 5.0 ml with the same solvent. Expose this solution to a source of ultraviolet light for 2.5 h and evaporate the solvent. Dissolve 40 mg of the remaining substance in the solvent mixture and dilute to 20.0 ml with the solvent mixture. Chromatographic system: Acolumn (15 cm × 3.9 mm) packed with stationary phase endcapped octylsilyl silica gel for chromatography R (5 µm). Column temperature: 30 °C. Autosampler temperature: 5 °C. Detector: A spectrophotometer set at 214 nm. Flow rate: 1.4 ml/min. Volume of injection: 10 µl. Procedure: The run time is 3 times the retention time of quinapril. Identification of impurities:  Use the chromatogram supplied with quinapril for system suitability RS and the chromatogram obtained with reference solution (2) to identify the peaks due to impurities A, C, D, E and G; use the chromatogram obtained with reference solution (3) to identify the peak due to impurity M. Relative retention with reference to quinapril (retention time = about 12 min): Impurity A = about 0.1; impurity C = about 0.3; impurity D = about 0.4; impurity M = about 0.7; impurities G + H = about 0.9; impurity E = about 2.3. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurities C and D is at least 1.5; the resolution between the peaks due to impurity G and quinapril is at least 1.5. Limits: Correction factor: For the calculation of content, multiply the peak area of impurity E by 1.5. Impurities C, D: For each impurity, the area is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). Impurity A: The area of the peak due to impurity A is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.3%). Impurities E, M: For each impurity, the corrected area, if necessary, is not more than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.15%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). The sum of the peak areas of all impurities is not more than 10 times the area of the principal peak in the chromatogram obtained with reference solution (1) (1.0 %). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained

with reference solution (1) (0.05%); disregard any peak due to impurities G + H. Note: Impurity A: (3S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid. Impurity B: (2S)-2-[[(1S)-1-(ethoxycarbonyl)-3-phenylpropyl] amino]propanoic acid. Impurity C: (3S)-2-[(2S)-2-[[(1S)-1-carboxy-3-phenylpropyl]amino] propanoyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid. Impurity D: Ethyl (2S)-2-[(3S,11aS)-3-methyl-1,4-dioxo1,3,4,6,11,11a-hexahydro-2H-pyrazino[1,2-b] isoquinolin-2yl]-4-phenylbutanoate. Impurity E: (3S)-2-[(2S)-2-[[(1S)-3-cyclohexyl-1-(ethoxycarbonyl) propyl]amino]propanoyl]-1,2,3,4-tetrahydroisoquinoline-3carboxylic acid. Impurity J: (1R,3S)-2-[(2S)-2-[[(1S)-1-(ethoxycarbonyl)-3phenylpropyl](hydroxy)amino]propanoyl]-1-hydroxy-1,2,3,4tetrahydroisoquinoline-3-carboxylic acid. Impurity M: (1R,3S)-2-[(2S)-2-[[(1S)-1-(ethoxycarbonyl)3-phenylpropyl]amino]propanoyl]-1-hydroperoxy-1,2,3,4tetrahydroisoquinoline-3-carboxylic acid.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). Solvent: Dimethyl sulfoxide R. 1.0 g complies with limit test for heavy metals, method 8. Prepare the standard using 2.0 ml of lead standard solution (10 ppm Pb) R. If the substance precipitates after addition of acetate buffer pH 3.5 R, dilute to 100 ml with dimethyl sulfoxide R; the substance re-dissolves completely. Treat the reference solution in the same way. Water Not more than 1.0% (Appendix 10.3). Determined on 0.500 g. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.200 g of the substance to be examined in 50 ml of water. Titrate with 0.1 N sodium hydroxide VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N sodium hydroxide VS is equivalent to 23.75 mg of C25H31ClN2O5. Storage Store in an airtight container, protected from light at a temperature of 2 °C to 8 °C. Action and use Angiotensin converting enzyme inhibitor. Preparation Tablets.

827

VP V

QUINIDINE BISULFATE

QUINIDINE BISULFATE Quinidini bisulfas

C20H24N2O2,H2SO4  

M. 422.5

Quinidine bisulfate is (8R,9S)-6’-methoxycinchonan-9-ol hydrogen sulfate. It contains not less than 98.5% and not more than 101.5% of C20H24N2O2,H2SO4 with reference to the anhydrous substance.

Characteristics Colourless crystals; odourless or almost odourless. Freely soluble in water and in ethanol 96%; practically insoluble in ether. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Diethylamine - ether - toluene (15 : 36 : 60). Test solution: A 1.0% solution of the substance being examined in methanol R. Reference solution (1): A 1.0% solution of quinidine sulfate RS. Reference solution (2): A solution contains 1.0% each of quinidine sulfate RS and quinine sulfate RS. Procedure: Apply separately to the plate 4 µl of each solution. After removal of the plate, dry it in a current of air for 15 minutes and repeat the development. Dry the plate at 105 °C for 30 min, allow it to cool and spray with iodoplatinate reagent R. The principal spot in the chromatogram obtained with test solution is similar in position, colour and size to that in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) shows two clearly separated spots. B. It complies with the test for pH. C. It gives reaction of sulfates (Appendix 8.1). pH 2.6 to 3.6 (Appendix 6.2). Determined on a 1.0% w/v solution of the substance being examined. Specific optical rotation +246° to +258°, calculated with reference to the anhydrous substance (Appendix 6.4). Determine on a 2% w/v solution of the substance to be examined in 0.1 M hydrochloric acid R, using a 2-dm layer. Other cinchona alkaloids Examine by liquid chromatography (Appendix 5.3). 828

Mobile phase: Dissolve 6.8 g of potassium dihydrogen phosphate R and 3.0 g of hexylamine R in 700 ml of water, adjusting the pH to 2.8 with 1 M phosphoric acid R, add 60 ml of acetonitrile R and dilute to 1000 ml with water. Test solution: Dissolve 20 mg of the substance to be examined in 5 ml of mobile phase, with gentle heating if necessary, and dilute to 10 ml with mobile phase. Reference solution (1): Dissolve 20 mg of quinine sulfate RS in 5 ml of mobile phase, with gentle heating if necessary, and dilute to 10 ml with mobile phase. Reference solution (2): Dissolve 20 mg of quinidine sulfate RS in 5 ml of mobile phase, with gentle heating if necessary, and dilute to 10 ml with mobile phase. Reference solution (3): A mixture of equal volumes of reference solution (1) and reference solution (2). Reference solution (4): Dilute 1.0 ml of reference solution (1) to 10.0 ml with the mobile phase. Dilute 1.0 ml of the resulting solution to 50.0 ml with the mobile phase. Reference solution (5): Dissolve 10 mg of thiourea in mobile phase and dilute to 10 ml with the same solution. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm) (Hypersil ODS 5 µm is suitable). Detector: A spectrophotometer set at 250 nm for reference solution (5) and at 316 nm for the other solutions. Flow rate: 1.5 ml/min. Volume of injection: 10 µl. Procedure: Inject reference solutions (2) and (5). In the chromatogram obtained with reference solution (2) the capacity factor of the peak due to quinidine is 3.5 to 4.5, V0 being calculated from the peak due to thiourea in the chromatogram obtained with reference solution (5), if necessary adjust the concentration of acetonitrile in the mobile phase. Inject reference solutions (1), (2), (3) and (4). The chromatogram obtained with reference solution (1) shows a principal peak due to quinidine and a peak due to dihydroquinine with a retention time relative to quinine of about 1.4. The chromatogram obtained with reference solution (2) shows a principal peak due to quinidine and a peak due to dihydroquinidine, with a retention time relative to quinidine of about 1.2. The chromatogram obtained with reference solution (3) shows four peaks due to quinine, dihydroquinine, quinidine and dihydroquinidine which are identified by comparison of their retention times with those of the corresponding peaks in the chromatograms obtained with reference solutions (1) and (2). System suitability: In the chromatogram obtained with reference solution (3), the resolution between the peaks due to quinine and quinidine is at least 1.5 and the resolution between the peaks due to quinine and dihydroquinidine is at least 1.0. The signal-to-noise ratio of the principal peak is not less than 5 in the chromatogram obtained with reference solution (4).

VP V

QUININE DIHYDROCHLORIDE

The run time is 2.5 times the retention time of the principal peak. Limits: Calculate the percentage content of related substances by normalisation, disregarding any peaks the areas of which are less than that of the peak in the chromatogram obtained with reference solution (4) (0.2%). Dihydroquinidine: Not more than 15%. Any related substance eluting before quinidine: For each impurity, not more than 5%. Any other related substance: For each impurity, not more than 2.5%.

QUININE DIHYDROCHLORIDE Quinini dihydrochloridum

Sulfated ash Not more than 0.1% (Appendix 9.9, method 1).

Quinine dihydrochloride is (8S,9R)-6’-methoxycinchonan -9-ol dihydrochloride. It contains not less than 99.0% and not more than 101.0% of C20H24N2O2,2HCl with reference to the dried substance.

Water Not more than 5.0% (Appendix 10.3). Determined on 1 g. Titratable cation 75.3% to 79.6%, calculated with reference to the anhydrous substance, when determined by the following method: To the combined aqueous solutions reserved in the Assay add 0.1 ml of phenolphthalein solution R1 and titrate with 0.1 N hydrochloric acid VS. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 16.32 mg of [C20H26N2O2]2+. Assay Dissolve 0.45 g of the substance to be examined in 15 ml of water, add 25 ml of 0.1 N sodium hydroxide VS and extract with three quantities of chloroform R, each of 25 ml. Wash each chloroform extract successively with the same 20 ml of water. Combine the aqueous solution and reserve for the test for Titratable cation. Dry the chloroform extracts with anhydrous sodium sulfate R, evaporate to dryness at a pressure of 2 kPa and dissolve the residue in 50 ml of anhydrous acetic acid R. Carry out non-aqueous titration (Appendix 10.6, method 1), using crystal violet solution R as indicator. 1 ml of 0.1 N perchloric acid VS is equivalent to 21.13 mg of C20H24N2O2,H2SO4. Storage Store in an airtight container, protected from light. Action and use Antiarrhythmic. Preparation Tablets.

C20H24N2O2,2HCl M.397.3

Characters A white or almost white powder. Very soluble in water; soluble in ethanol (96 %). Identification A. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel G. Mobile phase: Diethylamine - ether - toluene (15 : 36 : 60). Test solution: A 1.0% solution of the substance being examined in methanol R. Reference solution (1): A 1.0% solution of quinine sulphate RS in methanol R. Reference solution (2): A solution containing 1.0% each of quinidine sulfate RS and quinine sulfate RS in methanol R. Procedure: Apply separately to the plate 4 µl of each solution. After removal of the plate, dry it in a current of air for 15 min and repeat the development. Dry the plate at 105 °C for 30 minutes, allow it to cool and spray with iodoplatinate reagent R. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to that in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) shows two clearly separated spots. B. Complies with the test for pH. C. Yields reaction A characteristic of chlorides (Appendix 8.1). pH The pH of a 3% solution of the substance being examined is 2.0 to 3.0 (Appendix 6.2). Specific optical rotation -223° to -229°, calculated with reference to the dried substance (Appendix 6.4). Determined on a 3% solution of the substance being examined in 0.1 M hydrochloric acid R.

829

VP V

QUININE DIHYDROCHLORIDE INJECTION

Barium To 15 ml of a 2.0% solution of the substance being examined add 1 ml of 1 M sulfuric acid R. The solution remains clear for at least 15 min. Sulphate Not more than 0.12% (Appendix 9.4.14). Use 0.125 g of the substance being examined. Other cinchona alkaloids Carry out the test for Other cinchona alkaloids as directed under Quinine bisulphate. Loss on drying Not more than 3.0% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 1). Titratable cation 79.7% to 84.2%, calculated with reference to the dried substance, when determined by the following method: Dissolve 0.4 g of the substance being examined in 10 ml of water, add 40 ml of methanol R and titrate with 0.1 N sodium hydroxide VS, using phenolphthalein solution R as indicator. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 16.32 mg of [C20H26N2O2]2+. Assay Dissolve 0.3 g, accurately weighed, of the substance being examined in a mixture of 50 ml of anhydrous acetic acid R and 20 ml of acetic anhydride R, add 10 ml of mercury(II) acetate solution R. Titrate with 0.1 N perchloric acid VS, using crystal violet solution R as indicator. Each ml of 0.1 N perchloric acid VS is equivalent to 19.87 mg of C20H24N2O2,2HCl. Storage Protected from light. Action and use Antimalarial. Preparation Injection. QUININE DIHYDROCHLORIDE INJECTION Injectio Quinini dihydrochloridi Quinine dihydrochloride injection is a sterile solution of quinine dihydrochloride in water for injection. The injection complies with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements.

830

Content of quinine dihydrochloride, C20H24N2O2,2HCl, 95.0% to 105.0% of the stated amount. Characters A clear, almost colourless or pale yellow solution. The colour of solution is not more intense than a reference solution prepared by diluting 10 ml of a 0.80 mg/ml solution of potassium dichromate R with water to produce 20 ml. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Diethylamine - acetone - toluene (10 : 20 : 80). Test solution: Extract a volume of the injection containing about 0.5 g of quinine dihydrochloride with 50 ml of a mixture of chloroform - ethanol (96%) (2 : 1), take the organic layer, filter. Reference solution (1): A solution contains 1.0% of quinine sulfate RS in a mixture of chloroform - ethanol (96%) (2 : 1). Reference solution (2): A solution contains 1.0% each of quinidine sulfate RS and quinine sulfate RS in a mixture of chloroform - ethanol (96%) (2 : 1). Procedure: Apply separately to the plate 2 µl of each solution. After developing over a path of 15 cm, remove the plate, allow it to dry in air and spray with 0.05 M ethanolic sulfuric acid and then with Dragendorff reagent R. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) shows two clearly separated spots. B. To 0.5 ml of the injection add 0.5 ml of 16% nitric acid solution R and 0.5 ml of 5% silver nitrate solution R, a curdy white precipitate is formed. The precipitate dissolves in 6 M ammonia R. pH Not less than 2.5 (Appendix 6.2). Other cinchona alkaloids Examine by liquid chromatography (Appendix 5.3). Mobile phase, Reference solution (3) and (4), Chromatographic system and Procedure: Proceed as directed in the test for Other cinchona alkaloids under the monograph “Quinine bisulfate”. Test solution: Dilute the injection with the mobile phase to obtain a solution containing 0.2% of quinine dihydrochloride. Reference solution (1): Dissolve 20 mg of quinine sulfate RS, with gentle heating if necessary, in 5 ml of the mobile phase and dilute to 10 ml with the mobile phase. Reference solution (2): Dissolve 20 mg of quinidine sulfate RS, with gentle heating if necessary, in 5 ml of the mobile phase and dilute to 10 ml with the mobile phase. Assay Carry out the method for non-aqueous titration (Appendix 10.6).

QUININE HYDROCHLORIDE

VP V

To an accurately measured volume of the injection containing 0.6 g of quinine dihydrochloride, add 20 ml of water and 5 ml of 5 M sodium hydroxide R, extract with three 25 ml quantities of chloroform R and wash the combined extracts with 20 ml of water. Dry the chloroform extracts with anhydrous sodium sulfate R, evaporate to dryness at a pressure of 2 kPa, dissolve the residue in 50 ml of anhydrous acetic acid R and add 20 ml of acetic anhydride R, titrate with 0.1 N perchloric acid VS using crystal violet solution R as indicator. 1 ml of 0.1 N perchloric acid VS is equivalent to 19.87 mg of C20H24N2O2,2HCl.

Storage Quinine dihydrochloride injection should be protected from light. Action and use Antimalarial. Usual strength 25%, 50%.

Note: The injection should be diluted before administration. The preparation is possibly administered by slow intravenous injection under direction of the doctor.

QUININE HYDROCHLORIDE Quinini hydrochloridum

Reference solution: Dissolve 0.10 g of quinine sulfate RS in methanol R and dilute to 10 ml with the same solvent. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm. Dry in a current of air for 15 min and develop over a path of 15 cm again. Dry at 105 °C for 30 min and allow to cool, spray with iodoplatinate reagent R. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. B. Dissolve about 10 mg of the substance to be examined in water and dilute to 10 ml with the same solvent. To 5 ml of this solution add 0.2 ml of bromine water R and 1 ml of 2 M ammonia R. A green colour develops. C. Dissolve 0.10 g of the substance to be examined in 3 ml of 1 M sulfuric acid R and dilute to 100 ml with water. When examined in ultraviolet light at 366 nm, an intense blue fluorescence appears which disappears almost completely on the addition of 1 ml of hydrochloric acid R. D. It gives the reactions of chlorides (Appendix 8.1). E. It complies with the test for pH.

Appearance of solution Solution S: Dissolve 1.0 g in carbon dioxide-free water R prepared from distilled water R and dilute to 50 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution Y6 (Appendix 9.3, method 2). pH 6.0 to 6.8 (Appendix 6.2). Dilute 10 ml of solution S to 20 ml with carbon dioxidefree water R.

C20H24N2O2,HCl,2H2O  

M. 396.9

Quinine hydrochloride is (R)-[(2S,4S,5R)-5-ethenyl1-azabicyclo[2.2.2]oct-2-yl](6-methoxyquinolin-4-yl) methanol hydrochloride. It contains not less than 99.0% and not more than 101.0% of C20H24N2O2,HCl, calculated with reference to the dried substance.

Characters White or almost white or colourless, fine, silky needles, often in clusters. Soluble in water, freely soluble in ethanol (96%). Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Diethylamine - ether - toluene (10 : 24 : 40). Test solution: Dissolve 0.10 g of the substance to be examined in methanol R and dilute to 10 ml with the same solvent.

Specific optical rotation -245° to -258°, calculated with reference to the dried substance (Appendix 6.4). Dissolve 0.500 g of the substance to be examined in 0.1 M hydrochloric acid R and dilute to 25.0 ml with the same acid. Other cinchona alkaloids Examine by liquid chromatography (Appendix 5.3), use the normalisation procedure. Mobile phase: Dissolve 6.8 g of potassium dihydrogen phosphate R and 3.0 g of hexylamine R in 700 ml of water, adjust to pH 2.8 with 1 M phosphoric acid R, add 60 ml of acetonitrile R and dilute to 1000 ml with water. Test solution: Dissolve 20 mg of the substance to be examined in 5 ml of the mobile phase, with gentle heating if necessary, and dilute to 10 ml with the mobile phase. Reference solution (1): Dissolve 20 mg of quinine sulfate RS in 5 ml of the mobile phase, with gentle heating if necessary, and dilute to 10 ml with the mobile phase. Reference solution (2): Dissolve 20 mg of quinidine sulfate RS (impurity A) in 5 ml of the mobile phase, with 831

VP V

QUININE SULFATE

gentle heating if necessary, and dilute to 10 ml with the mobile phase. Reference solution (3): To 1 ml of reference solution (1) add 1 ml of reference solution (2). Reference solution (4): Dilute 1.0 ml of reference solution (1) to 10.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 50.0 ml with the mobile phase. Reference solution (5): Dissolve 10 mg of thiourea R in the mobile phase and dilute to 10 ml with the mobile phase. Chromatographic system: A column (15 cm to 25 cm × 4.6 mm) packed with stationary phase C (5 - 10 µm). Detector: Spectrophotometer at 250 nm for reference solution (5) and at 316 nm for the other solutions. Flow rate: 1.5 ml/min. Volume of injection: 10 µl. Procedure: The run time is 2.5 times the retention time of quinine. Identification of peaks: Use the chromatogram obtained with reference solution (1) to identify the peaks due to quinine and impurity C; use the chromatogram obtained with reference solution (2) to identify the peaks due to impurity A and dihydroquinidine; the chromatogram obtained with reference solution (3) shows 4 peaks due to impurity A, quinine, dihydroquinidine and impurity C, which are identified by comparison of their retention times with those of the corresponding peaks in the chromatograms obtained with reference solutions (1) and (2). Relative retention with reference to quinine: Impurity C = about 1.4. Relative retention with reference to impurity A: Dihydroquinidine = about 1.5. System suitability: In the chromatogram obtained with reference solution (3), the resolution between the peaks due to quinine and impurity A is at least 3.0 and the resolution between the peaks due to dihydroquinidine and quinine is at least 2.0. In the chromatogram obtained with reference solution (4), the signal-to-noise ratio of the principal peak is not less than 4. In the chromatogram obtained with reference solution (2), mass distribution ratio: 3.5 to 4.5 for the peak due to impurity A, tR’ being calculated from the peak due to thiourea in the chromatogram obtained with reference solution (5); if necessary, adjust the concentration of acetonitrile in the mobile phase. Limits: Impurity C: Not more than 10%. Any impurity eluted before quinine: For each impurity, not more than 5%. Any other impurity: For each impurity, not more than 2.5%. Disregard any peak with an area less than the area of the principal peak in the chromatogram obtained with reference solution (4) (0.2%). Note: Impurity A: (S)-[(2R,4S,5R)-5-ethenyl-1-azabicyclo[2.2.2]oct2-yl](6-methoxyquinolin-4-yl)methanol (quinidine).

832

Impurity B: (R)-[(2S,4S,5R)-5-ethenyl-1-azabicyclo[2.2.2]oct2-yl] (quinolin-4-yl)methanol (cinchonidine). Impurity C: (R)-[(2S,4S,5R)-5-ethyl-1-azabicyclo[2.2.2]oct-2yl](6-methoxyquinolin-4-yl)methanol (dihydroquinine).

Barium Add 1 ml of 1 M sulfuric acid R to 15 ml of solution S, allow to stand for 15 min. Any opalescence in the solution is not more intense than that in a mixture of 15 ml of solution S and 1 ml of distilled water. Sulfates Not more than 0.05% (Appendix 9.4.14). Determined on 15 ml of solution S. Loss on drying 6.0% to 10.0% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.250 g of the substance to be examined in 50 ml of ethanol (96%) R and add 5.0 ml of 0.01 M hydrochloric acid R. Titrate with 0.1 N sodium hydroxide VS, determining the end-point potentiometrically (Appendix 10.2). Read the volume of 0.1 N sodium hydroxide VS added between the 2 inflexion points. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 36.09 mg of C20H25ClN2O2,HCl. Storage Store in an airtight container, protected from light. Action and use Antiprotozoal (malaria). Preparation Tablets. QUININE SULFATE Quinini sulfas

(C20H24N2O2)2,H2SO4,2H2O  

M. 783.0  

QUININE SULFATE TABLETS

VP V

Quinine sulfate is bis[(R)-[(2S,4S,5R)-5-ethenyl-1azabicyclo[2.2.2]oct-2-yl](6-methoxyquinolin-4-yl) methanol] sulfate. It contains not less than 99.0% and not more than 101.0% of (C20H24N2O2)2,H2SO4, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder or fine, colourless needles. Slightly soluble in water, sparingly soluble in boiling water and in ethanol (96%). Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Diethylamine - ether - toluene (10 : 24 : 40). Test solution: Dissolve 0.10 g of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Reference solution: Dissolve 0.10 g of quinine sulfate RS in methanol R and dilute to 10 ml with the same solvent. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm. Dry in a current of air for 15 min and repeat the development. Dry at 105 °C for 30 min and allow to cool, spray with iodoplatinate reagent R. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. B. Dissolve about 5 mg of the substance to be examined in 5 ml of water, add 0.2 ml of bromine water R and 1 ml of 2 M ammonia R. A green colour develops. C. Dissolve 0.1 g of the substance to be examined in 3 ml of 1 M sulfuric acid R and dilute to 100 ml with water. When examined in ultraviolet light at 366 nm, an intense blue fluorescence appears which disappears almost completely on the addition of 1 ml of hydrochloric acid R. D. Dissolve about 45 mg of the substance to be examined in 5 ml of dilute hydrochloric acid R. The solution gives reaction (A) of sulfates (Appendix 8.1). E. It complies with the test for pH. Appearance of solution Solution S: Dissolve 0.500 g in 0.1 M hydrochloric acid R and dilute to 25.0 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution GY6 (Appendix 9.3, method 2). pH 5.7 to 6.6 (Appendix 6.2). Determined on a 10 g/l suspension of the substance to be examined in water. Specific optical rotation -237° to -245°, calculated with reference to the dried substance (Appendix 6.4). Determined on solution S.

Other cinchona alkaloids See the Other cinchona alkaloids in “Quinine hydrochloride” monograph. Loss on drying 3.0% to 5.0% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.300 g of the substance to be examined in a mixture of 10 ml of chloroform R and 20 ml of acetic anhydride R. Titrate with 0.1 N perchloric acid VS, determining the endpoint potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 24.90 mg of (C20H24N2O2)2,H2SO4. Storage Store in an airtight container, protected from light. Action and use Antiprotozoal (malaria). Preparation Tablets. QUININE SULFATE TABLETS Tabellae Quinini sulfatis Quinine sulfate tablets contain quinine sulfate. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of quinine sulfate, (C20H24N2O2)2, H2SO4,2H2O, 95.0% to 105.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Diethylamine - acetone - toluene (10 : 20 : 80). Test solution: Shake a quantity of of the powdered tablets containing about 0.1 g of quinine sulfate with 10 ml of a mixture of chloroform - ethanol (96%) (2 : 1), filter. Reference solution (1): A solution containing 1.0 % of quinine sulfate RS in a mixture of chloroform - ethanol (96%) (2 : 1). Reference solution (2): A solution containing 1.0% each of quinidine sulfate RS and quinine sulfate RS in a mixture of chloroform - ethanol (96%) (2 : 1). Procedure: Apply separately to the plate 2 µl of each solution. After developing over a path of 15 cm, remove the plate, allow it to dry in air and spray with 0.05 M ethanolic sulfuric acid and then with Dragendorff reagent 833

VP V

RAMIPRIL

R. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) shows two clearly separated spots. B. Shake a quantity of the powdered tablets containing 0.25 g of quinine sulfate with 25 ml of a mixture of chloroform ethanol (96%) (2 : 1), filter. Evaporate the filtrate to dryness and wash the residue with 10 ml of ether R and dry the residue at 60 °C at a pressure not exceeding 15 Pa for 2 hours. The pH of a 1% solution of the residue in water is 5.7 to 6.6. C. Extract a quantity of the powdered tablets containing 0.1 g of quinine sulfate with 20 ml of water and filter. The filtrate yields the reactions characteristic of sulfates (Appendix 8.1).

powder. To a quantity of the powdered tablets, accurately weighed, containing about 0.4 g of quinine sulfate, add in 40 ml of acetic anhydride R, dissolve as completely as possible, using heat. Cool and titrate with 0.1 N perchloric acid VS, using crystal violet solution R as indicator. 1 ml of 0.1 N perchloric acid VS is equivalent to 26.10 mg of (C20H24N2O2)2,H2SO4,2H2O.

Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 900 ml of 0.1 M hydrochloric acid R. Rotation speed: 100 rpm. Time: 45 min. Procedure: After the specified time, withdraw a sample of the medium and filter, discarding the first portion of the filtrate. Dilute a suitable volume of the filtrate, if necessary, with 0.1 M hydrochloric acid R to produce a solution containing 35 µg of quinine sulfate per ml. Measure the absorbance of the resulting solution (Appendix 4.1) at 348 nm in a 1 cm cell using 0.1 M hydrochloric acid R in the reference cell. Calculate the total content of (C20H24N2O2)2,H2SO4,2H2O, in the medium taking 136 as the value of A(1%, 1 cm) at the maximum at 348 nm. Tolerances: Not less than 70% (Q) of the stated amount of quinine sulfate, (C20H24N2O2)2,H2SO4,2H2O is dissolved in 45 min.

RAMIPRIL Ramiprilum

Other cinchona alkaloids Examine by liquid chromatography (Appendix 5.3). Mobile phase, Reference solution (3) and (4), Chromatographic system and Procedure: Proceed as directed in the test for Other cinchona alkaloids under the monograph “Quinine bisulfate”. Test solution: Weigh accurately a quantity of the powdered tablet containing 50 mg of quinine sulfate, add 20 ml of the mobile phase. Heat gently to dissolve, cool, dilute to 25.0 ml with the mobile phase and filter. Reference solution (1): Dissolve 20 mg of quinine sulfate RS, with gentle heating if necessary, in 5 ml of the mobile phase and dilute to 10 ml with the mobile phase. Reference solution (2): Dissolve 20 mg of quinidine sulfate RS, with gentle heating if necessary, in 5 ml of the mobile phase and dilute to 10 ml with the mobile phase. Assay Carry out the method for non-aqueous titration (Appendix 10.6). Weigh 20 tablets, calculate the average mass, and finely 834

Storage Store in an airtight container, protected from light. Action and use Antimalarial. Usual strength 250 mg, 500 mg.

C23H32N2O5  



M: 416.5 

Ramipril is (2S,3aS,6aS)-1-[(S)-2-[[(S)-1-(ethoxycarbonyl) phenylpropyl]amino]propanoyl]octahydrocyclopenta[b] pyrrole-2-carboxylic acid. It contains not less than 98.0% and not more than 101.0% of C23H32N2O5, calculated with reference to the dried substance.

Characters A white or almost white, crystalline powder, sparingly soluble in water, freely soluble in methanol. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of ramipril RS. B. It complies with the test for Specific optical rotation. Appearance of solution Dissolve 0.1 g in methanol R and dilute to 10 ml with the same solvent. The solution is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). Specific optical rotation +32.0° to +38.0°, calculated with reference to the dried substance (Appendix 6.4). Dissolve 0.250 g in a mixture of 14 volumes of 25% solution of hydrochloric acid R and 86 volumes of methanol R and dilute to 25.0 ml with the same mixture of solvents.

RAMIPRIL

VP V

Related substances Examine by liquid chromatography (Appendix 5.4). Mobile phase A: Dissolve 2.0 g of sodium perchlorate R in a mixture of 0.5 ml of triethylamine R and 800 ml of water; adjust to pH 3.6 with phosphoric acid R and add 200 ml of acetonitrile R, mix. Mobile phase B: Dissolve 2.0 g of sodium perchlorate R in a mixture of 0.5 ml of triethylamine R and 300 ml of water; adjust to pH 2.6 with phosphoric acid R and add 700 ml of acetonitrile R, mix. Test solution: Dissolve 20.0 mg of the substance to be examined in mobile phase A and dilute to 20.0 ml with the same mobile phase. Reference solution (1): Dissolve 5 mg each of ramipril impurity A RS, ramipril impurity B RS, ramipril impurity C RS and ramipril impurity D RS in 5 ml of the test solution and dilute to 10 ml with mobile phase B. Reference solution (2): Dilute 5.0 ml of the test solution to 100.0 ml with mobile phase B. Dilute 5.0 ml of the solution to 50.0 ml with mobile phase B. Reference solution (3): Dilute 1.0 ml of reference solution (2) to 10.0 ml with mobile phase B. Chromatographic system: A column (25 cm × 4.0 mm) packed with stationary phase C (3 µm). Column temperature: 65 °C. Detector: A spectrophotometer set at 210 nm. Volume of injection: 10 µl. Flow rate: 1.0 ml/min: Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

Comments

0-6

90

10

Isocratic

6-7

90 → 75

10 → 25

Linear gradient

7 - 20

75 → 65

25 → 35

Linear gradient

20 - 30

65 → 25

35 → 75

Linear gradient

30 - 40

25

75

Isocratic

40 – 45

25 → 90

75 → 10

Linear gradient

45 - 55

90

10

Re-equilibration

Equilibrate the column with a mixture of 90% mobile phase A and 10% mobile phase B for at least 35 min. If a suitable baseline cannot be obtained, use another grade of triethylamine. Inject reference solution (3). Adjust the sensitivity of the system so that the chromatogram obtained shows a visible peak. Inject reference solution (1), reference solution (2) and the test solution. The test is not valid unless: in the chromatogram obtained with reference solution (1) the resolution between the peaks corresponding to ramipril impurity A and ramipril is at least 3.0; in the chromatogram

obtained with reference solution (3) the principal peak has a signal-to-noise ratio of at least 3; and in the chromatogram obtained with the test solution, the symmetry factor of the principal peak is 0.8 to 2.0. The retention times are: Impurity A about 14 min, ramipril about 18 min, impurity B about 22 min, toluene about 24 min, impurity C about 26 min and impurity D about 28 min. In the chromatogram obtained with the test solution, multiply the area of any peak corresponding to impurity C by a correction factor of 2.4. Limits: In the chromatogram obtained with the test solution: The area of each peak corresponding to ramipril impurity A, ramipril impurity B, ramipril impurity C and ramipril impurity D is not greater than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.5%); the area of any peak, apart from the principal peak and any peak corresponding to the impurities A, B, C and D, is not greater than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.1%); the sum of the areas of all peaks, apart from the principal peak, is not greater than twice the area of the principal peak in the chromatogram obtained with reference solution (2) (1.0%). Disregard any peak with an area less than that of the principal peak in the chromatogram obtained with reference solution (3).

Notes: Impurity A (ramipril methyl ester): (2S,3aS,6aS)-1-[(S)-2[[(S)-1-(methoxycarbonyl)-3-phenylpropyl]amino]propanoyl] octahydrocyclopenta[b]pyrrole-2-carboxylic acid. Impurity B (ramipril isopropyl ester): (2S,3aS,6aS)-1-[(S)-2[[(S)-1-[(1-methylethoxy)carbonyl]-3-phenylpropyl]amino propanoyl]octahydrocyclopenta[b]pyrrole-2-carboxylic acid. Impurity C (hexahydroramipril): (2S,3aS,6aS)1-[(S)-2-[[(S)3-cyclohexyl-1-(ethoxycarbonyl) propyl]amino] propanoyl] octahydrocyclopenta[b]pyrrole-2-carboxylic acid). Impurity D (ramipril diketopiperazine): Ethyl (2S)-2[(3S,5aS,8aS,9aS)-3-methyl-1,4-dioxodecahydro-2Hcyclopenta[4,5]pyrrolo[1,2-a]pyrazin-2-yl]-4-phenylbutanoate .

Palladium Not more than 20 ppm. Examine by atomic absorption spectrometry (Appendix 4.4, method 1). Test solution: Dissolve 0.200 g of the substance to be examined in a mixture of 0.3 volumes of nitric acid R and 99.7 volumes of water and dilute to 100.0 ml with the same mixture of solvents. Reference solutions: Use solutions containing 0.02 µg, 0.03 µg and 0.05 µg of palladium per millilitre, freshly prepared by dilution of palladium standard solution (0.5 ppm Pd) R with a mixture of 0.3 volumes of nitric acid R and 99.7 volumes of water. Blank solution: Dissolve 0.150 g of magnesium nitrate R in a mixture of 0.3 volumes of nitric acid R and 99.7 volumes of water and dilute to 100.0 ml with the same mixture of solvents. 835

VP V

RAMIPRIL TABLETS

Procedure: Inject separately 20 µl of the test solution and the reference solution and 10 µl of the blank solution. Measure the absorbance at 247.6 nm using a palladium hollowcathode lamp as a source of radiation, a transmission band of preferably 1 nm and a graphite tube.

Loss on drying Not more than 0.2% (Appendix 9.6). Determined on 1.000 g by drying in an oven under high vacuum at 60 °C for 4 h. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.300 g in 25 ml of methanol R and add 25 ml of water. Titrate with 0.1 N sodium hydroxide VS, determining the end-point potentiometrically (Appendix 10.2). Carry out a blank titration. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 41.65 mg of C23H32N2O5. Storage Store protected from light. Action and use Angiotensin converting enzyme inhibitor. Preparations Capsules, tablets.

Procedure: Examine by liquid chromatography (Appendix 5.3). Mobile phase, Chromatographic system, Procedure: as described under Assay Test solution: After the specified time, withdraw a sample of the medium and filter, discard the first 5 ml of the filtrate. Dilute if necessary with 0.1 M hydrochloric acid R to obtain a solution with ramipril concentration of about 2.5 µg/ml. Reference solution: A 0.00025% solution of ramipril RS in 0.1 M hydrochloric acid R. Calculate the content of ramipril dissolved using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C23H32N2O5 in ramipril RS. Tolerance: Not less than 70% (Q) of the stated amount of ramipril, C23H32N2O5, is dissolved in 45 min.

Uniformity of content (Appendix 11.2). Examine by liquid chromatography (Appendix 5.3). Mobile phase, Chromatographic system, Procedure: as described under Assay. Reference solution: A 0.025% solution of ramipril RS in 0.1 M hydrochloric acid R. Test solution: To one tablet, add 5 ml of 0.1 M hydrochloric acid R, mix with the aid of ultrasound for 10 min and dilute with 0.1 M hydrochloric acid R to produce a 0.025% solution of ramipril. Centrifuge or filter to obtain a clear solution. Assay

RAMIPRIL TABLETS Tabellae Ramiprili Ramipril tablets contain ramipril. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of ramipril, C23H32N2O5, 90.0% to 105.0% of the stated amount. Identification Shake a quantity of the powdered tablets containing the equivalent of about 25 mg of ramipril with 50 ml of acetone R, centrifuge for 10 minutes, filter the clear supernatant liquid through a 0.45-µm filter. Evaporate the filtrate to dryness on a water bath and dry the residue for 3 hours at 60 °C. The infrared absorption spectrum of the residue (Appendix 4.2) is concordant with the reference spectrum of ramipril. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 500 ml of 0.1 M hydrochloric acid R. Rotation speed: 75 rpm. Time: 45 min. 836

For tablets containing 2 mg or more of Ramipril Examine by liquid chromatography (Appendix 5.3). Mobile phase: Mix 420 volumes of acetonitrile R and 580 volumes of a solution containing 1.4% of sodium perchlorate R and 0.58% of phosphoric acid R adjusted to pH 2.5 with triethylamine R. Adjust the obtained mixture to pH 2.1 with phosphoric acid R. Reference solution: A 0.025% solution of ramipril RS in 0.1 M hydrochloric acid R. Test solution: Weigh 20 tablets, calculate the average weight and powder finely. Weigh accurately a quantity of powdered tablets containing the equivalent of about 25 mg of ramipril in a 100 ml volumetric flask, add 70 ml of 0.1 M hydrochloric acid R and mix with the aid of ultrasound for 10 minutes, add sufficient 0.1 M hydrochloric acid R to volume, mix well, filter. Chromatographic system: A column (12.5 cm × 4.6 mm) packed with stationary phase C (5 µm) (Nucleosil 100 - C18 is suitable). Detector: A spectrophotometer set at 210 nm. Flow rate: 1.0 ml/min. Volume of injection: 50 µl. Procedure: Inject alternately the test and the reference solution.

RANITIDINE HYDROCHLORIDE

VP V

Calculate the content of ramipril, C23H32N2O5, in the tablets using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C23H32N2O5 in ramipril RS.

For tablets containing less than 2 mg of ramipril Use the average of the 10 individual results obtained in the test for Uniformity of content. Storage Store in a well-closed container, protected from light. Action and use Anti-hypertensive. Usual strength 1.25 mg, 2.5 mg; 5 mg. RANITIDINE HYDROCHLORIDE Ranitidini hydrochloridum

C13H22N4O3S,HCl  

M: 350.9

Ranitidine hydrochloride is N-[2-[[[5-[(dimethylamino) methyl]furan-2-yl]methyl]sulfanyl]ethyl]-N’-methyl-2nitroethene-1,1-diamine hydrochloride. It contains not less than 98.5% and not more than 101.5% of C13H22N4O3S,HCl, calculated with reference to the dried substance.

Characters White or pale yellow, crystalline powder, it shows polymorphism. Freely soluble in water, sparingly soluble or slightly soluble in anhydrous ethanol, very slightly soluble in methylene chloride. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of ranitidine hydrochloride RS. If the spectra obtained in the solid state show differences, dissolve 10 mg of the substance to be examined and 10 mg of the reference substance separately in 0.5 ml of methanol R in an agate mortar. Evaporate to dryness under a stream of nitrogen R. Dry the residues under vacuum for 30 min. Add 3 drops of liquid paraffin R to the residues and triturate until the mull shows a milky appearance. Compress the mulls between 2 plates transparent to infrared radiation and record new spectra. B. It gives reaction (A) of chlorides (Appendix 8.1).

Appearance of solution Solution S: Dissolve 1.0 g in carbon dioxide-free water R and dilute to 100.0 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution BY5 (Appendix 9.3, method 2). pH 4.5 to 6.0 (Appendix 6.2). Determined on solution S. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Acetonitrile - buffer solution (2 : 98). Mobile phase B: Acetonitrile - buffer solution (22 : 78). Buffer solution: Dissolve 6.8 g of potassium dihydrogen phosphate R in 950 ml of water. Adjust to pH 7.1 with 10 M sodium hydroxide R and dilute to 1000 ml with water. Test solution: Dissolve 13 mg of the substance to be examined in mobile phase A and dilute to 100.0 ml with the same solvent. Reference solution (1): Dissolve 6.5 mg of ranitidine for impurity A identification RS in mobile phase A and dilute to 50.0 ml with the same solvent. Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with mobile phase A. Reference solution (3): Dissolve the contents of a vial of ranitidine impurity J RS in 1.0 ml of the test solution. Reference solution (4): In order to prepare impurities D and H in situ, dissolve 6.5 mg of the substance to be examined in 2.5 ml of 1 M sodium hydroxide R and heat at 60 °C for 5 min, then dilute to 50.0 ml with mobile phase A. Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase octadecylsilyl amorphous organosilica polymer R (3.5 µm). Column temperature: 35 °C. Detector: A spectrophotometer set at 230 nm. Flow rate: 1.5 ml/min. Volume of injection: 10 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 10

100 → 0

0 → 100

10 - 15

0

100

Inject the test solution, reference solutions (1), (2) and (3) and mobile phase A as a blank. Identification of impurities: Use the chromatogram supplied with  ranitidine for impurity A identification RS and the chromatogram obtained with reference solution (2) to identify the peak due to impurity A; use the chromatogram obtained with reference solution (3) to identify the peak due to impurity J; use the chromatogram obtained with reference solution (4) to identify the peaks due to impurities D and H. 837

VP V

RANITIDINE TABLETS

Relative retention with reference to ranitidine (retention time = about 7 min): impurity H = about 0.1; impurity G = about 0.2; impurity F = about 0.4; impurity B = about 0.5; impurity C = about 0.6; impurity E = about 0.7; impurity D = about 0.8; impurity J = about 0.9; impurity I = about 1.3; impurity A = about 1.7. System suitability: In the chromatogram obtained with reference solution (3), the resolution between the peaks due to impurity J and ranitidine is at least 1.5. The chromatogram obtained with the blank solution does not show any peak with the same relative retention as the peak due to impurity A in the chromatogram obtained with reference solution (1). Limits: Correction factor: For the calculation of content, multiply the peak area of impurity J by 2. Impurity A: The area of the peak due to impurity A is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.5%). Impurities B, C, D, E, F, G, H, I, J: For each impurity, the area, corrected if necessary, is not more than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.2%). Any other impurity: For each impurity, the area is not more than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.10%). The sum of the peak areas of all impurities other than A is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.5%). Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Note: Impurity A: N,N’-bis[2-[[[5-[(dimethylamino)methyl]furan-2yl]methyl]sulfanyl]ethyl]-2-nitroethene-1,1-diamine. Impurity B: 2-[[[5-[(dimethylamino)methyl]furan-2-yl]methyl] sulfanyl]ethanamine. Impurity C: N-[2-[[[5-[(dimethylamino)methyl]furan-2-yl]methyl] sulfinyl]ethyl]-N’-methyl-2-nitroethene-1,1-diamine, Impurity D: N-[2-[[[5-[(dimethylamino)methyl]furan-2-yl]methyl] sulfanyl]ethyl]-2-nitroacetamide. Impurity E: N-[2-[[[5-[(dimethyloxidoamino)methyl]furan-2-yl] methyl]sulfanyl]ethyl]-N’-methyl-2-nitroethene-1,1-diamine. Impurity F: [5-[(dimethylamino)methyl]furan-2-yl]methanol. Impurity G: 3-(methylamino)-5,6-dihydro-2H-1,4-thiazin-2one-oxime. Impurity H: N-methyl-2-nitroacetamide. Impurity I: 2,2’-methylenebis[N-[2-[[[5-[(dimethylamino)methyl] furan-2-yl]methyl]sulfanyl]ethyl]-N’-methyl-2-nitroethene-1,1diamine]. Impurity J: 1,1’-N-[methylenebis(sulfanediylethylene)]bis(N’methyl-2-nitroethene-1,1-diamine). Impurity K: N-methyl-1-methylthio-2-nitroethenamine.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 838

1.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R.

Loss on drying Not more than 0.75% (Appendix 9.6). (1.000 g; 60 °C, in vacuo). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.280 g of the substance to be examined in 35 ml of water. Titrate with 0.1 N sodium hydroxide VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N sodium hydroxide VS is equivalent to 35.09 mg of C13H23ClN4O3S. Storage In airtight container, protected from light. Action and use Histamine H2 receptor antagonist; treatment of peptic ulcer disease. Preparation Tablets. RANITIDINE TABLETS Tabellae Ranitidini Ranitidine tablets contain ranitidine hydrochloride. They are film coated. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of ranitidine, C13H22N4O3S, 95.0% to 105.0% of the stated amount. Identification A. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (2) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). B. In the test for Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is the same as that of the principal peak in the chromatogram obtained with the reference solution. C. Shake a quantity of the powdered tablets containing 0.1 g of ranitidine with 2 ml of water and filter. The filtrate yields the reactions characteristic of chlorides (Appendix 8.1). Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Water - 18 M ammonia - 2-propanol - ethyl acetate (2 : 4 : 15 : 25).

RIBOFLAVIN

VP V

Test solution (1): Shake a quantity of the powdered tablets containing 0.45 g of ranitidine with 20 ml of methanol R, filter (Whatman No 1 paper is suitable). Test solution (2): Dilute 1.0 ml of test solution (1) to 10 ml with methanol R. Reference solution (1): Dissolve 50 mg of ranitidine hydrochloride RS in 20 ml of methanol R. Reference solution (2): Dilute 1.0 ml of test solution (1) to 200 ml with methanol R. Reference solution (3): Dilute 1.0 ml of test solution (1) to 20 ml with methanol R and dilute 3 ml of this solution to 50 ml with methanol R . Reference solution (4): Dilute 1.0 ml of test solution (1) to 20 ml with methanol R and dilute 1 ml of this solution to 50 ml with methanol R. Reference solution (5): A solution contains 0.10% of ranitidine impurity B RS in methanol R. Reference solution (6): A solution contains 0.10% of ranitidine impurity B RS in test solution (1). Procedure: Apply separately to the plate 10 µl of each solution. After developing over a path of 15 cm, remove the plate, allow it to dry in air and and expose to iodine vapour in a closed chamber until the spots are visible. Any secondary spot in the chromatogram obtained with test solution (1) is not more intense than the spot in the chromatogram obtained with reference solution (2) (0.5%), not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (3) (0.3%) and not more than three other such spots are more intense than the spot in the chromatogram obtained with reference solution (4) (0.1%). The test is not valid unless the chromatogram obtained with reference solution (6) shows two clearly separated spots corresponding to ranitidine impurity B and ranitidine.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: 0.1 M ammonium acetate - methanol (15 : 85). Reference solution: A 0.0112% solution of ranitidine hydrochloride RS in the mobile phase. Test solution: Shake 10 tablets with 400 ml of the mobile phase until the tablets have completely disintegrated (about 15 min), dilute to 500.0 ml with the mobile phase, filter (Whatman GF/C paper is suitable) and dilute the filtrate with the mobile phase to produce a solution containing the equivalent of 0.01% of ranitidine. Resolution solution: A solution containing 0.0112% of ranitidine hydrochloride RS and 0.0002% of dimethyl5-[2(1-methylamino-2-nitrovinylamino) ethylsulphinylmethyl]furfurylamine RS in the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (10 µm). Detector: A spectrophotometer set at 322 nm. Flow rate: 2 ml/min. Volume of injection: 20 µl.

Procedure: System suitability: Inject the reference solution, the relative standard deviation for 6 replicate injections is not more than 2.0%. Inject the resolution solution, the peak due to ranitidine in the chromatogram obtained with resolution solution shows baseline separation from the peak due to dimethyl 5-[2-(1-methylamino2-nitrovinylamino) ethylsulphinylmethyl]-furfurylamine. Inject separately the reference solution and the test solution. Calculate the content of ranitidine, C13H22N4O3S, in the tablets using the areas for the principal peaks in the chromatograms obtained with the test solution and the reference solution, and the declared content of C13H22N4O3S in ranitidine hydrochloride RS. Conversion factor from ranitidine hydrochloride (C13H22N4O3S,HCl) to ranitidine (C13H22N4O3S) is 0.8961.

Storage Store in an airtight container at a cool place, protected from light. Action and use Histamine H2-receptor antagonist. Usual strength 150 mg; 300 mg. RIBOFLAVIN Riboflavinum

C17H20N4O6  

M. 376.4 

Riboflavin is 7,8-dimethyl-10-[(2S,3S,4R)-2,3,4,5-tetrahydroxypentyl]benzo[g]pteridine-2,4(3H,10H)-dione. It contains not less than 97.0% and not more than 103.0% of C17H20N4O6, calculated with reference to the dried substance. This monograph applies to riboflavin produced by fermentation.

Characters Yellow or orange-yellow, crystalline powder, it shows polymorphism. Very slightly soluble in water, practically insoluble in ethanol (96%). Solutions deteriorate on exposure to light, especially in the presence of alkali. Identification A. Examine by thin-layer chromatography (Appendix 5.4). 839

VP V

RIBOFLAVIN

Coating substance: Silica gel (2 µm - 10 µm). Mobile phase: Water. Test solution: Suspend 25 mg of the substance to be examined in 10 ml of water, shake for 5 min and filter the suspension to remove the undissolved material. Reference solution: Suspend 25 mg of riboflavin RS in 10 ml of water, shake for 5 min and filter the suspension to remove the undissolved material. Procedure: As follows, drying in a current of cold air after each individual application: 1st application: 2 µl of methylene chloride R then 2 µl of the test solution; 2nd application: 2 µl of methylene chloride R then 2 µl of the reference solution. Develop over a path of 6 cm. Allow the plate to dry in air and examine in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. B. It complies with the test for Specific optical rotation. C. Dissolve about 1 mg of the substance to be examined in 100 ml of water. The solution has, by transmitted light, a pale greenish-yellow colour, and, by reflected light, an intense yellowish-green fluorescence which disappears on the addition of mineral acids or alkalis.

Specific optical rotation -115° to -135°, calculated with reference to the dried substance (Appendix 6.4). Determine on a 0.5% solution of the substance to be examined in 0.05 M sodium hydroxide free from carbonate. Measure the optical rotation within 30 min of dissolution. Absorbance Test solution: Dilute 25 ml of the final solution prepared for the Assay with 25 ml of water. The ultraviolet absorption spectrum (Appendix 4.1) of the solution exhibits four absorption maxima at 223 nm, 267 nm, 373 nm and 444 nm. The ratios of the absorbances measured at 373 nm to that measured at 267 nm is 0.31 to 0.33 and the ratios of the absorbances measured at 444 nm to that measured at 267 nm is 0.36 to 0.39. Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use and protect from light. Mobile phase A: Phosphoric acid - water (1 : 1000). Mobile phase B: Acetonitrile. Solution A: A 1.36% solution of sodium acetate R. Test solution: With the aid of ultrasound, dissolve 0.120 g of the substance to be examined in 10 ml of 0.1 M sodium hydroxide and dilute to 100.0 ml with solution A. Reference solution (1): Dilute 1.0 ml of the test solution to 10.0 ml with solution A. Dilute 1.0 ml of this solution to 100.0 ml with solution A. Reference solution (2): With the aid of ultrasound, dissolve the contents of a vial of riboflavin for peak identification RS (containing impurities C and D) in 1.0 ml of a mixture of mobile phase B - mobile phase A (1 : 9). 840

Reference solution (3): In order to prepare impurities A and B in situ, dissolve 10 mg of the substance to be examined in 1 ml of 0.5 M sodium hydroxide R. Expose to daylight for 1.5 h. Add 0.5 ml of acetic acid R and dilute to 100 ml with water. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 267 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0-5

90

10

5 - 20

90 → 80

10 → 20

20 - 25

80

20

25 - 35

80 → 50

20 → 50

35 - 45

50

50

Identification of impurities: Use the chromatogram supplied with riboflavin for peak identification RS and the chromatogram obtained with reference solution (2) to identify the peaks due to impurities C and D. Relative retention with reference to riboflavin (retention time = about 16 min): Impurity C = about 0.2; impurity D = about 0.5; impurity A = about 1.4; impurity B = about 1.9. System suitability: In the chromatogram obtained with reference solution (3), the resolution between the peaks due to to impurities A and B is at least 5. The chromatogram obtained with reference solution (2) is similar to the chromatogram supplied with riboflavin for peak identification RS. Limits: Correction factors: For the calculation of content, multiply the peak areas of the following impurities by the corresponding correction factor: impurity A = 0.7; impurity B = 1.4; impurity C = 2.3; impurity D = 1.4. Impurity A: The corrected area of the peak due to impurity A is not more than 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.025%). Impurities B, C, D: For each impurity, the corrected area is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). The sum of the peak areas of all impurities is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%).

RIBOFLAVIN SODIUM PHOSPHATE

VP V

Disregard any peak, other than those due to impurity A, with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A: 7,8,10-trimethylbenzo[g]pteridine-2,4(3H,10H)-dione (lumiflavine). Impurity B: 7,8-dimethylbenzo[g]pteridine-2,4(1H,3H)-dione. Impurity C: 6,7-dimethyl-8-[(2S,3S,4R)-2,3,4,5-tetrahydroxypentyl] pteridine-2,4(3H,8H)-dione. Impurity D: 8-(hydroxymethyl)-7-methyl-10-[(2S,3S,4R)-2,3,4,5tetrahydroxypentyl]benzo[g]pteridine-2,4(3H,10H)-dione.

Loss on drying Not more than 1.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on the residue obtained in the test for Loss on drying. Assay Carry out the assay protected from light. In a brown-glass 500 ml volumetric flask, suspend 65.0 mg of the substance to be examined in 5 ml of water ensuring that it is completely wetted and dissolve in 5 ml of 2 M sodium hydroxide R. As soon as dissolution is complete, add 100 ml of water and 2.5 ml of glacial acetic acid R and dilute to 500.0 ml with water. To 20.0 ml of this solution, add 3.5 ml of a 1,4% solution of sodium acetate R and dilute to 200.0 ml with water. Measure the absorbance at the absorption maximum at 444 nm. Calculate the content of riboflavin, C17H20N4O6, taking 328 as the value of A(1%, 1 cm) at the maximum at 444 nm.

Identification To a quantity of the powdered tablets containing about 1 mg of riboflavin, add 100 ml of water, shake vigorously, filter. The filtrate has a pale greenish-yellow colour and an intense yellowish-green fluorescence which disappears on the addition of mineral acids or alkalis. Assay Carry out the assay protected from light. Weigh 20 tablets, calculate the average mass, powder finely. Weigh accurately a quantity of the powdered tablets equivalent to about 10 mg of riboflavin, add a mixture of 5 ml of glacial acetic acid R and 100 ml of water. Heat on a water bath for 1 hour, shaking occasionally to dissolve riboflavin. Add 50 ml of water, cool, add 30 ml of 0.1 M sodium hydroxide and mix. Add sufficient water to produce 1000.0 ml. Mix and filter, discarding the first portion of the filtrate. Measure the absorbance of the filtrate at the maximum at 444 nm (Appendix 4.1) in a 1-cm cell. Calculate the content of riboflavin C17H20N4O­6, taking 328 as the value of A (1%, 1 cm) at 444 nm. Storage Store in a dry and cool place, protected from light. Action and use Component of vitamin B. Usual strength 2 mg. RIBOFLAVIN SODIUM PHOSPHATE Riboflavini natrii phosphas

Storage In an airtight container, protected from light. Action and use Component of vitamin B. Preparation Tablets. RIBOFLAVIN TABLETS Tabellae Riboflavini Vitamin B2 tablets Riboflavin tablets contain riboflavin. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of riboflavin, C17H20N4O6, 90.0% to 115.0% of the stated amount.

C17H20N4NaO9P

M. 478.3

Riboflavin sodium phosphate is a mixture containing riboflavin 5’-(sodium hydrogen phosphate) as the main component and other riboflavin sodium monophosphates. It contains 73.0% to 79.0% of of riboflavin (C17H20N4O6; Mr 376.4), calculated with reference to the dried substance. It contains a variable amount of water.

841

VP V

RIBOFLAVIN SODIUM PHOSPHATE

Characters Yellow or orange-yellow, crystalline, hygroscopic powder. Soluble in water, very slightly soluble in ethanol (96%). Identification A. Dissolve 50.0 mg of the substance to be examined in phosphate buffer solution pH 7.0 R and dilute to 100.0 ml with the same solution. Dilute 2.0 ml of this solution to 100.0 ml with phosphate buffer solution pH 7.0 R. The ultraviolet absorption spectrum (Appendix 4.1) of the solution in the range 230 nm to 350 nm exhibits four absorption maximum at 266 nm, and specific absorbance at the absorption maximum A (1%, 1 cm) is 580 to 640. B. In the test for Related substances, the principal peak in the chromatogram obtained with the test solution is similar in position and approximate size to the principal peak in the chromatogram obtained with reference solution (2). C. Dissolve about 10 mg in dilute sodium hydroxide solution R and dilute to 100 ml with the same solution. Expose 1 ml of this solution to ultraviolet light at 254 nm for 5 min, add sufficient acetic acid R to make the solution acidic to blue litmus paper R and shake with 2 ml of methylene chloride R. The lower layer shows yellow fluorescence. D. Add 10 ml of nitric acid R to 0.5 g of the substance to be examined and evaporate the mixture to dryness on a waterbath. Ignite the residue until it becomes white, dissolve the residue in 5 ml of water and filter. The filtrate gives reaction (A) of sodium and reaction (B) of phosphates (Appendix 8.1). pH 5.0 to 6.5 (Appendix 6.2). Dissolve 0.5 g in carbon dioxide-free water R and dilute to 50 ml with the same solvent. Specific optical rotation +38.0° to +43.0°, calculated with reference to the dried substance (Appendix 6.4). Dissolve 0.300 g in 18.2 ml of 25% hydrochloric acid solution R and dilute to 25.0 ml with water. Impurity E Add 10 ml of methylene chloride R to about 35 mg of the substance to be examined, shake for 5 min and filter. The filtrate is not more intensely coloured than reference solution BY6 (Appendix 6.4, method 2). Note: Impurity E: 7,8,10-trimethylbenzo[g]pteridine-2,4(3H,10H)dione (lumiflavin).

Related substances

Examine by liquid chromatography (Appendix 5.3). Carry out the test protected from light. Mobile phase: Methanol - 0.735% solution of potassium dihydrogen phosphate (15 : 85). 842

Test solution: Dissolve 0.100 g of the substance to be examined in 50 ml of water and dilute to 100.0 ml with the mobile phase. Dilute 8.0 ml of this solution to 50.0 ml with the mobile phase. Reference solution (1): Dissolve 60 mg of riboflavin CRS (impurity D) in 1 ml of hydrochloric acid R and dilute to 250.0 ml with water. Dilute 4.0 ml of this solution to 100.0 ml with the mobile phase. Reference solution (2): Dissolve 0.100 g of riboflavin sodium phosphate CRS in 50 ml of water and dilute to 100.0 ml with the mobile phase. Dilute 8.0 ml of this solution to 50.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 266 nm. Flow rate: 2 ml/min. Volume of injection: 100 µl. Procedure: Run time: Until the peak due to riboflavin can be clearly evaluated. Relative retention with reference to riboflavin 5’-monophosphate (retention time = about 20 min): impurity A = about 0.2; impurity B = about 0.3; impurity C = about 0.5; riboflavin 3’-monophosphate = about 0.7; riboflavin 4’-monophosphate = about 0.9; impurity D = about 2. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to riboflavin 4’-monophosphate and riboflavin 5’-monophosphate is at least 1.5. Calculate the percentage content of free riboflavin (impurity D) and of riboflavin in the form of the diphosphates of riboflavin (impurities A, B, C) from the areas of the peaks in the chromatogram obtained with the test solution and the amount of free riboflavin in reference solution (1). Limits: Impurity D: Not more than 6.0%, calculated with reference to the dried substance. Sum of impurities A, B and C: Not more than 6.0% calculated with reference to the dried substance. Note: Impurity A: Riboflavin 3’,4’-diphosphate. Impurity B: Riboflavin 3’,5’-diphosphate. Impurity C: Riboflavin 4’,5’-diphosphate. Impurity D: Riboflavin.

Inorganic phosphate Not more than 1.5%. Dissolve 0.10 g of the substance to be examined in water and dilute to 100 ml with the same solvent (Solution A). To 5 ml of solution A, add 10 ml of water, 5 ml of buffered copper sulfate solution pH 4.0 R, 2 ml of a 3% solution of ammonium molybdate R, 1 ml of a freshly prepared solution containing 2% of 4-methylaminophenol sulfate R and 5% of sodium metabisulfite R, and 1 ml of a 3% v/v solution of perchloric acid R and dilute to 25.0 ml with water. Measure, within 15 min of its preparation, the absorbance (Appendix 4.1) of the solution at 800 nm, using as the

RIFAMPICIN

VP V

compensation liquid a solution prepared in the same manner but without the substance to be examined. The absorbance is not greater than that of a reference solution prepared in the same maner as the test solution that use 15 ml of phosphate standard solution (5 ppm PO4) R replacing to 5 ml of solution A and 10 ml of water.

Heavy metals Not more than 10 ppm (Appendix 9.4.8). To 2.0 g of the substance to be examined in a silica crucible add 2 ml of nitric acid R, dropwise, followed by 0.25 ml of sulfuric acid R. Heat cautiously until white fumes are evolved and ignite. Extract the cooled residue with 2 quantities, each of 2 ml, of hydrochloric acid R and evaporate the extracts to dryness. Dissolve the residue in 2 ml of 2 M acetic acid R and dilute to 20 ml with water. 12 ml of the solution complies with method 1. Prepare the reference solution using 10 ml of lead standard solution (1 ppm Pb) R. Loss on drying Not more than 8.0% (Appendix 9.6). (1.000 g; 105 °C; at a pressure not exceeding 0.7 kPa; 5 h). Assay Carry out the assay protected from light. Dissolve 0.100 g of the substance to be examined in 150 ml of water, add 2 ml of glacial acetic acid R and dilute to 1000.0 ml with water. To 10.0 ml of this solution, add 3.5 ml of a 1.4% solution of sodium acetate R and dilute to 50.0 ml with water. Measure the absorbance (Appendix 4.1) at the absorption maximum at 444 nm. Calculate the content of riboflavin, C17H20N4O6, taking 328 as the value of A(1%, 1 cm) at the maximum at 444 nm. Storage In an airtight container, protected from light.

Characters A reddish-brown or brownish-red, crystalline powder. Slightly soluble in water, slightly soluble in acetone and in ethanol (96%), soluble in methanol. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of rifampicin RS. Examine the substances prepared as mulls in liquid paraffin R. B. Dissolve 50 mg of the substance to be examined in 50 ml of methanol R. Dilute 1 ml of the solution to 50 ml with phosphate buffer solution pH 7.4 R. The ultraviolet absorption spectrum (Appendix 4.1) of this solution, in the range between 220 nm and 500 nm, exhibits 4 absorption maxima, at 237 nm, 254 nm, 334 nm and 475 nm. The ratio of the absorbance at the maximum at 334 nm to that at 475 nm is about 1.75. C. Suspend about 25 mg of the substance to be examined in 25 ml of water, shake for 5 minutes and filter. To 5 ml of the filtrate add 1 ml of a 10% solution of ammonium persulphate R in phosphate buffer solution pH 7.4 R and shake for a few minutes. The colour changes from orangeyellow to violet-red and no precipitate is formed. pH The pH of a 1.0 % suspension of the substance to be examined in carbon dioxide-free water R is 4.5 to 6.5 (Appendix 6.2).

Preparation Vitamins B Injection. RIFAMPICIN Rifampicinum

C43H58N4O12

Rifampicin is (2S,12Z,14E,16S,17S,18R,19R,20R,21S, 22R,23S,24E)-5,6,9,17,19-pentahydroxy-23-methoxy2,4,12,16,18,20,22-heptamethyl-8-[[(4-methylpiperazin -1-yl)-imino]methyl]-1,11-dioxo-1,2-dihydro-2,7(epoxypentadeca [1,11,13]trienimino)naphto[2,1-b]furan21-yl acetate, a semisynthetic antibiotic obtained from rifamycin SV. It contains not less than 97.0% and not more than the equivalent of 102.0% of C43H58N4O12, calculated with reference to the dried substance.

M.823

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: A mixture of 35 volumes of acetonitrile R and 65 volumes of a solution containing 0.1% v/v of phosphoric acid R, 0.19% of sodium perchlorate R, 0.59% of citric acid R and 2.09% of potassium dihydrogen phosphate R. Solvent mixture: 1 M citric acid - 1 M potassium dihydrogen phosphate - 1 M dipotassium hydrogen phosphate acetonitrile - water (10 : 23 : 77 : 250 : 640). Test solution: Dissolve 20.0 mg of the substance to be examined in acetonitrile R and dilute to 10.0 ml with the same solvent, and mix. Dilute 5.0 ml to 50.0 ml with the solvent mixure, and mix. Reference solution: Dissolve 20.0 mg of rifampicin quinone RS in acetonitrile R and dilute to 100.0 ml with the same solvent. To 1.0 ml of the solution add 1.0 ml of 843

VP V

RIFAMPICIN CAPSULES

the test solution and dilute to 100.0 ml with the solvent mixture, and mix. Chromatographic system: A column (12 cm × 4.6 mm) packed with stationary phase B (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: Inject the reference solution. Adjust the sensitivity of the detector so that the height of the 2 principal peaks is not less than half the full scale of the recorder. The test is not valid unless the resolution between the 2 principal peaks is at least 4.0. Adjust the concentration of acetonitrile in the mobile phase, if necessary. Inject the test solution and continue the chromatography for at least twice the retention time of rifampicin. Limits: In the chromatogram obtained with the test solution: The area of any peak corresponding to rifampicin quinone is not greater than 1.5 times the area of the corresponding peak in the chromatogram obtained with the reference solution (1.5%). The area of any peak, apart from the principal peak and the peak corresponding to rifampicin quinone, is not greater than the area of the rifampicin peak in the chromatogram obtained with the reference solution (1.0%) and the sum of the areas of any such peaks is not greater than 3.5 times the area of the rifampicin peak in the chromatogram obtained with the reference solution (3.5%). Disregard any peak due to the solvent and any peak with an area less than 0.05 times that of the peak corresponding to rifampicin in the chromatogram obtained with the reference solution.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g of the substance to be examined complies with the limit test for heavy metals, method 3. Prepare the reference solution using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 1.0% (Appendix 9.6). (1.000 g; 80 °C; at a pressure not exceeding 670 Pa; 4 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 2.0 g. Assay Dissolve 0.100 g, accurately weighed, of the substance to be examined in methanol R and dilute to 100.0 ml with the same solvent, and mix. Dilute 2.0 ml of the solution to 100.0 ml with phosphate buffer solution pH 7.4 R, and mix. Measure the absorbance (Appendix 4.1) of this solution at the maximum at 475 nm, using phosphate buffer solution pH 7.4 R as the blank. 844

Calculate the content of C43H58N4O12, taking 187 as the value of A(1%; 1 cm) at the maximum at 475 nm.

Storage Store under nitrogen in an airtight container, protected from light at a temperature not exceeding 25 °C. Action and use Antituberculous. Preparations Capsules; tablets. RIFAMPICIN CAPSULES Capsulae Rifampicini Rifampicin capsules contain rifampicin. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of rifampicin, C43H58N4O12, 92.5% to 107.5% of the stated amount. Identification A. Shake a quantity of the contents of the capsules containing about 0.15 g of rifampicin with 5 ml of chloroform R, filter and evaporate the filtrate to dryness. The infrared absorption spectrum of the resulting residue (Appendix 4.2) is concordant with the reference spectrum of rifampicin. B. The ultraviolet absorption spectrum in the range 220 nm to 500 nm of the final solution obtained in the Assay (Appendix 4.1) exhibits four maxima at 237 nm, 254 nm, 334 nm, and 475 nm. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - buffer solution (35 : 65). Buffer solution: A solution containing 0.1% v/v of phosphoric acid R, 0.19% of sodium perchlorate R, 0.59% of citric acid R and 2.09% of potassium dihydrogen phosphate R. Prepare the following solutions immediately before use. Solvent mixture: A 21.01% solution of citric acid - 13.61% potassium dihydrogen phosphate solution - 17.42% of dipotassium hydrogen phosphate solution - acetonitrile water (10 : 23 : 77 : 250 : 640). Test solution: Shake a quantity of the capsule contents containing 200 mg of rifampicin with 100 ml of acetonitrile R, centrifuge and dilute 5 ml of the clear supernatant liquid to 50 ml with the solvent mixture. Reference solution: A solution containing 0.02% of rifampicin RS in acetonitrile R. Dilute accurately 1 ml of this solution to 100.0 ml with the solvent mixture.

RIFAMPICIN TABLETS

VP V

Resolution solution: A solution containing 0.01% of rifampicin RS and 0.01% rifampicin quinone RS in acetonitrile R. Dilute 5 ml of this solution to 50 ml with the solvent mixture. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase B (5 µm). Column temperature: 30 °C. Detector: A spectrophotometer set at 254 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the resolution solution, the test is not valid unless the resolution factor between the two principal peaks is at least 4.0. The symmetry factor of rifampicin peak is not more than 2.0, the number of theoretical plates is not less than 2000. Adjust the concentration of acetonitrile in the mobile phase if necessary. The relative retention times are 1.0; 0.55; 1.25 and 3.56 for rifampicin, rifampicin quinone, rifampicin N-oxide and 3-formylrifampicin SV, respectively. Inject the test solution and reference solution. Limits: To calculate the content of impurities, divide the area of the corresponding peak to the following response factor: 1.19 for rifampicin quinone; 1.03 for rifampicin N-oxide and 1.25 for 3-formylrifamycin SV. In the chromatogram obtained with the test solution, the area of any peak corresponding to rifampicin quinone is not greater than 4 times the area of the peak in the chromatogram obtained with the reference solution (4.0%); the area of any peak corresponding to rifampicin N-oxide is not greater 1.5 times than the area of the peak in the chromatogram obtained with reference solution (1.5%); the area of any peak corresponding to 3-formylrifamycin SV is not greater than the area of the peak in the chromatogram obtained with the reference solution (1.0%) and the area of any other secondary peak is not greater than the area of the peak in the chromatogram obtained with reference solution (1.0%).

Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 900 ml of 0.1 M hydrochloric acid R. Rotation speed: 100 rpm. Time: 45 min. Procedure: After the specified time, withdraw a sample of the medium and filter, discard the first portion of the filtrate. Dilute a suitable volume of the filtrate, if necessary, with 0.1 M hydrochloric acid R. Measure the absorbance of the resulting solution (Appendix 4.1) at the maximum wavelength at 336 nm in a 1-cm cell using 0.1 M hydrochloric acid R as the blank. Calculate the content of rifampicin, C43H58N4O12, in the medium taking 263 as the value of A (1%, 1 cm) at the maximum at 336 nm. Tolerance: Not less than 70% (Q) of the stated amount of rifampicin is dissolved in 45 min.

Assay Weigh 20 capsules, calculate the average mass of the content of the capsules, mix. Weigh accurately a quantity of the mixed contents containing 0.1 g of rifampicin and transfer into a 100-ml volumetric flask. Add 80 ml of methanol R, shake well and add sufficient methanol R to volume, mix and filter. Dilute 2.0 ml of the filtrate to 100 ml with phosphate buffer solution pH 7.4 R, and measure the absorbance of the resulting solution at the maximum wavelength at 475 nm (Appendix 4.1), using phosphate buffer solution pH 7.4 R as the blank. Calculate the content of rifampicin, C43H58N4O12, taking 187 as the value of A (1%, 1 cm) at the maximum at 475 nm. Storage Store in a well-closed container, in a cool and dry place, protected from light. Action and use Antituberculous. Usual strength 150 mg; 300 mg. RIFAMPICIN TABLETS Tabellae Rifampicini Rifampicin tablets are film-coated tablets containing rifampicin. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of rifampicin, C43H58N4O12, 92.5% to 107.5% of the stated amount. Identification A. Shake a quantity of the powdered tablets (with coating removed) containing about 0.15 g of rifampicin with 5 ml of chloroform R, filter, evaporate the filtrate to dryness. The infrared absorption spectrum of the resulting residue (Appendix 4.2) is concordant with the reference spectrum of rifampicin. B. The light absorption spectrum (Appendix 4.1) in the range 220 nm to 500 nm of the final solution obtained in the Assay exhibits four maxima at 237 nm, 254 nm, 334 nm, and 475 nm. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - buffer solution (35 : 65). Buffer solution: A solution containing 0.1% (v/v) of phosphoric acid R, 0.19% of sodium perchlorate R, 0.59% of citric acid R and 2.09% of potassium dihydrogen phosphate R. 845

VP V

RIFAMPICIN AND ISONIAZID CAPSULES

Prepare the following solutions immediately before use. Solvent mixture: 21.01% citric acid solution - 13.61% potassium dihydrogen phosphate solution - 17.42% dipotassium hydrogen phosphate solution - acetonitrile water (10 : 23 : 77 : 250 : 640). Test solution: Shake a quantity of the powdered tablets (removed the coating) containing 200 mg of rifampicin with 100 ml of acetonitrile R, centrifuge and dilute 5 ml of the clear supernatant liquid to 50 ml with the solvent mixture. Reference solution: A solution containing 0.02% of rifampicin RS in acetonitrile R. Transfer accurately 1 ml of this solution and dilute to 100.0 ml with the solvent mixture. Resolution solution: A solution containing 0.01% of rifampicin RS and 0.01% rifampicin quinone RS in acetonitrile R. Dilute 5 ml of this solution to 50 ml with the solvent mixture. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase B (5 µm). Column temperature: 30 °C. Detector: A spectrophotometer set at 254 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the resolution solution, the test is not valid unless the resolution factor between the two principal peaks is at least 4.0. The symmetry factor of rifampicin peak is not more than 2.0, the number of theoretical plates is not less than 2000. Adjust the concentration of acetonitrile in the mobile phase if necessary. The relative retention times are 1.0; 0.55; 1.25 and 3.56 for rifampicin, rifampicin quinone, rifampicin N-oxide and 3-formylrifampicin SV, respectively. Inject the test solution and reference solution. Limits: To calculate the content of impurities, divide the area of the corresponding peaks to the following response factor: 1.19 for rifampicin quinone; 1.03 for rifampicin N-oxide and 1.25 for 3-formylrifamycin SV. In the chromatogram obtained with the test solution, the area of any peak corresponding to rifampicin quinone is not greater than 4 times the area of the peak in the chromatogram obtained with the reference solution (4.0%); the area of any peak corresponding to rifampicin N-oxide is not greater than 1.5 times the area of the peak in the chromatogram obtained with reference solution (1.5%); the area of any peak corresponding to 3-formylrifamycin SV is not greater than the area of the peak in the chromatogram obtained with the reference solution (1.0%) and the area of any other secondary peak is not greater than the area of the peak in the chromatogram obtained with reference solution (1.0%).

846

Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 900 ml of 0.1 M hydrochloric acid R. Rotation speed: 100 rpm. Time: 45 min. Procedure: After the specified time, withdraw a sample of the medium and filter, discard the first portion of the filtrate. Dilute the filtrate with the phosphate buffer prepared by dissolving 3.02 g of potassium dihydrogen phosphate R and 6.2 g of dipotassium hydrogen phosphate R in 1000 ml of water, adjust the pH to 7.0 with phosphoric acid R to obtain a solution containing 20 µg of rifampicin per ml. Measure the absorbance of the resulting solution (Appendix 4.1) at maximum wavelength at 475 nm in a 1-cm cell using phosphate buffer as the blank. Calculate the content of rifampicin, C43H58N4O12, in the medium taking 187 as the value of A (1%, 1 cm) at the maximum at 475 nm. Tolerances: Not less than 70% (Q) of the stated amount of rifampicin is dissolved in 45 min. Assay Remove the coating of 20 tablets, calculate the average mass of the tablet cores and powder finely. Weigh accurately a quantity of the powdered tablets containing about 0.1 g of rifampicin, add 80 ml of methanol R, shake well and add sufficient methanol R to produce 100.0 ml, mix and filter. Dilute 2.0 ml of the filtrate to 100.0 ml with phosphate buffer solution pH 7.4 R, and measure the absorbance of the resulting solution at the maximum at 475 nm (Appendix 4.1), using phosphate buffer solution pH 7.4 R as the blank. Calculate the content of rifampicin, C43H58N4O12, taking 187 as the value of A (1%, 1 cm) at the maximum at 475 nm. Storage Store in a well-closed container, in a cool and dry place, protected from light. Action and use Antituberculous. Usual strength 150 mg; 300 mg. RIFAMPICIN AND ISONIAZID CAPSULES Capsulae Rifampicini et Isoniazidi Rifampicin and isoniazid capsules contain rifampicin and isoniazid. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of rifampicin, C43H58N4O12, 90.0% to 130.0% of the stated amount.

VP V

Content of isoniazid, C6H7N3O, 90.0% to 110.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silicagel GF254. Mobile phase: Acetone - glacial acetic acid (100 : 1). Test solution: Shake a quantity of the contents of the capsules equivalent to about 120 mg of rifampicin with 20 ml of methanol R, filter. Dilute the filtrate with an equal volume of acetone R, mix. Reference solution (1): Dissolve a quantity of rifampicin RS in methanol R to obtain a solution containing 6 mg per ml. Add an equal volume of acetone R and mix. Reference solution (2): Dissolve a quantity of isoniazid RS in methanol R to obtain a solution containing 3 mg per ml. Add an equal volume of acetone R and mix. Procedure: Apply separately to the plate 2 µl of each solution. After removal of the plate, allow it to dry in air and examine under ultraviolet light at 254 nm. The two principal spots in the chromatogram obtained with the test solution are similar in position, colour and size to those in the chromatogram obtained with the reference solutions. B. In the Assay, the retention times of the two principal peaks in the chromatogram obtained with the test solution correspond to those of the two principal peaks in the chromatogram obtained with the reference solution. Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 900 ml of 0.1 M hydrochloric acid R. Rotation speed: 100 rpm. Time: 45 min. Phosphate buffer solution: Weigh 15.3 g of dipotassium hydrogen phosphate R and 80.0 g of potassium dihydrogen phosphate R into a 1 litre volumetric flask, dissolve and dilute with water to volume, and mix. Isoniazid standard stock solution: Accurately weigh about 66 mg of isoniazid RS into a 100 ml volumetric flask. Dissolve in and dilute with 0.1 M hydrochloric acid R to volume, and mix. Mixed standard stock solution: Accurately weigh about 66 mg of rifampicin RS into a 200 ml volumetric flask, dissolve 10 ml of 0.1 M hydrochloric acid R, and mix. Add 50.0 ml of isoniazid standard stock solution, dilute with 0.1 M hydrochloric acid R to volume, and mix. (Note: Prepare this solution immediately before the test is performed, and place in the dissolution bath at the start of the test and take out at the end of the test). Determination of rifampicin dissolved Reference solution: Transfer 5.0 ml of the mixed standard stock solution and 10.0 ml of phosphate buffer solution to a 50 ml volumetric flask. Dilute with water to volume, and mix. (Note: Use the solution immediately, if possible, and if not, within 3 hours after final dilution).

RIFAMPICIN AND ISONIAZID CAPSULES

Test solution: After the specified time, withdraw a sample of the medium and filter, discarding the first portion of the filtrate. Allow to cool for about 10 minutes and transfer 5.0 ml of the filtrate and 10.0 ml of the phosphate buffer solution to a 50 ml volumetric flask. Dilute with water to volume, and mix. (Note: Use the solution immediately, if possible, and if not, within 3 hours after final dilution). Procedure: Measure the absorbances of the test solution and the reference solution at the maximum at about 475 nm (Appendix 4.1). The compensation liquid is prepared by transfering 5.0 ml of 0.1 M hydrochloric acid R and 10.0 ml of the phosphate buffer solution into a 50 ml volumetric flask, dilute with water to volume, and mix. Calculate the content of rifampicin dissolved, C43H58N4O12, using the absorbances of the reference solution and the test solution and the declared content of C43H58N4O12 of rifampicin RS. Tolerance: Not less than 75% (Q) of the stated amount of rifampicin, C43H58N4O12, is dissolved in 45 minutes.

Determination of isoniazid dissolved Examine by liquid chromatography (Appendix 5.3). Mobile phase: Water - phosphate buffer solution - methanol (850 : 100 : 50). Reference solution: Use the reference solution in the Determination of rifampicin dissolved. Test solution: Use the test solution in the Determination of rifampicin dissolved. Chromatographic system: A column (30 cm × 4.0 mm) packed with stationary phase C (10 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1.5 ml/min. Volume of injection: 50 µl. Procedure: Inject the reference solution and the test solution, calculate the content of isoniazid dissolved, C6H7N3O, using the areas of the principal peaks in the chromatograms obtained with the test solution and the reference solution, and the declared content of C6H7N3O in isoniazid RS. Tolerance: Not less than 80% of the stated amount of isoniazid, is dissolved in 45 minutes. Loss on drying Not more than 3.0% (Appendix 5.3). Determine on about 100 mg of the capsule contents in a capillary-stoppered bottle in vacuum at 60 °C for 3 hours. Assay Examine by liquid chromatography (Appendix 5.3). Buffer solution: Dissolve 1.4 g of disodium hydrogen phosphate R in 1 L of water, and adjust with phosphoric acid R to a pH of 6.8. Solution A: Acetonitrile - buffer solution (4 : 96). Solution B: Acetonitrile - buffer solution (55 : 45). Mobile phase: Use variable mixtures of solution A and solution B as directed in Chromatographic system. 847

RIFAMPICIN, ISONIAZID AND PYRAZINAMIDE TABLETS

Reference solution: Dissolve accurately weighed quantities of rifampicin RS and isoniazid RS in a mixture of buffer solution and methanol R (96 : 4) to obtain a solution having known concentrations of about 0.16 mg/ml and 0.08 mg/ml, respectively (Note: Use this solution within 10 minutes). Test solution: Weigh 20 capsules, calculate the average weight of the capsule contents and finely powder. Transfer an accurately weighed portion of the powder, equivalent to about 16 mg of rifampicin and 8 mg of isoniazid, to a 100 ml volumetric flask, and add about 90 ml of the buffer solution. Sonicate for about 10 minutes, allow to equilibrate to room temperature, dilute with the buffer solution to volume, and mix (Note: Use this solution within 2 hours). Chromatographic system A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 238 nm. Flow rate: 1.5 ml/min Volume of injection: 20 µl. Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

Comment

0

100

0

Equilibration

0-5

100

0

Isocratic

5-6

100 → 0

0 → 100

Linear gradient

6 - 15

0

100

Isocratic

Procedure: System suitability: Chromatograph the reference solution, and record the peak responses: the relative retention times are about 2.6 and 1.0 for rifampicin and isoniazid respectively; the column efficiency is not less than 50 000 and not less than 6 000 theoretical plates for rifampicin and isoniazid, respectively and the relative standard deviation of the peak areas for replicate injections is not more than 2.0%. Inject sepatately the reference solution and the test solution. Calculate the content of rifampicin, C43H58N4O12, and isoniazid, C6H7N3O, using the areas of the principal peaks in the chromatograms obtained with the test solution and the reference solution, and the declared contents of reference substances.

Storage Store in a well-closed container, in a cool and dry place, protected from light. Action and use Antituberculous. Usual strength 300 mg of rifampicin and 150 mg of isoniazid.

848

VP V

RIFAMPICIN, ISONIAZID AND PYRAZINAMIDE TABLETS Tabellae Rifampicini, isoniazidi et pyrazinamidi Rifampicin, isoniazid and pyrazinamide tablets contain rifampicin, isoniazid and pyrazinamide. They may be coated. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of rifampicin, C43H58N4O12, 90.0% to 110.0% of the stated amount. Content of isoniazid, C6H7N3O, 90.0% to 110.0% of the stated amount. Content of pyrazinamide, C5H5N3O, 90.0% to 110.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Aceton - glacial acetic acid (100 : 1). Rifampicin reference solution: Dissolve a quantity of rifampicin RS in methanol R to obtain a solution containing about 2.5R mg/ml of rifampicin (in which R is the ratio of the stated amount of rifampicin and isoniazid in the tablets). Dilute a volume of this solution with an equal volume of aceton R, mix. Isoniazid reference solution: Dissolve a quantity of isoniazid RS in methanol R to obtain a solution containing about 2.5 mg/ml of isoniazid. Dilute a volume of this solution with an equal volume of aceton R, mix. Pyrazinamide reference solution: Dissolve a quantity of pyrazinamide RS in methanol R to obtain a solution containing about 2.5P mg/ml of pyrazinamide (in which P is the ratio of the stated amount of pyrazinamide and isoniazid in the tablets). Dilute a volume of this solution with an equal volume of aceton R, mix. Test solution: To a quantity of the powdered tablets (coating layer removed) containing the equivalent of 50 mg of isoniazid add 20 ml of methanol R, shake for 5 min to dissolve, filter. Dilute a volume of the filtrate with an equal volume of aceton R, mix. Procedure: Apply separately to the plate 2 μl of each solution. After developing over 3/4 of the plate, remove the plate, allow it to dry in air and examine under ultraviolet light at 254 nm. The three principal spots in the chromatogram obtained with the test solution are similar in position, colour and size to those in the chromatogram obtained with the reference solutions. B. In the Assay, the retention time of the three principal peaks in the chromatogram obtained with the test solution correspond to those in the chromatogram obtained with the reference solution.

VP V

Loss on drying Not more than 3.0% (Appendix 9.6). (1.000 g of the powdered tablets, 60 °C, in vacuum, 3 h). Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 900 ml of simulated gastric fluid R, without pepsin. Rotation speed: 100 rpm. Time: 30 min. For rifampicin Reference stock solution: Transfer an accurately weighed quantity of rifampicin RS, isoniazid RS and pyrazinamide RS equivalent to 1/4 of the stated amount of those in the tablets to a 250 ml volumetric flask. Add about 200 ml of the medium, dissolve by sonicating and dilute to the volume with the medium. Place this flask into the dissolution bath immediately prior to starting the tablet dissolution and withdraw the flask from the dissolution bath at the same time that the solutions under test are withdrawn. Reference solution: Dilute a volume of the reference stock solution with the medium to obtain a solution containing 0.03 mg/ml of rifampicin. Test solution: After the specified time (30 min), withdraw a sample of the medium, and filter. Dilute the filtrate with the medium to obtain a solution containing 0.03mg/ml of rifampicin. Procedure: Measure the absorbance (Appendix 4.1) of the test solution and the reference solution at 475 nm, in 1 cm cell, using the medium as a blank. Calculate the content of rifampicin, C43H58N4O12, dissolved by using the absorbances of the test solution and the reference solution and the declared content of C43H58N4O12 in the rifampicin RS. Tolerance: Not less than 80% (Q) of the stated amount of rifampicin, C43H58N4O12, is dissolved in 30 min. For isoniazid and pyrazinamide Examine by liquid chromatography (Appendix 5.3). Mobile phase: Water - 1 M potassium dihydrogen phosphate - acetonitrile (860 : 100 : 40). Reference solution: Transfer 3.0 ml of the reference stock solution in the Dissolution for rifampicin to a 20 ml volumetric flask, add 3 ml of 1 M potassium hydrogen phosphate and dilute to volume with the mobile phase, mix. Test solution: After the specified time (30 min), withdraw a sample of the medium, and filter. Transfer 3.0 ml of the filtrate to a 20 ml volumetric flask, add 3 ml of 1 M potassium hydrogen phosphate and dilute to volume with the mobile phase, mix and filter. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 μm). Detector: A spectrophotometer set at 254 nm.

RIFAMPICIN, ISONIAZID AND PYRAZINAMIDE TABLETS

Flow rate: 1.5 ml/min. Volume of injection: 20 μl. Procedure: System suitability: Inject the reference solution, the resolution factor between the peaks due to isoniazid and pyrazinamide is at least 4. The tailing factor of the peaks due to isoniazid and pyrazinamide is not more than 2.0 and the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Chromatograph alternately the reference solution and the test solution. Calculate the content of isoniazid, C6H7N3O, and pyrazinamide, C5H5N3O, dissolved using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C6H7N3O and C5H5N3O in isoniazid RS and pyrazinamide RS, respectively. Tolerance: Not less than 80% (Q) of the stated amount of isoniazid, C6H7N3O, is dissolved in 30 min. Not less than 75% (Q) of the stated amount of pyrazinamide, C5H5N3O, is dissolved in 30 min.

Assay Examine by liquid chromatography (Appendix 5.3). Buffer: Dissolve 1.4 g of disodium hydrogen phosphate R in 1000 ml of water, adjust to pH 6.8 with phosphoric acid R. Mobile phase A: Buffer - acetonitrile (96 : 4). Mobile phase B: Acetonitrile - buffer (55 : 45). Reference solution: Dissolve an accurately weighed quantity of rifampicin RS, isoniazid RS and pyrazinamide RS in a mixture of buffer - methanol (94 : 4) to obtain a solution having known concentrations of about 0.08R mg/ml, 0.08 mg/ml và 0.08P mg/ml respectively (in which R is the ratio of the stated amount of rifampicin and isoniazid in the tablets. P is the ratio of the stated amount of pyrazinamide and isoniazid in the tablets). Use this solution within 10 min. Test solution: Weigh 20 tablets, remove the coating layer if necessary, calculate the average mass and powder finely. Transfer an accurately weighed quantity of the powdered tablets containing the equivalent of 8 mg of isoniazid to a 100 ml volumetric flask. Add 4 ml of methanol R, sonicate for 5 min. Add 80 ml of buffer and dissolve by sonicating for 10 min. Dilute to volume with buffer, mix and filter. Use this solution within 2 hours. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 μm). Detector: A spectrophotometer set at 238 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 μl. Procedure: Carry out a linear gradient elution using the following conditions:

849

VP V

RINGER - LACTATE INFUSION

Time (min)

Mobile Mobile phase A (v/v) phase B (v/v)

Elution

0

100

0

Equilibration

0-5

100

0

Isocratic

5-6

100 → 0

0 → 100

Linear gradient

6 - 15

0

100

Isocratic

Inject the reference solution. The relative retention times are about 0.7, 1.0 and 1.8 for isoniazid, pyrazinamide and rifampicin, respectively. The resolution between isoniazid peak and pyrazinamide peak is at least 4. The tailing factors of the peaks due to isoniazid, pyrazinamide and rifampicin are not more than 2.0. The relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of rifampicin, C43H58N4O12, isoniazid, C6H7N3O and pyrazinamide, C5H5N3O in the tablets using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C43H58N4O12, C6H7N3O and C5H5N3O in rifampicin RS, isoniazid RS and pyrazinamide RS, respectively.

Storage Store in an airtight container, in a cool and dry place, protected from light. Action and use Antiturbeculosis. Usual strength 150 mg of rifampicin, 75 mg of isoniazid, 400 mg of pyrazinamide. 120 mg of rifampicin, 50 mg of isoniazid, 300 mg of pyrazinamide. RINGER - LACTATE INFUSION Infusio Ringer - Lactate

Characters A clear, colourless solution. Identification A. When warmed with potassium permanganate R, yields acetaldehyde (lactate identification). B. In the Assay for lactate, the retention time of the principal peak in the chromatogram obtained with the test solution is the same as that of the principal peak in the chromatogram obtained with the reference solution. C. Identification of sodium and potassium: The residue obtained by evaporation, when introduced on a platinum wire into a flame, imparts a yellow colour to the flame. When viewed through a suitable blue glass, the flame is tinged with reddish purple. D. Yields reaction (B) characteristic of calcium salts (Appendix 8.1). E. Yields reaction (A) characteristic of chlorides (Appendix 8.1). pH 5.0 to 7.5 (Appendix 6.2). Bacterial endotoxins The endotoxin limit concentration is 0.25 EU/ml (Appendix 13.2). Assay For calcium chloride dihydrate To 50.0 ml of the infusion add 5.0 ml of 0.01 M magnesium sulphate VS and 5 ml of ammonia buffer pH 10.9 R and titrate with 0.01 M Trilon B solution VS using mordant black 11 solution R as indicator. From the volume of 0.01 M Trilon B solution VS required subtract the volume of 0.01 M magnesium sulfate VS added. 1 ml of 0.01 M Trilon B solution VS is equivalent to 1.470 mg of CaCl2,2H2O.

Ringer - lactate infusion is a sterile solution containing 0.027% of calcium chloride dihydrate, 0.04% of potassium chloride, 0.6% of sodium chloride, and 0.32% of sodium lactate in water for injections. The infusion complies with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements.

For K Examine by atomic emission spectrophotometry (Appendix 4.4, Method 1). Reference solution: Dilute stepwise potassium standard solution (600 ppm K) R with deionised water R to obtain an appropriate concentration of K. Test solution: Dilute stepwise the infusion with deionised water R to obtain an appropriate concentration of K. Procedure: Measure the intensities of the atomic emission of the reference solution and the test solution at 766.5 nm.

Content of sodium, Na, 0.27% to 0.32%. Content of potassium, K, 0.019% to 0.022%. Content of total chloride, Cl, 0.37% to 0.42%. Content of calcium chloride dihydrate, CaCl2,2H2O, 0.025% to 0.029%. Content of lactate, calculated as C3H6O3, 0.23% to 0.28%.

For Na Examine by atomic emission spectrophotometry (Appendix 4.4, Method 1). Reference solution: Dilute sodium standard solution (200 ppm Na) R with deionised water R to obtain an appropriate concentration of Na. Test solution: Dilute the infusion with deionised water R to obtain an appropriate concentration of Na.

850

RITONAVIR

VP V

Procedure: Measure the intensities of the atomic emission of the reference solution and the test solution at 589.0 nm.

RITONAVIR Ritonavirum

For total chloride To 20.0 ml of the infusion add 30 ml of water, 50.0 ml of 0.1 N silver nitrate VS and 2 ml of nitric acid R, filter, wash the precipitate with water. Combine the filtrate and the washing, slightly acidified with nitric acid R and titrate the excess of silver nitrate with 0.1 N ammonium thiocyanate VS using 10% ammonium iron (III) sulfate solution R as indicator. 1 ml of 0.1 N silver nitrate VS is equivalent to 3.545 mg of Cl. For lactate Carry out the method for liquid chromatography (Appendix 5.3). Mobile phase: A mixture of 90 volumes of water and 10 volumes of a 2% v/v solution of octylamine in acetonitrile, the pH of the final mixture being adjusted to 7.0 with a 10% v/v solution of orthophosphoric acid R. Reference solution: A solution containing 0.28% of lithium lactate RS in the mobile phase. Test solution: Use the infusion. Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase C (5 µm to 10 µm). Detector: A spectrophotometer set at 210 nm. Flow rate: 2.0 ml/min. Volume of injection: 20 µl. Procedure: Inject separately the reference solution and the test solution. Calculate the content of lactate, C3H6O3, in the infusion using the areas of the principal peaks in the chromatograms obtained with the test solution and the reference solution, and the declared equivalent content of C3H6O3 in lithium lactate RS. Storage Store in an airtight container, in a dry and cool place, protected from light. Action and use Solution correcting water, electrolyte and acid-base disturbances.

C37H48N6O5S2  

M: 721.0

Ritonavir is thiazol-5-ylmethyl [(1S,2S,4S)-1-benzyl-2hydroxy-4-[[(2S)-3-methyl-2-[[methyl[[2- (1-methylethyl) thiazol-4-yl]methyl]carbamoyl]amino]butanoyl]amino]-5phenylpentyl]carbamate. It contains not less than 98.5% and not more than 101.0% of C37H48N6O5S2, calculated with reference to the dried substance.

Characters White or almost white powder, it shows polymorphism. Practically insoluble in water, freely soluble in methanol and in dichloromethane, sparingly soluble in aceton, very slightly soluble in acetonitrile. Identification Apply one of the two following identifications: First identification: A. Second identification: B, C. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of ritonavir RS. If the spectra obtained show differences, dissolve separately the substance to be examined and the reference substance separately in a minimal amount of methanol R, crystallise by adding just enough water drop by drop, filter and dry for about 1 h and record the spectra again. B. The absorption spectrum of a 40 μg/ml solution of the substance to be examined in methanol R, in the range 220 nm to 280 nm (Appendix 4.1), exhibits one maximum at 240 nm; the specific absorbance at the absorption maximum is 116 to 128. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Dichloromethane - acetonitrile - methanol - ammonia (67 : 20 : 10 : 3). Test solution: Dissolve and dilute the substance to be examined in methanol R to obtain a solution containing 5 mg/ml. Reference solution: Dissolve and dilute ritonavir RS in methanol R to obtain a solution containing 5 mg/ml. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm. Dry the plate in a 851

VP V

RITONAVIR

current of cool air and examine under ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, appearance and size to the principal spot in the chromatogram obtained with the reference solution.

Specific optical rotation +7° to +10°, calculated with reference to the dried substance (Appendix 6.4). Determined on a 20.0 mg/ml solution of the substance to be examined in methanol R, measured at 20 °C. Related substances Examine by liquid chromatography (Appendix 5.3). Buffer phosphate pH 4,0 solution: Dissolve 7.8 g of sodium dihydrogen phosphate R and 1,88 g sodium hexansulfonate R in 800 ml water, adjust to pH 4.0 with a 10% solution of phosphoric acid R and dilute to 1000 ml with water. Mobile phase A: Acetonitrile - buffer phosphate pH 4.0 solution - water (35 : 28 : 37). Mobile phase B: Acetonitrile - buffer phosphate pH 4.0 solution - water (70 : 28 : 2). Test solution: A 0.5 mg/ml solution of the substance to be examined in mobile phase A. Reference solution (1): Dilute the test solution with mobile phase A to obtain a 0.5 µg/ml solution of the substance to be examined. Reference solution (2): To 5 ml the test solution, add 1 ml a 50% solution of sulfuric acid R, heat in a water-bath for 20 min. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase base-deactivated octadecylsilyl silica gel for chromatography R (5 µm). Column temperature: 35 °C. Detector: A spectrophotometer set at 240 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min) 0 - 20 20 - 30 30 - 40 40 - 45

Mobile phase A (% v/v) 70 70 → 0 0 0 → 70

Mobile phase B (% v/v) 30 30 → 100 100 100 → 30

Comments

Isocratic Linear gradient Isocratic Linear gradient Isocratic 45 - 50 70 30 re-equilibration System suitability: In the chromatogram obtained with reference solution (2), the resolution between the principal peak (retention time = about 22 min) and the peak having relative retention with reference to principal peak of about 0.8 is at least 3.5; the resolution between the principal peak and the peak having relative retention with reference

852

to principal peak of about 1.5 is at least 9.0. If necessary, adjust the amount of acetonitrile in both mobile phases A and B, or adjust the gradient program. Inject mobile phases A as the blank solution, reference solution (1) and the test solution. Limits: In the chromatogram obtained with the test solution: The area of any impurity peak is not more than 3 times the area of the principal peak obtained with reference solution (1) (0.3%). The areas of not more than two impurity peaks are more than twice the area of the principal peak obtained with reference solution (1) (0.2%). The areas of not more than four impurity peaks are more than the area of the principal peak obtained with reference solution (1) (0.1%). The sum of the areas of all impurity peaks is not more than 10 times the area of the principal peak obtained with reference solution (1) (1.0%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%).

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C; 2 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.25 g of the substance to be examined, accurately weighed, in 30 ml of glacial acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 36.05 mg of C37H48N6O5S2. Storage Store in an airtight container, protected from light. Action and use Protease inhibitor; antiviral HIV. Preparation Capsules.

ROTUNDINE

VP V

ROTUNDINE Rotundinum L-Tetrahydropalmatine

C21H25NO4

Determined on a solution containing 8 mg of the substance being examined per ml in ethanol (96%) R at 25°C.

Specific absorbance Measure the absorbance (Appendix 4.1) of a solution containing 30 µg of the substance being examined per ml in 0.5% solution of sulfuric acid R at 281nm. The value of A(1% ; 1cm) is 150 to 160.

M.355.43

Rotundine is 5,8,13,13α-tetrahydro-2,3,9,10-tetramethoxy6H-dibenzo[a,g]quinolizine. It contains not less than 98.5% and not more than the equivalent of 102.0% of C21H25NO4, calculated with reference to the dried substance.

Characters White to slightly yellow crystals; odourless, tasteless. It turns to yellow on exposure to light or heat. Soluble in chloroform, sparingly soluble in ethanol and in ether, practically in soluble in water, freely soluble in dilute sulfuric acid. Melting point: 141°C to 144°C (Appendix 6.7). Identification A.The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of rotundine RS. B. In the test for Assay, the retention time of the principal peak in the chromatogram obtained with test solution is concordant with retention time of the principal peak in the chromatogram obtained with reference solution. C. Solution S: Dissolve 0.1 g of the substance to be examined in a mixture of 10 ml of water and 1 ml of dilute sulfuric acid R. Add 1 drop of a 5% solution of potassium dichromate R to 2 ml of solution S, a yellow precipitate is produced. Add 1 drop of a saturated solution of sodium chloride R to 2 ml of solution S, a white precipitate is produced. Add 1 drop of a 5% solution of potassium ferricyanide R to 2 ml of solution S, a yellow precipitate is produced which gradually turns to green and finally changes to blue on gently heating. Appearance of solution Dissolve 0.15 g in 5 ml of 5% solution of sulfuric acid R. The solution is clear (Appendix 9.2) and not more intensely coloured than reference solution YG4 (Appendix 9.3, method 1). Specific optical rotation -290° to -300°, calculated with reference to the dried substance (Appendix 6.4).

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol - buffer solution (65 : 35). Buffer solution: A mixture of equal volumes of 0.05 M solution of potassium dihydrogen phosphate R and 0.05 M solution of sodium heptasulfonate R, containing 0.2% of triethylamine R, adjusted to pH 6.5 ± 0.05 with phosphoric acid R. Test solution: Dissolve 20 mg of the substance to be examined in 10 ml of methanol R, sonicate for 5 min and dilute to 100.0 ml with the mobile phase. Reference solution: Dilute 1.0 ml of the test solution to 200.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 280 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Inject the reference solution, adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained is at least 25% of the full scale of the recorder. The column efficiency determined on the rotundine peak is not less than 2.500 theoretical plates Inject the test solution and reference solution. The run time is twice the retention time of the principal peak. Limit: Total peak area of all impurities in the chromatogram obtained with the test solution is not more than the area of the principal peak in the chromatogram obtained with the reference solution (0.5%). Heavy metals Not more than 20 ppm (Appendix 9.4.8, method 3). Determine on the residues obtaining after Sulfated ash test. Prepare the standard using lead standard solution (10 ppm Pb) R. Loss on drying Not more than 5.0% (Appendix 9.6). (1.00 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 1). Determined on 1.0 g.

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ROTUNDINE TABLETS

Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. Test solution: Dissolve 25 mg of the substance to be examined in 10 ml of methanol R, sonicate for 5 min and dilute to 50.0 ml with the mobile phase. Dilute 5.0 ml of the test solution to 50.0 ml with the mobile phase. Reference solution: Dissolve 25 mg of rotundin RS in 10 ml of methanol R, sonicate for 5 min and dilute to 50.0 ml with the mobile phase. Dilute 5 ml of the test solution to 50 ml the with mobile phase. System suitability: In the chromatogram obtained with reference solution, the column efficiency determined from the rotundine peak is not less than 2 500 theoretical plates. Calculate the percentage content of C21H25NO4, using the chromatograms obtained with the test solution and reference solution and using the declared content of C21H25NO4 in rotundine RS. Storage Store in an airtight container, protected from light. Action and use Tranquilizer. Preparation Tablets. ROTUNDINE TABLETS Tabellae Rotundini Rotundine tablets contain rotundine. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of rotundine, C21H25NO4, 93.0% to 107.0% of the stated amount. Identification A. To a quantity of the powdered tablets containing 0.1 g of rotundine, add 10 ml of water and 1 ml of dilute sulfuric acid R, shake to dissolve rotundine and filter. The filtrate complies with the following tests: To 2 ml of the filtrate add 1 drop of 5% potassium dichromate solution R, a yellow precipitate is produced. To 2 ml of the filtrate add 1 drop of saturated sodium chloride solution R, a white precipitate is produced. To 2 ml of the filtrate add 1 drop of 5% potassium ferricyanide solution R, a yellow precipitate is produced, which gradually turns to green and finally change to blue on gentle heating. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of the rotundine peak in the chromatogram obtained with the reference solution. 854

VP V

C. Grind a quantity of the powdered tablets containing 90 mg rotundine with 20 ml of ether R, filter, evaporate to dryness. Dry the residue at 80 °C for 3 h in vacuum. The infrared absorption spectrum (Appendix 4.2) of the resulting residue is concordant with the spectrum of rotundine RS.

Related substances Examine by liquid chromatography (Appendix 5.3). Test solution: Weigh accurately a quantity of the powdered tablets containing about 20 mg of rotundine and transfer into a 100-ml volumetric flask, add 10 ml of methanol R and sonicate for 5 min, dilute with the mobile phase to volume, mix and filter. Reference solution: Transfer 1.0 ml of the test solution into a 100-ml volumetric flask, dilute to volume with the mobile phase, mix. Proceed with the chromatographic system as described in the Assay and record the chromatogram for twice the retention time of the principal peak. Limits: The sum of the areas of all secondary peaks in the chromatogram obtained with the test solution is not greater than the area of the principal peak in the chromatogram obtained with the reference solution (1.0%). Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 900 ml of 0.1 M hydrochloric acid R. Rotation speed: 100 rpm. Time: 45 min. Procedure: After the specified time, withdraw a sample of the medium, filter and dilute the filtrate twice with 0.1 M hydrochloric acid R (for 60 mg strength). Measure the absorbance of this solution (Appendix 4.1) at 281 nm in a 1-cm cell, using 0.1 M hydrochloric acid R as the blank. Calculate the content of rotundine, C21H25NO4, taking 155 as the value of A (1%, 1 cm) at the wavelength at 281 nm. Tolerance: Not less than 70% (Q) of the stated amount of rotundine, C21H25NO4, is dissolved in 45 min. Assay Examine by liquid chromatography (Appendix 5.3). Buffer solution pH 6.5: A mixture of 0.05 M potassium dihydrogen phosphate and 0.05 M sodium heptane sulfonate (1 : 1) containing 0.02% triethylamine R, adjust to pH 6.5 ± 0.05 with phosphoric acid R. Mobile phase: Buffer solution pH 6.5 - methanol (35 : 65). Make adjustments if necessary. Test solution: Weigh 20 tablets, calculate the average mass and finely powder. Weigh accurately a quantity of the powder containing about 25 mg of rotundine, transfer into a 50-ml volumetric flask, add 10 ml of methanol R, sonicate for 5 min to dissolve, dilute to volume with the mobile phase, and mix, filter. Transfer 5.0 ml of the filtrate into a 50-ml volumetric flask, dilute to volume with the mobile phase, mix.

ROXITHROMYCIN

VP V

Reference solution: Prepare as the test solution using 25 mg of rotudine RS instead of the powdered tablets. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C. Detector: A spectrophotometer set at 280 nm Flow rate: 1 ml/min. Volume of injection: 20 µl Procedure: Inject the reference solution. In the chromatogram obtained, the number of theoretical plates, determined on the rotundine peak, is not less than 2500. The symmetry factor of the rotundine peak is not more than 2 and the relative standard deviation for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and test solution. Calculate the content of rotundine, C21H25NO4, in the tablets using the areas for the principal peaks in the chromatograms obtained with the test solution, the reference solution and the declared content of C21H25NO4 in rotundine RS.

Storage Store in a well-closed container, at a cool and dry place, protected from light. Action and use Sedative. Usual strength 30 mg; 60 mg. ROXITHROMYCIN Roxithromycinum

C41H76N2O15

M. 837.0

Roxithromycin is (3R,4S,5S,6R,7R,9R,11S,12R,13S,14R)4-[(2,6-dideoxy-3-C-methyl-3-O-methyl-α-L-ribohexopyranosyl)oxy]-14-ethyl-7,12,13-trihydroxy-10-[(E)-[(2methoxyethoxy) methoxy] imino] -3,5,7,9,11,13-hexomethyl6-[[3,4,6-trideoxy-3-(dimethylamino)-β-D-xylohexapyranosyl] oxy] oxacyclotetradecan-2-one. It contains not less than 96.0% and not more than the equivalent of 102.0% of C41H76N2O15, calculated with reference to the dried substance.

Characters White, polymorphism, crystalline powder. Very slightly soluble in water, freely soluble in acetone, in ethanol (96%) and in methylene chloride. It is slightly soluble in dilute hydrochloric acid. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of roxithromycin RS. If the spectra obtained shows differences, prepare further spectra using 9% solutions in methylene chloride R. B. Examine the chromatograms obtained in the Assay. The retention time of principal peak in the chromatogram obtained with the test solution (2) corresponds to that in the chromatogram obtained with reference solution (1). Appearance of solution Dissolve 0.2 g of the substance to be examined in methanol R and dilute to 20 ml with the same solvent, and mix. The solution is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). Specific optical rotation -93° to -96°, calculated with reference to the dried substance (Appendix 6.4). Dissolve 0.500 g of the substance to be examined in acetone R and dilute to 50.0 ml with the same solvent, and mix. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Acetonitrile - 5.97% solution of ammonium dihydrogen phosphate adjusted to pH 4.3 with dilute sodium hydroxide solution (26 : 74). Mobile phase B: Water - acetonitrile (30 : 70). Solvent mixture: Acetonitrile - 4.86% solution of ammonium dihydrogen phosphate adjusted to pH 5.3 with dilute sodium hydroxide solution (30 : 70). Test solution: Dissolve 50.0 mg, accurately weighed, of the substance to be examined in the solvent mixture and dilute to 25.0 ml with the same mixture, and mix. Reference solution (1): Dissolve 50.0 mg of roxithromycin RS in the solvent mixture and dilute to 25.0 ml with the same mixture, and mix. Reference solution (2): Dilute 1.0 ml of reference solution (1) to 100.0 ml with the solvent mixture, and mix. Reference solution (3): Dissolve 20.0 mg of roxithromycin for system suitability RS in the solvent mixture and dilute to 10.0 ml with the same mixture, and mix. Reference solution (4): Dilute 1.0 ml of toluene R to 100.0 ml with acetonitrile R, and mix. Dilute 0.2 ml of this solution to 200.0 ml with the solvent mixture, and mix. Chromatographic system: A column (15 cm × 4.6 mm) packed with spherical endcapped octadecylsilyl silica gel for chromatography R (5 µm) with a 10 nm pore size and a carbon loading of about 19 %. 855

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ROXITHROMYCIN POWDER FOR SUSPENSION

Column temperature: 15 °C. Detector: A spectrophotometer set at 205 nm. Flow rate: 1.1 ml/min. Volume of injection: 20 µl, using an injector maintained at 8 °C. Procedure: Carry out a linear gradient elution using the following conditions: Time (min) 0 - 50 50 - 51 51 - 80 80 - 81 81 - 100

Mobile phase A (% v/v) 100 100 → 90 90 90 → 100 100

Mobile phase B (% v/v) 0 0 → 10 10 10 → 0 0

Inject the test solution and the reference solution (2), (3), (4). The relative retention with reference to roxithromycin (retention time = about 22 minutes) of erythromycin 9-(E)-[O-[[(2-methoxyethoxy)methoxy] methyl]oxime] (impurity G) is about 1.15. System suitability: The peak-to-valley ratio (Hp/Hv) is not less than 2.0, where Hp is the height above the baseline of the peak due to impurity G and Hv is the height above the baseline of the lowest point of the curve separating this peak from the peak due to roxithromycin in the chromatogram obtained with reference solution (3). Limits: In the chromatogram obtained with the test solution: Impurity G: the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (1.0%). The area of any secondary peak is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.5%). The sum of the areas of all the secondary peaks is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (2) (3.0%). Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Disregard any peak due to toluene (use reference solution (4) to identify it).

Heavy metals Not more than 10 ppm (Appendix 9.4.8). Dissolve 2.0 g of the substance to be examined in a mixture of water and acetone R (15 : 85), dilute to 20 ml with the same mixture of solvents, and mix. 12 ml of the solution complies with the limit test for heavy metals, method 2. Prepare the reference solution using lead standard solution (1 ppm Pb) obtained by diluting lead standard solution (100 ppm Pb) R with a mixture of water and acetone R (15 : 85). Water Not more than 3.0% (Appendix 10.3). Determined on 0.200 g. 856

Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances, with the following modifications. Mobile phase: Acetonitrile - 4.91% solution of ammonium dihydrogen phosphate adjusted to pH 5.3 with dilute sodium hydroxide solution (307 : 693). Chromatographic system: Column size: 25 cm × 4.6 mm. Flow rate: 1.5 ml/min. Procedure: Inject the test solution and reference solution (1) and (3). The retention time of roxithromycin is about 12 min. System suitability: The peak-to-valley ratio (Hp/Hv) is not less than 1.5, where Hp is height above the baseline of the peak due to impurity G and Hv is the height above the baseline of the lowest point of the curve separating this peak from the peak due to roxithromycin in the chromatogram obtained with reference solution (3). Calculate the content of C41H76N2O15 in the substance to be examined using the areas for the principal peaks in the chromatograms obtained with the test solution and the reference solution (1), and the declared content of C41H76N2O15 in roxithromycin RS. Storage Store in an airtight container. Action and use Macrolide antibacterial. Preparations Tablets; powders. ROXITHROMYCIN POWDER FOR SUSPENSION Pulveres Roxithromycini ad suspensionum peroralum Roxithromycin powder is the powder for oral suspension containing roxithromycin. It may be contain flavors, colorants, preservatives, dispersing agents. The suspension after constitution complies with the requirements stated under “Suspensions” (Appendix 1.5). The powder complies with the requirements stated under “Powder” (Appendix 1.7) and with the following requirements.

Content of roxithromycin, C41H76N2O15, 90.0% to 110.0% of the stated amount. Characters A loose dry, homogeneously coloured powder.

ROXITHROMYCIN TABLETS

VP V

Identification In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of roxithromycin peak in the chromatogram obtained with the reference solution. Alkalinity Weigh accurately a quantity of the powder containing 15 mg of roxithromycin, add 10 ml of carbon dioxide-free water R, shake thoroughly, pH of the resulting suspension is 7.0 to 9.0 (Appendix 6.2). Loss on drying Not more than 2.0% (Appendix 9.6). Weigh accurately about 1.0 g of the powder, dry at 80 °C in a vacuum to constant weight. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of acetate buffer solution pH 5.5 (600 ml for 50 mg strength, 500 ml for 25mg strength). Acetate buffer solution pH 5.5: Dissolve 5.44 g of sodium acetate R in 1000 ml of water, adjust to pH 5.5 with glacial acetic acid R. Rotation speed: 50 rpm. Time: 30 min. Procedure: Examine by liquid chromatography (Appendix 5.3). Mobile phase and chromatographic system are described in the Assay with the volume of injection being 50 µl. Test solution: After the specified time, withdraw a sample of the medium and filter, discard the first portion of the filtrate. Reference solution: Weigh accurately a quantity of roxithromycin RS, dissolve in the medium to obtain a solution having a known concentration of about 0.08 mg/ml (and 0.05 mg/ml for the 25 mg strength) Tolerance: Not less than 80% (Q) of the stated amount of roxithromycin, C41H76N2O15, is dissolved in 30 min. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: 0.067 M ammonium dihydrogen phosphate adjusted to pH 6.5 with triethylamine - acetonitrile (65 : 35). Resolution solution: Dissolve a quantity of roxithromycin RS and erythromycin RS in the mobile phase to obtain a solution having a concentration of each substance of about 1.0 mg/ml. Reference solution: Dissolve a quantity of roxithromycin RS in the mobile phase to obtain a solution having a concentration of about 0.5 mg/ml. Test solution: Weigh 20 units, calculate the average mass of the content and finely powder. Weigh accurately a quantity of the powder containing about 50 mg of roxithromycin, transfer into a 100-ml volumetric flask, add 70 ml of the mobile phase and sonicate for 20 min. Dilute with the mobile phase to volume, mix and filter.

Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 210 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the resolution solution, reference solution. In the chromatograms obtained, the retention time of roxithromycin is about 14 min. The test is not valid unless the resolution factor between roxithromycin peak and erythromycin peak is not less than 15.0, the resolution factor between roxithromycin peak and the impurity peak (with the relative retention time of about 0.95) is not less than 1.0, the resolution factor between roxithromycin peak and the impurity peak (with the relative retention time of about 1.2) is not less than 2.0. The relative standard deviation for replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of roxithromycin, C41H76N2O15, in the tablets using the areas of the peaks in the chromatograms obtained with the test solution, the reference solution and the declared content of C41H76N2O15 in roxithromycin RS.

Storage Store in an airtight container in a dry and cool place, protected from light. Action and use Macrolide antibacterial. Usual strength 25 mg; 50 mg; 75 mg. ROXITHROMYCIN TABLETS Tabellae Roxithromycini Roxithromycin tablets are film-coated tablets, containing roxithromycin. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20), section Coated tablets and with the following requirements.

Content of roxithromycin, C41H76N2O15, 90.0% to 110.0% of the stated amount. Identification In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of the principal peak in the chromatogram obtained with the reference solution. Dissolution (Appendix 11.4) 857

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ROXITHROMYCIN TABLETS

Apparatus: Basket. Medium: 900 ml of acetate buffer solution pH 5.5 (600 ml for 50 mg strength). Acetate buffer solution pH 5.5: Dissolve 5.44 g of sodium acetate R in 1000 ml of water, adjust the pH to 5.5 with glacial acetic acid R. Rotation speed: 100 rpm. Time: 45 min. Procedure: Examine by liquid chromatography (Appendix 5.3). Mobile phase and chromatographic system are described in the test for Assay with the volume of injection being 50 µl. Test solution: After the specified time, withdraw a sample of the medium and filter, discard the first portion of the filtrate. Reference solution: Weigh accurately a quantity of roxithromycin RS, dissolve in the medium to obtain a solution having a known concentration of about 0.16 mg/ml (and 0.08 mg/ml for the 75 mg and 50 mg strength). Tolerance: Not less than 80% (Q) of the stated amount of roxithromycin, C41H76N2O15, is dissolved in 45 min.

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase, resolution solution, chromatographic system: Proceed as directed in the Assay. Test solution: Remove the coating from 10 tablets and powder the cores finely. Weigh accurately a quantity of the powdered tablets containing 100 mg of roxithromycin in a 50-ml volumetric flask, add 30 ml of the mobile phase and sonicate for 20 min. Dilute with the mobile phase to volume, mix and filter. Reference solution: Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Procedure: Inject separately the reference solution and the test solution. Adjust the sensitivity so that the height of the principal peak in the chromatogram obtained is at least 20% of the full scale of the recorder. Record the chromatogram of the test solution for four times the retention time of the principal peak. In the chromatogram obtained with the test solution, the area of any secondary peak is not greater than 1.5 times the area of the principal peak in the chromatogram obtained with the reference solution (1.5%), the sum of the areas of all secondary peaks is not greater than 4.5 times the area of the principal peak in the chromatogram obtained with the reference solution (4.5%). Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with the reference solution (0.1%) and any peak due to the excipients with the relative retention time not more than 0.3. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: 0.067 M ammonium dihydrogen phosphate adjusted to pH 6.5 with triethylamine - acetonitrile (65 : 35). 858

Resolution solution: Dissolve a quantity of roxithromycin RS and erythromycin RS in the mobile phase to obtain a solution having concentration of each substance of about 1.0 mg/ml. Reference solution: Dissolve a quantity of roxithromycin RS in the mobile phase to obtain a solution having concentration of about 1.0 mg/ml. Test solution: Weigh 20 tablets, calculate the average mass (with the coating removed if necessary) and finely powder. Weigh accurately a quantity of the powder containing about 50 mg of roxithromycin, transfer into a 50-ml volumetric flask, add 30 ml of the mobile phase and sonicate 20 min. Dilute with the mobile phase to volume, mix and filter. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 210 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl Procedure: System suitability: Inject the resolution solution, reference solution. In the chromatograms obtained, the retention time of roxithromycin is about 14 min. The test is not valid unless the resolution factor between roxithromycin peak and erythromycin peak is not less than 15.0, the resolution factor between roxithromycin peak and the impurity peak with the relative retention time of about 0.95 is not less than 1.0, the resolution factor between roxithromycin peak and the impurity peak with the relative retention time of about 1.2 is not less than 2.0. The relative standard deviation for replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of roxithromycin, C41H76N2O15, in the tablets using the areas of the peak in the chromatograms obtained with the test solution, the reference solution and the declared content of C41H76N2O15 in roxithromycin RS.

Storage Store in a well-closed container, in a cool and dry place, protected from light. Action and use Macrolide antibacterial. Usual strength 50 mg; 75 mg; 150 mg.

VP V

RUTIN Rutinum Rutoside, Vitamin P

C27H30O16,3H2O M.665.0 Rutin is 3,3’,4’,5,7-pentahydroxyflavone 3-rutinoside, a glucoside extracted from flower buds of Sophora japonica L. (Fam. Fabaceae), or from other plants. It contains not less than 95.0% and not more than the equivalent of 101.0% of C27H30O16, calculated with reference to the anhydrous substance.

Characters Yellow or greenish-yellow, crystalline powder. Soluble in methanol and in solutions of alkali hydroxides, sparingly soluble in ethanol, practically insoluble in water and in dichloromethane. Identification Apply one of the two following identifications: First identification: A. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of rutin RS. B. Dissolve 50.0 mg of the substance to be examined in methanol R, dilute to 250.0 ml with the same solvent, mix, and filter if necessary. Dilute 5.0 ml of the solution to 50.0 ml with methanol R, and mix. The ultraviolet absorption spectrum (Appendix 4.1) of this solution, in the range between 210 nm and 450 nm, exhibits 2 absorption maxima, at 257 nm and 358 nm. The value of A(1%; 1 cm) at the maximum at 358 nm is 305 to 330, calculated with reference to the anhydrous substance. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: n-Butanol - anhydrous acetic acid - water - methyl ethyl ketone - ethyl acetate (5 : 10 : 10 : 30 : 50). Test solution: Dissolve 25 mg of the substance to be examined in methanol R and dilute to 10.0 ml with the same solvent, and mix. Reference solution: Dissolve 25 mg of rutin RS in methanol R and dilute to 10.0 ml with the same solvent. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 10 cm. Remove the plate, dry it in air. Spray with a mixture of 7.5 ml of a 1% solution

RUTIN

of potassium ferricyanide R and 2.5 ml of a 10.5% solution of ferric chloride R and examine within 10 minutes. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to that in the chromatogram obtained with the reference solution. D. Dissolve 10 mg of the substance to be examined in 5 ml of ethanol (96%) R, add 1 g of zinc R and 2 ml of 25% solution of hydrochloric acid R. A red colour develops.

Light absorbing impurities Dissolve 0.200 g of the substance to be examined in 40 ml of 2-propanol R. Stir for 15 minutes, dilute to 50.0 ml with 2-propanol R and filter. The absorbances of the filtrate at wavelengths between 450 nm and 800 nm are not more than 0.10. Substances insoluble in methanol Not more than 3%. Shake 2.5 g of the substance to be examined for 15 min with 50 ml of methanol R at 20 - 25 °C. Filter under reduced pressure through a sintered-glass filter (1.6) previously dried for 15 min at 100 - 105 °C, allowed to cool in a desiccator and tared. Wash the filter 3 times with 20 ml of methanol R. Dry the filter for 30 min at 100 - 105 °C. Allow to cool in a desiccator and weigh. The residue weighs a maximum of 75 mg. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: A mixture of 5 volumes of tetrahydrofuran R and 95 volumes of a 1.56% solution of sodium dihydrogen phosphate R previously adjusted to pH 3.0 with phosphoric acid R. Mobile phase B: A mixture of 40 volumes of tetrahydrofuran R with 60 volumes of a 1.56% solution of sodium dihydrogen phosphate R previously adjusted to pH 3.0 with phosphoric acid R. Test solution: Dissolve 0.10 g of the substance to be examined in 20 ml of methanol R and dilute to 100.0 ml with mobile phase B, and mix. Reference solution (1): Dissolve 10.0 mg of rutin RS in 10.0 ml of methanol R. Reference solution (2): Dilute 1.0 ml of reference solution (1) to 50.0 ml with mobile phase B, and mix. Chromatographic system: A column (25 cm × 4.0 mm) packed with stationary phase B (5 µm). Column temperature: 30 °C. Detector: A spectrophotometer set at 280 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions:

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RUTIN TABLETS

Time (min)

Mobile phase A (% v/v)

Mobile phase A (% v/v)

0 - 10

50 → 0

50 → 100

10 - 15

0

100

15 - 16

0 → 50

100 → 50

16 - 20

50

50

Relative retention with reference to rutin (retention time = about 7 minutes): impurity B (kaempferol 3-rutinoside) = about 1.1; impurity A (isoquercitroside) = about 1.2; impurity C (quercetin) = about 2.5. System suitability: In the chromatogram obtained with the reference solution (1), the resolution between the peaks due to rutin and impurity B is at least 2.5. Inject the test solution and reference solution (2). Locate the impurities by comparison with the chromatogram provided with rutin RS. Limits: Correction factors: For the calculation of contents, multiply the peak areas of the following impurities by the corresponding correction factor: impurity A = 0.8; impurity C = 0.5. In the chromatogram obtained with the test solution: The corrected area of each peak corresponding to impurity A, B, C is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (2%). The sum of the areas of all the secondary peaks is not more than 2 times the area of the principal peak in the chromatogram obtained with reference solution (2) (4%). Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.1%).

Water 7.5% to 9.5% (Appendix 10.3). Determined on 0.100 g. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.200 g, accurately weighed, of the substance to be examined in 20 ml of dimethylformamide R. Titrate with 0.1 M tetrabutylammonium hydroxide VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 M tetrabutylammonium hydroxide VS is equivalent to 30.53 mg of C27H30O16. Storage Store in an airtight container, protected from light. Action and use Strengthening blood-vessels. Preparation Tablets. 860

RUTIN TABLETS Tabellae Rutini Rutin tablets contain rutin. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements:

Content of rutin, C27H30O16,3H2O, 90.0% to 110.0% of the stated amount. Identification A. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is the same as that of the rutin peak in the chromatogram obtained with the reference solution. B. To a quantity of the powdered tablets equivalent to about 0.05 g of rutin add 20 ml of hot ethanol 90% R. Shake well to dissolve rutin. Filter. To 2 ml of the filtrate add a few drops of hydrochloric acid R and a small quantity of zinc powder R. The solution becomes red. To a further 2 ml of the filtrate add a few drops of a 3% solution of iron (III) chloride R, a greenish-brown colour is produced. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol - phosphate buffer pH 3.0 tetrahydrofuran (10 : 70 : 20). Reference solution: Weigh accurately about 50 mg of rutin RS in a 50 ml volumetric flask, dissolve with methanol R and dilute to volume with the same solvent. Dilute 2.0 ml of the solution to 20.0 ml with mobile the phase, mix. Test solution: Weigh 20 tablets, calculate the average mass, finely powder. Weigh accurately a quantity of the powdered tablets containing about 50 mg of rutin in a 50 ml volumetric flask, add 35 ml of methanol R and sonicate for 15 min. Add methanol R to volume, mix and filter, discard the first portion of the filtrate. Dilute 2.0 ml of the filtrate to 20.0 ml with the mobile phase, mix. Chromatographic system: A column (25 cm × 4.0 mm) packed with stationary phase C (5 µm). Column temperature: 40 °C. Detector: A spectrophotometer set at 254 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution, the test is not valid unless the relative standard deviation of the peak area for rutin in 6 replicate injections is not more than 2.0%. Inject separately the reference solution and the test solution. Calculate the content rutin, C27H30O16,3H2O, using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C27H30O16,3H2O in rutin RS.

VP V

Storage Store in an airtight container, protected from light. Usual strength 20 mg, 50 mg, 100 mg. RUTIN AND ASCORBIC ACID TABLETS Tabellae Rutini et Acidi ascorbici Rutin C tablets Rutin and ascorbic acid tablets are sugar-coated tablets containing equal quantities of rutin and ascorbic acid. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20), section Coated tablets and with the following requirements.

Content of rutin, C27H30O16,3H2O, 90.0% to 110.0% of the stated amount. Content of ascorbic acid, C6H8O6, 90.0% to 120.0% of the stated amount. Identification To a quantity of the powdered tablets (with coating removed) containing about 0.2 g of ascorbic acid, add 20 ml of water, shake for 5 min, filter. Use the residue for rutin identification and the filtrate (A) for ascorbic acid identification. A. Wash the residue with three 10-ml quantities of water. Transfer the filter with the residue in a beaker, add 10 ml of hot ethanol R, stir thoroughly and filter (filtrate B). To 3 ml of the filtrate B add 5 drops of hydrochloric acid R and 10 mg of zinc powder R. The solution becomes red. B.To 5 ml of the filtrate B add 1 drop of a 3% solution of iron (III) chloride R, a greenish-brown colour is produced. C. To 5 ml of the filtrate A, add 0.5 ml of a 2% solution of silver nitrate R, a grey precipitate is formed. D. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Ethanol - water (120 : 20). Test solution: Dilute 5 ml of the filtrate A to 10 ml with water. Reference solution: A solution contains 0.5% of ascorbic acid RS in water. Procedure: Apply separately to the plate 2 µl of each solution. After developing over a path of 15 cm, remove the plate, allow it to dry in air and examine under ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and colour to the principal spot in the chromatogram obtained with the reference solution.

RUTIN AND ASCORBIC ACID TABLETS

For rutin Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol - phosphate buffer solution pH 3.0 - tetrahydrofuran (10 : 70 : 20). Reference solution: Weigh accurately 50 mg of rutin RS, transfer into a 50-ml volumetric flask, dissolve in methanol R to volume and mix. Dilute 2.0 ml of the resulting solution to 20.0 ml with the mobile phase, mix. Test solution: Weigh accurately a quantity of the powdered tablets containing about 50 mg of rutin, transfer into a 50-ml volumetric flask, add 35 ml of methanol R, sonicatefor 15 min, dilute to volume with methanol R, and mix, filter through a filter paper, discard the first portion of the filtrate. Dilute 2.0 ml of the resulting solution to 20.0 ml with the mobile phase, mix. Chromatographic system: A column (25 cm × 4.0 mm) packed with stationary phase C (5 µm). Column temperature: 40 °C. Detector: A spectrophotometer set at 254 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution. The test is not valid unless the relative standard deviation for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution, test solution. Calculate the content of rutin, C27H30O16,3H2O, in the tablets using the areas of the principal peaks in the chromatograms obtained with the test solution, the reference solution and the declared content of C27H30O16,3H2O in rutin RS. For ascorbic acid Weigh accurately a quantity of the powdered tablets containing about 100 mg of ascorbic acid, add 50 ml of a mixture of freshly boiled and cooled water R and 1 M acetic acid R (10 : 1), mix. Add 1 ml of starch solution R, and titrate with 0.1 N iodine VS until a blue colour persists for at least 30 seconds. 1 ml of 0.1 N iodine VS is equivalent to 8.806 mg of C6H8O6. Storage Store in an airtight container, in a dry and cool place protected from light and from contact with metal. Action and use Strengthen blood vessels. Usual strength 50 mg of rutin; 50 mg of ascorbic acid.

Assay Weigh and powder 20 tablets (with coating removed). Calculate the average mass and powder finely.

861

VP V

SALBUTAMOL

SALBUTAMOL Salbutamolum

C13H21NO3  

M. 239.3  

Salbutamol is (1RS)-2-[(1,1-dimethylethyl)amino]-1-[4hydroxy-3-(hydroxymethyl)phenyl]ethanol. It contains not less than 98.0% and not more than 101.0% of C13H21NO3, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder. Sparingly soluble in water, soluble in ethanol (96%). Melting point: about 155 °C, with decomposition. Identification Apply one of the two following identifications: First identification: A. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of salbutamol RS. B. Dissolve 80.0 mg in a 1% solution of hydrochloric acid R and dilute to 100.0 ml with the same solution. Dilute 10.0 ml of the solution to 100.0 ml with a 1% solution of hydrochloric acid R. The ultraviolet absorptiom spectrum (Appendix 4.1) of this solution in the range between 230 nm and 350 nm, exhibits an absorption maximum at 276 nm. A ( 1%, 1 cm) at the absorption maximum is 66 to 75. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Concentrated ammonia - water - ethyl acetate - 2-propanol - methyl isobutyl ketone (3 : 18 : 35 : 45 : 50). Test solution: Dissolve 10 mg of the substance to be examined in methanol R and dilute to 50 ml with the same solvent. Reference solution: Dissolve 10 mg of salbutamol RS in methanol R and dilute to 50 ml with the same solvent. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of three quarters of the plate. Allow the plate to dry in air and spray with a 0.1% solution of methylbenzothiazolone hydrazone hydrochloride R in methanol 90% R, followed by a 2% solution of potassium ferricyanide R in a mixture of concentrated ammonia - water (1 : 3), followed by a further spraying with a 0.1% solution of methylbenzothiazolone hydrazone hydrochloride R in methanol 90% R. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to 862

the principal spot in the chromatogram obtained with the reference solution. D. Dissolve about 10 mg of the substance to be examined in 50 ml of a 2% solution of sodium tetraborate R. Add 1 ml of a 3% solution of 4-aminophenazone R, 10 ml of methylene chloride R and 10 ml of a 2% solution of potassium ferricyanide R. Shake and allow to separate. An orange-red colour develops in the methylene chloride layer.

Appearance of solution Solution S: Dissolve 0.50 g in methanol R and dilute to 25.0 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution BY5 (Appendix 9.3, method 2). Optical rotation -0.10° to +0.10° (Appendix 6.4). Determined on solution S. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - buffer solution pH 3.65 (22 : 78). Buffer solution pH 3.65: A solution containing 2.87 g/L of sodium heptanesulfonate R and 2.5 g/L of potassium dihydrogen phosphate R previously ajusted to pH 3.65 with dilute phosphoric acid R. Test solution: Dissolve 0.100 g of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Reference solution (1): Dissolve 2.0 mg of salbutamol RS, 2 mg of salbutamol impurity B RS, 3.0 mg of salbutamol impurity D RS, 3.0 mg of salbutamol impurity F RS and 3.0 mg of salbutamol impurity G RS in the mobile phase and dilute to 10.0 ml with the mobile phase. Dilute 2.0 ml of this solution to 100.0 ml with the mobile phase. Reference solution (2): Dissolve the contents of a vial of salbutamol impurity I RS in 1.0 ml of the mobile phase. Reference solution (3): Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 20.0 ml with the mobile phase. Chromatographic system: A column (15 cm × 3.9 mm) packed with stationary phase end-capped octylsilyl silica gel for chromatography R (5 µm) with a specific surface area of 335 m2/g, a pore size of 10 nm and a carbon loading of 11.7%. Detector: A spectrophotometer set at 220 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Procedure: The run time is 25 times the retention time of salbutamol. Identification of impurities: Use the chromatogram obtained with reference solution (1) to identify the peaks due to impurities B, D, F and G; use the chromatogram

SALBUTAMOL

VP V

obtained with reference solution (2) to identify the peak due to impurity I. Relative retention with reference to salbutamol (retention time = about 2 min): impurity B = about 1.3; impurity A = about 1.7; impurity C = about 2.0; impurity D = about 2.7; impurity H = about 3.0; impurity E = about 3.1; impurity G = about 4.1; impurity F = about 6.2; impurity I = about 23.2. System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to salbutamol and impurity B is at least 3.0. Limits: Impurity D: The area of the peak due to impurity D is not more than the area of the corresponding peak in the chromatogram obtained with reference solution (1) (0.3%). Impurity F: The area of the peak due to impurity F is not more than the area of the corresponding peak in the chromatogram obtained with reference solution (1) (0.3%). Impurity G: The area of the peak due to impurity G is not more than the area of the corresponding peak in the chromatogram obtained with reference solution (1) (0.3%). Impurities A, B, C, E, H, I: For each impurity, the area is not more than 1.5 times the area of the peak due to salbutamol in the chromatogram obtained with reference solution (1) (0.3%). Any other impurity: For each impurity, the area is not more than 0.5 times the area of the peak due to salbutamol in the chromatogram obtained with reference solution (1) (0.10%). Total all impurities is not more than 1.0%. Disregard any peak with an area less than the area of the principal peak in the chromatogram obtained with reference solution (3) (0.05%). Note: Impurity A: 5-[(1RS)-2-[(1,1-dimethylethyl)amino]-1-methoxyethyl]2-hydroxyphenyl]methanol. Impurity B: (1RS)-2-[(1,1-dimethylethyl)amino]-1-(4-hydroxyphenyl) ethanol. Impurity C: (1RS)-2-[(1,1-dimethylethyl)amino]-1-(4-hydroxy3-methylphenyl)ethanol. Impurity D: 5-[(1RS)-2-[(1,1-dimethylethyl)amino]-1-hydroxyethyl]2-hydroxybenzaldehyde. Impurity E: (1RS)-2-[benzyl(1,1-dimethylethyl)amino]-1-[4hydroxy-3-(hydroxymethyl)phenyl]ethanol. Impurity F: 1,1’-[oxybis[methylene(4-hydroxy-1,3-phenylene)]] bis[2-[(1,1-dimethylethyl)amino]ethanol]. Impurity G: 2-[benzyl(1,1-dimethylethyl)amino]-1-[4-hydroxy3-(hydroxymethyl)phenyl]ethanone. Impurity H: 4-[2-[(1,1-dimethylethyl)amino]ethyl]-2-methylphenol. Impurity I: (1RS)-2-[(1,1-dimethylethyl)amino]-1-[4-(benzyloxy)3-(hydroxymethyl)phenyl]ethanol.

Impurity J Not more than 0.2% Dissolve 50.0 mg in a 0.1% solution of hydrochloric acid R and dilute to 25.0 ml with the same solvent. The absorbance of the solution measured at 310 nm is not greater than 0.10.

Note: Impurity J: 2-[(1,1-dimethylethyl)amino]-1-[4-hydroxy-3(hydroxymethyl)phenyl]ethanone (salbutamone).

Boron Not more than 50 ppm. Test solution: Add 5 ml of a solution containing 1.3% of anhydrous sodium carbonate R and 1.7% of potassium carbonate R to 50 mg of the substance to be examined. Evaporate to dryness on a water-bath and dry at 120 °C. Ignite the residue rapidly until the organic matter has been destroyed, allow to cool and add 0.5 ml of water and 3.0 ml of a freshly prepared 1.25 g/L solution of curcumin R in glacial acetic acid R. Warm gently to effect solution, allow to cool and add 3.0 ml of a mixture prepared by adding 5 ml of sulfuric acid R, slowly and with stirring, to 5 ml of glacial acetic acid R. Mix and allow to stand for 30 min. Dilute to 100.0 ml with ethanol (96%) R, filter and use the filtrate. Reference solution: Dissolve 0.572 g of boric acid R in 1000.0 ml of water. Dilute 1.0 ml of the solution to 100.0 ml with water. To 2.5 ml of this solution add 5 ml of a solution containing 1.3% of anhydrous sodium carbonate R and 1.7% of potassium carbonate R, and treat this mixture in the same manner as the test solution. Measure the absorbance (Appendix 4.1) of the test solution and of the reference solution at the absorption maximum at about 555 nm. The absorbance of the test solution is not greater than that of the reference solution. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.200 g of the substance to be examined in 30 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 23.93 mg of C13H21NO3. Storage In an airtight container, protected from light. Action and use Beta2-adrenoceptor agonist; bronchodilator. Preparations Inhalation, Nebuliser solution.

863

VP V

SALBUTAMOL SULFATE

SALBUTAMOL SULFATE Salbutamoli sulfas

(C13H21NO3)2H2SO4  

M. 576.7  

Salbutamol sulfate is bis[(1RS)-2-[(1,1-dimethylethyl) amino]-1-[4-hydroxy-3-(hydroxymethyl)phenyl]ethanol] sulfate. It contains not less than 98.0% and not more than 101.0% of (C13H21NO3)2,H2SO4, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder. Freely soluble in water, practically insoluble or very slightly soluble in ethanol (96%) and in methylene chloride. Identification Apply one of the two following identifications: First identification: B, E. Second identification: A, C, D, E. A. Dissolve 80.0 mg in a 1% solution of hydrochloric acid R and dilute to 100.0 ml with the same solvent. Dilute 10.0 ml of the solution to 100.0 ml with the same solvent. Examined between 230 nm and 350 nm (Appendix 4.1), the solution shows an absorption maximum at 276 nm. The specific absorbance at the absorption maximum is 55 to 64. B. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of salbutamol sulfate RS. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel. Mobile phase: Ammonia - water - ethyl acetate - 2-propanol - methyl isobutyl ketone (3 : 18 : 35 : 45 : 50). Test solution: Dissolve 12 mg of the substance to be examined in water and dilute to 10 ml with the same solvent. Reference solution: Dissolve 12 mg of salbutamol sulfate RS in water and dilute to 10 ml with the same solvent. Procedure: Apply separately to the plate 1 µl of each solution. Develop over 18 cm of the plate. Allow the plate to dry at room temperature. Spray with a 0.1% solution of methylbenzothiazolone hydrazone hydrochloride R in a 90% v/v solution of methanol R, followed by a 2% solution of potassium ferricyanide R in a mixture of 1 volume of 18 M ammonia R and 3 volumes of water, followed by a further spraying with a 0.1% solution of methylbenzothiazolone hydrazone hydrochloride R in a 90% v/v solution of methanol R. 864

The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. D. Dissolve about 10 mg in 50 ml of a 2% solution of disodium tetraborate R. Add 1 ml of a 3% solution of aminopyrazolone R, 10 ml of methylene chloride R and 10 ml of a 2% solution of potassium ferricyanide R. Shake and allow to separate. An orange-red colour develops in the methylene chloride layer. E. It gives reaction (A) of sulfates (Appendix 8.1).

Appearance of solution Solution S: Dissolve 0.250 g in carbon dioxide-free water R and dilute to 25.0 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution BY6 (Appendix 9.3, method 2). Optical rotation -0.10° to +0.10° (Appendix 6.4). Determined on solution S. Acidity or alkalinity To 10 ml of solution S add 0.15 ml of methyl red solution R and 0.2 ml of 0.01 N sodium hydroxide VS, the solution is yellow. Not more than 0.4 ml of 0.01 N hydrochloric acid VS is required to change the colour of the indicator to red. Impurity J Not more than 0.2%. Dissolve 60.0 mg in a 0.1% solution of hydrochloric acid R and dilute to 25.0 ml with the same solvent. The absorbance (Appendix 4.1) of the solution measured at 310 nm is not greater than 0.10. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Mix 22 volumes of acetonitrile R and 78 volumes of phosphate buffer solution pH 3.65 (a solution containing 0.287% of sodium heptanesulfonate R and 0.25% of potassium dihydrogen phosphate R previously adjusted to pH 3.65 with dilute phosphoric acid R). Test solution: Dissolve 0.100 g of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Reference solution (1): Dissolve 2.4 mg of salbutamol sulfate RS, 2.0 mg of salbutamol impurity B RS, 3.0 mg of salbutamol impurity D RS, 3.0 mg of salbutamol impurity F RS and 3.0 mg of salbutamol impurity G RS in the mobile phase and dilute to 10.0 ml with the mobile phase. Dilute 2.0 ml of the solution to 100.0 ml with the mobile phase. Reference solution (2): Dissolve the contents of one vial of salbutamol impurity I RS with 1 ml of the mobile phase. Chromatographic system: A column (15 cm × 3.9 mm) packed with spherical end-

SALBUTAMOL TABLETS

VP V

capped octylsilyl silica gel for chromatography R (5 µm) with a specific surface area of 335 m2/g, a pore size of 10 nm and a carbon loading of 11.7%. Detector: A spectrophotometer set at 220 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Run time: 25 times the retention time of salbutamol. System suitability: Inject reference solution (1), resolution between the peaks due to salbutamol and impurity B is minimum 3.0. Relative retention with reference to salbutamol (retention time = about 1.9 min): impurity B = about 1.3, impurity A = about 1.7, impurity C = about 2.0, impurity D = about 2.7, impurity H = about 3.0, impurity E = about 3.1, impurity F = about 6.2, impurity I = about 23.2. Identification of impurities: Use the chromatogram obtained with reference solution (2) to identify the peak due to impurity I. Limits: In the chromatogram obtained with the test solution: The areas of the peaks due to impurity D, impurity F, impurity G is not more than the areas of the corresponding peaks due to impurity D, impurity F, impurity G in the chromatogram obtained with reference solution (1) (0.3%). The area of one peak due to each impurity A, B, C, E, H, I is not more than 1.5 times the area of the peak due to salbutamol in the chromatogram obtained with reference solution (1) (0.3%). The sum of the areas of all the peaks is maximum 1.0%, disregard any peak with an area less than 0.25 times the area of the peak due to salbutamol in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A: [5-[(1RS)-2-[(1,1-dimethylethyl)amino]-1methoxyethyl]-2-hydroxyphenyl] methanol. Impurity B: (1RS)-2-[(1,1-dimethylethyl)amino]-1-(4-hydroxyphenyl) ethanol. Impurity C: (1RS)-2-[(1,1-dimethylethyl)amino]-1-(4-hydroxy3-methylphenyl)ethanol. Impurity D: 5-[(1RS)-2-[(1,1-dimethylethyl)amino]-1-hydroxyethyl]2-hydroxybenzaldehyde. Impurity E: (1RS)-2-[benzyl(1,1-dimethylethyl)amino]-1-[4hydroxy-3-(hydroxymethyl)phenyl] ethanol. Impurity F: 1,1’-[oxybis[methylene(4-hydroxy-1,3-phenylene)]] bis[2-[(1,1-dimethylethyl) amino]ethanol]. Impurity G: 2-[benzyl(1,1-dimethylethyl)amino]-1-[4-hydroxy3-(hydroxymethyl)phenyl] ethanone. Impurity H: 4-[2-[(1,1-dimethylethyl)amino]ethyl]-2-methylphenol. Impurity I: (1RS)-2-[(1,1-dimethylethyl)amino]-1-[3-(hydroxymethyl)4-benzyloxyphenyl] ethanol.   Impurity J: 2-[(1,1-dimethylethyl)amino]-1-[4-hydroxy-3(hydroxymethyl)phenyl]ethanone (salbutamone).

Boron Not more than 50 ppm. Test solution: To 50 mg of the substance to be examined add 5 ml of a solution containing 1.3% of anhydrous

sodium carbonate R and 1.7% of potassium carbonate R. Evaporate to dryness on a water-bath and dry at 120 °C. Ignite the residue rapidly until the organic matter has been destroyed, allow to cool and add 0.5 ml of water and 3.0 ml of a freshly prepared 0.125% solution of curcumin R in glacial acetic acid R. Warm gently to effect solution, allow to cool and add 3.0 ml of a mixture prepared by adding 5 ml of sulfuric acid R, slowly and with stirring, to 5 ml of glacial acetic acid R. Mix and allow to stand for 30 min. Dilute to 100.0 ml with ethanol (96%) R, filter and use the filtrate. Reference solution: Dissolve 0.572 g of boric acid R in 1000.0 ml of water. Dilute 1.0 ml to 100.0 ml with water. To 2.5 ml of the solution add 5 ml of a solution containing 1.3% of anhydrous sodium carbonate R and 1.7% of potassium carbonate R, and treat this mixture in the same manner as the test solution, start from “Evaporate to dryness on a water-bath and dry at 120 °C …”. Measure the absorbance (Appendix 4.1) of the test solution and the reference solution at the maximum at about 555 nm. The absorbance of the test solution is not greater than that of the reference solution.

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 100 °C - 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.400 g in 5 ml of anhydrous formic acid R and add 35 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 57.67 mg of (C13H21NO3)2,H2SO4. Storage Protected from light. Action and use Beta-adrenoceptor agonist. Preparations Tablets, injection, inhalation, Nebuliser solution. SALBUTAMOL TABLETS Tabellae Salbutamoli Salbutamol tablets contain salbutamol sulfate. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

865

VP V

SALBUTAMOL TABLETS

Content of salbutamol, C13H21NO3, 92.5% to 107.5% of the stated amount. Identification A. Shake a quantity of the powdered tablets containing the equivalent of 2.5 mg of salbutamol with 50 ml of a 2% solution of sodium tetraborate R, add 1 ml of a 3% solution of aminopyrazolone R, 10 ml of a 2% solution of potassium ferricyanide R and 10 ml of chloroform R, shake and allow to separate. An orange-red colour is produced in the chloroform layer. B. Examine by thin-layer chromatography (Appendix 5.4). Carry out the method described under Related substances applying separately to the plate 2 µl of each of the following solutions. Test solution: Shake a quantity of the powdered tablets containing the equivalent of 10 mg of salbutamol with 10 ml of methanol (80%) (v/v) and filter. Reference solution: Dissolve 12 mg of salbutamol sulfate RS in 10 ml of methanol (80%) (v/v). The principal spot in the chromatogram obtained with the test solution is similar in colour, size and Rf value to the principal spot in the chromatogram obtained with the reference solution. C. Shake a quantity of the powdered tablets containing the equivalent of 4 mg of salbutamol with 10 ml of water and filter. The filtrate yields the reactions characteristic of sulfates (Appendix 8.1). Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Ethyl acetate - propane-2-ol - water - 13.5 M ammonia (50 : 30 : 16 : 4). Test solution: Shake a quantity of the powdered tablets equivalent to 10 mg of salbutamol with 1 ml of water for 15 minutes. Centrifuge, decant to get the transparent liquid. Reference solution: Prepare a solution containing 0.0060% of salbutamol sulfate RS in water. Procedure: Apply separately to the plate 20 µl of each solution. After removal of the plate, allow it to dry at room temperature. Place the plate in an atmosphere saturated with diethylamine R for a few minutes and spray with diazotised sulfanilic acid solution R. Any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with the reference solution, discard any pink spot at the application point. Uniformity of content (Appendix 11.2) Examine by liquid chromatography (Appendix 5.3). Mobile phase, chromatographic system and procedure: Proceed as directed in the Assay. Test solution: Place one tablet in a 25 ml volumetric flask, add 20 ml of the mobile phase and shake until complete disintegration. Add the mobile phase to volume, mix and filter. 866

Reference solution: Prepare a solution of salbutamol sulfate RS in the mobile phase to obtain a concentration equivalent to that of the test solution.

Assay For tablets containing the equivalent of 2 mg or less of salbutamol Use the average of the 10 individual results obtained in the test for Uniformity of content. For tablets containing the equivalent of more than 2 mg of salbutamol Carry out the method for liquid chromatography (Appendix 5.3). Mobile phase: Sodium dihydrogen phosphate solution pH 3.1 - methanol (85 : 15). Sodium dihydrogen phosphat solution pH 3.1: Dissolve 11.04 g of sodium dihydrogen phosphate R in 1000 ml of water, adjust the pH to 3.1 with phosphoric acid R. Reference solution: Prepare a solution of salbutamol sulfate RS in the mobile phase to obtain a concentration of 96 µg/ml. Test solution: Weigh accurately a quantity of the powdered tablets containing 4 mg of salbutamol sulfate in a 50 ml volumetric flask, add 40 ml of the mobile phase, shake to dissolve and add the mobile phase to volume. Mix and filter. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 276 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution, the number of theoretical plates, determined on the salbutamol sulfate peak, is more than 3000. Inject separately the reference solution and the test solution. Calculate the content of salbutamol, C13H21NO3, in each tablet using the areas of the principal peaks in the chromatograms obtained with the test solution and the reference solution and the declared content of C13H21NO3 in salbutamol sulfate RS. The factor to convert salbutamol sulfate into salbutamol base is 0.83. Storage Store in a well closed container, protected from light. Action and use Beta-adrenoceptor agonist. Usual strength 2 mg, 4 mg, 8 mg.

VP V

SALICYLIC ACID

SALICYLIC ACID Acidum salicylicum

Reference solution (6): Dilute a mixture of 0.1 ml of each of reference solutions (1), (2) and (3) to 10.0 ml with the mobile phase. CO2H Chromatographic system: A column (15 cm × 4.6 mm) packed with octadecylsilyl silica gel for chromatography R (5 µm). OH Detector: A spectrophotometer set at 270 nm. C7H6O3 M. 138.1   Flow rate: 0.5 ml/min. Salicylic acid is 2-hydroxybenzenecarboxylic acid. It Volume of injection: 10 µl. contains not less than 99.0% and not more than 100.5% of Procedure: Inject reference solutions (4) and (5). When the C7H6O3, calculated with reference to the dried substance. chromatograms are recorded in the prescribed conditions, Characters the retention times relative to phenol are: 4-hydroxybenzoic A white, crystalline powder or white or colourless, acicular acid about 0.70 and 4-hydroxyisophthalic acid about 0.90. crystals. Adjust the sensitivity of the system so that the height of the Slightly soluble in water, freely soluble in ethanol (96%) principal peak in the chromatogram obtained with reference and in ether, sparingly soluble in chloroform. Its solution solution (6) is at least 70% of the full scale of the recorder. is acidic. The test is not valid unless: in the chromatogram obtained with reference solution (5), the third peak corresponds Identification to the phenol peak in the chromatogram obtained with Apply one of the two following identifications: reference solution (4) and the resolution between the peaks First identification: A, B. corresponding to 4-hydroxyisophthalic acid and to phenol Second identification: B, C. A. The infrared absorption spectrum (Appendix 4.2) of the is at least 1.0. If this resolution is not obtained adjust the substance to be examined is concordant with the spectrum quantity of acetic acid in the mobile phase. Limits: of salicylic acid RS. B. Melting point: 158 °C to 161 °C (Appendix 6.7). The areas of the peaks due to 4-hydroxybenzoic acid, C. Dissolve about 30 mg in 5 ml of 0.05 M sodium 4-hydroxyisophthalic acid and phenol are not greater than the hydroxide R, neutralise if necessary and dilute to areas of the corresponding peaks in the chromatogram obtained 20 ml with water. 1 ml of the solution gives reaction A of with reference solution (6) (0.1% for 4-hydroxybenzoic acid; salicylates (Appendix 8.1). 0.05% for 4-hydroxyisophthalic acid and 0.02% for phenol). The area of any peak, apart from the principal peak and the Appearance of solution peaks due to 4-hydroxybenzoic acid, 4-hydroxyisophthalic Dissolve 1.0 g in 10.0 ml of ethanol (96%) R. The solution is clear (Appendix 9.2) and colourless (Appendix 9.3, acid and phenol, is not greater than that of the peak due to 4-hydroxyisophthalic acid in the chromatogram obtained method 2). with reference solution (6) (0.05%). The sum of the areas of all the peaks, apart from the Related substances principal peak, is not greater than twice the area of the Examine by liquid chromatography (Appendix 5.3). Mobile phase: Glacial acetic acid - methanol - water (1 : peak due to 4-hydroxybenzoic acid in the chromatogram 40 : 60). obtained with reference solution (6) (0.2%). Test solution: Dissolve 0.50 g of the substance to be Disregard any peak with an area less than 0.01 times that examined in the mobile phase and dilute to 100.0 ml with of the principal peak in the chromatogram obtained with the same solvent. reference solution (6). Reference solution (1): Dissolve 10 mg of phenol RS in the mobile phase and dilute to 100.0 ml with the same solvent. Chlorides Reference solution (2): Dissolve 5 mg of salicylic acid Not more than 100 ppm (Appendix 9.4.5). impurity B (4-hydroxyisophthalic acid) RS in the mobile Solution S: Dissolve 2.5 g in 50 ml of boiling water, cool and filter. phase and dilute to 20.0 ml with the same solvent. Reference solution (3): Dissolve 50 mg of 4-hydroxybenzoic 10 ml of solution S diluted to 15 ml with water complies acid R in the mobile phase and dilute to 100.0 ml with the with the limit test for chlorides. same solvent. Reference solution (4): Dilute 1.0 ml of reference solution Sulfates Not more than 0.020% (Appendix 9.4.14). (1) to 10.0 ml with the mobile phase. Reference solution (5): Dilute a mixture of 1.0 ml of each Dissolve 1.0 g in 5 ml of dimethylformamide R and add 4 ml of reference solutions (1), (2) and (3) to 10.0 ml with the of water. Mix thoroughly. Add 0.2 ml of dilute hydrochloric acid R and 0.5 ml of a 25% solution of barium chloride R. mobile phase. 867

VP V

SILVER NITRATE

After 15 min any opalescence in the solution is not more intense than that in a standard prepared as follows: to 2 ml of sulfate standard solution (100 ppm SO4) R add 0.2 ml of dilute hydrochloric acid R, 0.5 ml of a 25% m/m solution of barium chloride R, 3 ml of water and 5 ml of dimethylformamide R, mix.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). Dissolve 2.0 g in 15 ml of ethanol (96%) R and add 5 ml of water. 12 ml of the solution complies with limit test 2 for heavy metals. Prepare the reference solution using lead standard solution (2 ppm Pb) prepared by diluting lead standard solution (100 ppm Pb) R with a mixture of 1 volume of water and 3 volumes of ethanol (96%) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; dry in a desiccator in vacuum using silica gel as desiccant). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 2.0 g. Assay Dissolve 0.120 g in 30 ml of ethanol (96%) R and add 20 ml of water. Titrate with 0.1 N sodium hydroxide VS, using 0.1 ml of phenol red solution R as indicator. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 13.81 mg of C7H6O3. Storage Store in an airtight container, protected from light. Action and use Keratolytic, antiseborrhea, antipsoriatic, caustic.

Acidity or alkalinity To 2 ml of solution S add 0.1 ml of bromocresol green solution R. The solution is blue. To 2 ml of solution S add 0.1 ml of phenol red solution R. The solution is yellow. Foreign salts Not more than 0.3%. To 30 ml of solution S, add 7.5 ml of dilute hydrochloric acid R, shake vigorously, heat for 5 min on a water-bath and filter. Evaporate 20 ml of the filtrate to dryness on a water-bath and dry at 100  °C - 105 °C to constant mass. The residue weighs not more than 2 mg.

Aluminium, lead, copper and bismuth Dissolve 1.0 g in a mixture of 4 ml of concentrated ammonia R and 6 ml of water. The solution is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). Assay Dissolve 0.300 g in 50 ml of water and add 2 ml of 2 M nitric acid R and 1 ml of ferric ammonium sulphate solution R. Titrate with 0.1 N ammonium sulfocyanide VS until a reddish-yellow colour is obtained. 1 ml of 0.1 N ammonium sulfocyanide VS is equivalent to 16.99 mg of AgNO3.

Action and use Antiseptic. M. 169.9

Silver nitrate contains not less than 99.0% and not more than the equivalent of 100.5% of AgNO3.

Characters A white, crystalline powder or transparent, colourless crystals, very soluble in water, soluble in ethanol (96%). Identification A. Dissolve about 10 mg in 5 ml of water, add 3 drops of a 10% solution of hydrochloric acid R. A curdy, white precipitate is produced which is insoluble in a 16% solution of nitric acid R but soluble in dilute ammonia R. B. Dissolve about 10 mg in 2 ml of water, add 2 ml of sulphuric acid R, shake well and allow to cool. Carefully 868

Appearance of solution Solution S: Dissolve 2.0 g in water and dilute to 50 ml with the same solvent. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2).

Storage Store in a non-metallic container, protected from light.

SILVER NITRATE Argenti nitras AgNO3

add along the wall of the tube 1 ml of a 8% solution of ferrous sulphate R, a brown ring is produced at the interface of the two liquids.

Preparation Sterile silver nitrate solution. SILVER VITELLINATE Argentum vitellinicum Argyrol Silver vitellinate is a compound of silver and vitelline (phosphoprotein). It contains not less than 20.0% of Ag.

Characters Dark brown or lustrous dark-green flakes or plates, easily decomposing in air, hygroscopic. Soluble in water and in glycerol, soluble slowly and completely in ethanol (70%), insoluble in ethanol (96%) and in ether.

SIMVASTATIN

VP V

Identification A. Ignite about 0.50 g, dissolve the residue in 5 ml of a 16% solution of nitric acid R, add 1 ml of a 10% solution of hydrochloric acid R. A curdy, white precipitate is produced which is soluble in ammonia R. B. When burned, it will be carbonized and giving an odour of burning horn or burning fur. C. The solution of silver vitellinate in a 0.9% sodium chloride solution is stable (different from protargol). Appearance of solution Dissolve 0.2 g in 10 ml of water, the solution is clear (Appendix 9.2) and brownish red in colour. When viewed through, the solution exhibits a slight dichromaticity. When viewed in reflected light, the solution exhibits a green colour. Allow to stand for 2 hours, the solution is free from residue. Alkalinity Not more than 3.2%, expressed as sodium hydroxide. Weigh accurately about 1.000 g in a porcelain crucible, ignite. Allow to cool, extract the residue with successive 10-ml quantities of boiling water until the extract shows no pink colour with phenolphthalein solution R. Combine the extracts, add 2 - 3 drops of phenolphthalein solution R, titrate with 0.1 N sulfuric acid VS until the pink colour disappears. 1 ml of 0.1 N sulfuric acid VS is equivalent to 4.0 mg of NaOH. Assay Weigh 0.200 g of the substance to be examined, dissolve in 10 ml of water in a 200 ml or 250 ml conical flask. Slowly add 10 ml of 1 N sulfuric acid R. Allow to cool, add little by little 2 g of powdered potassium permanganate R, stirring continuously. Heat to gentle boiling the mixture for 5 minutes. Add dropwise, the ferrous sulfate solution R until a light yellow colour is obtained. Add 50 ml of water, 5 ml of a 25% solution of nitric acid R. Allow to cool, add 1 ml of ferric ammonium sulfate solution R. Titrate with 0.1 N ammonium sulfocyanide VS until a red colour is produced. 1 ml of 0.1 N ammonium sulfocyanide VS is equivalent to 10.79 mg of Ag. Storage Store in a dry, airtight, coloured glass container, protected from light and moisture. Incompatibility Alkaloids, adrenaline, tannin.

SIMVASTATIN Simvastatinum

C25H38O5  

M: 418.6

Simvastatin is (1S,3R,7S,8S,8aR)-8-[2-[(2R,4R)-4-hydroxy-6oxotetrahydro-2H-pyran-2-yl]ethyl]-3,7-dimethyl-1,2,3,7,8,8ahexahydronaphthalen-1-yl 2,2-dimethylbutanoate. It contains not less than 97.0% and not more than 102.0% of C25H38O5, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder. Practically insoluble in water, very soluble in methylene chloride, freely soluble in ethanol (96%). Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of simvastatin RS. B. It complies with the test for Specific optical rotation. Appearance of solution Solution S: Dissolve 0.20 g in methanol R and dilute to 20.0 ml with the same solvent. The solution is clear (Appendix 9.2) and not more intensely coloured than reference solution BY7 (Appendix 9.3, method 2). Specific optical rotation +285° to +300°, calculated with reference to the dried substance (Appendix 6.4). Dissolve 0.125 g in acetonitrile R and dilute to 25.0 ml with the same solvent. Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use. Solvent mixture: A 0.14% solution of potassium dihydrogen phosphate R adjusted to pH 4.0 with phosphoric acid R acetonitrile R (40 : 60). Filter. Mobile phase A: Acetonitrile R - a 0.1% v/v solution of phosphoric acid R (50 : 50). Mobile phase B: A 0.1% v/v solution of phosphoric acid R in acetonitrile R. 869

VP V

SIMVASTATIN

Test solution: Dissolve 75.0 mg of the substance to be examined in the solvent mixture and dilute to 50.0 ml with the solvent mixture. Reference solution (1): Dissolve 1.0 mg of simvastatin RS and 1.0 mg of lovastatin RS (impurity E) in the solvent mixture and dilute to 50.0 ml with the solvent mixture. Reference solution (2): Dilute 0.5 ml of the test solution to 100.0 ml with the solvent mixture. Reference solution (3): Dissolve 75.0 mg of simvastatin RS in the solvent mixture and dilute to 50.0 ml with the solvent mixture. Reference solution (4): Dissolve 5 mg of simvastatin for peak identification RS (containing impurities A, B, C, D, E, F and G) in 5.0 ml of the solvent mixture. Chromatographic system: A column (33 mm × 4.6 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (3 µm). Detector: A spectrophotometer set at 238 nm. Flow rate: 3.0 ml/min. Volume of injection: 5 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 4.5

100

0

4.5 - 4.6

100 → 95

0→5

4.6 - 8.0

95 → 25

5 → 75

8.0 - 11.5

25

75

Identification of impurities: Use the chromatogram supplied with simvastatin for peak identification RS and the chromatogram obtained with reference solution (4) to identify the peaks due to impurities A, B, C, D, E + F and G. Relative retention with reference to simvastatin (retention time = about 2.6 min): impurity A = about 0.5; impurities E + F = about 0.6; impurity G = about 0.8; impurities B and C = about 2.4; impurity D = about 3.8. System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to impurity E and simvastatin is at least 4.0. Limits: Sum of peak areas of impurities E and F is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (2) (1.0%). Sum of peak areas of impurities B and C is not more than 1.6 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.8%). Impurities A, D, G: For each impurity, the area is not more than 0.8 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.4%). Any other impurity: For each impurity, the area is not more than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.10%). 870

The sum of the peak areas of all impurities, other than E and F, is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (2) (1.0%). Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Note: ImpurityA: (3R,5R)-7-[(1S,2S,6R,8S,8aR)-8-[(2,2-dimethylbutanoyl) oxy]-2,6-dimethyl-1,2,6,7,8,8a-hexahydronaphthalen1-yl]-3,5dihydroxyheptanoic acid (tenivastatin). ImpurityB:(1S,3R,7S,8S,8aR)-8-[2-[(2R,4R)-4-(acetyloxy)-6-oxotetrahydro2H-pyran-2-yl]ethyl]-3,7-dimethyl-1,2,3,7,8,8ahexahydronaphthalen1-yl 2,2- dimethylbutanoate. Impurity C: (1S,3R,7S,8S,8aR)-3,7-dimethyl-8-[2-[(2R)-6-oxo-3,6dihydro-2H-pyran-2- yl]ethyl]-1,2,3,7,8,8a-hexahydronaphthalen1-yl 2,2-dimethylbutanoate. ImpurityD:(2R,4R)-2-[2-[(1S,2S,6R,8S,8aR)-8-[(2,2-dimethylbutanoyl) oxy]-2,6-dimethyl-1,2,6,7,8,8a-hexahydronaphthalen-1-yl]ethyl]6-oxotetrahydro-2H-pyran-4-yl(3R,5R)-7-[(1S,2S,6R,8S,8aR)8-[(2,2-dimethylbutanoyl)oxy]-2,6-dimethyl-1,2,6,7,8, 8a-hexahydronaphthalen1-yl]-3,5-dihydroxyheptanoate. Impurity E: Lovastatin. Impurity F: (1S,3R,7S,8S,8aR)-8-[2-[(2R,4R)-4-hydroxy-6oxotetrahydro-2H-pyran-2-yl]ethyl]-3,7-dimethyl-1,2,3,7,8,8ahexahydronaphthalen-1-yl (2R)-2-methylbutanoate (epilovastatin). Impurity G: (1S,3R,7S,8S,8aR)-8-[2-[(2R,4R)-4-hydroxy6-oxotetrahydro-2H-pyran-2-yl]ethyl]-3,7-dimethyl-1,2,3,7,8,8ahexahydronaphthalen-1-yl 2,2-dimethylbut-3-enoate.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; in vacuo; 60 °C; 3 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. Inject the test solution and reference solution (3). Calculate the percentage content of simvastatin, C25H38O5, using the areas of the simvastatin peaks in the chromatograms obtained with the test solution, reference solution (3) and the declared content of C25H38O5 in simvastatin RS. Storage Protected from light. If no antioxidant is present, store under nitrogen, in an airtight container.

VP V

Action and use HMG Co-A reductase inhibitor; lipid-regulating drug. Preparation Tablets. SIMVASTATIN TABLETS Tabellae Simvastatini Simvastatin tablets contain simvastatin. They may be coated. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of simvastatin, C25H38O5, 90.0% to 110.0% of the stated amount. Identification In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the peak due to simvastatin in the chromatogram obtained with the reference solution. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of buffer solution pH 7.0. Buffer solution pH 7.0: Dissolve 30 g of sodium laurylsufate R and 9.36 g of sodium dihydrogen phosphate R in 6000 ml of water, adjust to pH 7.0 with 10 M sodium hydroxide R. Rotation speed: 50 rpm. Time: 30 min. Procedure: Test solution: After the specified time, withdraw a sample of the medium, filter. Reference solution: Dissolve an accurately weighed quantity of simvastatin RS in the medium to obtain a solution having the same concentration as in the test solution. Examine by liquid chromatography (Appendix 5.3). Mobile phase and chromatographic system: Prepare as directed in the Assay. Volume of injection: 20 μl. Inject alternately the reference solution and the test solution. Calculate the content of simvastatin, C25H38O5, dissolved using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C25H38O5 in simvastatin RS. Tolerance: Not less than 75% (Q) of the stated amount of simvastatin, C25H38O5, is dissolved in 30 minutes. Uniformity of content (Appendix 11.2) Examine by liquid chromatography (Appendix 5.3). Solvent mixture, mobile phase, reference solution, chromatographic system: Prepare as directed in the Assay. Test solution: Transfer 1 tablet to a volumetric flask with nominal volume of 50 ml or 100 ml or 200 ml…

SIMVASTATIN TABLETS

(corresponding to 5 mg, 10 mg, 20 mg strengths… respectively), add the solvent mixture to dissolve and dilute to volume with the same solvent, mix and filter. Use the filtrate or dilute the filtrate with the solvent mixture to obtain a solution contaning 0.1 mg of simvastatin per ml, if necessary.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - buffer solution (65 : 35). Make adjustments if necessary. Buffer solution: Dissolve 4.4 g of sodium dihydrogen phosphate R in 900 ml of water, adjust to pH 4.5 with phosphoric acid R or 10 M solium hydroxide R, dilute to 1000 ml with water and mix. Solvent mixture: Transfer 3.0 ml of acetic acid glacial R to a 1000 ml baker containing 900 ml of water, adjust to pH 4.0 with 5 M sodium hydroxide R, mix. Transfer this solution to a 1000 ml volumetric flask and dilute to volume with water. Mix 20 volumes of this solution and 80 volumes of acetonitrile R. Reference solution: Dissolve an accurately weighed quantity of simvastatin RS in the solvent mixture to obtain a solution having a known concentration of 0.1 mg per ml. Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Transfer an accurately weighed quantity of the powdered tablets containing the equivalent of 20 mg of simvastatin to a 200 ml volumetric flask, add 120 ml of the solvent mixture and sonicate for 15 minutes. Cool to room temparature and dilute to volume with the solvent mixture, mix and filter. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 μm). Column temperature: 45 °C. Detector: A spectrophotometer set at 238 nm. Flow rate: 1.5 ml/min. Volume of injection: 10 μl. Procedure: Inject the reference solution. The test is not valid unless, the capacity factor is not less than 3.0; the column efficiency is not less than 4500 theoretical plates, the tailing factor for simvastatin peak is not more than 2.0 and the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of simvastatin, C25H38O5, in the tablets using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C25H38O5 in simvastatin RS. Storage Store in an airtight container, protected from light, in a cool and dry place or at room temperature.

871

VP V

SODIUM BENZOATE

Action and use Lipid-regulating drug. Usual strength 5 mg, 10 mg, 20 mg, 40 mg. SODIUM BENZOATE Natrii benzoas CO2Na

C7H5NaO2

M. 144.1

Sodium benzoate is sodium benzenecarboxylate. It contains not less than 99.0% and not more than 100.5% of C7H5NaO2, calculated with reference to the dried substance.

Characters A white, crystalline or granular powder or flakes, slightly hygroscopic. Freely soluble in water, sparingly soluble in ethanol (90%). Identification A. It gives reactions (B) and (C) of benzoates (Appendix 8.1). B. It gives reaction (A) of sodium (Appendix 8.1). Appearance solution Solution S: Dissolve 10.0 g in carbon dioxide-free water R and dilute to 100 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution Y6 (Appendix 9.3, method 2). Acidity or alkalinity To 10 ml of solution S add 10 ml of carbon dioxide-free water R and 0.2 ml of phenolphthalein solution R. Not more than 0.2 ml of 0.1 N sodium hydroxide VS or 0.1 N hydrochloric acid VS is required to change the colour of the indicator. Halogenated compounds All glassware used must be chloride-free and may be prepared by soaking overnight in a 35% solution of nitric acid R, rinsed with water and stored full of water. It is recommended that glassware be reserved exclusively for this test. Test solution: To 20.0 ml of solution S add 5 ml of water and dilute to 50.0 ml with ethanol (96%) R. Determination of ionised chlorine Not more than 0.02%. In three 25 ml volumetric flasks, prepare the following solutions. Solution (1): To 4.0 ml of the test solution add 3 ml of dilute sodium hydroxide solution R and 3 ml of ethanol (96%) R. This solution is used to prepare solution A. 872

Solution (2): To 3 ml of dilute sodium hydroxide solution R add 2 ml of water and 5 ml of ethanol (96%) R. This solution is used to prepare solution B. Solution (3): To 4.0 ml of chloride standard solution (8 ppm Cl) R add 6.0 ml of water. This solution is used to prepare solution C. In a fourth 25 ml volumetric flask, place 10.0 ml of water. To each flask add 5 ml of ferric ammonium sulfate solution R, mix and add dropwise and with swirling 2 ml of nitric acid R and 5 ml of mercuric thiocyanate solution R. Shake. Dilute the contents of each flask to 25.0 ml with water and allow the solutions to stand in a water-bath at 20 °C for 15 min. Measure at 460 nm in a 2 cm cell the absorbance (Appendix 4.1) of solution A using solution B as the compensation liquid, and the absorbance of solution C using the solution obtained with 10 ml of water as the compensation liquid. The absorbance of solution A is not greater than that of solution C.

Determination of total chlorine Not more than 0.03%. Solution (1): To 10.0 ml of the test solution add 7.5 ml of dilute sodium hydroxide solution R and 0.125 g of nickel-aluminium alloy R and heat on a water-bath for 10 min. Allow to cool to room temperature, filter into a 25 ml volumetric flask and wash the filter with 3 quantities, each of 2 ml, of ethanol (96%) R (a slight precipitate may form that disappears on acidification). Dilute the filtrate and washings to 25.0 ml with water. This solution is used to prepare solution A. Solution (2): In the same manner, prepare a similar solution replacing the solution (1) by a mixture of 5 ml of ethanol (96%) R and 5 ml of water. This solution is used to prepare solution B. Solution (3): To 6.0 ml of chloride standard solution (8 ppm Cl) R add 4.0 ml of water. This solution is used to prepare solution C. In four 25 ml volumetric flasks, place separately 10 ml of solution (1), 10 ml of solution (2), 10 ml of solution (3) and 10 ml of water. To each flask add 5 ml of ferric ammonium sulfate solution R, mix and add dropwise and with swirling 2 ml of nitric acid R and 5 ml of mercuric (II) thiocyanate solution R. Shake. Dilute the contents of each flask to 25.0 ml with water and allow the solutions to stand in a water-bath at 20 °C for 15 min. Measure at 460 nm in a 2 cm cell the absorbance (Appendix 4.1) of solution A using solution B as the compensation liquid, and the absorbance of solution C using the solution obtained with 10 ml of water as the compensation liquid. The absorbance of solution A is not greater than that of solution C. Heavy metals Not more than 10 ppm (Appendix 9.4.8). 2.0 g complies with limit test 3 for heavy metals. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R.

SODIUM BROMIDE

VP V

Loss on drying Not more than 2.0% (Appendix 9.6). (1.000 g; 100 °C - 105 °C).

Acidity or alkalinity Add 0.1 ml of bromothymol blue solution R to 10 ml of solution S. Not more than 0.5 ml of 0.01 N hydrochloric acid VS or 0.01 N sodium hydroxide VS is required to change the colour of the indicator.

Mobile phase: Dissolve 0.600 g of potassium hydroxide R in water for chromatography R and dilute to 1000.0 ml with the same solvent. Test solution (1): Dissolve 0.400 g of the substance to be examined in 50 ml of water for chromatography R and dilute to 100.0 ml with the same solvent. Test solution (2): Dilute 25.0 ml of test solution (1) to 50.0 ml with water for chromatography R. Reference solution (1): Add 1.0 ml of sulfate standard solution (10 ppm SO4) R and 12.0 ml of chloride standard solution (50 ppm Cl) R to 25.0 ml of test solution (1) and dilute to 50.0 ml with water for chromatography R. Reference solution (2): Dilute 10.0 ml of test solution (1) to 100.0 ml with water for chromatography R. To 2.0 ml of this solution add 8.0 ml of chloride standard solution (50 ppm Cl) R and dilute to 20.0 ml with water for chromatography R. Blank solution: water for chromatography R. Chromatographic system: A column (25 cm × 2 mm) packed with stationary phase strongly basic anion-exchange resin for chromatography R (13 µm). Detector: Conductivity detector equipped with a suitable ion suppressor. Flow rate: 0.4 ml/min. Volume of injection: 50 µl. Procedure: Inject the test solution (2), reference solutions (1) and (2) and the blank solution. The run time is 2.5 times the retention time of bromide. Retention time: Chloride = about 5 min; bromide = about 8 min; sulfate = about 16 min. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to chloride and bromide is at least 8.0. Limits: Correct the areas of the peaks obtained with test solution (2) and reference solution (1) using the areas of the peaks obtained with the blank solution: Chlorides: the area of the peak due to chloride in test solution (2) is not more than the difference between the areas of the peaks due to chloride in the chromatograms obtained with test solution (2) and reference solution (1) (0.6%). Sulfates: the area of the peak due to sulfate in test solution (2) is not more than the difference between the areas of the peaks due to sulfate in the chromatograms obtained with test solution (2) and reference solution (1) (100 ppm).

Bromates Add 1 ml of starch solution R to 10 ml of solution S, 0.1 ml of a 10% solution of potassium iodide R and 0.25 ml of 0.5 M sulfuric acid R and allow to stand protected from light for 5 min. No blue or violet colour develops.

Iodides Add 0.15 ml of 10.5% solution of ferric chloride R and 2 ml of methylene chloride R to 5 ml of solution S. Shake and allow to separate. The lower layer is colourless (Appendix 9.3, method 1).

Chlorides and sulfates Examine by liquid chromatography (Appendix 5.3).

Iron Not more than 20 ppm (Appendix 9.4.13). Dilute 5 ml of solution S to 10 ml with water.

Assay Dissolve 0.250 g in 20 ml of anhydrous acetic acid R, heating to 50 °C if necessary. Cool. Using 0.05 ml of naphtholbenzein solution R as indicator, titrate with 0.1 N perchloric acid VS until a green colour is obtained. 1 ml of 0.1 N perchloric acid VS is equivalent to 14.41 mg of C7H5NaO2. Storage Store in an airtight container. Action and use Preservative. SODIUM BROMIDE Natrii bromidum NaBr  

M. 102.9

Sodium bromide contains not less than 98.5% and not more than 101.0% of NaBr, calculated with reference to the dreid substance.

Characters White or almost white, granular powder or small, colourless, transparent or opaque crystals, slightly hygroscopic. Freely soluble in water, soluble in ethanol (96%). Identification A. It gives reaction (A) of bromides (Appendix 8.1). B. Solution S (see Appearance of solution) gives the reactions of sodium (Appendix 8.1). Appearance of solution Solution S: Dissolve 10.0 g in carbon dioxide-free water R and dilute to 100 ml with the same solvent. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2).

873

VP V

SODIUM CALCIUM EDETATE

Magnesium and alkaline-earth metals Not more than 0.02%, calculated as Ca (Appendix 9.4.16). 10.0 g complies with the test for magnesium and alkalineearth metals. The volume of 0.01 M sodium edetate VS used does not exceed 5.0 ml. Heavy metals Not more than 10 ppm (Appendix 9.4.8). 12 ml of solution S complies with limit test for heavy metals, method 1. Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on drying Not more than 3.0% (Appendix 9.6). (1.000 g; 105 °C; 3 h). Assay Dissolve 85.0 mg of the substance to be examined in water, add 5 ml of dilute nitric acid R and dilute to 50 ml with water. Titrate with 0.1 N silver nitrate VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N silver nitrate VS is equivalent to 10.29 mg of NaBr. Calculate the percentage content of NaBr using the following expression: a – 2.902 b Where: a: percentage content of NaBr and NaCl obtained in the assay and calculated as NaBr; b: percentage content of Cl obtained in the test for Chlorides and sulfates. Storage In an airtight container. SODIUM CALCIUM EDETATE Natrii Calcii Edetas

C10H12CaN2Na2O8,xH2O  

M. 374.3 (anhydrous)

Sodium calcium edetate is disodium [(ethylenedinitrilo) tetraacetato]calciate(2-). It contains a variable amount of water of crystallisation and contains not less than 98.0% and not more than 102.0% of C10H12CaN2Na2O8, calculated with reference to the anhydrous substance.

Characters White or almost white, hygroscopic powder. Freely soluble in water, practically insoluble in ethanol (96%). Identification Apply one of the two following identifications: 874

First identification: A, C, D. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of sodium calcium edetate RS. Examine the substances prepared as discs. B. Dissolve 2 g of the substance to be examined in 10 ml of water, add 6 ml of lead nitrate solution R, shake and add 3 ml of 16.6% solution of potassium iodide R. No yellow precipitate is formed. Make alkaline to red litmus paper R by the addition of 2 M ammonia R and add 3 ml of 4% solution of ammonium oxalate R. A white precipitate is formed. C. Dissolve 0.5 g of the substance to be examined in 10 ml of water and add 10 ml of potassium pyroantimonate solution R. A white, crystalline precipitate is formed. The formation of the precipitate is accelerated by rubbing the wall of the tube with a glass rod. D. Ignite the substance to be examined. The residue gives reaction (B) of calcium (Appendix 8.1).

Appearance of solution Solution S: Dissolve 5.0 g in water and dilute to 100 ml with the same solvent. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). pH 6.5 to 8.0 (Appendix 6.2). Dissolve 5.0 g in carbon dioxide-free water R and dilute to 25 ml with the same solvent. Impurity A Examine by liquid chromatography (Appendix 5.3). Carry out the test protected from light. Mobile phase: Dissolve 50.0 mg of ferric sulfate pentahydrate R in 50 ml of 0.5 M sulfuric acid R and add 750 ml of water; adjust to pH 1.5 with 0.5 M sulfuric acid R or 1 M sodium hydroxide R, add 20 ml of ethylene glycol R and dilute to 1000 ml with water. Solvent mixture: Dissolve 10.0 g of ferric sulfate pentahydrate R in 20 ml of 0.5 M sulfuric acid R and add 780 ml of water. Adjust to pH 2.0 with 1 M sodium hydroxide R and dilute to 1000 ml with water. Test solution: Dissolve 0.100 g of the substance to be examined in the solvent mixture and dilute to 25.0 ml with the solvent mixture. Reference solution: Dissolve 40.0 mg of nitrilotriacetic acid R (impurity A) in the solvent mixture and dilute to 100.0 ml with the solvent mixture. To 1.0 ml of this solution add 0.1 ml of the test solution and dilute to 100.0 ml with the solvent mixture. Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase spherical graphitised carbon for chromatography R1 (5 µm) with a specific surface area of 120 m2/g and a pore size of 25 nm.

SODIUM CAMPHOSULFONATE

VP V

Detector: A spectrophotometer set at 273 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Filter the solutions and inject immediately. The run time of the test solution is 4 times the retention time of the iron complex of impurity A. Retention time: Iron complex of impurity A = about 5 min; iron complex of edetic acid = about 10 min. System suitability: In the chromatogram obtained with reference solution, the resolution between the peaks due to the iron complex of impurity A and the iron complex of edetic acid is at least 7; the signal-to-noise ratio of the peak due to impurity A is not less than 50. Limit: impurity A: The area of the peak due to impurity A is not more than the area of the corresponding peak in the chromatogram obtained with the reference solution (0.1%). Note: Impurity A: nitrilotriacetic acid.

Disodium edentate Not more than 1.0%. Dissolve 5.0 g of the substance to be examined in 250 ml of water. Add 10 ml of ammonium chloride buffer solution pH 10.0 R and about 50 mg of mordant black 11 triturate R. Not more than 1.5 ml of 0.1 M magnesium chloride is required to change the colour of the indicator to violet. Chlorides Not more than 0.1%. Test solution: Dissolve 0.7 g of the substance to be examined in water and dilute to 20 ml with the same solvent. Add 30 ml of dilute nitric acid R, allow to stand for 30 min and filter. Dilute 10 ml of the filtrate to 50 ml with water. Reference solution: To 0.40 ml of 0.01 M hydrochloric acid R, add 6 ml of dilute nitric acid R and dilute to 50 ml with water. Filter both solutions if necessary. Add 1 ml of 1.7% solution of silver nitrate R to the test solution and to the reference solution and mix. After standing for 5 min protected from light, any opalescence in the test solution is not more intense than that in the reference solution.

Water 5.0% to 13.0% (Appendix 10.3). Determined on 0.200 g. Assay Dissolve 0.500 g of the substance to be examined in water and dilute to 200 ml with the same solvent. To 20.0 ml of this solution, add 80 ml of water and adjust to pH 2 with dilute nitric acid R. Titrate with 0.01 M bismuth nitrate VS, using 0.1 ml of xylenol orange solution R as indicator. The colour of the solution changes from yellow to red. 1 ml of 0.01 M bismuth nitrate VS is equivalent to 3.74 mg of C10H12CaN2Na2O8. Storage In an airtight container, protected from light. Action and use Chelating agent. Preparation Infusion. SODIUM CAMPHOSULFONATE Natrii camphosulfonas Me

Me SO3Na O

C10H15O4SNa

M. 254.3

Sodium camphosulfonate is the sodium salt of 10-camphosulfonic acid (7,7-dimethyl-2-oxobicyclo-[2.2.1] heptane 1-methanesulfonic acid) obtained by synthesis from natural (or synthetic) camphor and sulfuric acid and anhydride acetic.

Iron Not more than 80 ppm (Appendix 9.4.13). Dilute 2.5 ml of solution S to 10.0 ml with water. Add 0.25 g of calcium chloride R to the test solution and the standard before the addition of the mercaptoacetic acid R.

Character A white, crystalline powder, hygroscopic; slight odour of camphor; slightly bitter taste. It curdles and becomes yellow in moist air. Very soluble in water, soluble in ethanol, sparingly soluble in ether, in benzene and in cyclohexane, practically insoluble in carbontetrachloride.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 6. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R.

Identification A. Melting point: 283 °C to 286 °C (Appendix 6.7). B. Specific optical rotation. +17.25° to +19.25° (sodium camphosulfonate obtained from natural camphor). 875

VP V

SODIUM CHLORIDE

-1.5 to +1.5° (sodium camphosulfonate obtained from synthetic camphor, racemic). Dissolve 1.0 g in water and dilute to 25.0 ml with the same solvent (Appendix 6.4). C. Heat 1 g with a few granules of sodium hydroxide R, the odour of camphor is detectable.

Appearance of solution Dissolve 2 g in 10 ml of water. The solution is clear (Appendix 9.2) and not more intensely coloured than 0.00005 N iodine R. pH Dissolve 1 g in 10 ml of carbon dioxide-free water R. The pH of the solution is 6.0 to 8.0 (Appendix 6.2). Barium Dissolve 0.1 g in 5 ml of water, add 2 drops of a 10% solution of hydrochloric acid R and 2.5 ml of a saturated solution of calcium sulfate R. The solution is clear. Chlorides Not more than 0.01% (Appendix 9.4.5) Dissolve 0.5 g in water and dilute to 15 ml with the same solvent. The solution complies with the limit test for chlorides. Sulfates Not more than 0.05% (Appendix 9.4.14). Dissolve 0.3 g in water and dilute to 15 ml with the same solvent. The solution complies with the limit test for sulfate. Heavy metals Not more than 20 ppm (Appendix 9.4.8). Dissolve 2.0 g in water and dilute to 20 ml with the same solvent. 12 ml of the solution complies with limit test for heavy metals, method 1. Prepare the standard solution using the lead standard solution (2 ppm Pb) R. Loss on drying Not more than 2.7% (Appendix 9.6). (1.000 g; 100 °C - 105 °C; 2 h). Storage Store in a well-closed container, protected from light. Action and use Central nervous stimulant (prior action on medullary bulb). Used as a respiratory and cardiac stimulant. Preparations Injection, dropwise oral solution.

876

SODIUM CHLORIDE Natrii chloridum NaCl  

M. 58.44

Sodium chloride contains not less than 99.0% and not more than 100.5% of NaCl, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder or colourless crystals or white or almost white pearls. Freely soluble in water, practically insoluble in anhydrous ethanol. Identification A. It gives the reactions of chlorides (Appendix 8.1). B. It gives the reactions of sodium (Appendix 8.1). If the substance is in the form of pearls crush before use. Appearance of solution Solution S: Dissolve 20.0 g of the substance to be examined in carbon dioxide-free water R prepared from distilled water R and dilute to 100.0 ml with the same solvent. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). Acidity or alkalinity Add 0.1 ml of bromothymol blue solution R to 20 ml of solution S. Not more than 0.5 ml of 0.01 N hydrochloric acid VS or 0.01 N sodium hydroxide VS is required to change the colour of the indicator. Bromides Not more than 100 ppm. To 0.5 ml of solution S add 4.0 ml of water, 2.0 ml of phenol red solution R2 and 1.0 ml of a 0.01% solution of chloramine T R and mix immediately. After exactly 2 min, add 0.15 ml of 0.1 M sodium thiosulfate, mix and dilute to 10.0 ml with water. The absorbance (Appendix 4.1) of the solution measured at 590 nm, using water as the compensation liquid, is not greater than that of a standard prepared at the same time and in the same manner, using 5.0 ml of a 3.0 mg/L solution of potassium bromide R. Ferrocyanides Dissolve 2.0 g of the substance to be examined in 6 ml of water. Add 0.5 ml of a mixture of 5 ml of a 1% solution of ferric ammonium sulfate R in a 0.25% solution of sulfuric acid R and 95 ml of a 1% solution of ferrous sulfate R. No blue colour develops within 10 min. Iodides Moisten 5 g of the substance to be examined by the dropwise addition of a freshly prepared mixture of 0.15 ml of 10% solution of sodium nitrite R, 2 ml of 0.5 M sulfuric acid R, 25 ml of iodide-free starch solution R and 25 ml of water. After 5 min, examine in daylight. The mixture shows no blue colour.

VP V

Nitrites Add 10 ml of water to 10 ml of solution S. The absorbance (Appendix 4.1) is not greater than 0.01 at 354 nm. Phosphates Not more than 25 ppm (Appendix 9.4.12). Dilute 2 ml of solution S to 100 ml with water. Sulfates Not more than 0.02% (Appendix 9.4.14). Dilute 7.5 ml of solution S to 30 ml with distilled water R. Aluminium Not more than 0.2 ppm (Appendix 9.4.9). If intended for use in the manufacture of peritoneal dialysis solutions, haemodialysis solutions or haemofiltration solutions. Test solution: Dissolve 20.0 g of the substance to be examined in 100 ml of water and add 10 ml of acetate buffer solution pH 6.0 R. Reference solution: Mix 2 ml of aluminium standard solution (2 ppm Al) R, 10 ml of acetate buffer solution pH 6.0 R and 98 ml of water. Blank solution: Mix 10 ml of acetate buffer solution pH 6.0 R and 100 ml of water.

SODIUM CHLORIDE EYE DROPS (0.9%)

Reference solutions: Dissolve 1.144 g of potassium chloride R, previously dried at 100 °C to 105 °C for 3 h, in water and dilute to 1000.0 ml with the same solvent (600 µg of K per millilitre). Dilute as required. Wavelength: 766.5 nm.

Heavy metals Not more than 5 ppm (Appendix 9.4.8). 12 ml of solution S complies with limit test for heavy metals, method 1. Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C; 2 h). Bacterial endotoxins Less than 5 EU/mg (Appendix 13.2). If intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins.

Arsenic Not more than 1 ppm (Appendix 9.4.2, method A). Determined on 5 ml of solution S.

Assay Dissolve 50.0 mg of the substance to be examined in water and dilute to 50 ml with the same solvent. Titrate with 0.1 N silver nitrate VS determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N silver nitrate VS is equivalent to 5.844 mg of NaCl.

Barium Add 5 ml of distilled water R and 2 ml of 1 M sulfuric acid R to 5 ml of solution S. After 2 h, any opalescence in the solution is not more intense than that in a mixture of 5 ml of solution S and 7 ml of distilled water R.

Labelling The label states: where applicable, that the substance is suitable for use in the manufacture of parenteral preparations or use in the manufacture of peritoneal dialysis solutions, haemodialysis solutions or haemofiltration solutions.

Iron Not more than 2 ppm (Appendix 9.4.13). Determined on 10 ml of solution S. Prepare the standard using a mixture of 4 ml of iron standard solution (1 ppm Fe) R and 6 ml of water.

Storage Store in an airtight container.

Magnesium and alkaline-earth metals Not more than 0.01%, calculated as Ca (Appendix 9.4.16). Determined on 10.0 g and 150 mg of eriochrome black T R. The volume of 0.01 M sodium edetate VS used is not more than 2.5 ml.

Preparations Infusion, eye drops.

Potassium Not more than 0.05%. If intended for use in the manufacture of parenteral preparations or haemodialysis, haemofiltration or peritoneal dialysis solutions. Examine by Atomic emission spectrometry (Appendix 4.4, method 1). Test solution: Dissolve 1.00 g of the substance to be examined in water and dilute to 100.0 ml with the same solvent.

Action and use Used in treatment of electrolyte deficiency.

SODIUM CHLORIDE EYE DROPS (0.9%) Collyrium Natrii chloridi Sodium chloride eye drops are a sterile solution of sodium chloride in water. The eye drops comply with the requirements stated under “Eye drops” (Appendix 1.14) and with the following requirements.

Content of sodium chloride, NaCl, 90.0% to 110.0% of the stated amount.

877

VP V

SODIUM CHLORIDE INJECTION

Characters A clear and colourless solution.

Action and use Treatment of electrolyte deficiency.

Identification It gives reactions of sodium and chloride (Appendix 8.1).

Usual strength 0.9%.

pH 6.0 to 8.0 (Appendix 6.2). Assay Transfer 10.0 ml of the eye drops to a 100 ml conical flask. Titrate with 0.1 N silver nitrate VS until the pink precipitate is produced, using 3 drops of potassium chromate solution R as indicator. Each ml of 0.1 N silver nitrate VS is equivalent to 5.844 mg of NaCl. Storage Store at a temperature not exceeding 25 °C. Action and use For cleaning eyes. Usual strength 0.9%. SODIUM CHLORIDE INJECTION Injectio Natrii chloridi Sodium chloride injection is a sterile solution of sodium chloride in water for injections. The preparation complies with requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Content of sodium chloride, NaCl, 95.0% to 105.0% of the stated amount. Characters A clear and colourless solution. Identification It gives reactions of sodium and chloride (Appendix 8.1). pH 4.5 to 7.0 (Appendix 6.2). Assay Transfer 10.0 ml of the injection to a 100 ml conical flask. Titrate with 0.1 N silver nitrate VS until the pink precipitate is produced, using 3 drops of potassium chromate solution R as indicator. Each ml of 0.1 N silver nitrate VS is equivalent to 5.844 mg of NaCl. Storage Packed in a 5 ml airtight glass vial. Store at a temperature not exceeding 25 °C. 878

ISOTONIC SODIUM CHLORIDE INFUSION Infusio Natrii chloridi isotonica Isotonic sodium chloride infusion is a sterile solution of sodium chloride in water for injections. It should not have any preservative agent. The preparation complies with the requirements stated under “Injections, infusions”, “Intravenous infusions” section (Appendix 1.19) and with the following requirements.

Content of sodium chloride, NaCl, 95.0% to 105.0% of the stated amount. Characters A clear and colourless solution. Identification It gives reactions of sodium and chloride (Appendix 8.1). pH 4.5 to 7.0 (Appendix 6.2). Particulate contamination For the preparation supplied in containers with capacity of more than 100 ml, carry out the test for particulate contamination (Appendix 11.8). It complies with test A for particulate contamination: sub-visible particles. Bacterial endotoxins Not more than 0.5 EU/ml. Carry out the test for bacterial endotoxins (Appendix 13.2). Assay Transfer 10.0 ml of the infusion to a 100 ml conical flask. Titrate with 0.1 N silver nitrate VS until the pink precipitate is produced, using 3 drops of potassium chromate solution R as indicator. Each ml of 0.1N silver nitrate VS is equivalent to 5.844 mg of NaCl. Storage Store in airtight neutral glass or plastic containers with capacity of 300 ml or 500 ml, at a temperature not exceeding 25 °C. Action and use Treatment of electrolyte deficiency. Usual strength 0.9%.

SODIUM HYDROGEN CARBONATE

VP V

SODIUM CITRATE Natrii citras

C6H5Na3O7,2H2O

30 min. Any pink colour in the solution is not more intense than that in a standard prepared at the same time in the same manner using 4 ml of a 0.005% solution of oxalic acid in place of a solution of 0.50 g and 4 ml of water.

M. 294.1

Sodium citrate is trisodium 2-hydroxypropane-1,2,3tricarboxylate. It contains not less than 99.0% and not more than 101.0% of C6H5Na3O7, calculated with reference to the anhydrous substance.

Characters A white, crystalline powder or white, granular crystals, slightly deliquescent in moist air. Freely soluble in water, practically insoluble in ethanol (96%). Identification Solution S: Dissolve 10.0 g in carbon dioxide-free water R and dilute to 100.0 ml with the same solvent. A. To 1 ml of solution S add 4 ml of water. The solution gives the reaction of citrates (Appendix 8.1). B. 1 ml of solution S gives reaction A of sodium (Appendix 8.1). Appearance of solution Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). Acidity or alkalinity To 10 ml of solution S add 0.1 ml of phenolphthalein solution R. Not more than 0.2 ml of 0.1 N hydrochloric acid VS or 0.1 N sodium hydroxide VS is required to change the colour of the solution. Readily carbonisable substances To 0.20 g of the powdered substance to be examined add 10 ml of sulfuric acid R and heat in a water bath at 90 ± 1 °C for 60 min. Cool rapidly. The solution is not more intensely coloured than reference solution Y2 or GY2 (Appendix 9.3, method 2). Chlorides Not more than 50 ppm (Appendix 9.4.5). Dilute 10 ml of solution S to 15 ml with water. The solution complies with the limit test for Chlorides. Oxalates Not more than 0.03%. Dissolve 0.50 g in 4 ml of water, add 3 ml of hydrochloric acid R and 1 g of granulated zinc R and heat on a water bath for 1 min. Allow to stand for 2 min, decant the liquid into a test tube containing 0.25 ml of a 1.0% solution of phenylhydrazine hydrochloride R and heat to boiling. Cool rapidly, transfer to a graduated cylinder and add an equal volume of hydrochloric acid R and 0.25 ml of a 5% solution of potassium ferricyanide R. Shake and allow to stand for

Sulfates Not more than 0.015% (Appendix 9.4.14). To 10 ml of solution S add 2 ml of 25% solution of hydrochloric acid R and dilute to 15 ml with water. The solution complies with the limit test for Sulfates. Heavy metals Not more than 10 ppm (Appendix 9.4.8). 12 ml of solution S complies with limit test 1 for heavy metals. Prepare the standard using lead standard solution (1 ppm Pb) R. Water 11.0% to 13.0% (Appendix 10.3). Determined on 0.300 g. After adding the substance to be examined, stir for 15 min before titrating. Pyrogens If intended for use in large-volume preparations for parenteral use, the competent authority may require that it complies with the test for pyrogens. Inject per kilogram of the rabbit’s mass 10 ml of a freshly prepared solution in water for injections R containing 10.0 mg of the substance to be examined and 7.5 mg of pyrogen-free calcium chloride R per millilitre (Appendix 13.4). Assay Dissolve 0.150 g in 20 ml of anhydrous acetic acid R, heating to about 50 °C. Allow to cool. Using 0.25 ml of naphtholbenzein solution R as indicator, titrate with 0.1 N perchloric acid VS until a green colour is obtained. 1 ml of 0.1 N perchloric acid VS is equivalent to 8.602 mg of C6H5Na3O7. Storage Store in an airtight container. Action and use Systemic alkalinising substance. SODIUM HYDROGEN CARBONATE Natrii hydrocarbonas Sodium bicarbonate NaHCO3

M. 84.01

Sodium hydrogen carbonate contains not less than 99.0% and not more than 101.0% of NaHCO3.

Characters A white, crystalline powder. Soluble in water, practically insoluble in ethanol (96%). When heated in the dry state or in solution, it gradually changes into sodium carbonate. 879

VP V

SODIUM HYDROGEN CARBONATE INJECTION

Identification Solution S: Dissolve 5.0 g in 90 ml of carbon dioxide-free water R and dilute to 100.0 ml with the same solvent. A. To 5 ml of solution S add 0.1 ml of phenolphthalein solution R. A pale pink colour is produced. When heated: gas is evolved and the solution becomes red. B. It gives the reactions of carbonates and bicarbonates (Appendix 8.1). C. Solution S gives the reaction (A) of sodium (Appendix 8.1).

Dissolve 0.5 g in 5 ml of dilute hydrochloric acid R and dilute to 10 ml with water. The solution complies with the limit test for iron.

Appearance of solution Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2).

Storage Store in an airtight container.

Carbonates The pH of freshly prepared solution S is not more than 8.6 (Appendix 6.2). Chlorides Not more than 0.015% (Appendix 9.4.5). To 7 ml of solution S add 2 ml of nitric acid R and dilute to 15 ml with water. The solution complies with the limit test for Chlorides. Sulfates Not more than 0.015% (Appendix 9.4.14) To a suspension of 1.0 g in 10 ml of water add hydrochloric acid R until neutral and about 1 ml in excess. Dilute to 15 ml with water. The solution complies with the limit test for Sulfates. Ammonium Not more than 20 ppm (Appendix 9.4.1). 10 ml of solution S diluted to 15 ml with water complies with limit test A for ammonium. Prepare the standard using a mixture of 5 ml of water and 10 ml of ammonium standard solution (1 ppm NH4) R. Arsenic Not more than 2 ppm (Appendix 9.4.2). 0.5 g complies with limit test A for Arsenic. Calcium Not more than 0.01% (Appendix 9.4.3). To a suspension of 1.0 g in 10 ml of water add hydrochloric acid R until neutral and dilute to 15 ml with water. The solution complies with the limit test for Calcium. Heavy metals Not more than 10 ppm (Appendix 9.4.8). Dissolve 2.0 g in a mixture of 2 ml of hydrochloric acid R and 18 ml of water. 12 ml of the solution complies with limit test 1 for heavy metals. Prepare the standard using lead standard solution (1 ppm Pb) R. Iron Not more than 20 ppm (Appendix 9.4.13). 880

Assay Dissolve 1.500 g in 50 ml of carbon dioxide-free water R. Titrate with 1 N hydrochloric acid VS, using 0.2 ml of methyl orange solution R as indicator. 1 ml of 1 N hydrochloric acid VS is equivalent to 84.0 mg of NaHCO3.

Action and use Antacid, use in treatment of electrolyte deficiency. SODIUM HYDROGEN CARBONATE INJECTION Injectio Natrii bicarbonas Sodium bicarbonate injection is a sterile solution of sodium bicarbonate in water for injections. It may contain suitable stabilizers. The preparation complies with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Content of sodium bicarbonate, NaHCO3, 95.0% to 105.0% of the stated amount. Characters A clear and colourless solution. Identification The preparation gives reactions of sodium and bicarbonates (Appendix 8.1). pH 7.5 to 8.5 (Appendix 6.2). Pyrogens Complies with the test for pyrogens (Appendix 13.4). For the injections containing 2.5% or less of sodium bicarbonate, inject 10 ml per kg of the rabbit’s weight. For the injections containing more than 2.5% of sodium bicarbonate, dilute with pyrogen-free water for injection to obtain a solution of 2.5% of sodium bicarbonate and inject 10 ml per kg of the rabbit’s weight. Assay Take an accurately measured volume of the injection containing 1 g of sodium hydrocacbonate and titrate with 0.5 N hydrochloric acid VS using methyl orange solution R as indicator. Each ml of 0.5 N hydrochloric acid VS is equivalent to 42.0 mg of NaHCO3.

SODIUM SALICYLATE

VP V

Storage Store in airtight neutral glass vial, in a cool place and protected from light. Action and use Antacid. Usual strength 1.4%; 4.2%; 7.5%; 8.4%. SODIUM HYDROGEN CARBONATE POWDER Pulveres Natrii hydrocarbonas Sodium bicarbonate powder contains sodium bicarbonate for oral use. The powder complies with the requirements stated under “Powders” (Appendix 1.7) and with the following requirements.

Content of sodium bicarbonate, NaHCO3, not less than 0.985 g per 1 g. Characters White, loose and dry powder with salty taste. Identification Solution S: Dissolve 5.0 g of the powder in 90 ml of carbon dioxide-free water R and dilute to 100 ml with the same solvent. A. To 5 ml of solution S add 0.1 ml of phenolphthalein solution R, a pale pink colour is produced. Heat, gas is evolved and the solution becomes red. B. The powder gives the reactions of carbonates and bicarbonates (Appendix 8.1). C. Solution S gives reaction of sodium (Appendix 8.1). Cabonates The pH of solution S freshly prepared is not more than 8.6 (Appendix 6.2). Assay Dissolve 1.500 g of the powder in 50 ml of carbon dioxidefree water R. Titrate with 1 N hydrochloric acid VS, using 0.2 ml of methyl orange solution R as indicator. 1 ml of 1 N hydrochloric acid VS is equivalent to 84.0 mg of NaHCO3. Storage Store in a well-closed container, at a temperature not exceeding 30 °C. Action and use Antacid. Usual strength 5 g, 10 g, 20 g, 50 g, 100 g.

SODIUM SALICYLATE Natrii salicylas CO2Na OH

C7H5NaO3  

M. 160.1

Sodium salicylate is sodium 2-hydroxybenzenecarboxylate. It contains not less than 99.0% and not more than 101.0% of C7H5NaO3, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder or small, colourless crystals or shiny flakes. Freely soluble in water, sparingly soluble in ethanol (96%). Identification Apply one of the two following identifications: First identification: A, C. Second identification: B, C. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of sodium salicylate RS. B. Solution S (see Appearance of solution) gives the reactions of salicylates (Appendix 8.1). C. It gives reaction of sodium (Appendix 8.1). Appearance of solution Solution S: Dissolve 5.0 g in carbon dioxide-free water R and dilute to 50 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution BY6 (Appendix 9.3, method 2). Acidity Add 0.1 ml of phenol red solution R to 20 ml of solution S. The solution is yellow. Not more than 2.0 ml of 0.01 N sodium hydroxide VS is required to change the colour of the indicator to violet-red. Chlorides Not more than 0.02% (Appendix 9.4.5). Add 5 ml of water and 10 ml of dilute nitric acid R to 5 ml of solution S and filter. Dilute 10 ml of the filtrate to 15 ml with water. Sulfates Not more than 0.06% (Appendix 9.4.14). Dilute 2.5 ml of solution S to 15 ml with water. Heavy metals Not more than 20 ppm (Appendix 9.4.8). Dissolve 1.6 g of the substance to be examined in 16 ml of a mixture of water and ethanol (96%) (5 : 10). 12 ml of the 881

SODIUM STARCH GLYCOLATE (TYPE A)

solution complies with limit test for heavy metals, method 2. Prepare the standard using lead standard solution (2 ppm Pb) obtained by diluting lead standard solution (100 ppm Pb) R with a mixture of water and ethanol (96%) (5 : 10).

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Assay Dissolve 0.130 g of the substance to be examined in 30 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 16.01 mg of C7H5NaO3. Storage In an airtight container, protected from light. Action and use Anti-inflammatory; analgesic. Preparation Tablets. SODIUM STARCH GLYCOLATE (TYPE A) Carboxymethylamylum natricum A Sodium starch glycolate type A is sodium salt of a crosslinked partly O-carboxymethylated potato starch. It contains not less than 2.8% and not more than 4.2% of Na (Ar: 22.99), calculated with reference to the substance washed with ethanol (80%) and dried.

Characters White or almost white, fine, free-flowing powder, very hygroscopic. Practically insoluble in methylene chloride. It gives a translucent suspension in water. Examined under a microscope: Granules, irregularly shaped, ovoid or pear-shaped (30 µm to 100 µm in size) or rounded (10 µm to 35 µm in size); the granules have an eccentric hilum and clearly visible concentric striations. Compound granules consisting of 2 to 4 components occur occasionally. Between crossed nicol prisms, the granules show a distinct black cross intersecting at the hilum; small crystals are visible at the surface of the granules. The granules show considerable swelling in contact with water. Identification A. It complies with the test for pH. B. To 4.0 g of the substance to be examined, add 20 ml of carbon dioxide-free water R, shake without heating. The mixture has the appearance of a gel. Add 100 ml of carbon dioxide-free water R and shake. A suspension forms that settles after standing.  882

VP V

C. To an acidified solution, add iodinated potassium iodide solution R1. The solution becomes blue or violet. D. Solution S2 gives reaction of sodium (Appendix 8.1). Solution S2: Place 2.5 g of the substance to be examined in a silica or platinum crucible and add 2 ml of a 50% solution of sulfuric acid R. Heat on a water-bath, then cautiously over a naked flame, raising the temperature progressively, then incinerate in a muffle furnace at 600 °C ± 25 °C. Continue heating until all black particles have disappeared. Allow to cool, add a few drops of 1 M sulfuric acid R, heat and incinerate as above. Allow to cool, add a few drops of ammonium carbonate solution R, evaporate to dryness and incinerate cautiously. Allow to cool and dissolve the residue in 50 ml of water.

Appearance of solution S1 Solution S1: Centrifuge the suspension obtained in identification test B at 2500 g for 10 min. Collect carefully the supernatant. Solution S1 is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). pH 5.5 to 7.5 (Appendix 6.2). Disperse 1.0 g of the substance to be examined in 30 ml of water. Sodium glycolate Not more than 2.0%. Carry out the test protected from light. Test solution: Place 0.20 g of the substance to be examined in a beaker, add 5 ml of acetic acid R and 5 ml of water. Stir until dissolution is complete (about 10 min). Add 50 ml of acetone R and 1 g of sodium chloride R, mix well. Filter through a fast filter paper impregnated with acetone R, rinse the beaker and filter with acetone R. Combine the filtrate and washings and dilute to 100.0 ml with acetone R. Allow to stand for 24 h without shaking. Use the clear supernatant. Reference solution: Dissolve 0.310 g of glycollic acid R (previously dried in vacuo over diphosphorus pentoxide R at room temperature overnight) in water and dilute to 500.0 ml with the same solvent. To 5.0 ml of this solution add 5 ml of acetic acid R and allow to stand for about 30 min. Add 50 ml of acetone R and 1 g of sodium chloride R, mix well. Filter through a fast filter paper impregnated with acetone R, rinse the beaker and filter with  acetone R. Combine the filtrate and washings and dilute to 100.0 ml with acetone R. Allow to stand for 24 h without shaking. Use the clear supernatant. Heat 2.0 ml of the test solution on a water-bath for 20 min. Cool to room temperature and add 20.0 ml of 2,7-dihydroxynaphthalene solution R. Shake and heat in a water-bath for 20 min. Cool under running water, transfer to a volumetric flask and dilute to 25.0  ml with  sulfuric acid R, maintaining the flask under running water. Within 10 min, measure the absorbance at 540 nm (Appendix 4.1) using water as the blank solution. The absorbance of the

VP V

solution prepared with the test solution is not greater than that of a solution prepared at the same time and in the same manner with 2.0 ml of the reference solution.

Sodium chloride Not more than 7.0%. Place 0.500 g of the substance to be examined in a beaker and suspend in 100 ml of water. Add 1 ml of nitric acid R. Titrate with 0.1 M silver nitrate VS, determining the endpoint potentiometrically (Appendix 10.2), using a silverbased indicator electrode. 1 ml of 0.1 M silver nitrate VS is equivalent to 5.844 mg of NaCl. Iron Not more than 20 ppm (Appendix 9.4.13). Determined on 10 ml of solution S2. Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 4. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 10.0% (Appendix 9.6). (1.000 g; 130 °C; 1.5 h). Microbial contamination It complies with the test for Escherichia coli and Salmonella (Appendix 13.6). Assay Shake 1.000 g of the substance to be examined with 20 ml of ethanol (80%) v/v R, stir for 10 min and filter. Repeat the operation until chloride has been completely extracted (verify the absence of chloride ion using a 1.7% solution of silver nitrate R). Dry the residue at 105 °C to constant mass. To 0.700 g of the dried residue, add 80 ml of glacial acetic acid R and heat under a reflux condenser for 2 h. Cool the solution to room temperature. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). Carry out a blank titration. 1 ml of 0.1 N perchloric acid VS is equivalent to 2.299 mg of Na. Storage Store in an airtight container, protected from light. Action and use Excipients.

SODIUM STARCH GLYCOLATE (TYPE B)

SODIUM STARCH GLYCOLATE (TYPE B) Carboxymethylamylum natricum B Sodium starch glycolate type B is sodium salt of a crosslinked partly O-carboxymethylated potato starch. It contains not less than 2.0% and not more than 3.4% of Na (Ar: 22.99), calculated with reference to the substance washed with ethanol (80%) and dried.

Characters White or almost white, fine, free-flowing powder, very hygroscopic. Practically insoluble in methylene chloride. It gives a translucent suspension in water. Examined under a microscope: Granules, irregularly shaped, ovoid or pear-shaped (30 µm to 100 µm in size) or rounded (10 µm to 35 µm in size); the granules have an eccentric hilum and clearly visible concentric striations. Compound granules consisting of 2 to 4 components occur occasionally. Between crossed nicol prisms, the granules show a distinct black cross intersecting at the hilum; small crystals are visible at the surface of the granules. The granules show considerable swelling in contact with water. Identification A. It complies with the test for pH. B. To 4.0 g of the substance to be examined, add 20 ml of carbon dioxide-free water R, shake without heating. The mixture has the appearance of a gel. Add 100 ml of carbon dioxide-free water R and shake. A suspension forms that settles after standing.  C. To an acidified solution, add iodinated potassium iodide solution R1. The solution becomes blue or violet. D. Solution S2 gives reaction of sodium (Appendix 8.1). Solution S2: Place 2.5 g of the substance to be examined in a silica or platinum crucible and add 2 ml of a 50% solution of sulfuric acid R. Heat on a water-bath, then cautiously over a naked flame, raising the temperature progressively, then incinerate in a muffle furnace at 600 °C ± 25 °C. Continue heating until all black particles have disappeared. Allow to cool, add a few drops of 1 M sulfuric acid R, heat and incinerate as above. Allow to cool, add a few drops of ammonium carbonate solution R, evaporate to dryness and incinerate cautiously. Allow to cool and dissolve the residue in 50 ml of water. Appearance of solution S1 Solution S1: Centrifuge the suspension obtained in identification test B at 2500 g for 10 min. Collect carefully the supernatant. Solution S1 is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). pH 3.0 to 5.0 (Appendix 6.2). Disperse 1.0 g of the substance to be examined in 30 ml of water. 883

VP V

SODIUM STARCH GLYCOLATE (TYPE C)

Sodium glycolate Not more than 2.0%. Carry out the test protected from light. Test solution: Place 0.20 g of the substance to be examined in a beaker. Add 5 ml of acetic acid R and 5 ml of water. Stir until dissolution is complete (about 10 min). Add 50 ml of acetone R and 1 g of sodium chloride R, mix well. Filter through a fast filter paper impregnated with acetone R, rinse the beaker and filter with acetone R. Combine the filtrate and washings and dilute to 100.0 ml with acetone R. Allow to stand for 24 h without shaking. Use the clear supernatant. Reference solution: Dissolve 0.310 g of glycollic acid R (previously dried in vacuo over diphosphorus pentoxide R at room temperature overnight) in water and dilute to 500.0 ml with the same solvent. To 5.0 ml of this solution add 5 ml of acetic acid R and allow to stand for about 30 min. Add 50 ml of acetone R and 1 g of sodium chloride R, mix well. Filter through a fast filter paper impregnated with acetone R, rinse the beaker and filter with  acetone R. Combine the filtrate and washings and dilute to 100.0 ml with acetone R. Allow to stand for 24 h without shaking. Use the clear supernatant. Heat 2.0 ml of the test solution on a water-bath for 20 min. Cool to room temperature and add 20.0 ml of 2,7-dihydroxynaphthalene solution R. Shake and heat in a water-bath for 20 min. Cool under running water, transfer to a volumetric flask and dilute to 25.0  ml with  sulfuric acid R, maintaining the flask under running water. Within 10 min, measure the absorbance at 540 nm (Appendix 4.1) using water as the blank solution. The absorbance of the solution prepared with the test solution is not greater than that of a solution prepared at the same time and in the same manner with 2.0 ml of the reference solution. Sodium chloride Not more than 7.0%. Place 0.500 g of the substance to be examined in a beaker and suspend in 100 ml of water. Add 1 ml of nitric acid R. Titrate with 0.1 M silver nitrate VS, determining the endpoint potentiometrically (Appendix 10.2), using a silverbased indicator electrode. 1 ml of 0.1 M silver nitrate VS is equivalent to 5.844 mg of NaCl. Iron Not more than 20 ppm (Appendix 9.4.13). Determined on 10 ml of solution S2. Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 4. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 10.0% (Appendix 9.6). (1.000 g; 130 °C; 1.5 h). 884

Microbial contamination It complies with the test for Escherichia coli and Salmonella (Appendix 13.6). Assay Shake 1.000 g of the substance to be examined with 20 ml of ethanol (80%) R, stir for 10 min and filter. Repeat the operation until chloride has been completely extracted (verify the absence of chloride using a 1.7% solution of silver nitrate R). Dry the residue at 105 °C to constant mass. To 0.700 g of the dried residue, add 80 ml of glacial acetic acid R and heat under a reflux condenser for 2 h. Cool the solution to room temperature. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). Carry out a blank titration. 1 ml of 0.1 N perchloric acid VS is equivalent to 2.299 mg of Na. Storage Store in an airtight container, protected from light. Action and use Excipients. SODIUM STARCH GLYCOLATE (TYPE C) Carboxymethylamylum natricum C Sodium starch glycolate type C is sodium salt of a partly  O-carboxymethylated starch, cross-linked by physical dehydration. It contains not less than 2.8% and not more than 5.0% of Na (Ar: 22.99), calculated with reference to the substance washed with ethanol (80%) and dried.

Characters White or almost white, fine, free-flowing powder, very hygroscopic. Soluble in water, practically insoluble in methylene chloride. It gives a translucent suspension in water. Examined under a microscope: Granules, irregularly shaped, ovoid or pear-shaped (30 µm to 100 µm in size) or rounded (10 µm to 35 µm in size); the granules have an eccentric hilum and clearly visible concentric striations. Compound granules consisting of 2 to 4 components occur occasionally. Between crossed nicol prisms, the granules show a distinct black cross intersecting at the hilum; small crystals are visible at the surface of the granules. The granules show considerable swelling in contact with water. Identification A. It complies with the test for pH. B. To 4.0 g of the substance to be examined, add 20 ml of  carbon dioxide-free water R, shake without heating. The mixture has the appearance of a gel. Add 100 ml of carbon dioxide-free water R and shake. The gel remains stable (difference from types A and B). Keep the gel for the tests for Appearance of gel and pH.

SODIUM SULFATE

VP V

C. To 5 ml of the gel obtained in identification test B add 0.05 ml of iodine solution R1. A dark blue colour is produced. D. Solution S gives reaction of sodium (Appendix 8.1). Solution S: Place 2.5 g of the substance to be examined in a silica or platinum crucible and add 2 ml of a 50% solution of sulfuric acid R. Heat on a water-bath, then cautiously over a naked flame, raising the temperature progressively, then incinerate in a muffle furnace at 600 °C ± 25 °C. Continue heating until all black particles have disappeared. Allow to cool, add a few drops of sulfuric acid R, heat and incinerate as above. Allow to cool, add a few drops of ammonium carbonate solution R, evaporate to dryness and incinerate cautiously. Allow to cool and dissolve the residue in 50 ml of water.

of ethanol (80%) v/v R for 10 min and filter. Repeat the operation 4 times. Dry the residue to constant mass at 100 °C and set aside for the assay. Combine the filtrates. Evaporate to dryness, take up the residue with water and dilute to 25.0 ml with the same solvent. To 10.0 ml of the solution add 30 ml of water and 5 ml of dilute nitric acid R. Titrate with 0.1 M silver nitrate VS, determining the endpoint potentiometrically (Appendix 10.2), using a silver indicator electrode. 1 ml of 0.1 M silver nitrate VS is equivalent to 5.844 mg of NaCl.

Appearance of gel The gel obtained in identification test B is colourless (Appendix 9.3, method 2).

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 4. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R.

pH 5.5 to 7.5 (Appendix 6.2). Determine on the gel obtained in identification test B. Sodium glycolate Not more than 2.0%. Carry out the test protected from light. Test solution: Place 0.20 g of the substance to be examined in a beaker. Add 5 ml of acetic acid R and 5 ml of water. Stir until dissolution is complete (about 10 min). Add 50 ml of acetone R and 1 g of sodium chloride R, mix well. Filter through a fast filter paper impregnated with acetone R, rinse the beaker and filter with acetone R. Combine the filtrate and washings and dilute to 100.0 ml with acetone R. Allow to stand for 24 h without shaking. Use the clear supernatant. Reference solution: Dissolve 0.310 g of glycollic acid R (previously dried in vacuo over diphosphorus pentoxide R at room temperature overnight) in water and dilute to 500.0 ml with the same solvent. To 5.0 ml of this solution add 5 ml of acetic acid R and allow to stand for about 30 min. Add 50 ml of acetone R and 1 g of sodium chloride R, then dilute to 100.0 ml with acetone R. Heat 2.0 ml of the test solution on a water-bath for 20 min. Cool to room temperature and add 20.0 ml of 2,7-dihydroxynaphthalene solution R. Shake and heat on a water-bath for 20 min. Cool under running water, transfer to a volumetric flask and dilute to 25.0  ml with  sulfuric acid R, maintaining the flask under running water. Within 10 min, measure the absorbance at 540 nm (Appendix 4.1) using water as the blank solution. The absorbance of the solution prepared with the test solution is not greater than that of a solution prepared at the same time and in the same manner with 2.0 ml of the reference solution. Sodium chloride Not more than 1%. Shake 1.00 g of the substance to be examined with 20 ml

Iron Not more than 20 ppm (Appendix 9.4.13). Determined on solution S.

Loss on drying Not more than 7.0% (Appendix 9.6). (1.000 g; 105 °C; 4 h). Microbial contamination It complies with the test for Escherichia coli and Salmonella (Appendix 13.6). Assay To 0.500 g of the dried and crushed residue obtained in the test for Sodium chloride add 80 ml of anhydrous acetic acid R and heat under a reflux condenser for 2 h. Cool the solution to room temperature. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). Carry out a blank titration. 1 ml of 0.1 N perchloric acid VS is equivalent to 2.299 mg of Na. Storage Store in an airtight container, protected from light. Action and use Excipients. SODIUM SULFATE Natrii sulfas Sodium sulfate decahydrate Na2SO4,10H2O  

M. 322.2 

Sodium sulfate contains not less than 98.5% and not more than 101.0% of Na2SO4, calculated with reference to the dried substance.

885

VP V

ANHYDROUS SODIUM SULFATE

Characters White or almost white, crystalline powder or colourless, transparent crystals. Freely soluble in water, practically insoluble in ethanol (96%). It partly dissolves in its own water of crystallisation at about 33 °C. Identification A. It gives reaction of sodium (Appendix 8.1). B. It gives the reactions of sulfates (Appendix 8.1). C. It complies with the test for Loss on drying. Appearance of solution Solution S: Dissolve 5.0 g in carbon dioxide-free water R and dilute to 100 ml with the same solvent. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). Acidity or alkalinity Add 0.1 ml of bromothymol blue solution R to 10 ml of solution S. Not more than 0.5 ml of 0.01 N hydrochloric acid VS or 0.01 N sodium hydroxide VS is required to change the colour of the indicator. Chlorides Not more than 0.02% (Appendix 9.4.5). 5 ml of solution S diluted to 15 ml with water complies with the limit test for chlorides. Calcium Not more than 0.02% (Appendix 9.4.3), if intended for use in the manufacture of parenteral preparations. 10 ml of solution S diluted to 15 ml with distilled water R complies with the limit test for calcium. Iron Not more than 40 ppm (Appendix 9.4.13), if intended for use in the manufacture of parenteral preparations. 5 ml of solution S diluted to 10 ml with water complies with the limit test for iron. Magnesium Not more than 0.01%, if intended for use in the manufacture of parenteral preparations. Add 1 ml of glycerol (85%) R, 0.15 ml of titan yellow solution R, 0.25 ml of 4% solution of ammonium oxalate R and 5 ml of 2 M sodium hydroxide R to 10 ml of solution S and shake. Any pink colour in the test solution is not more intense than that in a standard prepared at the same time in the same manner using a mixture of 5 ml of magnesium standard solution (10 ppm Mg) R and 5 ml of water. Heavy metals Not more than 20 ppm (Appendix 9.4.8). 12 ml of solution S complies with limit test for heavy metals, method 1. Prepare the standard using lead standard solution (1 ppm Pb) R.

886

Loss on drying 52.0% to 57.0% (Appendix 9.6). (1.000 g; 30 °C for 1 h, then at 130 °C). Assay Dissolve 0.250 g of the substance to be examined in 40 ml of water. Add a mixture of 0.2 ml of 0.1 M hydrochloric acid R and 80 ml of methanol R. Titrate with 0.1 M lead nitrate VS, determining the end-point potentiometrically (Appendix 10.2) using a lead-selective electrode as indicator electrode and a silver-silver chloride electrode as reference electrode. 1 ml of 0.1 M lead nitrate VS is equivalent to 14.20 mg of Na2SO4. Storage Store in an airtight container. Action and use Laxative. Labelling The label states, where applicable, that the substance is suitable for use in the manufacture of parenteral preparations. ANHYDROUS SODIUM SULFATE Natrii sulfas anhydricum Na2SO4

M. 142.0

Sodium sulfate contains not less than 98.5% and not more than 101.0% of Na2SO4, calculated with reference to the dried substance.

Characters White or almost white powder, hygroscopic. Freely soluble in water. Identification A. It gives reaction of sodium (Appendix 8.1). B. It gives the reactions of sulfates (Appendix 8.1). C. It complies with the test for Loss on drying. Appearance of solution Solution S: Dissolve 2.2 g in carbon dioxide-free water R and dilute to 100 ml with the same solvent. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). Acidity or alkalinity Add 0.1 ml of bromothymol blue solution R to 10 ml of solution S. Not more than 0.5 ml of 0.01 N hydrochloric acid VS or 0.01 N sodium hydroxide VS is required to change the colour of the indicator. Chlorides Not more than 0.045% (Appendix 9.4.5).

SODIUM THIOSULFATE

VP V

5 ml of solution S diluted to 15 ml with water complies with the limit test for chlorides.

Calcium Not more than 0.045% (Appendix 9.4.3), if intended for use in the manufacture of parenteral preparations. 10 ml of solution S diluted to 15 ml with distilled water complies with the limit test for calcium. Iron Not more than 90 ppm (Appendix 9.4.13), if intended for use in the manufacture of parenteral preparations. 5 ml of solution S diluted to 10 ml with water complies with the limit test for iron. Magnesium Not more than 0.02%, if intended for use in the manufacture of parenteral preparations. Add 1 ml of glycerol (85%) R, 0.15 ml of titan yellow solution R, 0.25 ml of 4% solution of ammonium oxalate R and 5 ml of 2 M sodium hydroxide R to 10 ml of solution S and shake. Any pink colour in the test solution is not more intense than that in a standard prepared at the same time in the same manner using a mixture of 5 ml of magnesium standard solution (10 ppm Mg) R and 5 ml of water. Heavy metals Not more than 45 ppm (Appendix 9.4.8). 12 ml of solution S complies with limit test for heavy metals, method 1. Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 130 °C). Assay Dissolve 0.100 g of the substance to be examined in 40 ml of water. Add a mixture of 0.2 ml of 0.1 M hydrochloric acid R and 80 ml of methanol R. Carry out a potentiometric titration (Appendix 10.2), using 0.1 M lead nitrate VS and as indicator electrode a lead-selective electrode and as reference electrode a silver-silver chloride electrode. 1 ml of 0.1 M lead nitrate VS is equivalent to 14.20 mg of Na2SO4. Storage Store in an airtight container. Action and use Laxative. Labelling The label states, where applicable, that the substance is suitable for use in the manufacture of parenteral preparations.

SODIUM THIOSULFATE Natrii thiosulfas Sodium hyposulfit Na2S2O3,5H2O  

M. 248.2

Sodium thiosulfate contains not less than 99.0% and not more than 101.0% of Na2S2O3,5H2O.

Characters Transparent, colourless crystals, efflorescent in dry air. Very soluble in water, practically insoluble in ethanol (96%). It dissolves in its water of crystallisation at about 49 °C. Identification Solution S: Dissolve 10.0 g in carbon dioxide-free water R and dilute to 100 ml with the same solvent. A. It decolourises iodinated potassium iodide solution R. B. To 0.5 ml of solution S add 0.5 ml of water and 2 ml of 0.1 N silver nitrate R. A white precipitate is formed which rapidly becomes yellowish and then black. C. To 2.5 ml of solution S add 2.5 ml of water and 1 ml of hydrochloric acid R. A precipitate of sulfur is formed and gas is evolved which gives a blue colour to starch iodate paper R. D. 1 ml of solution S gives reaction of sodium (Appendix 8.1). Appearance of solution Dissolve 10.0 g in 50 ml of distilled water R, add 1 ml of 0.1 M sodium hydroxide R and dilute to 100 ml with distilled water R. The freshly prepared solution is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). pH 6.0 to 8.4 (Appendix 6.2). Determined on the freshly prepared solution S. Sulfates and sulfites Not more than 0.2% (Appendix 9.4.14). Dilute 2.5 ml of freshly prepared solution S to 10 ml with distilled water R. To 3 ml of this solution first add 2 ml of iodinated potassium iodide solution R and continue the addition dropwise until a very faint persistent yellow colour appears. Dilute to 15 ml with distilled water R. Sulfides Add 0.5 ml of a freshly prepared 0.5% solution of sodium nitroprusside R to 10 ml of solution S. The solution does not become violet. Heavy metals Not more than 10 ppm (Appendix 9.4.8). Add 0.05 ml of sodium sulfide solution R to 10.0 ml of solution S. After 2 min, any brown colour in the solution is not more intense than that in a reference solution prepared at the same time and in the same manner using 10 ml of lead standard solution (1 ppm Pb) R and 0.05 ml of sodium sulfide solution R. 887

VP V

SODIUM THIOSULFATE TABLETS

Assay Dissolve 0.500 g of the substance to be examined in 20 ml of water and titrate with 0.1 N iodine VS, using 1 ml of starch solution R, added towards the end of the titration, as indicator. 1 ml of 0.1 N iodine VS is equivalent to 24.82 mg of Na2S2O3,5H2O. Storage In an airtight container. Action and use Used in treatment of cyanide poisoning. Preparations Injection, tablets. SODIUM THIOSULFATE TABLETS Tabellae Natrii thiosulfas Sodium thiosulfate tablets contain sodium thiosulfate. They are made gastro-resistant by enteric coating. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of sodium thiosulfate, Na2S2O3,5H2O, 95.0% to 105.0% of the stated amount. Identification Solution S: Remove the coating layer and powder finely. Dissolve a quantity of the powdered tablets containing about 1.0 g of sodium thiosulfate in 10 ml of carbon dioxide-free water R and filter. A. Solution S gives reactions of sodium (Appendix 8.1). B. To 1 ml of solution S add a few drops of 0.1 N iodine R, the colour of iodine disappears. C. To 1 ml of solution S add 1 ml of hydrochloric acid R. A precipitate of sulphur is formed; and the smell of sulphur dioxide is evolved. D. To 1 ml of solution S add 2 ml of 0.1 N silver nitrate R. A white precipitate is formed which rapidly becomes yellowish and then black. Disintegration The tablets comply with “Disintegration test for gastroresistant tablets” (Appendix 11.7). Assay Weigh 20 tablets (with coating layer removed), calculate the average mass and powder finely. Dissolve an accurately weighed quantity of the powdered tablets containing about 0.5 g of sodium thiosulfate with 20 ml of water and titrate with 0.1 N iodine VS, using 1 ml of starch solution R, added towards the end of the titration, as indicator. 1 ml of 0.1 N iodine VS is equivalent to 24.82 mg of Na2S2O3,5H2O. 888

Storage Stored in a well-closed container, in a cool and dry place, protected from light. Action and use Used in treatment of cyanide poisoning. Antifungal. Usual strength 0.33 g. SODIUM VALPROATE Valproas natrii H 3C

CO2Na

H 3C

C8H15NaO2  

M: 166.2

Sodium valproate is sodium 2-propylpentanoate. It contains not less than 98.5% and not more than 101.0% of C8H15NaO2, calculated with reference to the dried substance.

Characters White or almost white crystalline powder, it shows polymorphism, hygroscopic. Very soluble in water, freely soluble in ethanol (96%). Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of sodium valproate RS. If the spectra obtained in the solid state show differences, record new spectra using discs prepared by placing 50 µl of a 10% solution of the substance to be examined in methanol R on a disc of potassium bromide R and evaporating the solvent in vacuo. Examine immediately. B. Solution S: Dissolve 1.25 g of the substance to be examined in 20 ml of distilled water R in a separating funnel, add 5 ml of dilute nitric acid R and shake. Allow the mixture to stand for 12 h. Use the aqueous lower layer. 2 ml of solution S gives reaction of sodium (Appendix 8.1). Appearance of solution Dissolve 2.0 g in water and dilute to 10 ml with the same solvent. The solution is not more opalescent than reference suspension II (Appendix 9.2) and not more intensely coloured than reference solution Y6 (Appendix 9.3). Acidity or alkalinity Dissolve 1.0 g in 10 ml of water. Add 0.1 ml of phenolph thalein solution R as indicator. Not more than 0.75 ml of

SODIUM VALPROATE TABLETS

VP V

0.1 M hydrochloric acid or 0.1 M sodium hydroxide is required to change the colour of the indicator.

Related substances Examine by gas chromatography (Appendix 5.2). Test solution: Dissolve 0.500 g of the substance to be examined in 10 ml of water. Add 5 ml of dilute sulfuric acid R and shake with 3 quantities, each of 20 ml, of heptane R. Dilute the combined upper layers to 100.0 ml with heptane R. Reference solution (1): Dissolve 5 mg of valproic acid for system suitability RS (containing impurity K) in 1.0 ml of heptane R. Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with heptane R. Chromatographic system: A fused-silica capillary column (30 m long and 0.53 mm in internal diameter) coated with a film of macrogol 20 000 2-nitroterephthalate R (film thickness 0.5 µm). Carrier gas: Helium for chromatography. Flow rate: 8 ml/min. Temperature programme:

Column

Time (min)

Temperature (°C)

0-5 5 - 15 15 - 28.3 28.3 - 30

80 80 → 150 150 → 190 190

Injection port

220

Detector

220

Detector: A flame-ionisation detector. Volume of injection: 1 µl. Procedure: Inject the test solution and reference solutions (1), (2). Relative retention with reference to valproic acid (retention time = about 17 min): Impurity K = about 0.97. System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to impurity K and valproic acid is at least 2.0. Limits: In the chromatogram obtained with the test solution: Impurity K: The area of the peak due to impurity K is not more than 0.15 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.15%). Any other impurity: For each impurity, the area is not more than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). The sum of the peak areas of all impurities is not more than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.2%). Disregard any peak with an area less than 0.03 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.03%).

Impurity B: (2RS)-2-ethylpentanoic acid. Impurity C: (2RS)-2-(1-methylethyl)pentanoic acid. Impurity D: 2,2-dipropylpentanoic acid. Impurity F: 2-propylpentanamide. Impurity G: 2,2-dipropylpentanamide. Impurity I: 2-propylpentanenitrile. Impurity J: 2,2-dipropylpentanenitrile. Impurity K: (2RS)-2-ethyl-2-methylpentanoic acid. Impurity L: (2RS)-2-methylpentanoic acid.

Chlorides Not more than 200 ppm (Appendix 9.4.5). Add 10 ml of water to 5 ml of solution S and determine. Sulfates Not more than 200 ppm (Appendix 9.4.14). Determined on solution S. Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 2.0% (Appendix 9.6). (1.000 g; 105 °C). Assay Dissolve 0.150 g of the substance to be examined in 25 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 16.62 mg of C8H15NaO2. Storage Preserve in an airtight container, protected from light. Action and use Antiepileptic. Preparation Tablets. SODIUM VALPROATE TABLETS Tabellae natrii valproatis Sodium valproate tablets contain sodium valproate. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of sodium valproate, C8H15NaO2, 95.0% to 105.0% of the stated amount.

Note: Impurity A: pentanoic acid (valeric acid).

889

VP V

SORBITOL

Identification A. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution correspond to that in the chromatogram obtained with the reference solution. B. Shake a quantity of the powdered tablets equivalent to 0.25 g of sodium valproate with 3 ml of water, contrifuge. To 2 ml of the supernatant liquid add 0.5 ml of a 10% solution of cobalt nitrate R. A purple precipitate is produced which is soluble in dichloromethane R. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of phosphate buffer pH 6.8 R. Rotation speed: 50 rpm. Time: 45 min. Procedure: Examine by liquid chromatography (Appendix 5.3). Chromatographic system and mobile phase: prepare as directed in the Assay. Test solution: After the specified time (45 min), withdraw a sample of the medium, filter and dilute the filtrate with phosphate buffer pH 6.8 R to obtain a solution containing 0.02 mg/ml of sodium valproate. Reference solution: A 0.02 mg/ml solution of sodium valproate RS in phosphate buffer pH 6.8 R. Tolerance: Not less than 70% (Q) of the stated amount of sodium valproate, C8H15NaO2, is dissolved in 45 minutes. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: A 0.32% solution of potassium dihydrogen phosphate previously adjusted to pH 3.0 with phosphoric acid R - acetonitrile (45 : 55). Reference solution: A solution of sodium valproate RS in the mobile phase having a known concentration of 0.5 mg/ml. Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Transfer an accurately weighed quantity of the powdered tablets containing the equivalent of 100 mg of sodium valproate to a 200 volumetric flask, add 160 ml of the mobile phase and dissolve by sonicating. Allow to cool, dilute to volume with the mobile phase, mix and filter. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 μm). Detector: A spectrophotometer set at 220 nm Flow rate: 2.0 ml/min. Volume of injection: 20 μl. Procedure: System suitability: Inject the reference solution, the tailing factor of the principal peak is not more than 1.5, the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject separately the reference solution and the test solution. Calculate the content of sodium valproate, C8H15NaO2, in the tablets using the peak areas in the chromatograms 890

obtained with the test solution and the reference solution and the declared content of C8H15NaO2 in sodium valproate RS.

Storage Store in an airtight container, in a cool and dry place. Action and use Antiepileptic. Usual strength 200 mg, 300 mg, 500 mg. SORBITOL Sorbitolum

C6H14O6 

M. 182.2

Sorbitol is D-glucitol (D-sorbitol). It contains not less than 97.0% and not more than 102.0% of C6H14O6, calculated with reference to the anhydrous substance.

Characters White or almost white, crystalline powder, it shows polymorphism. Very soluble in water, practically insoluble in ethanol (96%). Identification Apply one of the two following identifications: First identification: A. Second identification: B, C, D. A. In the Assay, the principal peak in the chromatogram obtained with the test solution is similar in retention time and size to the principal peak in the chromatogram obtained with reference solution (1). B. Dissolve 0.5 g of the substance to be examined with heating in a mixture of 5 ml of acetic anhydride R and 0.5 ml of pyridine R, allow to stand for 10 min. Pour the solution into 25 ml of water and allow to stand in iced water for 2 h and filter. The precipitate, recrystallised from a small volume of ethanol (96%) R and dried in vacuo, melts at 98 °C to 104 °C (Appendix 6.7). C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Propanol - ethyl acetate - water (70 : 20 : 10). Test solution: Dissolve 25 mg of the substance to be examined in water and dilute to 10 ml with the same solvent. Reference solution (1): Dissolve 25 mg of sorbitol RS in water and dilute to 10 ml with the same solvent. Reference solution (2): Dissolve 25 mg of mannitol RS and 25 mg of sorbitol RS in water and dilute to 10 ml with the same solvent.

SORBITOL

VP V

Procedure: Apply separately to the plate 2 µl of each solution. Develop over a path of 17 cm. Allow the plate to dry in air and spray with 4-aminobenzoic acid solution R. Dry the plate in a current of cold air until the acetone is removed totally; heat at 100 °C for 15 min. Allow to cool and spray with a 0.2% solution of sodium periodate R; dry in a current of cold air; heat at 100 °C for 15 min. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with the reference solution (2) shows two clearly separated spots. D. Specific optical rotation: +4.0° to +7.0°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 5.00 g of the substance to be examined and 6.4 g of disodium tetraborate R in 40 ml of water. Allow to stand for 1 h, shaking occasionally, and dilute to 50.0 ml with water. Filter if necessary.

Appearance of solution Dissolve 5.0 g in water and dilute to 50 ml with the same solvent. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). Conductivity Not more than 20 µS·cm-1 (Appendix 6.10). Dissolve 20.0 g in carbon dioxide-free water R and dilute to 100.0 ml with the same solvent. Measure the conductivity of the solution while gently stirring with a magnetic stirrer. Reducing sugars Not more than 0.2%, expressed as glucose equivalent. Dissolve 5.0 g of the substance to be examined in 6 ml of water with the aid of gentle heat. Cool and add 20 ml of cupri-citric solution R and a few glass beads. Heat so that boiling begins after 4 min and maintain boiling for 3 min. Cool rapidly and add 100 ml of a 2.4% v/v solution of glacial acetic acid R and 20.0 ml of 0.05 N iodine VS. With continuous shaking, add 25 ml of a mixture hydrochloric acid - water (6 : 94) and, when the precipitate has dissolved, titrate the excess of iodine with 0.05 N sodium thiosulfate VS using 1 ml of starch solution R, added towards the end of the titration, as indicator. Not less than 12.8 ml of 0.05 N sodium thiosulfate VS is required. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Degassed water. Test solution: Dissolve 5.0 g of the substance to be examined in 20 ml of water and dilute to 100.0 ml with the same solvent. Reference solution (1): Dissolve 0.50 g of sorbitol RS in 2 ml of water and dilute to 10.0 ml with the same solvent. Reference solution (2): Dilute 2.0 ml of the test solution to 100.0 ml with water.

Reference solution (3): Dilute 5.0 ml of reference solution (2) to 100.0 ml with water. Reference solution (4): Dissolve 0.5 g of sorbitol R and 0.5 g of mannitol R (impurity A) in 5 ml of water and dilute to 10.0 ml with the same solvent. Chromatographic system: A column (0.3 m × 7.8 mm) packed with stationary phase strong cation exchange resin (calcium form) R (9 µm). Column temperature: 85 °C ± 1 °C. Flow rate: 0.5 ml/min. Detection: Refractometer maintained at a constant temperature. Volume of injection: 20 µl. Procedure: Inject the test solution and reference solutions (2), (3) and (4). The run time is twice times the retention time of sorbitol. Relative retention with reference to sorbitol (retention time = about 27 min): Impurity C = about 0.6; impurity A = about 0.8; impurity B = about 1.1. System suitability: In the chromatogram obtained with reference solution (4), the resolution between the peaks due to impurity A and sorbitol is at least 2. Limits: For each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (2%). The sum of the peak areas of all impurities is not more than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (3%). Disregard any peak with an area less than the area of the principal peak in the chromatogram obtained with reference solution (3) (0.1%). Note: Impurity A: D-mannitol. Impurity B: D-iditol. Impurity C: 4-O-α-D-glucopyranosyl-D-glucitol (D-maltitol).

Lead Not more than 0.5 ppm (Appendix 9.4.4). Nickel Not more than 1 ppm (Appendix 9.4.10). Dissolve the substance to be examined in 150.0 ml of the prescribed mixture of solvents. Water Not more than 1.5% (Appendix 10.3). Determined on 1.00 g. Microbial contamination (Appendix 13.6) If intended for use in the manufacture of parenteral preparations: Total aerobic microbial count is not more than 102 CFU/g. If not intended for use in the manufacture of parenteral preparations: Total aerobic microbial count is not more than 103 CFU/g. 891

VP V

SORBITOL POWDER

and total combined yeasts/moulds count is not more than 102 CFU/g. Absence of Escherichia coli, Salmonella.

Bacterial endotoxins (Appendix 13.2) Not more than 4 EU/g for parenteral preparations having a concentration of less than 100 g/l of sorbitol and not more than 2.5 EU/g for parenteral preparations having a concentration of 100 g/l or more of sorbitol if intended for use in the manufacture of parenteral preparations without a further appropriate procedure for the removal of bacterial endotoxins. Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. Inject the test solution and reference solution (1). Calculate the content of D-sorbitol using the areas in the chromatograms obtained with the test solution, the reference solution (1) and the declared content of C6H14O6 in sorbitol RS. Storage Store in an airtight container. Action and use Used for parenteral nutrition. Aperient. Labelling The label states: where applicable, the maximum concentration of bacterial endotoxins and that the substance is suitable for use in the manufacture of parenteral preparations. SORBITOL POWDER Pulveres Sorbitoli Sorbitol powder contains sorbitol. The powder complies with the requirements stated under “Powder” (Appendix 1.7) and with the following requirements.

Content of sorbitol, C6H14O6, 95.0% to 105.0% of the labelled amount. Characters A white powder with sweet taste. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Propanol - ethyl acetate - water (70 : 20 : 10) Test solution: Dissolve 50 mg of the powder in water and dilute to 20.0 ml with the same solvent. Reference solution: Dissolve 50 mg of sorbitol RS in water and dilute to 20.0 ml with the same solvent. 892

Procedure: Apply separately to the plate 2 µl of each solution. Develop over a path of 17 cm. Allow the plate to dry in air and spray with 4-aminobenzoic acid solution R. Dry the plate in a current of cold air until the acetone is removed totally. Heat the plate at 100 °C for 15 min. Allow to cool and spray with a 0.2% solution of sodium periodate R. Dry the plate in a current of cold air. Heat the plate at 100 °C for 15 min. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. B. Dissolve a quantity of the powder equivalent to 7 g of sorbitol in 10 ml of water. To 1 ml of the resulting solution add 2 ml of a 8% solution of iron (II) sulfate R and 1 ml of 20% sodium hydroxyd solution R, a blue colour develops which turns gradually to green but no turbidity is formed. C. To 3 ml of a freshly prepared 10% solution of pyrocatechol R, add 6 ml of sulfuric acid R while cooling in iced water. To 3 ml of the cooled mixture add 0.3 ml of a solution of the powder containing the equivalent of 10% of sorbitol in carbon dioxide free water R. Heat gently for 30 seconds, a pink colour develops.

Loss on drying Not more than 2.0% (Appendix 9.6). (0.5 g; over phosphorus pentoxide, in vacuum at 80 °C for 3 h). Assay Weigh accurately a quantity of the powder equivalent to about 0.4 g of sorbitol in a 100 ml volumetric flask, add water to volume, mix well. Pipette 10.0 ml of the solution in a 250 ml conical flask, add 20 ml of a 2.14% solution of sodium periodate R and 2 ml of 1 M sulfuric acid R, heat on a water-bath for exactly 15 minutes. Cool and add 3 g of sodium hydrogen carbonate R little by little and 25.0 ml of 0.1 M sodium arsenite VS. Mix well and add 5 ml of a 20% solution of potassium iodid R and allow to stand for 15 minutes. Titrate with 0.1 N iodine VS until the first trace of yellow colour appears. Carry out a blank titration. 1 ml of 0.1 N iodine VS is equivalent to 1.822 mg of C6H14O6. Storage Store in a dry and cool place, protected from light. Action and use Osmotic laxative. Usual strength 5 g.

SPARTEINE SULFATE

VP V

SPARTEINE SULFATE Sparteini sulfas H

H N . H 2SO 4 . 5 H 2O

N H

H

C15H26N2,H2SO4,5H2O M.422.4 Sparteine sulfate is the sulfate salt of dodecahydro methano-7,14-2H,6H-dipyrido[1,2-a:1’,2’-e][diazocin1,5]-(7S,7aR,14S,14aS). It contains not less than 98.0% and not more than 101.0% of C15H28O4N2S, calculated with reference to the dried substance.

Characters Colourless crystals. Freely soluble in water and in ethanol (96%), practically insoluble in ether. Identification Apply one of the two following identifications: First identification: C, D, E. Second identification: A, B, E. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of sparteine sulfate RS. B. It complies with test for Specific optical rotation. C. Examine the chromatograms obtained in the test for Related substances. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). D. Dissolve 1 g of the substance to be examined in 10 ml of water. To 5 ml of this solution add 1 ml of a 5% solution of mercury (II) chloride R, no precipitate is produced. With vigorously shaking, add dropwise 0.5 ml of hydrochloric acid R, a precipitate is produced. Separate the upper clear solution, add 1 ml of 10 N sodium hydroxide R, a milky turbid layer is produced. Warm in a water bath, small oildrops are produced on the surface. E. It gives the reactions of sulfates (Appendix 8.1). Appearance of solution Solution S: Dissolve 1.0 g of the substance to be examined in carbon dioxide-free water R, and dilute to 50.0 ml with the same solvent, and mix. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). pH The pH of solution S is 3.0 to 4.0 (Appendix 6.2).

Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Diethylamine - ethyl acetate - cyclohexane (5 : 25 : 70). Test solution: Dissolve 0.1 g of the substance to be examined in methanol R and dilute to 5 ml with the same solvent, and mix. Reference solution (1): Dissolve 0.1 g of sparteine sulfate RS in methanol R and dilute to 5 ml with the same solvent, and mix. Reference solution (2): Dilute 1.0 ml of reference solution (1) to 100.0 ml with methanol R, and mix. Reference solution (3): Dilute 5 ml of reference solution (2) to 10.0 ml with methanol R, and mix. Procedure: Apply separately to the plate 5 µl of each of the solutions. Develop over a path of 15 cm. Remove the plate, dry it at 100 °C to 105 °C for 5 minutes and allow to cool. Spray with potassium iodobismuthate solution R until appear the spots. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the principal spot in the chromatogram obtained with reference solution (2), and not more than one such spot is more intense than the principal spot in the chromatogram obtained with reference solution (3). Loss on drying 19.5% to 22.0% (Appendix 9.6). (1.0 g; 100 °C - 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 1). Determined on the dried substance obtained in the test for Loss on drying. Assay Dissolve 0.300 g, accurately weighed, of the substance to be examined in 30 ml of anhydrous acetic acid R. Add 1 ml of acetic anhydride (TT). Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 33.24 mg of C15H28O4N2S. Storage Store in an airtight container. Action and use Antiarrhythmics. Preparation Injection.

Specific optical rotation -26° to -30°, calculated with reference to the dried substance (Appendix 6.4). Determined on solution S. 893

VP V

SPARTEINE SULFATE INJECTION

SPARTEINE SULFATE INJECTION Injectio Sparteini sulfatis Sparteine sulfate injection is a sterile solution of sparteine sulfate in water for injection. The injection complies with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Content of sparteine sulfate, C15H26N2, H2SO4, 5H2O, 95.0% to 105.0% of the stated amount. Characters A clear, colourless solution. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Diethylamine - ethyl acetate - cyclohexane (5 : 25 : 70). Test solution: Evaporate a volume of the injection containing the equivalent of 0.1 g of sparteine sulfate, dissolve the residue obtained in 5 ml of methanol R. Reference solution: Dissolve 0.1 g of sparteine sulfate RS in 5 ml of methanol R. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm. Dry the plate at 100 °C to 105 °C for 5 min. Allow to cool and spray with potassium iodobismuthate solution R until the spots appear. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. B. Evaporate a volume of the injection containing the equivalent of 1 g of sparteine sulfate, dissolve the residue obtained in 10 ml of water. To 5 ml of the solution add 1 ml of a 5% solution of mercury(II) chloride R, no precipitate is formed. With vigorous shaking, add 0.5 ml of hydrochloric acid R dropwise, a precipitate is produced. To the rest of the solution add 1 ml of 10 M sodium hydroxide R, a milky turbid layer is formed. Warm in a water bath, small oildrops are produced on the surface. C. Yields the reactions of sulfates (Appendix 8.1). pH 3.0 to 4.0 (Appendix 6.2). Assay Pipet exactly a volume of the injection containing the equivalent of about 0.5 g of sparteine sulfate and transfer into a separator, add 8 ml of ammonia R. Extract with four 20-ml quantities of ether R. Filter the ether extracts through about 2 g of anhydrous sodium sulfate R supported on absorbent cotton, wash the filter with 10 ml of ether R. Combine the extracts and washing, evaporate on a waterbath until the volume is reduced to 10 ml, continue to evaporate the ether in a current of air. Dissolve 894

the residue in 20 ml of ethanol (90%) R, titrate with 0.1 N hydrochloric acid VS using methyl red solution R as indicator. Carry out a blank determination. 1 ml of 0.1 N hydrochloric acid VS is equivalent to 0.04224 g of C15H26N2,H2SO4,5H2O.

Storage Store in a cool place, protected from light. Action and use Cardiotonic. Usual strength 5%. SPECTINOMYCIN HYDROCHLORIDE Spectinomycini hydrochloridum

C14H24N2O7,2HCl,5H2O

M. 495.35

Spectinomycin hydrochloride is (2R,4aR,5aR,6S, 7S,8R,9S, 9aR,10aS)-4a,7,9-trihydroxy-2-methyl-6,8-bis(methylamino)decahydro-4H-pyrano[2,3-b][1,4]benzodioxin-4-one dihydrochloride pentahydrate (spectinomycin dihydrochloride pentahydrate). It contains not less than 603 µg spectinomycin (C14H24N2O7) per mg.

Characters White or almost white, slightly hygroscopic powder. Freely soluble in water, very slightly soluble in ethanol (96%). Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of spectinomycin hydrochloride RS (do not dry specimen). pH 3.8 to 5.6 (Appendix 6.2). Determined on a solution containing 10 mg/ml of the substance to be examined. Water 16.0% to 20.0% (Appendix 10.3). Determined on 0.100 g. Sulfated ash Not more than 1.0% (Appendix 9.9, method 2). Determined on 1.0 g. The charred residue being moistened with 2 ml of nitric acid R and 5 drops of sulfuric acid R.

SPIRAMYCIN

VP V

Bacterial endotoxins Not more than 0.09 EU/mg (Appendix 13.2). If intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins.

SPIRAMYCIN Spiramycinum

Sterility Test procedure using membrane filtration method (Appendix 13.7). If the substance is sterile, it complies with the test for sterility. Assay Examine by gas chromatography (Appendix 5.2). Internal standard solution: Dissolve triphenylantimony R in dimethylformamide R to obtain a solution containing about 2 mg/ml. Standard solution: Transfer about 30 mg of spectinomycin hydrochloride RS, accurately weighed, to a glassstoppered, 25-ml conical flask. Add 10.0 ml of internal standard solution and 1.0 ml of hexamethyldisilazane R, and shake intermittently for 1 h. Test solution: Proceed as directed under standard solution using the substance to be examined. Chromatographic system: A glass column (60 cm × 3 mm) packed with silanised diatomaceous earth for gas chromatography R impregnated with 5% of poly[methyl(95)phenyl(5)siloxane. Carrier gas: Helium for chromatography. Flow rate: 45 ml/min. Temperature: Column 190 °C and detector 220 °C and injection port 215 °C. Detector: A flame-ionisation detector. Volume of injection: 1 µl. Procedure: System suitability: In the chromatogram obtained with standard solution, the resolution between the major peaks is at least 2.0. The relative standard deviation of the peak response ratios, RS, from replicate injections of the standard preparation is not more than 3.5%. Calculate the ratio of the response of the spectinomycin peak to the response of the internal standard peak in the chromatogram from the test solution (Ru) and similarly calculate the ratio in the chromatogram from the standard solution (Rs). Calculate the quantity, in µg, of C14H24N2O7 in the substance to be examined by the formula: P(WS)(Ru/Rs) in which: P is the potency of spectinomycin hydrochloride RS (µg/mg). WS is the weight spectinomycin hydrochloride RS in the standard solution (mg). Storage In an airtight container. If the substance is sterile, store in a sterile, airtight, tamper-proof container. Action and use Antibacterial.

Spiramycinisamacrolideantibioticproducedbythegrowthofcertain strains of Steptomyces ambofaciens or obtained by any other means. The main component is (4R,5S,6S,7R,9R,10R,11E,13E,16R)6-[[3,6-dideoxy-4-O-(2,6-dideoxy-3-C-methyl-α-L-ribohexopyranosyl)-3-(dimethylamino)-β-D-glucopyranosyl]oxy]-4 hydroxy - 5 -methoxy-9,16-dimethyl-7-(2-oxoethyl)-10-[[2,3,4,6tetradeoxy - 4 - (dimethylamino) - D -erythro-hexopyranosyl] oxy] oxacyclohexadeca-11,13-dien-2-one (spiramycin I; M.843). Spiramycin II (4-O-acetylpiramycin I) and spiramycin III (4-O-propanoylspiramycin I) are also present. The potency is at least 4100 IU/mg, calculated with reference to the dried substance.

Characters A white or slightly yellowish powder, slightly hygroscopic. Slightly soluble in water, freely soluble in acetone, in ethanol (96%) and in methanol. Identification A. Dissolve 0.10 g of the substance to be examined in methanol R and dilute to 100.0 ml with the same solvent, and mix. Dilute 1.0 ml of the solution to 100.0 ml with methanol R, and mix. The ultraviolet absorption spectrum (Appendix 4.1) of this solution, in the range between 220 nm and 350 nm, exhibits an absorption maximum at 232 nm. The value of A(1%; 1 cm) is about 340. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: The upper layer of a mixture of isopropanol, a 15% solution of ammonium acetate previously adjusted to pH 9.6 with a 40% solution of sodium hydroxide and ethyl acetate (4 : 8 : 9). Test solution: Dissolve 40 mg of the substance to be examined in methanol R and dilute to 10 ml with the same solvent, and mix. 895

VP V

SPIRAMYCIN

Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use. Mobile phase: Acetonitrile - buffer solution pH 2.2 containing 0.93% of sodium perchlorate (30 : 70). 896

μV 1.0E+05 8.0E+04 6.0E+04 4.0E+04 2.0E+04 0.0E+00

10.00

20.00

Spiramycin III

Composition Examine by liquid chromatography (Appendix 5.3). Not less than 80.0% of spiramycin I, not more than 5.0% of spiramycin II and not more than 10.0% of spiramycin III; the sum of the contents of spiramycin I, spiramycin II and spiramycin III is not less than 90.0%, all contents are calculated with reference to the dried substance. Mobile phase, test solution, reference solution (1) and chromatographic system: As directed in the test for Related substances. Inject alternately the test solution and reference solution (1). Calculate the percentage contents of spiramycin I, spiramycin II and spiramycin III in RS .

Spiramycin II

Specific optical rotation -80° to -85°, calculated with reference to the dried substance (Appendix 6.4). Dissolve 1.00 g of the substance to be examined in 0.2 M acetic acid R and dilute to 50.0 with the same solvent.

Spiramycin

pH Dissolve 0.5 g of the substance to be examined in 5 ml of methanol R and dilute to 100 ml with carbon dioxide-free water R, and mix. The pH of the solution is 8.5 to 10.5 (Appendix 6.2).

Solvent mixture: A mixture of acetonitrile R and water (3 : 7). Test solution: Dissolve 25.0 mg of the substance to be examined in the solvent mixture and dilute to 100.0 ml with the same mixture, and mix. Reference solution (1): Dissolve 25.0 mg of spiramycin RS in the solvent mixture and dilute to 100.0 ml with the same mixture, and mix. Reference solution (2): Dilute 2.0 ml of reference solution (1) to 100.0 ml with the solvent mixture, and mix. Reference solution (3): Dilute 5.0 ml of reference solution (1) to 100.0 ml with the solvent mixture, and mix. Reference solution (4): Dissolve 5 mg of spiramycin RS in 25 ml of the mobile phase, then heat in a water bath at 60 °C for 30 minutes. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase B (5 µm) with a specific surface of 350 m2/g and a pore size of 0.01 µm. Detector: A spectrophotometer set at 232 nm. Flow rate: 0.8 ml/min. Volume of injection: 20 µl. Procedure: Inject reference solution (2). Adjust the sensitivity of the system so that the height of the principal peak is at least 50% of the full scale of the recorder. Inject reference solution (3) and reference solution (4). The test is not valid unless in the chromatogram obtained with reference solution (3), there is no significant peak with a retention time, relative to spiramycin I, of about 1.1; in the chromatogram obtained with reference solution (4), the resolution between impurity A (neospiramycin I, eluting first) and spiramycin I (eluting at 13 to 17 minutes) is at least 6.3. If necessary, adjust the concentration of acetonitrile in the mobile phase (increasing the concentration to decrease the retention time or decreasing the concentration to increase the retention time). Inject the test solution and reference solution (2). Record the chromatogram of the test solution for 3 times the retention time of spiramycin I. Limits: In the chromatogram obtained with the test solution: Neospiram.I (Impurity A)

Reference solution (1): Dissolve 40 mg of spiramycin RS in methanol R and dilute to 10 ml with the same solvent, and mix. Reference solution (2): Dissolve 40 mg of erythromycin A RS in methanol R and dilute to 10 ml with the same solvent, and mix. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm. Remove the plate, dry it in air, spray with anisaldehyde solution in ethanol R and heat at 110 °C for 5 minutes. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to that in the chromatogram obtained with reference solution (1). If in the chromatogram obtained with the test solution one or two spots occur with Rf values slightly higher than that of the principal spot, these spots are similar in position and colour to the secondary spots in the chromatogram obtained with reference solution (1) and differ from the spots in the chromatogram obtained with reference solution (2). C. Dissolve 0.5 g of the substance to be examined in 10 ml of 0.05 M sulfuric acid R and add 25 ml of water. Adjust to about pH 8 with 0.1 M sodium hydroxide R and dilute to 50 ml with water, and mix. To 5 ml of this solution add 2 ml of a mixture of 1 volume of water and 2 volumes of sulfuric acid R. A brown colour develops.

30.00

40.00 [min]

The area of any peak, apart from the peaks corresponding to spiramycin I, spiramycin II and spiramycin III, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (2) (2%). Disregard any peak with an area less than 0.05 times the

PREGELATINISED STARCH

VP V

area of the principal peak in the chromatogram obtained with reference solution (2).

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g of the substance to be examined complies with the limit test for heavy metals, method 6. Prepare the reference solution using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 3.5% (Appendix 9.6). (0.500 g; 80 °C; diphosphorus pentoxide at a pressure not exceeding 670 Pa; 6 h). Sulphated ash Not more than 0.1% (Appendix 9.8, method 2). Determined on 1.0 g of the substance to be examined. Assay Carry out the Microbiological assay of antibiotics (Appendix 13.9). Use the diffusion method; micro-organism: Bacillus subtilis ATCC 6633; solvent: methanol R; Diluent: buffer No.2; medium: medium No.1 adjusted to pH of 8.0 ± 0.1; concentration range of the solutions to be tested: 40 IU/ml - 160 IU/ml; incubation temperature: 32 °C - 35 °C. Storage Store in an airtight container. Action and use Macrolide antibiotic. Preparation Tablets. SPIRAMYCIN TABLETS Tabellae Spiramycini Spiramycin tablets are film-coated tablets containing spiramycin. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) section Coated tablets and with the following requirements.

Content of spiramycin, 90.0% to 110.0% of the stated amount. Identification A. Dissolve a quantity of the powdered tablets containing about 400 000 IU of spiramycin in 100 ml of methanol R, filter. Dilute 1 ml of the filtrate to 100 ml with methanol R. The ultraviolet absorption (Appendix 4.1) of the solution in the range 220 nm to 350 nm exhibits an absorption maximum at 232 nm. B. Dissolve a quantity of the powdered tablets containing about 2 000 000 IU of spiramycin in 10 ml of 0.05 M

sulfuric acid R and add 25 ml of water, shake well and filter. Adjust the filtrate to pH 8 with 0.1 M sodium hydroxide and dilute to 50 ml with water. To 5 ml of this solution add 2 ml of a mixture of water - sulfuric acid (1 : 2). A brown colour is produced. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Isopropanol - 15% ammonium acetate solution previously adjusted to pH 9.6 with a 40% sodium hydroxide solution - ethyl acetate (4 : 8 : 9). Mix well and then allow to stand. Use the upper layer. Test solution: Shake a quantity of the powdered tablets containing 160 000 IU of spiramycin in 10 ml of methanol R, filter. Reference solution (1): A 0.4% solution of spiramycin RS in methanol R. Reference solution (2): A 0.4% solution of erythromycin A RS in methanol R. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm. Remove the plate, allow it to dry at room temperature. Spray with anisaldehyde solution in ethanol R and heat at 110 °C for 5 min. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1) and differs from the principal spot in the chromatogram obtained with reference solution (2).

Assay Weigh 20 tablets, calculate the average mass and powder finely. Weigh accurately a quantity of the powdered tablets containing 400 000 IU of spiramycin, transfer into a 100-ml volumetric flask. Add 50 ml of methanol R, sonicate for 15 min, allow it to cool to room temperature, dilute to volume with methanol R, centrifuge and use the supernatant liquid. Carry out the Microbiological assay of antibiotics (Appendix 13.9). Storage Store in a well-closed container, in a cool and dry place, protected from light. Action and use Antibacterial. Usual strength 375 000 IU; 1500 000 IU; 3 000 000 IU. PREGELATINISED STARCH Amylum pregelificatum Pregelatinised starch is prepared by chemical or mechanical processing in the presence of water to rupture all or part of the starch granules, and subsequent drying. It contains no added substances but it may be modified to render it compressible and to improve its flow characteristics. 897

VP V

MAIZE STARCH

Characters White or yellowish-white powder. It swells in cold water. Identification A. Examined under a microscope using a mixture of equal volumes of glycerin R and water it presents irregular, translucent, white or yellowish-white flakes or pieces with an uneven surface. Under polarised light (between crossed nicol prisms), starch granules with a distinct black cross intersecting at the hilum may be seen. B. Disperse 0.5 g of the substance to be examined in 2 ml of water without heating and add 0.05 ml of iodine solution R1. A reddish-violet to blue colour is produced. pH 4.5 to 7.0 (Appendix 6.2). Progressively add 3.0 g of the substance to be examined to 100.0 ml of carbon dioxide-free water R, stirring continuously until a homogeneous solution is obtained. Determine the pH of this solution. Iron Not more than 20 ppm (Appendix 9.4.13). Dissolve the residue obtained in the test for Sulfated ash in 20 ml of dilute hydrochloric acid R. Filter. The filtrate complies with the test. Oxidising substances It complies with the test for Oxidising substances(Appendix 7.10). Use a mixture of equal volumes of water and methanol R as solvent. Sulfur dioxide Not more than 80 ppm. Shake 20.0 g of the substance to be examined with 200 ml of a 20% solution of anhydrous sodium sulfate R, and filter. To 100.0 ml of the clear filtrate, add 3 ml of starch solution R, and titrate with 0.01 N iodine solution VS to the first permanent blue color. The volume of 0.01 N iodine solution VS is not more than 2.7 ml. Loss on drying Not more than 15.0% (Appendix 9.6). (1.000 g; 130 °C; 90 min). Foreign matter Examined under a microscope using a mixture of equal volumes of glycerin R and water, not more than traces of matter other than starch granules are present. Sulfated ash Not more than 0.6% (Appendix 9.9, method 2). Determined on 1.0 g. Microbial contamination (Appendix 13.6) Total aerobic microbial count (TAMC) is not more than 103 CFU/g and total combined yeasts/moulds count (TYMC) is not more than 102 CFU/g. 898

Absence of Salmonella and Escherichia coli. Storage Store in an airtight container. Action and use Excipients. Labelling The label states the type of starch used as starting material Pregelatinised Starch. MAIZE STARCH Amylum mays Maize starch is obtained from the caryopsis of Zea mays L., (Fam. Poaceae).

Characters Matt, white to slightly yellowish, very fine powder that creaks when pressed between the fingers. Practically insoluble in cold water and in ethanol (96%). The presence of granules with cracks or irregularities on the edge is exceptional. Identification A. Examined under a microscope, using not less than 20 × magnification and using a mixture of glycerol - water (1 : 1) to prepare samples, it appears as either angular polyhedral granules of irregular sizes with diameters ranging from about 2 µm to about 23 µm or as rounded or spheroidal granules of irregular sizes with diameters ranging from about 25 µm to about 35 µm. The central hilum consists of a distinct cavity or 2- to 5-rayed cleft and there are no concentric striations. Between orthogonally orientated polarising plates or prisms, the starch granules show a distinct black cross intersecting at the hilum. B. Suspend 1 g of the substance to be examined in 50 ml of water, boil for 1 min and cool. A thin, cloudy mucilage is formed. C. Add 0.05 ml of iodine solution R1 to 1 ml of the mucilage obtained in Identification test B. An orange-red to dark blue colour is produced, which disappears on heating. pH 4.0 to 7.0 (Appendix 6.2). Shake 5.0 g with 25.0 ml of carbon dioxide-free water R for 60 s. Allow to stand for 15 min. Foreign matter Examine under a microscope using a mixture of glycerol - water (1 : 1) to prepare samples. Not more than traces of matter other than starch granules are present. No starch grains of any other origin are present. Oxidising substances Not more than 20 ppm, calculated as H2O2 (Appendix 7.10).

RICE STARCH

VP V

Sulfur dioxide Not more than 50 ppm (Appendix 7.9, method 2). Iron Not more than 10 ppm (Appendix 9.4.13). Shake 1.5 g with 15 ml of 1 M hydrochloric acid R, filter. Determine on the filtrate. Loss on drying Not more than 15.0% (Appendix 9.6). (1.00 g; 130 °C; 90 min). Sulfated ash Not more than 0.6% (Appendix 9.9, method 2). Determined on 1.0 g.

C. Add 0.05 ml of iodine solution R1 to 1 ml of the mucilage obtained in Identification test B. An orange-red to dark blue colour is produced, which disappears on heating.

pH 5.0 to 8.0 (Appendix 6.2). Shake 5.0 g of the substance to be examined with 25.0 ml of carbon dioxide-free water R for 60 s. Allow to stand for 15 min. Foreign matter Examine under a microscope using a mixture of glycerol - water (1 : 1) to prepare samples. Not more than traces of matter other than starch granules are present. No starch grains of any other origin are present.

Microbial contamination (Appendix13.6) Total aerobic microbial count is not more than 103 CFU/g. Total combined yeasts/moulds count is not more than 102 CFU/g. Absence of Salmonella and Escherichia coli.

Oxidising substances Not more than 20 ppm, calculated as H2O2 (Appendix 7.10).

Storage Store in an airtight container.

Iron Not more than 10 ppm (Appendix 9.4.13). Shake 1.5 g of the substance to be examined with 15 ml of 1 M hydrochloric acid R, filter. Determine on the filtrate.

Action and use Excipients. POTATO STARCH Amylum Solani Potato starch is obtained from the tuber of Solanum tuberosum L., (Fam. Solanaceae).

Characters Very fine, white or almost white powder which creaks when pressed between the fingers. Practically insoluble in cold water and in ethanol (96%). Potato starch does not contain starch grains of any other origin. It may contain a minute quantity, if any, of tissue fragments of the original plant. Identification A. Examined under a microscope using a mixture of glycerol - water (1 : 1) to prepare samples: There are irregularly shaped, occasional compound granules having 2 - 4 components, all granules show clearly visible concentric striations. It presents granules, ovoid or pearshaped, usually 30 - 100 µm in size but occasionally exceeding 100 µm, have an eccentric hilum or rounded granules, 10 - 35 µm in size and the rounded granules acentric or slightly eccentric hilum. Between orthogonally orientated polarising plates or prisms, the granules show a distinct black cross intersecting at the hilum. B. Suspend 1 g of the substance to be examined in 50 ml of water, boil for 1 min and cool. A thin, cloudy mucilage is formed.

Sulfur dioxide Not more than 50 ppm (Appendix 7.9, method 2).

Loss on drying Not more than 20.0% (Appendix 9.6). (1.00 g; 130 °C; 90 min). Sulfated ash Not more than 0.6% (Appendix 9.9, method 2). Determined on 1.0 g. Microbial contamination (Appendix13.6) Total aerobic microbial count is not more than 103 CFU/g. Total combined yeasts/moulds count is not more than 102 CFU/g. Absence of Salmonella and Escherichia coli. Storage Store in an airtight container. Action and use Excipients. RICE STARCH Amylum oryzae Rice starch is obtained from the caryopsis of Oryza sativa L., (Fam. Poaceae).

Characters Very fine, white or almost white powder, which creaks when pressed between the fingers. Practically insoluble in cold water and in ethanol (96%). 899

VP V

TAPIOCA STARCH

Identification A. Examined under a microscope using a mixture of glycerol - water (1 : 1) to prepare samples, it presents polyhedral, simple grains 1 µm to 10 µm, mostly 4 µm to 6 µm, in size. These simple grains often gather in ellipsoidal, compound grains 50 - 100 µm in diameter. The grains have a poorly visible central hilum and there are no concentric striations. Between orthogonally orientated polarising plates or prisms, the starch grains show a distinct black cross intersecting at the hilum. B. Suspend 1 g of the substance to be examined in 50 ml of water, boil for 1 min and cool. A thin, cloudy mucilage is formed. C. Add 0.05 ml of iodine solution R1 to 1 ml of the mucilage obtained in Identification test B. An orange-red to dark blue colour is produced, which disappears on heating. pH 5.0 to 8.0 (Appendix 6.2). Shake 5.0 g of the substance to be examined with 25.0 ml of carbon dioxide-free water R for 60 s. Allow to stand for 15 min. Foreign matter Examine under a microscope using a mixture of glycerol - water (1 : 1) to prepare samples. Not more than traces of matter other than starch granules are present. No starch grains of any other origin are present. Iron Not more than 10 ppm (Appendix 9.4.13). Shake 1.5 g of the substance to be examined with 15 ml of 1 M hydrochloric acid R. Filter. Determine on the filtrate. Oxidising substances Not more than 20 ppm, calculated as H2O2 (Appendix 7.10). Sulfur dioxide Not more than 50 ppm (Appendix 7.9, method 2). Loss on drying Not more than 15.0% (Appendix 9.6). (1.00 g; 130 °C; 90 min). Sulfated ash Not more than 0.6% (Appendix 9.9, method 2). Determined on 1.0 g. Microbial contamination (Appendix13.6) Total aerobic microbial count is not more than 103 CFU/g. Total combined yeasts/moulds count is not more than 102 CFU/g. Absence of Salmonella and Escherichia coli. Storage Store in an airtight container. Action and use Excipients. 900

TAPIOCA STARCH Amylum Manihoti Tapioca Starch is obtained from the rhizomes of Manihot utilissima Pohl (Fam. Euphorbiaceae).

Characteristics Very fine powder which creaks when pressed between the fingers. Practically insoluble in cold water and in ethanol (96%). Identification A. Examined under a microscope: Principally simple granules, subspherical, muller-shaped or rounded polyhedral; smaller granules 5 µm to 10 µm, larger granules 20 µm to 35 µm in diameter; hilum, central, punctate, linear or triradiate; striations, faint, concentric; compound granules, few, of two to three unequal components. B. Suspend 1 g of the substance to be examined in 50 ml of water, boil for 1 min and cool. A thin, cloudy mucilage is formed. C. Add 0.05 ml of iodine solution R1 to 1 ml of the mucilage obtained in Identification test B. A dark blue colour is produced which disappears on heating and reappears on cooling. Acidity Add 10 g of the starch to 100 ml of ethanol (70%) previously neutralised to 0.5 ml of phenolphthalein solution, shake for 1 h, filter. Titrate 50 ml of the filtrate with 0.1 N sodium hydroxide VS. Not more than 2.0 ml is required to change the colour of the solution. Foreign matter Not more than traces of cell membranes and protoplasm are present. Loss on drying Not more than 15.0% (Appendix 9.6). (1 g; 100 °C to 105 °C). Sulfated ash Not more than 0.6% (Appendix 9.9, method 2). Determined on 1 g. Microbial contamination (Appendix13.6) Total aerobic microbial count is not more than 103 CFU/g. Total combined yeasts/moulds count is not more than 102 CFU/g. Absence of Escherichia coli. Storage Store in an airtight container. Action and use Excipient.

STAVUDINE

VP V

WHEAT STARCH Amylum tritici Wheat starch is obtained from the caryopsis of Triticum aestivum L. (T. vulgare Vill.) (Fam. Poaceae).

Characters Very fine, white or almost white powder that creaks when pressed between the fingers. Practically insoluble in cold water and in ethanol (96%). Wheat starch does not contain starch grains of any other origin. It may contain a minute quantity, if any, of tissue fragments of the original plant. Identification A. Examined under a microscope using a mixture of glycerol - water (1 : 1) to prepare samples, it presents large and small granules, and, very rarely, intermediate sizes. The large granules, 10 - 60 µm in diameter, are discoid or, more rarely, reniform when seen face-on. The central hilum and striations are invisible or barely visible and the granules sometimes show cracks on the edges. Seen in profile, the granules are elliptical and fusiform and the hilum appears as a slit along the main axis. The small granules, rounded or polyhedral, are 2 - 10 µm in diameter. Between orthogonally orientated polarising plates or prisms, the granules show a distinct black cross intersecting at the hilum. B. Suspend 1 g in 50 ml of water, boil for 1 min and cool. A thin, cloudy mucilage is formed. C. Add 0.05 ml of iodine solution R1 to 1 ml of the mucilage obtained in Identification test B. An orange-red to dark blue colour is produced, which disappears on heating. pH 4.5 to 7.0 (Appendix 6.2). Shake 5.0 g with 25.0 ml of carbon dioxide-free water R for 60 s. Allow to stand for 15 min. Foreign matter Examine under a microscope using a mixture of glycerol - water (1 : 1) to prepare samples. Not more than traces of matter other than starch granules are present. No starch grains of any other origin are present. Total protein (Appendix 10.9) Not more than 0.3% of total protein (corresponding to 0.048% of N2, conversion factor: 6.25). Determined on 6.0 g by sulfuric acid R digestion as described in Appendix 10.9 modified as follows: Place accurately 6.0 g into Kjeldahl A flask. Wash any adhering particles from the neck into the flask with 25 ml of sulfuric acid R; continue the heating until a clear solution is obtained; add 45 ml of 40% solution of sodium hydroxide R. Oxidising substances Not more than 20 ppm, calculated as H2O2 (Appendix 7.10).

Sulfur dioxide Not more than 50 ppm (Appendix 7.9, method 2). Iron Not more than 10 ppm (Appendix 9.4.13). Shake 1.5 g of the substance to be examined with 15 ml of 1 M hydrochloric acid R. Filter. Determine on the filtrate. Loss on drying Not more than 15.0% (Appendix 9.6). (1.00 g; 130 °C; 90 min). Sulfated ash Not more than 0.6% (Appendix 9.9, method 2). Determined on 1.0 g. Microbial contamination (Appendix13.6) Total aerobic microbial count is not more than 103 CFU/g. Total combined yeasts/moulds count is not more than 102 CFU/g. Absence of Salmonella and Escherichia coli. Storage Store in an airtight container. Action and use Excipient. STAVUDINE Stavudinum

C10H12N2O4  

M: 224.2

Stavudine is 1-(2,3-dideoxy-β-D-glycero-pent-2-enofuranosyl)5-methylpyrimidine-2,4(1H,3H)-dione. It contains not less than 97.0% and not more than 102.0% of C10H12N2O4, calculated with reference to the anhydrous substance.

Characters White or almost white powder, it shows polymorphism. Soluble in water, sparingly soluble in ethanol (96%), slightly soluble in methylene chloride. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of stavudine RS. If the spectra obtained with the substance to be examined and stavudine RS show differences, dissolve separately the substance to be examined and stavudine RS in anhydrous ethanol R, evaporate to dryness and record new spectra using the residues. 901

VP V

STAVUDINE

B. Specific optical rotation: -39.5° to -45.9°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.100 g of the substance to be examined in water and dilute to 10.0 ml with the same solvent.

Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use or maintain at 2 °C to 8 °C until use. Mobile phase A: Acetonitrile for chromatography - a 0.077% solution of ammonium acetate (35 : 965). Mobile phase B: Acetonitrile for chromatography - a 0.077% solution of ammonium acetate (25 : 75). Test solution: Dissolve 25.0 mg of the substance to be examined in water and dilute to 50.0 ml with the same solvent. Reference solution (1): Dilute 0.5 ml of the test solution to 100.0 ml with water. Reference solution (2): Dilute 20.0 ml of reference solution (1) to 100.0 ml with water. Reference solution (3): Dissolve 5 mg of stavudine for system suitability RS (containing impurities A to H) in water and dilute to 10.0 ml with the same solvent. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 2 ml/min. Volume of injection: 10 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 10

100

0

10 - 20

100 → 0

0 → 100

20 - 30

0

100

Identification of impurities: Use the chromatogram supplied with stavudine for system suitability RS to identify the peaks due to impurities A to H. Relative retention with reference to stavudine (retention time = 9.5 min to 12.5 min): Impurity A = about 0.3; impurity B = about 0.50; impurity C = about 0.53; impurity E = about 1.1. System suitability: In the chromatogramobtained with reference solution (3): Peak-to-valley ratio (Hp/Hv) is at least 1.5, where Hp = height above the baseline of the peak due to impurity C and Hv = height above the baseline of the lowest point of the curve separating this peak from the peak due to impurity B. Peak-to-valley ratio (Hp/Hv) is at least 1.5, where Hp = height above the baseline of the peak due to impurity E and Hv = height above the baseline of the lowest point of the curve separating this peak from the peak due to stavudine. 902

Limits: Correction factor: For the calculation of content, multiply the peak area of impurity A by 0.7. The corrected area of the peak due to impurity A is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.10%). The sum of the peak areas of all impurities is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (1) (1.0%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Note: Impurity A: 5-methylpyrimidine-2,4(1H,3H)-dione (thymine). Impurity B: 1-(2-deoxy-β-D-threo-pentofuranosyl)-5- methylpyrimidine2,4(1H,3H)-dione (3’-epithymidine). Impurity C: 1-(2-deoxy-β-D-erythro-pentofuranosyl)-5- methylpyrimidine-2,4(1H,3H)-dione (thymidine). Impurity D: 1-[(2R)-5-oxo-2,5-dihydrofuran-2-yl]-5-methylpyrimidine-2,4(1H,3H)-dione. Impurity E: 1-(2,3-dideoxy-α-D-glycero-pent-2-enofuranosyl)5-methylpyrimidine-2,4(1H,3H)-dione (stavudine anomer a). Impurity F: 1-(3,5-anhydro-2-deoxy-β-D-threo-pentofuranosyl)5-methylpyrimidine-2,4(1H,3H)-dione. Impurity G: 5’-O-[[(2S,5R)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-2,5-dihydrofuran-2-yl]methyl]-3’epithymidine. Impurity H: 1-[2-deoxy-5-O-(1-methylethyl)-β-D-erythro- pentofuranosyl]-5-methylpyrimidine-2,4(1H,3H)-dione.

Water Not more than 0.5% (Appendix 10.3). Determined on 0.500 g. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use or maintain at 2 °C to 8 °C until use. Mobile phase: Acetonitrile for chromatography - a 0.077% solution of ammonium acetate (5 : 95). Test solution: Dissolve 10.0 mg of the substance to be examined in water and dilute to 100.0 ml with the same solvent. Dilute 10.0 ml of the solution to 50.0 ml with water. Reference solution (1): Dissolve 10.0 mg of stavudine RS in water and dilute to 100.0 ml with the same solvent. Dilute 10.0 ml of the solution to 50.0 ml with water. Reference solution (2): Dissolve 5 mg of thymine R and 7.5 mg of thymidine R in water and dilute to 100.0 ml with the same solvent. Dilute 10.0 ml of the solution to 50.0 ml with water.

STAVUDINE TABLETS

VP V

Chromatographic system: A column (3.3 cm × 4.0 mm) packed with stationary phase C (3 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 0.7 ml/min. Volume of injection: 25 µl. Procedure: Retention time of stavudine is 2.8 min to 5.0 min. System suitability: In the chromatogram obtained with reference solution (1), the symmetry factor for the peak due to stavudine is not more than 1.6. In the chromatogram obtained with reference solution (2), the resolution between the peaks due to thymine and thymidine is at least 3.5. Calculate the content of stavudine, C10H12N2O4, using the areas of the stavudine peaks in the chromatograms obtained with the test solution, reference solution (1) and the declared content of C10H12N2O4 in stavudine RS.

Time: 45 min. Procedure: Test solution: After the specified time (45 min), withdraw a sample of the medium, filter. Reference solution: Dissolve an accurately weighed quantity of stavudine RS in the medium to obtain a solution having the same concentration of stavudine to that in the test solution. Examine by liquid chromatography (Appendix 5.3). Chromatographic system: Prepare as directed in the Assay. Calculate the content of stavudine, C10H12N2O4, dissolved using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C10H12N2O4 in stavudine RS. Tolerance: Not less than 70% (Q) of the stated amount of stavudine, C10H12N2O4, is dissolved in 45 min.

Storage Protected from light and humidity.

Related substances Examine by liquid chromatography (Appendix 5.3). Reference solution: Dissolve an accurately weighed quantity of stavudine RS and thymine RS in water to obtain a solution contaning about 0.2 mg/ml of stavudine, C10H12N2O4 and 0.2 mg/ml of thymine. Test solution: Transfer an accurately weighed quantity of the powdered tablets equivalent to about 50 mg of stavudine to a 100 ml volumetric flask, add 80 ml of water and sonicate for 10 min. Dilute to volume with water, mix and filter. Mobile phase A: 0.1 M ammonium acetate R. Mobile phase B: Acetonitrile R. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase B (5 μm). Detector: A spectrophotometer set at 270 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 μl. Procedure: Carry out a linear gradient elution using the following conditions:

Action and use Antiviral (HIV). Preparation Tablets. STAVUDINE TABLETS Tabellae Stavudini Stavudine tablets contain stavudine. They may be coated. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of stavudine, C10H12N2O4, 90.0% to 110.0% of the stated amount. Identification A. Shake a quantity of the powdered tablets containing the equivalent of about 0.05 g of stavudine with 100 ml of water, filter. Dilute 5 ml of the filtrate to 50 ml with water. Dilute 5 ml of this solution to 25 ml with water. The ultraviolet absorption spectrum (Appendix 4.1) of the solution in the range 200 nm to 300 nm corresponds to that of a reference solution of stavudine RS having the same concentration dissolved in the same solvent. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to the retention time of stavudine peak in the chromatogram obtained with the reference solution. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of 0.01 M hydrochloric acid R. Rotation speed: 75 rpm.

Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0

95

5

5

95

5

25

20

80

30

20

80

31

95

5

35

95

5

System suitability: Inject the reference solution, the number of theoretical plates of the peaks due to stavudine and thymine is not less than 7000; the tailing factor of the peaks due to stavudine and thymine is not more than 2.0. Inject the test solution. 903

VP V

STEARYL ALCOHOL

Calculate the content of impurities by using the normalisation procedure (Appendix 5). Limits: The area of the peak due to thymine is not more than 3.0%. The area of any secondary peaks, apart from the principal peak and thymine peak is not more than 0.5%. The total impurities, apart from the principal peak, is not more than 4.0%. Disregard any peak observed in the blank and disregard any peak less than 0.05%.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - 0.1 M ammonium acetate (5 : 95). Reference solution: Dissolve an accurately weighed quantity of stavudine RS in water to obtain a solution having a known concentration of about 0.2 mg/ml. Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Transfer an accurately weighed quantity of the powdered tablets containing the equivalent of 50 mg of stavudine to a 100 volumetric flask, add 80 ml of water and sonicate for 10 min. Dilute to volume with water, mix and filter. Dilute 10.0 ml of the filtrate to 25.0 ml with water. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase B (5 μm). Detector: A spectrophotometer set at 270 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 μl. Procedure: System suitability: Inject the reference solution, the number of theoretical plates calculated for the principal peak is not less than 3000, the tailing factor is not more than 2.0, the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of stavudine, C10H12N2O4, in the tablets using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C10H12N2O4 in stavudine RS. Storage Store in an airtight container, in a cool and dry place, at a temperature not exceeding 30 °C, protected from light. Action and use Antiviral HIV. Usual strength 30 mg and 40 mg.

904

STEARYL ALCOHOL Alcohol stearylicus Stearyl alcohol is mixture of solid alcohols, mainly octadecan-1-ol (C18H38O) of animal or vegetable origin. It contains not less than 95.0% of C18H38O (Mr. 270.5).

Characters White or almost white, unctuous flakes, granules or mass. Practically insoluble in water, soluble in ethanol (96%). When melted, it is miscible with fatty oils, with liquid paraffin and with melted wool fat. Identification In the test for Assay, the principal peak in the chromatogram obtained with the test solution is similar in retention time to the principal peak in the chromatogram obtained with reference solution (2). Appearance of solution Dissolve 0.50 g of the substance to be examined in 20 ml of boiling ethanol (96%) R. Allow to cool. The solution is clear (Appendix 9.2) and not more intensely coloured than reference solution B6 (Appendix 9.3, method 2). Melting point 57 °C to 60 °C (Appendix 6.7). Acid value Not more than 1.0 (Appendix 7.2). Hydroxyl value 197 to 217 (Appendix 7.4, method A). Iodine value Not more than 2.0 (Appendix 7.5, method A). Dissolve 2.00 g of the substance to be examined in methylene chloride R, warming if necessary and dilute to 25 ml with the same solvent. Saponification value Not more than 2.0 (Appendix 7.7). Assay Examine by gas chromatography (Appendix 5.2). Use the normalisation procedure. Test solution: Dissolve 0.100 g of the substance to be examined in ethanol (96%) R and dilute to 10.0 ml with the same solvent. Reference solution (1): Dissolve 50 mg of cetyl alcohol R in ethanol (96%) R and dilute to 10 ml with the same solvent. Reference solution (2): Dissolve 50 mg of stearyl alcohol RS in ethanol (96%) R and dilute to 5 ml with the same solvent. Reference solution (3): Mix 1 ml of reference solution (1) and 1 ml of reference solution (2) and dilute to 10 ml with ethanol (96%) R.

STREPTOMYCIN SULFATE

VP V

Chromatographic system: A column (30 m × 0.32 mm) coated with poly(dimethyl) siloxane R (1 µm). Carrier gas: Helium for chromatography. Flow rate: 1 ml/min. Split ratio: 1 : 100. Temperature programme:

Column

Time (min)

Temperature (°C)

0 - 20 20 - 40

150 → 250 250

Injection port

250

Detector

250

Detector: A flame-ionisation detector. Volume of injection: 1 µl. Procedure: Inject the test solution and the reference solutions (2) and (3). System suitability: In the chromatogram obtained with reference solution (3), the resolution between the peaks due to cetyl alcohol and stearyl alcohol is at least 5.0. Calculate the percentage content of C18H38O, using the chromatograms obtained with the test solution and reference solution (2) and using the declared content of C18H38O in stearyl alcohol RS.

Storage Store in an airtight container, protected from light. Action and use Excipient. STREPTOMYCIN SULFATE Streptomycini sulfas

(C21H39N7O12)2,3H2SO4  

M: 1457.0

Streptomycin sulfate is bis[N,N’-bis(aminoiminomethyl)4-O-[5-deoxy-2-O-[2-deoxy-2-(methylamino)-α-L-

glucopyranosyl]-3-C-formyl-α-L-lyxofuranosyl]-Dstreptamine] trisulfate, a substance produced by the growth of certain strains of Streptomyces griseus or obtained by any other means. Stabilisers may be added. The potency is not less than 720 IU/mg, calculated with reference to the dried substance.

Production It is produced by methods of manufacture designed to eliminate or minimise substances lowering blood pressure. The method of manufacture is validated to demonstrate that the product if tested would comply with the following test: Abnormal toxicity (Appendix 13.5) Inject into each mouse 1 mg of the substance to be examined dissolved in 0.5 ml of water for injections R. Characters A white or almost white powder, hygroscopic, very soluble in water, practically insoluble in ethanol. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Use a plate coated with a 0.75 mm layer of the following mixture: Mix 0.3 g of carbomer R with 240 ml of water and allow to stand, with moderate shaking, for 1 h; adjust to pH 7 by the gradual addition, with continuous shaking, of 2 M sodium hydroxide R and add 30 g of silica gel H R. Heat the plate at 110 °C for 1 h, allow to cool and use immediately. Mobile phase: A 7% solution of potassium dihydrogen phosphate R. Visualisation reagent: A mixture of equal volumes of a 0.2% solution of 1,3-dihydroxynaphthalene R in ethanol (96%) R and a 46% solution of sulfuric acid R (m/v). Test solution: Dissolve 10 mg of the substance to be examined in 10 ml of water. Reference solution (1): Dissolve 10 mg of streptomycin sulfate RS in 10 ml of water. Reference solution (2): Dissolve 10 mg of kanamycin monosulfate RS, 10 mg of neomycin sulfate RS and 10 mg of streptomycin sulfate RS in 10 ml of water. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 12 cm. Dry the plate in a current of warm air, and spray with the visualisation reagent. Heat at 150 °C for 5 min to 10 min. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) shows three clearly separated spots. B. Dissolve 5 mg to 10 mg of the substance to be examined in 4 ml of water and add 1 ml of 1 M sodium hydroxide R. Heat in a water-bath for 4 min. Add a slight excess of 10% solution of hydrochloric acid R and 0.1 ml of a 10.5% solution of ferric chloride R. A violet colour develops. 905

VP V

STREPTOMYCIN SULFATE

C. Dissolve 0.1 g of the substance to be examined in 2 ml of water, add 1 ml of 1-naphthol solution R and 2 ml of a mixture of equal volumes of strong sodium hypochlorite solution R and water. A red colour develops. D. Dissolve about 10 mg of the substance to be examined in 5 ml of water and add 1 ml of 1 M hydrochloric acid R. Heat in a water-bath for 2 min. Add 2 ml of a 0.5% solution of 1-naphthol R in 1 M sodium hydroxide R and heat in a water-bath for 1 min. A faint yellow colour develops. E. It gives the reactions of sulfates (Appendix 8.1).

Appearance of solution Solution S: Dissolve 2.5 g of the substance to be examined in carbon dioxide-free water R and dilute to 10 ml with the same solvent. Solution S is not more intensely coloured than intensity 3 of the range of reference solutions of the most appropriate colour (Appendix 9.3, method 2). Allow the solution S to stand protected from light, at a temperature of about 20 °C for 24 h. Solution S is not more opalescent than reference suspension II (Appendix 9.2). pH 4.5 to 7.0 (Appendix 6.2). Determined on solution S. Methanol Not more than 0.3%. Examine by gas chromatography (Appendix 5.2). Test solution: Dissolve 1.00 g of the substance to be examined in water and dilute to 25.0 ml with the same solvent. Reference solution: Dilute 12.0 mg of methanol R to 100 ml with water. Chromatographic system: A column (1.5 m to 2.0 m × 2 mm to 4 mm) packed with ethylvinylbenzene-divinylbenzene copolymer R (150 µm to 180 µm) Carrier gas: Nitrogen for chromatography at a constant flow rate of 30 ml/min to 40 ml/min. Detector: A flame-ionisation detector. Maintain the column at a constant temperature between 120 °C and 140 °C and the injection port and the detector at a temperature at least 50 °C higher than that of the column. Procedure: Inject the test solution and the reference solution. The area of the peak corresponding to methanol in the chromatogram obtained with the test solution is not greater than the area of the peak in the chromatogram obtained with the reference solution. Streptomycin B Not more than 3.0%. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Glacial acetic acid - methanol - toluene (25 : 25 : 50). 906

Visualisation reagent: A mixture of equal volumes of a 0.2% solution of 1,3-dihydroxynaphthalene R in ethanol (96%) R and a 20% (v/v) solution of sulfuric acid R. Prepare immediately before use. Test solution: Dissolve 0.2 g of the substance to be examined in a freshly prepared mixture of sulfuric acid methanol (3 : 97) and dilute to 5 ml with the same mixture of solvents. Heat under a reflux condenser for 1 h, cool, rinse the condenser with methanol R and dilute to 20 ml with the same solvent (1.0% solution). Reference solution: Dissolve 36 mg of mannose R in 5 ml of a freshly prepared mixture of sulfuric acid - methanol (3 : 97). Heat under a reflux condenser for 1 h, cool, rinse the condenser with methanol R and dilute to 50 ml with methanol R. Dilute 5 ml of the solution to 50 ml with methanol R (0.03% solution expressed as streptomycin B; 1 mg of mannose R is equivalent to 4.13 mg of streptomycin B). Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 13 cm to 15 cm. Allow the plate to dry in air and spray with visualisation reagent and heat at 110 °C for 5 min. Any spot corresponding to streptomycin B in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution.

Loss on drying Not more than 7.0% (Appendix 9.6). (1.000 g; 60 °C; over diphosphorus pentoxide at a pressure not exceeding 0.1 kPa; 24 h). Sulfated ash Not more than 1.0% (Appendix 9.9, method 2). Determined on 1.0 g. Sulfate 18.0% to 21.5% of sulfate (SO4), calculated with reference to the dried substance. Dissolve 0.250 g of the substance to be examined in 100 ml of water and adjust the solution to pH 11 with ammonia R. Add 10.0 ml of 0.1 M barium chloride VS and about 0.5 mg of phthalein purple R. Titrate with 0.1 M sodium edetate VS adding 50 ml of ethanol (96%) R when the colour of the solution begins to change and continue the titration until the violet-blue colour disappears. Carry out a blank titration. 1 ml of 0.1 M barium chloride VS is equivalent to 9.606 mg of sulfate (SO4). Colorimetric test Dry the substance to be examined and streptomycin sulfate RS at 60 °C over diphosphorus pentoxide R at a pressure not exceeding 0.1 kPa for 24 h. Dissolve 0.100 g of the dried substance to be examined in water and dilute to 100.0 ml with the same solvent. Prepare a reference solution in the same manner using 0.100 g of the dried streptomycin sulfate RS. Place 5.0 ml of each solution

VP V

separately in two volumetric flasks and in a third flask place 5 ml of water. To each flask add 5.0 ml of 0.2 M sodium hydroxide and heat for exactly 10 min in a waterbath. Cool in ice water for exactly 5 min, add 3 ml of a 1.5% solution of ferric ammonium sulfate R in 0.5 M sulfuric acid R, dilute to 25.0 ml with water and mix. Exactly 20 min after the addition of the ferric ammonium sulfate solution measure the absorbance of the test solution and the reference solution in a 2 cm cell at the maximum at 525 nm (Appendix 4.1), using as compensation liquid the solution prepared from 5 ml of water. The absorbance of the test solution is not less than 90.0% of that of the reference solution.

Bacterial endotoxins Less than 0.25 EU/mg (Appendix 13.2). If intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins. Assay Carry out the microbiological assay of antibiotics (Appendix 13.9). Storage Store in an airtight container. If the substance is sterile, store in a sterile, airtight, tamper-proof container. Action and use Aminoglycoside antibacterial; antituberculosis drug. Preparation Injection powder. STREPTOMYCIN FOR INJECTION Streptomycin pro injectione Streptomycin for injection is a sterile powder of streptomycin sulfate supplied in a sealed glass container. It is dissolved in water for injections immediately before use. The powder complies with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Content of streptomycin, C21H39N7O12, 95.0% to 115.0% of the stated amount. Characters A white or almost white powder. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Mix 0.3 g of carbomer R with 240 ml of water, allow to stand for 1 h and occasionally shake, adjust the pH to 7.0 by the gradual addition, with constant shaking, of 2 M sodium hydroxide R and add 30 g of silica gel H R. Spread a uniform layer of the resulting suspension

STREPTOMYCIN FOR INJECTION

0.75 mm thick. Heat the plate at 110 °C for 1 h, allow to cool and use immediately. Mobile phase: A 7% solution of potassium dihydrogen orthophosphate R. Visualization reagent: A mixture of equal volumes of a 0.2% naphthalene-1,3-diol solution in ethanol (96%) and 46% sulfuric acid solution R. Test solution: Dissolve a quantity of the powder to be examined in water to obtain a solution containing 0.08% of streptomycin. Reference solution (1): A solution containing 0.1% of streptomycin sulfate RS in water. Reference solution (2): A solution containing 0.1% of kanamycin monosulfate RS, 0.1% of streptomycin sulfate RS and 0.1% of neomycin sulfate RS and in water. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 12 cm. Remove the plate, dry it in a current of warm air, spray with the visualization reagent and heat at 150 °C for 5 min to 10 min. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) shows three clearly separated principal spots. B. Dissolve 5 mg to 10 mg of the powder in a test tube with 4 ml of water, add 1 ml of 1 M sodium hydroxide R and heat in a water bath for 4 min. Add a slight excess of 10 % hydrochloric acid solution R and 0.1 ml of a 10.5% solution of iron (III) chloride R. A violet colour is produced. C. The powder gives reaction of sulfates (Appendix 8.1).

Acidity or alkalinity pH of a solution containing 25% of streptomycin is 4.5 to 7.0 (Appendix 6.2). Loss on drying Not more than 7.0% (Appendix 9.6). (Use 1.0 g, dry at 60 °C at a pressure not exceeding 0.1 kPa for 24 h using phosphorus pentoxide). Streptomycin B Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Glacial acetic acid - methanol - toluene (25 : 25 : 50). Reagent: A mixture of equal volumes of a 0.2% solution of naphthalene-1,3-diol in ethanol (96%) and 20% (v/v) sulfuric acid solution R. Test solution: Dissolve a quantity of the powder being examined containing 0.16 g of streptomycin in 5 ml of a mixture of sulfuric acid - methanol (3 : 97) freshly prepared. Heat under a reflux condenser for 1 h, cool, rinse the condenser with methanol R and add sufficient 907

VP V

STRYCHNINE SULFATE

methanol R to produce 20 ml (solution containing 1% of streptomycin). Reference solution: Dissolve 36 mg of mannose in 5 ml of a mixture of sulfuric acid - methanol (3 : 97) freshly prepared. Heat under a reflux condenser for 1 h, cool, rinse the condenser with methanol R and add sufficient methanol R to produce 50 ml. Dilute 5 ml of the resulting solution to 50 ml with methanol R; this solution contains 0.03% of streptomycin B (1 mg of mannose is equivalent to 4.13 mg of streptomycin B). Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm. Remove the plate, dry it in air, spray with the reagent and heat at 110 °C for 5 min. Any spot corresponding to streptomycin B in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution.

Bacterial endotoxins Carry out the Test for bacterial endotoxins (Appendix 13.2). Dissolve a quantity of the powder to be examined with BET water to obtain a solution containing 10 mg of streptomycin per ml (solution A). The endotoxin limit concentration of solution A is 2.5 EU per ml. Perform the test using the maximum valid dilution of solution A calculated from the declared sensitivity of the lysate used in the test. Assay Determine quickly the contents of 20 containers and calculate the average mass and mix quickly. Dissolve a quantity of the mixed contents containing 0.33 g of streptomycin in sufficient water to produce 100.0 ml and carry out the Biological assay of antibiotics (Appendix 13.9). Calculate the content of streptomycin in the injection, taking each 1000 IU found to be equivalent to 1 mg of streptomycin. Storage Store in a cool place, protected from light. Usual strength 1000 mg stated in terms of streptomycin.

O

S O4 -- ,. 5 H2O

H O 2

(C21H22N2O2)2,H2SO4,5H2O

908

Identification Apply one of the two following identifications: First identification: B, C, D. Second identification: A, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of strychnine sulfate RS. B. Examine the chromatograms obtained in the test for Related substances. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). C. Dissolve 50 mg of the substance to be examined in 5 ml of water, add 0.1 ml of ammonia R and extract with 5 ml of chloroform R. Evaporate the chloroform extract to dryness on a water bath. To the residue add 0.1 ml of sulphuric acid R and a crystal of potassium dichromate R. The violet, red and yellow colours develop around the crystal when shaked. D. It gives the reactions of sulfates (Appendix 8.1). Appearance of solution Solution S: Dissolve 1.0 g of the substance to be examined in carbon dioxide-free water R and dilute to 50.0 ml with the same solvent, and mix. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2).

Specific optical rotation -25° to -29°, calculated with reference to the anhydrous substance (Appendix 6.4). Determined on solution S.

N H

N

Characters A white, crystalline powder or colourless needles; odourless. Freely soluble in boiling water, sparingly soluble in ethanol and in cold water, slightly soluble in chloroform, practically insoluble in ether.

Acidity or alkalinity To 25 ml of solution S add 0.1 ml of methyl red solution R, the solution is red. Not more than 0.5 ml of 0.02 N sodium hydroxide VS is required to change the colour of the solution to yellow.

STRYCHNINE SULFATE Strychnini sulfas H

Strychnine sulfate is strychnidin-10-one sulfate pentahydrate, a substance extracted from the seeds of Strychnos nux-vomica L. and species of other Strychnos sp. (Fam. Loganiaceae). It contains not less than 97.0% and not more than 100.5% of (C21H22N2O2)2,H2SO4, calculated with reference to the anhydrous substance.

M. 857.0

Brucine Dissolve 0.1 g of the substance to be examined in a mixture of 1 ml of nitric acid R and 0.2 ml of water. If the solution is red or orange, which is not more intense coloured than a reference solution prepared by mixing 1 ml of nitric acid R and 0.2 ml of a 0.054% solution of brucine.

SUCRALFATE

VP V

Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Diethylamine - ethyl acetate - cyclohexane (15 : 25 : 60). Test solution: Dissolve 0.1 g of the substance to be examined in methanol R and dilute to 10 ml with the same solvent, and mix. Reference solution (1): Dissolve 0.1 g of strychnine sulfate RS in methanol R and dilute to 10 ml with the same solvent, and mix. Reference solution (2): Dilute 0.5 ml of reference solution (1) to 100 ml with methanol R and mix. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm. Remove the plate, dry it at 100 - 105 °C for 15 min, and allow to cool. Spray with potassium iodobismuthate solution R until the orangered spots appear. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the principal spot in the chromatogram obtained with the reference solution (2). Water 10.0% to 13.0% (Appendix 10.3). Determined on 0.10 g. Sulfated ash Not more than 0.1% (Appendix 9.9, method 1). Determined on 1.0 g. Assay Dissolve 0.500 g of the substance to be examined in 25 ml of anhydrous acetic acid R, add 1 ml of acetic anhydride R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2) or using malachite green R as indicator. 1 ml of 0.1 N perchloric acid VS is equivalent to 76.70 mg of (C21H22N2O2)2,H2SO4. Storage Store in an airtight container, protected from light. Action and use Central nervous system stimulator, spinal cord stimulator; digestion stimulator. Preparation Injection.

SUCRALFATE Sucralfatum

C12H30Al8O51S8[Al(OH)3]n[H2O]n′ in which n = 8 to 10 and n′ = 22 to 31. Sucralfate is β-D-fructofuranosyl-α-D-glucopyranoside octakis(dihydroxyaluminium sulfate) with 8 to 10 molecules of aluminium hydroxide and 22 to 31 molecules of water. It contains not less than 30.0% and not more than 36.0% of β-D-fructofuranosyl-α-D-glucopyranoside octakis sulfate (sucrose octasulfate) (C12H14O35S88-; M. 975) and contains not less than 16.5% and not more than 18.5% of aluminium (Al; Ar: 26.98).

Characters White or almost white, amorphous powder. Practically insoluble in water, in ethanol (96%) and in methylene chloride. It dissolves in dilute solutions of mineral acids and alkali hydroxides. Identification A. The infrared absorption spectrophotometry (Appendix 4.2) of the substance to be examined is concordant with the spectrum of sucralfate RS. B. Dissolve 2 g of the substance to be examined add 10 ml of 0.1 M hydrochloric acid R and boil. Cool and neutralise with 0.1 M sodium hydroxide R. To 5 ml of the solution add 0.15 ml of freshly prepared a 12.5% solution of copper sulphate R and 2 ml of freshly prepared dilute sodium hydroxide solution R. The solution is blue and clear and remains so after boiling. To the hot solution add 4 ml of dilute hydrochloric acid R and boil for 1 min. Add 4 ml of dilute sodium hydroxide solution R; an orange precipitate is formed immediately. C. Dissolve about 15 mg of the substance to be examined in a mixture of 0.5 ml of dilute hydrochloric acid R and 2 ml of water. The solution gives the reaction of aluminium (Appendix 8.1). Impurity A Examine by liquid chromatography (Appendix 5.3). Mobile phase: A 7% solution of ammonium sulfate R adjusted to pH 3.5 with phosphoric acid R. Test solution: Dissolve 450.0 mg of the substance to be examined in a mixture of equal volumes of a 8.8% solution 909

VP V

SUCRALFATE

of  sodium hydroxide R and a 19.62% solution of sulfuric acid R and dilute to 10.0 ml with the same mixture of solvents. Without delay, while shaking at a moderate rate, add a volume (V), accurately measured in millilitres, of 0.1 M  sodium hydroxide R to adjust the solution to approximately pH 2.3. Dilute the solution with (15.0 - V) ml of water. Shake for 1 min. If the pH is not between 2.3 and 3.5, repeat the test using a different volume (V) of 0.1 M sodium hydroxide R. Reference solution (1): Dissolve 40.0 mg of potassium sucrose octasulfate RS in the mobile phase and dilute to 5.0 ml with the mobile phase. Reference solution (2): Dilute 1.0 ml of reference solution (1) to 10.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.0 mm) packed with stationary phase aminopropylsilyl silica gel for chromatography R (5 µm). Detector: Differential refractometer. Flow rate: 1 ml/min. Volume of injection: 50 µl. Procedure: Inject the test solution and reference solution (2). Relative retention with reference to sucrose octasulfate (retention time = about 6 min) of impurity A is about 0.6. System suitability: In the chromatogram obtained with reference solution (2), the number of theoretical plates is not less than 400 and the symmetry factor is not more than 4.0, determined from the peak due to potassium sucrose octasulfate. Limit: Impurity A: The area of the peak due to impurity A is not more than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (5.0%). Note: Impurity A: β-D-fructofuranosyl-α-D-glucopyranoside heptakis (hydrogensulfate).

Neutralising capacity Disperse 0.25 g in 100.0 ml of 0.1 N hydrochloric acid VS, previously heated at 37 °C, stir continuously for 1 h in a water-bath at 37 °C and cool. Titrate 20.0 ml of this solution with 0.1 N sodium hydroxide VS to pH 3.5; not more than 14.0 ml of 0.1 N sodium hydroxide VS is required. Chlorides Not more than 0.50 % (Appendix 9.4.5). Dissolve 0.10 g in 5 ml of dilute nitric acid R and dilute to 50 ml with water. Dilute 5 ml of this solution to 15 ml with water. Arsenic Not more than 4 ppm (Appendix 9.4.2, method A). Introduce 0.25 g of the substance to be examined and 5 ml of sulfuric acid R into a combustion flask. Carefully add a few millilitres of hydrogen peroxide solution (100 vol) R and 910

heat to boiling until a clear, colourless solution is obtained. Continue heating to eliminate the water and as much sulfuric acid as possible and dilute to 25 ml with water.

Heavy metals Not more than 10 ppm (Appendix 9.4.8). 2.0 g complies with limit test for heavy metals, method 6. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Assay Aluminium Disperse 1.0 g of the substance to be examined in 10 ml of  6 M hydrochloric acid R. Heat with continuous stirring in a water-bath at 70 °C for 5 min. Cool to room temperature, transfer quantitatively to a volumetric flask, dilute to 250.0 ml with water, and mix. Filter the solution, discarding the first  portion of the filtrate. To 10.0  ml of the solution, add 10.0 ml of 0.1 M sodium edetate VS and 30 ml of a mixture of equal volumes of ammonium acetate solution R and dilute acetic acid R. Heat in a waterbath at 70 °C for 5 min, then cool. Add 25 ml of ethanol (96%) R and 1 ml of a freshly prepared a 0.025% solution of  dithizone R in ethanol (96%) R. Titrate the excess of sodium edetate with 0.1 M zinc sulfate VS until the colour changes to pink. 1 ml of 0.1 M sodium edetate VS is equivalent to 2.698 mg of Al. Sucrose octasulfate Examine by liquid chromatography (Appendix 5.3) as described in the test for Impurity A with the following modifications. Mobile phase: A 13.2% solution of ammonium sulfate R, adjusted to pH 3.5 with phosphoric acid R. Inject the test solution and reference solution (1). Calculate the content of C12H14O35S8 using the areas for the peaks in the chromatograms obtained with the test solution, reference solution (1), the declared content of C12H14O35S8 in potassium sucrose octansulfate RS and by multiplying the potassium sucrose octasulfate content by 0.757. Storage Store in an airtight container, protected from light. Action and use Treatment of gastric and duodenal ulcers. Preparations Tablets, suspension powder.

SUCROSE

VP V

SUCROSE

C. Dilute 1 ml of solution S (see Appearence of solution) to 100 ml with water. To 5 ml of the solution add 0.15 ml of freshly prepared 12.5% solution of copper sulfate R and 2 ml of freshly prepared 2 M sodium hydroxide R. The solution is blue and clear and remains so after boiling. To the hot solution add 4 ml of 2 M hydrochloric acid R and boil for 1 min. Add 4 ml of 2 M sodium hydroxide R. An orange precipitate is formed immediately.

Saccharum

C12H22O11

M. 342.3

Sucrose is β-D-Fructofuranosyl α-D-glucopyranoside. It contains no additives.

Characters White or almost white, crystalline powder, or lustrous, colourless or white or almost white crystals. Very soluble in water, slightly soluble in ethanol (96%), practically insoluble in anhydrous ethanol. Identification Apply one of the two following identifications: First identification: A. Second identification: B, C. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of sucrose RS. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Cold saturated boric acid solution - 60% v/v solution of glacial acetic acid - ethanol - acetone ethyl acetate (10 : 15 : 20 : 60 : 60). Test solution: Dissolve 10 mg of the substance to be examined in a mixture of 2 volumes of water and 3 volumes of methanol R and dilute to 20 ml with the same mixture of solvents. Reference solution (1): Dissolve 10 mg of sucrose RS in a mixture of 2 volumes of water and 3 volumes of methanol R and dilute to 20 ml with the same mixture of solvents. Reference solution (2): Dissolve 10 mg each of fructose RS, glucose RS, lactose RS and sucrose RS in a mixture of 2 volumes of water and 3 volumes of methanol R and dilute to 20 ml with the same mixture of solvents. Procedure: Apply separately to the plate 2 µl of each solution. Develop in an unsaturated tank over a path of 15 cm. Dry the plate in a current of warm air. Spray with a solution of 0.5 g of thymol R in a mixture of 5 ml of sulfuric acid R and 95 ml of ethanol (96%) R. Heat the plate at 130 °C for 10 min. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with the reference solution (2) shows 4 clearly separated spots.

Appearence of solution Solution S: Dissolve 50.0 g of the substance to be examined in water and dilute to 100.0 ml with the same solvent. Solution S is clear (Appendix 9.2). Conductivity Not more than 35 µS·cm-1 at 20 °C. Test solution: Dissolve 31.3 g of the substance to be examined in carbon dioxide-free water R prepared from distilled water R and dilute to 100 ml with the same solvent. Measure the conductivity of the solution (C1), while gently stirring with a magnetic stirrer, and that of the water used for preparing the solution (C2). The readings must be stable within 1% over a period of 30 s. Calculate the conductivity of the solution of the substance to be examined from the following expression: C1 – 0.35C2 Specific optical rotation +66.3° to +67.0° (Appendix 6.4). Dissolve 26.0 g of the substance to be examined in water and dilute to 100.0 ml with the same solvent. Colour value Not more than 45. Dissolve 50.0 g of the substance to be examined in 50.0 ml of water. Mix, filter (diameter of pores 0.45 µm) and degas. Measure the absorbance (Appendix 4.1) at 420 nm, using a cell of minimum 4 cm (a cell length of 10 cm or more is preferred). Calculate the colour value using the following expression: A × 1000 b×c Where: A: Absorbance measured at 420 nm; b: Path length in centimetres; c: Concentration of the solution, g/ml, calculated from the refractive index (Appenidx 6.1) of the solution; use table and interpolate the values if necessary. STT

nD20

C (g/ml)

1

1.4138

0.570

2

1.4159

0.585

3

1.4179

0.600

4

1.4200

0.615

5

1.4221

0.630

6

1.4243

0.645

7

1.4264

0.661

911

VP V

SULBACTAM SODIUM

Repeatability: the absolute difference between 2 results is not greater than 3.

Dextrins If intended for use in the manufacture of large-volume parenteral preparations, it complies with the test for dextrins. To 2 ml of solution S add 8 ml of water, 0.05 ml of 2 M hydrochloric acid R and 0.05 ml of 0.1 M iodine R. The solution remains yellow. Reducing sugars To 5 ml of solution S in a test-tube about 150 mm long and 16 mm in diameter add 5 ml of water, 1.0 ml of 1 M sodium hydroxide R and 1.0 ml of a 0.1% solution of methylene blue R. Mix and place in a water-bath. After exactly 2 min, take the tube out of the bath and examine the solution immediately. The blue colour does not disappear completely. Ignore any blue colour at the air/ solution interface. Sulfites Not more than 15 ppm, calculated as SO2. Test solution: Dissolve 5.0 g in 40 ml of water, add 2.0 ml of 0.1 N sodium hydroxide R and dilute to 50.0 ml with water. To 10.0 ml of the solution, add 1 ml of 3 N hydrochloric acid R, 2.0 ml of decolorised fuchsin solution R and 2.0 ml of a 0.5% (v/v) solution of formaldehyde R. Allow to stand for 30 min and measure the absorbance at the maximum at 583 nm. Reference solution: Dissolve 76 mg of sodium metabisulphite R in water and dilute to 50.0 ml with the same solvent. Dilute 5.0 ml of this solution to 100.0 ml with water. To 3.0 ml of this solution add 4.0 ml of 0.1 M sodium hydroxide R and dilute to 100.0 ml with water. To 10.0 ml of this solution add immediately 1 ml of 3 N hydrochloric acid R, 2.0 ml of decolorised fuchsin solution R and 2.0 ml of a 0.5% (v/v) solution of formaldehyde R. Allow to stand for 30 min and measure the absorbance at the maximum at 583 nm. Blank solution: Prepare a blank solution in the same manner as described under Test solution beginning at the word “To 10.0 ml of the solution, add 1 ml of 3N hydrochloric acid R…” but using 10.0 ml of water instead of 10.0 ml of the solution of the substance to be examined. The absorbance of the test solution is not greater than that of the reference solution. The test is not valid unless the reference solution shows a clearly visible violet-red colour. Loss on drying Not more than 0.1% (Appendix 9.6). (2.000 g; 105 °C; 3 h). Bacterial endotoxins Less than 0.25 EU/mg (Appendix 13.2), if intended for use in the manufacture of large-volume parenteral preparations. 912

Labelling The label states, where applicable, that the substance is suitable for use in the manufacture of large-volume parenteral preparations. SULBACTAM SODIUM Sulbactamum natricum

C8H10NNaO5S  



M. 255.2

Sulbactam sodium is sodium (2S,5R)-3,3-dimethyl-7oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide. It contains not less than 97.0% and not more than 102.0% of C8H10NNaO5S, calculated with reference to the dried substance. Semi-synthetic product derived from a fermentation product.

Characters A white or almost white, hygroscopic, crystalline powder. Freely soluble in water, sparingly soluble in ethyl acetate, very slightly soluble in ethanol (96%). It is freely soluble in dilute acids. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum obtained with sulbactam sodium RS. B. It gives reaction of sodium (Appendix 8.1).

Appearance of solution Dissolve 2.0 g in water and dilute to 20 ml with the same solvent. The solution is clear (Appendix 9.2). Absorbance Dissolve 1.0 g in water and dilute to 100.0 ml with the same solvent. The absorbance of the solution determined at 430 nm (Appendix 4.1) is not greater than 0.10. pH Dissolve 1.0 g in carbon dioxide-free water R. The pH of this solution is 4.5 to 7.2 (Appendix 6.2), if the substance is sterile, the pH of this solution is 5.2 to 7.2. Specific optical rotation +219° to +233°, calculated with reference to the dried substance (Appendix 6.4). Dissolve 0.50 g in water and dilute to 50.0 ml with the same solvent.

SULBACTAM SODIUM

VP V

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: A 5.44 g/L solution of potassium dihydrogen phosphate R adjusted to pH 4.0 with dilute phosphoric acid R. Mobile phase B: Acetonitrile for chromatography R. Solution A: A 2.72 g/L solution of potassium dihydrogen phosphate R adjusted to pH 4.0 with dilute phosphoric acid R. Solution B: Dilute 2 ml of acetonitrile for chromatography R to 100.0 ml with solution A. Test solution: Suspend 77.0 mg of the substance to be examined in 2 ml of acetonitrile for chromatography R and sonicate for about 5 min. Dilute to 100.0 ml with solution A. Reference solution (1): Suspend 70.0 mg of sulbactam RS in 2 ml of acetonitrile for chromatography R and sonicate for about 5 min. Dilute to 100.0 ml with solution A. Reference solution (2): Dilute 1.0 ml of reference solution (1) to 100.0 ml with solution B. Dilute 1.0 ml of this solution to 10.0 ml with solution B. Reference solution (3): Dissolve 15.0 mg of 6-aminopenicillanic acid R in solution A and dilute to 50.0 ml with solution A. Resolution solution: Mix 1 ml of reference solution (1) and 1 ml of reference solution (3) and dilute to 25.0 ml with solution B. Reference solution (4): Dissolve 8 mg of sulbactam for peak identification RS (containing impurities A, C, D, E and F) in 1 ml of acetonitrile for chromatography R, sonicate for about 5 min and dilute to 10 ml with solution B. Chromatographic system: A column (10 cm × 4 mm) packed with octadecylsilyl silica gel for chromatography R (5 - 10 µm). Temperature: 40 °C . Detector: A spectrophotometer set at 215 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 7.5

98 → 50

2 → 50

7.5 - 8.5

50

50

8.5 - 9.0

50 → 98

50 → 2

9.0 - 12.5

98

2

Inject test solution, solution B, reference solution (2), resolution solution and reference solution (4). Relative retention with reference to sulbactam (retention time = about 2.5 min): impurity A = about 0.4; impurity B = about 0.6; impurity C = about 1.6; impurity D = about 2.0; impurity E = about 2.1; impurity F = about 2.5.

Dentification of impurities: Use the chromatogram supplied with sulbactam for peak identification RS and the chromatogram obtained with reference solution (4) to identify the peaks due to impurities A, C, D, E and F. System suitability: In the chromatogram obtained with the standard solution, resolution between the peaks due to impurity B and sulbactam is minimum 7.0. Limits: Correction factors: for the calculation of content, multiply the peak areas of the following impurities by the corresponding correction factor: impurity A = 0.6; impurity B = 0.5; impurity D = 0.5; impurity F = 0.6; Impurity A: not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.5%). Impurities B, D, F: For each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.1%). Impurities C, E: For each impurity, not more than twice the area of the principal peak in the chromatogram obtained with reference solution (2) (0.2%). Unspecified impurities: For each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.10%). Total: not more than 10 times the area of the principal peak in the chromatogram obtained with reference solution (2) (1.0%). Disregard any peak with an area less than or equal 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Note: Impurity A: (2S)-2-amino-3-methyl-3-sulfinobutanoic acid. Impurity B: (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia-1azabicyclo[3.2.0]heptane-2-carboxylic acid (6-aminopenicillanic acid). Impurity C: 2S,5R,6R)-6-bromo-3,3-dimethyl-7-oxo-4-thia1-azabicyclo[3.2.0]heptane-2-carboxylic acid 4,4-dioxide (6-bromopenicillanic acid sulfone). Impurity D: (2S,5R,6R)-6-bromo-3,3-dimethyl-7-oxo-4-thia-1azabicyclo[3.2.0]heptane-2-carboxylic acid (6-bromopenicillanic acid). Impurity E: (2S,5R)-6,6-dibromo-3,3-dimethyl-7-oxo-4-thia1-azabicyclo[3.2.0]heptane-2-carboxylic acid 4,4-dioxide (6,6-dibromopenicillanic acid sulfone). Impurity F: (2S,5R)-6,6-dibromo-3,3-dimethyl-7-oxo-4-thia-1azabicyclo[3.2.0]heptane-2-carboxylic acid (6,6-dibromopenicillanic acid).

2-Ethylhexanoic acid Not more than 0.5% (Appendix 10.17). Heavy metals Not more than 20 ppm (Appendix 9.4.8). Dissolve 1.0 g in water and dilute to 20 ml with the same solvent. 12 ml of the solution complies with limit test for 913

VP V

SULFACETAMIDE SODIUM

heavy metals, method 1. Prepare the reference solution using 10.0 ml of lead standard solution (2 ppm Pb) R.

Water Not more than 1.0% (Appendix 10.3). Determined on 1.00 g. Bacterial endotoxins Less than 0.17 EU/mg (Appendix 13.2). If intended for use in the manufacture of parenteral preparations without a further appropriate procedure for the removal of bacterial endotoxins. Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. Inject the test solution and reference solution (1). Calculate the percentage content of C8H10NNaO5S in the substance to be examined, using the areas of the peaks in the chromatograms obtained with the test solution and reference solution (1) and the declared content of C8H10NNaO5S in sulbactam RS. Calculate the percentage content of sulbactam sodium by multiplying the percentage content of sulbactam by 1.094 and using the declared content of sulbactam RS. Storage Store in an airtight container. If the substance is sterile, store in a sterile, airtight, tamper-proof container. Action and use Antibacterial.

Identification Apply one of the two following identifications: First identification: A, E. Second identification: B, C, D, E. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of sulfacetamide sodium RS. B. Dissolve 0.1 g in phosphate buffer solution pH 7.0 R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of this solution to 100.0 ml with phosphate buffer solution pH 7.0 R. The ultraviolet absorption spectrum (Appendix 4.1) of the resulting solution in the range 230 nm to 350 nm, exhibits an absorption maximum at 255 nm. The specific absorbance at this maximum is 660 to 720, calculated with reference to the anhydrous substance C. Dissolve 1 g in 10 ml of water, add 6 ml of 2 M acetic acid R and filter. Wash the precipitate with a small quantity of water and dry at 100 °C to 105 °C for 4 h. Melting point: 181 °C to 185 °C (Appendix 6.7). D. Dissolve about 1 mg of the precipitate obtained in identification C, with heating, in 1 ml of water. The solution gives the reaction of primary aromatic amines (Appendix 8.1) with formation of an orange-red precipitate. E. Solution S (see Appearance of solution) gives the reactions of sodium (Appendix 8.1). Appearance of solution Solution S: Dissolve 1.25 g in carbon dioxide-free water R and dilute to 25 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution GY4 (Appendix 9.3, method 2).

Preparation Injections.

pH 8.0 to 9.5 (Appendix 6.2). Determined on solution S.

SULFACETAMIDE SODIUM Sulfacetamidum natricum

Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use and carry out the test protected from light. Mobile phase: Glacial acetic acid - methanol - water for chromatography (1 : 10 : 89). Test solution: Dissolve 0.200 g of the substance to be examined in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (1): Dissolve 5 mg of sulfacetamide sodium RS and 5 mg of sulfanilamide R (impurity A) in 1.0 ml of the mobile phase. Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Chromatographic system: A column (12.5 cm × 4 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 0.8 ml/min.

C8H9N2NaO3S,H2O  

M. 254.2

Sulfacetamide sodium is sodium acetyl[(4-aminophenyl) sulfonyl]azanide. It contains not less than 99.0% and not more than 101.0% of C8H9N2NaO3S, calculated with reference to the anhydrous substance.

Characters White or yellowish-white, crystalline powder. Freely soluble in water, slightly soluble in anhydrous ethanol.

914

SULFADIAZINE

VP V

Volume of injection: 10 µl. Procedure: The run time is 7 times the retention time of sulfacetamide. Relative retention with reference to sulfacetamide (retention time = about 5 min): impurity A = about 0.5. System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to impurity A and sulfacetamide is at least 5.0. Limits: Correction factor: for the calculation of the content, multiply the peak area of impurity A by 0.5. Impurity A: The corrected area of the peak due to impurity A is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (2) (0.2%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.10%). The sum of the peak areas of all impurities is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.5%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Note: Impurity A: 4-aminobenzenesulfonamide (sulfanilamide). Impurity B: N-(4-sulfamoylphenyl)acetamide. Impurity C: N-[[4-(acetylamino)phenyl]sulfonyl]acetamide. Impurity D: 4,4’-sulfonyldianiline (dapsone).

Sulfates Not more than 0.02% (Appendix 9.4.14). Dissolve 2.5 g in distilled water R and dilute to 25 ml with the same solvent. Add 25 ml of 2 M acetic acid R, shake for 30 min and filter. 15 ml of the filtrate complies with the limit test for sulfates. Heavy metals Not more than 20 ppm (Appendix 9.4.8). 12 ml of the filtrate obtained in the test for Sulfates complies with limit test for heavy metals, method 1. Prepare the standard using lead standard solution (1 ppm Pb) R. Water 6.0% to 8.0% (Appendix 10.3). Determined on 0.200 g. Assay Dissolve 0.500 g of the substance to be examined in a mixture of 50 ml of water and 20 ml of 2 M hydrochloric acid R. Add 3 g of potassium bromid R, cool the solution in a bath of iced water and carry out the determination of nitrite titration (Appendix 10.4), determining the endpoint electrometrically. 1 ml of 0.1 M sodium nitrite VS is equivalent to 23.62 mg of C8H9N2NaO3S.

Storage Protected from light. Action and use Antibacterial. Preparation Eye drops. SULFADIAZINE Sulfadiazinum O O S

N N H

N

H 2N

C10H10N4O2S  

M. 250.3

Sulfadiazine is 4-amino-N-(pyrimidin-2-yl)benzene sulfonamide. It contains not less than 99.0% and not more than 101.0% of C10H10N4O2S, calculated with reference to the dried substance.

Characters White, yellowish-white or pinkish-white, crystalline powder or crystals. Practically insoluble in water, slightly soluble in acetone, very slightly soluble in ethanol (96%). It dissolves in solutions of alkali hydroxides and in dilute mineral acids. Identification Apply one of the two following identifications: First identification: A. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of sulfadiazine RS. B. Place 3 g in a dry tube. Immerse the lower part of the tube, inclined at 45°, in a silicone oil bath and heat to about 270 °C. The substance to be examined decomposes and a white or yellowish-white sublimate is formed, which, after recrystallisation from toluene R and drying at 100 °C, melts at 123 °C to 127 °C (Appendix 6.7). C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: 6 M ammonia - water - nitromethane dioxan (3 : 5 : 40 : 50). Solvent mixture: Concentrated ammonia - methanol (4 : 96). Test solution: Dissolve 20 mg of the substance to be examined in 3 ml of the solvent mixture and dilute to 5.0 ml with the solvent mixture. Reference solution: Dissolve 20 mg of sulfadiazine RS in 3 ml of the solvent mixture and dilute to 5.0 ml with the solvent mixture. 915

VP V

SULFADIAZINE

Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of three quarters of the plate. Dry at 105 °C and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. D. Dissolve about 5 mg in 10 ml of a 1 M hydrochloric acid R. Dilute 1 ml of the solution to 10 ml with water. The solution, without further acidification, gives the reaction of primary aromatic amines (Appendix 8.1).

Appearance of solution Dissolve 0.8 g in a mixture of 5 ml of dilute sodium hydroxide solution R and 5 ml of water. The solution is not more intensely coloured than reference solution Y5, BY5 or GY5 (Appendix 9.3, method 2). Acidity To 1.25 g, finely powdered, add 25 ml of carbon dioxidefree water R, shake well. Heat at about 70 °C for 5 min. Cool in iced water for about 15 min and filter. To 20 ml of the filtrate add 0.1 ml of bromothymol blue solution R. Not more than 0.2 ml of 0.1 M sodium hydroxide is required to change the colour of the indicator. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - 0.28% solution of phosphoric acid (10 : 90). Solvent mixture: 1 M sodium hydroxide - acetonitrile water (2 : 20 : 60). Test solution: Dissolve 50.0 mg of the substance to be examined in the solvent mixture and dilute to 100.0 ml with water. Reference solution (1): Dissolve 5.0 mg of sulfadiazine impurity A RS and 5.0 mg of sulfanilic acid (impurity B) in the solvent mixture and dilute to 10.0 ml with water. Dilute 1.0 ml of the solution to 100.0 ml with the mobile phase. Dilute 3.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (3): Dissolve the contents of a vial of acetylsulfadiazine RS (impurity E) in 1 ml of the mobile phase. Reference solution (4): Dissolve 5 mg of sulfadiazine for identification of impurity F RS in the solvent mixture and dilute to 10.0 ml with water. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 260 nm. Flow rate: 1.2 ml/min. Volume of injection: 20 µl. 916

Procedure: The run time is 7 times the retention time of sulfadiazine. Identification of impurities: Use the chromatogram obtained with reference solution (1) to identify the peaks due to impurities A and B; use the chromatogram obtained with reference solution (3) to identify the peak due to impurity E; use the chromatogram obtained with reference solution (4) to identify the peak due to impurity F. Relative retention with reference to sulfadiazine (retention time = about 8.5 min): Impurity A = about 0.26; impurity B = about 0.30; impurity E = about 2.1; impurity F = about 6.0. System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to impurities A and B is at least 2.0. Limits: Correction factor: for the calculation of content, multiply the peak area of impurity E by 0.7. Impurities A, B: For each impurity, not more than the area of the corresponding peak in the chromatogram obtained with reference solution (1) (0.3%). Impurity E: The corrected area of the peak due to impurity E is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (2) (0.2%). Impurity F: The area of the peak due to impurity F is not more than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.15%). Any other impurity: For each impurity, not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Total: not more than 0.5%. Disregard any peak with an area less than 0.3 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.03%). Note: Impurity A: Pyrimidin-2-amine. Impurity B: 4-Aminobenzenesulfonic acid (sulfanilic acid). Impurity C: [(4-Aminophenyl)sulfonyl]guanidine (sulfaguanidine). Impurity D: 4-Aminobenzenesulfonamide (sulfanilamide). Impurity E: N-[4-(pyrimidin-2-ylsulfamoyl)phenyl]acetamide (acetylsulfadiazine). Impurity F: Unknown structure.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). Solvent: Dimethyl sulfoxide R. 1.0 g complies with limit test for heavy metals, method 8. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g.

SULFADIMIDINE

VP V

Assay Dissolve 0.200 g of the substance to be examined in a mixture of 20 ml of 2 M hydrochloric acid R and 50 ml of water. Cool the solution in iced water. Carry out the determination of primary aromatic aminonitrogen (Appendix 10.4), determining the end-point electrometrically. 1 ml of 0.1 M sodium nitrite VS is equivalent to 25.03 mg of C10H10N4O2S. Storage Protected from light.

Appearance of solution Dissolve 0.5 g of the substance to be examined in 10 ml of 1 M sodium hydroxide R. The solution is not more intensely coloured than reference solution Y5, BY5 or GY5 (Appendix 9.3, method 2).

Action and use Sulfonamide antibacterial. Preparation Cream.

Acidity To 1.25 g, finely powdered, of the substance to be examined add 25 ml of carbon dioxide-free water R, and mix. Heat at about 70 °C for 5 minutes. Cool in iced water for about 15 minutes and filter. To 20 ml of the filtrate add 0.1 ml of bromothymol blue solution R. Not more than 0.2 ml of 0.1 N sodium hydroxide VS is required to change the colour of the indicator.

SULFADIMIDINE Sulfadimidinum CH3 O O S

a silicone oil bath and heat to about 270 °C. The substance to be examined decomposes and a white or yellowishwhite sublimate is formed which after recrystallisation from toluene R and drying at 100 °C, melts at 150 °C to 154 °C (Appendix 6.7). D. Dissolve about 5 mg of the substance to be examined in 10 ml of 1 M hydrochloric acid R. Dilute 1 ml of the solution to 10 ml with water. The solution, without further acidification, gives the reaction of primary aromatic amines (Appendix 8.1).

N N H

N

CH3

H 2N

C12H14N4O2S

M. 278.3

Sulfadimidine is 4-amino-N-(4,6-dimethylpyrimidin-2-yl) benzenesulphonamide. It contains not less than 99.0% and not more than 101.0% of C12H14N4O2S, calculated with reference to the dried substance. Characters White, or almost white, crystalline powder or crystals. Very slightly soluble in water, soluble in acetone, slightly soluble in ethanol (96%). It dissolves in solutions of alkali hydroxides and in dilute mineral acids. It melts at about 197 °C, with decomposition. Identification Apply one of the two following identifications: First identification: A, B. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of sulfadimidine RS. B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (1) is similar in position and size to the principal spot in the chromatogram obtained with reference solution (1). C. Place 3 g of the substance to be examined in a dry test tube. Immerse the lower part of the tube, inclined at 45°, in

Related substances Not more than 0.5%. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: 10% ammonia solution - water nitromethane - dioxan (3 : 5 : 40 : 50). Test solution (1): Dissolve 20 mg of the substance to be examined in a mixture of ammonia and methanol (2 : 48) and dilute to 5.0 ml with the same mixture of solvents, and mix. Test solution (2): Dissolve 0.10 g of the substance to be examined in 0.5 ml of ammonia R and dilute to 5.0 ml with methanol R, and mix. If the solution is not clear, heat gently until dissolution is complete. Reference solution (1): Dissolve 20 mg of sulfadimidine RS in a mixture of ammonia and methanol (2 : 48) and dilute to 5.0 ml with the same mixture of solvents, and mix. Reference solution (2): Dilute 2.50 ml of test solution (1) to 100 ml with a mixture of ammonia and methanol (2 : 48), and mix. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm. Dry the plate at 100 °C to 105 °C and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with test solution (2) is not more intense than the principal spot in the chromatogram obtained with reference solution (2). Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g of the substance to be examined complies with the limit test for heavy metals, method 4. Prepare the reference solution using 2 ml of lead standard solution (10 ppm Pb) R. 917

VP V

SULFADOXINE

Loss on drying Not more than 0.5% (Appendix 9.6). (1.00 g; 100 °C - 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.250 g, accurately weighed, of the substance to be examined in a mixture of 20 ml of 2 M hydrochloric acid R and 50 ml of water. Add 3 g of potassium bromide R, and cool the solution in iced water. Titrate slowly with 0.1 M sodium nitrite VS, determining the end-point amperometrically (Appendix 10.1). 1 ml of 0.1 M sodium nitrite VS is equivalent to 27.83 mg of C12H14N4O2S. Storage Protected from light. Action and use Sulfonamide antibacterial.

Appearance of solution Dissolve 1.0 g of the substance to be examined in 10 ml of 1 M sodium hydroxide solution R. The solution is not more intensely coloured than reference solution Y5, BY5 or GY5 (Appendix 9.3, method 2).

Preparations Injections, tablets. SULFADOXINE Sulfadoxinum H3CO O O S

N H

Acidity To 1.25 g, finely powdered, of the substance to be examined add 25 ml of carbon dioxide-free water R, and mix. Heat at about 70 °C for 5 minutes. Cool in iced water for about 15 minutes and filter. To 20 ml of the filtrate add 0.1 ml of bromothymol blue solution R. Not more than 0.2 ml of 0.1 N sodium hydroxide VS is required to change the colour of the indicator.

OCH3 N N

H 2N

C12H14N4O4S

M. 310.3

Sulfadoxine is 4-amino-N-(5,6-dimethoxypyrimidin-4-yl) benzenesulfonamide. It contains not less than 99.0% and not more than 101.0% of C12H14N4O4S, calculated with reference to the dried substance.

Characters White or yellowish-white crystalline powder or crystals. Very slightly soluble in water, slightly soluble in ethanol (96%) and in methanol. It dissolves in solutions of alkali hydroxides and in dilute mineral acids. It melts at about 198 °C, with decomposition. Identification Apply one of the two following identifications: First identification: A, C. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the 918

substance to be examined is concordant with the spectrum of sulfadoxine RS. B. Examine the chromatograms obtained in the test for Related substances. The principal spot in the chromatogram obtained with test solution (2) is similar in position and size to the principal spot in the chromatogram obtained with reference solution (1). C. Dissolve 0.5 g of the substance to be examined in 1 ml of a 40% v/v solution of sulfuric acid R, heating gently. Continue heating until a white crystalline precipitate appears (about 2 minutes). Allow to cool and add 10 ml of 2 M sodium hydroxide R. Cool again. Add 25 ml of ether R and shake for 5 minutes. Separate the ether layer, dry over anhydrous sodium sulfate R and filter. Evaporate the solvent by heating in a water-bath. The residue melts at 80 °C to 82 °C or at 90 °C to 92 °C (Appendix 6.7). D. Dissolve about 5 mg of the substance to be examined in 10 ml of 1 M hydrochloric acid R. Dilute 1 ml of the solution to 10 ml with water. The solution, without further acidification, gives the reaction of primary aromatic amines (Appendix 8.1).

Related substances Not more than 0.5%. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: 10% ammonia solution - water nitromethane - dioxan (3 : 5 : 40 : 50). Test solution (1): Dissolve 0.10 g of the substance to be examined in a mixture of ammonia R and methanol R (2 : 48) and dilute to 5 ml with the same mixture of solvents, and mix. Test solution (2): Dilute 2 ml of test solution (1) to 10 ml with a mixture of ammonia R and methanol R (2 : 48), and mix. Reference solution (1): Dissolve 20 mg of sulfadoxine RS in a mixture of ammonia R and methanol R (2 : 48) and dilute to 5 ml with the same mixture of solvents, and mix. Reference solution (2): Dilute 2.5 ml of test solution (2) to 100 ml with a mixture of ammonia R and methanol R (2 : 48), and mix.

VP V

Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm. Dry the plate at 100 to 105°C and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with test solution (1) is not more intense than the principal spot in the chromatogram obtained with reference solution (2).

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g of the substance to be examined complies with the limit test for heavy metals, method 4. Prepare the reference solution using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.00 g; 100 °C - 105 °C). Sulphated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.250 g, accurately weighed, of the substance to be examined in a mixture of 20 ml of 2 M hydrochloric acid R and 50 ml of water. Add 3 g of potassium bromide R, and cool the solution in iced water. Titrate slowly with 0.1 M sodium nitrite VS, determining the end-point amperometrically (Appendix 10.1). 1 ml of 0.1 M sodium nitrite VS is equivalent to 31.03 mg of C12H14N4O4S. Storage Protected from light. Action and use Sulfonamide antibacterial. Preparations Injection; tablets (in combination with pyrimethamine). SULFADOXINE AND PYRIMETHAMINE TABLETS Tabellae Sulfadoxini et Pyrimethamini Sulfadoxine and pyrimethamine tablets contain sulfadoxine and pyrimethamine. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of sulfadoxine, C12H14N4O4S, 90.0% to 110.0% of the stated amount. Content of pyrimethamine, C12H13ClN4, 90.0% to 110.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4).

SULFADOXINE AND PYRIMETHAMINE TABLETS

Coating substance: Silica gel GF254 (coating thickness 0.25 mm). Mobile phase: Heptane - chloroform - 5% (v/v) solution of methanol in ethanol - glacial acetic acid (4 : 4 : 4 : 1). Test solution: To a quantity of the powdered tablets equivalent to about 100 mg sulfadoxine add 10 ml of a 2% (v/v) solution of ammonia in methanol, shake vigorously for 3 minutes and filter. Reference solution (1): A 10 mg/ml solution of sulfadoxine RS in a 2% (v/v) solution of ammonia in methanol. Reference solution (2): A 0.5 mg/ml solution of pyrimethamine RS in a 2% (v/v) solution of ammonia in methanol. Procedure: Apply separately to the plate 10 µl of each solution. After developing over 3/4 of the plate, remove the plate, allow it to dry in air and examine under ultraviolet light at 254 nm. The two principal spots in the chromatogram obtained with the test solution are similar in position, colour to the two principal spots in the chromatograms obtained with the reference solutions. B. In the Assay, the retention times of the two principal peaks in the chromatogram obtained with the test solution are the same as those of two principal peaks in the chromatogram obtained with the reference solution.

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 1000 ml of phosphate buffer pH 6.8 R. Rotation speed: 75 rpm. Time: 30 min. Procedure: After the specified time, withdraw a sample of the medium and filter, discarding the first 20 ml of the filtrate. Dilute the filtrate to an appropriate concentration with the mobile phase if necessary. Carry out the method for liquid chromatography (Appendix 5.3). Mobile phase, chromatographic system, reference solution as described in the Assay. Tolerance: Not less than 60% (Q) of the stated amount of sulfadoxine, C12H14N4O4S is dissolved in 30 minutes. Not less than 60% (Q) of the stated amount of pyrimethamine, C12H13ClN4 is dissolved in 30 minutes. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Buffer solution pH 5.9 - acetonitrile (4 : 1). Buffer solution pH 5.9: To a mixture of 200 ml of acetonitrile R and 700 ml of water add 1 ml of triethylamine R, adjust the pH to 5.9 with a 1% solution of acetic acid R, add water to produce 1000 ml, mix and filter. Standard stock solution of sulfadoxine: Transfer about 500 mg, accurately weighed, of sulfadoxine RS to a 100 ml volumetric flask, dissolve in 35 ml of acetonitrile R, dilute with mobile phase to volume, and mix. Standard stock solution of pyrimethamine: Transfer about 25 mg, accurately weighed, of sulfadoxine RS to a 100 ml 919

VP V

SULFAGUANIDINE

volumetric flask, dissolve in 35 ml of acetonitrile R, dilute with mobile phase to volume, and mix. Reference solution: Pipette 2.0 ml of standard stock solution of sulfadoxine and 2.0 ml of standard stock solution of pyrimethamine into a 100 ml volumetric flask, dilute with mobile phase to volume, and mix. Test solution: Weigh 20 tablets, calculate the average mass, finely powder. Transfer an accurately weighed quantity of the powder, equivalent to about 500 mg of sulfadoxine and 25 mg of pyrimethamine, to a 100 ml volumetric flask, add 35 ml of acetonitrile R, sonicate for 30 minutes, dilute with the mobile phase to volume, mix, and filter. Pipette 2.0 ml of the filtrate in a 100 ml volumetric flask, dilute to volume with mobile phase. Chromatographic system A column (30 cm × 3.9 mm) packed with stationary phase B (5 µm). Detector: A spectrophotometer set at 230 nm. Flow rate: 2.0 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution. The test is not valid unless the resolution between the peaks due to sulfadoxine and pyrimethamineis at least 1.5, the relative standard deviation of the peak areas of each component in 6 replicate injections is not more than 2.0%. Calculate the content of sulfadoxine, C12H14N4O4S, and pyrimethamine, C12H13ClN4, using the areas of the principal peaks obtained with the reference solution and the test solution and the declared contents of C12H14N4O4S and C12H13ClN4 in sulfadoxine RS and pyrimethamine RS, respectively.

Storage Store in a well-closed container, at a cool and dry place, protected from light. Action and use Antimalarial. Usual strength 500 mg of sulfadoxine and 25 mg of pyrimethamin. SULFAGUANIDINE Sulfaguanidinum O O S

NH N H

NH2

H 2N

C7H10N4O2S

920

M. 214.2

Sulfaguanidine is (4-aminophenylsulfonyl) guanidine. It contains not less than 99.0% and not more than 101.0% of C7H10N4O2S, calculated with reference to the dried substance.

Characters A white or almost white, fine crystalline powder. Very slightly soluble in water and in ethanol (96%), slightly soluble in acetone, practically insoluble in methylene chloride. It dissolves in dilute solutions of mineral acids. Identification Apply one of the two following identifications: First identification: A, B. Second identification: B, C, D, E. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of sulfaguanidine RS. B. Melting point: 189 °C to 193°C (Appendix 6.7), determined on the dried substance. C. Examine the chromatograms obtained in the test for Related substances. The principal spot in the chromatogram obtained with test solution (2) is similar in position and size to the principal spot in the chromatogram obtained with reference solution (1). D. Dissolve about 5 mg of the substance to be examined in 10 ml of 1 M hydrochloric acid R. Dilute 1 ml of the solution to 10 ml with water. The solution, without further acidification, gives the reaction of primary aromatic amines (Appendix 8.1). E. Suspend 0.1 g of the substance to be examined in 2 ml of water, add 1 ml of 1-naphthol solution R and 2 ml of a mixture of equal volumes of water and strong sodium hypochlorite solution R. A red colour develops. Acidity Solution S: To 2.5 g of the substance to be examined, add 40 ml of carbon dioxide-free water R. Heat at about 70 °C for 5 minutes. Cool while stirring in iced water for about 15 minutes, filter and dilute to 50 ml with carbon dioxidefree water R, and mix. To 20 ml of solution S add 0.1 ml of bromothymol blue solution R. Not more than 0.2 ml of 0.1 N sodium hydroxide VS is required to change the colour of the indicator. Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Anhydrous formic acid - methanol methylene chloride (10 : 20 : 70). Test solution (1): Dissolve 50 mg of the substance to be examined in 5 ml of acetone R. Test solution (2): Dilute 2 ml of test solution (1) to 10 ml with acetone R, and mix. Reference solution (1): Dissolve 10 mg of sulfaguanidine RS in 5 ml of acetone R.

SULFAMETHOXAZOLE

VP V

Reference solution (2): Dilute 5 ml of test solution (2) to 200 ml with acetone R, and mix. Reference solution (3): Dilute 5 ml of reference solution (2) to 10 ml with acetone R, and mix. Reference solution (4): Dissolve 10 mg of sulfanilamide RS in 5 ml of test solution (2). Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with test solution (1) is not more intense than the principal spot in the chromatogram obtained with reference solution (2) (0.5%) and at most one such spot is more intense that the spot in the chromatogram obtained with reference solution (3) (0.25%). The test is not valid unless the chromatogram obtained with reference solution (4) shows two clearly separated principal spots.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 12 ml of solution S complies with the limit test for heavy metals, method 6. Prepare the reference solution using lead standard solution (1 ppm Pb) R. Loss on drying Not more than 8.0% (Appendix 9.6). (1.00 g; 100 - 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.175 g, accurately weighed, of the substance to be examined in 50 ml of 2 M hydrochloric acid R. Add 3 g of potassium bromide R, and cool the solution in iced water. Titrate slowly with 0.1 M sodium nitrite VS, determining the end-point amperometrically (Appendix 10.1). 1 ml of 0.1 M sodium nitrite VS is equivalent to 21.42 mg of C7H10N4O2S. Storage Protected from light. Action and use Sulfonamide antibacterial. Preparation Tablets.

Content of sulfaguanidine, C7H10N4O2S, 95.0% to 105.0% of the stated amount. Identification A. To a quantity of the powdered tablets equivalent to about 0.2 g of sulfaguanidine add 5 ml of 10% sodium hydroxide solution R and boil, ammonia vapour is detectable. B. To a quantity of the powdered tablets equivalent to about 50 mg of sulfaguanidine add 2 ml of 10% hydrochloric acid solution R, shake well, and filter. Cool the filtrate in iced water, add 4 ml of 1% sodium nitrite solution R, and shake well. To 1 ml of the resulting solution add 5 ml of 2-naphthol solution R, a dark red precipitate is produced. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Dichloromethane - methanol - anhydrous formic acid (70 : 20 : 10). Reference solution: Dissolve 10 mg of sulfaguanidine RS in 5 ml of acetone R. Test solution: To a quantity of the powdered tablets equivalent to about 20 mg sulfaguanidine add 10 ml of acetone R, shake well and filter. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. Assay Weigh 20 tablets, calculate the average mass, finely powder. Weigh accurately a quantity of the powdered tablets equivalent to about 0.200 g of sulfaguanidine, add 15 ml of a 25% solution of hydrochloric acid R and 50 ml of water and shake well. Carry out the assay by nitrite titration (Appendix 10.4). 1 ml of 0.1 M sodium nitrite VS is equivalent to 21.42 mg of C7H10N4O2S. Storage Store in a well-closed container, protected from light. Usual strength 500 mg. SULFAMETHOXAZOLE Sulfamethoxazolum

SULFAGUANIDINE TABLETS Tabellae Sulfaguanidini Sulfaguanidine tablets contain sulfaguanidine. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements:

C10H11N3O3S

M. 253.3 921

SULFAMETHOXAZOLE

Sulfamethoxazole is 4-amino-N-(5-methylisoxazol-3-yl) benzenesulfonamide. It contains not less than 99.0% and not more than 101.0% of C10H11N3O3S, calculated with reference to the dried substance.

Characters White, or almost white, crystalline powder. Practically insoluble in water, freely soluble in acetone, sparingly soluble in ethanol (96%). It dissolves in dilute solutions of sodium hydroxide and in dilute acids. Identification Apply one of the two following identifications: First identification: A, B. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of sulfamethoxazole RS. B. Melting point: 169 °C to 172 °C (Appendix 6.7). C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: 10% ammonia solution - water nitromethane - dioxan (3 : 5 : 41 : 51). Test solution: Dissolve 20 mg of the substance to be examined in a mixture of ammonia and methanol (2 : 48) and dilute to 5 ml with the same mixture of solvents, and mix. Reference solution: Dissolve 20 mg of sulfamethoxazole RS in a mixture of ammonia and methanol (2 : 48) and dilute to 5 ml with the same mixture of solvents, and mix. Procedure: Apply separately to the plate 5 µl of each solution. Develop over 3/4 of the plate. Dry the plate at 100 °C to 105 °C and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. D. Dissolve about 5 mg of the substance to be examined in 10 ml of 1 M hydrochloric acid R. Dilute 1 ml of the solution to 10 ml with water. The solution, without further acidification, gives the reaction of primary aromatic amines (Appendix 8.1). Appearance of solution Dissolve 1.0 g of the substance to be examined in 10 ml of 1 M sodium hydroxide R. The solution is clear (Appendix 9.2) and not more intensely coloured than reference solution Y5, BY5 or GY5 (Appendix 9.3, method 2). Acidity Shake 1.25 g, finely powdered, of the substance to be examined with 25 ml of water. Heat at about 70 °C for 5 min. Cool in iced water for about 15 min and filter. To 20 ml of the filtrate add 0.1 ml of bromothymol blue solution R. Not more than 0.3 ml of 0.1 M sodium hydroxide VS is required to change the colour of the indicator.

922

VP V

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: A mixture of methanol R and 0.1 M potassium dihydrogen phosphate R (35 : 65), adjusted to pH 6.0 with a 10% solution of potassium hydroxide R. Test solution: Dissolve 50.0 mg of the substance to be examined in 45 ml of the mobile phase, sonicate at about 45°C for 10 min, cool and dilute to 50.0 ml with the mobile phase, and mix. Reference solution (1): Dilute 1.0 ml of the test solution to 10.0 ml with the mobile phase, and mix. Dilute 1.0 ml of this solution to 100.0 ml with the mobile phase, and mix. Reference solution (2): Dissolve 5 mg of the substance to be examined and 5 mg of sulfamethoxazole impurity A RS in the mobile phase, dilute to 50.0 ml with the mobile phase, and mix. Reference solution (3): Dissolve 5.0 mg of sulfamethoxazole impurity F RS in the mobile phase, dilute to 50 ml with the mobile phase, and mix. Dilute 1.0 ml of this solution to 100.0 ml with the mobile phase, and mix. Chromatographic system: A column (25 cm × 4.0 mm) packed with stationary phase B (5 µm). Column temperature: 30 °C. Detector: A spectrophotometer set at 210 nm. Flow rate: 0.9 ml/min. Volume of injection: 20 µl. Procedure: Inject the reference solutions and the test solution. Continue the chromatography for at least 3 times the retention time of sulfamethoxazole. Relative retentions with reference to sulfamethoxazole (retention time = about 10 min): impurity D = about 0.3; impurity E = about 0.35; impurity F = about 0.45; impurity C = about 0.5; impurity A = about 1.2; impurity B = about 2.0. The test is not valid unless in the chromatogram obtained with reference solution (2), the resolution between the peaks due to sulfamethoxazole and impurity A is at least 3.5. Limits: In the chromatogram obtained with the test solution: The area of any peak corresponding to impurities A, B, C, D, E is not greater the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%); the area of the peak corresponding to impurity F is not greater than the area of the principal peak in the chromatogram obtained with reference solution (3) (0.1%); the area of any other impurity peak is not greater the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%). The sum of the areas of all impurity peaks is not greater than 3 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.3%). Disregard any peak with an area equal to or less than 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.025%). Note: Impurity A: N-[4-[(5-methylisoxazol-3-yl)sulfamoyl] phenyl] acetamide.

SULFAMETHOXAZOLE TABLETS

VP V Impurity B: 4 - [[(4 - aminophenyl)sulfonyl]amino] - N - (5 methylisoxazol–3-yl)benzenesulfonamide. Impurity C: 5-methylisoxazol-3-amine. Impurity D: 4-aminobenzenesulfonic acid (sulfanilic acid). Impurity E: 4-aminobenzenesulfonamide (sulfanilamide). Impurity F: 4-amino-N-(3-methylisoxazol-5-yl) benzene-sulfonamide.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with the limit test for heavy metals, method 4. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 100 °C to 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.200 g, accurately weighed, of the substance to be examined in a mixture of 50 ml of 10% solution of hydrochloric acid R and 50 ml of water. Add 3 g of potassium bromide R, and cool the solution in iced water. Titrate slowly with 0.1 M sodium nitrite VS, determining the end-point amperometrically (Appendix 10.1). 1 ml of 0.1 M sodium nitrite VS is equivalent to 25.33 mg of C10H11N3O3S. Storage Protected from light. Action and use Sulfonamide antibacterial. Preparations Co-trimoxazole oral suspension; Co-trimoxazole tablets. SULFAMETHOXAZOLE TABLETS Tabellae Sulfamethoxazoli Sulfamethoxazole tablets contain sulfamethoxazole. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements:

Content of sulfamethoxazole, C10H11N3O3S, 95.0% to 105.0% of the stated amount. Identification A. To a quantity of the powdered tablets equivalent to 100 mg of sulfamethoxazole, add 2 ml of 10% hydrochloric acid solution R, shake vigorously, and filter. Cool the filtrate in iced water, add 0.2 ml of 10% sodium nitrite solution R, and after 1 min to 2 min, add 1 ml of alkaline 2-naphthol solution R, a dark red precipitate and dark red colour are produced.

C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Chloroform - methanol - dimethylformamide (20 : 2 : 1) or chloroform - methanol (19 : 1). Test solution: Dissolve a quantity of the powdered tablets equivalent to 0.4 g of sulfamethoxazole in 20 ml of methanol R and filter. Reference solution: A 2% solution of sulfamethoxazole RS in methanol R. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm. After removal of the plate, allow it to dry at room temperature and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution (spray with Dragendorff reagent R when using silica gel G as the coating substance).

Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 900 ml of 0.1 M hydrochloric acid R. Rotation speed: 100 rpm. Time: 30 min. Procedure: After the specified time, withdraw a sample of the medium and filter, discarding the first 20 ml of the filtrate. Filter a portion of the test solution. Dilute the filtrate with the medium to obtain an appropriate concentration. Determine the absorbance (Appendix 4.1) at the maximum at about 265 nm, in a 1-cm cell, using 0.1 M hydrochloric acid R as a blank, in comparison with the reference solution having the same concentration of sulfamethoxazole RS in 0.1 M hydrochloric acid R. Tolerance: Not less than 80% (Q) of the stated amount of sulfamethoxazole, C10H11N3O3S, is dissolved in 30 min. Assay Weigh 20 tablets, calculate the average mass, finely powder. Weigh accurately a quantity of the powdered tablets equivalent to about 0.300 g of sulfamethoxazole and dissolve in 20 ml of 10% hydrochloric acid solution R, add 3 g of potassium bromide R. Cool the solution in iced water and carry out the Assay by nitrite titration (Appendix 10.4), determining the end-point potentiometrically (Appendix 10.2). Use a platinum-calomel electrode combination. 1 ml of 0.1 M sodium nitrite VS is equivalent to 25.33 mg of C10H11N3O3S. Storage Protected from light. Action and use Sulfonamide antibacterial. Usual strength 500 mg.

923

VP V

SULFAMETHOXYPYRIDAZINE

SULFAMETHOXYPYRIDAZINE Sulfamethoxypyridazinum

C11H12N4O3S

more intensely coloured than reference solution Y4 or BY4 (Appendix 9.3, method 2).

M. 280.3

Sulfamethoxypyridazine is 4-amino-N-(6-methoxypyridazin -2-yl)benzenesulfonamide. It contains not less than 99.0% and not more than 101.0% of C11H12N4O3S, calculated with reference to the dried substance.

Characters A white or slightly yellowish, crystalline powder, colouring slowly on exposure to light. Practically insoluble in water, sparingly soluble in acetone, slightly soluble in ethanol (96%), very slightly soluble in methylene chloride. It dissolves in dilute solutions of alkali hydroxides and in dilute mineral acids. It melts at about 180 °C, with decomposition. Identification Apply one of the two following identifications: First identification: A, B. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of sulfamethoxypyridazine RS. B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (2) is similar in position and size to the principal spot in the chromatogram obtained with reference solution (1). C. Dissolve about 0.5 g of the substance to be examined in a 1 ml of a 40% v/v solution of sulfuric acid R, heating gently. Continue heating until a crystalline precipitate appears (about 2 min). Cool and add 10 ml of 2 M sodium hydroxide R. Cool again, add 25 ml of ether R and shake the solution for 5 min. Separate the ether layer, dry over anhydrous sodium sulfate R and filter. Evaporate the ether by heating in a water bath. An oily residue is obtained which becomes crystalline on cooling, if necessary, scratch the wall of the container with a glass rod. The residue melts at 102  °C to 106 °C (Appendix 6.7). D. Dissolve about 5 mg of the substance to be examined in 10 ml of 1 M hydrochloric acid R. Dilute 1 ml of the solution to 10 ml with water. The solution, without further acidification, gives the reaction of primary aromatic amines (Appendix 8.1). Appearance of solution Dissolve 1.0 g of the substance to be examined in a mixture of 10 ml of 1 M sodium hydroxide R and 15 ml of water. The solution is clear (Appendix 9.2) and not 924

Acidity To 1.25 g, finely powdered, of the substance to be examined add 25 ml of carbon dioxide-free water R, and mix. Heat at about 70 °C for 5 min. Cool in iced water for about 15 min and filter. To 20 ml of the filtrate add 0.1 ml of bromothymol blue solution R. Not more than 0.5 ml of 0.1 M sodium hydroxide VS is required to change the colour of the indicator. Related substances Not more than 0.5%. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Dilute ammonia - water - 2-propanol - ethyl acetate (1 : 9 : 30 : 50). Test solution (1): Dissolve 0.10 g of the substance to be examined in acetone R and dilute to 5 ml with the same solvent, and mix. Test solution (2): Dilute 1 ml of test solution (1) to 10 ml with acetone R and mix. Reference solution (1): Dissolve 20 mg of sulfamethoxypyridazine RS in acetone R and dilute to 10 ml with the same solvent, and mix. Reference solution (2): Dilute 1.0 ml of test solution (2) to 20 ml with acetone R and mix. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm. Dry the plate at 100 °C to 105 °C and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with test solution (1) is not more intense than the spot in the chromatogram obtained with reference solution (2). Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with the limit test for heavy metals, method 4. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.00 g; 100  °C to 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.250 g, accurately weighed, of the substance to be examined in 50 ml of 2 M hydrochloric acid R. Add 3 g of potassium bromide R, and cool the solution in iced water. Titrate slowly with 0.1 M sodium nitrite VS, determining the end-point amperometrically (Appendix 10.1). 1 ml of 0.1 M sodium nitrite VS is equivalent to 28.03 mg of C11H12N4O3S.

SULFASALAZINE

VP V

Storage Protected from light.

Detector: A spectrophotometer set at 320 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions:

Action and use Sulfonamide antibacterial. Preparation Injections. SULFASALAZINE Sulfasalazinum

C18H14N4O5S 

M. 398.4 

Sulfasalazine is 2-hydroxy-5-[2-[4-(pyridin-2-ylsulfa moyl)phenyl]diazenyl]benzoic acid. It contains not less than 97.0% and not more than 101.5% of C18H14N4O5S, calculated with reference to the dried substance.

Characters Bright yellow or brownish-yellow, fine powder. Practically insoluble in water and methylene chloride, very slightly soluble in ethanol (96%). It dissolves in dilute solutions of alkali hydroxides. Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of sulfasalazine RS. Examine the substances prepared as discs. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Dissolve 1.13 g of sodium dihydrogen phosphate R and 2.5 g of sodium acetate R in 900 ml of water; adjust to pH 4.8 with glacial acetic acid R and dilute to 1000.0 ml with water. Mobile phase B: Mobile phase A - methanol (10 : 40). Test solution: Dissolve 25.0 mg of the substance to be examined in 0.1 M ammonia R and dilute to 25.0 ml with the same solvent. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with 0.1 M ammonia R. Reference solution (2): Dissolve 1.0 mg of sulfasalazine derivative for resolution RS in 10.0 ml of reference solution (1). Dilute 1.0 ml of this solution to 10.0 ml with reference solution (1). Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm).

Time (minutes)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

1 - 15

60 → 45

40 → 55

15 - 25

45

55

25 - 60

45 → 0

55 → 100

60 - 65

0

100

Relative retention with reference to sulfasalazine: Impurity H = about 0.16; impurity I = about 0.28; impurity C = about 0.80; impurity F = about 0.85; impurity G = about 1.39; impurity E = about 1.63; impurity B = about 1.85; impurity D = about 1.90; impurity A = about 2.00. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to sulfasalazine and sulfasalazine derivative for resolution is at least 3.0. Limits: Impurities A, B, C, D, E, F, G, I: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (1%). The sum of the peak areas of all impurities is not more than 4 times the area of the principal peak in the chromatogram obtained with reference solution (1) (4%). Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%); disregard any peak with a retention time less than 6 min (due to impurities H and J). Note: Impurity A: 4,4′-[(4-hydroxy-1,3-phenylene)bis(diazenediyl)] bis[N-(pyridin-2-yl)benzenesulfonamide]. Impurity B: 2-hydroxy-3,5-bis[2-[4-(pyridin-2-ylsulfamoyl) phenyl]diazenyl]benzoic acid. Impurity C: 2-hydroxy-5-[2-[4-(2-iminopyridin-1(2H)-yl)phenyl] diazenyl]benzoic acid. Impurity D: 4-[2-(2-hydroxyphenyl)diazenyl]-N-(pyridin-2-yl) benzenesulfonamide. Impurity E: 2-hydroxy-4′-(pyridin-2-ylsulfamoyl)-5-[2-[4-(pyridin2-ylsulfamoyl)phenyl]diazenyl]biphenyl-3-carboxylic acid. Impurity F: 2-hydroxy-3-[2-[4-(pyridin-2-ylsulfamoyl)phenyl] diazenyl]benzoic acid. Impurity G: 5-[2-[4′,5-bis(pyridin-2-ylsulfamoyl)biphenyl-2-yl] diazenyl]-2-hydroxybenzoic acid. Impurity I: 2-hydroxy-5-[2-(4-sulfophenyl)diazenyl]benzoic acid.

Impurities H and J Examine by liquid chromatography (Appendix 5.3). Mobile phase: Mobile phase B - mobile phase A (30 : 70) 925

VP V

SULFATHIAZOLE

(mobile phase A and mobile phase B as described in the test for Related substances). Test solution: Dissolve 25.0 mg of the substance to be examined in 0.1 M ammonia R and dilute to 25.0 ml with the same solvent. Reference solution (1): Dissolve 5.0 mg of salicylic acid R (impurity H) and 5.0 mg of sulfapyridine RS (impurity J) in 0.1 M ammonia R and dilute to 10.0 ml with the same solvent. Reference solution (2): Dilute 2.0 ml of reference solution (1) to 100.0 ml with 0.1 M ammonia R. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 300 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: The run time: 10 min. Inject the test solution and reference solution (2). Retention time of impurity H is about 6 min; impurity J is about 7 min. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurities H and J is at least 2.0. Limits: Impurities H and J: For each impurity, the area is not more than 0.5 times the area of the corresponding peak in the chromatogram obtained with reference solution (2) (0.5%). Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%).

Chlorides Not more than 140 ppm (Appendix 9.4.5). Dissolve 1.25 g of the substance to be examined in 50 ml of distilled water R. Heat at about 70 °C for 5 min, allow to cool and filter. Add 1 ml of nitric acid R to 20 ml of the filtrate, allow to stand for 5 min and filter to obtain a clear solution. Sulfates  Not more than 0.04% (Appendix 9.4.14). Add 1 ml of 2 M hydrochloric acid R to 20 ml of the filtrate prepared for the test for Chlorides, allow to stand for 5 min and filter. Heavy metals Not more than 10 ppm (Appendix 9.4.8). 2.0 g complies with limit test for heavy metals, method 4. Prepare the standard using 2.0 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 1.0% (Appendix 9.6). (1.000 g; 105 °C; 2 h).

926

Sulfated ash Not more than 0.5% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.150 g of the substance to be examined in 0.1 M sodium hydroxide R and dilute to 100.0 ml with the same solvent. Transfer 5.0 ml of this solution to a 1000 ml volumetric flask containing about 750  ml of  water. Add 20.0 ml of 0.1 M acetic acid R and dilute to 1000.0 ml with water. Prepare a reference solution at the same time and in the same manner using 0.150 g of sulfasalazine RS. Measure the absorbance of the 2 solutions at the absorption maximum at 359 nm. Calculate the content of C18H14N4O5S using the absorbances for the test solution, the reference solution and the content of C18H14N4O5S in sulfasalazine RS. Storage Store in an airtight container, protected from light. Action and use Treatment of ulcerative colitis. Preparation Tablets. SULFATHIAZOLE Sulfathiazolum

C9H9N3O2S2

M. 255.3

Sulfathiazole is 4-amino-N-(thiazol-2-yl) benzenesulfonamide. It contains not less than 99.0% and not more than 101.0% of C9H9N3O2S2, calculated with reference to the dried substance.

Characters A white or slightly yellowish, crystalline powder. Practically insoluble in water and in methylene chloride, slightly soluble in ethanol (96%). It dissolves in dilute solutions of alkali hydroxides and in dilute mineral acids. Identification Apply one of the two following identifications: First identification: A, B. Second identification: B, C, D, E. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of sulfathiazole RS. If the spectra obtained show differences, dissolve the substance to be examined and

SULPIRIDE

VP V

the reference substance separately in ethanol (96%) R, evaporate to dryness in vacuo and record the spectra again using the residues. B. Melting point: 200  °C to 203  °C (Appendix 6.7). Melting may occur at about 175 °C, followed by solidification and a second melting between 200 °C and 203 °C. C. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (2) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). D. Dissolve about 10 mg of the substance to be examined in a mixture of 10 ml of water and 2 ml of 0.1 M sodium hydroxide R and add 0.5 ml of a 12.5% solution of copper (II) sulfate R. A greyish-blue or purple precipitate is formed. E. Dissolve about 5 mg of the substance to be examined in 10 ml of 1 M hydrochloric acid R. Dilute 1 ml of the solution to 10 ml with water. The solution, without further acidification, gives the reaction of primary aromatic amines (Appendix 8.1).

100 °C to 105 °C for 10 minutes and spray with a 0.1% solution of dimethylaminobenzaldehyde R in ethanol (96%) R containing 1% v/v of hydrochloric acid R. Any secondary spot in the chromatogram obtained with test solution (1) is not more intense than the spot in the chromatogram obtained with reference solution (2).

Appearance of solution Dissolve 1.0 g of the substance to be examined in 10 ml of 1 M sodium hydroxide R. The solution is clear (Appendix 9.2) not more intensely coloured than reference solution GY5 (Appendix 9.3, method 2).

Assay Dissolve 0.200 g, accurately weighed, of the substance to be examined in 50 ml of 2 M hydrochloric acid R. Add 3 g of potassium bromide R, and cool the solution in iced water. Titrate slowly with 0.1 M sodium nitrite VS, determining the end-point amperometrically (Appendix 10.1). 1 ml of 0.1 M sodium nitrite VS is equivalent to 25.53 mg of C9H9N3O2S2.

Acidity To 1.0 g, finely powdered, of the substance to be examined add 50 ml of carbon dioxide-free water R, and mix. Heat at about 70 °C for 5 minutes. Cool rapidly to 20 °C and filter. To 25 ml of the filtrate add 0.1 ml of bromothymol blue solution R. Not more than 0.1 ml of 0.1 M sodium hydroxide VS is required to change the colour of the indicator. Related substances Not more than 0.5%. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel H. Mobile phase: Ammonia - butanol (18 : 90). Test solution (1): Dissolve 0.1 g of the substance to be examined in a mixture of ammonia and ethanol (96%) (1 : 9) and dilute to 10 ml with the same mixture of solvents, and mix. Test solution (2): Dilute 1 ml of test solution (1) to 5 ml with a mixture of ammonia R and ethanol (96%) R (1 : 9), and mix. Reference solution (1): Dissolve 20 mg of sulfathiazole RS in a mixture of ammonia R and ethanol (96%) R (1 : 9), dilute to 10 ml with the same mixture of solvents, and mix. Reference solution (2): Dissolve 50 mg of sulfanilamide RS in a mixture of ammonia and ethanol (96%) (1 : 9), dilute to 100 ml with the same mixture of solvents, and mix. Dilute 1 ml of this solution to 10 ml with the same mixture of solvents, and mix. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm. Dry the plate at

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with the limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.00 g; 100 °C to 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g.

Storage Protected from light. Action and use Sulfonamide antibacterial. SULPIRIDE Sulpiridum

C15H23N3O4S  

M. 341.4

Sulpiride is (RS)-N-[(1-ethylpyrrolidin-2-yl)methyl]2-methoxy-5-sulfamoylbenzamide. It contains not less than 98.5% and not more than 101.0% of C15H23N3O4S, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder. Practically insoluble in water, sparingly soluble in methanol, slightly soluble in ethanol (96%) and in methylene chloride. It dissolves in dilute solutions of mineral acids and alkali hydroxides. 927

VP V

SULPIRIDE

Identification Apply one of the two following identifications: First identification: A, B. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of sulpiride RS. Examine the substances prepared as discs. B. Melting point: 177 °C to 181 °C (Appendix 6.7). C. In test for Impurity A, examine plate in ultraviolet light at 254 nm, the principal spot in the chromatogram obtained with test solution (2) is similar in position and size to the principal spot in the chromatogram obtained with reference solution (1). D. To about 1 mg in a porcelain dish, add 0.5 ml of sulfuric acid R and 0.05 ml of formaldehyde solution R. Examined in ultraviolet light at 365 nm, the solution shows blue fluorescence. Appearance of solution Dissolve 1.0 g in 2 M acetic acid R and dilute to 10 ml with the same acid. The solution is clear (Appendix 9.2) and not more intensely coloured than reference solution Y6 (Appendix 9.3, method 1). Impurity A Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel F254. Mobile phase: Concentrated ammonia - dioxan - methanol - methylene chloride (2 : 10 : 14 : 90). Test solution (1): Dissolve 0.20 g of the substance to be examined in methanol R, sonicate until complete dissolution and dilute to 10.0 ml with the same solvent. Test solution (2): Dilute 1 ml of test solution (1) to 10.0 ml with methanol R. Reference solution (1): Dissolve 20 mg of sulpiride RS in methanol R and dilute to 10.0 ml with the same solvent. Reference solution (2): Dissolve 5 mg of sulpiride impurity A RS in methanol R and dilute to 25.0 ml with the same solvent. Dilute 1.0 ml of this solution to 10.0 ml with methanol R. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of a half of the plate. Allow the plate to dry in air. Examine in ultraviolet light at 254 nm for identification test C and then spray with ninhydrin solution R; heat at 100 °C to 105 °C for 15 min and examine in daylight. In the chromatogram obtained with the test solution (1), any spot due to impurity A is not more intense than the corresponding spot in the chromatogram obtained with reference solution (2) (0.1%). Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Buffer solution pH 3.3 - methanol acetonitrile (80 : 10 : 10). 928

Buffer solution pH 3.3: A solution containing 0.68% of potassium dihydrogen phosphate R and 0.1% of sodium octanesulfonate R, adjusted to pH 3.3 with phosphoric acid R. Test solution: Dissolve 0.100 g of the substance to be examined in the mobile phase and dilute to 100.0 ml with the mobile phase. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (2): Dissolve 5 mg of sulpiride RS and 5 mg of sulpiride impurity B RS in the mobile phase and dilute to 50.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase end-capped octylsilyl silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 240 nm. Flow rate: 1.5 ml/min. Volume of injection: 10 µl. Procedure: The run time is twice the retention time of sulpiride. Identification of impurities: Use the chromatogram obtained with reference solution (2) to identify the peak due to impurity B. Relative retention with reference to sulpiride (retention time = about 15 min): Impurity B = about 0.7. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurity B and sulpiride is at least 2.5. Limits: In the chromatogram obtained with the test solution: Any unknown impurity: For each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%). The sum of the peak areas of all impurities is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.3%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A: [(2RS)-1-ethylpyrrolidin-2-yl]methanamine. Impurity B: Methyl 2-methoxy-5-sulfamoylbenzoate. Impurity C: Ethyl 2-methoxy-5-sulfamoylbenzoate. Impurity D: 2-methoxy-5-sulfamoylbenzoic acid. Impurity E: 2-methoxy-5-sulfamoylbenzamide. Impurity F: 1-ethyl-2-[[(2-methoxy-5-sulfamoylbenzoyl)amino] methyl]pyrrolidine 1-oxide. Impurity G: (RS)-N-[(1-ethylpyrrolidin-2-yl)methyl]-2-hydroxy5-sulfamoylbenzamide.

Chlorides Not more than 0.01% (Appendix 9.4.5). Shake 1.0 g of the substance to be examined with 20 ml of water. Filter through a No 4 sintered-glass filter. To 10 ml of the filtrate add 5 ml of water. Iron Not more than 10 ppm (Appendix 9.4.13).

SULPIRIDE CAPSULES

VP V

Ignite 1.0 g in a silica crucible. To the residue add 1 ml of 1 M hydrochloric acid R, 3 ml of water and 0.1 ml of nitric acid R. Heat on a water-bath for a few minutes. Place the solution in a test-tube. Rinse the crucible with 4 ml of water. Collect the rinsings in the test-tube and dilute to 10 ml with water.

Heavy metals Not more than 10 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 1 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.250 g of the substance to be examined in 80 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 34.14 mg of C15H23N3O4S. Storage Store in an airtight container. Action and use Antipsycholic. Preparation Tablets. SULPIRIDE CAPSULES Capsulae Sulpiridi Sulpiride capsules contain sulpiride. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of sulpiride, C15H23N3O4S, 95.0% to 105.0% of the stated amount. Identification Shake a quantity of the contents of the capsules containing 0.2 g of sulpiride with 20 ml of methanol R for 5 minutes, filter and evaporate the filtrate to dryness. The infrared absorption spectrum of the residue (Appendix 4.2), is concordant with the reference spectrum of sulpiride RS.

Apparatus: Paddle. Medium: 900 ml of 0.1 M hydrochloric acid R. Rotation speed: 50 rpm. Time: 30 min. Procedure: Test solution: After the specified time, withdraw a sample of the medium and filter, discarding the first portion of the filtrate. Reference solution: Weigh accurately about 55.0 mg of sulpiride RS in a 100 ml volumetric flask, add 70 ml of 0.1 M hydrochloric acid R, shake to dissolve and add 0.1 M hydrochloric acid R to volume, mix. Dilute 5.0 ml of the resulting solution to 50.0 ml with 0.1 M hydrochloric acid R. Measure the absorbances (Appendix 4.1) of the test solution and the reference solution at (291 ± 1) nm using 0.1 M hydrochloric acid R in the reference cell. Calculate the total content of sulpiride, C15H23N3O4S, in the medium using the absorbances measured and the declared contents of C15H23N3O4S in sulpiride RS. Tolerance: Not less than 75% (Q) of the stated amount of sulpiride, C15H23N3O4S, is dissolved in 30 minutes.

Assay Test solution: Weigh 20 capsules, calculate the average weight of the capsule contents and finely powder. Dissolve a quantity of the powder, accurately weighed, containing 0.1 g of sulpiride with about 50 ml of 0.1 M sodium hydroxide R for 5 minutes with the aid of ultrasound, add sufficient 0.1 M sodium hydroxide R to produce 100.0 ml and filter, discarding the first 10 ml of the filtrate. Dilute 5.0 ml of the filtrate to 100.0 ml with 0.1 M sodium hydroxide R. Reference solution: Dissolve 0.1 g of sulpiride RS, accurately weighed, in 50 ml of 0.1 M sodium hydroxide R with the aid of ultrasound for 5 minutes. Add sufficient 0.1 M sodium hydroxide R to produce 100.0 ml. Dilute 5.0 ml of the resulting solution to 100.0 ml with 0.1 M sodium hydroxide R. Measure the absorbances of the solutions at the maximum at 291 nm (Appendix 4.1), using 0.1 M sodium hydroxide R in the reference cell. Calculate the total content of sulpiride, C15H23N3O4S, in the capsules using the absorbances measured and the declared contents of C15H23N5O4S in sulpiride RS. Storage Store in a well-closed container, on a cool and dry place, protected from light. Action and use Antipsychotic. Usual strength 50 mg.

Dissolution (Appendix 11.4) 929

VP V

SULTAMICILLIN

SULTAMICILLIN Sultamicillinum

C25H30N4O9S2 

M. 594.7

Sultamicillin is methylene (2S,5R,6R)-6-[[(2R)-aminophenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1azabicyclo[3.2.0]heptane-2-carboxylate (2S,5R)-3,3dimethyl-4,4,7-trioxo-4λ6-thia-1-azabicyclo[3.2.0] heptane-2-carboxylate. It contains not less than 96.0% and not more than 102.0% of C25H30N4O9S2, calculated with reference to the anhydrous substance. Semi-synthetic product derived from a fermentation product.

Characters White or almost white, slightly hygroscopic, crystalline powder. Practically insoluble in water and in ethanol (96%), very slightly soluble in methanol. Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of sultamicillin RS. Specific optical rotation +190° to +210°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.500 g of the substance to be examined in  dimethylformamide R and dilute to 50.0 ml with the same solvent. Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use or keep at 2 °C to 8 °C for not more than 6 h. Mobile phase A: A 4.68 g/L solution of sodium dihydrogen phosphate R adjusted to pH 3.0 with phosphoric acid R. Mobile phase B: Acetonitrile R1. Solution A: Methanol R1 - acetonitrile R1 (20 : 80). Solution B: Dissolve 1.56 g of sodium dihydrogen phosphate R in 900 ml of water. Add 7.0 ml of phosphoric acid R and dilute to 1000 ml with water. Blank solution: Solution B - solution A (30 : 70). Test solution: Dissolve 50.0 mg of the substance to be examined in 35 ml of solution A and sonicate for about 1 min. Add 13 ml of solution B, mix and sonicate for about 1 min. Dilute to 50.0 ml with solution B and mix. Reference solution (1): Dissolve 70.0 mg of sultamicillin tosilate RS in 35 ml of solution A and sonicate for about 1 min. Add 13 ml of solution B, mix and sonicate for about 1 min. Dilute to 50.0 ml with solution B and mix. 930

Reference solution (2): Suspend 15 mg of sultamicillin tosilate RS in 20 ml of a 0.04% solution of  sodium hydroxide R and sonicate in an ultrasonic bath for about 5 min. Add 20 ml of a 0.36 g/L solution of hydrochloric acid R and dilute to 100 ml with water. Reference solution (3): Dilute 1.0 ml of reference solution (1) to 100.0 ml with the blank solution. Reference solution (4): Dissolve 17.3 mg of ampicillin trihydrate RS (impurity C) and 15.0 mg of sulbactam RS (impurity A) in water and dilute to 50 ml with the same solvent. Dilute 1.0 ml of this solution to 100.0 ml with water. Reference solution (5): Dissolve 5 mg of sultamicillin for peak identification RS (containing impurity G) in 7.0 ml of solution A and sonicate for about 1 min. Dilute to 10.0 ml with solution B, mix and sonicate for about 1 min. Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase C (3.5 µm). Column temperature: 25 °C. Detector: A spectrophotometer set at 215 nm. Flow rate: 1.0 ml/min. Volume of injection: 5 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

1 - 15

95 → 30

5 → 70

15 - 16

30

70

16 - 16.5

30 → 95

70 → 5

16.5 - 20

95

5

Inject the blank, the test solution and reference solutions (2), (3), (4) and (5). Identification of impurities: Use the chromatogram supplied with sultamicillin for peak identification RS and the chromatogram obtained with reference solution (5) to identify the peak due to impurity G. Relative retention with reference to sultamicillin (retention time = about 9.3 min): Impurity A = about 0.41; ampicillin penicilloic acid = about 0.47; impurity B = about 0.50; impurity C = about 0.55; impurity D = about 0.94; impurity E = about 1.09; impurity F = about 1.26; impurity G = about 1.42. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to ampicillin penicilloic acid and impurity B is at least 2.5 and the resolution between the peaks due to impurities B and C is at least 2.5. Limits: In the chromatogram obtained with the test solution: Impurity G: The area of the peak due to impurity G is not more than the area of the peak due to sultamicillin in the chromatogram obtained with reference solution (3) (1.0%). Impurity A: The area of the peak due to impurity A is

SULTAMICILLIN

VP V

not more than the area of the corresponding peak in the chromatogram obtained with reference solution (4) (0.3%). Impurity B: The area of the peak due to impurity B is not more than the area of the corresponding peak in the chromatogram obtained with reference solution (3) (0.3%). Impurity C: The area of the peak due to impurity C is not more than the area of the corresponding peak in the chromatogram obtained with reference solution (4) (0.3%). Impurities D, E, F: For each impurity, not more than 0.3 times the area of the peak due to sultamicillin in the chromatogram obtained with reference solution (3) (0.3%). Any other impurity: For each impurity, not more than 0.3 times the area of the peak due to sultamicillin in the chromatogram obtained with reference solution (3) (0.3%). Total peak area of all impurities is not more than 3 times the area of the peak due to sultamicillin in the chromatogram obtained with reference solution (3) (3.0%). Disregard any peak with an area less than 0.1 times the area of the peak due to sultamicillin in the chromatogram obtained with reference solution (3) (0.1%). Note: Impurity A: (2S,5R)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0] heptane-2-carboxylic acid 4,4-dioxide (sulbactam). Impurity B: 4-methylbenzenesulfonic acid (p-toluenesulfonic acid). Impurity C: (2S,5R,6R)-6-[[(2R)-2-amino-2-phenylacetyl]amino]3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2carboxylic acid (ampicillin). Impurity D: [[(2R)-aminophenylacetyl]amino][(4S)-4-[[[[[(2S,5R)3,3-dimethyl-4,4,7-trioxo-4λ6-thia-1-azabicyclo[3.2.0]hept-2-yl] carbonyl]oxy]methoxy]carbonyl]-5,5-dimethylthiazolidin-2-yl] acetic acid (penicilloic acids of sultamicillin). Impurity E: Methylene (2S,5R,6R)-3,3-dimethyl-6-[[(2R)[(1-methyl-4-oxopentylidene)amino]phenylacetyl]amino]-7oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate (2S,5R)3,3-dimethyl-7-oxo-4-oxa-1-azabicyclo[3.2.0]heptane-2carboxylate. Impurity F: Methylene (2S,5R,6R)-6-[[(2R)-[[[(2S,5R,6R)-6[[(2R)-aminophenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia1-azabicyclo[3.2.0]hept-2-yl]carbonyl]amino]phenylacetyl] amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane2-carboxylate(2S,5R)-3,3-dimethyl-4,4,7-trioxo-4λ 6-thia-1azabicyclo[3.2.0]heptane-2-carboxylate (ampicillin sultamicillin amide). Impurity G: Methylene (2S,5R,6R)-6-[[(2R)-[[[[(2R)-amino phenylacetyl]amino][(4S)-4-[[[[[(2S,5R)-3,3-dimethyl-4,4,7trioxo-4λ 6-thia-1-azabicyclo[3.2.0]hept-2-yl]carbonyl]oxy] methoxy]carbonyl]-5,5-dimethylthiazolidin-2-yl]acetyl] amino]phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1azabicyclo[3.2.0]heptane-2-carboxylate (2S,5R)-3,3-dimethyl4,4,7-trioxo-4λ6-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate (sultamicillin dimer).

Ethyl acetate Not more than 2.5%. Examine by head-space gas chromatography (Appendix 5.2).

Test solution: Dissolve 0.200 g of the substance to be examined in 7.0 ml of a mixture of water - dimethylformamide (1 : 99). Reference solution: Dissolve 0.200 g of ethyl acetate R in 240 ml of a mixture of water - dimethylformamide (1 : 99) and dilute to 250.0 ml with the same mixture of solvents. Dilute 5.0 ml of this solution to 7.0 ml with a mixture of water - dimethylformamide (1 : 99). Close the vials immediately with a tight rubber membrane stopper coated with polytetrafluoroethylene and secure with an aluminium crimped cap. Shake to obtain a homogeneous solution. Chromatographic system: A fused-silica capillary column (50 m long and 0.32 mm in internal diameter) coated with poly(dimethyl)siloxane R (film thickness: 1.8 µm or 3 µm). Carrier gas: Helium for chromatography. Linear velocity: 35 cm/s. Split ratio: 1 : 5. Static head-space conditions: − Equilibration temperature: 105 °C. − Equilibration time: 45 min. − Transfer-line temperature: 110 °C. − Pressurisation time: 30 s. Temperature programme: Time (min) Column

Temperature (°C)

0-6

70

6 - 16

70 → 220

16 - 18

220

Injection port

140

Detector

250

Detector: A flame-ionisation detector. Volume of injection: 1 ml. Relative retention with reference to dimethylformamide (retention time = about 14 min): Ethyl acetate = about 0.7.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). Test solution: Dissolve 2.0 g in a mixture of methanol  -  acetonitrile  (40 : 60) and dilute to 20.0 ml with the same mixture of solvents. 12 ml of the solution complies with limit test for heavy metals, method 2. Prepare the standard using lead standard solution (2 ppm Pb) obtained by diluting lead standard solution (100 ppm Pb) R with a mixture methanol - acetonitrile (40 : 60). Water Not more than 1.0% (Appendix 10.3). Determined on 0.50 g. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. 931

VP V

SULTAMICILLIN TOSILATE DIHYDRATE

Assay Examine by liquid chromatography (Appendix 5.3) as described in the test for Related substances. Inject the test solution and reference solution (1). Calculate the content of sultamicillin, C25H30N4O9S2, using the peak areas due to sultamicillin in the chromatograms obtained with the test solution, reference solution (1) and the declared content of C25H30N4O9S2 in sultamicillin tosilate RS and by multiplying the sultamicillin tosilate content by 0.7752. Storage In an airtight container. Action and use Beta-lactamase inhibitor. Preparation Tablets, capsules, powder for suspension. SULTAMICILLIN TOSILATE DIHYDRATE Sultamicillini tosilas dihydricus

C25H30N4O9S2,C7H8O3S,2H2O 



M. 803

Sultamicillin tosilate dihydrate is 4-methylbenzenesulfonate of methylene (2S,5R,6R)-6-[[(2R)-aminophenylacetyl] amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0] heptane-2-carboxylate (2S,5R)-3,3-dimethyl-4,4,7-trioxo -4λ 6 -thia-1-azabicyclo[3.2.0]heptane-2-carboxylate dihydrate. It contains not less than 95.0% and not more than 102.0% of C32H38N4O12S3, calculated with reference to the anhydrous substance. Semi-synthetic product derived from a fermentation product.

Characters White or almost white, crystalline powder. Practically insoluble in water, sparingly soluble in ethanol (96%). Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of sultamicillin tosilate RS. Specific optical rotation +178° to +195°, calculated with reference to the anhydrous substance (Appendix 6.4). 932

Dissolve 1.000 g of the substance to be examined in  dimethylformamide R and dilute to 50.0 ml with the same solvent.

Related substances Examine by liquid chromatography (Appendix 5.3).  Prepare the solutions immediately before use or keep at 2 °C to 8 °C for not more than 6 h. Mobile phase A: A 4.68 g/L solution of sodium dihydrogen phosphate R adjusted to pH 3.0 with phosphoric acid R. Mobile phase B: Acetonitrile R1. Solution A: Methanol R1 - acetonitrile R1 (20 : 80). Solution B: Dissolve 1.56 g of sodium dihydrogen phosphate R in 900 ml of water. Add 7.0 ml of phosphoric acid R and dilute to 1000 ml with water. Blank solution: Solution B - solution A (30 : 70). Test solution: Dissolve 70.0 mg of the substance to be examined in 35 ml of solution A and sonicate for about 1 min. Add 13 ml of solution B, mix and sonicate for about 1 min. Dilute to 50.0 ml with solution B and mix. Reference solution (1): Dissolve 70.0 mg of sultamicillin tosilate RS in 35 ml of solution A and sonicate for about 1 min. Add 13 ml of solution B, mix and sonicate for about 1 min. Dilute to 50.0 ml with solution B and mix. Reference solution (2): Suspend 15 mg of sultamicillin tosilate RS in 20 ml of a 0.04% solution of  sodium hydroxide R and sonicate in an ultrasonic bath for about 5 min. Add 20 ml of a 0.36 g/L solution of hydrochloric acid R and dilute to 100.0 ml with water. Reference solution (3): Dissolve 0.200 g of the substance to be examined in 70.0 ml of solution A and sonicate for about 1 min. Add 25 ml of solution B, mix and sonicate for about 1 min. Dilute to 100.0 ml with solution B and mix. Dilute 1.0 ml of this solution to 100.0 ml with the blank solution. Reference solution (4): Dissolve 32.3 mg of  ampicillin trihydrate RS  (impurity B) and 7.0 mg of  sulbactam RS (impurity A) in water and dilute to 1000 ml with the same solvent. Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase C (3.5 µm). Column temperature: 25 °C. Detector: A spectrophotometer set at 215 nm. Flow rate: 1.0 ml/min. Volume of injection: 5 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 15

95 → 30

5 → 70

15 - 16

30

70

16 - 16.5

30 → 95

70 → 5

16.5 - 20

95

5

SULTAMICILLIN TOSILATE DIHYDRATE

VP V

Inject the blank, the test solution and the reference solutions (2), (3) and (4). Relative retention time with reference to sultamicillin (retention time = about 9.3 min): Impurity A = about 0.41; ampicillin penicilloic acid = about 0.47; tosilate = about 0.50; impurity B = about 0.55; impurity C = about 0.94; impurity D = about 1.09; impurity F = about 1.23; impurity E = about 1.26; impurity G = about 1.42. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to ampicillin penicilloic acid and tosilate is at least 2.5 and the resolution between the peaks due to tosilate and impurity B is at least 2.5. Limits: In the chromatogram obtained with the test solution: Impurity B: The area of the peak due to impurity B is not more than the area of the corresponding peak in the chromatogram obtained with reference solution (4) (2.0%). Impurity A: The area of the peak due to impurity A is not more than the area of the corresponding peak in the chromatogram obtained with reference solution (4) (0.5%). Impurities C, D, E, F, G: For each impurity, not more than 0.5 times the area of the peak due to sultamicillin in the chromatogram obtained with reference solution (3) (0.5%). Any other impurity: For each impurity, not more than 0.5 times the area of the peak due to sultamicillin in the chromatogram obtained with reference solution (3) (0.5%). The sum of the peak areas of all impurities is not more than 4 times the area of the peak due to sultamicillin in the chromatogram obtained with reference solution (3) (4.0%). Disregard any peak with an area less than 0.1 times the area the peak due to sultamicillin in the chromatogram obtained with reference solution (3) (0.1%). Note: Impurity A: (2S,5R)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0] heptane-2-carboxylic acid 4,4-dioxide(sulbactam). Impurity B: (2S,5R,6R)-6-[[(2R)-2-amino-2-phenylacetyl]amino]3,3-dimethyl-7-oxo-4-thia-1-azabicyclo [3.2.0]heptane-2-carboxylic acid (ampicillin). Impurity C: [[(2R)-aminophenylacetyl]amino][(4S)-4-[[[[[(2S,5R)3,3-dimethyl-4,4,7-trioxo-4λ6-thia-1-azabicyclo [3.2.0]hept-2-yl]carbonyl]oxy]methoxy]carbonyl]-5,5dimethylthiazolidin-2-yl]acetic acid (penicilloic acids of sultamicillin). Impurity D: Methylene (2S,5R,6R)-3,3-dimethyl-6-[[(2R)[(1-methyl-4-oxopentylidene)amino]phenylacetyl]amino]-7oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate (2S,5R)3,3-dimethyl-7-oxo-4-oxa-1-azabicyclo[3.2.0]heptane-2carboxylate. Impurity E: Methylene bis[(2S,5R)-3,3-dimethyl-4,4,7-trioxo4λ6-thia-1-azabicyclo [3.2.0]heptane-2-carboxylate] (sulbactam methylene ester). Impurity F: Methylene (2S,5R,6R)-6-[[(2R)-[[[(2S,5R,6R)-6[[(2R)-aminophenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia1-azabicyclo[3.2.0]hept-2-yl]carbonyl]amino]-phenylacetyl] amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0] heptane-2-carboxylate(2S,5R)-3,3-dimethyl-4,4,7-trioxo-4

λ6-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate (ampicillin sultamicillin amide). Impurity G: Methylene (2S,5R,6R)-6-[[(2R)-[[[[(2R)-aminophenylacetyl]amino][(4S)-4-[[[[[(2S,5R)-3,3-dimethyl-4,4,7trioxo-4l 6-thia-1-azabicyclo[3.2.0]hept-2-yl]-carbonyl]oxy] methoxy]carbonyl]-5,5-dimethylthiazolidin-2-yl]acetyl]amino] phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo [3.2.0]heptane-2-carboxylate(2S,5R)-3,3-dimethyl-4,4,7-trioxo4l6-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate (sultamicillin dimer).

Ethyl acetate Not more than 2.0%. Examine by gas chromatography (Appendix 5.2). Test solution: Dissolve 0.200 g of the substance to be examined in 7.0 ml of a mixture of water - dimethylformamide (1 : 99). Reference solution: Dissolve 0.200 g of ethyl acetate R in 240 ml of a mixture of water - dimethylformamide (1 : 99) and dilute to 250.0 ml with the same mixture of solvents. Dilute 5.0 ml of this solution to 7.0 ml with a mixture of water - dimethylformamide (1 : 99). Close the vials immediately with a tight rubber membrane stopper coated with polytetrafluoroethylene and secure with an aluminium crimped cap. Shake to obtain a homogeneous solution. Chromatographic system: A fused-silica capillary column (50 m long and 0.32 mm in internal diameter) coated with poly(dimethyl) siloxane R (film thickness: 1.8 µm or 3 µm). Carrier gas: Helium for chromatography. Linear velocity: 35 cm/s. Flow rate: 1.2 ml/min. Split ratio: 1 : 5. Static head-space conditions: − Equilibration temperature: 105 °C. − Equilibration time: 45 min. − Transfer-line temperature: 110 °C. − Pressurisation time: 30 s. Temperature programme:

Column

Time (min)

Temperature (°C)

0-6

70

6 - 16

70 → 220

16 - 18

220

Injection port

140

Detector

250

Detector: A flame-ionisation detector. Volume of injection: 1 ml. Relative retention with reference to dimethylformamide (retention time = about 14 min): Ethyl acetate = about 0.7.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). Test solution: Dissolve 2.0 g of the substance to be examined in a mixture of methanol - acetonitrile (40 : 60) 933

TALC

and dilute to 20.0 ml with the same mixture of solvents. 12 ml of the solution complies with limit test for heavy metals, method 2. Prepare the standard using lead standard solution (2 ppm Pb) obtained by diluting  lead standard solution (100 ppm Pb) R with a mixture methanol - acetonitrile (40 : 60).

Water 4.0% to 6.0% (Appendix 10.3). Determined on 0.200 g. Sulfated ash Not more than 0.2% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Liquid chromatography (Appendix 5.3) as described in the test for Related substances with the following modifications: Inject the test solution and reference solution (1). Calculate the content of sultamicillin tosilate, using the peak areas due to sultamicillin in the chromatograms obtained with the test solution, the reference solution (1) and the declared content of C32H38N4O12S3 in sultamicillin tosilate RS. Storage In an airtight container. Preparations Tablets, capsules, powder for suspension. TALC Talcum Talc is a powdered, selected, natural, hydrated magnesium silicate. Pure talc has the formula [Mg3Si4O10(OH)2; M. 379.3]. It may contain variable amounts of associated minerals among which chlorites (hydrated aluminium and magnesium silicates), magnesite (magnesium carbonate), calcite (calcium carbonate) and dolomite (calcium and magnesium carbonate) are predominant.

Production Talc derived from deposits that are known to contain associated asbestos is not suitable for pharmaceutical use. The manufacturer is responsible for demonstrating by the test for amphiboles and serpentines that the product is free from asbestos. The presence of amphiboles and of serpentines is revealed by X-ray diffraction or by infrared spectrophotometry (see A and B). If detected, the specific morphological criteria of asbestos are investigated by a suitable method of optical microscopy to determine whether tremolite asbestos or chrysotile is present, as described below. A. Examine by infrared spectrophotometry (Appendix 4.2). In the range 740 cm-1 to 760 cm-1 using scale expansion, any absorption band at 758 ± 1 cm-1 may indicate the 934

VP V

presence of tremolite or of chlorite. If the absorption band remains after ignition of the substance at 850 °C for at least 30 min, it indicates the presence of the tremolite. In the range 600 cm-1 to 650 cm-1 using scale expansion, any absorption band or shoulder may indicate the presence of serpentines. B. Examine by X-ray diffraction employing the following conditions: − Cu Kα monochromatic 40 kV radiation, 24 mA to 30 mA. − Incident slit: 1° − Detection slit: 0.2° − Goniometer speed: 1/10° 2θ/min − Scanning range: 10° to 13° 2θ and 24° to 26° 2θ. − The sample is not oriented. Place the sample on the sample holder; pack and smooth its surface with a polished glass microscope slide. Record the diffractograms. The presence of amphiboles is detected by a diffraction peak at 10.5 ± 0.1° 2θ, the presence of serpentines is detected by diffraction peaks at 24.3 ± 0.1° 2θ and at 12.1 ± 0.1° 2θ. If, by one of the 2 methods, amphiboles and/or serpentine are detected, examine by a suitable method of optical microscopy to determine the asbestos character. Examined by optical microscopy, the presence of asbestos is shown if the following criteria are met: A range of length to width ratios of 20:1 to 100:1, or higher for fibres longer than 5 µm; Capability of splitting into very thin fibrils; and if 2 or more of the following 4 criteria are met: Parallel fibres occurring in bundles, Fibre bundles displaying frayed ends, Fibres in the form of thin needles, Matted masses of individual fibres and/or fibres showing curvature.

Characters A light, homogeneous, white or almost white powder, greasy to the touch (non abrasive), practically insoluble in water, in ethanol (96%) and in dilute solutions of acids and alkali hydroxides. Identification Apply one of the two following identifications: First identification: A. Second identification: B, C. A. Examine by infrared absorption spectrophotometry (Appendix 4.2). The spectrum shows absorption bands at 3677 ± 2 cm-1, at 1018 ± 2 cm-1 and at 669 ± 2 cm-1. B. In a platinum crucible, melt a mixture of 0.2 g of anhydrous sodium carbonate R and 2.0 g of potassium carbonate R. To the melted mass add 0.1 g of the substance to be examined and heat until the mixture is completely melted. Allow to cool and transfer the melted mass into an evaporating dish with 50 ml of hot water. Add hydrochloric acid R until effervescence ceases. Add

VP V

10 ml of hydrochloric acid R and evaporate to dryness on a water-bath. Allow to cool. Add 20 ml of water, heat to boiling and filter (the residue is used for identification test C). To 5 ml of the filtrate add 1 ml of ammonia R and 1 ml of 10.7% solution of ammonium chloride and filter. To the filtrate add 1 ml of 9% solution of disodium hydrogen phosphate. A white, crystalline precipitate is formed. C. The residue obtained in identification test B gives the reaction of silicates (Appendix 8.1).

Acidity or alkalinity Boil 2.5 g with 50 ml of carbon dioxide-free water R under reflux. Filter under vaccum. To 10 ml of the filtrate add 0.1 ml of bromothymol blue solution R1; not more than 0.4 ml of 0.01 N hydrochloric acid VS is required to change the colour of the indicator to green. To 10 ml of the filtrate add 0.1 ml of phenolphthalein solution R1; not more than 0.3 ml of 0.01 N sodium hydroxide VS is required to change the colour of the indicator to pink. Water-soluble substances Not more than 0.2%. To 10.0 g add 50 ml of carbon dioxide-free water R, heat to boiling and maintain boiling under a reflux condenser for 30 min. Allow to cool, filter through a medium-speed filter paper and dilute to 50.0 ml with carbon dioxide-free water R. Take 25.0 ml of the filtrate, evaporate to dryness and heat at 105 °C for 1 h. The residue weighs not more than 10 mg. Aluminium Not more than 2.0 %. Determined by atomic absorption spectrometry (Appendix 4.4, method 1). Solution S2: Perchlorates mixed with heavy metals are known to be explosive. Take proper precautions while performing this procedure. Weigh 0.5 g of the substance to be examined in a 100 ml polytetrafluoroethylene dish, add 5 ml of hydrochloric acid R, 5 ml of lead-free nitric acid R and 5 ml of perchloric acid R. Stir gently then add 35 ml of hydrofluoric acid R and evaporate slowly to dryness on a hot plate. To the residue, add 5 ml of hydrochloric acid R, cover with a watch-glass, heat to boiling and allow to cool. Rinse the watch-glass and the dish with water. Transfer into a volumetric flask, rinse the dish with water and dilute to 50.0 ml with the same solvent. Test solution: To 5.0 ml of solution S2 add 10 ml of a 2.534% solution of caesium chloride, 10.0 ml of hydrochloric acid R and dilute to 100.0 ml with water. Reference solutions: Into 4 identical 100 ml volumetric flasks, each containing 10.0 ml of hydrochloric acid R and 10 ml of a 2.534% solution of caesium chloride, introduce respectively 5.0 ml, 10.0 ml, 15.0 ml and 20.0 ml of aluminium standard solution (100 ppm Al) R and dilute to 100.0 ml with water.

TALC

Measure the absorbance at 309.3 nm, using an aluminium hollow-cathode lamp as the radiation source and a nitrous oxide-acetylene flame.

Calcium Not more than 0.9%. Determined by atomic absorption spectrometry (Appendix 4.4, method 1). Test solution: To 5.0 ml of solution S2 add 10.0 ml of hydrochloric acid R, 10 ml of lanthanum chloride solution R and dilute to 100.0 ml with water. Reference solutions: Into 4 identical 100ml volumetric flasks, each containing 10.0 ml of hydrochloric acid R and 10 ml of lanthanum chloride solution R, introduce respectively 1.0 ml, 2.0 ml, 3.0 ml and 4.0 ml of calcium standard solution (100 ppm Ca) R and dilute to 100.0 ml with water. Measure the absorbance at 422.7 nm using a calcium hollow-cathode lamp as the radiation source and a nitrous oxide-acetylene flame. Iron Not more than 0.25 %. Determined by atomic absorption spectrometry (Appendix 4.4, method 1). Solution S1: Weigh 10.0 g of the substance to be examined into a conical flask fitted with a reflux condenser, add 50 ml of 0.5 M hydrochloric acid gradually while stirring and heat on a water-bath for 30 min. Allow to cool. Transfer the mixture to a beaker and allow the undissolved material to settle. Filter the supernatant through mediumspeed filter paper into a 100 ml volumetric flask, retaining as much as possible of the insoluble material in the beaker. Wash the residue and the beaker with 3 quantities, each of 10 ml, of hot water. Wash the filter with 15 ml of hot water, allow the filtrate to cool and dilute to 100.0 ml with the same solvent. Test solution: To 2.5 ml of solution S1, add 50.0 ml of 0.5 M hydrochloric acid R and dilute to 100.0 ml with water. Reference solutions: Into 4 identical 100 ml volumetric flasks, each containing 50.0 ml of 0.5 M hydrochloric acid, introduce respectively 2.0 ml, 2.5 ml, 3.0 ml and 4.0 ml of iron standard solution (250 ppm Fe) R and dilute to 100.0 ml with water. Measure the absorbance at 248.3 nm using an iron hollowcathode lamp as the radiation source and an air-acetylene flame. Make a correction using a deuterium lamp. Lead Not more than 10 ppm. Determine by atomic absorption spectrometry (Appendix 4.4, method 1). Test solution: Use solution S1. Reference solutions: Into 4 identical 100 ml volumetric flasks, each containing 50.0 ml of 0.5 M hydrochloric acid R, introduce respectively 5.0 ml, 7.5 ml, 10.0 ml and 12.5 ml of lead standard solution (10 ppm Pb) R1 and dilute to 100.0 ml with water. 935

VP V

TAMOXIFEN CITRATE

Measure the absorbance at 217.0 nm using a lead hollowcathode lamp as the radiation source and an air-acetylene flame.

Magnesium 17.0 % to 19.5 %. Determine by atomic absorption spectrometry (Appendix 4.4, method 1). Test solution: Dilute 0.5 ml of solution S2 to 100.0 ml with water. To 4.0 ml of the solution, add 10.0 ml of hydrochloric acid R, 10 ml of lanthanum chloride solution R and dilute to 100.0 ml with water. Reference solutions: Into 4 identical 100 ml volumetric flasks, each containing 10.0 ml of hydrochloric acid R and 10 ml of lanthanum chloride solution R, introduce respectively 2.5 ml, 3.0 ml, 4.0 ml and 5.0 ml of magnesium standard solution (10 ppm Mg) R1 and dilute to 100.0 ml with water. Measure the absorbance at 285.2 nm using a magnesium hollow-cathode lamp as the radiation source and an airacetylene flame. Loss on ignition Not more than 7.0 %. Determine on 1.00 g by ignition to constant weight at 1050 °C - 1100 °C. Microbial contamination If intended for topical administration, total aerobic microbial count (Appendix 13.6) is not more than 102 CFU/g. If intended for oral administration, the total aerobic microbial count (Appendix 13.6) is not more than 103 CFU/g and total combined yeasts/moulds count is not more than 102 CFU/g. Storage Store in a well-closed container. Action and use Excipients. TAMOXIFEN CITRATE Tamoxifeni Citras

C26H29NO,C6H8O7  

936

M. 563.6

Tamoxifen citrate is 2-[4-[(Z)-1,2-diphenylbut-1-enyl] phenoxy]-N,N-dimethylethanamine dihydrogen 2- hydroxypropane-1,2,3-tricarboxylate. It contains not less than 99.0% and not more than 101.0% of C26H29NO,C6H8O7, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder, it shows polymorphism. Soluble in methanol, slightly soluble in water and acetone. Identification Apply one of the two following identifications: First identification: A. Second identification: B, C. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of tamoxifen citrate RS. If the spectra obtained show differences, dissolve separately the substance to be examined and the reference substance in acetone R, evaporate to dryness and record new spectra using the residues. B. Ultraviolet absorptiom spectrum (Appendix 4.1). Dissolve 20 mg of the substance to be examined in methanol R and dilute to 50.0 ml with the same solvent. Dilute 5.0 ml of the solution to 100.0 ml with methanol R. The ultraviolet absorptiom spectrum of the solution, in the range between 220 nm and 350 nm, exhibits two absorption maxima at 237 nm and at 275 nm. The ratio of the absorbance measured at 237 nm to at 275 nm is 1.45 to 1.65. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel F254. Mobile phase: Triethylamine - toluene (10 : 90). Test solution: Dissolve 10 mg of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Reference solution (1): Dissolve 10 mg of tamoxifen citrate RS in methanol R and dilute to 10 ml with the same solvent. Reference solution (2): Dissolve 10 mg of tamoxifen citrate RS and 10 mg of clomifene citrate RS in methanol R and dilute to 10 ml with the same solvent. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 3/4 of the plate. Allow the plate to dry in air and examine in ultravioletlight at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) shows two clearly separated spots. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Mix 40 volumes of acetonitrile R and 60 volumes of water containing 0.09% of sodium dihydrogen phosphate R and 0.48% of N,N-dimethyloctylamine R; adjust to pH 3.0 with phosphoric acid R.

TARTRAZINE

VP V

Prepare the following solutions immediately before use and protect from light. Test solution: Dissolve 15.0 mg of the substance to be examined in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (1): Dissolve 15 mg of tamoxifen citrate for performance test RS (containing impurities A and F) in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 240 nm. Flow rate: 1.2 ml/min. Volume of injection: 10 µl. Procedure: Equilibrate the column with the mobile phase for 30 min. The run time of the test solution is twice the retention time of tamoxifen. System suitability: Inject reference solution (1), the obtained chromatogram is similar to the chromatogram supplied with tamoxifen citrate for performance test RS and baseline separation between the peaks due to impurity F and tamoxifen. The resolution between the peaks due to impurities A and F is at least 3. Limits: In the chromatogram obtained with the test solution: Impurities A, B, C, D, E, F, G, H: For each impurity, the area is not more than 0.3 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.3%). Total peak area of all impurities, other than impurity A, is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.5%). Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%) and any peak due to the citrate (retention time = about 2.5 min). Note: Impurity A: 2-[4-[(E)-1,2-diphenylbut-1-enyl]phenoxy]-N,Ndimethylethanamine ((E)- isomer). Impurity B: 1-[4-[2-(dimethylamino)ethoxy]phenyl]-1,2diphenylbutan-1-ol. Impurity C: 2-[4-[(EZ)-1,2-diphenylethenyl]phenoxy]-N,Ndimethylethanamine. Impurity D: 2-[4-[(EZ)-1,2-diphenylprop-1-enyl]phenoxy]N,N-dimethylethanamine. Impurity E: 2-[2-[(EZ)-1,2-diphenylbut-1-enyl]phenoxy]-N,Ndimethylethanamine. Impurity F: 2-[4-[(Z)-1,2-diphenylbut-1-enyl]phenoxy]-Nmethylethanamine. Impurity G: (2RS)-1-[4-[2-(dimethylamino)ethoxy]phenyl]-2phenylbutan-1-one.

Impurity H: 2-[4-[(RS)-[4-[(Z)-1-[4-[2-(dimethylamino)ethoxy] phenyl]-2-phenylbut-1- enyl]phenyl](phenyl)methyl]phenoxy]N,N-dimethylethanamine.

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 65 °C; 4 h; in vacuo). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.400 g of the substance to be examined in 75 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS using 0.1 ml of naphtholbenzein solution R as indicator. 1 ml of 0.1 N perchloric acid VS is equivalent to 56.36 mg of C32H37NO8. Storage Store in an airtight container, protected from light. Action and use Selective estrogen receptor modulator. Preparation Tablets. TARTRAZINE Tartrazinum

C16H9N4Na 3O9S2

M. 534.4

Tartrazine is trisodium hydroxy-5-(sulfonato-4-phenyl)1-[(sulfonato-4-phenyl)azo]-4-1H-pyrazolcarboxylate-3. It contains not less than 85.0% of C16H9N4Na3O9S2, calculated with reference to the dried substance.

Characters A dark orange powder. Freely soluble in water, sparingly soluble in ethanol, practically insoluble in acetone and in methylene chloride. A 0.1% w/v solution is bright yellow. Identification A. Dilute 1 ml of solution S (see Appearance of solution) to 100 ml with 0.1 M hydrochloric acid R, and mix. The ultraviolet absorptiom spectrum (Appendix 4.1) of the 937

TARTRAZINE

solution, in the range between 230 nm and 550 nm, exhibits absorption maxima at 256 ± 5 nm and at 430 ± 3 nm. Dilute 1 ml of solution S to 100 ml with 0.1 N sodium hydroxide R. The ultraviolet absorption spectrum (Appendix 4.1) of the solution, in the range between 230 nm and 550 nm, exhibits absorption maxima at (260 ± 5) nm and at (396 ± 3) nm. B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (2) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1).

Appearance of solution Solution S: Dissolve 50 mg of the substance to be examined in water and dilute to 50 ml with the same solvent. The solution is clear (Appendix 9.2). Related substances Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel G. Mobile phase: Ammonia - water - ethanol - n-butanol (10 : 25 : 25 : 50). Dissolution solvent: A mixture of water and methanol (1 : 1). Test solution (1): Dissolve 40 mg of the substance to be examined in the dissolution solvent and dilute to 10 ml with the same solvent, and mix. Test solution (2): Dilute 2 ml of test solution (1) to 10 ml with the dissolution solvent and mix. Reference solution (1): Dissolve 40 mg of tartrazine RS in the dissolution solvent and dilute to 50 ml with the same solvent, and mix. Reference solution (2): Dilute 1.0 ml of reference solution (1) to 20 ml with the dissolution solvent and mix. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm. Remove the plate, allow it to dry in air, and examine in daylight. Any secondary spot in the chromatogram obtained with test solution (1) is not more intense than the spot in the chromatogram obtained with reference solution (2). Ether-extractable substances Not more than 0.5%. Dry the substance to be examined to constant weight in vacuo at 60 °C. Transfer 2.0 g, accurately weighed, of the dried substance to a 200 ml brown-coloured flask, add anhydrous ether R to volume, shake by mechanical means for 30 min, and filter. Evaporate 100.0 ml of the filtrate in vacuo at 20 °C. Dry the residue to constant weight in a desiccator. The weight of the dried residue is not more than 5 mg. Water-insoluble substances Not more than 0.2%. Dissolve 2.0 g of the substance to be examined in 200 ml of water by heating to about 90 °C. Allow to cool, and filter through a tared sintered-glass filter No.4. Wash the residue with water until the filtrate is colourless. Dry the 938

VP V

residue to constant weight at 100 °C to 105 °C. The weight of the dried residue is not more than 4 mg.

Primary aromatic amines Not more than 40 ppm. Dissolve the residue obtained in the test for ether-extractable substances in 10 ml of toluene R. To 2.5 ml of the solution add 6 ml of water and 4 ml of 0.1 N hydrochloric acid R. Shake vigorously, and allow the layers to separate, discarding the organic layer. To the aqueous layer add 0.4 ml of a freshly prepared 0.25% solution of sodium nitrite R, mix and allow to stand for 1 min. Add 0.8 ml of a 0.5% solution of ammonium sulfamate R, mix and allow to stand for 1 min. Add 2 ml of a 0.5% solution of N-(1-naphthyl)-ethylenediamine dihydrochloride R, mix and allow to stand for 1 hour. The solution is not more intensely coloured than a reference solution prepared at the same time in the same manner, but using a mixture of 1 ml of a 0.001% solution of naphthylamine R, 5 ml of water and 4 ml of 0.1 N hydrochloric acid R in place of the aqueous layer. Dissoluble chromium Not more than 50 ppm. Examine by atomic absorption spectrometry (Appendix 4.4, method 1). Test solution: Dissolve 0.500 g of the substance to be examined in 25 ml of water by heating to about 90 °C. Allow to cool, dilute to 25.0 ml with water and filter through a tared sintered-glass filter No.4. Reference solutions: Prepare the solutions (0.5 ppm; 1 ppm and 2 ppm Cr) using chromium standard solution (100 ppm Cr) R. Measure the absorbance at 357.9 nm using a chromium hollow-cathode lamp as source of radiation and an airacetylene flame. Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with the limit test for heavy metals, method 3. Prepare the standard using 2.0 ml of lead standard solution (10 ppm Pb) R. Assay Dry the substance to be examined to constant weight in vacuo at 60 °C. Dissolve about 0.150 g, accurately weighed, of the dried substance in freshly prepared 2 M ammonium acetate R and dilute to 100.0 ml with the same solvent, and mix. Dilute 2.0 ml of the solution to 200.0 ml with freshly prepared 2 M ammonium acetate R, and mix. Prepare reference solution in the same manner, using about 0.150 g, accurately weighed, of tartrazine RS previously dried to constant weight in vacuo at 60 °C. The ultraviolet absorption spectra (Appendix 4.1) of the test solution and the reference solution exhibit an absorption maximum at about 426 nm. The difference in the wavelength of the maxima is not more than 5 nm.

TELMISARTAN

VP V

Measure the absorbance of the solutions at the wavelength of the maximum. Calculate the content of C16H9N4Na3O9S2 in the substance to be examined using the measured absorbances of the test solution and the reference solution, and the declared content of C16H9N4Na 3O9S2 in tartrazine RS.

Storage Store in an airtight container, protected from light. Action and use Coloured agent. TELMISARTAN Telmisartanum

C33H30N4O2  

M: 514.6

Telmisartan is 4’-[[4-methyl-6-(1-methyl-1H-benzimidazol2-yl)-2-propyl-1H-benzimidazol-1-yl]methyl]biphenyl2-carboxylic acid. It contains not less than 99.0% and not more than 101.0% of C33H30N4O2, calculated with reference to the dried substance.

Characters White or slightly yellowish, crystalline powder, it shows polymorphism. Practically insoluble in water, slightly soluble in methanol, sparingly soluble in methylene chloride and dissolves in 1 M sodium hydroxide. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of telmisartan RS. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in hot anhydrous ethanol R, evaporate to dryness and record new spectra using the residues. Appearance of solution The solution is not more intensely coloured than reference solution Y4 (Appendix 9.3, method 2). Dissolve 0.5 g in 1 M sodium hydroxide R and dilute to 10 ml with the same solvent.

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Dissolve 2.0 g of potassium dihydrogen phosphate R and 3.5 g of sodium pentanesulfonate R in water, adjust to pH 3.0 with dilute phosphoric acid R and dilute to 1000 ml with water. Mobile phase B: Methanol R2 - acetonitrile R1 (20 : 80). Test solution: To 25 mg of the substance to be examined add about 5 ml of methanol R and 100 µl of 1 M sodium hydroxide R. Dissolve with the aid of ultrasound and dilute to 50 ml with methanol R. Reference solution (1): Dilute 1.0 ml of the test solution to 10.0 ml with methanol R. Dilute 1.0 ml of this solution to 100.0 ml with methanol R. Reference solution (2): Dissolve the contents of a vial of telmisartan for system suitability RS (containing impurities A, B, C, E and F) in 2 ml of methanol R. Reference solution (3): To 5 mg of telmisartan for peak identification RS (containing impurity D) add about 5 ml of methanol R and 100 µl of 1 M sodium hydroxide R. Dissolve with the aid of ultrasound and dilute to 10 ml with methanol R. Chromatographic system: A column (12.5 cm × 4.0 mm) packed with stationary phase C (5 µm) with a pore size of 10 nm. Column temperature: 40 °C. Detector: A spectrophotometer set at 230 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0-3

70

30

3 - 28

70 → 20

30 → 80

Identification of impurities: Use the chromatogram supplied with telmisartan for system suitability RS and the chromatogram obtained with reference solution (2) to identify the peaks due to impurities A, B, C, E and F; use the chromatogram supplied with telmisartan for peak identification RS and the chromatogram obtained with reference solution (3) to identify the peak due to impurity D. Relative retention time with reference to telmisartan (retention time = about 15 min): Impurity A = about 0.2; impurity E = about 0.6; impurity F = about 0.7; impurity B = about 0.9; impurity C = about 1.5; impurity D = about 1.6. System suitability: The chromatogram obtained with reference solution (2) is similar to the chromatogram supplied with telmisartan for system suitability RS. The resolution between the peaks due to impurity B and telmisartan is at least 3.0. Limits: In the chromatogram obtained with the test solution: Impurities C, D: For each impurity, not more than twice the 939

VP V

TELMISARTAN TABLETS

area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%). Impurities A, B: For each impurity, not more than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.15%). Any other impurity: For each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). Total peak area of all impurities is not more than 10 times the area of the principal peak in the chromatogram obtained with reference solution (1) (1.0%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%).

Note: Impurity A: 4-methyl-6-(1-methyl-1H-benzimidazol-2-yl)-2propyl-1H-benzimidazole. Impurity B: 4’-[[7-methyl-5-(1-methyl-1H-benzimidazol-2-yl)2-propyl-1H-benzimidazol-1-yl]methyl]biphenyl-2-carboxylic acid. Impurity C: 1,1-dimethylethyl 4’-[[4-methyl-6-(1-methyl-1Hbenzimidazol-2-yl)-2-propyl-1H-benzimidazol-1-yl]methyl] biphenyl-2-carboxylate. Impurity D: unidentified impurity. Impurity E:1-[(2’-carboxybiphenyl-4-yl)methyl]-4-methyl-2-pro pyl-1H-benzimidazol-6-carboxylic acid. Impurity F: 4’-[[4-methyl-6-(1-methyl-1H-benzimidazol-2-yl)-2propyl-1H-benzimidazol-1-yl]methyl]biphenyl-2-carboxamide. Impurity G: 4’-[[4-methyl-6-(1-methyl-1H-benzimidazol-2-yl)2-propyl-1H-benzimidazol-1-yl]methyl]biphenyl-2-carbonitrile. Impurity H: 1,1-dimethylethyl 4’-(bromomethyl)biphenyl-2carboxylate.

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.190 g of the substance to be examined in 5 ml of anhydrous formic acid R. Add 75 ml of acetic anhydride R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 25.73 mg of C33H30N4O2.

TELMISARTAN TABLETS Tabellae Telmisartani Telmisartan tablets contain telmisartan. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements:

Content of telmisartan, C33H30N4O2, 90.0% to 110.0% of the stated amount. Identification A. In the Dissolution, the ultraviolet absorption spectrum (Appendix 4.1) of the test solution corresponds to that of the reference solution. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that of the telmisartan peak in the chromatogram obtained with the reference solution. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Dissolve 0.5 g of potassium dihydrogen phosphate R in 1000 ml of water, add 2 ml of triethylamine R, adjust to pH 3.2 with phosphoric acid R. Mobile phase B: Acetonitrile R. Diluent: Dissolve 2 ml of triethylamine R in 800 ml of water, add 200 ml of methanol R. Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Disperse an accurately weighed quantity of the powder contaning the equivalent of 100 mg of telmisartan in the diluent to produce 100.0 ml. Sonicate for about 45 min and filter. Reference solution: A solution of telmisartan RS in the diluent having a known concetntration of 0.005 mg per ml. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 μm). Detector: A spectrophotometer set at 298 nm. Flow rate: 1.8 ml/min. Volume of injection: 20 μl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0

78

22

Storage Store in an airtight container, protected from light.

6

80

20

7

70

30

Action and use Angiotensin II (AT1) receptor antagonist.

15

60

40

25

60

40

26

40

60

35

20

80

40

78

22

Preparation Tablets. 940

TENOXICAM

VP V

System suitability: Inject the reference solution. The test is not valid unless, the number of theoretical plates calculated for the telmisartan peak is not less than 3000; the tailing factor is not more than 2 and the relative standard deviation of the peak area for 6 replicate injections is not more than 5.0%. In the chromatograph obtained with the test solution, the area of any secondary peak is not greater than the area of the principal peak in the chromatogram obtained with the reference solution (0.5%); the sum of the areas of any secondary peaks is not more than four times the area of the principal peak in the chromatogram obtained with the reference solution (2%). Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with the reference solution (0.05%).

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of phosphate buffer pH 7.5. Rotation speed: 75 rpm. Time: 30 min. Phosphate buffer pH 7.5: Dissolve 13.61 g of potassium dihydrogen phosphate R in 800 ml of water, adjust to pH 7.5 with 2 M sodium hydroxide R. Dilute to 1000 ml with water. Procedure: Test solution: After the specified time, withdraw a sample of the medium and filter, discarding the first portion of filtrate. Dilute the filtrate quantitatively with the medium to obtain a solution containing 0.011 mg of telmisartan per ml. Reference solution: Transfer an accurately weighed quantity of about 44 mg of telmisartan RS to a 100 ml volumetric flask. Add 1 ml of 0.1 M sodium hydroxide R and dilute to volume with methanol R. Dilute the filtrate quantitatively with the medium to obtain a solution containing 0.011 mg of telmisartan per ml. Measure the absorbance (Appendix 4.1) of the resulting solutions at the maximum at about 296 nm, in a 1 cm cell, using the medium as a blank. Calculate the content of telmisartan, C33H30N4O2, dissolved using the absorbances of the reference solution, the test solution and the content of C33H30N4O2 in telmisartan RS. Tolerance: Not less than 75% (Q) of the stated amount of telmisartan, C33H30N4O2, is dissolved in 30 minutes. Assay Examine by liquid chromatography (Appendix 5.3). Buffer solution: Dissolve 2.72 g of potassium dihydrogen phosphate R in 1000 ml of water, add 2 ml of triethylamine R and adjust to pH 2.4 with phosphoric acid R. Mobile phase: Buffer solution - acetonitrile (60 : 40). Diluent: Dissolve 2 ml of triethylamine R in 800 ml of water, add 200 ml of methanol R. Reference solution: Dissolve an accurately weighed quantity of about 40 mg of telmisartan RS in the diluent to produce 100.0 ml. Dilute 5.0 ml of this solution to 50.0 ml with the same solvent.

Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Transfer an accurately weighed quantity of the powder containing the equivalent of 40 mg of telmisartan to a 100 ml volumetric flask, add 80 ml of the diluent and sonicate for 45 minutes. Cool to room temparature and dilute to volume with the same solvent, mix and filter. Dilute 5.0 ml of the filtrate to 50.0 ml with the diluent. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 μm). Detector: A spectrophotometer set at 298 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 μl. Procedure: System suitability: Inject the reference solution, the test is not valid unless the number of theoretical plates is not less than 3000; the tailing factor for telmisartan peak is not more than 2.0 and the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of telmisartan, C33H30N4O2, in the tablets using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C33H30N4O2 in telmisartan RS.

Storage Store in an airtight container, in a cool and dry place, protected from light. Action and use Angiotensin II (AT1) receptor antagonist. Usual strength 20 mg; 40 mg; 80 mg. TENOXICAM Tenoxicamum

C13H11N3O4S2  

M. 337.4

Tenoxicam is 4-hydroxy-2-methyl-N-(pyridin-2-yl)-2Hthieno[2,3-e]1,2-thiazine-3-carboxamide 1, 1-dioxide. It contains not less than 99.0% and not more than 101.0% of C13H11N3O4S2, calculated with reference to the anhydrous substance.

Characters Yellow, crystalline powder, it shows polymorp. Practically insoluble in water, sparingly soluble in methylene chloride, very slightly soluble in anhydrous ethanol. It dissolves in solutions of acids and alkalis. 941

VP V

TENOXICAM

Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of tenoxicam RS. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in the minimum volume of methylene chloride R, evaporate to dryness and record new spectra using the residues. Appearance of solution Dissolve 0.10 g of the substance to be examined in methylene chloride R and dilute to 20 ml with the same solvent. This solution is clear (Appendix 9.2). Related substances Examine by liquid chromatography (Appendix 5.3). Carry out the test protected from light. Mobile phase A: A mixture of 25 volumes of methanol R2 and 75 volumes of water and adjust to apparent pH 3.2 with 1.5 M phosphoric acid R. Mobile phase B: A mixture of 25 volumes of water and 75 volumes of methanol R2 and adjust to apparent pH 3.2 with 1.5 M phosphoric acid R. Solvent mixture: Mix equal volumes of acetonitrile R and water. Adjust to apparent pH 3.2 with 1.5 M phosphoric acid R. Test solution: Dissolve 35 mg of the substance to be examined in the solvent mixture, sonicate and dilute to 50.0 ml with the solvent mixture. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 10.0 ml with the solvent mixture. Reference solution (2): Dissolve 7 mg of pyridin-2-amine R (impurity A) in the solvent mixture and dilute to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 100.0 ml with the solvent mixture. Reference solution (3): Dissolve the contents of a vial of tenoxicam impurity mixture RS (impurities B, G and H) in 1.0 ml of the test solution. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase cyanosilyl silica gel for chromatography R (3.5 µm). Column temperature: 35 °C. Detector: A spectrophotometer set at 230 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions:

942

Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0-5

96

4

5 - 16

96 → 76

4 → 24

16 - 25

76

24

Identification of impurities: Use the chromatogram obtained with reference solution (2) to identify the peak due to impurity A. Use the chromatogram supplied with tenoxicam impurity mixture RS and the chromatogram obtained with reference solution (3) to identify the peaks due to impurities B, G and H; for identification of impurities G and H, which may be inverted in the elution order, take into account the heights of the corresponding peaks in the chromatogram supplied with tenoxicam impurity mixture RS. Relative retention time with reference to tenoxicam (retention time = about 12 min): impurity A = about 0.1; impurity G = about 0.85; impurity H = about 0.9; impurity B = about 1.3. System suitability: In the chromatogram obtained with reference solution (3), the resolution between the peaks due to impurity H (or impurity G if peaks are inverted) and tenoxicam is at least 1.3 and the resolution between the peaks due to impurities G and H is at least 1.3; if necessary, optimise the apparent pH of the mobile phases within the range 3.0 - 3.4. Limits: Correction factors: for the calculation of content, multiply the peak areas of the following impurities by the corresponding correction factor: impurity A = 0.2; impurity B = 2.0; Impurities A, B: For each impurity, the corrected area is not more than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.15%). Any other impurity: For each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). The sum of the peak areas of all impurities is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.3%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A: Pyridin-2-amine. Impurity B: Methyl 4-hydroxy-2-methyl-2H-thieno[2,3-e]1,2thiazine-3-carboxylate 1,1- dioxide. Impurity C: N-methylthiophene-2-carboxamide. Impurity D: N-methyl-N'-(pyridin-2-yl)-ethanediamide. Impurity E: 2-methylthieno[2,3-d]isothiazol-3(2H)-one 1,1-dioxide. Impurity F: 4-hydroxy-N,2-dimethyl-N-(pyridin-2-yl)-2Hthieno[2,3-e]1,2-thiazine-3- carboxamide 1,1-dioxide. Impurity G: 4-hydroxy-2-methyl-2H-thieno[2,3-e]1,2-thiazine3-carboxamide 1,1-dioxide. Impurity H: 3-[(methylamino)sulfonyl]thiophene-2-carboxylic acid.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 0.5 g complies with limit test for heavy metals, method 3. Prepare the standard using 5 ml of lead standard solution (2 ppm Pb) R.

VP V

Water Not more than 0.5% (Appendix 10.3). Determined on 1.000 g. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.250 g of the substance to be examined in 5 ml of anhydrous formic acid R. Add 70 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 33.74 mg of C13H11N3O4S2. Storage Protected from light. Action and use Cyclo-oxygenase inhibitor; analgesic; anti-inflammatory. Preparations Injection, tablets. TENOXICAM TABLETS Tabellae Tenoxicami Tenoxicam tablets contain tenoxicam. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of tenoxicam, C13H11N3O4S2, 92.5% to 105.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel F254. Mobile phase: Anhydrous formic acid - acetone dichloromethane (4 : 30 : 70). Test solution: To a quantity of the powdered tablets containing 20 mg of tenoxicam add 20 ml of dichloromethane R, sonicate for 15 minutes, centrifuge and use the supernatant liquid. Reference solution: A 0.1% solution of tenoxicam RS in dichloromethane R. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 10 cm. After removal of the plate, allow to dry in air and examine under ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of the tenoxicam peak in the chromatogram obtained with reference solution.

TENOXICAM TABLETS

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of phosphate buffer pH 6.8. Phosphate buffer pH 6.8: Dissolve 6.8 g of potassium dihydrogen orthophosphate R in 500 ml of water, add 23 ml of 1 M sodium hydroxide R, dilute to 1000 ml and, if necessary, adjust the pH to 6.8 using either 1 M sodium hydroxide solution R or a 10% (w/v) solution of phosphoric acid R. Rotation speed: 50 rpm. Time: 45 min. Procedure: After the specified time, withdraw a sample of the medium and filter, discard the first 20 ml of the filtrate, dilute the filtrate with the medium if necessary to obtain an appropriate concentration. Measure the absorbance at the maximum at 368 nm (Appendix 4.1), using a 1 cm cell and the medium as a blank, in comparison with a reference solution having the same concentration of tenoxicam as the test solution. Calculate the total content of tenoxicam, C13H11N3O4S2, in the medium using the absorbances measured and the declared content of C13H11N3O4S2 in tenoxicam RS. Tolerance: Not less than 70% (Q) of the stated amount of tenoxicam, C13H11N3O4S2, is dissolved in 45 minutes. Related substance Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 0.12 g of sodium lauryl sulphate R in 700 ml of methanol R, mix with 1000 ml of 0.05 M potassium dihydrogen orthorphosphate R and adjust the pH to 2.8 with orthophosphoric acid R. Test solution: Shake a quantity of the powdered tablets containing 0.1 g of tenoxicam with 100 ml of acetonitrile (50%) for 70 minutes, mixing occasionally with the aid of ultrasound. Allow to stand for 10 minutes, dilute 5 ml of the clear supernatant liquid to 20 ml with the mobile phase and filter. Reference solution: Dilute 1.0 ml of the test solution to 200.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4 mm) packed with stationary phase B (Nucleosil C8, 5 µm is suitable), and a pre-column packed with stationary phase B. Detector: A spectrophotometer set at 254 nm. Flow rate: 0.7 ml/min. Volume of injection: 20 µl. Procedure: Equilibrate the column with the mobile phase for 3 hours. Inject the test solution and the reference solution. In the chromatogram obtained with the test solution the area of any secondary peak is not greater than the area of the principal peak in the chromatogram obtained with the reference solution (0.5%) and the sum of the areas of any such peaks is not greater than four times the area of the principal peak in the chromatogram obtained with the reference solution (2%). 943

VP V

TERBUTALINE SULFATE

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase and chromatographic system as described under Related substances. Test solution: Shake 10 whole tablets with 200 ml of acetonitrile (50%) for 70 minutes, mixing occasionally with the aid of ultrasound. Allow to stand for 10 minutes, dilute a volume of the clear supernatant liquid with sufficient mobile phase to produce a solution containing 0.025% of tenoxicam and filter. Reference solution: Dilute 5 ml of a 0.1% solution of tenoxicam RS in acetonitrile (50%) to 20 ml with the mobile phase. Procedure: Inject the reference solution. The test is not valid unless the relative standard deviation of the peak areas of tenoxicam for 6 replicate injections is less than 2.0%. Inject alternatively the reference solution and the test solution. Calculate the content of tenoxicam, C13H11N3O4S, using the areas of the principal peaks in the chromatograms obtained with the reference solution, the test solution and the declared contents of C13H11N3O4S in tenoxicam RS. Storage Store in a well-closed container, protected from light. Action and use Non steroidal anti-inflammatory. Usual strength 10 mg, 20 mg. TERBUTALINE SULFATE Terbutalini sulfas

(C12H19NO3)2,H2SO4  

M. 548.7

Terbutaline sulfate bis[(1RS)-1-(3,5-dihydroxyphenyl)2-[(1,1-dimethylethyl)amino]ethanol] sulfate. It contains not less than 98.0% and not more than 101.0% of (C12H19NO3)2,H2SO4, calculated with reference to the anhydrous substance.

Characters White or almost white, crystalline powder, it shows polymorphism. Freely soluble in water, slightly soluble in ethanol (96%).

944

Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of terbutaline sulfate RS. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in  aldehyde-free methanol R, evaporate to dryness and record new spectra using the residues. B. 5 ml of solution S (see Acidity) gives reaction (A) of sulfates (Appendix 8.1). Appearance of solution Dissolve 2.0 g in carbon dioxide-free water and dilute to 50 ml with the same solvent. The solution is clear (Appendix 9.2) and its absorbance at 400 nm (use a 1 cm cell) is not greater than 0.11 (Appendix 4.1). Acidity Solution S: Dissolve 1.0 g of the substance to be examined in carbon dioxide-free water R and dilute to 50 ml with the same solvent. Add 0.05 ml of methyl red solution R to 10 ml of solution S. Not more than 1.2 ml of 0.01 N sodium hydroxide VS is required to change the colour of the indicator to yellow. Optical rotation -0.10° to +0.10° (Appendix 6.4). Determined on solution S. Related substances Examine by liquid chromatography (Appendix 5.3). 0.05 M ammonium formate buffer solution: Dissolve 3.15 g of ammonium formate R in about 980 ml of water; adjust to pH 3.0 by adding about 8 ml of anhydrous formic acid R and dilute to 1000 ml with water. Mobile phase: Dissolve 4.23 g of sodium hexanesulfonate R in 770 ml of 0.05 M ammonium formate buffer solution, then add 230 ml of methanol R. Mix well. Test solution: Dissolve 75.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Reference solution (1): Dissolve 7.5 mg of terbutaline impurity C RS and 22.5 mg of terbutaline sulfate RS in the mobile phase and dilute to 50.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 100.0 ml with the mobile phase. Reference solution (2): Dilute 1.0 ml of the test solution to 50.0 ml with the mobile phase. Dilute 2.0 ml of this solution to 20.0 ml with the mobile phase. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase base-deactivated  octadecylsilyl silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 276 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl.

TERFENADINE

VP V

Procedure: The run time is 6 times the retention time of terbutaline. Retention time of impurity C is about 9 min; terbutaline is about 11 min. System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to impurity C and terbutaline is at least 2.0. If necessary adjust the composition of the mobile phase, decrease the content of methanol to increase the retention time. Limits: In the chromatogram obtained with the test solution: The area of the peak due to impurity C is not more than twice the area of the corresponding peak in the chromatogram obtained with reference solution (1) (0.2%). Any other impurity: For each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.2%). The sum of the peak areas of all impurities other than C is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (2) (0.4%). Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.02%). Note: Impurity C: 1-(3,5-dihydroxyphenyl)-2-[(1,1-dimethylethyl) amino]ethanone.

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C; 3 h). Assay Dissolve 0.400 g of the substance to be examined in 70 ml of anhydrous acetic acid R with heating. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 54.87 mg of C24H40N2O10S. Storage Store in an airtight container, protected from light. Action and use Beta2-adrenoceptor agonist; bronchodilator. Preparations Tablets, injection.

TERFENADINE Terfenadinum

C32H41NO2

M. 471.7

Terfenadine is (1RS)-1-[4-(1,1-dimethylethyl)phenyl]-4[4-(hydroxydiphenylmethyl) piperidin-1-yl]butan-1-ol. It contains not less than 98.5% and not more than 101.0% of C32H41NO2, calculated with reference to the dried substance.

Characters A white, crystalline powder, it shows polymorphism. Very slightly soluble in water and in dilute hydrochloric acid, freely soluble in methylene chloride, soluble in methanol. Identification Apply one of the two following identifications: First identification: A Second identification: B, C, D A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of terfenadine RS. B. Melting point: 146 °C to 152 °C (Appendix 6.7). C. Dissolve 50.0 mg of the substance to be examined in methanol R and dilute to 100.0 ml with the same solvent, and mix. The ultraviolet absorptiom spectrum (Appendix 4.1) of the solution, in the range between 230 nm and 350 nm, exhibits an absorption maximum at 259 nm and shoulders at 253 nm and 270 nm. The value of A(1%; 1 cm) at the maximum is 13.5 to 14.9. D. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel HF254. Mobile phase: Methanol - dichloromethane (10 : 90). Test solution: Dissolve 50 mg of the substance to be examined in dichloromethane R and dilute to 10 ml with the same solvent, and mix. Reference solution: Dissolve 50 mg of terfenadine RS in dichloromethane R and dilute to 10 ml with the same solvent, and mix. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm. Remove the plate, allow it to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. 945

VP V

TERFENADINE TABLETS

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dilute 600 ml of acetonitrile R to 1000 ml with diethylammonium phosphate buffer solution pH 6.0 R, and mix. Test solution: Dissolve 15 mg of the substance to be examined in the mobile phase and dilute to 10.0 ml with the mobile phase, and mix. Reference solution (1): Dilute 1.0 ml of the test solution to 10.0 ml with the mobile phase, and mix. Dilute 1.0 ml of this solution to 20.0 ml with the mobile phase, and mix. Reference solution (2): Dilute 10.0 ml of reference solution (1) to 25.0 ml with the mobile phase, and mix. Reference solution (3): Dissolve 0.1 g of potassium iodide R in the mobile phase and dilute to 100 ml with the mobile phase, and mix. Dilute 1 ml to 100 ml with the mobile phase, and mix. Resolution solution: Dissolve 15 mg of terfenadine impurity A RS in the mobile phase and dilute to 10.0 ml with the mobile phase, and mix. To 5.0 ml of the solution, add 5.0 ml of the test solution and dilute to 50.0 ml with the mobile phase, and mix. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase B (5 µm). Detector: A spectrophotometer set at 217 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Inject each solution and continue the chromatography for 5 times the retention time of terfenadine. The test is not valid unless, in the chromatogram obtained with the resolution solution, the resolution between the peaks due to terfenadine and impurity A is greater than 5.0 and the mass distribution ratio of the peak corresponding to terfenadine is greater than 2.0. Determine the mass distribution ratio using, as unretained compound, potassium iodide. Limits: In the chromatogram obtained with the test solution: The area of any peak, apart from the principal peak, is not greater than that of the peak in the chromatogram obtained with reference solution (2) (0.2%). The sum of the areas of all the peaks, apart from the principal peak, is not greater than the area of the peak in the chromatogram obtained with reference solution (1) (0.5%). Disregard any peak with an area less than 2.5% of that of the peak in the chromatogram obtained with reference solution (2). Note: Impurity A: 1-[4-(1,1-dimethylethyl)phenyl]-4-[4-(hydroxydiphenylmethyl)piperidin-1-yl]butan-1-one.

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 60 °C; under a pressure not exceeding 0.5 kPa). 946

Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.400 g, accurately weighed, of the substance to be examined in 50 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 47.17 mg of C32H41NO2. Storage Store in an airtight container, protected from light. Action and use Histamine H1-receptor antagonist. Preparation Tablets. TERFENADINE TABLETS Tabellae Terfenadini Terfenadine tablets contain terfenadine. The tablets comply with the requirements stated under "Tablets" (Appendix 1.20) and with the following requirements.

Content of terfenadine, C32H41NO2, 95.0% to 105.0% of the stated amount. Identification A. Shake a quantity of the powdered tablets containing 0.2 g of terfenadine with 20 ml of dichloromethane R, add 10 ml of 0.1 M sodium hydroxide R and shake again. Allow to separate collect the dichloromethane layer, wash the dichloromethane layer with 10 ml of water, shake with 2 g of anhydrous sodium sulfate R and filter. Add 0.2 ml of the filtrate to 0.3 g of potassium bromide R in a mortar, mix with a pestle, warm to remove the solvent and prepare a disc from the resulting mixture. The infrared absorption spectrum (Appendix 4.2) is concordant with the reference spectrum of terfenadine. B. In the Assay, the retension time of the principal peak in the chromatogram obtained with the test solution is similar to that of the principal peak in the chromatogram obtained with the reference solution. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 1000 ml of 0.1M hydrochloric acid R. Rotation speed: 50 rpm. Time: 45 min. Test solution: After the specified time, withdraw a sample of the medium and filter. Dilute the filtrate sample, if

TERPIN HYDRATE

VP V

necessary, with 0.1 M  hydrochloric acid  R to obtain a solution containing about 0.006% of terfenadine. Reference solution: Dilute 1 volume of a 0.06% solution of terfenadine RS in methanol R to 10 volumes with 0.1 M hydrochloric acid R. Procedure: Carry out the method for liquid chromatography (Appendix 5.3) with the chromatographic conditions described under Assay and a detection wavelength of 217 nm. Calculate the total content of terfenadine, C32H41NO2, in the medium using peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C32H41NO2 in terfenadine RS. Tolerance: Not less than 70% (Q) of the stated amount of terfenadine, C32H41NO2, is dissolved in 45 min.

Impurity A Not more than 0,2%. Examine by liquid chromatography (Appendix 5.3). Test solution: Disperse a quantity of the powdered tablets containing 0.15 g of terfenadine in 75 ml of the mobile phase with the aid of ultrasound for 15 minutes, cool to room temperature, dilute to 100 ml with the mobile phase, mix and filter through a glass microfibre filter (Whatman GF/C is suitable). Reference solution: A 0.0003% solution of terfenadine impurity A RS  (1-[4-(1,1-dimethylethyl)phenyl]-4-[4(hydroxydiphenylmethyl)piperidin-1-yl]butan-1-one) in the mobile phase. Resolution solution: Mix 1 volume of the test solution and 9 volumes of a 0.015% solution of terfenadine impurity A RS in the mobile phase. The chromatographic conditions described under Assay may be used but using a detection wavelength of 217 nm. Procedure: Inject the above solutions and allow the chromatography to proceed for 5 times the retention times of terfenadine. System suitability: The test is not valid unless, in the chromatogram obtained with the resolution solution, the resolution factor between the peaks due to terfenadine and terfenadine impurity A is at least 5.0. Limits: In the chromatogram obtained with the test solution: The area of any peak corresponding to terfenadine impurity A is not greater than the area of the principal peak in the chromatogram obtained with the reference solution. In the chromatogram obtained with the test solution peak due to excipients with long retention time may be present. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dilute 3 volumes of acetonitrile R to 5 volumes with diethylammonium phosphate buffer solution pH 6.0 R. Test solution: Weigh and finely powder 20 tablets. Disperse a quantity of the powdered tablets containing 0.15 g of terfenadine in 75 ml of the mobile phase with the aid of

ultrasound for 15 minutes, cool to room temperature, dilute to 100.0  ml with mobile phase, mix and filter through a glass microfibre filter (Whatman GF/C is suitable). Reference solution: A 0.15% solution of terfenadine RS in the mobile phase. Resolution solution: A solution containing 0.015% each of terfenadine RS and terfenadine impurity A RS in the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase B (5 µm) (Lichrosorb RP8 is suitable). Flow rate: 1.0 ml/min. Detector: A spectrophotometer set at 254 nm. Volume of injection: 20 µl. Procedure: Inject the above solutions and allow the chromatography to proceed for 5 times the retention times of terfenadine. The test is not valid unless, in the chromatogram obtained with the resolution solution, the resolution factor between the peaks due to terfenadine and terfenadine impurity A is at least 5.0. Calculate the content of C32H41NO2 in the tablets using the peak areas of terfenadine in the chromatogram obtained with the test solution and the reference solution and the declared content of C32H41NO2 in terfenadine RS.

Storage Storage in a close container and protect from light. Action and use Histamine H1 receptor antagonist; antihistamine. Strength use 60 mg. TERPIN HYDRATE Terpinum hydratum HO

H 3C

CH 3

CH 3 OH

. H 2O

C10H20O2, H2O M.190.3 Terpin hydrate is cyclohexanemethanol,4-hydroxy-α,α,4-trimethyl monohydrate or p-menthane-1,8-diol monohydrate. It contains not less than 98.0% and not more than the equivalent of 100.5% of C10H20O2, calculated with reference to the anhydrous substance.

Characters A white, crystalline powder or colourless, trasparent crystals; odourless. It sublimates and is transfomated into needles when dried carefully at 100 °C. It slowly losses its water 947

VP V

TERPIN HYDRATE

of crystallisation and its melting point is decreased on exposure to hot and dry air. Sparingly soluble in water, in chloroform and in ether, soluble in hot water and in ethanol (96%), freely soluble in hot ethanol (96%).

and spray with a 1% solution of vanillin R in sulfuric acid R. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (2).

Identification Apply one of the two following identifications: First identification: A, D Second identification: B, C, D, E A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of terpin hydrate RS. B. Melting point: 115 °C to 117 °C (Appendix 6.7). Heat the bath to 110 °C, then insert the cappilary tube containing the substance to be examined and continue heating with a rate of 4 °C to 6 °C per minute. C. In the test for Related substances, the principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). D. Heat 5 ml of a 2% solution of the substance to be examined, add few drops of sulfuric acid R. The solution becomes turbid and an aromatic odour of terpineol is detectable. E. To about 10 mg of the substance to be examined in a porcelain crucible add 5 drops of iron (III) chloride solution in ethanol R, and evaporate to dryness. Vermilion, violet and green colours appear simultaneously at different parts of the crucible.

Water 8.0% to 10.0% (Appendix 10.3). Determined on 0.20 g.

Appearance of solution Solution S: Dissolve 2.50 g in ethanol (96%) R, and dilute to 50 ml with the same solvent, and mix. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). Acidity or alkalinity To 10 ml of solution S add 0.1 ml of bromothymol blue solution R. Not more than 0.2 ml of 0.02 N hydrochloric acid VS or of 0.02 N sodium hydroxide VS is required to change the colour of the indicator. Related substances Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel G. Mobile phase: Chloroform - ethyl acetate (1 : 9). Test solution: Dissolve 0.25 g of the substance to be examined in methanol R and dilute to 5 ml with the same solvent, and mix. Reference solution (1): Dissolve 0.25 g of terpin hydrate RS in methanol R and dilute to 5 ml with the same solvent, and mix. Reference solution (2): Dilute 1.0 ml of reference solution (1) to 100.0 ml with methanol R and mix. Procedure: Apply separately to the plate 3 µl of each solution. Develop over a path of 15 cm. Remove the plate, dry it at 100 °C to 105 °C for 5 min. Allow the plate to cool 948

Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by gas chromatography (Appendix 5.2). Internal standard solution: Dissolve an accurately weighed quantity of biphenyl in chloroform R to obtain a solution containing about 20 mg/ml. Test solution: Dissolve 170 mg, accurately weighed, of the substance to be examined in 5 ml of ethanol (96%) R, add 5 ml of the internal standard solution, dilute to 100.0 ml with chloroform R, and mix Reference solution: Dissolve 170 mg, accurately weighed, of terpin hydrate RS in 5 ml of ethanol (96%) R, add 5 ml of the internal standard solution, dilute to 100.0 ml with chloroform R, and mix Chromatographic system: A stainless steel or glass column (1.2 m × 3.5 mm) packed with silanised diatomaceous earth for gas chromatography (Chromosorb AW; 80 to 100 mesh) impregnated with 6% w/w of dimethylpolysiloxane for gas chromatography R. Carrier gas: Nitrogen for chromatography at a suitable flow rate to obtain a retention time of about 7 min for terpin and of about 11 min for biphenyl. A flame-ionisation detector. Maintain the temperature of the column at 120 °C and that of the injection port and of the detector at 260 °C. Volume of injection: 1 μl. Procedure: Inject the reference solution. The resolution between the peaks corresponding to terpin and biphenyl is at least 2.0, and the relative standard deviation for replicate injections is not more than 2.0%. Inject the test solution. Calculate the content of C10H20O2­ in the substance to be examined, using the ratios of the peak area of terpin to the peak area of internal standard obtained from the test solution and the reference solution, and the declared content of C10H20O2 in terpin hydrate RS. Storage Store in an airtight container, protected from light. Action and use Expectorant. Preparations It is usually combined in anticough preparations such as terpin benzoat tablet, terpin codein tablet.

TETRACAINE HYDROCHLORIDE

VP V

TETRACAINE HYDROCHLORIDE Tetracaini hydrochloridum

C15H24N2O2,HCl  



M. 300.8

Tetracaine hydrochloride is 2-(dimethylamino)ethyl 4-(butylamino)benzoate hydrochloride. It contains not less than 99.0% and not more than 101.0% of C15H24N2O2,HCl, calculated with reference to the dried substance.

Characters White or almost white, slightly hygroscopic, crystalline powder. Freely soluble in water, soluble in ethanol (96%). It melts at about 148 °C or it may occur in either of 2 other crystalline forms which melt respectively at about 134 °C and 139 °C. Mixtures of these forms melt within the range 134 °C to 147 °C. Identification Apply one of the two following identifications: First identification: A, B, D. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of tetracaine hydrochloride RS. B. Add 1 ml of ammonium thiocyanate solution R to 10 ml of solution S (see Appearance of solution). A white, crystalline precipitate is formed which, after recrystallisation from water and drying at 80 °C for 2 h, melts at about 131 °C. C. Add 0.5 ml of fuming nitric acid R to about 5 mg of the substance to be examined. Evaporate to dryness on a water-bath, allow to cool and dissolve the residue in 5 ml of acetone R. Add 1 ml of 0.1 M potassium hydroxide in ethanol R. A violet colour develops. D. Solution S gives reaction (A) of chlorides (Appendix 8.1). Appearance of solution Solution S: Dissolve 5.0 g in carbon dioxide-free water R and dilute to 50 ml with the same solvent. Dilute 2 ml of solution S to 10 ml with water. The solution is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). pH 4.5 to 6.5 (Appendix 6.2). Dilute 1 ml of solution S to 10 ml with carbon dioxide-free water R. Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use or store them at a temperature of 2 °C to 8 °C.

Mobile phase A: Dissolve 1.36 g of potassium dihydrogen phosphate R in water, add 0.5 ml of phosphoric acid R and dilute to 1000 ml with water. Mobile phase B: Acetonitrile R. Solvent mixture: Acetonitrile - water (20 : 80). Test solution: Dissolve 50 mg of the substance to be examined in the solvent mixture and dilute to 50 ml with the solvent mixture. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 10.0 ml with the solvent mixture. Reference solution (2): Dissolve the contents of a vial of tetracaine for system suitability RS (containing impurities A, B and C) in 2 ml of the solvent mixture. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 µm). Column temperature: 30 °C. Detector: A spectrophotometer set at 300 nm. Flow rate: 1.5 ml/min. Volume of injection: 10 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time Mobile phase A Mobile phase B (min) (% v/v) (% v/v) 0-3 80 20 3 - 18 80 → 40 20 → 60 18 - 23 40 60 Identification of impurities: Use the chromatogram supplied with tetracaine for system suitability RS and the chromatogram obtained with reference solution (2) to identify the peaks due to impurities A, B and C. Relative retention with reference to tetracaine (retention time = about 8 min): Impurity A = about 0.3; impurity B = about 1.7; impurity C = about 2.1. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to tetracaine and impurity B is at least 5.0. Limits: Correction factors: For the calculation of content, multiply the peak areas of the following impurities by the corresponding correction factor: impurity B = 0.6; impurity C = 0.7; Impurity A: The area of the peak due to impurity A is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Impurities B, C: For each impurity, the corrected area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). The sum of the peak areas of all impurities is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). 949

VP V

TETRACYCLINE HYDROCHLORIDE

Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A: 4-aminobenzoic acid. Impurity B: 4-(butylamino)benzoic acid. Impurity C: Methyl 4-(butylamino)benzoate.

Heavy metals Not more than 10 ppm (Appendix 9.4.8). 12 ml of solution S complies with limit test for heavy metals, method 1. Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on drying Not more than 1.0% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.250 g of the substance to be examined in 50 ml of ethanol (96%) R and add 5.0 ml of 0.01 N hydrochloric acid VS. Carry out a potentiometric titration (Appendix 10.2), using 0.1 N sodium hydroxide VS. Read the volume added between the 2 points of inflexion. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 30.08 mg of C15H24N2O2,HCl. Storage In an airtight container, protected from light. Action and use Local anaesthetic.

Specific optical rotation -240° to -255°, calculated with reference to the dried substance (Appendix 6.4). Dissolve 0.250 g of the substance to be examined in 0.1 M hydrochloric acid R, dilute to 25.0 ml with the same acid, and mix.

TETRACYCLINE HYDROCHLORIDE Tetracyclini hydrochloridum



M. 480.9

Tetracycline hydrochloride is (4S,4aS,5aS,6S,12aS)-4(dimethylamino)-3,6,10,12,12a-pentahydroxy-6-methyl1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-tetracene2-carboxamide hydrochloride. It contains not less than 95.0% and not more than 102.0% of C22H24N2O8,HCl, calculated with reference to the dried substance. 950

Identification A. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Octadecylsilyl silica gel F254. Mobile phase: Acetonitrile - methanol - 6.3% solution of oxalic acid previously adjusted to pH 2.0 with ammonia (20 : 20 : 60). Test solution: Dissolve 5 mg of the substance to be examined in 10 ml of methanol R. Reference solution (1): Dissolve 5 mg of tetracycline hydrochloride RS in 10 ml of methanol R. Reference solution (2): Dissolve 5 mg of tetracycline hydrochloride RS, 5 mg of demeclocycline hydrochloride RS and 5 mg of oxytetracycline hydrochloride RS in 10 ml of methanol R. Procedure: Apply to the plate 1 µl of each solution. Develop over a path of 15 cm. Remove the plate, allow it to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) shows 3 clearly separated spots. B. To about 2 mg of the substance to be examined add 5 ml of sulfuric acid R. A violet-red colour develops. Add 2.5 ml of water to the solution. The colour becomes yellow. C. It gives reaction (A) of chlorides (Appendix 8.1). pH Dissolve 0.1 g of the substance to be examined in 10 ml of carbon dioxide-free water R. The pH of the solution is 1.8 to 2.8 (Appendix 6.2).

Preparations Injection, cream, local solution.

C22H24N2O8,HCl

Characters A yellow, crystalline powder. Soluble in water, slightly soluble in ethanol (96%), practically insoluble in acetone. It dissolves in solutions of alkali hydroxides and carbonates. Solutions in water become turbid on standing, owing to the precipitation of tetracycline.

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Weigh 80.0 g of tert-butanol R and transfer to a 1000 ml volumetric flask with the aid of 200 ml of water. Add 100 ml of a 3.5% solution of dipotassium hydrogen phosphate R adjusted to pH 9.0 with 2 M phosphoric acid R, 200 ml of a 1.0% solution of tetrabutylammonium hydrogen sulfate R adjusted to pH 9.0 with 2 M sodium hydroxide R and 10 ml of a 4.0% solution of sodium edetate R adjusted to pH 9.0 with 2 M sodium hydroxide R, dilute to 1000.0 ml with water, and mix. Filter and degas.

TETRACYCLINE HYDROCHLORIDE

VP V

Test solution: Dissolve 25.0 mg of the substance to be examined in 0.01 M hydrochloric acid R and dilute to 25.0 ml with the same acid, and mix. Reference solution (1): Dissolve 25.0 mg of tetracycline hydrochloride RS in 0.01 M hydrochloric acid R and dilute to 25.0 ml with the same acid, and mix. Reference solution (2): Dissolve 15.0 mg of 4-epitetracycline hydrochloride RS in 0.01 M hydrochloric acid R and dilute to 50.0 ml with the same acid, and mix. Reference solution (3): Dissolve 10.0 mg of anhydrotetracycline hydrochloride RS in 0.01 M hydrochloric acid R and dilute to 100.0 ml with the same acid, and mix. Reference solution (4): Dissolve 10.0 mg of 4-epianhydrotetracycline hydrochloride RS in 0.01 M hydrochloric acid R and dilute to 50.0 ml with the same acid, and mix. Reference solution (5): Mix 1.0 ml of reference solution (1), 2.0 ml of reference solution (2) and 5.0 ml of reference solution (4) and dilute to 25.0 ml with 0.01 M hydrochloric acid R, and mix. Reference solution (6): Mix 20.0 ml of reference solution (2), 10.0 ml of reference solution (3) and 5.0 ml of reference solution (4) and dilute to 200.0 ml with 0.01 M hydrochloric acid R, and mix. Reference solution (7): Dilute 1.0 ml of reference solution (3) to 50.0 ml with 0.01 M hydrochloric acid R, and mix. Chromatographic system: A column (25 cm × 4.6 mm) packed with styrenedivinylbenzene copolymer (8 µm), maintained at 60 °C. Detector: A spectrophotometer set at 254 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Inject the test solution, reference solutions (5), (6) and (7). System suitability: In the chromatogram obtained with reference solution (5), the resolution between the peaks due to impurity A (1st peak) and tetracycline (2nd peak) is not less than 2.5; and the resolution between the peaks due to tetracycline and impurity D (4-epianhydrotetracycline, 3rd peak) is not less than 8.0. If necessary, adjust the concentration of tert-butanol in the mobile phase. The signal-to-noise ratio is not less than 3 for the principal peak in the chromatogram obtained with reference solution (7). The symmetry factor is not more than 1.25 for the peak due to tetracycline in the chromatogram obtained with reference solution (5). Limits: In the chromatogram obtained with the test solution: The area of the peak due to impurity A is not greater than that of the corresponding peak in the chromatogram obtained with reference solution (6) (3.0%). The area of the peak due to impurity B (eluting on the tail of the principal peak) is not greater than 0.5 times the area of the peak due to impurity A in the chromatogram obtained with reference solution (6) (1.5%).

The area of the peak due to impurity C (anhydrotetracycline) is not greater than that of the corresponding peak in the chromatogram obtained with reference solution (6) (0.5%). The area of the peak due to impurity D is not greater than that of the corresponding peak in the chromatogram obtained with reference solution (6) (0.5%).

Heavy metals Not more than 50 ppm (Appendix 9.4.8). 0.5 g complies with the limit test for heavy metals, method 3. Prepare the standard using 2.5 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 2.0% (Appendix 9.6). (1.000 g; 60 °C; diphosphorus pentoxide; at a pressure not exceeding 670 Pa; 3 h). Sulfated ash Not more than 0.5% (Appendix 9.9, method 2). Determined on 1.0 g. Bacterial endotoxins Less than 0.5 EU/mg (Appendix 13.2), if intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins. Assay Examine by liquid chromatography (Appendix 5.3), as described in the test for Related substances. Inject the test solution and reference solution (1). Calculate the content of C22H24N2O8,HCl in the substance to be examined, using the principal peak areas obtained with the test solution, reference solution (1) and the declared content of C22H24N2O8,HCl in tetracycline hydrochloride RS. Storage Store in an airtight container, protected from light. If the substance is sterile, store in a sterile, tamper-proof container. Labelling The label states, where applicable, that the substance is free from bacterial endotoxins. Action and use Antibacterial. Preparations Capsules; tablets, eye ointment.

951

TETRACYCLINE HYDROCHLORIDE CAPSULES

TETRACYCLINE HYDROCHLORIDE CAPSULES Capsulae Tetracyclini hydrochloridi Tetracycline hydrochloride capsules contain tetracycline hydrochloride. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of tetracycline hydrochloride, C22H24N2O8,HCl, 95.0% to 105.0% of the stated amount. Identification In the Assay, the principal peak in the chromatogram obtained with test solution has the same retention time as the principal peak in the chromatogram obtained with reference solution. Loss on drying Not more than 4.0% (Appendix 9.6). Use 0.1 g of the mixed contents of the capsules at 60 °C at a pressure not exceeding 5 mmHg for 3 hours. Dissolution (Appendix 11.4) Apparatus: Paddle. The distance between the paddle and the vessel bottom is (45 ± 5) mm. Medium: 900 ml of water. Rotation speed: 75 rpm. Time: 60 min for capsules containing less than 500 mg; 90 min for capsules containing 500 mg and more. Procedure: After the specified time, withdraw a sample of the medium and filter, discarding the first 20 ml of the filtrate. Measure the absorbance of the filtrate, suitably diluted with the dissolution medium if necessary, at the maximum at 276 nm (Appendix 4.1). At the same time, measure the absorbance of a solution of tetracycline hydrochloride RS having the equivalent concentration in the same medium. From the obtained absorbances and the declared content of C22H24N2O8,HCl in tetracycline hydrochloride RS, calculate the content of C22H24N2O8,HCl dissolved. Tolerance: Not less than 80% (Q) of the stated amount of C22H24N2O8,HCl is dissolved in required time. Limit of 4-epianhydrotetracycline Not more than 3.0%. Examine by liquid chromatography (Appendix 5.3). Mobile phase, Diluting solvent, Chromatographic system as described in the Assay. Reference solution: Dissolve an acccurately weighed quantity of 4-epianhydrotetracycline hydrochloride RS in diluting solvent to obtain a a known concentration of about 15 µg/ml. Procedure: Chromatograph the reference solution and compare with the chromatogram of the test solution obtained in the Assay. Calculate the percentage of 4-epianhydrotetracycline in 952

VP V

capsules using the areas for the 4-epianhydrotetracycline peaks in the chromatograms obtained with the test solution and reference solution, and the declared contents of 4-epianhydrotetracycline hydrochloride RS.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: 0.1 M ammonium oxalate solution dimethylformamide - 0.2 M diammonium hydrogen phosphate solution (68 : 27 : 5), adjust the pH of the mixture to between 7.6 to 7.7 with 3 M ammonia solution R or 3 M phosphoric acid R. Diluting solvent: 0.1 M ammonium oxalate solution dimethylformamide (68 : 27). Reference solution: Dissolve an acccurately weighed quantity of tetracycline hydrochloride RS in diluting solvent and dilute quantitatively with diluting solvent to obtain a known concentration of about 0.5 mg/ml. Test solution: Weigh 20 capsules, calculate the average mass of the capsule contents and transfer an accurately weighed portion of the powder, equivalent to about 50 mg of tetracycline hydrochloride, to a 100 ml volumetric flask. Add about 50 ml of diluting solvent, mix with the aid of ultrasound for about 5 min. Allow to cool and dilute with diluting solvent to volume, mix and filter. Resolution solution: Prepare a solution in diluting solvent containing about 100 µg of tetracycline hydrochloride RS and 25 µg of 4-epianhydrotetracycline hydrochloride RS per ml. Chromatographic system: Guard column: A column (3 cm × 4.6 mm) packed with stationary phase B (10 µm) Analytical column: A column (25 cm × 4.6 mm) packed with stationary phase B (5 µm to 10 µm). Detector: A spectrophotometer set at 280 nm. Flow rate: 2 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the resolution solution, the relative retention times are about 0.9 for 4-epianhydrotetracycline and 1.0 for tetracycline, and the resolution between the peaks due to 4-epianhydrotetracycline and tetracycline is not less than 1.2. Inject the reference solution, the relative standard deviation for replicate injections is not more than 2.0%. Inject sepatately the reference solution and the test solution. Calculate the content of tetracycline hydrochloride, C22H24N2O8,HCl, in capsules using the areas for the principal peaks in the chromatograms obtained with the test solution and reference solution, and the declared contents of tetracycline hydrochloride RS. Storage Store in a well-closed container, at a cool and dry place, protected from light.

VP V

Action and use Antibacterial. Usual strength 250 mg (250,000 IU); 500 mg (500,000 IU). TETRACYCLINE HYDROCHLORIDE EYE OINTMENT Unguentum Tetracyclini hydrochloridi Tetracycline hydrochloride eye ointment contains tetracycline hydrochloride in a suitable basis. The preparation comply with the requirements stated under “Topical semi-solid preparations” (Appendix 1.12), “Eye ointment” section and with the following requirements.

Content of tetracycline hydrochloride, C22H24N2O8,HCl, 90.0% to 125.0% of the stated amount. Characters A homogeneous yellowish ointment which is suitably soft. It may be easily applied to mucous membrane or skin. The ointment should not melt at 37 °C or show any phase separation in ordinary conditions. Identification A. To 5 g of the ointment add 5 ml of water, heat on a water bath until melted, stir with a glass rod, cool until the ointment base solidifies. Decant the water layer (solution A) for the following tests: Place about 1 ml of solution A in a porcelain dish and evaporate on a water bath to dryness. Add 1 or 2 drops of sulphuric acid R, a deep purple colour is produced. Add 1 drop of a 3% solution of ferric chlorid R, the colour changes to brown or reddish brown. Place about 2 ml of solution A in a test tube, add 1 drop of 32% nitric acid solution R and a few drops of 2% silver nitrate solution R, a white precipitate is produced B. In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the principal peak in the chromatogram obtained with the reference solution. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: 0.1 M ammonium oxalate solution dimethylformamide - 0.2 M diammonium hydrogen phosphate solution (68 : 27 : 5), adjust the pH of the mixture to between 7.6 and 7.7 with 3 M ammonia solution R or 3 M phosphoric acid R. Diluting solvent: 0.1 M ammonium oxalate solution dimethylformamide (68 : 27). Reference solution: Dissolve an acccurately weighed quantity of tetracycline hydrochloride RS in methanol R to obtain a solution having a concentration of about

TETRACYCLINE HYDROCHLORIDE TABLETS

1 mg/ml. Dilute 6.0 ml of the resulting solution to 50.0 ml with diluting solvent and mix. Test solution: Weigh accurately a quantity of the ointment equivalent to about 0.3 g of tetracycline hydrochloride and transfer to a 100 ml ground-glass-stoppered conical flask, add 20 ml cyclohexane R, shake vigorously. Add 35 ml of methanol R and sonicate for 20 minutes. Filter this solution into a 100 ml volumetric flask and rinse the conical flask with 40 ml of methanol R, filter the rinsing into the volumetric flask and dilute to volume with methanol R, mix. Dilute 2.0 ml of the resulting solution to 50.0 ml with diluting solvent and mix. Chromatographic system: Guard column: A column (3 cm × 4.6 mm) packed with stationary phase B (10 µm). Analytical column: A column (25 cm × 4.6 mm) packed with stationary phase B (5 µm to 10 µm). Detector: A spectrophotometer set at 280 nm. Flow rate: 2 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution, the relative standard deviation for replicate injections is not more than 2.0%. Inject sepatately the reference solution and the test solution. Calculate the content of tetracycline hydrochloride, C22H24N2O8,HCl, in the ointment using the areas for the principal peaks in the chromatograms obtained with the test solution and reference solution, and the declared contents of C22H24N2O8,HCl in tetracycline hydrochloride RS.

Storage Store at a cool place, protected from light. Action and use Antibacterial. Usual strength 1%. TETRACYCLINE HYDROCHLORIDE TABLETS Tabellae Tetracyclini hydrochloridi Tetracycline hydrochloride tablets contain tetracycline hydrochloride. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of tetracycline hydrochloride, C22H24N2O8,HCl, 95.0% to 105.0% of the stated amount. Identification In the Assay, the principal peak in the chromatogram obtained with the test solution has the same retention time as the principal peak in the chromatogram obtained with the reference solution. 953

VP V

THEOPHYLLINE

Loss on drying Not more than 4.0% (Appendix 9.6). (0.100 g of the powdered tablets, at 60 °C at a pressure not exceeding 5 mmHg for 3 hours). Dissolution (Appendix 11.4) Apparatus: Paddle. The distance between the paddle and the vessel bottom is (45 ± 5) mm. Medium: 900 ml of water. Rotation speed: 75 rpm. Time: 60 min. Procedure: After the specified time, withdraw a sample of the medium and filter, discarding the first 20 ml of the filtrate. Measure the absorbance of the filtrate, suitably diluted with the dissolution medium if necessary, at the maximum at 276 nm (Appendix 4.1). At the same time, measure the absorbance of a solution of tetracycline hydrochloride RS having the equivalent concentration in the same medium. From the obtained absorbances and the declared content of C22H24N2O8,HCl in tetracycline hydrochloride RS, calculate the content of C22H24N2O8,HCl dissolved. Tolerance: Not less than 80% (Q) of the stated amount of C22H24N2O8,HCl is dissolved in 60 min. Limit of 4-epianhydrotetracycline Not more than 3.0%. Examine by liquid chromatography (Appendix 5.3). Mobile phase, Diluting solvent, Chromatographic system as described in the Assay. Reference solution: Dissolve an acccurately weighed quantity of 4-epianhydrotetracycline hydrochloride RS in diluting solvent to obtain a known concentration of about 15 µg/ml. Procedure: Chromatograph the reference solution and compare with the chromatogram of the test solution obtained in the Assay. Calculate the percentage of 4-epianhydrotetracycline in tablets using the areas for the 4-epianhydrotetracycline peaks in the chromatograms obtained with the test solution and reference solution, and the declared contents of 4-epianhydrotetracycline hydrochloride RS. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: 0.1 M ammonium oxalate solution dimethylformamide - 0.2 M diammonium hydrogen phosphate solution (68 : 27 : 5), adjust the pH of the mixture to between 7.6 and 7.7 with 3 M ammonia solution R or 3 M phosphoric acid R. Diluting solvent: 0.1 M ammonium oxalate solution dimethylformamide (68 : 27). Reference solution: Dissolve an acccurately weighed quantity of tetracycline hydrochloride RS in diluting solvent and dilute quantitatively with diluting solvent to obtain a known concentration of about 0.5 mg/ml. Test solution: Weigh 20 tablets, calculate the average mass 954

and finely powder. Transfer an accurately weighed portion of the powder, equivalent to about 50 mg of tetracycline hydrochloride, to a 100 ml volumetric flask. Add about 50 ml of diluting solvent, mix with the aid of ultrasound for about 5 minutes. Allow to cool and dilute with diluting solvent to volume, mix and filter. Resolution solution: Prepare a solution in diluting solvent containing about 100 µg of tetracycline hydrochloride RS and 25 µg of 4-epianhydrotetracycline hydrochloride RS per ml. Chromatographic system: Guard column: A column (3 cm × 4.6 mm) packed with stationary phase B (10 µm). Analytical column: A column (25 cm × 4.6 mm) packed with stationary phase B (5 µm to 10 µm). Detector: A spectrophotometer set at 280 nm. Flow rate: 2 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the resolution solution, the relative retention times are about 0.9 for 4-epianhydrotetracycline and 1.0 for tetracycline, and the resolution between the peaks due to 4-epianhydrotetracycline and tetracycline is not less than 1.2. Inject the reference solution, the relative standard deviation for replicate injections is not more than 2.0%. Inject sepatately the reference solution and the test solution. Calculate the content of tetracycline hydrochloride, C22H24N2O8,HCl, in the tablets using the areas for the principal peaks in the chromatograms obtained with the test solution and reference solution, and the declared contents of C22H24N2O8,HCl in tetracycline hydrochloride RS.

Storage Store in a well-closed container or blisters, in a cool and dry place, protected from light. Action and use Antibacterial. Usual strength 0.125 g (125,000 IU); 0.25 g (250,000 IU). THEOPHYLLINE

Theophyllinum

C7H8N4O2  

M. 180.2

C7H8N4O2,H2O  

M. 198.2

THEOPHYLLINE

VP V

Theophylline is 1,3-dimethyl-3,7-dihydro-1H-purine-2,6dione in anhydrous form or hydrate form. It contains not less than 99.0% and not more than 101.0% of C7H8N4O2, calculated with reference to the dried substance (for anhydrous form) and to the anhydrous substance (for hydrate form).

Characters White or almost white, crystalline powder. Slightly soluble in water, sparingly soluble in ethanol (96%). It dissolves in solutions of alkali hydroxides, in ammonia and in mineral acids. Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D, E. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined (for hydrate form, dry the substance to be examined at 100 °C to 105 °C before use) is concordant with the spectrum of theophylline RS. B. Melting point (Appendix 6.7): 270 °C to 274 °C, determined after drying at 100 °C to 105 °C. C. Heat 10 mg with 1.0 ml of a 36% solution of potassium hydroxide R in a water-bath at 90 °C for 3 min, then add 1.0 ml of diazotised sulfanilic acid solution R. A red colour slowly develops. Carry out a blank test. D. It complies with the test for Loss on drying (for anhydrous form) or Water (for hydrate form). E. It gives the reaction of xanthines (Appendix 8.1). Appearance of solution Solution S: Dissolve 0.5 g of the substance to be examined with heating in carbon dioxide-free water R, cool and dilute to 75 ml with the same solvent. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). Acidity Add 0.1 ml of methyl red solution R to 50 ml of solution S, the solution is red. Not more than 1.0 ml of 0.01 N sodium hydroxide VS is required to change the colour of the indicator to yellow. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile for chromatography - solution A (7 : 93). Solution A: A 0.136% solution of sodium acetate R containing 5.0 ml/L of glacial acetic acid R. Test solution: Dissolve 40.0 mg of the substance to be examined in the mobile phase and dilute to 20.0 ml with the mobile phase. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase.

Reference solution (2): Dissolve 10 mg of theobromine R in the mobile phase, add 5 ml of the test solution and dilute to 100 ml with the mobile phase. Dilute 5 ml of this solution to 50 ml with the mobile phase. Chromatographic system: A column (25 cm × 4 mm) packed with stationary phase C (7 µm). Detector: A spectrophotometer set at 272 nm. Flow rate: 2.0 ml/min. Volume of injection: 20 µl. Procedure: The run time is 3.5 times the retention time of theophylline. Relative retention with reference to theophylline (retention time = about 6 min): Impurity C = about 0.3; impurity B = about 0.4; impurity D = about 0.5; impurity A = about 2.5. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to theobromine and theophylline is at least 2.0. Limits: Impurities A, B, C, D: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%). Total peak area of all impurities is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A: 1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione (caffeine). Impurity B: 3-methyl-3,7-dihydro-1H-purine-2,6-dione. Impurity C: N-(6-amino-1,3-dimethyl-2,4-dioxo-1,2,3,4tetrahydropyrimidin-5-yl)formamide. Impurity D: N-methyl-5-(methylamino)-1H-imidazole-4carboxamide (theophyllidine). Impurity E: 1,3-dimethyl-7,9-dihydro-1H-purine-2,6,8(3H)trione. Impurity F: 7-(2-hydroxyethyl)-1,3-dimethyl-3,7-dihydro-1Hpurine-2,6-dione (etofylline).

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying (for anhydrous form) Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Water (for hydrate form) 8.0% to 9.5% (Appendix 10.3). Determined on 0.20 g. 955

VP V

THEOPHYLLINE TABLETS

Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.150 g (for anhydrous form) or 0.160 g (for hydrate form) of the substance to be examined in 100 ml of water, add 20 ml of 0.1 N silver nitrate VS and shake. Add 1 ml of bromothymol blue solution R1. Titrate with 0.1 N sodium hydroxide VS. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 18.02 mg of C7H8N4O2. Storage Store in an airtight container. Action and use Non-selective phosphodiesterase inhibitor (xanthine); treatment of reversible airways obstruction. Preparation Tablets. THEOPHYLLINE TABLETS Tabellae Theophyllini Theophylline tablets contain theophylline. The tablets comply with the requirements stated under "Tablets" (Appendix 1.20) and with the following requirements.

Content of theophylline, C7H8N4O2, 94.0% to 106.0% of the stated amount. Identification A. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of the theophylline peak in the chromatogram obtained with the reference solution. B. Dissolve a quantity of the powdered tablets containing 0.2 g of theophylline with 10 ml of a mixture of 60 volumes of chloroform R and 40 volumes of methanol R, filter, evaporate the filtrate to dryness. The residue gives the reaction of xanthines (Appendix 8.1). Dissolution (Appendix 11.4). Apparatus: Paddle. Medium: 900 ml of water. Rotation speed: 50 rpm. Time: 45 min. Procedure: After the specified time, withdraw a suitable volume of the medium, filter and discard the first 20 ml of the filtrate. Dilute the filtrate with water if necessary, measure the absorbance of the resulting solution at the maximum wavelength at 272 nm (Appendix 4.1) in a 1-cm cell, using water as a blank. In comparison with a 956

solution of theophylline RS having the same concentration with the test solution in water. Calculate the content of theophylline dissolved using the absorbances measured and the declared content of C7H8N4O2 in theophylline RS. Tolerance: Not less than 80% (Q) of the stated amount of theophylline, C7H8N4O2, is dissolved in 45 min.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Water - methanol (75 : 25). Test solution: Weigh 20 tablets, calculate the average mass and finely powder. Weigh accurately a quantity of the powder containing 100 mg of theophylline, transfer into a 100-ml volumetric flask, add 1 ml of methanol R, mix and add 50 ml of water. Sonicate for 10 min to dissolve, add sufficient water to volume, mix, filter. Dilute 5.0 ml of the filtrate to 50.0 ml with water, mix. Reference solution: A 0.01% solution of theophylline RS in water. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (10 µm). Detector: A spectrophotometer set at 270 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution. The symmetry factor of the theophylline peak is not more than 2 and the relative standard deviation for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of theophylline, C7H8N4O2, in tablets using the areas of the principal peaks in the chromatograms obtained with the test solution, the reference solution and the declared content of C7H8N4O2 in theophylline RS. Storage Store in a well-closed container. Action and use Xanthine bronchodilator. Usual strength 100 mg. THIAMINE HYDROCHLORIDE Thiamini hydrochloridum

C12H17ClN4OS,HCl  

M. 337.3

THIAMINE HYDROCHLORIDE

VP V

Thiamine hydrochloride is 3-[(4-amino-2methylpyrimidin-5-yl)methyl]-5-(2-hydroxyethyl)-4methylthiazolium chloride hydrochloride. It contains not less than 98.5% and not more than 101.0% of C12H17ClN4OS,HCl, calculated with reference to the anhydrous substance.

Characters White or almost white, crystalline powder or colourless crystals. Freely soluble in water, soluble in glycerol, slightly soluble in ethanol (96%). Identification Apply one of the two following identifications: First identification: A, C. Second identification: B, C. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of thiamine hydrochloride RS. If the spectra obtained show differences, dissolve the substance to be examined and the reference substance separately in water, evaporate to dryness and record new spectra using the residues. B. Dissolve about 20 mg in 10 ml of water, add 1 ml of 2 M acetic acid R and 1.6 ml of 1 M sodium hydroxide R, heat on a water-bath for 30 min and allow to cool. Add 5 ml of 2 M sodium hydroxide R, 10 ml of a 5% solution of potassium ferricyanide R and 10 ml of butanol R and shake vigorously for 2 min. The upper alcoholic layer shows an intense light-blue fluorescence, especially in ultraviolet light at 365 nm. Repeat the test using 0.9 ml of 1 M sodium hydroxide R and 0.1 g of anhydrous sodium sulfite R instead of 1.6 ml of 1 M sodium hydroxide R. Practically no fluorescence is seen. C. It gives reaction (A) of chlorides (Appendix 8.1). Appearance of solution Solution S: Dissolve 2.5 g of the substance to be examined in carbon dioxide-free water R and dilute to 25 ml with the same solvent. Dilute 2.5 ml of solution S to 5 ml with water. The solution is clear (Appendix 9.2) and not more intensely coloured than reference solution Y7 or GY7 (Appendix 9.3, method 2). pH 2.7 to 3.3 (Appendix 6.2). Dilute 2.5 ml of solution S to 10 ml with water. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: A 0.3764% solution of sodium hexanesulfonate R adjusted to pH 3.1 with phosphoric acid R. Mobile phase B: Methanol R2. Test solution: Dissolve 0.35 g of the substance to be examined in 15.0 ml of a 5% v/v solution of glacial acetic acid R and dilute to 100.0 ml with water. Reference solution (1): Dissolve the contents of a vial of thiamine for system suitability RS (containing impurities

A, B and C) in 1.0 ml of a 0.75% v/v solution of glacial acetic acid R. Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with water. Dilute 1.0 ml of this solution to 10.0 ml with water. Chromatographic system: A column (25 cm × 4.0 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (5 µm). Column temperature: 45 °C. Detector: A spectrophotometer set at 248 nm. Flow rate: 1.0 ml/min. Volume of injection: 25 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0-2

90

10

2 - 27

90 → 70

10 → 30

27 - 35

70 → 50

30 → 50

35 - 42

50

50

Identification of impurities: Use the chromatogram supplied with thiamine for peak identification RS and the chromatogram obtained with reference solution (1) to identify the peaks due to impurities A, B, and C. Relative retention with reference to thiamine (retention time = about 30 min): Impurity A = about 0.3; impurity B = about 0.9; impurity C = about 1.2. System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to impurity B and thiamine is at least 3.0 and the resolution between the peaks due to thiamine and impurity C is at least 2.0. Limits: In the chromatogram obtained with the test solution: Impurity B: The area of the peak due to impurity B is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.3%). Impurities A, C: For each impurity, the area is not more than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.15%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.10%). Total peak area of all impurities is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.5%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Note: Impurity A: 3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-4methyl-5-[2-(sulfonatooxy)ethyl]thiazolium (thiamine sulfate ester).

957

VP V

THIAMINE HYDROCHLORIDE INJECTION Impurity B: 3-[(4-aminopyrimidin-5-yl)methyl]-5-(2hydroxyethyl)-4-methylthiazolium (desmethylthiamine). Impurity C: 3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-5-(2chloroethyl)-4-methylthiazolium (chlorothiamine). Impurity D: 3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-5-(2hydroxyethyl)-4-methylthiazol-2(3H)-one (oxothiamine). Impurity E: 3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-5-(2hydroxyethyl)-4-methylthiazol-2(3H)-thione (thioxothiamine). Impurity F: 3-[(4-amino-2-ethylpyrimidin-5-yl)methyl]-5-(2hydroxyethyl)-4-methylthiazolium (ethylthiamine). ImpurityG:5-[2-(acetyloxy)ethyl]-3-[(4-amino-2-methylpyrimidin5-yl)methyl]-4-methylthiazolium (acetylthiamine). Impurity H: (3RS)-3-[[[(4-amino-2-methylpyrimidin-5-yl) methyl]thiocarbamoyl]sulfanyl]-4-oxopentyl acetate (ketodithio carbamate).

THIAMINE HYDROCHLORIDE INJECTION Injectio Thiamini hydrochloridi Vitamin B1 injection

Sulfates Not more than 0.03% (Appendix 9.4.14). Dilute 5 ml of solution S to 15 ml with distilled water.

Identification A. To a volume of the injection containing about 20 mg of thiamine hydrochloride add water to produce 10 ml. Carry out the procedure described in identification B in monograph for Thiamine hydrochloride, beginning at the words “add 1 ml of 2 M acetic acid R… ”. B. In the Assay, the principal peak in the chromatogram obtained with test solution has the same retention time as the thiamine peak in the chromatogram obtained with reference solution. C. It gives reaction A of chlorides (Appendix 8.1).

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 12 ml of solution S complies with limit test for heavy metals, method 1. Prepare the standard using lead standard solution (2 ppm Pb) R. Water Not more than 5.0% (Appendix 10.3). Determined on 0.400 g. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.110 g of the substance to be examined in 5 ml of anhydrous formic acid R and add 50 ml of acetic anhydride R. Titrate immediately with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2) and carrying out the titration within 2 min. Carry out a blank titration in the same manner. 1 ml of 0.1 N perchloric acid VS is equivalent to 16.86 mg of C12H17ClN4OS,HCl. Storage In a non-metallic container, protected from light. Action and use Vitamin B group. Preparations Injection, tablets.

958

Thiamine hydrochloride injection is a sterile solution of thiamine hydrochloride in water for injections. The injection complies with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Content of thiamine hydrochloride, C12H17ClN4OS,HCl, 95.0% to 110.0% of the stated amount. Characters A clear, colourless solution.

pH 2.5 to 4.0 (Appendix 6.2). Bacterial endotoxins Not more than 3.5 EU/mg thiamine hydrochloride (Appendix 13.2). Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 1 g of sodium heptane sulphonate R in a mixture of 180 ml of methanol R and 10 ml of triethylamine R, dilute to 1000 ml with water and adjust the pH to 3.2 with phosphoric acid R. Reference solution: A solution containing 0.05 mg/ml of thiamin hydrochloride RS in 0.005 M hydrochloric acid R. Test solution: To a volume of the injection, exactly measured, containing about 100 mg of thiamine hydrochloride add 0.1 M hydrochloric acid R to produce 100 ml and mix. Dilute 5.0 ml of the solution to 100.0 ml with water. Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 244 nm. Flow rate: 2.0 ml/min. Volume of injection: 20 µl. Procedure: Inject the reference solution and the test solution. Calculate the content of thiamine hydrochloride, C12H17ClN4OS,HCl, using the areas or heights of the principal peaks in the chromatograms obtained with the

THIAMINE NITRATE

VP V

reference solution and test solution and the declared contents of C12H17ClN4OS,HCl in thiamine hydrochloride RS.

Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution Y7 (Appendix 9.3, method 2).

Storage Store at a cool dry place, protected from direct light.

pH The pH of solution S is 6.8 to 7.6 (Appendix 6.2).

Action and use Vitamin.

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: A 0.3764% solution of sodium hexanesulfonate R adjusted to pH 3.1 with phosphoric acid R. Mobile phase B: Methanol for HPLC. Test solution: Dissolve 0.35 g of the substance to be examined in 15.0 ml of a 5% v/v solution of glacial acetic acid R and dilute to 100.0 ml with water. Reference solution (1): Dissolve the contents of a vial of thiamine for system suitability RS (containing impurities A, B and C) in 1.0 ml of a 0.75% v/v solution of glacial acetic acid R. Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with water. Dilute 1.0 ml of this solution to 10.0 ml with water. Chromatographic system: A column (25 cm × 4.0 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (5 µm). Column temperature: 45 °C. Detector: A spectrophotometer set at 248 nm. Flow rate: 1.0 ml/min. Volume of injection: 25 µl. Procedure: Carry out a linear gradient elution using the following conditions:

Usual strength 2.5%. THIAMINE NITRATE Thiamini mononitras

C12H17N5O4S M.327.4 Thiamine nitrate is 3-[(4-amino-2-methylpyrimidin-5-yl) methyl]-5-(2-hydroxyethyl)-4-methylthiazolium nitrate. It contains not less than 98.0% and not more than 101.0% of C12H17N5O4S, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder or small, colourless crystals. Sparingly soluble in water, freely soluble in boiling water, slightly soluble in ethanol (96%) and in methanol. Identification Apply one of the two following identifications: First identification: A, C. Second identification: B, C. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of thiamine nitrate RS. B. Dissolve about 20 mg of the substance to be examined in 10 ml of water, add 1 ml of 2 M acetic acid R and 1.6 ml of 1 M sodium hydroxide R, heat on a water-bath for 30 min and allow to cool. Add 5 ml of 2 M sodium hydroxide solution R, 10 ml of a 5% solution of potassium ferricyanide R and 10 ml of n-butanol R and shake vigorously for 2 min The upper butanol layer shows an intense light-blue fluorescence, especially in ultraviolet light at 365 nm. Repeat the test using 0.9 ml of 1 M sodium hydroxide R and 0.2 g of sodium sulfite R instead of 1.6 ml of 1 M sodium hydroxide R. Practically no fluorescence is seen. C. About 5 mg gives the reaction of nitrates (Appendix 8.1). Appearance of solution Solution S: Dissolve 1.0 g in carbon dioxide-free water R, and dilute to 50 ml with the same solvent.

Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0-2

90

10

2 - 27

90 → 70

10 → 30

27 - 35

70 → 50

30 → 50

35 - 42

50

50

Identification of impurities: Use the chromatogram supplied with thiamine for system suitability RS and the chromatogram obtained with reference solution (1) to identify the peaks due to impurities A, B and C. Relative retention with reference to thiamine (retention time = about 30 min): impurity A = about 0.3; impurity B = about 0.9; impurity C = about 1.2. System suitability: In the chromatogram obtained with reference solution (1), the resolution between the peaks due to impurity B and to thiamine is at least 3.0 and the resolution between the peaks due to thiamine and to impurity C is at least 2.0. Limits: In the chromatogram obtained with the test solution: Impurity B: The area of the peak due to impurity B is not more than 6 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.6%). Impurity C: The area of the peak due to impurity C is not 959

VP V

THIAMINE TABLETS

more than 4 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.4%). Impurity A: The area of the peak due to impurity A is not more than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.15%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.10%). The sum of the peak areas of all impurities is not more than 10 times the area of the principal peak in the chromatogram obtained with reference solution (2) (1.0%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%). Note: Impurity A: 3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-4methyl-5-[2-(sulphonatooxy) ethyl]thiazolium (thiamine sulfate ester). Impurity B: 3-[(4-aminopyrimidin-5-yl)methyl]-5-(2-hydroxyethyl) -4-methylthiazolium (desmethylthiamine). Impurity C: 3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-5-(2chloroethyl)-4-methylthiazolium (chlorothiamine). Impurity E: 3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-5-(2hydroxyethyl)-4-methylthiazol-2(3H)-thione (thioxothiamine).

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with the limit test for heavy metals, method 4. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Loss on drying Not more than 1.0% (Appendix 9.6). (1.000 g; 100 °C to 105 °C). Assay Dissolve 0.140 g of the substance to be examined in 5 ml of anhydrous formic acid R and add 50 ml of acetic anhydride R. Titrate immediately with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2) and carrying out the titration within 2 min. Carry out a blank titration. 1 ml of 0.1 N perchloric acid VS is equivalent to 16.37 mg of C12H17N5O4S. Storage Store in an airtight non-metallic container, protected from light. Action and use Vitamin B1. Preparations Tablets; injection. 960

THIAMINE TABLETS Tabellae Thiamini Vitamin B1 Tablets Thiamine tablets contain thiamine hydrochloride or thiamine nitrate. They may be film coated. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of thiamine hydrochloride, C12H17ClN4OS,HCl or thiamine nitrate, C12H17N5O4S, 90.0% to 110.0% of the stated amount. Identification To a quantity of the powdered tablets equivalent to about 50 mg vitamin B1 add 25 ml of water, shake and filter (solution A). A. To 10 ml of solution A, proceed as directed under identification B in the monograph for Thiamine hydrochlorid, beginning with “add 1 ml of 2 M acetic acid R…”. B. In the Assay, the principal peak in the chromatogram obtained with test solution has the same retention time as the thiamine hydrochloride peak (or thiamine nitrate peak) in the chromatogram obtained with the reference solution. C. Solution A yields reaction (A) of chlorides (Appendix 8.1) or solution A yields reaction (A) of nitrates (Appendix 8.1). Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 1 g of sodium heptane sulfonate R in a mixture of 180 ml of methanol R and 10 ml of triethylamine R, dilute to 1000 ml with water and adjust the pH to 3.2 with phosphoric acid R. Reference solution: A solution contains 0.05 mg/ml of thiamin hydrochloride RS or thiamine nitrate RS in 0.005 M hydrochloric acid R. Test solution: Weigh 20 tablets, calculate the average mass, finely powder. To a quantity of the powder accurately weighed equivalent to about 100 mg of thiamine hydrochloride or thiamine nitrate add 70 ml of 0.005 M hydrochloric acid R and sonicate for 10 min. Dilute with 0.005 M hydrochloric acid R to produce 100.0 ml. Mix and filter, discarding the first portion of the filtrate. Dilute 5.0 ml of the filtrate to 100.0 ml with 0.005 M hydrochloric acid R. Chromatographic system: A column (10 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 244 nm. Flow rate: 2.0 ml/min. Volum of injection: 20 µl. Procedure: Inject the reference solution and the test solution. Calculate the content of thiamine hydrochloride, C12H17ClN4OS,HCl, or thiamine nitrate C12H17N5O4S,

VP V

using the areas (or heights) of the principal peaks obtained from the reference solution and test solution and the declared contents of C12H17ClN4OS,HCl in thiamine hydrochloride RS or C12H17N5O4S in thiamine nitrate RS.

Storage Store at a cool place, protected from light. Action and use Vitamin. Usual strength 10 mg. VITAMIN B1, B­6 AND B12 TABLETS Tabellae Vitamini B1, B­6 et B12 Vitamin B1, B6 and B12 tablets contain thiamine hydrochloride (or thiamine nitrate), pyridoxine hydrochloride and cyanocobalamin. They may be coated. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of thiamine hydrochloride, C12H17ClN4OS,HCl or thiamine nitrate, C12H17N5O4S, 90.0% to 120.0% of the stated amount. Content of pyridoxine hydrochloride, C8H11NO3,HCl, 90.0% to 120.0% of the stated amount. Content of cyanocobalamin, C63H88CoN14O14P, 90.0% to 150.0% of the stated amount. Identification A. In the Assay for vitamin B1 and B­6, the retention times of the two principal peaks in the chromatogram obtained with the test solution are the same as those of the peaks due to thiamine and pyridoxine in the chromatogram obtained with the reference solution. B. In the Assay for vitamin B12, the retention time of the principal peak in the chromatogram obtained with the test solution is the same as that of the peak due to cyanocobalamin in the chromatogram obtained with the reference solution. Assay Assay for vitamin B1 and B­6 Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 1.40 g of sodium hexanesulfonate R in 1000 ml of a mixture of water - methanol - glacial acetic acid (73 : 27: 1). Make adjustements if necessary. Solvent mixture: Water - acetonitrile - glacial acetic acid (94 : 5 : 1). Reference solution: A solution contains 0.05 mg/ml each of thiamine hydrochloride RS or thiamine nitrate RS and pyridoxine hydrochloride RS in solvent mixture.

VITAMIN B1, B­6 AND B12 TABLETS

Test solution: Remove the coating of 20 tablets. Weigh, calculate the average mass and finely powder. In a 100 ml volumetric flask, weigh accurately a quantity of the powdered tablets equivalent to about 50 mg of pyridoxine hydrochloride, add 70 ml of the solvent mixture, shake thoroughly for 15 minutes, add the solvent mixture to volume and mix. Filter, discard the first portion of the filtrate. Dilute 5.0 ml of the filtrate to 50.0 ml with the solvent mixture. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (10 µm). Detector: A spectrophotometer set at 280 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution, the test is not valid unless the relative standard deviation of the area of the principal peaks for six replicate injections of the reference solution is at most 3.0%. Inject separately the test solution and the reference solution. Calculate the content of thiamine hydrochloride, C12H17ClN4OS,HCl or thiamine nitrate, C12H17N5O4S and pyridoxine hydrochloride, C8H11NO3,HCl, in the tablets using the areas (or heights) of thiamine and pyridoxine peaks in the chromatograms obtained with the test solution and reference solution and the concentration of C12H17ClN4OS,HCl (or C12H17N5O4S) and C8H11NO3,HCl in the reference solution.

Assay for vitamin B12 Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol - water (35 : 65). Make adjustements if necessary. Reference solution: A solution containing 10 µg/ml of cyanocobalamin RS. Test solution: Remove the coating of 20 tablets. Weigh, calculate the average mass and powder finely. In a 50 ml volumetric flask, weigh accurately a quantity of the powdered tablets equivalent to about 250 µg of cyanocobalamin, add 25.0 ml of water, shake thoroughly (or sonicate) for 15 min, filter (or centrifuge) and use the filtrate (or the clear surpernatant). Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 550 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution, the test is not valid unless the relative standard deviation of the area of the principal peak for six replicate injections of the reference solution is at most 3.0%. Inject separately the test solution and reference solution. 961

VP V

THIAMPHENICOL

Calculate the content of cyanocobalamin, C63H88CoN14O14P, in the tablets using the areas (or heights) of cyanocobalamin peak in the chromatograms obtained with the test solution and reference solution and the concentration of C63H88CoN14O14P in the reference solution.

Storage Store at a cool dry place, protected from light. Action and use Vitamin. Usual strength 125 mg of vitamin B1, 125 mg of vitamin B6, 125 µg of vitamin B12. THIAMPHENICOL Thiamphenicolum

Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 10 cm. Dry the plate in air. Examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution. C. To 50 mg of the substance to be examined in a porcelain crucible, add 0.5 g of anhydrous sodium carbonate R. Heat over an open flame for 10 min. Allow to cool. Dissolve the residue in 5 ml of 2 M nitric acid R and filter. Add 1 ml of water to 1 ml of the filtrate. The solution gives reaction (A) of chlorides (Appendix 8.1).

Acidity or alkalinity Shake 0.1 g with 20 ml of carbon dioxide-free water and add 0.1 ml of bromothymol blue solution R. Not more than 0.1 ml of 0.02 N hydrochloric acid VS or 0.02 N sodium hydroxide VS is required to change the colour of the indicator. Specific optical rotation -21° to -24°, calculated with reference to the dried substance (Appendix 6.4). Dissolve 1.25 g in dimethylformamide R and dilute to 25.0 ml with the same solvent.

C12H15Cl2NO5S  

M. 356.2

Thiamphenicol is 2,2-dichloro-N-[(1R,2R)-2-hydroxy1-(hydroxymethyl)-2-[4-(methylsulfonyl)phenyl]ethyl] acetamide. It contains not less than 98.0% and not more than 100.5% of C12H15Cl2NO5S, calculated with reference to the dried substance.

Characters Fine, white or yellowish-white, crystalline powder or crystals. Slightly soluble in water and in ethyl acetate, very soluble in dimethylacetamide, freely soluble in acetonitrile and in dimethylformamide, soluble in methanol, sparingly soluble in acetone and in anhydrous ethanol. A solution in anhydrous ethanol is dextrorotatory and a solution in dimethylformamide is laevorotatory. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of thiamphenicol RS. Dry the substance to be examined and the standard substance at 100 °C to 105 °C for 2 h; examine as halide discs of potassium bromide R. B. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel GF254. Mobile phase: Methanol - ethyl acetate (3 : 97). Test solution: Dissolve 0.1 g of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Reference solution: Dissolve 0.1 g of thiamphenicol RS in methanol R and dilute to 10 ml with the same solvent. 962

Melting point 163 °C to 167 °C (Appendix 6.7). Absorbance Test solution (1): Dissolve 20 mg of the substance to be examined in water, heating to about 40 °C, and dilute to 100.0 ml with the same solvent. Test solution (2): Dilute 5.0 ml of test solution (1) to 100.0 ml with water. The ultraviolet absorption spectrum (Appendix 4.1) of test solution (1), in the range between 240 nm and 300 nm, exhibits two absorption maxima at 266 nm and at 273 nm. Specific absorbances at the absorption maxima are 25 to 28 and 21.5 to 23.5, respectively. The ultraviolet absorption spectrum of test solution (2), in the range between 200 nm and 240 nm, exhibits an absorption maximum at 224 nm. Specific absorbance at the absorption maximum is 370 to 400. Chlorides Not more than 200 ppm (Appendix 9.4.5). Shake 0.5 g of the substance to be examined with 30 ml of water for 5 min and filter. Heavy metals Not more than 10 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 1 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 1.0% (Appendix 9.6). (1.000 g; 105 °C).

THIOPENTAL SODIUM AND SODIUM

VP V

Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 2.0 g. Assay Dissolve 0.300 g of the substance to be examined in 30 ml of ethanol (96%) R, add 20 ml of a 50% solution of potassium hydroxide R, mix and heat under a reflux condenser for 4 h. Cool, add 100 ml of water, neutralise with 2 M nitric acid R and add 5 ml of the same acid in excess. Titrate with 0.1 N silver nitrate VS, determining the end-point potentiometrically (Appendix 10.2), using a silver indicator electrode and a mercurous sulfate reference electrode or any other appropriate electrode. Carry out a blank test in the same manner. 1 ml of 0.1 N silver nitrate VS is equivalent to 17.81 mg of C12H15Cl2NO5S. Storage In an airtight container, protected from light. Action and use Antibacterial. THIOPENTAL SODIUM AND SODIUM CARBONATE Natrii thiopentalum et natrii Carbonas

C11H17N2NaO2S2  

M. 264.3

Thiopental sodium and sodium carbonate is mixture of sodium 5-ethyl-5-[(1RS)-1-methylbutyl]-4,6-dioxo-1,4,5,6tetrahydropyrimidine-2-thiolate and anhydrous sodium carbonate. It contains 84.0% to 87.0% of C11H18N2O2S and 10.2% to 11.2% of Na, calculated with reference to the dried substance.

Characters Yellowish-white, hygroscopic powder. Freely soluble in water, partly soluble in anhydrous ethanol. Identification Apply one of the two following identifications: First identification: A, B, E. Second identification: B, C, D, E. A. The infrared absorption spectrum (Appendix 4.2) of the residue obtained on identification test B is concordant with the spectrum of thiopental RS.

B. Acidify 10 ml of solution S (see Appearance of solution) with dilute hydrochloric acid R. An effervescence is produced. Shake with 20 ml of 1,1-dimethylethyl methyl ether R. Separate the ether layer, wash with 10 ml of water, dry over anhydrous sodium sulfate R and filter. Evaporate the filtrate to dryness and dry the residue at 100 °C to 105 °C. Determine the melting point (Appendix 6.7) of the residue. Mix equal parts of the residue and thiopental RS and determine the melting point of the mixture. The difference between the melting points (which are about 160 °C) is not greater than 2 °C. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Ammonia - ethanol (96%) - methylene chloride (5 : 15 : 80); use the lower layer. Test solution: Dissolve 0.1 g of the residue obtained in Identification test B in water and dilute to 100 ml with the same solvent. Reference solution: Dissolve 85 mg of thiopental RS in 10 ml of 2 M sodium hydroxide R and dilute to 100 ml with water. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of three quarters of the plate. Examine immediately in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. D. It gives the reaction of non-nitrogen substituted barbiturates (Appendix 8.1). E. It gives reaction of sodium (Appendix 8.1).

Appearance of solution Solution S: Dissolve 5.0 g in carbon dioxide-free water R and dilute to 50 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution GY3 (Appendix 9.3, method 2). Related substances Examine by liquid chromatography (Appendix 5.3). Prepare the solutions immediately before use. Mobile phase: Acetonitrile R1 - 1 g/L solution of phosphoric acid R (35 : 65). Test solution: Dissolve 20.0 mg of the substance to be examined in the mobile phase and dilute to 20.0 ml with the mobile phase. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 5.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (2): Dissolve 2 mg of thiopental for system suitability RS (containing impurities A, B, C and D) in the mobile phase and dilute to 2.0 ml with the mobile phase. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (5 µm). 963

VP V

TICARCILLIN SODIUM

Detector: A spectrophotometer set at 225 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: The run time is twice the retention time of thiopental. Identification of impurities: Use the chromatogram supplied with thiopental for system suitability RS (containing impurities A, B, C and D) and the chromatogram obtained with reference solution (2) to identify the peaks due to impurities A, B, C and D. Relative retention with reference to thiopental (retention time = about 20 min): impurity A = about 0.3; impurity B = about 0.4; impurity C = about 0.9; impurity D = about 1.3. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurities A and B is at least 1.5; the resolution between the peaks due to impurity C and thiopental is at least 1.5. Limits: Correction factor: for the calculation of content, multiply the peak area of impurity B by 1.5; Impurity C: The area of the peak due to impurity C is not more than 6 times the area of the principal peak in the chromatogram obtained with reference solution (1) (3.0%). Impurity B: The corrected area of the peak due to impurity B is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (1) (1.0%). Impurity D: The area of the peak due to impurity D is not more than 0.6 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.3%). Any other impurity: For each impurity, the area is not more than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). The sum of the peak areas of all impurities is not more than 10 times the area of the principal peak in the chromatogram obtained with reference solution (1) (5.0%). Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A: 5-[(1RS)-1-methylbutyl]-2-thioxo-2,3-dihydropyrimidine4,6(1H,5H)-dione. Impurity B: 5-ethyl-5-[(1RS)-1-methylbutyl]pyrimidine-2,4,6(1H,3H,5H)trione. ImpurityC:5-ethyl-5-(1-ethylpropyl)-2-thioxo-2,3-dihydropyrimidine4,6(1H,5H)-dione. ImpurityD:mixtureof(2RS,3RS)-2-(carbamothioylcarbamoyl)-2-ethyl3-methylhexanoic acid and (2RS,3SR)-2-(carbamothioylcarbamoyl)-2ethyl-3-methylhexanoic acid.

Chlorides Not more than 0.033% (Appendix 9.4.5). Add 35 ml of water and 10 ml of 2 M nitric acid R to 964

5 ml of solution S. Shake with 3 quantities, each of 25 ml, of 1,1-dimethylethyl methyl ether R and discard the upper layer. Eliminate the organic solvent from the lower layer by heating on a water-bath. 15 ml of the solution complies with the test for chlorides.

Loss on drying Not more than 2.5% (Appendix 9.6). (0.50 g; 100 °C; in vacuo; 4 h). Assay Sodium Dissolve 0.400 g of the substance to be examined in 30 ml of water. Add 0.1 ml of methyl red solution R and titrate with 0.1 N hydrochloric acid VS until a red colour is obtained. Boil gently for 2 min. Allow to cool and, if necessary, continue the titration with 0.1 N hydrochloric acid VS until the red colour is again obtained. 1 ml of 0.1 N hydrochloric acid VS is equivalent to 2.299 mg of Na. Thiopental Dissolve 0.150 g of the substance to be examined in 5 ml of water. Add 2 ml of 1 M sulfuric acid R and shake with 4 quantities, each of 10 ml, of chloroform R. Combine the chloroform layers, filter and evaporate the filtrate to dryness on a water-bath. Dissolve the residue in 30 ml of previously neutralised dimethylformamide R and add 0.1 ml of a 0.2% solution of thymol blue R in methanol R. Titrate immediately with 0.1 M lithium methoxide VS until a blue colour is obtained. Protect the solution from atmospheric carbon dioxide during the titration. 1 ml of 0.1 M lithium methoxide VS is equivalent to 24.23 mg of C11H18N2O2S. Storage In an airtight container, protected from light. Action and use Anaesthetic. Preparation Thiopental injection. TICARCILLIN SODIUM Ticarcillinum natricum

C15H14N2Na2O6S2  

M. 428.4

Ticarcillin sodium is disodium (2S,5R,6R)-6-[[(2RS)2-carboxylato-2-(thiophen-3-yl)acetyl]amino]-3,3-

TICARCILLIN SODIUM

VP V

dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2carboxylate. It contains not less than 89.0% and not more than 102.0% of C15H14N2Na2O6S2, calculated with reference to the anhydrous substance.

Characters White or slightly yellow, hygroscopic powder. Freely soluble in water, soluble in methanol. Identification Apply one of the two following identifications: First identification: A, D, E. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of ticarcillin monosodium RS. Test solution: Dissolve 50 mg of the substance to be examined in 1 ml of water, add 0.1 ml of a 25% solution of hydrochloric acid R, swirl and allow to stand in iced water for 10 min. Filter the precipitate and rinse with 2 ml of water. Dissolve in a mixture of water - acetone R (1 : 9). Evaporate the solvent almost to dryness, then dry in an oven at 60 °C for 30 min. Standard solution: Repeat the operations using ticarcillin monosodium RS. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: TLC silanised silica gel. Mobile phase: Acetone - a 15.4% solution of ammonium acetate R (10 : 90), adjusted to pH 5.0 with glacial acetic acid R. Test solution: Dissolve 25 mg of the substance to be examined in methanol R and dilute to 5 ml with the same solvent. Reference solution (1): Dissolve 25 mg of ticarcillin monosodium RS in methanol R and dilute to 5 ml with the same solvent. Reference solution (2): Dissolve 25 mg of carbenicillin sodium RS and 25 mg of ticarcillin monosodium RS in methanol R and dilute to 5 ml with the same solvent. Procedure: Apply separately to the plate 1 µl of each solution. Develop over a path of 12 cm. Dry the plate in a current of hot air and expose to iodine vapour. The principal spot in the chromatogram obtained with the test solution is similar in colour and size to the principal spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) shows two clearly separated spots. C. Place about 2 mg of the substance to be examined in a test-tube about 15 cm long and 15 mm in diameter. Moisten with 0.05 ml of water and add 2 ml of formaldehyde solution in sulfuric acid R. Mix the contents of the tube by swirling; the solution is brown. Place the test-tube in a water-bath for 1 min; a dark reddish-brown colour develops. D. It gives reaction of sodium (Appendix 8.1). E. It complies with the test for Specific optical rotation.

Appearance of solution Solution S: Dissolve 2.50 g in carbon dioxide-free water and dilute to 50 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution Y5 (Appendix 9.3, method 2). pH 5.5 to 7.5 (Appendix 6.2). Determined on solution S. Specific optical rotation +172° to +187°, calculated with reference to the anhydrous substance (Appendix 6.4). Dissolve 0.250 g of the substance to be examined in water and dilute to 25.0 ml with the same solvent. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: A 0.13% solution of ammonium phosphate R adjusted to pH 7.0 with phosphoric acid R. Mobile phase B: Mobile phase A - methanol (50 : 50). Test solution: Dissolve 25.0 mg of the substance to be examined in mobile phase A and dilute to 25.0 ml with mobile phase A. Reference solution (1): Dissolve 20.0 mg of decarboxyticarcillin RS (impurity A) in mobile phase A and dilute to 100.0 ml with mobile phase A. Dilute 5.0 ml of this solution to 50.0 ml with mobile phase A. Reference solution (2): Dilute 1 ml of the test solution to 50 ml with mobile phase A. Chromatographic system: A column (25 cm × 4 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 220 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 30

100 → 30

0 → 70

30 - 40

30

70

System suitability: In the chromatogram obtained with reference solution (2), the resolution between the 2 principal peaks (diastereoisomers) is at least 2.0. Inject the test solution and reference solution (1). Limits: In the chromatogram obtained with the test solution: Impurity A: The area of the peak due to impurity A is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (1) (4%). Any other impurity (except impurity A peak and principal peak): For each impurity, the area is not more than 1.25 times the area of the principal peak in the chromatogram obtained with reference solution (1) (2.5%). 965

VP V

TIMOLOL MALEATE

N,N-Dimethylaniline Not more than 20 ppm (Appendix 10.16, method 2).

TIMOLOL MALEATE Timololi maleas

2-Ethylhexanoic acid Not more than 0.5% (Appendix 10.17). Water Not more than 5.5% (Appendix 10.3). Determined on 0.150 g. Bacterial endotoxins Not more than 0.05 EU/mg (Appendix 13.2). If intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol - a 0.13% solution of ammonium phosphate R adjusted to pH 7.0 with phosphoric acid R (20 : 80). Test solution: Dissolve 50.0 mg of the substance to be examined in the mobile phase and dilute to 100.0 ml with the mobile phase. Dilute 10.0 ml of this solution to 50.0 ml with the mobile phase. Standard solution: Dissolve 50.0 mg of ticarcillin monosodium RS in the mobile phase and dilute to 100.0 ml with the mobile phase. Dilute 10.0 ml of this solution to 50.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 220 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the standard solution, the test is not valid unless the resolution between the 2 principal peaks is at least 2.5. The relative standard deviation for the 2 peaks due to ticarcillin after 6 injections is not more than 1.0%. Calculate the percentage content of ticarcillin sodium as the sum of the areas of the 2 peaks, multiplying the content of ticarcillin monosodium by 1.054. Storage In an airtight container, at a temperature of 2 °C to 8 °C. If the substance is sterile, store in a sterile, airtight, tamperproof container. Action and use Penicillin antibacterial. Preparation Tablets.

966

C13H24N4O3S,C4H4O4   

M. 432.5

Timolol maleate is (2S)-1-[(1,1-dimethylethyl)amino]-3[[4-(morpholin-4-yl)-1,2,5-thiadiazol-3- yl]oxy]propan-2ol (Z)-butenedioate. It contains not less than 98.5% and not more than 101.0% of C13H24N4O3S,C4H4O4, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder or colourless crystals. Soluble in water and in ethanol (96%). Melting point is about 199 °C, with decomposition. Identification Apply one of the two following identifications: First identification: A, B. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of timolol maleate RS. B. Dissolve 1.000 g in 1 M hydrochloric acid R and dilute to 10.0 ml with the same acid. Specific optical rotation (Appendix 6.4) of this solution is -6.2° to -5.7°. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Concentrated ammonia - methanol methylene chloride (1 : 20 : 80). Test solution: Dissolve 5 mg of the substance to be examined in methanol R and dilute to 5 ml with the same solvent. Reference solution: Dissolve 5 mg of timolol maleate RS in methanol R and dilute to 5 ml with the same solvent. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of two-thirds of the plate. Allow the plate to dry in air and expose to iodine vapour for 2 h. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. D. Triturate 0.1 g with a mixture of 1 ml of dilute sodium hydroxide solution R and 3 ml of water. Shake with 3 quantities, each of 5 ml, of ether R. To 0.1 ml of the aqueous layer add a solution containing 10 mg of resorcinol R in 3 ml of sulfuric acid R. Heat on a waterbath for 15 min; no violet-red colour develops. Neutralise the remainder of the aqueous layer with dilute sulfuric

TIMOLOL MALEATE

VP V

acid R and add 1 ml of bromine water R. Heat on a waterbath for 15 min, then heat to boiling and cool. To 0.2 ml of this solution add a solution containing 10 mg of resorcinol R in 3 ml of sulfuric acid R. Heat on a water-bath for 15 min; a violet-red colour develops. Add 0.2 ml of a 100 g/L solution of potassium bromide R and heat for 5 min on a water-bath; the colour becomes violet-blue.

Appearance of solution Solution S: Dissolve 0.5 g in carbon dioxide-free water R and dilute to 25 ml with the same solvent. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution B8 (Appendix 9.3, method 2) pH 3.8 to 4.3 (Appendix 6.2). Determined on solution S. Enantiomeric purity Examine by liquid chromatography (Appendix 5.3). Carry out the test protected from actinic light. Mobile phase: Diethylamine - 2-propanol - hexane (2 : 40 : 960). Solvent mixture: Methylene chloride - 2-propanol (10 : 30). Test solution: Dissolve 30.0 mg of the substance to be examined in the solvent mixture and dilute to 10.0 ml with the solvent mixture. Reference solution (1): Dissolve 30 mg of timolol maleate RS in the solvent mixture and dilute to 10 ml with the solvent mixture. Reference solution (2): Dissolve 3.0 mg of (R)-timolol RS (impurity A) in the solvent mixture and dilute to 10.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 10.0 ml with the solvent mixture. Reference solution (3): Dilute 1 ml of reference solution (1) to 100 ml with the solvent mixture. Mix 1 ml of this solution with 1 ml of reference solution (2). Reference solution (4): Dilute 1.0 ml of the test solution to 100.0 ml with the solvent mixture. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase cellulose  derivative of  silica gel  for chiral separation R (5 µm). Detector: A spectrophotometer set at 297 nm. Flow rate: 1 ml/min. Volume of injection: 5 µl. Elution order: Impurity A is eluted first. Procedure: System suitability: In the chromatogram obtained with reference solution (3), the resolution between the peaks due to impurity A and the (S)-enantiomer is at least 4.0. The retention times of the principal peaks due to the (S)enantiomer in the chromatograms obtained with the test solution and reference solution (1) are identical. Limit: Impurity A: The area of the peak due to impurity A is not more than the area of the principal peak in the chromatogram obtained with reference solution (4) (1.0%).

Note: Impurity A: (2R)-1-[(1,1-dimethylethyl)amino]-3-[[4-(morpholin4-yl)-1,2,5-thiadiazol-3- yl]oxy]propan-2-ol ((R)-timolol).

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Methanol - 4.32 g/l solution of sodium octanesulfonate R previously adjusted to pH 3.0 with glacial acetic acid R (1 : 1). Mobile phase B: Methanol R. Test solution: Dissolve 0.100 g of the substance to be examined in mobile phase A and dilute to 20 ml with mobile phase A. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with mobile phase A. Dilute 1.0 ml of this solution to 10.0 ml with mobile phase A. Reference solution (2): Dissolve the contents of a vial of timolol for system suitability RS (containing impurities B, C, D and F) in 1.0 ml of mobile phase A. Reference solution (3): Dissolve 2 mg of the substance to be examined and 20 mg of maleic acid R in 10 ml of acetonitrile R. Evaporate 1 ml of the solution to dryness under a stream of nitrogen R in an amber glass vial. Heat the open vial at 105 °C for 1 h. Dissolve the residue with 1.0 ml of mobile phase A. Chromatographic system: A column (15 cm × 3.9 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 295 nm. Flow rate: 1.2 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 10

97.5

2.5

10 - 11

97.5 → 70

2.5 → 30

11 - 14.5

70

30

Identification of impurities: Use the chromatogram supplied with timolol for system suitability RS and the chromatogram obtained with reference solution (2) to identify the peaks due to impurities B, C, D and F. Use the chromatogram obtained with reference solution (3) to identify the peak due to impurity E. Relative retention with reference to timolol (retention time = about 7.5 min): maleic acid = about 0.1; impurity D = about 0.3; impurity E = about 0.4; impurity B = about 0.7; impurity F = about 0.8; impurity C = about 2.1. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurity F and impurity B is at least 1.5. Limits: Correction factor: for the calculation of content, multiply the peak area of impurity D by 0.6. 967

VP V

TIMOLOL TABLETS

Impurities B, C, D, E, F: For each impurity, the area, corrected if necessary, is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). The sum of the peak areas of all impurities is not more than 4 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.4%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%) and disregard any peak due to maleic acid. Note: Impurity B: (2RS)-3-[(1,1-dimethylethyl)amino]-2-[[4-(morpholin4-yl)-1,2,5-thiadiazol-3- yl]oxy]propan-1-ol. Impurity C: (2RS)-N-(1,1-dimethylethyl)-2,3-bis[[4-(morpholin4-yl)-1,2,5-thiadiazol-3- yl]oxy]propan-1-amine. Impurity D: 4-(morpholin-4-yl)-1,2,5-thiadiazol-3-ol. Impurity E: (2Z)-4-[(1S)-1-[[(1,1-dimethylethyl)amino]methyl]2-[[4-(morpholin-4-yl)-1,2,5-thiadiazol-3-yl]oxy]ethoxy]-4oxobut-2-enoic acid. Impurity F: 4-(4-chloro-1,2,5-thiadiazol-3-yl)morpholine. Impurity G: 4-(morpholin-4-yl)-1,2,5-thiadiazol-3(2H)-one 1-oxide. Impurity H: 2-[(2RS)-3-[(1,1-dimethylethyl)amino]-2hydroxypropyl]-4-(morpholin-4-yl)-1,2, 5-thiadiazol-3(2H)-one. Impurity I: (2RS)-1-(ethylamino)-3-[[4-(morpholin-4-yl)-1,2,5thiadiazol-3-yl]oxy]propan-2-ol. Impurity J: 1,1’-[1,2,5-thiadiazol-3,4-diylbis(oxy)]bis[3-[(1,1dimethylethyl)amino]propan-2- ol].

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.350 g of the substance to be examined in 60 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 43.25 mg of C17H28N4O7S. Storage Protected from light. Action and use Beta-adrenoceptor antagonist. Preparations Eye drops, tablets.

968

TIMOLOL TABLETS Tabellae Timololi Timolol tablets contain timolol maleate. The tablets comply with the requirements stated under "Tablets" (Appendix 1.20) and with the following requirements.

Content of timolol maleate, C13H24N4O3S, C4H4O4, 90.0% to 110.0% of the stated amount. Identification Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Ammonia - methanol - chloroform (1 : 20 : 80). Test solution: Shake a quantity of the powdered tablets containing 30 mg of timolol maleate with 2 ml of 0.1 M hydrochloric acid R, add 5 ml of methanol R and continue shaking for about 20 min. Add sufficient  methanol  R to produce 50 ml, centrifuge and use the supernatant liquid. Reference solution: Dissolve, with shaking, 60 mg of timolol maleate RS in 4 ml of 0.1 M hydrochloric acid R and add sufficient methanol R to produce 100 ml. Procedure: Apply 10 µL of each solution. Develop over 3/4 of the plate. After removal of the plate, allow it to dry in air and examine under ultraviolet light (254 nm). The principal spot in the chromatogram obtained with the test solution corresponds in position and colour to that in the chromatogram obtained with the reference solution. Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 500 ml of 0.1 M hydrochloric acid R. Rotation speed: 100 rpm. Time: 20 min. Test solution: After the specified time, withdraw a sample of the medium and filter, discarding the first 20 ml of the filtrate. Reference solution: Dissolve timolol maleate RS in 0.1 M hydrochloric acid R to obtain a solution having concentration the same as that of the test solution. Procedure: Carry out the method for liquid chromatography (Appendix 5.3) with the chromatographic conditions described under Assay. Adjust the volume of injection if necessary. Tolerance: Not less than 80% (Q) of the stated amount of timolol maleat, C13H24N4O3S,C4H4O4, is dissolved in 20 min. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol - phosphate buffer solution pH 2.8 (2 : 3). Phosphate buffer solution pH 2.8: Dissolve 11.04 g of sodium dihydrogen phosphate R in 1000 ml of water, adjust pH to 2.8 with phosphoric acid R.

TINIDAZOLE

VP V

Reference solution: Weigh accurately 50 mg of timolol maleat RS, put into a 500 ml volumetric flask, add 50 ml of 0.05 M sodium dihydrogen phosphate R, sonicate until completely dissolve, add 100 ml of acetonitrile R, dilute to volume with water, mix well. Test solution: Weigh and finely powder 20 tablets. Weigh accurately a quantity of the powdered tablets containing 10 mg of timolol maleat, put into a 100 ml volumetric flask, add 10 ml of 0.05 M sodium dihydrogen phosphate R, sonicate for 5 min, add 20 ml of acetonitrile R, dilute to volume with water, mix well. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 295 nm. Flow rate: 1.8 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution for 6 times. The relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%; the symmetry factor of the principal peak is not more than 2.0. Inject the reference solution and the test solution. Calculate the content of C13H24N4O3S, C4H4O4 in the tablets using the peak areas of timolol maleat in the chromatograms obtained with the test solution and the reference solution and the declared content of C13H24N4O3S,C4H4O4 in timolol maleat RS.

Storage Storage in a close container in a dry place and protect from light. Action and use Beta-adrenoceptor antagonist. Strength use 5 mg; 10 mg; 20 mg.

Appearance of solution Dissolve 1.0 g in acetone R and dilute to 20 ml with the same solvent. The solution is clear (Appendix 9.2) and not more intensely coloured than reference solution Y5 (Appendix 9.3, method 2).

TINIDAZOLE Tinidazolum

C8H13N3O4S  

Identification Apply one of the two following identifications: First identification: A, C. Second identification: B, C, D, E. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of tinidazole RS. B. Dissolve 10.0 mg of the substance to be examined in methanol R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of this solution to 10.0 ml with methanol R. The ultraviolet absorptiom spectrum (Appendix 4.1) of the solution, in the range between 220 nm and 350 nm, exhibits absorption maximum at 310 nm. Specific absorbance at the absorption maximum is 340 to 360. C. Melting point: 125 °C to 128 °C (Appendix 6.7). D. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Heat at 110 °C for 1 h and allow to cool. Mobile phase: Butanol - ethyl acetate (25 : 75). Test solution: Dissolve 20 mg of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Reference solution: Dissolve 20 mg of tinidazole RS in methanol R and dilute to 10 ml with the same solvent. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of two thirds of the plate. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. E. To about 10 mg, add about 10 mg of zinc powder R, 0.3 ml of hydrochloric acid R and 1 ml of water. Heat in a water-bath for 5 min and cool. The solution gives the reaction of primary aromatic amines (Appendix 8.1).

M. 247.3

Tinidazole is 1-[2-(ethylsulfonyl)ethyl]-2-methyl-5-nitro1H-imidazole. It contains not less than 98.0% and not more than 101.0% of C8H13N3O4S, calculated with reference to the dried substance.

Characters Almost white or pale yellow, crystalline powder. Practically insoluble in water, soluble in acetone and in methylene chloride, sparingly soluble in methanol.

Related substances Examine by liquid chromatography (Appendix 5.3). Protect solutions from light. Mobile phase: Acetonitrile - methanol - water (10 : 20 : 70). Test solution: Dissolve 10.0 mg of the substance to be examined in 10.0 ml of methanol R and dilute to 100.0 ml with the mobile phase. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (2): Dissolve 5.0 mg of tinidazole impurity A RS and 5.0 mg of tinidazole impurity B RS 969

VP V

TINIDAZOLE TABLETS

in 10.0 ml of methanol R and dilute to 100.0 ml with the mobile phase. Dilute 2.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (3): Dilute 1.0 ml of reference solution (2) to 50.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 3.0 mm) packed with stationary phase B (5 µm). Detector: A spectrophotometer set at 320 nm. Flow rate: 0.5 ml/min. Volume of injection: 20 µl. Procedure: The run time is 1.5 times the retention time of tinidazole. Relative retention with reference to tinidazole (retention time = about 6 min): Impurity A = about 0.6; impurity B = about 0.7. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurities A and B is at least 2.0. Limits: Impurities A, B: For each impurity, the area is not more than the area of the corresponding peak in the chromatogram obtained with reference solution (3) (0.2%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). The sum of the peak areas of all impurities is not more than 4 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.4%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A: 2-methyl-5-nitro-1H-imidazole. Impurity B: 1-[2-(ethylsulfonyl)ethyl]-2-methyl-4-nitro-1Himidazole.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 4. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.150 g of the substance to be examined in 25 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 970

1 ml of 0.1 N perchloric acid VS is equivalent to 24.73 mg of C8H13N3O4S.

Storage Protected from light. Action and use Antiprotozoal; antibacterial. Preparation Tablets. TINIDAZOLE TABLETS Tabellae Tinidazoli Tinidazole tablets contain tinidazole. They are film coated. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of tinidazole, C8H13N3O4S, 93.0% to 107.0% of the stated amount. Identification A. Place a quantity of powdered tablets equivalent to about 0.1 g of tinidazole in a test tube and heat gently, an irritating odour of sulfur dioxide is produced which turns a strip of filter paper wetted with mercury (II) nitrate solution R black. B. Dissolve a quantity of powdered tablets equivalent to about 0.1 g of tinidazole in 5 ml of a 5% solution of sulfuric acid R, shake and filter. To the filtrate add 2 ml of saturated picric acid solution R, a yellow precipitate is produced. C. The ultraviolet absorption spectrum (Appendix 4.1) of the test solution obtained in the Assay exhibits two maxima at 317 nm and 229 nm, a minimum at 263 nm. Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 900 ml of 0.1 M hydrochloric acid R. Rotation speed: 100 rpm. Time: 30 min. Procedure: After the specified time, withdraw a sample of the medium and filter, discarding the first 20 ml of the filtrate. Pipet 2 ml of the filtrate and transfer it to a 100 ml volumetric flask, add water to volume and mix. Measure the absorbance of the resulting solution at 317 nm (Appendix 4.1). Calculate the content of tinidazole, C8H13N3O4S, dissolve from each tablet, taking 365 as the value of A(1%, 1 cm) at 317 nm. Tolerance: Not less than 80% (Q) of the stated amount of tinidazole, C8H13N3O4S is dissolved in 30 minutes. Assay Weigh 20 tablets (after removing the coating film, if necessary), calculate the average mass, finely powder. In a

VP V

200 volumeric flask, dissolve a quantity of the powdered tablets, accurately weighed, equivalent to about 50 mg of tinidazole in water by warming. Shake thoroughly for 10 minutes. Cool to room temperature, dilute to volume with water, mix well and filter through a dry filter paper. Discard the first 20 ml of the filtrate. Transfer 5.0 ml of the filtrate to a 100 volumeric flask, add water to volume and mix. Prepare a reference solution having a concentration of 0.0012% of tinidazole in water. Measure the absorbance of the test solution and the reference solution at 317 nm (Appendix 4.1) using a 1 cm cell and water as a blank. Calculate the content of tinidazole, C8H13N3O4S, using the absorbances of the test solution, the reference solution and the declared content of C8H13N3O4S in tinidazole RS.

Storage Store in a well-closed container, protected from light. Action and use Antiprotozoan. Usual strength 500 mg. TITANIUM DIOXIDE Titani dioxidum TiO2 M.79.9 Titanium dioxide contains not less than 98.0% and not more than 100.5% of TiO2.

Characters A white or almost white powder. Practically insoluble in water. It does not dissolve in dilute mineral acids but dissolves slowly in hot concentrated sulfuric acid. Identification Solution S1: Shake 20.0 g of the substance to be examined with 30 ml of hydrochloric acid R for 1 min. Add 100 ml of distilled water R and heat the mixture to boiling. Filter the hot mixture until a clear filtrate is obtained. Wash the filter with 60 ml of distilled water R and dilute the combined filtrate and washings to 200 ml with distilled water R. Solution S2: Mix 0.500 g of the substance to be examined with 5 g of anhydrous sodium sulfate R in a 300 ml longnecked combustion flask. Add 10 ml of water and mix. Add 10 ml of sulfuric acid R and boil vigorously until a clear solution is obtained. Cool, add slowly a cooled mixture of 30 ml of water and 10 ml of sulfuric acid R, cool again and dilute to 100.0 ml with water. A. When strongly heated, it becomes pale yellow; the colour disappears on cooling. B. To 5 ml of solution S2 add 0.1 ml of hydrogen peroxide solution (30%) R. An orange-red colour appears.

TITANIUM DIOXIDE

C. To 5 ml of solution S2 add 0.5 g of zinc R in granules. After 45 min, the mixture has a violet-blue colour.

Appearance of solution Solution S­ 2 is not more opalescent than reference suspension II (Appendix 9.2) and is colourless (Appendix 9.3, method 2). Acidity or alkalinity Shake 5.0 g of the substance to be examined with 50 ml of carbon dioxide-free water R for 5 min. Centrifuge or filter until a clear solution is obtained. To 10 ml of the solution add 0.1 ml of bromothymol blue solution R. Not more than 1.0 ml of 0.01 N hydrochloric acid VS or 0.01 N sodium hydroxide VS is required to change the colour of the indicator. Water-soluble substances Not more than 0.5%. To 10.0 g of the substance to be examined add a solution of 0.5 g of ammonium sulfate R in 150 ml of water, and boil for 5 min. Cool, dilute to 200 ml with water and filter until a clear solution is obtained. Evaporate 100 ml of the solution to dryness in a tared evaporating dish, and ignite at 600 °C to constant weight. The residue weighs not more than 25 mg. Antimony Not more than 0.01%. To 10 ml of solution S2 add 10 ml of hydrochloric acid R and 10 ml of water. Cool to 20 °C, if necessary, and add 0.15 ml of a 10% solution of sodium nitrite R. After 5 min, add 5 ml of a 1% solution of hydroxylamine hydrochloride R and 10 ml of a freshly prepared 0.01% solution of rhodamine B R. Mix thoroughly after each addition. Shake vigorously with 10.0 ml of toluene R for 1 min. Allow to separate and centrifuge for 2 min if necessary. Any pink colour in the toluene phase is not more intense than that in the toluene phase of a standard prepared at the same time in the same manner using a mixture of 5.0 ml of antimony standard solution (1 ppm Sb) R, 10 ml of hydrochloric acid R and 15 ml of a solution containing 0.5 g of anhydrous sodium sulfate R and 2 ml of sulfuric acid R instead of the mixture of 10 ml of solution S2, 10 ml of hydrochloric acid R and 10 ml of water. Arsenic Not more than 5 ppm (Appendix 9.4.2). Place 0.50 g of the substance to be examined in a 250 ml round-bottomed flask, fitted with a thermometer, a funnel with stopcock and a vapour-outlet tube connected to a flask containing 30 ml of water. Add 50 ml of water, 0.5 g of hydrazine sulfate R, 0.5 g of potassium bromide R and 20 g of sodium chloride R. Through the funnel, add dropwise 25 ml of sulfuric acid R, heat and maintain the temperature of the liquid at 110 °C to 115 °C for 20 min. Collect the vapour in the flask containing 30 ml of water. 971

VP V

TOBRAMYCIN

Dilute to 50 ml with water. 20 ml of the solution complies with limit test A for arsenic.

In which: m is the mass in grams of the substance to be examined used for the preparation of solution S­2.

Barium To 10 ml of solution S1 add 1 ml of dilute sulfuric acid R. After 30 min, any opalescence in the solution is not more intense than that in a mixture of 10 ml of solution S1 and 1 ml of distilled water R.

Storage Store in an airtight container, protected from light.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). Dilute 10 ml of solution S1 to 20 ml with water. 12 ml of the solution complies with the limit test for heavy metals, method 1. Prepare the standard using lead standard solution (1 ppm Pb) R. Iron Not more than 0.02%. To 8 ml of solution S2 add 4 ml of water. Mix and add 0.05 ml of bromine water R. Allow to stand for 5 min and remove the excess of bromine with a current of air. Add 3 ml of a 9.7% solution of potassium thiocyanate R. Any colour in the solution is not more intense than that in a standard prepared at the same time in the same manner using a mixture of 4 ml of iron standard solution (2 ppm Fe) R and 8 ml of sulfuric acid (20%) R. Assay To 300 g of zinc R in granules add 300 ml of a 2% solution of mercuric nitrate R and 2 ml of nitric acid R, shake for 10 min and wash with water. Pack the amalgamated zinc into a glass tube about 400 mm long and about 20 mm in diameter fitted with a tap and a filter plate. Pass through the column 100 ml of 1 M sulfuric acid R followed by 100 ml of water, making sure that the amalgam is always covered with liquid. Pass slowly at a rate of about 3 ml/min through the column a mixture of 100 ml of 1 M sulfuric acid R and 100 ml of water followed by 100 ml of water. Collect the eluate in a 500 ml conical flask containing 50.0 ml of a 15% solution of ferric ammonium sulfate R in a mixture of 1 volume of sulfuric acid R and 3 volumes of water. Add 0.1 ml of ferroin sulfate solution R and titrate immediately with 0.1 M ammonium and cerium nitrate VS until a greenish colour is obtained (n1 ml). Pass slowly at a rate of about 3 ml/min through the column a mixture of 50 ml of 1 M sulfuric acid R and 50 ml of water, followed by 20.0 ml of solution S2, a mixture of 50 ml of 1 M sulfuric acid R and 50 ml of water and finally 100 ml of water. Collect the eluate in a 500 ml conical flask containing 50.0 ml of a 15% solution of ferric ammonium sulfate R in a mixture of 1 volume of sulfuric acid R and 3 volumes of water. Rinse the lower end of the column with water, add 0.1 ml of ferroin sulfate solution R and titrate immediately with 0.1 M ammonium and cerium nitrate VS until a greenish colour is obtained (n2 ml). Calculate the percentage content of TiO2 from the expression: 3.99(n2 - n1)/m 972

Action and use Protective; pharmaceutical aid. TOBRAMYCIN Tobramycinum

C18H37N5O9 M.467.5 Tobramycin is 4-O-(3-amino-3-deoxy-α-D-glucopyranosyl)-2-deoxy-6-O-(2,6-diamino-2,3,6-trideoxyα-D-ribo-hexopyranosyl)-L-streptamine, produced by Streptomyces tenebrarius or obtained by any other means. The potency is not less than 900 µg of C18H37N5O9 per mg, calculated with reference to the anhydrous.

Characters White or almost white powder. Freely soluble in water, very slightly soluble in ethanol (96%). Identification A. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel G. Mobile phase: Methanol - ammonia - chloroform (60 : 30 : 25). Test solution: Dissolve 30 mg of the substance to be examined in water and dilute to 5 ml with the same solvent. Reference solution (1): Dissolve 30 mg of tobramycin RS in water and dilute to 5 ml with the same solvent. Reference solution (2): A mixture of equal volumes of the test solution and reference solution (1). Procedure: Apply separately to the plate 3 µl of each solution. Develop over a path of three quarters of the plate. Remove the plate from the chamber, allow the solvent to evaporate, and heat the plate at 105 °C for 15 min. Immediately locate the spots on the plate by spraying with a 1% solution of ninhydrin R in a mixture of 1-butanol R and pyridin R (100 : 1). Tobramycin appears as a pink spot, the principal spot in the chromatogram obtained with the test solution is similar in position to the principal spot in the chromatogram obtained with reference solution (1). The chromatogram obtained with reference solution (2) only shows 1 spot which is similar in position

VP V

to the principal spot in the chromatogram obtained with reference solution (1). B. In the test for Assay, the principal peak in the chromatogram obtained with the test solution is similar in retention time to the tobramycin peak in the chromatogram obtained with the standard solution.

pH 9.0 to 11.0 (Appendix 6.2). Dissolve 1.0 g of the substance to be examined in 10 ml of carbon dioxide-free water R. Related substances Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel G. Mobile phase: A 29.2% solution of sodium chloride ethanol (96%) - water (50 : 30 : 20). Diluted hypochlorite solution: Dilute 20 ml of sodium hypochlorite solution R with water to 100 ml. Starch-potassium iodide reagent: Dissolve 1.1 g of potassium iodide R in 60 ml of water, boil for 15 min, and slowly add a suspension of 1.5 g of soluble starch R in 10 ml of water. Add 25 ml of water, and boil for 10 min. Allow to cool, and dilute with water to 100 ml. Test solution:  Transfer 50 mg of of the substance to be examined to a 10-ml volumetric flask. Dissolve in 7 ml of water, and adjust with 0.5 M sulfuric acid R to a pH of 5.5 ± 0.4. Dilute with water to volume. Reference solution: Dilute the test solution with water to obtain a 0.05 mg/ml solution of tobramycin. Procedure: Apply separately to the plate 1 µl of each solution. Develop over a path of about three-quarters of the plate. Remove the plate from the chromatographic chamber, evaporate the solvent in a current of hot air, then heat the plate at 110 °C for 10 min. Lightly spray the hot plate with diluted hypochlorite solution. Dry the plate in a current of cold air until a sprayed area of the plate below the origin gives at most a faint blue color with a drop of starch-potassium iodide reagent. Then spray the plate with starch-potassium iodide reagent. Bluish-purple spots are immediately visible. Other than the principal tobramycin spot, no spot observed in the test solution is more intense than the principal spot from the reference solution (1.0%). Water Not more than 8.0% (Appendix 10.3). Determined on 0.30 g. Suflated ash Not more than 0.3% (Appendix 9.9, method 2). Determined on 1.0 g. Bacterial endotoxins (Appendix 13.2). Not more than 2.00 EU/mg. If intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins.

TOBRAMYCIN

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 2.0 g of tris(hydroxymethyl) aminomethane in 800 ml of water. Add 20 ml of 0.5 M sulfuric acid R, and dilute to 2000 ml with acetonitrile R. Cool, and pass through a filter of 0.2 µm pore size. 2,4-dinitrofluorobenzene reagent: A 10 mg/ml solution of 2,4-dinitrofluorobenzene in ethanol (96%) R. This solution may be used for 5 days if refrigerated when not in use. Tris(hydroxymethyl)aminomethane reagent: Prepare a 15 mg/ml solution of tris(hydroxymethyl)aminomethane in water. This solution may be used for 1 month if refrigerated when not in use. Transfer 40 ml of this solution to a 200-ml volumetric flask, add dimethyl sulfoxide R to volume. Use this reagent within 4 h (If kept immersed in an ice-water bath below 10 °C, the reagent may be used for up to 8 h). Test solution: Weigh accurately 55 mg of the substance to be examined to a 50-ml volumetric flask. Add 1 ml of 0.5 M sulfuric acid R and enough water to dissolve it, and dilute with water to volume. Dilute 10.0 ml of the resulting solution to 50.0 ml with water. Standard solution: Weigh accurately 55 mg of tobramycin RS to a 50-ml volumetric flask. Add 1 ml of 0.5 M sulfuric acid R and enough water to dissolve it, and dilute with water to volume. Dilute 10.0 ml of the resulting solution to 50.0 ml with water. Preparation of derivatized solution: Heat all solutions at the same temperature and for the same duration of time as indicated. Move all flasks to and from the 60° constant temperature bath at the same time. To separate 50-ml volumetric flasks transfer 4.0 ml of the standard solution, 4.0 ml of the test solution, and 4.0 ml of water. To each flask add 10 ml of 2,4-dinitrofluorobenzene reagent and 10 ml of tris(hydroxymethyl)aminomethane reagent, shake, and insert the stopper. Place the flasks in a constant temperature bath at 60 °C ± 2 °C, and heat for 50 min ± 5 min. Remove the flasks from the bath, and allow to stand for 10 min. Add acetonitrile R to about 2 ml below the mark, allow to cool to room temperature, then dilute with acetonitrile R to volume. The solutions thus obtained are the derivatized standard solution, the derivatized test solution, and the blank solution, respectively. Resolution solution: Prepare a 0.24 mg/ml solution of p-naphtholbenzen R in acetonitrile R. Dilute 2 ml of this solution to 10.0 ml with the derivatized standard solution. Chromatographic system: A column (30 cm × 3.9 mm) packed with stationary phase C. Detector: A spectrophotometer set at 365 nm. Flow rate: 1.2 ml/min. Volume of injection: 20 µl. Procedure: Inject the blank solution to identify the peaks due to solvent and reagent. 973

VP V

TOBRAMYCIN EYE DROPS

Inject the resolution solution, the relative retention times for p-naphtholbenzein and tobramycin are about 0.6 and 1.0, respectively. The resolution between the peaks due to p-naphtholbenzein and tobramycin is at least 4.0. Inject the derivatized standard solution: the relative standard deviation of the peak areas for 6 replicate injections is not more than 2.0%. Inject the derivatized standard solution and the derivatized test solution. Calculate the percentage content of tobramycin, C18H17N5O9, using the chromatograms obtained with the derivatized standard solution the derivatized test solution and using the declared content of C18H17N5O9 in tobramycin RS.

Storage Store in an airtight container. If the substance is sterile, store in a sterile, airtight container. Action and use Aminoglycoside antibacterial.

of warm air, spray with a mixture of equal volumes of a 0.2% solution of naphthalene-1,3-diol in ethanol (96%) R and a 46% solution of sulfuric acid R and heat at 105 °C for 5 to 10 min. The principal spot in the chromatogram obtained with the test solution corresponds in position, colour and size to that in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) shows three clearly separated principal spots. B. To 2 ml of the eye drops add 5 ml of a 0.1 % solution of ninhydrine R in ethanol (96%) R and heat in a water-bath for 3 min. A violet-blue is produced.

pH pH 7.0 to 8.0 (Appendix 6.2). Assay Carry out the method for Microbiological assay of antibiotic (Appendix 13.9). 1000 IU is equivalent to 1 mg of tobramycin, C18H37N5O9.

Preparations Eye drops; Injection.

Storage Store in a closed-container at a dry place and protect from light.

TOBRAMYCIN EYE DROPS Collyrium Tobramycini

Action and use Aminoglycoside antibacterial.

Tobramycin eye drops are a sterile solution of tobramycin in purified water. It may contain suitable buffers, dispersants, preservatives, and tonicity agents. The eye drops comply with the requirements stated under “Eye drops” (Appendix 1.14) and with the following requirements.

Content of tobramycin, C18H37N5O9, 90.0% to 120.0% of the stated amount. Characters A clear, colourless solution. Identification A.Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Methanol - ammonia - methylene chloride (50 : 33 : 17). Test solution: Dilute a suitable volume of the eye drops (if necessary) with water to produce a solution containing 3 mg/ml of tobramycin. Reference solution (1): A 3 mg/ml solution of tobramycin RS. Reference solution (2): Dissolve 3.0 mg of neomycin sulfate RS and 3.0 mg of kanamycin monosulfate RS in 1 ml of reference solution (1). Procedure: Apply 5 µL of each solution. Develop over a path of 15 cm. After removal of the plate, dry it in a current 974

Strength dosage Solution (0.3%). TOBRMYCIN INJECTION Injectio Tobramycini Tobramycin injection is a sterile solution of tobramycin in water for Injection containing of sulfuric acid. It comply with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Content of tobramycin, C18H37N5O9, 90.0 % to 120.0 % of the stated amount. Characters A colourless solution. Identification A. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel G. Mobile phase: Methanol - concentrated ammonia chloroform (60 : 40 : 20). Test solution: Dilute a suitable volume of the injection with water to produce a solution containing 4 mg of tobramycin per ml. Reference solution (1): Dissolve 20.0 mg of tobramycin RS in water and dilute to 5 ml with the same solvent.

VP V

Reference solution (2): Dissolve 4.0 mg of kanamycin sulfate RS, 4.0 mg of neomycin sulfate RS in 1 ml of reference solution (1). Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm. After removal of the plate, dry it in a current of warm. Spray with a mixture of equal volumes of a 0.2% solution of 1,3-dihydronaphtalene in ethanol (96%) and a 46.0% solution of sulfuric acid R. Dry it at 105 °C for 5 - 10 min. The principal spot in the chromatogram obtained with the test solution corresponds in position, colour and size to that in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) shows three clearly separated principal spots. B. To 1 volume of the injection to be examined containing about 6 mg of tobramycin, add 2 ml of water, add further 5 ml of a 0.1% solution of ninhydrin in ethanol (96%) and heat in a water-bath for 3 minutes. A violet-blue colour is produced. C. Yield the reaction characteristic of sulfate (Appendix 8.1).

All-rac-ALPHA TOCOPHEROL

20 EU of endotoxin per ml. Carry out the test using the maximum valid dilution of solution A calculated from the declared sensitivity of the lysate used in the test.

Assay Carry out the Biological assay of antibiotics (Appendix 13.9). 1000 IU equivalent to 1 mg of tobramycin, C18H37N5O9. Storage Store in a cool place, protected from light. Action and use Antibacterial. Usual strength 20 mg/ml; 80mg/ml. All-rac-ALPHA TOCOPHEROL Alpha tocopherolum

pH 3.5 to 6.0. (Appendix 6.2). Related substances Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel H. Mobile phase: Ammonia - butan-2-on - ethanol (96%) (1 : 1 : 1). Test solution: Dilute a suitable volume of the injection with 0.01 M ammonia R to produce a solution containing 40 mg of tobramycin per 4 ml. Shake with 10 ml of ether (R), and use the aqueous layer. Reference solution: Dissolve 1 ml of the test solution to 50 ml with 0.01 M ammonia R. Procedure: Apply separately to the plate 4 µl of each solution. Develop over a path of 15 cm. After removal of the plate, allow it to dry in air, heat at 110 °C for 10 minutes and immediately spray the hot plate with a solution prepared immediately before use by diluting sodium hypochlorite solution (3% of Cl) with water to contain 0.5% of Cl. Dry in a current of cold air until a sprayed area of the plate below the line of application gives at most a very faint blue colour with a drop of potassium iodide and starch solution R, avoid prolonged exposure to cool air. Spray the plate with potassium iodide and starch solution R. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution (2%). Bacterial endotoxins Carry out the Test for bacterial endotoxins (Appendix 13.2). Dilute the injection, if necessary, with water BET to give a solution containing 10 mg of tobramycin per ml (solution A). The endotoxin limit concentration of solution A is

C29H50O2 

M. 430.7

All-rac-alpha tocopherol is all-rac-2,5,7,8-tetramethyl-2(4,8,12-trimethyltridecyl)-3,4-dihydro-2H-1-benzopyran6-ol. It contains not less than 96.0% and not more than 101.5% of C29H50O2.

Characters Clear, colourless or yellowish-brown, viscous, oily liquid. Practically insoluble in water, freely soluble in acetone, in anhydrous ethanol, in dichloromethane and in fatty oils. Identification Apply one of the two following identifications. First identification: A, B Second identification: B, C. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of α-tocopherol RS. B. Optical rotation: -0.01° to +0.01° (Appendix 6.4). Dissolve 2.5 g in anhydrous ethanol R and dilute to 25.0 ml with the same solvent. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel F254. Mobile phase: Cyclohexane - ether (80 : 20). Test solution: Dissolve 10 mg of the substance to be examined in 2 ml of cyclohexane R. Reference solution: Dissolve 10 mg of α-tocopherol RS in 2 ml of cyclohexane R. 975

VP V

All-rac-ALPHA TOCOPHERYL ACETATE

Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in a current of air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution.

Related substances Examine by gas chromatography (Appendix 5.2). Use the normalisation procedure. Internal standard solution: Dissolve 1.0 g of squalane R in cyclohexane R and dilute to 100.0 ml with the same solvent. Test solution (1): Dissolve 0.100 g of the substance to be examined in 10.0 ml of the internal standard solution. Test solution (2): Dissolve 0.100 g of the substance to be examined in 10.0 ml of cyclohexane R. Reference solution (1): Dissolve 0.100 g of α-tocopherol RS in 10.0 ml of the internal standard solution. Reference solution (2): Dissolve 10 mg of the substance to be examined and 10 mg of α-tocopheryl acetate R in cyclohexane R and dilute to 100.0 ml with the same solvent. Reference solution (3): Dissolve 10 mg of all-racα-tocopherol for peak identification RS (containing impurities A, B and D) in cyclohexane R and dilute to 1 ml with the same solvent. Reference solution (4): Dilute 1.0 ml of test solution (2) to 100.0 ml with cyclohexane R. Dilute 1.0 ml of this solution to 10.0 ml with cyclohexane R. Chromatographic system: A capillary column (30 m long and 0.25 mm in internal diameter) coated with stationary phase of poly(dimethyl) siloxane R (film thickness 0.25 µm) Carrier gas: Helium for chromatography R. Detection: Flame ionisation. Temperature of column: 280 °C, temperature of injection port and detector: 290 °C. Flow rate: 1 ml/min. Split ratio: 1 : 100. Volum injection: 1 µl. Proceduce: Injection separately test solution (2) and reference solutions (2), (3) and (4). Run time: Twice the retention time of all-rac-α-tocopherol. Identification of impurities: Use the chromatogram supplied with all-rac-α-tocopherol for peak identification RS and the chromatogram obtained with reference solution (3) to identify the peaks due to impurities A, B, C and D. Relative retention: With reference to all-rac-α-tocopherol (retention time = about 13 min): squalane = about 0.5; impurity A = about 0.7; impurity B = about 0.8; impurities C and D = about 1.05. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to all-rac-α-tocopherol and α-tocopheryl acetate is minimum 3.5. 976

Limits:  Impurity A: Maximum 0.5%, Impurity B: Maximum 1.5%, Sum of impurities C and D: Maximum 1.0%, Any other impurity: For each impurity, maximum 0.25%. Total: Maximum 2.5%. Disregard limit: The area of the principal peak in the chromatogram obtained with reference solution (4) (0.1%). Notes: Impurity A: all-rac-trans-2,3,4,6,7-pentamethyl-2-(4,8,12-trimethyltridecyl)-2,3-dihydrobenzofuran-5-ol. Impurity B: all-rac-cis-2,3,4,6,7-pentamethyl-2-(4,8,12-trimethyltridecyl)-2,3-dihydrobenzofuran-5-ol. Impurity C: 4-methoxy-2,3,6-trimethyl-5-[(all-RS,E)-3,7,11,15tetramethylhexadec-2-enyl]phenol. Impurity D: (all-RS,all-E)-2,6,10,14,19,23,27,31-octamethyldotriaconta-12,14,18-triene.

Assay Examine by gas chromatography (Appendix 5.2) as described in the test for Related substances. Injection test solution (1) and reference solution (1). Calculate the percentage content of C29H50O2 from the declared content of α-tocopherol RS. Storage Under an inert gas, protected from light. Action and use Vitamin. All-rac-ALPHA TOCOPHERYL ACETATE Alpha tocopheryli acetas

C31H52O3

M. 472.7

All-rac-alpha tocopheryl acetate is 2,5,7,8-tetramethyl-2(4,8,12-trimethyltridecyl)-3,4-dihydro-2H-1-benzopyran6-yl acetate. It contains not less than 96.5% and not more than 101.0% of C31H52O3.

Characters Clear, colourless or slightly greenish-yellow, viscous, oily liquid. Practically insoluble in water, freely soluble in acetone, in anhydrous ethanol and in fatty oils. Identification Apply one of the two following identifications. First identification: A, B

All-rac-ALPHA TOCOPHERYL ACETATE

VP V

Second identification: B, C. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of α-tocopheryl acetate RS. B. Optical rotation: -0.01° to +0.01° (Appendix 6.4). Dissolve 2.50 g in anhydrous ethanol R and dilute to 25.0 ml with the same solvent. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel F254. Mobile phase: Cyclohexane - ether (80 : 20). Test solution: Dissolve 10 mg of the substance to be examined in 2 ml of cyclohexane R. Reference solution: Dissolve 10 mg of α-tocopheryl acetate RS in 2 ml of cyclohexane R. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in a current of air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution.

Related substances Examine by gas chromatography (Appendix 5.2). Use the normalisation procedure. Internal standard solution: Dissolve 1.0 g of squalane R in cyclohexane R and dilute to 100.0 ml with the same solvent. Test solution (1): Dissolve 0.100 g of the substance to be examined in 10.0 ml of the internal standard solution. Test solution (2): Dissolve 0.100 g of the substance to be examined in 10.0 ml of cyclohexane R. Reference solution (1): Dissolve 0.100 g of α-tocopheryl acetate RS in 10.0 ml of the internal standard solution. Reference solution (2): Dissolve 10 mg of the substance to be examined and 10 mg of α-tocopherol R in cyclohexane R and dilute to 100.0 ml with the same solvent. Reference solution (3): Dissolve 10 mg of all-rac-αtocopheryl acetate for peak identification RS (containing impurities A, B and E) in cyclohexane R and dilute to 1 ml with the same solvent. Reference solution (4): Dilute 1.0 ml of test solution (2) to 100.0 ml with cyclohexane R. Dilute 1.0 ml of this solution to 10.0 ml with cyclohexane R. Chromatographic system: A capillary column (30 m long and 0.25 mm in internal diameter) coated with stationary phase of poly(dimethyl) siloxane R (film thickness 0.25 µm). Carrier gas: Helium for chromatography R. Detection: Flame ionisation. Temperature of column: 280 °C, temperature of injection port and detector: 290 °C. Flow rate: 1 ml/min. Split ratio: 1 : 100. Volum injection: 1 µl.

Run time: Twice the retention time of all-rac-α-tocopheryl acetate. Proceduce: Injection separately test solution (2) and reference solutions (1), (2), (3) and (4). Identification of impurities: Use the chromatogram supplied with all-rac-α-tocopheryl acetate for peak identification RS and the chromatogram obtained with reference solution (3) to identify the peaks due to impurities A, B, D and E. Relative retention: With reference to all-rac-α-tocopheryl acetate (retention time = about 15 min): squalane = about 0.4; impurity A = about 0.7; impurity B = about 0.8; impurities C = about 0.9; impurities D = about 1.05 and impurities E = about 1.05. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurities C and all-rac-α-tocopheryl acetate is minimum 3.5. In the chromatogram obtained with reference solution (1), the area of any peak corresponding to impurity C is not greater than 0.2% of the area of the peak corresponding to all-rac-α-tocopheryl acetate. Limits:  Impurity A and C: For each impurity, maximum 0.5%. Impurity B: Maximum 1.5% Sum of impurities D and E: Maximum 1.0%. Any other impurity: For each impurity, maximum 0.25%. Total: Maximum 2.5%. Disregard limit: The area of the principal peak in the chromatogram obtained with reference solution (4) (0.1%). Notes: Impurity A: all-rac-trans-2,3,4,6,7-pentamethyl-2-(4,8,12trimethyltridecyl)-2,3-dihydrobenzofuran-5-yl acetate. Impurity B: all-rac-cis-2,3,4,6,7-pentamethyl-2-(4,8,12trimethyltridecyl)-2,3-dihydrobenzofuran-5-yl acetate. Impurity C: all-rac-α-tocopherol. Impurity D: 4-methoxy-2,3,6-trimethyl-5-[(all-RS,E)-3,7,11,15tetramethylhexadec-2-enyl]phenyl acetate. Impurity E: (all-RS,all-E)-2,6,10,14,19,23,27,31octamethyldotriaconta-12,14,18-triene.

Assay Examine by gas chromatography (Appendix 5.2) as described in the test for Related substances. Injection test solution (1) and reference solution (1). Calculate the percentage content of C31H52O3 from the declared content of all-rac-α-tocopheryl acetate RS. Storage Protected from light. Action and use Vitamin.

977

VP V

VITAMIN E SOFT CAPSULES

VITAMIN E SOFT CAPSULES Molles capsulae Vitamini E Vitamin E soft capsules contain one of the following forms of vitamin E: d- or dl-alpha tocopherol (C29H50O2); d- or dl-alpha tocopheryl acetate (C31H52O3). The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

Content of vitamin E, 95.0% to 120.0% of the stated amount. Identification Test solution for alpha tocopheryl acetate Use low-actinic glassware. Transfer an accurately weighed quantity of the capsule contents, equivalent to about 220 mg of d- or dl-alpha tocopheryl acetate, to a 150 ml glass-stoppered conical flask, add 25 ml of ethanol R to dissolve. Add 20 ml of a mixture of sulphuric acid (10%) R and ethanol R (1 : 72), and boil on a water bath under a reflux condenser for 3 hours, protected from light. Cool, transfer to a 200 ml volumetric flask, dilute to volume with a mixture of sulphuric acid (10%) R and ethanol R (1 : 72), and mix. A. Prepare a solution containing about 10 mg of alpha tocopherol in 10 ml of ethanol R, or use 10 ml of the test solution for alpha tocopheryl acetate. Add, with swirling, 2 ml of nitric acid R, and heat at about 75 °C for 15 minutes: a bright red or orange colour develops. B. Prepare a solution containing about 100 mg, of alpha tocopherol in 50 ml of ether R, or transfer 100.0 ml of the test solution for alpha tocopheryl acetate to a separator, and add 200 ml of water. Extract first with 75 ml, then with 25 ml, of ether R, and combine the ether extracts in an other separator. To the ether solution, add 20 ml of a 10% solution of potassium ferricyanide R in 0.2 M sodium hydroxide R, and shake for 3 minutes. Wash the ether solution with four 50 ml portions of water, discard the washings, and dry the ether layer over anhydrous sodium sulphate R. Evaporate the dried ether on a water bath under reduced pressure or in an atmosphere of nitrogen untill about 7 or 8 ml remain, then complete the evaporation, removing the last traces of ether without the application of heat. Immediately dissolve the residue in 5.0 ml of isooctane R and determine the optical rotation (Appendix 6.4). Calculate the specific optical rotation, using concentration (C%) of tocopherol, determined in the Assay. The d-isomers have a specific optical rotation of not less than +24°. The dl-forms show essentially no optical rotation. C. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. Assay Examine by liquid chromatography (Appendix 5.3) 978

Mobile phase: Methanol - water (49 : 1). Test solution: Weigh accurately 20 capsules. With a sharp blade, or by other appropriate means, carefully open the capsules without loss of shell material, and transfer the combined capsule contents to a 100 ml beaker. Remove any adhering substance from the empty capsules by washing with several small portions of ether R or n-hexane R. Discard the washings, and allow the empty capsules to dry in a current of hot air until the odour of the solvents is no longer perceptible, cool in a desiccator. Weigh accurately the empty capsules, and determine the average weight of the contents. Transfer an accurately weighed quantity of the combined capsule contents, equivalent to 50 mg of vitamin E, to a 50 ml volumetric flask, dissolve in and dilute to volume with ethanol R, and mix. Reference solution: Weigh accurately about 50 mg of vitamin E [alpha tocopherol (C29H50O2) RS or alpha tocopheryl acetate (C31H52O3) RS] in a 50 ml volumetric flask, dissolve in and dilute to volume with ethanol R, and mix. Resolution solution: Dissolve 50 mg of alpha tocopherol RS and 50 mg of alpha tocopheryl acetate RS in 50 ml of ethanol R, and mix. Chromatographic system: A stainless steel column (15 to 30 cm × 4.6 mm) packed with stationary phase C (5 or 10 µm). Detector: A spectrophotometer set at 284 nm for alpha tocopheryl acetate and at 292 nm for alpha tocopherol. Flow rate: 1.0 to 2.0 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the resolution solution, the test is not valid unless the resolution factor between the peaks corresponding to alpha tocopherol and alpha tocopheryl acetate is at least 2.6. Inject the reference solution, the relative standard deviation for replicate injections is not more than 0.8% Inject the test solution. Calculate the content of vitamin E using the areas for the principal peaks in the chromatograms obtained with the test solution and the reference solution, and the concentration of vitamin E in the reference solution. The potency of vitamin E is expressed in USP Units. 1 mg of dl-alpha tocopherol = 1.1 USP Vitamin E Units. 1 mg of dl-alpha tocopheryl acetate = 1 USP Vitamin E Unit. 1 mg of d-alpha tocopherol = 1.49 USP Vitamin E Units. 1 mg of d-alpha tocopheryl acetate = 1.36 USP Vitamin E Units.

Storage Store in an airtight container, protected from light. Action and use Vitamin. Usual strength 100 IU; 400 IU.

TOLBUTAMIDE

VP V

TOLBUTAMIDE Tolbutamidum

C12H18N2O3S   

M. 270.3

Tolbutamide is 1-butyl-3-[(4-methylphenyl)sulfonyl]urea. It contains not less than 99.0% and not more than 101.0% of C12H18N2O3S, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder. Practically insoluble in water, soluble in acetone and in ethanol (96%). It dissolves in dilute solutions of alkali hydroxides. Identification Apply one of the two following identifications: First identification: A, B. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of tolbutamide RS. B. Melting point: 126 °C to 130 °C (Appendix 6.7). C. Dissolve 25.0 mg in methanol R and dilute to 100.0 ml with the same solvent. The ultraviolet absorptiom spectrum (Appendix 4.1) of the solution, in the range between 245 nm and 300 nm, exhibits three absorption maxima at 258 nm, 263 mn, and 275 nm; a shoulder at 268 nm. Dilute 10.0 ml of the above solution to 250.0 ml with methanol R. The ultraviolet absorptiom spectrum (Appendix 4.1) of the solution, in the range between 220 nm and 235 nm, exhibits an absorption maximum at 228 nm. Specific absorbance at the absorption maximum at 228 nm is 480 to 520. D. To 0.2 g add 8 ml of a 50% solution of sulfuric acid R and heat under a reflux condenser for 30 min. Allow to cool. Crystals are formed which, after recrystallisation from hot water and drying at 105 °C, melt (Appendix 6.7) at 135 °C to 140 °C. Appearance of solution Dissolve 0.2 g in 5 ml of dilute sodium hydroxide solution R and add 5 ml of water. The solution is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). pH 4.5 to 5.5 (Appendix 6.2). To 2.0 g add 50 ml of carbon dioxide-free water R and heat at 70 °C for 5 min. Cool rapidly and filter. Related substances Examine by liquid chromatography (Appendix 5.3).

Prepare the solutions immediately before use. Mobile phase: Acetonitrile R1 - buffer solution pH 3.5 (35 : 65). Buffer solution pH 3.5: A 1.36 g/L solution of potassium dihydrogen phosphate R, adjusted to pH 3.5 with phosphoric acid R. Test solution: Dissolve 50 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Reference solution (1): Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 10.0 ml with the mobile phase. Reference solution (2): Dissolve 10 mg of toluenesulfonamide R (impurity A) and 10 mg of toluenesulfonylurea R (impurity B) in the mobile phase and dilute to 10.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 100.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 230 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: The run time is 1.5 time the retention time of tolbutamide. Identification of impurities: Use the chromatogram obtained with reference solution (2) to identify the peak due to impurities A and B. Relative retention with reference to tolbutamide (retention time = about 18 min): impurity B = about 0.2; impurity A = about 0.3. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impuritites A and B is at least 2.0. Limits: Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). The sum of the peak areas of all impurities is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.3%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A: (4-methylphenyl)sulfonamide (toluenesulfonamide). Impurity B: 1-[(4-methylphenyl)sulfonyl]urea (toluenesulfonylurea). Impurity C: 1-azepan-1-yl-3-[(4-methylphenyl)sulfonyl]urea (tolazamide).

Heavy metals Not more than 10 ppm (Appendix 9.4.8). Dissolve 1.0 g in a mixture of water - acetone (15 : 85) and dilute to 20 ml with the same mixture of solvents. 12 ml 979

TOLBUTAMIDE TABLETS

of the solution complies with limit test for heavy metals, method 2. Prepare the reference solution using lead standard solution (0.5 ppm Pb) obtained by diluting lead standard solution (100 ppm Pb) R with a mixture of water - acetone (15 : 85).

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.250 g of the substance to be examined in a mixture of 20 ml of water and 40 ml of ethanol (96%) R. Titrate with 0.1 N sodium hydroxide VS, using 1 ml of phenolphthalein solution R as indicator. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 27.03 mg of C12H18N2O3S. Storage In an airtight container. Action and use Treatment of diabetes mellitus. Preparation Tablets. TOLBUTAMIDE TABLETS Tabellae Tolbutamidi Tolbutamide tablets contain tolbutamide. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of tolbutamide, C12H18N2O3S, 95.0% to 105.0% of the stated amount. Identification Extract a quantity of the powdered tablets containing 1 g of tolbutamide with 10 ml of chloroform R, filter in to a small beaker, evaporate the filtrate to dryness, scratching the wall of the beaker, if necessary, to induce crystallisation. Collect the crystals in a filter and dry it at 100 °C to 105 °C for 30 min. The infrared absorption spectrum (Appendix 4.2) of the residue is concordant with the spectrum of tolbutamide RS. Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 900 ml of a solution containing 2.04% of disodium hydrogen orthophosphate R and 0.135% of potassium dihydrogen orthophosphate R 980

VP V

Rotation speed: 100 rpm. Time: 45 min. Procedure: After the specified time, withdraw a sample of the medium and filter, discarding the first 20 ml of the filtrate. Measure the absorbance of the filtered sample, suitably diluted with the medium if necessary, at the maximum at 228 nm (Appendix 4.1). Calculate the total content of tolbutamide, C12H18N2O3S, dissolved from each tablet, taking 417 as the value of A(1%, 1 cm) at the maximum at 228 nm. Tolerance: Not less than 75% (Q) of the stated amount of tolbutamide, C12H18N2O3S, is dissolved in 45 min.

Related substances Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Anhydrous formic acid - methanol chloroform (2 : 8 : 90). Test solution: Shake a quantity of the powdered tablets containing 0.5 g of tolbutamide with 10 ml of acetone R and filter (Whatman filter paper N°1). Reference solution (1): A 0.015% solution of toluene-psulphonamide R in acetone R. Reference solution (2): A mixture of equal volumes of the test solutions and reference solution (1). Procedure: Apply separately to the plate 5 µl of each of the test solution and reference solution (1) and 10 µl of reference solution (2). Develop over a path of 15 cm. After removal of the plate, allow it to dry in air and heat at 110 °C for 10 min. While still hot, place the plate in a chromatography tank with an evaporating dish containing 5% potassium permanganate solution R, add an equal volume of hydrochloric acid R and close the tank. Leave the plate in the tank for 2 min, then place it in a current of cold air until the excess of chlorine is removed and an area of coating below the line of application gives at most a very faint blue colour with potassium iodide and starch solution R (avoid prolonged exposure to cold air). Spray the plate with potassium iodide and starch solution R and allow to stand for 5 min. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (1) (0.3%). The test is not valid unless the chromatogram obtained with reference solution (2) shows two clearly separated spots. Assay Weigh 20 tablets, calculate the average mass, finely powder. To a quantity of the powder, accurately weighed, containing 0.5 g of tolbutamide add 50 ml of ethanol 96% R previously neutralised to phenolphthalein solution R, warm to dissolve, add 25 ml of water and titrate with 0.1 N sodium hydroxide VS using phenolphthalein solution R as indicator. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 27.03 mg of C12H18N2O3S.

TRAMADOL HYDROCHLORIDE

VP V

Storage Store in a well-closed container, in a cool place and protected from light.

Optical rotation -0.10° to +0.10° (Appendix 6.4). Determined on solution S.

Action and use Antidiabetic (sulphonylurea).

Content of chloride 11.6% to 12.1%. Dissolve 150 mg of the substance to be examined in 40 ml of water, with stirring, add 7.5 ml of 4 N nitric acid R and 15.0 ml of 0.1 N silver nitrate VS. Titrate with 0.1 N ammonium thiocyanate VS, determining the endpoint potentiometrically and using a silver-glass electrode system (Appendix 10.2). Carry out a blank titration in the same manner. 1 ml of 0.1 N ammonium thiocyanate VS is equivalent to 3.545 mg of chloride.

Usual strength 0.25 g; 0.5 g. TRAMADOL HYDROCHLORIDE Tramadoli hydrochloridum

C16H25NO2,HCl  

M. 299.8

Tramadol hydrochloride is (1RS,2RS)-2-[(dimethylamino) methyl]-1-(3-methoxyphenyl)cyclohexanol hydrochloride. It contains not less than 98.0% and not more than 102.0% of C16H25NO2,HCl, calculated with reference to the anhydrous substance.

Characters White or almost white, crystalline powder. Freely soluble in water and in methanol, very slightly soluble in acetone. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of tramadol hydrochloride RS. B. In the test for Assay, retention time of the principal peak in the chromatogram obtained with the test solution corresponds to retention time of the peak due to tramadol in the chromatogram obtained with reference solution. C. A 1% solution of the substance to be examined gives reaction (A) of chlorides (Appendix 8.1). Appearance of solution Solution S: Dissolve 1.0 g in water and dilute to 20 ml with the same solvent. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). Acidity Add 0.2 ml of methyl red solution R and 0.2 ml of 0.01 N hydrochloric acid VS to 10 ml of solution S. The solution is red. Not more than 0.4 ml of 0.01 N sodium hydroxide VS is required to change the colour of the indicator to yellow.

Impurity B Not more than 0.2%. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel. Mobile phase: Toluene - 2-propanol - a 25% solution of ammonia (80 : 19 : 1). Test solution: A 50 mg/ml of the substance to be examined in methanol R. Reference solution: A 0.1 mg/ml of tramadol impurity B RS in methanol R. Procedure: Place the plate in a chromatographic chamber saturated with ammonia vapor from stronger ammonia water, and allow to stand at least 20 min. Apply separately to the plate 10 µl of each solution. Develop over a path of 10 cm. Remove the plate, spray with Dragendorff reagents R, and dry for 5 min. Spray the dried plate with 50 mg/ml solution of sodium nitrite R until the spot from tramadol impurity B in the reference solution is visible. In the chromatogram obtained with the test solution, any secondary spot corresponding to tramadol impurity B is not more intense than a principal spot in the chromatogram obtained with the reference solution (0.2%). Note: Impurity B: (2-[(dimethylamino)methyl]cyclohexanone hydrochloride).

Organic impurities Examine by liquid chromatography (Appendix 5.3) Mobile phase, system suitability solution, test solution, chromatographic system and system suitability as described in the test for Assay. Procedure: The run time is 4 times the retention time of tramadol. Calculate the percentage of each impurity base on the peak area of each impurity and sum of all the peak area in the chromatogram obtained with the test solution. Limits: Impurity A: Not more than 0.2%. Any other impurity: For each impurity, not more than 0.1%. Total impurities: Not more than 0.4%. 981

VP V

TRANEXAMIC ACID Note: ImpurityA: (RS,SR)-1-(3-methoxyphenyl)-2-(dimethylaminomethyl) cyclohexanol hydrochloride.

TRANEXAMIC ACID Acidum tranexamicum H

Water Not more than 0.5% (Appendix 10.3). Determined on 1.0 g. Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by liquid chromatography (Appendix 5.3). Solution A: Dissolve 2 ml of trifluoroacetic acid R in 1000 ml of water. Mobile phase: Acetonitrile - solution A (30 : 70) . If necessary, adjust the proportions of the constituents of the mobile phase to meet the system suitability. Test solution: Dissolve the substance to be examined, weighed accurately, in the mobile phase and dilute with mobile phase to obtain a 1.5 mg/ml solution of tramadol hydrochloride. Standard solution: Dissolve tramadol hydrochloride RS, weighed accurately, in the mobile phase and dilute with the mobile phase to obtain a 1.5 mg/ml solution of tramadol hydrochloride. System suitability solution: A solution contains 0.05 mg/ml of tramadol hydrochloride RS and 0.05 mg/ml tramadol impurity A RS in the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 270 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Procedure: Inject the system suitability solution. The relative retention for impurity A is 0.9 and tramadol is 1.0; the resolution between the peaks due to impurity A and tramadol is at least 2.0; the relative standard deviation for 6 replicate injections is not more than 2.0%. Inject the standard solution and the test solution. Calculate the content of C16H25NO2,HCl, using the areas of the tramadol peaks in the chromatograms obtained with the test solution, the standard solution and the content of C16H25NO2,HCl in tramadol RS. Storage Store in an airtight container, protected from light. Action and use Opioid analgesic. Preparations Tablets, capsules.

982

H2N

C8H15NO2  

CO2H

H



M. 157.2

Tranexamic acid is trans-4-(aminomethyl)cyclohexan carboxylic acid, it contains not less than 99.0% and not more than 101.0% of C8H15NO2, calculated with reference to the anhydrous substance.

Characters A white or almost white crystalline powder. Freely soluble in water and in glacial acetic acid, practically insoluble in acetone and in ethanol (96%). Identification The infrared absorption spectrophotometry (Appendix 4.2) of the substance to be examined is concordant with the spectrum of tranexamic acid RS. pH 7.0 to 8.0 (Appendix 6.2). Dissolve 2.5 g of the subtance to be examined in carbon dioxide-free water R and dilute to 50 ml with the same solvent. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 11.0 g of anhydrous sodium dihydrogen phosphate R in 500 ml of water, add 5 ml of triethylamine R and 1.4 g of sodium laurilsulfate R. Adjust to pH 2.5 with diluted phosphoric acid solution R and dilute to 600 ml with water. Add 400 ml of methanol R and mix. Test solution: Dissolve 0.20 g of the substance to be examined in water and dilute to 20.0 ml with the same solvent. Reference solution: Dilute 5.0 ml of the test solution to 100.0 ml with water. Dilute 1.0 ml of this solution to 10.0 ml with water. Resolution solution: Dissolve 5 mg of tranexamic acid impurity C RS in water and dilute to 50.0 ml with the same solvent. To 1.0 ml of this solution, add 1.0 ml of the test solution and dilute to 50.0 ml with water. Chromatographic system: A column (0.25 m × 4.6 mm) or (0.25 m × 6.0 mm) packed with stationary phase C (5 µm). Flow rate: 0.9 ml/min. Detector: A spectrophotometer set at 220 nm. Volume of injection: 20 µl. Procedure: The run time is 3 times the retension time of tranexamic acid.

TRANEXAMIC ACID CAPSULES

VP V

The relative retention times with reference to peak of tranexamic acid (retension time is about 13 minutes) of impurity C is about 1.1, of impurity D is about 1.3, of impurity B is about 1.5, of impurity A is about 2.1. System suitability: Inject resolution solution. The test is not valid unless the resolution between the peaks due to tranexamic acid and impurity C is at least 2.0. Limits: When calculating the contents, multiply the peak areas of impurities by the corresponding correction factor as follow: Impurity B is 1.2; impurity C is 0.005; impurity D is 0.006. In the chromatogram obtained with the test solution: The area of peak corresponding to impurity A is not greater than 0.2 times the area of principal peak in the chromatogram obtained with the reference solution (0.1%). The area of peak corresponding to impurity B is not greater than 0.4 times the area of principal peak in the chromatogram obtained with the reference solution (0.2%). The peak area of any other impurity is not greater than 0.2 times the area of principal peak in the chromatogram obtained with the reference solution (0.1%). The total of the peak areas of all the other impurities, is not greater than 0.4 times the area of the principal peak in the chromatogram obtained with the reference solution (0.2%). Disregard any peak with an area less than 0.05 times that of the principal peak in the chromatogram obtained with the reference solution (0.025%). Notes : Impurity A: Trans, trans-4,4-(iminodimethylene). di(cyclohexanecarboxylic) acid. Impurity B: Cis-4-(aminomethyl)cyclohexanecarboxylic acid. Impurity C: (RS)-4-(aminomethyl)cyclohex-1-enecarboxylic acid. Impurity D: 4-aminomethylbenzoic acid.

Halides expressed as chloride Not more than 140 ppm (Appendix 9.4.5). Dissolve 1.2 g of the substance to be examined in water and dilute to 50 ml with the same solvent. 15 ml of this solution complies with the limit test for chlorides. Heavy metals Not more than 10 ppm (Appendix 9.4.8). Dissolve 2.0 g of the substance to be examined in water and dilute to 20 ml with the same solvent. 12 ml of this solution complies with limit test for heavy metals, method 1. Prepare the standard using lead standard solution (1 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g, 105 °C; 2 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g.

Assay Dissolve 0.140 g of the substance to be examined in 20 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS. Determine the end-point by potentiometric titration (Appendix 10.2). 1 ml of 0.1 N perchloric acid solution VS is equivalent to 15.72 mg of C8H15NO2. Action and use Antifibrinolytic; haemostatic. Preparations Injection, tablets. TRANEXAMIC ACID CAPSULES Capsulae Acidi tranexamici Tranexamic acid capsules contain tranexamic acid. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements:

Content of tranexamic acid, C8H15NO2, 95.0% to 105.0% of the stated amount. Identification A. Weigh a quantity of the capsule contents containing about 0.1 g of tranexamic acid, add 10 ml of water, shake well, filter. Add 1 ml of 2% ninhydrin solution R and heat on a water-bath for 3min, a dark bluish violet is produced. B. In the Assay, the retention time of principal peak in the chromatogram obtained with the test solution is similar to that of the tranexamic acid peak in the chromatogram obtained with the reference solution. Dissolution Apparatus: Paddle. Medium: 900 ml of water. Rotation speed: 50 rpm. Time: 15 min. Procedure: Perform the liquid chromatography (Appendix 5.3) with the chromatographic systems as described in the Assay. Test solution: After specified time, withdraw a sample of the medium and filter. Dilute the filtrate with water to obtain a solution having a concentration of tranexamic acid of about 0.56 mg per ml. Reference solution: Weigh accurately about 56 mg of tranexamic acid RS, transfer into a 100-ml volumetric flask, dissolve in water and dilute to volume with water. Inject alternately the reference solution and the test solution with a volume of injection of 50 µl. Calculate the content of tranexamic acid dissolved using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C8H15NO2 in tranexamic acid RS. 983

VP V

TRANEXAMIC ACID TABLETS

Tolerances: Not less than 80% (Q) of the stated amount of tranexamic acid, C8H15NO2, is dissolved in 15 min.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 11.0 g of anhydrous sodium dihydrogen phosphate R in 500 ml of water, add 5 ml of triethylamine R and 1.4 g of sodium lauryl sulfate R. Adjust pH to 2.5 with phosphoric acid R or 10% phosphoric acid solution R, add water to 600 ml and further add 400 ml of methanol R shake well. Make adjustments if necessary. Reference solution: Transfer 50 mg of tranexamic acid RS, to a 25-ml volumetric flask, dissolve and dilute to volume with water. Test solution: Weigh 20 capsules, calculate the average weight of the contents and finely powder. Weigh a quantity of the powder contents containing 100 mg of tranexamic acid, transfer into a 50-ml volumetric flask, add 30 ml of water, shake well and add sufficient water to volume, mix well. Centrifuge and use the supernatant. Resolution solution: Transfer 1 ml of a 0.01% solution of 4-(aminomethyl) benzoic acid R into a 50-ml volumetric flask containing 5 ml of the reference solution and dilute to volume with water. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Column temperature: 35 °C. Detector: A spectrophotometer set at 220 nm. Flow rate: Adjust to obtain the retention time of tranexamic acid of about 16 min. Volume of injection: 20 µl. Procedure: System stability: Inject the resolution solution, the elution order is tranexamic acid and 4-(aminomethyl) benzoic acid, respectively and the resolution factor between the two peaks is not less than 3. Inject separately 6 times of the reference solution, the relative standard deviation of the peak areas of tranexamic acid for six replicate injections is not more than 1.0%. Inject alternately the reference solution and the test solution. Calculate the content of tranexamic acid, C8H15NO2, in the capsules using the peak areas in the chromatograms obtained with the reference solution, the test solution and the declared content of C8H15NO2 in tranexamic acid RS. Storage Store in a tight container, in a cool place, protected from light. Action and use Antifibrinolytic. Preparations 500 mg, 1000 mg.

984

TRANEXAMIC ACID TABLETS Tabellae Acidi Tranexamini Tranexamic acid tablets contain tranexamic acid. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of tranexamic acid, C8H15NO4, 95.0% to 105.0% of the stated amount. Identification A. Weigh a quantity of the powdered tablets containing about 0.1 g of tranexamic acid, add 10 ml of water, shake well, filter. Add 1 ml of a 2% ninhydrin solution R and heat on a water-bath for 3 min, a dark bluish violet colour is produced. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of the tranexamic acid peak in the chromatogram obtained with the reference solution. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of water. Rotation speed: 50 rpm. Time: 15 min. Procedure: Examine by the liquid chromatography (Appendix 5.3) with the chromatographic system as described in the Assay. Test solution: After the specified time, withdraw a sample of the medium, filter. Dilute with water to obtain a solution having a concentration of about 0.56 mg per ml. Reference solution: Weigh accurately about 56 mg of tranexamic acid RS, transfer into a 100-ml volumetric flask. Dissolve and dilute to volume with water. Inject alternately the reference solution and test solution with the injection volume of 50 µl. Calculate the content of tranexamic acid, C8H15NO4, dissolved using the peak areas in the chromatograms obtained with the reference solution, the test solution and the declared content of C8H15NO4 in tranexamic acid RS. Tolerance: Not less than 80% (Q) of the stated amount of tranexamic acid, C8H15NO4, is dissolved in 15 min. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 11.0 g of anhydrous sodium dihydrgen phosphate R in 500 ml of water, add 5 ml of triethyleamine R and 1.4 g of sodium laurylsulfate R. Adjust the pH to 2.5 with phosphoric acid R or 10% phosphoric acid solution R, add water to obtain 600 ml and add 400 ml of methanol R, mix well. Make adjustments if necessary. Reference solution: Weigh accurately about 50 mg of tranexamic acid RS, transfer into a 25-ml volumetric flask, dissolve and dilute to volume with water.

TRIAMCINOLONE ACETONID

VP V

Test solution: Weigh 20 tablets, calculate the average mass and finely powder. Weigh accurately a quantity of the powdered tablets containing about 100 mg of tranexamic acid. Transfer into a 50-ml volumetric flask, add 30 ml of water, shake well to dissolve and dilute to volume with water, shake and filter. Centrifuge and use the supernatant. Resolution solution: Transfer 1 ml of a 0.01% solution of 4-(aminomethyl) benzoic acid R into a 50-ml volumetric flask containing 5 ml of the reference solution and dilute to volume with water. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Column temperature: 35 oC. Detector: A spectrophotometer set at 220 nm. Flow rate: Adjust to obtain the retention time of the tranexamic acid of about 16 min. Volume of injection: 20 µl. Procedure: System suitability: Inject the resolution solution, in the chromatogram obtained, the elution order is tranexamic acid and 4-(aminomethyl) benzoic acid, respectively and the resolution factor between the two peaks is not less than 3. Inject the reference solution, the relative standard deviation of peak areas for six replicate injections is not more than 1.0%. Inject alternately the reference solution and the test solution. Calculate the content of tranexamic acid, C8H15NO4, dissolved using the peak areas in the chromatograms obtained with the reference solution, the test solution and the declared content of C8H15NO4 in tranexamic acid RS.

Storage Store in a well-closed container, in a cool place, protected from light. Action and use Antifibrinolytic. Usual strength 500 mg, 1000 mg. TRIAMCINOLONE ACETONIDE Triamcinoloni acetonidum

C24H31FO6 



M. 434.5

Triamcinolone acetonide is 9-fluoro-11b,21-dihydroxy16a,17-(1-methylethylidenedioxy)pregna-1,4-diene-3,20-

dione. It contains not less than 97.0% and not more than 103.0% of C24H31FO6, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder. It shows polymorphism. Practically insoluble in water, sparingly soluble in ethanol (96%). Identification Apply one of the two following identifications: First identification: A, B. Second identification: C, D. A.The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum obtained with triamcinolone acetonide RS. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in the minimum volume of methanol R and evaporate to dryness. Using the residues, prepare halogen salt discs or mulls in liquid paraffin R and record new spectra. B. Examine by thin-layer chromatography (Appendix 5.4). Prepare the solutions immediately before use and protect from light. Coating substance: Silica gel F254. Mobile phase: Add a mixture of 1.2 volumes of water and 8 volumes of methanol R to a mixture of 15 volumes of ether R and 77 volumes of methylene chloride R. Test solution: Dissolve 10 mg of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Reference solution (1): Dissolve 20 mg of triamcinolone acetonide RS in methanol R and dilute to 20 ml with the same solvent. Reference solution (2): Dissolve 10 mg of triamcinolone hexacetonide RS in reference solution (1) and dilute to 10 ml with reference solution (1). Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in air. Examine in ultraviolet light at 254 nm, immediately after development. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) shows two clearly separated spots. C. Examine by thin-layer chromatography (Appendix 5.4). Prepare the solutions immediately before use and protect from light. Coating substance: Silica gel F254. Mobile phase: Add a mixture of 1.2 volumes of water and 8 volumes of methanol R to a mixture of 15 volumes of ether R and 77 volumes of methylene chloride R. Test solution (1): Dissolve 10 mg of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. 985

VP V

TRIAMCINOLONE ACETONID

Test solution (2): In a separating funnel, dissolve 10 mg of the substance to be examined in 1.5 ml of glacial acetic acid R, add 0.5 ml of a 2% solution of chromium trioxide R and allow to stand for 60 min. Add 5 ml of water, 2 ml of methylene chloride R and shake vigorously for 2 min. Allow to separate and use the lower layer. Reference solution (1): Dissolve 10 mg of triamcinolone acetonide RS in methanol R and dilute to 10 ml with the same solvent. Reference solution (2): In a separating funnel, dissolve 10 mg of triamcinolone hexacetonide RS in 1.5 ml of glacial acetic acid R, add 0.5 ml of a 2% solution of chromium trioxide R and allow to stand for 60 min. Add 5 ml of water, 2 ml of methylene chloride R and shake vigorously for 2 min. Allow to separate and use the lower layer. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in air. Examine in ultraviolet light at 254 nm, immediately after development. The principal spot in each of the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the corresponding reference solution. The principal spot in the chromatogram obtained with test solution (2) and reference solution (2) have an Rf value distinctly higher than that of the principal spot in the chromatogram obtained with test solution (1) and reference solution (1). D. Mix about 5 mg with 45 mg of heavy magnesium oxide R and ignite in a crucible until an almost white residue is obtained (usually less than 5 min). Allow to cool, add 1 ml of water, 0.05 ml of phenolphthalein solution R1 and about 1 ml of dilute hydrochloric acid R to render the solution colourless. Filter. To a freshly prepared mixture of 0.1 ml of alizarin S solution R and 0.1 ml of zirconyl nitrate solution R, add 1.0 ml of the filtrate. Mix, allow to stand for 5 min and compare the colour of the solution to that of a blank prepared in the same manner. The test solution is yellow and the blank is red.

Specific optical rotation +100° to +107°, calculated with reference to the dried substance (Appendix 6.4). Dissolve 0.100 g in dioxan R and dilute to 10.0 ml with the same solvent. Related substances Examine by liquid chromatography (Appendix 5.3). Carry out the test protected from light. Mobile phase: In a 1000 ml volumetric flask, mix 525 ml of methanol R with 400 ml of water and allow to equilibrate, adjust the volume to 1000 ml with water and mix again. Test solution: Dissolve 25.0 mg of the substance to be examined in 7 ml of methanol R and dilute to 10.0 ml with water. 986

Reference solution (1): Dissolve 2 mg of triamcinolone acetonide RS and 2 mg of triamcinolone R (impurity A) in the mobile phase and dilute to 100.0 ml with the mobile phase. Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with the mobile phase. Chromatographic system: A column (25cm × 4.6 mm) packed with stationary phase C (5 µm). Flow rate: 1.5 ml/min. Detector: A spectrophotometer set at 254 nm. Volume of injection: 20 µl. Procedure: Equilibrate the column with the mobile phase about 10 min. Inject reference solution (1). The retention time: triamcinolone about 5 min and triamcinolone acetonide about 17 min. The test is not valid unless the resolution between the peaks corresponding to triamcinolone and triamcinolone acetonide is at least 15; if necessary, adjust the concentration of methanol in the mobile phase. Inject separately the test solution and reference solution (2). Continue the chromatography for 3.5 times the retention time of triamcinolone acetonide. In the chromatogram obtained with the test solution: the area of any peak corresponding to triamcinolone (impurity A) is not greater than 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.25%). The sum of the areas of all the peaks, apart from the principal peak, is not greater than half the area of the principal peak in the chromatogram obtained with reference solution (2) (0.5%). Disregard any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.05%).

Water Not more than 2.0% (Appendix 10.3). Determined on 0.500 g. Assay Carry out the assay protected from light. Dissolve about 50.0 mg in ethanol (96%) R and dilute to 50.0 ml with the same solvent. Dilute 2.0 ml of the solution to 100.0 ml with ethanol (96%) R. Measure the absorbance (Appendix 4.1) at the maximum at 238.5 nm. Calculate the content of triamcinolone acetonide, C24H31FO6, using A (1%, 1 cm), taking 355 as the value of A (1%, 1 cm) at 283.5 nm. Storage Store in an airtight container, protected from light. Action and use Glucocorticoid. Preparation Cream. 

MEDIUM-CHAIN TRIGLYCERIDES

VP V

TRIAMCINOLONE ACETONIDE CREAM Cremoris Triamcinoloni acetonidi The triamcinolone acetonide cream contains triamcinolone acetonide in the suitable cream base. The cream complies with the requirements stated under "Semi-solid preparation for cutaneous and mucosal application" (Appendix 1.12) and the following requirements.

Content of triamcinolone acetonide, C24H31FO6, from 90.0% to 110.0% of the stated amount. Characters White or almost white homogenous cream. Identification A. In the Assay, the retention time of principal peak in the chromatogram obtained with the test solution is similar to that of triamcinolone acetonide peak in the chromatogram obtained with the reference solution. B. Examine by thin layer chromatography (Appendix 5.4) Coating substance: Silica gel G. Mobile phase: Ethyl acetate. Test solution: Transfer a quantity of the cream containing about 5 mg of triamcinolone acetonide into a conical flask, add 5 ml of chloroform R and 15 g of sodium sulfate anhydrous R. Swirl to dissolve, filter and clarify the filtrate, if necessary, by adding anhydrous sodium sulfate R and filter again. Evaporate the filtrate to near dryness and dissolve the residue in 10 ml of chloroform R. Reference solution (1): A solution of triamcinolone acetonide RS having a concentration of about 0.05% in chloroform R. Reference solution (2): A mixture of equal volume of the test solution and reference solution (1). Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of about 12 cm. Remove the plate, allow it to dry in air and spray with a mixture of equal volume of 20% sodium hydroxide solution R and a 0.2% solution of tetrazolium blue in methanol R. The principal spot in the chromatogram obtained with the test solution is similar in position, colour to that in the chromatogram obtained with reference solution (1). The chromatogram obtained with reference solution (2) exhibits only one principal spot. Assay Examine by liquid chromatography (Appendix 5.3) Mobile phase: Acetonitrile - water (30 : 70). Make adjustments if necessary. Reference solution: Weigh accurately about 37 mg of triamcinolone acetonide RS, transfer into a 50-ml volumetric flask. Dissolve in isopropanol R and dilute to volume with the same solvent. Dilute 10.0 ml of the resulting solution to 100.0 ml with isopropanol R. Mix a volume of the obtained solution with an equal volume of the mobile phase.

Test solution: Weigh a quantity of the cream containing about 1.5 mg of triamcinolone acetonide in 50-ml screwcap. Add 20.0 ml of isopropanol R and cap securely. Heat in a water-bath at 60 °C for 5 min, swirl cautiously for 30 seconds. Repeat heating and swirling 3 times. Cool in ice water for 15 to 20 min. Centrifuge for 15 min at -5 °C, dilute a volume of the supernatant with an equal volume of the mobile phase. Cool in ice water for 10 to 15 min. Filter through a glass wool and then through a filter paper. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 254 nm. Flow rate: 1 ml/min. Volume of injection: 20 µl. Procedure: Inject alternately the reference solution, the test solution. Calculate the content of triamcinolone acetonide, C24H31FO6, in cream using the peak areas in the chromatograms obtained with the test solution, reference solution and the declared content of C24H31FO6 in triamcinolone acetonide RS.

Storage Store in a tight container, in a dry and cool place, protected from light. Action and use Steroids anti-inflammatory for topical use. Usual strength 0.1% topical cream. MEDIUM-CHAIN TRIGLYCERIDES Triglycerida saturata media Medium-chain triglycerides is a mixture of triglycerides of saturated fatty acids, mainly of caprylic acid (octanoic acid) and of capric acid (decanoic acid). Not less than 95.0% of saturated fatty acids with 8 and 10 carbon atoms. Medium-chain triglycerides are obtained from the oil extracted from the hard, dried fraction of the endosperm of Cocos nucifera L. or from the dried endosperm of Elaeis guineensis Jacq. Medium-chain triglycerides are obtained from the endosperm of Cocos nucifera L. may be called fraction coconut oil.

Characters Colourless or slightly yellowish, oily liquid. Practically insoluble in water, miscible with ethanol (96%), with methylene chloride, with petroleum ether (boiling range 50 - 70 °C) and with fatty oils. Identification Apply one of the two following identifications: 987

VP V

MEDIUM-CHAIN TRIGLYCERIDES

First identification: B, C. Second identification: A, D. A. Heat 3.0 g of the substance to be examined under a reflux condenser for 30 min with 50 ml of a mixture of equal volumes of ethanol (96%) R and 2 M potassium hydroxide R in ethanol R. Reserve 10 ml of the mixture for identification test D. Add 30 ml of water to 40 ml of the mixture, evaporate the ethanol on a water bath and acidify the hot solution with 25 ml of dilute hydrochloric acid R. After cooling, shake with 50 ml of peroxide-free ether R. Wash the ether layer with 3 quantities, each of 10 ml, of a 0.9% solution of sodium chloride, dry over anhydrous sodium sulfate R and filter. Evaporate the ether and determine the acid value of the residue (Appendix 7.2), using 0.300 g residue. The acid value is 350 to 390. B. It complies with the test for Saponification value. C. It complies with the test for Composition of fatty acids. D. Evaporate 10 ml of the alcoholic mixture obtained in Identification test A to dryness on a water-bath. Transfer the residue into a test-tube, add 0.3 ml of sulfuric acid R and close the test-tube with a stopper through which a U-shaped glass tube is inserted. One end of the U-tube is dipped into 3 ml of a 1 % solution of tryptophan R in a mixture of equal volumes of sulfuric acid R and water. Heat the test-tube in a silicone-oil bath at 180 °C for 10 min and collect the liberated fumes in the tryptophan reagent. Heat the tryptophan reagent on a water-bath for 1 min. A violet colour develops.

Appearance The substance to be examined is clear (Appendix 9.2), and not more intensely coloured than reference solution Y3 (Appendix 9.3, method 1). Alkaline impurities Dissolve 2.00 g in a mixture of 1.5 ml of ethanol (96%) R and 3.0 ml of ether R. Add 0.05 ml of bromophenol blue solution R. Not more than 0.15 ml of 0.01 N hydrochloric acid VS is required to change the colour of the indicator to yellow. Relative density 0.93 to 0.96 (Appendix 6.5). Refractive index 1.440 to 1.452 (Appendix 6.1). Viscosity 25 mPa·s to 33 mPa·s (Appendix 6.3). Acid value Not more than 0.2 (Appendix 7.2). Hydroxyl value Not more than 10 (Appendix 7.4, method A). Iodine value Not more than 1.0 (Appendix 7.5). 988

Peroxide value Not more than 1.0 (Appendix 7.6, method A). Saponification value 310 to 360 (Appendix 7.7). Unsaponifiable matter Not more than 0.5% (Appendix 7.8). Determined on 5.0 g. Composition of fatty acids Examine by gas chromatography (Appendix 12.9, method C). Chromatographic system: A fused-silica capillary column (30 m long and 0.32 mm in internal diameter) coated with a film of macrogol 20 000 R (film thickness 0.5 µm). Carrier gas: Helium for chromatography R. Flow rate: 1.3 ml/min. Split ratio: 1 : 100. Detector: A flame-ionisation detector. Volume of injection: 1 µl. Temperature programme:

Column

Time (min)

Temperature (°C)

0-1

70

1 - 35

70 → 240

35 - 50

240

Injection port

250

Detector

250

System suitability : In the chromatogram obtained with reference solution (a), the resolution between the peaks due to methyl oleate and methyl stearate is at least 1.8. In the chromatogram obtained with reference solution (b), the signal-to-noise ratio of the peaks due to methyl myristate is not less than 5. In the chromatogram obtained with reference solution (a), the column efficiency determined from the methyl stearate peak is not less than 30 000 theoretical plates. Limits composition of the fatty-acid fraction of the substance: Caproic acid: Not more than 2.0%. Caprylic acid: 50.0% to 80.0%. Capric acid: 20.0% to 50.0%. Lauric acid : Not more than 3.0%. Myristic acid: Not more than 1.0%.

Chromium Not more than 0.05 ppm, if intended for use in parenteral nutrition. Examine by atomic absorption spectrometry (Appendix 4.4, method 2). Test solution: Dissolve 2.0 g of the substance to be examined in methyl isobutyl ketone R3 and dilute to 10.0 ml with the same solvent.

VP V

Solution A: Dilute 0.100 ml of chromium liposoluble standard solution (1000 ppm Cr) R to 10.0 ml with methyl isobutyl ketone R3. Stock solution: Dilute 0.100 ml of solution A to 10.0 ml with methyl isobutyl ketone R3. Standard solutions: Prepare 3 standard solutions by dissolving for each 2.0 g of the substance to be examined in the minimum volume of methyl isobutyl ketone R3, adding 0.5 ml, 1.0 ml and 2.0 ml, respectively, of stock solution and diluting to 10.0 ml with methyl isobutyl ketone R3. Measure the absorbance at 357.8 nm using a chromium hollow-cathode lamp as asource of radiatio and graphite furnace, carrier gas is argon R.

Copper Not more than 0.1 ppm, if intended for use in parenteral nutrition. Examine by atomic absorption spectrometry (Appendix 4.4, method 2). Test solution: Dissolve 2.0 g of the substance to be examined in methyl isobutyl ketone R3 and dilute to 10.0 ml with the same solvent. Solution A: Dilute 0.100 ml of copper liposoluble standard solution (1000 ppm Cu) R to 10.0 ml with methyl isobutyl ketone R3. Stock solution: Dilute 0.100 ml of solution A to 10.0 ml with methyl isobutyl ketone R3. Standard solutions: Prepare 3 standard solutions by dissolving for each 2.0 g of the substance to be examined in the minimum volume of methyl isobutyl ketone R3, adding 1.0 ml, 2.0 ml and 4.0 ml, respectively, of stock solution and diluting to 10.0 ml with methyl isobutyl ketone R3. Measure the absorbance at 324.7 nm using a copper hollow-cathode lamp as asource of radiatio and graphite furnace, carrier gas is argon R. Lead Not more than 0.1 ppm, if intended for use in parenteral nutrition. Examine by atomic absorption spectrometry (Appendix 4.4, method 2). Test solution: Dissolve 2.0 g of the substance to be examined in methyl isobutyl ketone R3 and dilute to 10.0 ml with the same solvent. Solution A: Dilute 0.100 ml of lead liposoluble standard solution (1000 ppm Pb) R to 10.0 ml with methyl isobutyl ketone R3. Stock solution: Dilute 0.100 ml of solution A to 10.0 ml with methyl isobutyl ketone R3. Standard solutions: Prepare 3 standard solutions by dissolving for each 2.0 g of the substance to be examined in the minimum volume of methyl isobutyl ketone R3, adding 1.0 ml, 2.0 ml and 4.0 ml, respectively, of stock solution and diluting to 10.0 ml with methyl isobutyl ketone R3. Measure the absorbance at 283.3 nm using a lead hollowcathode lamp as asource of radiatio and Graphite furnace coated inside with palladium carbide; calcination is carried

MEDIUM-CHAIN TRIGLYCERIDES

out in the presence of oxygen at a temperature below 800 °C, carrier gas is argon R.

Nickel Not more than 0.2 ppm, if intended for use in parenteral nutrition. Examine by atomic absorption spectrometry (Appendix 4.4, method 2). Test solution: Dissolve 2.0 g of the substance to be examined in methyl isobutyl ketone R3 and dilute to 10.0 ml with the same solvent. Solution A: Dilute 0.100 ml of nickel liposoluble standard solution (1000 ppm Ni) R to 10.0 ml with methyl isobutyl ketone R3. Stock solution: Dilute 0.100 ml of solution A to 10.0 ml with methyl isobutyl ketone R3. Standard solutions: Prepare 3 standard solutions by dissolving for each 2.0 g of the substance to be examined in the minimum volume of methyl isobutyl ketone R3, adding 1.0 ml, 2.0 ml and 4.0 ml, respectively, of stock solution and diluting to 10.0 ml with methyl isobutyl ketone R3. Measure the absorbance at 232 nm using a nickel hollowcathode lamp as asource of radiatio and graphite furnace, carrier gas is argon R. Tin Not more than 0.1 ppm, if intended for use in parenteral nutrition. Examine by atomic absorption spectrometry (Appendix 4.4, method 2). Test solution: Dissolve 2.0 g of the substance to be examined in methyl isobutyl ketone R3 and dilute to 10.0 ml with the same solvent. Solution A: Dilute 0.100 ml of tin liposoluble standard solution (1000 ppm Sn) R to 10.0 ml with methyl isobutyl ketone R3. Stock solution: Dilute 0.100 ml of solution A to 10.0 ml with methyl isobutyl ketone R3. Standard solutions: Prepare 3 standard solutions by dissolving for each 2.0 g of the substance to be examined in the minimum volume of methyl isobutyl ketone R3, adding 1.0 ml, 2.0 ml and 4.0 ml, respectively, of stock solution and diluting to 10.0 ml with methyl isobutyl ketone R3. Measure the absorbance at 286.3 nm using a tin hollowcathode lamp as asource of radiatio and graphite furnace coated inside with palladium carbide, carrier gas is argon R. Heavy metals Not more than 10 ppm (Appendix 9.4.8) if intended for use other than parenteral nutrition. 2.0 g complies with limit test for heavy metals, method 4. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Water Not more than 0.2% (Appendix 10.3). Determined on 10.00 g. 989

VP V

TRIHEXYPHENIDYL HYDROCHLORIDE

Total ash Not more than 0.1% (Appendix 9.8; Method 2). Determined on 2.0 g. Storage In a well-filled container, protected from light. Action and use Excipient. Labelling The label states, where applicable, that the substance is intended for use in parenteral nutrition.  TRIHEXYPHENIDYL HYDROCHLORIDE Trihexyphenidyli hydropchloridum

C20H31NO,HCl

M.337.9

Trihexyphenidyl hydrochloride is (1RS)-1-cyclohexyl1-phenyl-3-(piperidin-1-yl)propan-1-ol hydrochloride. It contains not less than 99.0% and not more than 101.0% of C20H31NO,HCl, calculated with reference to the dried substance.

Characters White, crystalline powder. Slightly soluble in water, sparingly soluble in ethanol (96%) and in methylene chloride. Melting point: about 250 °C, with decomposition. Identification Apply one of the two following identifications: First identification: A, D Second identification: B, C, D A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of trihexyphenidyl hydrochloride RS. B. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel G. Mobile phase: Diethylamine - hexane (5 : 95). Test solution: Dissolve 25 mg of the substance to be examined in a mixture of methanol R and methylene chloride R (20 : 80), and dilute to 10 ml with the same mixture of solvents, and mix. Reference solution: Dissolve 25 mg of trihexyphenidyl hydrochloride RS in a mixture of methanol R and methylene chloride R (20 : 80), and dilute to 10 ml with the same mixture of solvents, and mix. Procedure: Apply separately to the plate 5 µl of each solution. Develop over a path of 12 cm. Allow the plate 990

to dry in air, spray with a 0.01% solution of chloroplatinic acid R in hydrochloric acid (0.4%) R. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. C. Dissolve 0.5 g of the substance to be examined in 5 ml of warm methanol R and make just alkaline to red litmus paper R with sodium hydroxide solution R. A precipitate is formed which, after recrystallisation from methanol R, melts (Appendix 6.7) at about 113 °C to 115 °C. D. It gives reaction (A) of chlorides (Appendix 8.1).

pH 5.2 to 6.2 (Appendix 6.2). Dissolve 0.5 g of the substance to be examined with heating in 25 ml of carbon dioxide-free water R. Cool to room temperature and dilute to 50 ml with carbon dioxidefree water R. Optical rotation -0.10° to +0.10° (Appendix 6.4). Dissolve 1.25 g in a mixture of methanol R and methylene chloride R (20 : 80), and dilute to 25.0 ml with the same mixture of solvents. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: Mix 200 ml of water with 0.2 ml of triethylamine R. Adjust to pH 4.0 with phosphoric acid R and add 800 ml of acetonitrile R. Test solution: Dissolve 20.0 mg of the substance to be examined in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (1): Dilute 1.0 ml of the test solution to 200.0 ml with the mobile phase, and mix. Dilute 10.0 ml of this solution to 50.0 ml with the mobile phase, and mix. Reference solution (2): Dissolve 10.0 mg of trihexyphenidyl impurity A RS [1-phenyl-3-(piperidin-1-yl)propan-1-one] in the mobile phase and dilute to 10.0 ml with the mobile phase. Reference solution (3): Dilute 1.0 ml of reference solution (2) to 100.0 ml with the mobile phase. Reference solution (4): To 1.0 ml of reference solution (2), add 1.0 ml of the test solution and dilute to 100.0 ml with the mobile phase. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 210 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: The run time is three times the retention time of trihexyphenidyl. System suitability: In the chromatogram obtained with reference solution (4), the resolution between the peaks due to trihexyphenidyl and to impurity A is at least 4.0. Inject reference solution (1), reference solution (3) and the test solution.

VP V

Limits: In the chromatogram obtained with the test solution: The area of any peak corresponding to impurity A is not greater than the area of the principal peak in the chromatogram obtained with reference solution (3) (0.5%). The area of any other impurity is not greater than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%). The sum of the areas of all the secondary peaks is not greater than 0.5%. Disregard any peak with an area less than 0.2 times that of the principal peak in the chromatogram obtained with reference solution (1) (0.02%).

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 100 °C to 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.250 g of the substance to be examined in 50 ml of ethanol (96%) R and add 5.0 ml of 0.01 N hydrochloric acid VS. Carry out a potentiometric titration (Appendix 10.2), using 0.1 N sodium hydroxide VS. Read the volume added between the 2 points of inflexion. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 33.79 mg of C20H32ClNO. Storage Store in an airtight container at cold place, protected from light. Action and use Anticholinergic. Treatment of Parkinson’s disease. Preparation Tablets. TRIHEXYPHENIDYL TABLETS Tabellae Trihexyphenidyli Trihexyphenidyl tablets contain trihexyphenidyl hydrochloride. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of trihexyphenidyl hydrochloride, C20H31NO, HCl, 90.0% to 110.0% of the stated amount. Identification A. To a quantity of the powdered tablets containing 20 mg of trihexyphenidyl hydrochloride add 25 ml of chloroform R, shake thoroughly. Filter, and evaporate the filtrate, by gently heating, to about 10 ml, add 100 ml of n-hexane R,

TRIHEXYPHENIDYL TABLETS

a white precipitate is formed. Allow the mixture to stand for 30 min, and collect the precipitate on a solvent-resistant membrane filter of 1 µm pore size. Wash the crystals with a small portion of n-hexane R, and allow them to dry in air. The infrared absorption spectrum (Appendix 4.2) of the residue is concordant with the spectrum of trihexyphenidyl hydrochloride RS. B. The precipitate obtained in Identification test A give reaction (A) of chlorides (Appendix 8.1). C. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is the same as that of the trihexyphenidyl hydrochloride peak in the chromatogram obtained with the reference solution.

Dissolution (Appendix 11.4) Apparatus: Basket. Medium: 900 ml of pH 4.5 acetate buffer, prepared by dissolving 2.99 g of sodium acetate trihydrate R and 1.66 ml of glacial acetic acid R with water to obtain 1000 ml of solution having a pH of 4.50 ± 0.05. Rotation speed: 100 rpm. Time: 45 min. Bromocresol green solution: Dissolve 250 mg of bromocresol green R in a mixture of 15 ml of water and 5 ml of 0.1 M sodium hydroxide R, dilute with medium to 500 ml, and mix. Extract 250 ml of this solution with two 100-ml portions of chloroform R, and discard the chloroform extracts. Test solution: After the specified time, withdraw a sample of the medium and filter, discarding the first 20 ml of the filtrate. Reference solution: Prepare a solution of trihexyphenidyl hydrochloride RS in the medium having the same concentration as the test solution. Procedure: After the specified time, transfer an accurately measured volume of the test solution estimated to contain about 50 µg of trihexyphenidyl hydrochloride, to a 50-ml centrifuge tube. Add 5 ml of bromocresol green solution R and 10.0 ml of chloroform R, insert the stopper into the tube, and shake vigorously for not less than 20 seconds. Centrifuge the mixture to separate the layers, and aspirate and discard the upper aqueous layer. Filter the chloroform layer. Measure the absorbance of the filtrate at the wavelength of maximum absorbance at about 415 nm, in a 1-cm cell (Appendix 4.1) using as the compensation liquid an equal volume of medium treated in the same manner. Calculate the total content of trihexyphenidyl hydrochloride, C20H31NO,HCl, dissolve from each tablet in comparison with the reference solution prepared at the same time in the same manner. Tolerance: Not less than 75% (Q) of the stated amount of trihexyphenidyl hydrochloride, C20H31NO,HCl, is dissolved in 45 min.

991

VP V

TRIMETAZIDINE HYDROCHLORIDE

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: A mixture of acetonitrile - water triethylamine (920 : 80 : 0.2) adjusted to pH 4.0 with phosphoric acid R. Reference solution: Dissolve a quantity of trihexyphenidyl hydrochloride RS in mobile phase to obtain a concentration of about 0.2 mg/ml. Test solution: Weigh 20 tablets, calculate the average mass and powder finely. Weigh accurately a quantity of the powder equivalent to about 20 mg of trihexyphemidyl in a 100 ml volumetric flask, add 70 ml of the mobile phase and sonicate for 5 min. Dilute with the mobile phase to volume, mix and filter. Chromatographic system: A column (8 cm × 4.6 mm) packed with stationary phase C (3 µm). Detector: A spectrophotometer set at 210 nm. Flow rate: 2.0 ml/min. Volume of injection: 10 µl. Procedure: Inject the reference solution and the test solution. Calculate the content of trihexyphenidyl hydrochloride, C20H31NO,HCl, using the areas of the principal peaks obtained with the reference solution, the test solution and the declared contents of C20H31NO,HCl in trihexyphenidyl hydrochloride RS. Storage Store in a cool and dry place, protected from light. Action and use Treatment of Parkinson’s disease. Usual strength 2 mg; 5 mg. TRIMETAZIDINE HYDROCHLORIDE Trimetazidini hydrochloridum

C14H22N2O3,2HCl 



M. 339.3

Trimetazidine hydrochloride is sodium 1-(2,3,4-trimethoxybenzyl) piperazine dihydrochloride. It contains not less than 98.5 % and not more than 101.5 % of C14H22N2O3,2HCl, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder, slightly hygroscopic. Freely soluble in water, sparingly soluble in ethanol (96%). 992

Identification A.The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of trimetazidine hydrochloride RS. B. Dissolve 25 mg in 5 ml of water. 2 ml of the solution gives reaction (A) of chlorides (Appendix 8.1). Apperance of solution Dissolve 1.0 g of the substance to be examined in water and dilute to 10 ml with the same solvent. The solution is clear (Appendix 9.2) and not more intensely coloured than reference solution BY6 (Appendix 9.3, method 2). Related substances Examine by liquid chromatography (Appendix 5.3) Mobile phase A: Methanol - a 2.87 g/L solution of sodium heptanesulfonate adjusted to pH 3.0 with a 25% solution of acid phosphoric (357 : 643). Mobile phase B: Methanol. Test solution: Dissolve 0.200 g of the substance to be examined in water and dilute to 50.0 ml with the same solvent. Reference solution (1): Dissolve 20.0 mg of trimetazidine for system suitability RS in water and dilute to 5.0 ml with the same solvent. Reference solution (2): Dilute 2.0 ml of the test solution to 100.0 ml with water. Dilute 5.0 ml of this solution to 100.0 ml with water. Reference solution (3): Dilute 25.0 ml of reference solution (2) to 50.0 ml with water. Chromatographic system: A column (15 cm × 4.6 mm) packed with spherical octadecylsilyl silica gel for chromatography (5 µm). Temperature: 30 °C. Detector: A spectrophotometer set at 240 nm. Flow rate: 1.2 ml/min. Volume of injection: 10 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 50

95 → 75

5 → 25

50 - 52

75 → 95

25 → 5

Equilibrate the column with a mobile phase at the initial composition for at least 1 h. Inject the blank, test solution and reference solution (1), (2) and (3). Relative retention: With reference to trimetazidine (retention time = about 25 min): impurity D = about 0.2; impurity C = about 0.4; impurity H = about 0.6; impurities A and I = about 0.9; impurity E = about 0.95; impurity F = about 1.4; impurity B = about 1.8. System suitability:

TRIMETAZIDINE TABLETS

VP V

Peak-to-valley ratio: Minimum 3, where Hp = height above the baseline of the peak due to impurity E and Hv = height above the baseline of the lowest point of the curve separating this peak from the principal peak in the chromatogram obtained with reference solution (1); Signal-to-noise ratio: Minimum 10 for the principal peak in the chromatogram obtained with reference solution (3). Limits: Correction factors: for the calculation of contents, multiply the peak areas of the following impurities by the corresponding correction factor: impurity B = 0.55; impurity C = 0.37; impurity F = 0.71; Impurities A, B, C, D, E, F, H, I: for each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.1 %). Any other impurity: for each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.1 %). The sum of the peak areas of all impurities is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (2) (0.2%). Disregard any peak with an area less than the area of the principal peak in the chromatogram obtained with reference solution (3) (0.05%). Note: Impurity A: 1-(3,4,5-trimethoxybenzyl)piperazine. Impurity E: 1-(2,4,5-trimethoxybenzyl)piperazine. Impurity F: 1-(2,4,6-trimethoxybenzyl)piperazine. Impurity B: 1,4-bis(2,3,4-trimethoxybenzyl)piperazine. Impurity C: 2,3,4-trimethoxybenzaldehyde. Impurity D: (2,3,4-trimethoxyphenyl)methanol. Impurity G: piperazine. Impurity H: ethyl 4-(2,3,4-trimethoxybenzyl)piperazine-1carboxylate. Impurity I: 1-methyl-4-(2,3,4-trimethoxybenzyl)piperazine (N-methyltrimetazidine).

Piperazine (impurity G) Not more than 0.1 % (expressed as anhydrous piperazine) Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Concentrated ammonia - ethanol (96%) (20 : 80). Test solution: Dissolve 0.10 g of the substance to be examined in methanol R and dilute to 10 ml with the same solvent. Reference solution: Dissolve 22.6 mg of piperazine hydrate R in methanol R and dilute to 100 ml with the same solvent. Dilute 10 ml of the solution to 100 ml with methanol R. Procedure: Apply to the plate 10 µl of each solution. Develop over 2/3 of the plate. Remove the plate and heat at 100 °C - 105 °C for 30 min. Spray with iodoplatinate reagent R. In the chromatogram obtained with the test solution, any spot due to piperazine is not more intense than the spot in the chromatogram obtained with the reference solution.

Loss on drying Not more than 2.5 % (Appendix 9.6). (1.000 g; 105 °C; diphosphorus pentoxide R, a pressure not exceeding 15 kPa). Sulfated ash Not more than 0.1 % (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.120 g in 50.0 ml of water. Add 1 ml of nitric acid R and titrate with 0.1 N silver nitrate VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N silver nitrate VS is equivalent to 16.96 mg of C14H22N2O3,2HCl. Storage Store in an airtight container. Action and use Vasodilator. Preparation Tablets. TRIMETAZIDINE TABLETS Tabellae Trimetazidini Trimetazidine tablets contain trimetazidine hydrochloride. The tablets comply with the requirements stated under "Tablets" (Appendix 1.20) and with the following requirements:

Content of trimetazidine, C14H22N2O3,2HCl, from 94.0% to 106.0% of the stated amount. Identification A. Shake a quantity of powdered tablets containing about 10 mg of trimetazidine hydrochloride in 10 ml of a mixture of 3 volumes of ethanol (95%) R and 1 volume of water, filter. Evaporate to dryness on a water bath. Dissolve the residue in 2 ml of water. To 1 ml of the resulting solution, add 1 ml of p-benzoquinone solution R, boil for about 2 - 3 min, allow to cool, a red colour is produced. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that of principal peak in the chromatogram obtained with the reference solution. Uniformity of content (Appendix 11.2) Examine by liquid chromatography (Appendix 5.3). Mobile phase, chromatographic system and procedure are described in the Assay. Test solution: Transfer 1 tablet into a 100-ml volumetric flask, add about 70 ml of a mixture of ethanol R and 0.1 M hydrochloride acid R (1 : 1), shake to disintegrate, sonicate for 10 min, add to volume with the same solvent, shake well, filter. Dilute 5.0 ml of the solution to 100.0 ml with 0.1 M hydrochloride acid R. 993

VP V

TRIMETHOPRIM

Reference solution: Weigh accurately a quantity of trimetazidine hydrochloride RS corresponding to the quantity of trimetazidine in a tablet and transfer into a 100-ml volumetric flask, add about 70 ml of a mixture of ethanol and 0.1 M hydrochloride acid (1 : 1), sonicate for 10 min, dilute to volume with the same solvent, shake well and filter. Dilute 5.0 ml of the filtrate to 100.0 ml with 0.1 M hydrochloride acid R.

Dissolution (Appendix 11.4). Apparatus: Paddle. Medium: 900 ml of water. Rotation speed: 50 rpm. Time: 45 min. Procedure: Test solution: After the specified time, withdraw a sample of the medium, filter, discard the first few ml of the filtrate. Dilute the filtrate with 0.1 M hydrochloride acid R to obtain a solution containing about 10 µg of trimetazidine hydrochloride per ml. Reference solution: Weigh accurately about 20.0 mg of trimetazidine hydrochloride RS, transfer into a 100-ml volumetric flask, dissolve in the medium and add to volume with the medium, shake well. Dilute 5.0 ml of the obtained solution to 100.0 ml with 0.1 M hydrochloride acid R. Determine the dissolved content of trimetazidine hydrochloride by liquid chromatography (Appendix 5.3) with the mobile phase, chromatographic conditions described in the Assay. Tolerance: Not less than 80% (Q) of the labeled amount of trimetazidine hydrochloride, C14H22N2O3,2HCl, is dissolved in 45 min. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: A mixture of 0.05 M potassium dihydrogen phosphate R previously adjusted to pH 3.0 with phosphoric acid R and methanol R (17 : 3). Make adjustments if necessary. Test solution: Weigh 20 tablets, calculate the average weight and finely powder. Weigh accurately a quantity of the powdered tablets containing about 20 mg of trimetazidine hydrochloride. Transfer into a 100-ml volumetric flask, add about 70 ml of a mixture of ethanol R and 0.1 M hydrochloride acid R (1 : 1), sonicate for 10 min, add to volume with the same solvent, shake well and filter. Dilute 5.0 ml of the filtrate to 100.0 ml with 0.1 M hydrochloride acid R. Reference solution: Weigh accurately about 20.0 mg of trimetazidine hydrochloride RS into 100-ml volumetric flask, dissolve in a mixture of ethanol R and 0.1 M hydrochloride acid R (1 : 1), dilute to volume with the same solvent, shake well. Dilute 5.0 ml of the filtrate to 100.0 ml with 0.01 M hydrochloride acid R. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C (5 µm), maintained at 40 °C. 994

Detector: A spectrophotometer set at 230 nm. Flow rate: 1.0 ml/min, adjust the flow rate so that the retention time of trimetazidine hydrochloride of about 7 min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution, in the chromatogram obtained, the tailing factor of the trimetazidine peak is not more than 1.5; the column efficiency determined on the trimetazidine peak is not less than 5000 theoretical plates; the relative standard deviation of the peak areas for 6 replicate injections of the reference solution is not more than 2.0%. Inject separately the reference solution and the test solution. Calculate the content of trimetazidine hydrochloride, C14H22N2O3,2HCl in the tablets using the areas of the principal peaks in the chromatograms obtained with the reference solution, test solution and the content of C14H22N2O3,2HCl in trimetazidine hydrochloride RS.

Storage Store in air-tight container in a cool place. Action and use Vasodilator. Usual strength 20 mg. TRIMETHOPRIM Trimethoprimum

C14H18N4O3  

M. 290.3

Trimethoprim is 5-(3,4,5-trimethoxybenzyl)pyrimidine2,4-diamine. It contains not less than 98.5% and not more than 101.0% of C14H18N4O3, calculated with reference to the dried substance.

Characters White or yellowish-white powder. Very slightly soluble in water, slightly soluble in ethanol (96%). Identification Apply one of the two following identifications: First identification: A. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of trimethoprim RS. Examine the substances prepared as discs. B. Dissolve about 20 mg in 0.1 M sodium hydroxide R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml of this solution to 10.0 ml with 0.1 M sodium hydroxide R.

VP V

Examined the ultraviolet absorption spectrum (Appendix 4.1) of the solution in the range 230 nm to 350 nm. The solution shows an absorption maximum at 287 nm. The specific absorbance A (1%, 1 cm) at the absorption maximum is 240 to 250. C. Melting point: 199 °C to 203 °C (Appendix 6.7). D. Dissolve about 25 mg, heating if necessary, in 5 ml of 0.005 M sulfuric acid R and add 2 ml of a 1.6% solution of potassium permanganate R in 0.1 M sodium hydroxide R. Heat to boiling and add to the hot solution 0.4 ml of formaldehyde R. Mix, add 1 ml of 0.5 M sulfuric acid R, mix and heat again to boiling. Cool and filter. To the filtrate, add 2 ml of methylene chloride R and shake vigorously. The organic layer, examined in ultraviolet light at 365 nm, shows green fluorescence.

Appearance of solution Dissolve 0.5 g in 10 ml of a mixture of methylene chloride - methanol - water (5 : 4.5 :1). The solution is not more intensely coloured than reference solution BY7 (Appendix 9.3, method 2). Related substances A. Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol - a 1.4 g/L solution of sodium perchlorate R adjusted to pH 3.6 with phosphoric acid R (30 : 70). Test solution: Dissolve 25.0 mg of the substance to be examined in the mobile phase and dilute to 25.0 ml with the mobile phase. Reference solution (1): Dilute 1.0 ml of the test solution to 200.0 ml with the mobile phase. Reference solution (2): Dissolve the contents of a vial of trimethoprim for system suitability RS (containing impurity E) in 1 ml of the mobile phase. Chromatographic system: A column (25 cm × 4.0 mm) packed with stationary phase base-deactivated octadecylsilyl silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 280 nm. Flow rate: 1.3 ml/min. Volume of injection: 20 µl. Procedure: The run time is 11 times the retention time of trimethoprim. Relative retention with reference to trimethoprim (retention time = about 5 min): Impurity C = about 0.8; impurity E = about 0.9; impurity A = about 1.5; impurity D = about 2.0; impurity G = about 2.1; impurity B = about 2.3; impurity J = about 2.7; impurity F = about 4.0. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurity E and trimethoprim is at least 2.5. Limits: Correction factors: For the calculation of content, multiply the peak areas of the following impurities by the corresponding correction factor: impurity B = 0.43; impurity E = 0.53; impurity J = 0.66;

TRIMETHOPRIM

Any other impurity: For each impurity, the area, corrected if necessary, is not more than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%). The sum of the peak areas of all impurities is not more than 0.4 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%). Disregard any peak with an area less than 0.04 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.02%). Disregard any peak corresponding to impurity H (relative retention = about 10.3). B. Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 1.14 g of sodium hexane sulfonate R in 600 ml of a 1.36% solution of potassium dihydrogen phosphate R; adjust to pH 3.1 with phosphoric acid R and mix with 400 ml of methanol R. Test solution: Dissolve 25.0 mg of the substance to be examined in the mobile phase and dilute to 25.0 ml with the mobile phase. Reference solution (1): Dilute 1.0 ml of the test solution to 200.0 ml with the mobile phase. Reference solution (2): Dissolve 5.0 mg of trimethoprim RS and 5.0 mg of trimethoprim impurity B RS in the mobile phase and dilute to 100.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase nitrile silica gel for chromatography R (5 µm) with a specific surface area of 350 m2/g and a pore diameter of 10 nm. Detector: A spectrophotometer set at 280 nm. Flow rate: 0.8 ml/min. Volume of injection: 20 µl. Procedure: The run time is 6 times the retention time of trimethoprim. Relative retention with reference to trimethoprim (retention time = about 4 min): Impurity H = about 1.8; impurity I = about 4.9. System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to trimethoprim and impurity B is at least 2.0. Limits: Correction factors: For the calculation of content, multiply the peak areas of the following impurities by the corresponding correction factor: impurity H = 0.50; impurity I = 0.28; Any other impurity: For each impurity, the area, corrected if necessary, is not more than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%). Total peak area of all impurities is not more than 0.4 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.2%). Disregard any peak with an area less than 0.04 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.02%). Disregard any peak due to impurity B (relative retention = about 1.3). 995

VP V

COTRIMOXAZOLE TABLETS Note: Impurity A: N2-methyl-5-(3,4,5-trimethoxybenzyl)pyrimidine2,4-diamine. Impurity B: (2,4-diaminopyrimidin-5-yl)(3,4,5-trimethoxyphenyl) methanone. Impurity C: (RS)-(2,4-diaminopyrimidin-5-yl)(3,4,5trimethoxyphenyl)methanol. Impurity D: 2-amino-5-(3,4,5-trimethoxybenzyl)pyrimidin-4-ol. Impurity E: 4-amino-5-(3,4,5-trimethoxybenzyl)pyrimidin-2-ol. Impurity F: 5-(3-bromo-4,5-dimethoxybenzyl)pyrimidine-2,4diamine. Impurity G: 5-(4-ethoxy-3,5-dimethoxybenzyl)pyrimidine-2,4diamine. Impurity H: methyl 3,4,5-trimethoxybenzoate, Impurity J: 3,4,5-trimethoxybenzoic acid. Impurity I: 3-(phenylamino)-2-(3,4,5-trimethoxybenzyl)prop-2enenitrile.

Loss on drying Not more than 1.0% (Appendix 9.6). (1.000 g; 105 °C).

Impurity K Examine by gas chromatography (Appendix 5.2). Test solution: Dissolve 0.500 g of the substance to be examined in 35.0 ml of citrate buffer solution pH 5.0 R, add 10.0 ml of 1,1-dimethylethyl methyl ether R, shake thoroughly and centrifuge for 10 min. Use the upper layer. Reference solution: Dilute 5.0 ml of hydrochloric acid R to 50.0 ml with water, add 12.5 mg of aniline R and shake thoroughly. Add 10.0 µL of this solution and 10.0 ml of 1,1-dimethylethyl methyl ether R to 35.0 ml of citrate buffer solution pH 5.0 R, shake thoroughly and centrifuge for 10 min. Use the upper layer. Chromatographic system: A fused-silica capillary column (30 m × 0.53 mm) coated with a film of poly(dimethyl)siloxane R (film thickness 3 µm). Carrier gas: Helium for chromatography. Flow rate: 12 ml/min. Temperature: column: 80 °C; injection port: 230 °C; detector: 270 °C. Detector: Nitrogen-phosphorus detector. Volume of injection: 3 µl. Run time: 15 min. Procedure: System suitability: In the chromatogram obtained with the reference solution, the relative standard deviation of the peak areas for 6 replicate injections is not more than 5.0%. Limit: Impurity K: The area of the peak due to impurity K is not more than the area of the principal peak in the chromatogram obtained with the reference solution (5 ppm).

Preparation Tablets.

Note: Impurity K: Aniline.

Heavy metals Not more than 20 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. 996

Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.250 g of the substance to be examined in 50 ml of anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 29.03 mg of C14H18N4O3. Action and use Antibacterial.

COTRIMOXAZOLE TABLETS Tabellae Cotrimoxazoli Cotrimoxazole tablets contain trimethoprim and sulfamethoxazole in the proportions 1 part to parts 5 by weight The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of trimethoprim, C14H18N4O3, 93.0% to 107.0% of the stated amount of trimethoprim. Content of sulfamethoxazole, C10H11N3O3S, 93.0% to 107.0% of the stated amount of sulfamethoxazole. Identification A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Chloroform - methanol - dimethylformamide (100 : 10 : 5). Test solution: Shake a quantity of the powdered tablets containing 0.4 g of sulfamethoxazole with 20 ml of methanol R and filter. Reference solution (1): A 2.0% solution of sulfamethoxazole RS in methanol R. Reference solution (2): A 0.4% solution of trimethoprim RS in methanol R. Procedure: Apply separately to the plate 5 µl of each solution. After removal of the plate, allow it to dry at room temperature and spray with the dilute potassium iodobismuthate solution R. Heat the plate at 100 °C for 5 to 10 min in case no spot appears. One of the two principal spots in the chromatogram obtained with the test solution

VANCOMYCIN HYDROCHLORIDE

VP V

corresponds to the spot in the chromatogram obtained with reference solution (1) and the other corresponds to the spot in the chromatogram obtained with reference solution (2). B. In the Assay, the retention times of two principal peaks in the chromatogram obtained with the test solution are similar to those of two principal peaks in the chromatogram obtained with the reference solution.

Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of 0.1 M hydrochloric acid R. Rotation speed: 75 rpm. Time: 60 min. Procedure: After the specified time, withdraw a sample of the medium and filter. Dilute the filtrate with the mobile phase (if necessary) to obtain a suitable concentration. Determine the content of sulfamethoxazole and trimethoprim dissolved in the medium by liquid chromatography (Appendix 5.3) with the mobile phase, chromatographic system and reference solution as described in the Assay. Tolerance: Not less than 70% (Q) of the stated amount of trimethoprim, C14H18N4O3, is dissolved in 60 min. Not less than 70% (Q) of the stated amount of sulfamethoxazole, C10H11N3O3S, is dissolved in 60 min. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Mix 1400 ml of water, 400 ml of acetonitrile R and 2.0 ml of triethylamine R in a 2000-ml volumetric flask. Allow to stand at room temperature, adjust the pH to 5.9 ± 0.1 with 0.2 M sodium hydroxide R or with 1% acetic acid solution R, add sufficient water to produce 2000 ml, mix. Reference solution: Dissolve an accurate quantity of trimethoprim RS and sulfamethoxazole RS in methanol R to obtain a stock reference solution having concentration of each substance is 0.32 mg per ml and 1.6 mg per ml, respectively. Dilute 5.0 ml of the stock reference solution to 50.0 ml with the mobile phase, mix. The concentration of the final reference solution is 0.032 mg trimethoprim per ml and 0.16 mg of sulfamethoxazole per ml. Test solution: Weigh 20 tablets, calculate the average mass and finely powder. Weigh accurately a quantity of the powder containing about 160 mg of sulfamethoxazole and transfer into a 100-ml volumetric flask, add 50 ml of methanol R and sonicate for about 5 min. Allow to stand at room temperature, dilute to volume with the methanol R, mix and filter. Dilute 5.0 ml of the resulting solution to 50.0 ml with the mobile phase, mix. Filter through a 0.45-µm membrane. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (3 µm to 10 µm). Detector: A spectrophotometer set at 254 nm.

Flow rate: 2.0 ml/min. Volume of injection: 20 µl Procedure: System suitability: Inject the reference solution. In the chromatogram obtained, the elution order is trimethoprim and sulfamethoxazole, respectively. The resolution factor between the peaks due to sulfamethoxazole and to trimethoprim is not less than 5.0. The symmetry factors of trimethoprim and sulfamethoxazole are not more than 2.0 and the relative standard deviation for replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution. Calculate the content of trimethoprim, C14H18N4O3 and sulfamethoxazole, C10H11N3O3S, in the tablets using the peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C14H18N4O3 in trimethoprim RS and of C10H11N3O3S in sulfamethoxazole RS.

Storage Store in a well-closed container, protected from light. Action and use Antibacterial. Usual strength 80 mg of trimethoprim and 400 mg of sulfamethoxazole. VANCOMYCIN HYDROCHLORIDE Vancomycini hydrochloridum

C66H75Cl2N9O24,HCl

M. 1486.0

Vancomycin hydrochloride is the hydrochloride of a mixture of related glycopeptides, consisting principally of the monohydrochloride of (3S, 6R, 7R, 22R, 23S, 26S, aS, 36R, 38aR)-3-(2-amino-2oxoethyl)-44-[[2-O-(3-amino-2,3,6-trideoxy-3-C997

VANCOMYCIN HYDROCHLORIDE

methyl-α-L-lyxo-hexopyranosyl)-β-D-glucopyranosyl] oxy]-10,19-dichloro-7,22,28,30,32-pentahydroxy6-[[(2R)4-methyl-2-(methylamino)pentanoyl]amino]-2,5,24,38,39pentaoxo-2,3,4,5,6, 7,23,24,25,26, 36,37,38,38a – tetradecahydro22H-8,11:18,21-dietheno-23,36-(iminomethano)-13,16:31,35dimetheno-1H,13H -[1,6,9]oxadiazacyclohexadecino[4,5-m] [10,2,16] benzoxadiazacyclotetracosine-26-carboxylic acid (vancomycin B), substance produced by certain strains of Amycolatopsis orientalis or obtained by any other means. The potency is not less than 1050 IU/mg, calculated with reference to the anhydrous substance.

Characters A white or almost white powder, hygroscopic. Freely soluble in water, slightly soluble in ethanol (96%). Identification A. In the test for Vancomycin B, the retention time of the principal peak in the chromatogram obtained with test solution (1) is similar to that of the principal peak in the chromatogram obtained with the reference solution. B. It gives reaction (A) of chlorides (Appendix 8.1). Appearance of solution Dissolve 2.50 g of the substance to be examined in water and dilute to 25.0 ml with the same solvent, and mix. The solution is clear (Appendix 9.2). The absorbance (Appendix 4.1) of the solution measured at 450 nm is not greater than 0.10. pH Dissolve 0.50 g of the substance to be examined in carbon dioxide-free water R and dilute to 10 ml with the same solvent, and mix. The pH of the solution is 2.5 to 4.5 (Appendix 6.2). Vancomycin B Not less than 93.0%. Examine by liquid chromatography (Appendix 5.3). Mobile phase A: To 4.0 ml of triethylamine R add 1996 ml of water and adjust to pH 3.2 with phosphoric acid R. To 920 ml of this solution add 10 ml of tetrahydrofuran R and 70 ml of acetonitrile R, and mix. Mobile phase B: To 4.0 ml of triethylamine R add 1996 ml of water and adjust to pH 3.2 with phosphoric acid R. To 700 ml of this solution add 10 ml of tetrahydrofuran R and 290 ml of acetonitrile R, and mix. Test solution (1): Dissolve 10.0 mg of the substance to be examined in mobile phase A and dilute to 5.0 ml with mobile phase A, and mix. Test solution (2): Dilute 2.0 ml of test solution (1) to 50.0 ml with mobile phase A, and mix. Test solution (3): Dilute 0.5 ml of test solution (2) to 20.0 ml with mobile phase A, and mix. Reference solution: Dissolve 5 mg of vancomycin hydrochloride RS in 4 ml of water and dilute to 10 ml with the same solvent. Heat at 65 °C for 24 h. Allow to cool. 998

VP V

Use the solutions within 4 h of preparation. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 280 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Elute initially with mobile phase A. After 13 min, use gradient elution increasing the concentration of mobile phase B by 11% v/v per min. Finally, elute for 4 min using mobile phase B. Inject test solution (3). The test is not valid unless the principal peak in the chromatogram obtained has a signalto-noise ratio of at least 5. Inject test solution (2). The test is not valid unless the symmetry factor of the vancomycin peak is at most 1.6. Inject the reference solution. The test is not valid unless the resolution between the two principal peaks is at least 5.0. Inject test solution (1). Calculate the percentage content of vancomycin B hydrochloride using the expression: Ab × 100 Ab + (At/25) Where: Ab = area of the peak corresponding to vancomycin B in the chromatogram obtained with test solution (2). At = sum of the areas of the peaks corresponding to impurities in the chromatogram obtained with test solution (1).

Related substances Examine by liquid chromatography (Appendix 5.3), as described under the test for Vancomycin B. Inject separately test solution (1), test solution (2) and test solution (3). Calculate the percentage content of each impurity using the expression: (Ai/25) × 100 Ab + (At/25) Where: Ai = area of an impurity peak in the chromatogram obtained with test solution (1). Ab = area of the peak corresponding to vancomycin B in the chromatogram obtained with test solution (2). At = sum of the areas of the peaks corresponding to impurities in the chromatogram obtained with test solution (1). The content of no impurity is greater than 4.0% and the sum of the contents of impurities is not greater than 7.0%. Disregard any peak with an area less than that of the principal peak in the chromatogram obtained with test solution (3). Heavy metals Not more than 30 ppm (Appendix 9.4.8). 1.0 g complies with the limit test for heavy metals, method 3. Prepare the standard using 3.0 ml of lead standard solution (10 ppm Pb) R.

VANCOMYCIN POWDER FOR INJECTION

VP V

Water Not more than 5.0% (Appendix 10.3). Determined on 0.5 g.

test solution is similar to that of the principal peak in the chromatogram obtained with the reference solution. B. Yield reaction A characteristic of chlorides (Appendix 8.1).

Sulfated ash Not more than 1.0% (Appendix 9.9). Determined on 1.0 g.

Appearance of solution A solution containing 10.0 % of vancomycin hydrochloride is clear (Appendix 9.2). The absorbance of the solution at 450 nm is not greater than 0.10 (Appendix 4.1).

Sterility If intended for use in the manufacture of parenteral dosage forms without a further appropriate sterilisation procedure, it complies with the test for sterility (Appendix 13.7). Bacterial endotoxins Not more than than 0.25 EU/mg (Appendix 13.2). If intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins. Assay Carry out the microbiological assay of antibiotics (Appendix 13.9). Use vancomycin hydrochloride RS as the reference substance. Storage Store in an airtight container, protected from light. If the substance is sterile, store in a sterile, tamper-proof container. Labelling The label states, where applicable, that the substance is sterile and free from bacterial endotoxins. Action and use Antibacterial. Preparations Injection; capsules. VANCOMYCIN POWDER FOR INJECTION Vacomycini pulvis ad injectionem Vancomycin powder for Injection is a sterile material consisting of vancomycin hydrochloride with or without excipients. It is supplied in a sealed container . The contents of the sealed container comply with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Content of vancomycin hydrochloride, C66H75Cl2N9O24,HCl, 90.0 % to 115.0 % of the stated amount. Characters A white or almost white powder. Identification A. In the test for Vancomycin B, the retention time of the principal peak in the chromatogram obtained with the

pH pH of a solution containing 5% of vancomycin hydrochloride, 2.5 to 4.5 (Appendix 6.2). Vancomycin B Not less than 88.0%. Examine by liquid chromatography (Appendix 5.3). Solution A: To 4 ml of triethylamine add 1996 ml of water and adjust to pH 3.2 with orthophosphoric acid R. Mobile phase A: Solution A - acetonitrile - tetrahydrofuran (920 : 70 : 10). Mobile phase B: Solution A - acetonitrile - tetrahydrofuran (700 : 290 : 10). Test solution (1): Dissolve a quantity of the contents of the sealed container containing the equivalent of 50 000 IU of vancomycin in mobile phase A and dilute to 25.0 ml with the same solvent. Test solution (2): Dilute 2.0 ml of solution (1) to 50.0 ml with mobile phase A. Test solution (3): Dilute 0.5 ml of solution (2) to 20.0 ml with mobile phase A. Reference solution: Dissolve 5.0 mg of vancomycin hydrochloride RS in 10 ml of water. Heat the solution at 65 °C for 24 hours and allow to cool. Use the solutions within 4 hours of preparation. Chromatographic system: A stainless steel column (25 cm × 4.6 mm) packed with end-capped octadecylsilyl silica gel (5 µm). Detector: A spectrophotometer set at 280 nm. Flow rate: 1.0 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

Comment

0 - 13

100

0

Isocratic

13 - 21

100 → 0

0 → 100

Linear gradient

21 - 25

0

100

Isocratic

25 - 35

100

0

Re-equilibration

System suitability: Inject test solution (3), the test is not valid unless in the chromatogram obtained the signal-to-noise ratio of the principal peak is not less than 5. Inject test solution (2), the test is not valid unless, the symmetry factor of the vancomycin peak is not greater than 1.6. Inject the reference 999

VP V

VANILLIN

solution, the test is not valid unless, the resolution factor between the two principal peaks is at least 5.0. Inject test solution (1). Calculate the percentage of vancomycin B using the following expression: Ab × 100 Ab + (At/25) Where:Ab: Area of the peak corresponding to vancomycin B in the chromatogram obtained with test solution (2). At: Sum of the areas of the peaks corresponding to impurities in the chromatogram obtained with test solution (1).

Related substances Carry out the method for liquid chromatography (Appendix 5.3), using test solutions (1), (2) and (3) as described under Vancomycin B. Calculate the percentage content of each impurity using the following expression: (Ai/25) × 100 Ab + (At/25) Where: Ai : Area of an impurity in the chromatogram obtained with test solution (1). Ab: Area of the peak corresponding to vancomycin B in the chromatogram obtained with test solution (2). At: Sum of the areas of the peaks corresponding to impurities in the chromatogram obtained with test solution (1). The content of any impurity is not greater than 4.0 % and the sum of the contents of any such impurities is not greater than 12.0 %. Disregard any peak with an area less than the area of the principal peak in the chromatogram obtained with test solution (3) (0.1%).

Water Not more than 5.0 % (Appendix 10.3). Determined on 0.5 g. Bacterial endotoxins Carry out the test for bacterial endotoxins (Appendix 13.2). Dissolve the contents of the sealed container in trischloride buffer pH 7.4 R prepared using water BET to give a solution containing 1000 IU of vancomycin per ml (solution A). The endotoxin limit concentration of solution A is 2.5 EU of endotoxin per ml. Carry out the test using the maximum valid dilution of solution A calculated from the declared sensitivity of the lysate used in the test. Assay Weigh 20 containers, calculate the average weight of the contents of the containers. Mix the contents of the 20 containers and carry out the microbiological assay of antibiotics (Appendix 13.9). Storage Store in a cool place, protected from light. 1000

Action and use Antibacterial. Usual strength 0.5 g; 0.1 g. VANILLIN Vanillinum HO H3CO

C8H8O3

CHO

M. 152.1

Vanillin is 4-hydroxy-3-methoxybenzaldehyde. It contains not less than 99.0% and not more than 101.0% of C8H8O3, calculated with reference to the dried substance.

Characters White or slightly yellowish, crystalline powder or needles. Slightly soluble in water, freely soluble in ethanol (96%) and in methanol. It dissolves in dilute solutions of alkali hydroxides. Identification Apply one of the two following identifications: First identification: A. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of vanillin RS. B. Melting point: 81 °C to 84 °C (Appendix 6.7). C. In the test for Related substances, examine the chromatograms obtained in daylight after spraying, the principal spot in the chromatogram obtained with test solution (2) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (1). D. To 5 ml of a saturated solution of the substance to be examined add 0.2 ml of a 10.5% solution of ferric chloride R. A blue colour is produced. Heat to 80 °C. The solution becomes brown. On cooling, a white precipitate is formed. Appearance of solution Dissolve 1.0 g of the substance to be examined in ethanol (96%) R and dilute to 20 ml with the same solvent, and mix. The solution is clear (Appendix 9.2) and not more intensely coloured than reference solution B6 (Appendix 9.3, method 2). Related substances Not more than 0.5%. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254.

VASELINE

VP V

Mobile phase: Anhydrous acetic acid - methanol methylene chloride (0.5 : 1 : 98.5). Test solution (1): Dissolve 0.1 g of the substance to be examined in methanol R and dilute to 5 ml with the same solvent, and mix. Test solution (2): Dilute 1 ml of test solution (1) to 10 ml with methanol R, and mix. Reference solution (1): Dissolve 10 mg of vanillin RS in methanol R and dilute to 5 ml with the same solvent, and mix. Reference solution (2): Dilute 0.5 ml of test solution (1) to 100 ml with methanol R, and mix. Procedure: Apply separately to the plate 5 µl of each solution. Develop in an unsaturated tank over a path of 10 cm. Remove the plate, dry it in a current of cold air. Examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with test solution (1) is not more intense than the spot in the chromatogram obtained with reference solution (2). Spray with dinitrophenylhydrazineacetohydrochloric solution R and examine in daylight. Any secondary spot in the chromatogram obtained with test solution (1) is not more intense than the spot in the chromatogram obtained with reference solution (2).

Characters A off-white, translucent, soft unctuous mass, almost anhydrous. The thin-layers are transparent and almost colourless. It is slightly fluorescent in daylight when melted. The melting range is between 36 °C and 60 °C. Practically insoluble in water and in ethanol, soluble in chloroform and in ether. It is miscible with methylene chloride when melted. The solutions may be slightly opalescent.

Reaction with sulfuric acid Dissolve 50 mg of the substance to be examined in 5 ml of sulfuric acid R. After 5 min, the solution is not more intensely coloured than a mixture of 4.9 ml of yellow primary solution and 0.1 ml of red primary solution or a mixture of 4.9 ml of yellow primary solution and 0.1 ml of blue primary solution (Appendix 9.3, method 1).

Homogeneity The substance to be examined remains homogeneous when kept at 20 °C for 1 h.

Loss on drying Not more than 1.0% (Appendix 9.6). (1.000 g; in a desiccator; 4 h). Sulfated ash Not more than 0.05% (Appendix 9.9, method 2). Determined on 2.0 g. Assay Dissolve 0.120 g of the substance to be examined in 20 ml of ethanol (96%) R and add 60 ml of carbon dioxide-free water R. Titrate with 0.1 N sodium hydroxide VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N sodium hydroxide VS is equivalent to 15.21 mg of C8H8O3. Storage Store in an airtight container, protected from light. Action and use Excipient. VASELINE Vaselinum album Vaseline is a purified and decolorised mixture of hydrocarbons, obtained from petroleum.

Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined exhibits absorption maxima at the wavelengths of 2950 cm-1, 2920 cm-1, 2850 cm-1, 1460 cm-1, 1375 cm-1, 725 cm-1 and 715 cm-1. Prepare a film of the substance to be examined on a halogenide plate having a transmittance of 5% at 2915 cm-1. B. Melt 2 g of the substance to be examined and when a homogeneous phase is obtained, add 2 ml of water and 0.2 ml of 0.1 N iodine R. Shake, and allow to cool. The solid upper layer is violet-pink.

Acidity or alkalinity To 10 g of the substance to be examined add 20 ml of boiling water and shake vigorously for 1 minutes. Allow to cool and decant. To 10 ml of the aqueous layer add 0.1 ml of phenolphthalein solution R. The solution is colourless. Not more than 1.0 ml of 0.01 N sodium hydroxide VS is required to change the colour of the indicator to pink. Readily carbonisable substances To 0.5 g of the substance to be examined in a stoppered test-tube add 20.0 ml of sulfuric acid R. Heat in a water bath for 10 minutes, shaking for 5 seconds every two minutes. Allow to cool, and transfer to a dry separating funnel, and allow to stand for 10 minutes. Collect the lower layer, if necessary filter through a sintered glass filter No.4. Measure the absorbance (Appendix 4.1) of the solution in the range between 400 nm and 450 nm, using sulfuric acid R in the reference cell. The absorbance is not more than 0.40. Absorbance Dissolve 0.100 g of the substance to be examined in hexane R and dilute to 200.0 ml with the same solvent, and mix. Measure the absorbance (Appendix 4.1) of the solution in the range 250 to 275 nm and 300 to 350 nm, using hexane R in the reference cell. The absorbances are not more than 0.20 and 0.05, respectively. Saponification value Not more than 2 (Appendix 7.7). Determined on 2.00 g. 1001

VP V

VERAPAMIL HYDROCHLORIDE

Sulfated ash Not more than 0.03% (Appendix 9.9, method 1). Determined on 4.0 g.

Reference solution (1): Dissolve 20 mg of verapamil hydrochloride RS in methylene chloride R and dilute to 10 ml with the same solvent. Reference solution (2): Dissolve 5 mg of papaverine hydrochloride RS in reference solution (1) and dilute to 5 ml with the same solution. Procedure: Apply to the plate 5 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in air. Examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (1). The test is not valid unless the chromatogram obtained with reference solution (2) shows two clearly separated spots. D. It gives reaction (B) of chlorides (Appendix 8.1).

Storage Store in an airtight container at a cold place. Action and use Excipient. VERAPAMIL HYDROCHLORIDE Verapamili hydrochloridum

and enantiomer

C27H38N2O4,HCl

M. 491.1

Verapamil hydrochloride is (2RS)-2-(3,4-Dimethoxyphenyl)-5-[[2-(3,4-dimethoxyphenyl)ethyl](methyl) amino]-2-(1-methylethyl)pentanenitrile hydrochloride. It contains not less than 99.0% and not more than 101.0% of C27H38N2O4,HCl, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder. Soluble in water, freely soluble in methanol, sparingly soluble in ethanol (96%). Melting point about 144 °C. Identification Apply one of the two following identifications: First identification: A, D. Second identification: B, C, D. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of verapamil hydrochloride RS. B. Ultraviolet and visible absorption spectrophotometry (Appendix 4.1) Dissolve 20.0 mg in 0.01 M hydrochloric acid R and dilute to 100.0 ml with the same acid. Dilute 5.0 ml of this solution to 50.0 ml with 0.01 M hydrochloric acid R. Examined between 210 nm and 340 nm, the solution shows 2 absorption maxima, at 229 nm and 278 nm, and a shoulder at 282 nm. The ratio of the absorbance at the maximum at 278 nm to that at the maximum at 229 nm is 0.35 to 0.39. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel F254. Mobile phase: Diethylamine - cyclohexane (15 : 85). Test solution: Dissolve 10 mg of the substance to be examined in methylene chloride R and dilute to 5 ml with the same solvent. 1002

Appearance of solution Solution S: Dissolve 1.0 g in carbon dioxide-free water while gently heating and dilute to 20.0 ml with the same solvent. Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2). pH 4.5 to 6.0 (Appendix 6.2). Determined on solution S Specific optical rotation -0.10° to +0.10° determined on solution S (Appendix 6.4). Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Dissolve 6.97 g of dipotassium hydrogen phosphate R in 1000 ml of water, adjust to pH 7.2 with phosphoric acid R. Mobile phase B: Acetonitrile R. Solvent mixture: Mobile phase A - mobile phase B (63 : 37). Test solution: Dissolve 25.0 mg of the substance to be examined in the solvent mixture and dilute to 10.0 ml with the solvent mixture. Reference solution (1): Dissolve 5 mg of verapamil hydrochloride RS, 5 mg of verapamil impurity I RS and 5 mg of verapamil impurity M RS in the solvent mixture and dilute to 20 ml with the solvent mixture. Dilute 1 ml of this solution to 10 ml with the solvent mixture. Reference solution (2): Dilute 1.0 ml of the test solution to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 10.0 ml with the solvent mixture. Chromatographic system: A column (25 cm × 4.6 mm) packed with end-capped octadecylsilyl silica gel for chromatography (5 µm). Detector: A spectrophotometer set at 278 nm. Flow rate: 1.5 ml/min. Volume of injection: 10 µl. Procedure: Equilibrate the column with the solvent mixture for about 60 min.

VINBLASTINE SULFATE

VP V

Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 22

63

37

22 - 27

63 → 35

37 → 65

27 - 35

35

65

35 - 36

35 → 63

65 → 37

36 - 50

63

37

Relative retention with reference to verapamil (retention time = about 16 min): impurity I = about 21 min; impurity M = about 32 min, eluting as a doublet. System suitability: In the chromatogram obtained with reference solution (1), resolution between the peaks corresponding to verapamil and impurity I is at least 5.0 and impurity M elutes from the column. Inject separately the blank (the solvent mixture), the test solution and reference solution (2). In the chromatogram obtained with the test solution, the area of any peak, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (2) (0.1%); the sum of the areas of the peaks, apart from the principal peak, is not greater than 3 times the area of the prinpical peak in the chromatogram obtained with reference solution (2) (0.3%). Disregard any peak with an area less than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (2) (0.01%).

Heavy metals Not more than 10 ppm (Appendix 9.4.8). 1.0 g complies with limit test for heavy metals, method 3. Prepare the standard using 1 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.400 g in 50 ml of anhydrous ethanol R and add 5.0 ml of 0.01 M hydrochloric acid R. Titrate with 0.1 N sodium hydroxide VS, determining the end-point potentiometrically (Appendix 10.2). Measure the volume added between the 2 points of inflexion. 1 ml of 0.1 N sodium hydroxide VS is equivalent to 49.11 mg of C27H39ClN2O4. Storage Protected from light.

Action and use Calcium channel blocker. Preparation Tablets. VINBLASTINE SULFATE Vinblastini sulfas

C46H58N4O9, H2SO4

M. 909.0

Vinblastine sulfate is methyl (3aR,4R,5S,5aR,10bR, 13aR)4-(acetyloxy)-3a-ethyl-9-[(5S,7R,9S)-5-ethyl-5-hydroxy9-(methoxycarbonyl)-1,4,5,6,7,8,9,10-octahydro-2H-3,7methanoazacycloundecino[5,4-b]indol-9-yl]-5-hydroxy8-methoxy-6-methyl-3a,4,5,5a,6,11,12,13a-octahydro1H-indolizino[8,1-cd]carbazole-5-carboxylate sulfate. It contains not less than 95.0% and not more than 104.0% of C46H58N4O9,H2SO4, calculated with reference to the dried substance.

Characters A white or slightly yellowish, crystalline powder, very hygroscopic. Freely soluble in water, practically insoluble in ethanol (96%). Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the reference spectrum of vinblastine sulfate RS. B. In the test for Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (1). Appearance of solution Solution S: Dissolve 50.0 mg of the substance to be examined in carbon dioxide-free water R and dilute to 10.0 ml with the same solvent, and mix. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution Y7 (Appendix 9.3, method 1). pH Dilute 3 ml of solution S to 10 ml with carbon dioxidefree water R, and mix. The pH of this solution is 3.5 to 5.0 (Appendix 6.2). 1003

VP V

VINBLASTINE SULFATE FOR INJECTION

Related substances In the test for Assay, in the chromatogram obtained with the test solution: The area of any secondary peak is not greater than the area of the principal peak in the chromatogram obtained with reference solution (2) (2.0%) The sum of the areas of any such peaks is not greater than 2.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (5.0%). Disregard any peak with an area less than that of the peak in the chromatogram obtained with reference solution (3). Loss on drying Not more than 15.0% (Appendix 9.6). (0.050 g; in vacuo; 105 °C; 2 h). Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol - 1.5% (v/v) solution of diethylamine adjusted to pH 7.5 with phosphoric acid - acetoniltrile (50 : 38 : 12). Test solution: Dilute 1.0 ml of solution S to 5.0 ml with water, and mix. Reference solution (1): Dissolve 5.0 mg of vinblastine sulfate RS in water and dilute to 5.0 ml with the same solvent. Reference solution (2): Dilute 1.0 ml of reference solution (1) to 50.0 ml with water, and mix. Reference solution (3): Dilute 1.0 ml of reference solution (2) to 20.0 ml with water, and mix. Resolution solution: Dissolve 1.0 mg of vincristine sulfate RS in 1.0 ml of reference solution (1). Keep the solutions in iced water before use. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase B (5 µm) (Zorbax C8 is suitable). A precolumn packed with suitable silica gel places between the injector and the column. Detector: A spectrophotometer set at 262 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: Inject each of the solutions and record the chromatograms for three times the retention time of the peak corresponding to vinblastine. The assay is not valid unless, in the chromatogram obtained with the resolution solution, the resolution between the peaks corresponding to vincristine and vinblastine is not less than 4; the peak in the chromatogram obtained with reference solution (3) has a signal-to-noise ratio not less than 5. Calculate the content of C46H58N4O9, H2SO4 in the substance to be examined, using the principal peak areas obtained from the test solution and reference solution (1), and the declared content of C46H58N4O9,H2SO4 in vinblastine sulfate RS. 1004

Sterility If intended for use in the manufacture of parenteral dosage forms without a further appropriate sterilisation procedure, it complies with the test for sterility (Appendix 13.7). Storage Store in an airtight, glass container, protected from light, at a temperature not exceeding -20 °C. If the substance is sterile, store in an sterile, airtight, tamper-proof glass container. Labelling The label states, where applicable, that the substance is sterile. Action and use Cytotoxic. Preparation Injection. VINBLASTINE SULFATE FOR INJECTION Vinblastini Sulfatis Injectione Vinblastine sulfate for injection is a sterile material prepared from vinblastine sulfate. It may contain excipients. It is supplied in a sealed container. The injection complies with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Content of vinblastine sulfate, C46H58N4O9,H2SO4, 90.0% to 110.0% of the stated amount. Characters White powder. Identification A. In the test for Related substances, the principal peak in the chromatogram obtained with solution (1) has the same retention time as the vinblastine sulfate peak in the chromatogram obtained with solution (3). B. To a quantity of the powder containing the equivalent of 1 mg of vinblastine sulfate add 0.2 ml of a freshly prepared 1% solution of vanillin in hydrochloric acid R. A pink colour is produced in about 1 minute (distinction from vincristine sulfate). C. Dissolve a quantity of the powder for injection equivalent to 5 mg of vinblastine sulfate in 2 ml of water, the solution yields the reactions characteristic of sulfates (Appendix 8.1). pH Prepare a solution of the powder for injection in carbon dioxide-free water R to obtain a concentration of 0.15% of anhydrous vinblastine sulfate, pH of the solution is 3.5 to 5.0 (Appendix 6.2).

VINCRISTINE SULFATE

VP V

Bacterial endotoxins Not more than 10.0 EU per mg of vinblastine sulfate (Appendix 13.2). Appearance of solution Dissolve the contents of a sealed container in 10 ml of carbon dioxide-free water R. The resulting solution is clear (Appendix 9.2). Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: A mixture of 12 volumes of acetonitrile R, 38 volumes of a 1.5% (v/v) solution of diethylamine R adjusted to pH 7.5 with phosphoric acid R and 50 volumes of methanol R. Solution (1): Dissolve a quantity of the powder in water to produce a solution containing the equivalent of 0.1% of anhydrous vinblastine sulfate. Solution (2): A solution containing 0.10% each of vinblastine sulfate RS and vincristine sulfate RS in water. Solution (3): A solution containing 0.10% of vinblastine sulfate RS in water. Solution (4): A solution containing 0.0020% of vinblastine sulfate RS in water. Solution (5): A solution containing 0.00010% of vinblastine sulfate RS in water. Keep the solutions in ice before use. Chromatographic system: A column (25 cm × 4.6 mm) packed with end - capped octylsilyl silica gel for chromatography (5 µm) (Zorbax C8 is suitable). A guard column packed with a suitable silica gel placed between the pump and the injection device. Detector: A spectrophotometer set at 262 nm. Flow rate: 1 ml/min. Volume of injection: 10 µl. Procedure: System suitability: The test is not valid unless the resolution factor between the peaks due to vinblastine and vincristine in the chromatogram obtained with solution (2) is at least 4 and unless the signal-to-noise ratio in the peak in the chromatogram obtained with solution (5) is at least 5. Inject separately each solution, record the chromatograms for 3 times the retention time of the peak due to vinblastine. In the chromatogram obtained with solution (1), the area of any secondary peak is not greater than the area of the principal peak in the chromatogram obtained with solution (4) (2%) and the sum of the areas of any such peaks is not greater than 2.5 times the area of the principal peak in the chromatogram obtained with solution (4) (5%). Disregard any peak with an area less than that of the peak in the chromatogram obtained with solution (5) (0.1%). Loss on drying Not more than 17.0% (Appendix 9.6). (60 °C, at a pressure not exceeding 0.7 kPa, 16 h).

Uniformity of content The content of anhydrous vinblastine sulfate in each of 10 individual containers as determined in the Assay is not less than 90.0% and not more than 110.0% of the average except that in one container the content may be not less than 80.0% and not more than 120.0% of the average. Assay Dissolve the contents of a sealed container in a suitable volume of methanol R and dilute with sufficient methanol R to produce a solution containing 0.004% of anhydrous vinblastine sulfate. Measure the absorbance of the resulting solution at the maximum at 267 nm (Appendix 4.1). Calculate the content of C46H58N4O9,H2SO4 in the sealed container taking 185 as the value of A(1%, 1 cm) at the maximum at 267 nm. Repeat the procedure with a further 9 sealed containers. Calculate the average content of C46H58N4O9,H2SO4 per container from the 10 individual results thus obtained. Storage Stored at a temperature of 2 °C to 8 °C. Action and use Cytotoxic. Usual strength 10 mg. VINCRISTINE SULFATE Vincristini sulfas

C46H56N4O10,H2SO4

M. 923.1

Vincristine sulfate is methyl (3aR,4R,5S,5aR,10bR,13aR)4-(acetyloxy)-3a-ethyl-9-[(5S,7R,9S)-5-ethyl-5-hydroxy9-(methoxycarbonyl)-1,4,5,6,7,8,9,10-octahydro-2H-3,7methanoazacycloundecino[5,4-b]indol-9-yl]-6-formyl-5hydroxy-8-methoxy-3a,4,5,5a,6,11,12,13a-octahydro-1Hindolizino[8,1-cd] carbazole-5-carboxylate sulfate. It contains not less than 95.0% and not more than 104.0% of C46H56N4O10, H2SO4, calculated with reference to the dried substance.

Characters A white or slightly yellowish, crystalline powder, very hygroscopic. Freely soluble in water, slightly soluble in ethanol (96%). 1005

VP V

VINCRISTINE SULFATE FOR INJECTION

Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the reference spectrum of vincristine sulfate RS. Appearance of solution Solution S: Dissolve 50.0 mg of the substance to be examined in carbon dioxide-free water R and dilute to 10.0 ml with the same solvent, and mix. Keep the solution in iced water to carry out the test for Related substances. Solution S is clear (Appendix 9.2) and not more intensely coloured than reference solution Y7 (Appendix 9.3, method 1). pH Dilute 2 ml of solution S to 10 ml with carbon dioxidefree water R, and mix. The pH of this solution is 3.5 to 4.5 (Appendix 6.2). Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: A 1.5% v/v solution of diethylamine R adjusted to pH 7.5 with phosphoric acid R. Mobile phase B: Methanol R. Test solution: Dilute 1.0 ml of solution S to 5.0 ml with water, and mix. Reference solution (1): Dissolve 5.0 mg of vincristine sulfate RS in water and dilute to 5.0 ml with the same solvent. Reference solution (2): Dilute 1.0 ml of the test solution to 50.0 ml with water, and mix. Reference solution (3): Dilute 1.0 ml of reference solution (2) to 20.0 ml with water, and mix. Resolution solution: Dissolve 1.0 mg of vinblastine sulfate RS in 1.0 ml of reference solution (1). Keep the solutions in iced water before use. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase B (5 µm) (Zorbax C8 is suitable). A precolumn packed with stationary phase B. Detector: A spectrophotometer set at 297 nm. Flow rate: 2.0 ml/min. Volume of injection: 20 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 12

38

62

12 - 27

38 → 8

62 → 92

27 - 29

8 → 38

92 → 62

29 - 34

38

62

System suitability: In the chromatogram obtained with the resolution solution, the resolution between the peaks due to vincristine and vinblastine is not less than 4. Limits: In the chromatogram obtained with the test solution: 1006

The area of any secondary peak is not greater than the area of the principal peak in the chromatogram obtained with reference solution (2) (2.0%). The sum of the areas of any such peaks is not greater than 2.5 times the area of the principal peak in the chromatogram obtained with reference solution (2) (5.0%). Disregard any peak with an area less than that of the peak in the chromatogram obtained with reference solution (3) (0.1%).

Loss on drying Not more than 12.0% (Appendix 9.6). (0.0500 g; in vacuo; 105 °C; 2 h). Assay Examine by liquid chromatography (Appendix 5.3), as described in the test for Related substances. Mobile phase: Methanol - 1.5% v/v solution of diethylamine adjusted to pH 7.5 with phosphoric acid (7 : 3). Flow rate: 1.0 ml/min. Calculate the content of C46H56N4O10,H2SO4 in the substance to be examined, using the areas of the principal peaks in the chromatograms obtained with the test solution, reference solution (1) and the declared content of C46H56N4O10,H2SO4 in vincristine sulfate RS. Sterility If intended for use in the manufacture of parenteral dosage forms without a further appropriate sterilisation procedure, it complies with the test for sterility (Appendix 13.7). Storage Store in an airtight, glass container, protected from light, at a temperature not exceeding -20 °C. If the substance is sterile, store in an sterile, airtight, tamper-proof glass container. The label states, where applicable, that the material is sterile. Action and use Cytotoxic. Preparation Injection. VINCRISTINE SULFATE FOR INJECTION Vincristini Sulfatis Injectione Vincristine sulfate for injection is a sterile material prepared from vincristine sulfate. It may contain excipients. It is supplied in a sealed container. The injection complies with the requirements stated under “Injections, infusions” (Appendix 1.19) and with the following requirements.

Content of vincristine sulfate, C46H56N4O10,H2SO4, 90.0% to 110.0% of the stated amount.

VINPOCETINE

VP V

Characters White powder. Identification A. In the test for Related substances, the principal peak in the chromatogram obtained with solution (1) has the same retention time as the vincristine sulfate peak in the chromatogram obtained with solution (3). B. Shake a quantity of the powder containing the equivalent of 1 mg of anhydrous vincristine sulfate with 3 ml of chloroform R, filter and wash the filter with 2 ml of chloroform R. Evaporate the combined chloroform solutions to dryness at 40 °C. Add 0.2 ml of a freshly prepared 1% solution of vanillin in hydrochloric acid R to the residue. An orange colour is produced in about 1 minute (distinction from vinblastine sulfate). Appearance of solution Dissolve the contents of a sealed container in 10 ml of carbon dioxide-free water R. The resulting solution is clear (Appendix 9.2). Bacterial endotoxins Not more than 100.0 EU per mg of vincristine sulfate (Appendix 13.2). Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: A mixture of 30 volumes of a 1.5% (v/v) solution of diethylamine R adjusted to pH 7.5 with phosphoric acid R and 70 volumes of methanol R. Solution (1): Dissolve a quantity of the powder in water to produce a solution containing the equivalent of 0.10% of anhydrous vincristine sulfate. Solution (2): A solution containing 0.10% each of vincristine sulfate RS and vinblastine sulfate RS in water. Solution (3): A solution containing 0.10% of vincristine sulfate RS in water. Solution (4): A solution containing 0.0020% of vincristine sulfate RS in water. Solution (5): A solution containing 0.00010% of vincristine sulfate RS in water. Keep the solutions in ice before use. Chromatographic system: A column (25 cm × 4.6 mm) packed with end - capped octylsilyl silica gel for chromatography (5 µm) (Zorbax C8 is suitable). A guard column packed with a suitable silica gel placed between the pump and the injection device. Detector: A spectrophotometer set at 297 nm. Flow rate: 1 ml/min. Volume of injection: 10 µl. Procedure: System suitability: The test is not valid unless the resolution factor between the peaks due to vincristine and vinblastine in the chromatogram obtained with solution (2) is at least 4 and unless the signal-to-noise ratio in the peak in the chromatogram obtained with solution (5) is at least 10.

Inject separately each of solutions, record the chromatograms for 3 times the retention time of the peak due to vincristine. In the chromatogram obtained with solution (1) the area of any secondary peak is not greater than the area of the principal peak in the chromatogram obtained with solution (4) (2%) and the sum of the areas of any such peaks is not greater than 2.5 times the area of the principal peak in the chromatogram obtained with solution (4) (5%). Disregard any peak with an area less than that of the peak in the chromatogram obtained with solution (5) (0.1%).

Uniformity of content The content of anhydrous vincristine sulfate in each of 10 individual containers as determined in the Assay is not less than 90.0% and not more than 110.0% of the average except that in one container the content may be not less than 80.0% and not more than 120.0% of the average. Assay Dissolve the contents of a sealed container in a suitable volume of methanol R and dilute with sufficient methanol R to produce a solution containing 0.005% of anhydrous vincristine sulfate. Measure the absorbance of the resulting solution at the maximum at 297 nm (Appendix 4.1), using methanol R as a blank. Calculate the content of C46H56N4O10,H2SO4 in the sealed container taking 177 as the value of A(1%, 1 cm) at the maximum at 297 nm. Repeat the procedure with a further 9 sealed containers. Calculate the average content of C46H56N4O10,H2SO4 per container from the 10 individual results thus obtained. Storage Stored at a temperature of 2 °C to 8 °C. Action and use Cytotoxic. Usual strength 1 mg. VINPOCETINE Vinpocetinum

C22H26N2O2  

M: 350.5

Vinpocetine is ethyl (13aS,13bS)-13a-ethyl-2,3,5,6,13a,13bhexahydro-1H-indolo[3,2,1-de]pyrido[3,2,1-ij][1,5]naphthyridine-12-carboxylate. It contains not less than 98.5% and not more than 101.5% of C22H26N2O2, calculated with reference to the dried substance. 1007

VP V

VINPOCETINE

Characters White or slightly yellow, crystalline powder. Practically insoluble in water, soluble in methylene chloride, slightly soluble in anhydrous ethanol. Identification A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of vinpocetine RS. B. It complies with the test for Specific optical rotation. Specific optical rotation +127° to +134°, calculated with reference to the dried substance (Appendix 6.4). Dissolve 0.25 g in dimethylformamide R and dilute to 25.0 ml with the same solvent. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase: A 1.54% solution of ammonium acetate acetonitrile (45 : 55). Test solution: Dissolve 50.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase. Reference solution (1): Dilute 1.0 ml of the test solution to 50.0 ml with the mobile phase. Reference solution (2): Dissolve 5.0 mg of vinpocetine impurity B RS, 6.0 mg of vinpocetine impurity A RS, 5.0 mg of vinpocetine impurity C RS and 5.0 mg of vinpocetine impurity D RS in the mobile phase and dilute to 50.0 ml with the same solvent. Reference solution (3): Dilute 1.0 ml of reference solution (1) and 1.0 ml of reference solution (2) to 20.0 ml with the mobile phase. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase end-capped octadecylsilyl silica gel for chromatography R (5 µm). Detector: A spectrophotometer set at 280 nm. Flow rate: 1.0 ml/min. Volume of injection: 15 µl. Procedure: The run time is 3 times the retention time of vinpocetine. Relative retention with reference to vinpocetine (retention time = about 16 min): Impurity A = about 0.4; impurity D = about 0.68; impurity B = about 0.75; impurity C = about 0.83. System suitability: In the chromatogram obtained with reference solution (3), the resolution between the peaks due to impurities D and B is at least 2.0. Limits: Impurity A: The area of the peak due to impurity A is not more than the area of the corresponding peak in the chromatogram obtained with reference solution (3) (0.6%). Impurities B, D: For each impurity, the area is not more than the area of the corresponding peak in the chromatogram obtained with reference solution (3) (0.5%). 1008

Impurity C: The area of the peak due to impurity C is not more than 0.6 times the area of the corresponding peak in the chromatogram obtained with reference solution (3) (0.3%). Any other impurity: For each impurity, the area is not more than the area of the peak due to vinpocetine in the chromatogram obtained with reference solution (3) (0.10%). The sum of the peak areas of all impurities is not more than 10 times the area of the peak due to vinpocetine in the chromatogram obtained with reference solution (3) (1.0%). Disregard any peak with an area less than 0.5 times the area of the peak due to vinpocetine in the chromatogram obtained with reference solution (3) (0.05%). Note: Impurity A: ethyl (12S,13αS,13βS)-13α-ethyl-12-hydroxy2,3,5,6,12,13,13α,13β-octahydro-1H-indolo[3,2,1-de]pyrido[3,2,1ij][1,5]naphthyridine-12-carboxylate (ethyl vincaminate). Impurity B: methyl (13αS,13βS)-13α-ethyl-2,3,5,6,13α,13βhexahydro-1H-indolo[3,2,1-de]pyrido[3,2,1-ij][1,5] naphthyridine-12-carboxylate (apovincamine). Impurity C: ethyl (13αS,13βS)-13α-ethyl-10-methoxy2,3,5,6,13α,13β-hexahydro-1H-indolo [3,2,1-de]pyrido[3,2,1ij][1,5]naphthyridine-12-carboxylate (methoxyvinpocetine). Impurity D: ethyl (12RS,13αRS,13βRS)-13α-ethyl2,3,5,6,12,13,13α,13β-octahydro-1H-indolo[3,2,1-de]pyrido [3,2,1-ij][1,5]naphthyridine-12-carboxylate (dihydrovinpocetine).

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 100 °C; in vacuo, 3 h). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.300 g of the substance to be examined in 50 ml of a mixture of equal volumes of acetic anhydride R and anhydrous acetic acid R. Titrate with 0.1 N perchloric acid VS, determining the end-point potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 35.05 mg of C22H26N2O2. Storage Store in an airtight container. Action and use Vasodilator. Preparations Tablets, capsules.

VP V

VINPOCETINE TABLETS Tabellae Vinpocetini Vinpocetine tablets contain vinpocetine. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of vinpocetine, C22H26N2O2, 90.0% to 110.0% of the stated amount. Identification A. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel GF254. Mobile phase: Toluene - ethanol 96% - chloroform (4 : 2 : 8). Test solution: To a quantity of the powdered tablets, equivalent to about 10 mg of vinpocetine add 5 ml of chloroform R, shake for 30 minutes and filter. Reference solution: Dissolve about 10 mg of vinpocetine RS in 5 ml of cloroform R, shake for 30 minutes. Procedure: Apply separately to the plate 5 μl of the test solution and the reference solution. After developing over 3/4 of the plate, remove the plate, allow it to dry in air and examine under ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution is similar in position, shape and size to the principal spot in the chromatogram obtained with the reference solution. B. The ultraviolet absorption spectrum (Appendix 4.1) of the test solution prepared in the Assay in the range 220 nm to 360 nm corresponds to that of the reference solution. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 500 ml of 0.1 M hydrochloric acid R. Rotation speed: 75 rpm. Time: 30 min. Procedure: After the specified time, withdraw a sample of the medium, filter and discard the first portion of the filtrate. Measure the absorbance (Appendix 4.1) of the resulting solution at 269 nm, in a 1 cm cell, using the medium as a blank, in comparison with a reference solution having the same concentration in the medium. Tolerance: Not less than 70% (Q) of the stated amount of vinpocetine, C22H26N2O2, is dissolved in 30 minutes. Assay Examine by ultraviolet - visible absorption spectrophotometry (Appendix 4.1). Test solution: Weigh 20 tablets, calculate the average mass and finely powder. Transfer an accurately weighed quantity of the powdered tablets, equivalent to about 20 mg of vinpocetine, to a 100 ml volumetric flask, add 70 ml of ethanol (96%) R, shake to dissolve. Dilute to volume with ethanol (96%) R, mix and filter. Dilute 10.0 ml of the filtrate to 100.0 ml with ethanol (96%) R and mix.

SYNTHETIC VITAMIN A CONCENTRATE (OILY FROM)

Reference solution: Transfer an accurately weighed quantity of about 20 mg of vinpocetine RS to a 100 ml volumetric flask, add 70 ml of ethanol (96%) R, shake to dissolve. Dilute to volume with ethanol (96%) R, mix. Dilute 10.0 ml of this solution to 100.0 ml with ethanol (96%) R and mix. Measure the absorbance of the reference solution and the test solution at 314 nm, in a 1 cm cell, using ethanol (96%) R in the reference cell. Calculate the content of vinpocetine, C22H26N2O2, in the tablets using the measured absorbances of the test solution and the reference solution, and the declared content of C22H26N2O2 in vinpocetine RS.

Storage Store in an airtight container, in a cool and dry place, protected from light. Action and use Vasodilator. Usual strength 5 mg, 10 mg. SYNTHETIC VITAMIN A CONCENTRATE (OILY FROM) Vitaminum A syntheticum densatum oleosum Synthetic Retinol Concentrate (Oily Form) Oily concentrate retinol prepared from synthetic retinol ester as is or by dilution with a suitable vegetable fatty oil. It may contain suitable stabilisers such as antioxidants. It contains not less than 95.0% and not more than 110.0% of the vitamin A content stated on the label, which is not less than 500 000 IU/g.

Characters Yellow or brownish-yellow, oily liquid. Practically insoluble in water, soluble or partly soluble in anhydrous ethanol, miscible with organic solvents. Partial crystallisation may occur in highly concentrated solutions. Identification Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel F254. Mobile phase: Ether - cyclohexane (20 : 80). Test solution: Prepare a solution containing about 3.3 IU of vitamin A per microlitre in cyclohexane R containing 0.1 % of butylhydroxytoluene R. Reference solution: Prepare a 0.1% solution of retinol esters RS (i.e 3.3 IU of each ester per microlitre) in cyclohexane R containing 0.1 % of butylhydroxytoluene R. Procedure: Apply separately to the plate 3 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The test is not valid unless the chromatogram obtained with 1009

SYNTHETIC VITAMIN A CONCENTRATE (POWDER FORM)

VP V

the reference solution shows the individual spots of the corresponding esters. The elution order from bottom to top is: Retinol acetate, retinol propionate and retinol palmitate. The composition of the test solution is confirmed by the correspondence of the principal spot or spots with those obtained with the reference solution.

Identification Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel F254. Mobile phase: Ether - cyclohexane (20 : 80). Test solution: Introduce a quantity of the preparation to be examined containing about the equivalent of 17 000 IU of vitamin A into a 20 ml glass-stoppered test tube. Add about 20 mg of bromelains R, 2 ml of water and about 150 µl of 2-propanol R, swirling gently for 2 min to 5 min in a water-bath at 60 °C to 65 °C. Cool to below 30 °C and add 5 ml of 2-propanol R containing 1 g/L of butylhydroxytoluene R. Shake vigorously for 1 min, allow to stand for a few minutes and use the supernatant solution. Reference solution: Prepare a 10 mg/ml solution of retinol esters RS (i.e. 3.3 IU of each ester per microlitre) in 2-propanol R containing 1 g/L of butylhydroxytoluene R. Procedure: Apply separately to the plate 3 µl of each solution. Develop over a path of 15 cm. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. The test is not valid unless the chromatogram obtained with the reference solution shows the individual spots of the corresponding esters. The elution order from bottom to top is: Retinol acetate, retinol propionate and retinol palmitate. The composition of the test solution is confirmed by the correspondence of the principal spot or spots with those obtained with the reference solution.

Acid value Not more than 2.0 (Appendix 7.2). Determined on 2.0 g. Peroxide value Not more than 10.0 (Appendix 7.6, method A). Assay If partial crystallisation has occurred, homogenise the material at a temperature of about 65 °C, but avoid prolonged heating. Carry out the assay according to method 1 or 4 (Appendix 10.10). Storage In an airtight container, fully fill, protected from light. Once the container has been opened, its contents are to be used as soon as possible; any part of the contents not used at once should be protected by an atmosphere of inert gas. Labelling The label states: The number of International Units (IU) per gram. The name of the ester or esters. The name of any added stabilisers. The method of restoring the solution if partial crystallisation has occurred. Action and use Vitamin A. Preparation Capsules. SYNTHETIC VITAMIN A CONCENTRATE (POWDER FORM) Vitamin synthetici densati A pulvis Synthetic Retinol Concentrate (Powder Form) Powder concentrate obtained by dispersing a synthetic retinol ester in a matrix of gelatin or acacia or other suitable material. It may contain suitable stabilisers such as antioxidants. It contains not less than 95.0% and not more than 115.0% of the vitamin A content stated on the label, which is not less than 250 000 IU/g.

Characters Yellowish powder usually in the form of particles of almost uniform size. Practically insoluble in water, swells or forms an emulsion, depending on the formulation. 1010

Assay Carry out the assay according to method 4 (Appendix 10.10). Storage In an airtight container, protected from light. Once the container has been opened, its contents are to be used as soon as possible; any part of the contents not used at once should be protected by an atmosphere of inert gas. Labelling The label states: The number of International Units (IU) per gram. The name of the ester or esters. The name of the principal excipient or excipients used and the name of any added stabilisers. Action and use Vitamin A. VITAMIN A SOFT CAPSULES Molles capsulae Vitamini A Vitamin A soft capsules contain a solution of vitamin A in a suitable refined oil. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

VITAMIN A AND D SOFT CAPSULES

VP V

Content of vitamin A, 90.0% to 120.0% of the stated amount. Characters Soft capsules containg a clear oil with homogeneous colour. Identification A. In the Assay: If examined by spectrophotometry, the light absorption spectrum of the test solution has a maxium corresponding to the requirement of the Assay. If examined by liquid chromatography, the retention time of the principal peak in the chromatogram obtained with the test solution is the same as that of the principal peak in the chromatogram obtained with the reference solution. B. Dilute the content of the capsule in chloroform R to produce a solution of 10 IU/ml to 20 IU/ml of vitamin A. To 1 ml of the resulting solution add 2 ml of a 25% solution of antimony trichoride R, a blue colour is produced which is not stable. Assay Carry out the method described in Appendix 10.10. Vitamin A assay. Storage Store in a cool and dry place, protected from light. Action and use Vitamin. Usual strength 2000 IU, 5000 IU. Preparations Soft capsules. VITAMIN A AND D SOFT CAPSULES Molles capsulae Vitamini A et D Vitamin A and D soft capsules contain vitamin A and vitamin D3. The capsules comply with the requirements stated under “Capsules” (Appendix 1.13) and with the following requirements.

If examined by liquid chromatography, the retention time of the principal peak in the chromatogram obtained with the test solution is the same as that of the principal peak in the chromatogram obtained with the reference solution. B. Dilute the content of the capsules in chloroform R to produce a solution of 10 IU/ml to 20 IU/ml of vitamin A. To 1 ml of the resulting solution add 2 ml of antimony trichoride solution R, a blue colour is produced which is not stable. C. In the Assay for vitamin D3, the retention time of the principal peak in the chromatogram obtained with the test solution is the same as that of the principal peak in the chromatogram obtained with the reference solution.

Assay Assay for vitamin A Carry out the method described in Appendix 10.10. Vitamin A assay. Assay for vitamin D3 Examine by liquid chromatography (Appendix 5.3), avoiding exposure to actinic light. Mobile phase: Methanol - ethyl acetate - water (90 : 7 : 3). Reference solution: A 20 IU/ml solution of vitamin D3 (cholecalciferol) RS in ethanol R. Test solution: Weigh 20 capsules, calculate the average mass of the contents. In a 25 ml volumetric flask, weigh accurately a quantity of the oily contents of the capsules equivalent to about 500 IU of vitamin D3, add 20 ml of ethanol R, shake thoroughly (add 1 ml of ethyl acetate R to dissolve the oil before adding ethanol, if necessary). Add ethanol R to volume, mix. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm to 10 µm). Detector: A spectrophotometer set at 265 nm. Flow rate: 1.0 ml/min. Volume of injection: 100 µl. Procedure: Inject separately the test solution and reference solution. Calculate the content of vitamin D3 in the capsules using the areas (or heights) of vitamin D3 peaks in the chromatograms obtained with the test solution and reference solution and the concentration of vitamin D3 in the reference solution.

Content of vitamin A and vitamin D, 90.0% to 120.0% of the stated amount.

Storage Store in a cool and dry place, protected from light.

Characters Soft capsules containg a clear oil with homogeneous colour.

Action and use Vitamin.

Identification A. In the Assay for vitamin A (Appendix 10.10): If examined by ultraviolet and visible adsorption spectrophotometry (Appendix 4.1), the light absorption spectrum of the test solution has a maxium corresponding to the requirement of the Assay.

Usual strength 5000 IU of vitamin A and 400 IU of vitamin D.

1011

VP V

WATER FOR INJECTIONS

WATER FOR INJECTIONS Aqua pro injectione H2O  



M: 18.02

Water for injections in bulk is obtained from water that complies with the regulations on water intended for human consumption or from purified water by distillation with suitable equipments and use as a solvent for the preparation of medicines for parenteral administration. During production and subsequent storage, appropriate measures are taken to ensure that the microbial count is adequately controlled and monitored. Appropriate alert and action levels are set so as to detect adverse trends. Under normal conditions, an appropriate action level is a microbial count of 10 CFU per 100 ml when determined by filtration through a membrane (Appendix 13.6), using at least 200 ml of water for injections in bulk and incubating at 30°C to 35 °C for not less than 5 days. For aseptic processing, stricter alert levels may need to be applied.

Characters Clear, colourless liquid, odorless and tasteless. Total organic carbon Not more than 0.5 mg/l (Appendix 7.11). Conductivity Conductometer: Accuracy of 0.1 µS·cm-1 or better at the lowest range. Table 1 - Temperature and conductivity requirements Temperature (°C) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

1012

Conductivity (µS·cm-1) 0.6 0.8 0.9 1.0 1.1 1.3 1.4 1.5 1.7 1.8 1.9 2.1 2.2 2.4 2.5 2.7 2.7 2.7 2.7 2.9 3.1

Measure the conductivity without temperature compensation, recording simultaneously the temperature. Temperaturecompensated measurement may be performed after suitable validation. The water to be examined meets the requirements if the measured conductivity (Appendix 6.10) at the recorded temperature is not greater than the value in Table 1. For temperatures not listed in Table 1, find the closest temperature value that is not greater than the measured temperature. The corresponding conductivity value is the limit at that temperature. If the conductivity of the substance to be examined donot meet the requirements in the Table 1. Use the following procedure: Transfer a sufficient amount of the water to be examined (100 ml or more) to a suitable container, and stir the test sample. Adjust the temperature at 25 °C ± 1 °C, begin vigorously agitating the test sample while periodically observing the conductivity. When the change in conductivity (due to uptake of atmospheric carbon dioxide) is less than 0.1 µS·cm-1 per 5 min, note the conductivity (D1). The conductivity is not greater than 2.1 µS·cm-1. If the conductivity is greater than 2.1 µS·cm-1, take the following procedure: Add a recently prepared saturated solution of potassium chloride R to the test sample Table 2 - pH and conductivity requirements pH

Conductivity (µS·cm-1)

5.0

4.7

5.1

4.1

5.2

3.6

5.3

3.3

5.4

3.0

5.5

2.8

5.6

2.6

5.7

2.5

5.8

2.4

5.9

2.4

6.0

2.4

6.1

2.4

6.2

2.5

6.3

2.4

6.4

2.3

6.5

2.2

6.6

2.1

6.7

2.6

6.8

3.1

6.9

3.8

7.0

4.6

PURIFIED WATER

VP V

(0.3 ml per 100 ml of the test sample), while maintaining the sample temperature at 25 °C ± 1 °C and determine the pH (Appendix 6.2), to the nearest 0.1 pH unit. Perform this test within approximately 5 min of the conductivity determination. Using Table 2, determine the conductivity limit at the measured pH (D2). If D1 is less than D2, the water to be examined meets the requirements of the test for conductivity. If D1 is greater than D2 or the pH is outside the range of 5.0 - 7.0, the water to be examined does not meet the requirements of the test for conductivity.

Nitrates Not more than 0.2 ppm. Place 5 ml of the substance to be examined in a test-tube immersed in iced water, add 0.4 ml of a 10% solution of potassium chloride R, 0.1 ml of diphenylamine solution R and 5 ml of nitrogen-free sulfuric acid R, dropwise with shaking. Transfer the tube to a water-bath at 50 °C. After 15 min, any blue colour in the solution is not more intense than that in a reference solution prepared at the same time in the same manner using a mixture of 4.5 ml of nitratefree water R and 0.5 ml of nitrate standard solution (2 ppm NO3) R. Aluminium Not more than 10 ppb, if intended for use in the manufacture of dialysis solutions (Appendix 9.4.9). To 400 ml of the substance to be examined, add 10 ml of acetate buffer solution pH 6.0 R and 100 ml of distilled water. Reference solution: A mixture of 2 ml of aluminium standard solution (2 ppm Al) R, 10 ml of acetate buffer solution pH 6.0 R and 98 ml of distilled water. Blank solution: A mixture of 10 ml of acetate buffer solution pH 6.0 R and 100 ml of distilled water. Bacterial endotoxins Not more than 0.25 EU/ml (Appendix 13.2). Storage Store in sterile, avoid any other contamination. DISTILLED WATER Aqua destillata Distilled water is prepared by distillation from potable or purified water. It meets the requirements stated under “Purified water”.

PURIFIED WATER Aqua purificata H2O  

M. 18.02

Water for the preparation of medicines other than those that are required to be both sterile and apyrogenic, unless otherwise justified and authorised. PURIFIED WATER IN BULK

Purified water in bulk is prepared by distillation, by ion exchange, by reverse osmosis or by any other suitable method from water that complies with the regulations on water intended for human consumption laid down by the competent authority. Purified water in bulk is stored and distributed in conditions designed to prevent growth of micro-organisms and to avoid any other contamination. During production and subsequent storage, appropriate measures are taken to ensure that the microbial count is adequately controlled and monitored. Appropriate alert and action levels are set so as to detect adverse trends. Under normal conditions, an appropriate action level is a microbial count of 100 CFU/ml, determined by filtration through a membrane (Appendix 13.6), using a suitable volume of water for injections in bulk and incubating at 30 °C to 35 °C for not less than 5 days.

Characters Clear, colourless liquid, odorless and tasteless. Total organic carbon or oxidisable substances Apply one of the two following methods: A. Total organic carbon is not more than 0.5 mg/l (Appendix 7.11). B. Oxidisable substances: To 100 ml of the substance to be examined, add 10 ml of a 10% solution of sulfuric acid R and 0.1 ml of 0.02 M potassium permanganate R and boil for 5 min; the solution remains faintly pink. Conductivity Conductometer: Accuracy of 0.1 µS·cm-1 or better at the lowest range. Measure the conductivity without temperature compensation, recording simultaneously the temperature. Temperaturecompensated measurement may be performed after suitable validation. The water to be examined meets the requirements if the measured conductivity (Appendix 6.10) at the recorded temperature is not greater than the value in Table 1. For temperatures not listed in Table 1, calculate the maximal permitted conductivity by interpolation between the next lower and next higher data points in the table.

1013

VP V

PURIFIED WATER

Table 1 - Temperature and conductivity requirements Temperatuer (°C)

Conductivity (µS·cm-1)

0

2.4

10

3.6

20

4.3

25

5.1

30

5.4

40

6.5

50

7.1

60

8.1

70

9.1

75

9.7

80

9.7

90

9.7

100

10.2

Nitrates Not more than 0.2 ppm. Place 5 ml of the substance to be examined in a test-tube immersed in iced water, add 0.4 ml of a 10% solution of potassium chloride R, 0.1 ml of diphenylamine solution R and 5 ml of nitrogen-free sulfuric acid R, dropwise with shaking. Transfer the tube to a water-bath at 50 °C. After 15 min, any blue colour in the solution is not more intense than that in a reference solution prepared at the same time in the same manner using a mixture of 4.5 ml of nitratefree water R and 0.5 ml of nitrate standard solution (2 ppm NO3) R. Aluminium Not more than 10 ppb, if intended for use in the manufacture of dialysis solutions (Appendix 9.4.9). To 400 ml of the substance to be examined, add 10 ml of acetate buffer solution pH 6.0 R and 100 ml of distilled water R. Use reference solution: A mixture of 2 ml of aluminium standard solution (2 ppm Al) R, 10 ml of acetate buffer solution pH 6.0 R and 98 ml of distilled water R. Blank solution: A mixture of 10 ml of acetate buffer solution pH 6.0 R and 100 ml of distilled water. Heavy metals Not more than 0.1 ppm (Appendix 9.4.8). To 200 ml of the substance to be examined, add 0.15 ml of 0.1 M nitric acid R and heat in a glass evaporating dish on a water-bath until the volume is reduced to 20 ml. 12 ml of the concentrated solution complies with limit test for heavy metals, method 1. Prepare the reference solution using 10 ml of lead standard solution (1 ppm Pb) R and adding 0.075 ml of 0.1 M nitric acid R. Prepare the blank solution adding 0.075 ml of 0.1 M nitric acid R. 1014

If purified water in bulk complies with the requirement for conductivity prescribed for “Water for injections”, it is not necessary to carry out the test for heavy metals.

Bacterial endotoxins Not more than 0.25 EU/ml (Appendix 13.2), if intended for use in the manufacture of dialysis solutions without a further appropriate procedure for removal of bacterial endotoxins. PURIFIED WATER IN CONTAINERS

Purified water in containers that has been filled and stored in conditions designed to assure the required microbiological quality. Purified water in containers complies the requirements stated under “Purified water in bulk” and with the following requirements:

Characters Clear, colourless liquid, odorless and tasteless. Acidity or alkalinity Add 0.05 ml of methyl red solution R to 10 ml of the substance to be examined freshly boiled and cooled in a borosilicate glass flask. The solution is not coloured red. Add 0.1 ml of bromothymol blue solution R to 10 ml of the substance to be examined. The solution is not coloured blue. Oxidisable substances To 100 ml of the substance to be examined, add 10 ml of a 10% solution of sulfuric acid R and 0.1 ml of 0.02 M potassium permanganate VS and boil for 5 min. The solution remains faintly pink. Chlorides To 10 ml of the substance to be examined, add 1 ml of dilute nitric acid R and 0.2 ml of a 1.7% solution of silver nitrate R. The solution shows no change in appearance for at least 15 min. Sulfates To 10 ml of the substance to be examined, add 0.1 ml of dilute hydrochloric acid R and 0.1 ml of a 6.1% solution of barium chloride R. The solution shows no change in appearance for at least 1 h. Ammonium Not more than 0.2 ppm. To 20 ml of the substance to be examined, add 1 ml of alkaline potassium tetraiodomercurate solution R. After 5 min, examine the solution down the vertical axis of the tube. The solution is not more intensely coloured than a standard prepared at the same time by adding 1 ml of alkaline potassium tetraiodomercurate solution R to a mixture of 4 ml of ammonium standard solution (1 ppm NH4) R and 16 ml of ammonium-free water R.

VP V

Calcium and magnesium To 100 ml of the substance to be examined, add 2 ml of ammonium chloride buffer solution pH 10.0 R, 50 mg of erichorome black T R and 0.5 ml of 0.01 M sodium edetate R. A pure blue colour is produced. Residue on evaporation Not more than 0.001%. Evaporate 100 ml to dryness on a water-bath and dry in an oven to constant mass at 100 °C to 105 °C. The residue weighs a maximum of 1 mg. Microbial contamination Total aerobic microbial count is not more than 102 CFU/ml. Determined by filtration through a membrane, use casein soya bean digest agar (Appendix 13.6). Storage and labelling Store in airtight container. The container should not cause any changes of characteristics of water. The label states, where applicable, that the substance is suitable for use in the manufacture of dialysis solutions. STERILISED WATER FOR INJECTIONS Aqua sterilis pro injectione Sterilised water for injections is “water for injections” that has been distributed into suitable containers, closed and sterilised by heat in conditions which ensure that the product still complies with the test for bacterial endotoxins. The containers are made from glass, or suitable materials that meet the requiremnets in Vietnamese Pharmacopoiea. Sterilised water for injections is used to dissolve powder injections or dilute parenteral dosage forms before use. Each container contains a sufficient quantity of water for injections to permit the nominal volume to be withdrawn. Sterilised water for injections complies the requirements stated under “Injections, infusions” (Appendix 1.19).

Characters Clear, colourless liquid, odorless and tasteless. Acidity or alkalinity To 20 ml of the substance to be examined, add 0.05 ml of phenol red solution R. If the solution is yellow, it becomes red on the addition of 0.1 ml of 0.01 M sodium hydroxide VS; if red, it becomes yellow on the addition of 0.15 ml of 0.01 M hydrochloric acid VS. Conductivity Not more than 25 µS·cm-1 for containers with a nominal volume of 10 ml or less and not more than 5 µS·cm-1 for containers with a nominal volume greater than 10 ml. Maintaining the sample temperature at 25 °C ± 1 °C when determine.

STERILISED WATER FOR INJECTIONS

Oxidisable substances For containers with a nominal volume less than 50 ml: Boil 100 ml of the substance to be examined with 10 ml of dilute sulfuric acid R, add 0.4 ml of 0.02 M potassium permanganate VS and boil for 5 min; the solution remains faintly pink. For containers with a nominal volume equal to or greater than 50 ml: Boil 100 ml of the substance to be examined with 10 ml of dilute sulfuric acid R, add 0.2 ml of 0.02 M potassium permanganate VS and boil for 5 min; the solution remains faintly pink. Chlorides For containers with a nominal volume of 100 ml or less: Not more than 0.5 ppm (Appendix 9.4.5). 15 ml of the substance to be examined complies with the limit test for chlorides. Prepare the standard using a mixture of 1.5 ml of chloride standard solution (5 ppm Cl) R and 13.5 ml of water. Examine the solutions down the vertical axes of the tubes. For containers with a nominal volume greater than 100 ml: To 10 ml of the substance to be examined, add 1 ml of 2 M nitric acid R and 0.2 ml of a 1.7% solution of silver nitrate R. The solution shows no change in appearance for at least 15 min. Nitrates Not more than 0.2 ppm. Place 5 ml of the substance to be examined in a test-tube immersed in iced water, add 0.4 ml of a 10% solution of potassium chloride R, 0.1 ml of diphenylamine solution R and 5 ml of nitrogen-free sulfuric acid R, dropwise with shaking. Transfer the tube to a water-bath at 50 °C. After 15 min, any blue colour in the solution is not more intense than that in a reference solution prepared at the same time in the same manner using a mixture of 4.5 ml of nitrate-free water R and 0.5 ml of nitrate standard solution (2 ppm NO3) R. Sulfates To 10 ml of the substance to be examined, add 0.1 ml of dilute hydrochloric acid R and 0.1 ml of 6.1% solution of barium chloride R. The solution shows no change in appearance for at least 1 h. Aluminium Not more than 10 ppb, if intended for use in the manufacture of dialysis solutions (Appendix 9.4.9). To 400 ml of the substance to be examined, add 10 ml of acetate buffer solution pH 6.0 R and 100 ml of distilled water R. Reference solution: A mixture of 2 ml of aluminium standard solution (2 ppm Al) R, 10 ml of acetate buffer solution pH 6.0 R and 98 ml of distilled water R. Blank solution: A mixture of 10 ml of acetate buffer solution pH 6.0 R and 100 ml of distilled water. 1015

VP V

XYLOMETAZOLINE HYDROCHLORIDE

Ammonium For containers with a nominal volume less than 50 ml: Not more than 0.6 ppm. To 20 ml of the substance to be examined, add 1 ml of alkaline potassium tetraiodomercurate solution R; after 5 min, examine the solution down the vertical axis of the tube; the solution is not more intensely coloured than a standard prepared at the same time by adding 1 ml of alkaline potassium tetraiodomercurate solution R to a mixture of 4 ml of ammonium standard solution (3 ppm NH4) R and 16 ml of ammonium-free water R. For containers with a nominal volume equal to or greater than 50 ml: Not more than 0.2 ppm. To 20 ml of the substance to be examined, add 1 ml of alkaline potassium tetraiodomercurate solution R; after 5 min, examine the solution down the vertical axis of the tube; the solution is not more intensely coloured than a standard prepared at the same time by adding 1 ml of alkaline potassium tetraiodomercurate solution R to a mixture of 4 ml of ammonium standard solution (1 ppm NH4) R and 16 ml of ammonium-free water R. Calcium and magnesium To 100 ml of the substance to be examined, add 2 ml of ammonium chloride buffer solution pH 10.0 R, 50 mg of erichorome black T R and 0.5 ml of 0.01 M sodium edetate. A pure blue colour is produced. Residue on evaporation Evaporate 100 ml to dryness on a water-bath and dry in an oven to constant mass at 100 °C to 105 °C. For containers with a nominal volume of 10 ml or less: Not more than 4 mg (0.004%). For containers with a nominal volume greater than 10 ml: Not more than 3 mg (0.003%). Sterility It complies with the test for Sterility (Appendix 13.7). Bacterial endotoxins Not more than 0.25 EU/ml (Appendix 13.2). Storage Store in an airtight container, in a dry and cool place, avoid any other contamination. XYLOMETAZOLINE HYDROCHLORIDE Xylometazolini hydrochloridum

C16H24N2,HCl   1016

M. 280.8

Xylometazoline hydrochloride is 2-[4-(1,1-dimethylethyl)-2,6dimethylbenzyl]-4,5-dihydro-1H-imidazole hydrochloride. It contains not less than 99.0% and not more than 101.0% of C16H24N2,HCl, calculated with reference to the dried substance.

Characters White or almost white, crystalline powder. Freely soluble in water, in ethanol (96%) and in methanol. Identification Apply one of the two following identifications: First identification: A, E. Second identification: B, C, D, E. A. The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of xylometazoline hydrochloride RS. B. Thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Ammonia - methanol (5 : 100). Test solution: Dissolve 20 mg of the substance to be examined in methanol R and dilute to 5 ml with the same solvent. Reference solution: Dissolve 20 mg of xylometazoline hydrochloride RS in methanol R and dilute to 5 ml with the same solvent. Procedure: Spray with potassium iodide - starch solution R. Chlorine treatment: At the bottom of a chromatographic tank place a beaker containing a mixture of 1 volume of  water, 1 volume of a 25% solution of hydrochloric acid R and 2 volumes of a 1.5% solution of potassium permanganate R. Close the tank and allow to stand for 15 min. Place the dried plate in the tank and reclose the tank. Leave the plate in contact with the chlorine vapour for 5 min. Withdraw the plate and place it in a current of cold air until the excess of chlorine is removed and an area of the coating below the points of application does not give a blue colour with a drop of potassium iodide - starch solution R. Apply separately to the plate 5 µl of each solution. Develop over a path of 2/3 of the plate, allow the plate to dry in air. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. C. Dissolve about 0.5 mg in 1 ml of methanol R. Add 0.5 ml of a freshly prepared a 5% solution of sodium nitroprusside R and 0.5 ml of 0.5 M sodium hydroxide R. Allow to stand for 10 min and add 1 ml of a 8% solution of sodium hydrogen carbonate R. A violet colour develops. D. Dissolve 0.2 g in 1 ml of  water, add 2.5 ml of ethanol (96%) R and 2 ml of 1 M sodium hydroxide R. Mix thoroughly and examine in ultraviolet light at 365 nm. The solution shows no fluorescence or at most the same fluorescence as a blank solution prepared in the same manner. The identification is not valid unless a solution prepared in the same manner using naphazoline

XYLOMETAZOLINE HYDROCHLORIDE

VP V

hydrochloride RS instead of the substance to be examined shows a distinct bluish fluorescence. E. It gives reaction (A) of chlorides (Appendix 8.1).

Appearance of solution Solution S: Dissolve 2.5 g in carbon dioxide-free water R and dilute to 50.0 ml with the same solvent. The solution is clear (Appendix 9.2) and not more intensely coloured than reference solution Y6 (Appendix 9.3, method 2). Acidity or alkalinity Dissolve 0.25 g in carbon dioxide-free water R and dilute to 25 ml with the same solvent. Add 0.1 ml of methyl red solution R and 0.1 ml of 0.01 N hydrochloric acid VS. The solution is red. Not more than 0.2 ml of 0.01 N sodium hydroxide VS is required to change the colour of the indicator to yellow. Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase A: A 1.36 g/L solution of potassium dihydrogen phosphate R adjusted to pH 3.0 with phosphoric acid R. Mobile phase B: Acetonitrile R1. Test solution: Dissolve 50.0 mg of the substance to be examined in  water and dilute to 50.0 ml with the same solvent. Allow to stand for 1 h before injection. Reference solution (1): Dilute 5.0 ml of the test solution to 100.0 ml with  water. Dilute 2.0 ml of this solution to 100.0 ml with  water. Reference solution (2): Dissolve 5.0 mg of xylometazoline impurity A RS and 5.0 mg of the substance to be examined in  water and dilute to 50.0 ml with the same solvent. Dilute 10.0 ml of this solution to 50.0 ml with  water. Reference solution (3): Dilute 5.0 ml of reference solution (2) to 50.0 ml with water. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase endcapped octadecylsilyl silica gel for chromatography with polar incorporated groups R (5 µm). Detector: A spectrophotometer set at 220 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: Carry out a linear gradient elution using the following conditions: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0-5

70

30

5 - 20

70 → 15

30 → 85

20 - 35

15

85

System suitability: In the chromatogram obtained with reference solution (2), the resolution between the peaks due to impurity A and xylometazoline is at least 2.5. Limits: Impurity A: The area of the peak due to impurity A is not more than the area of the corresponding peak in the chromatogram obtained with reference solution (3) (0.2%). Any other impurity: For each impurity, the area is not more than the area of the principal peak in the chromatogram obtained with reference solution (1) (0.10%). The sum of the peak areas of all impurities is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.5%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.05%). Note: Impurity A: N-(2-aminoethyl)-2-[4-(1,1-dimethylethyl)-2,6dimethylphenyl]acetamide. Impurity B: 2-(chloromethyl)-5-(1,1-dimethylethyl)-1,3dimethylbenzene. Impurity C: [4-(1,1-dimethylethyl)-2,6-dimethylphenyl]acetonitrile. Impurity D: 1-(1,1-dimethylethyl)-3,5-dimethylbenzene. ImpurityE:Ethane-1,2-diaminemono(4-methylbenzenesulfonate). Impurity F: [4-(1,1-dimethylethyl)-2,6-dimethylphenyl]acetic acid.

Loss on drying Not more than 0.5% (Appendix 9.6). (1.000 g; 105 °C). Sulfated ash Not more than 0.1% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Dissolve 0.200 g of the substance to be examined in 25 ml of anhydrous acetic acid R and add 10 ml of acetic anhydride R. Titrate with 0.1 N perchloric acid VS, determining the endpoint potentiometrically (Appendix 10.2). 1 ml of 0.1 N perchloric acid VS is equivalent to 28.08 mg of C16H25ClN2. Storage Protected from light. Action and use Alpha-adrenoceptor agonist. Preparation Nasal drops.

Inject the test solution and the reference solutions. Relative retention with reference to xylometazoline (retention time = about 7.2 min): Impurity A = about 0.79. 1017

VP V

XYLOMETAZOLINE NASAL DROPS

XYLOMETAZOLINE NASAL DROPS Nasalia Xylometazolini Xylometazoline nasal drops are solution of xylometazoline hydrochloride in water, it may contain suitable excipients. The nasal drops comply with the requirements stated under “Nasal drops and liquid nasal sprays” (Appendix 1.15) and with the following requirements.

Content of xylometazoline hydrochloride, C16H24N2,HCl, 90.0% to 110.0% of the stated amount. Characters A clear and colourless solution. Identification A. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of xylometazoline hydrochloride peak in the chromatogram obtained with the reference solution. B. To a volume of the substance to be examined containing about 0.5 mg of xylometazoline hydrochloride, add 0.2 ml of 5% sodium nitroprusiate solution R and 0.1 ml of 5M sodium hydroxide R, and allow to stand for 10 min. Add 1 ml of 10% sodium hydrocarbonate solution. A violet colour is produced. pH 5.0 to 7.5 (Appendix 6.2). Assay Examine by liquid chromatography (Appendix 5.3). Buffer solution pH 2.7: Dissolve 2.5 g of anhydrous ammonium sulfate R in 1000 ml of water. Adjust to pH 2.7 with 1 M phosphoric acid R. Mobile phase: Buffer solution pH 2.7 - acetonitrile (65 : 35). Make adjustments if necessary. Reference solution: Weigh accurately about 100 mg of xylometazoline hydrochloride RS, transfer into a 100-ml volumetric flask, add 10 ml of methanol R, shake to dissolve and dilute to volume with water, mix. Dilute 5.0 ml of the resulting solution to 20.0 ml with water, mix. Test solution: Use the nasal drops or dilute to obtain a solution having a similar concentration to the reference solution. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (10 µm). Detector: A spectrophotometer set at 225 nm. Flow rate: 1.5 ml/min. Volume of injection: 20 µl. Procedure: System suitability: Inject the reference solution, the symmetric factor of the xylometazoline hydrochloride peak is not more than 3.5, the relative standard deviation for 6 replicate injections is not more than 2.0%. Inject alternately the reference solution and the test solution.Calculate the content of xylometazoline 1018

hydrochloride, C16H24N2,HCl, in the tablets using the areas of the principal peaks in the chromatograms obtained with the test solution, the reference solution and the declared content of C16H24N2,HCl in xylometazoline hydrochloride RS.

Storage Store in a cool place, protected from light. Action and use Nasal decongestant. Usual strength 0.05% and 0.1%. ZIDOVUDINE Zidovudinum

C10H13N5O4



M. 267.2

Zidovudine is 1-(3-azido-2,3-dideoxy-β-D-erythro-pentofuranosyl)-5- methylpyrimidine-2,4(1H,3H)-dione. It contains not less than 97.0% and not more than 102.0% of C10H13N5O4, calculated with reference to the dried substance.

Characters A white or brownish powder. Sparingly soluble in water, soluble in ethanol. It melts at about 124 °C. It shows polymorphism. Identification The infrared absorption spectrum (Appendix 4.2) of the substance to be examined is concordant with the spectrum of zidovudine RS. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in the minimum quantity of water and evaporate to dryness in a desiccator, under high vacuum over diphosphorus pentoxide R. Record new spectra using the residues. Appearance of solution Dissolve 0.5 g of the substance to be examined in 50 ml of water, heating if necessary. The solution is not more intensely coloured than reference solution BY5 (Appendix 9.3, method 2). Specific optical rotation +60.5 to +63.0°, calculated with reference to the dried substance (Appendix 6.4).

ZIDOVUDINE

VP V

Dissolve 0.50 g of the substance to be examined in ethanol R and dilute to 50.0 ml with the same solvent, and mix. Examine the obtained solution at 25 °C.

Related substances A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254 Mobile phase: Methanol - methylene chloride (10 : 90). Test solution: Dissolve 0.20 g of the substance to be examined in methanol R and dilute to 10 ml with the same solvent, and mix. Reference solution (1): Dissolve 20 mg each of thymine R, zidovudine impurity A RS and triphenylmethanol R in methanol R, add 1.0 ml of the test solution and dilute to 100 ml with methanol R, and mix. Reference solution (2): Dilute 5.0 ml of reference solution (1) to 10 ml with methanol R, and mix. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 12 cm. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. In the chromatogram obtained with the test solution, any spot corresponding to impurity A is not more intense than the corresponding spot in the chromatogram obtained with reference solution (2) (0.5%); and any spot, apart from the principal spot and any spot corresponding to impurity A and thymine (which is limited by liquid chromatography), is not more intense than the spot corresponding to zidovudine in the chromatogram obtained with reference solution (2) (0.5%). Spray the plate with a 1% solution of vanillin R in sulfuric acid R. In the chromatogram obtained with the test solution, any spot corresponding to triphenylmethanol is not more intense than the corresponding spot in the chromatogram obtained with reference solution (2) (0.5%). The test is not valid unless the chromatogram obtained with reference solution (1) shows 4 clearly separated spots, corresponding to thymine, impurity A, zidovudine and triphenylmethanol, in order of increasing Rf value. B. Examine by liquid chromatography (Appendix 5.3) as described in the Assay. Inject separately 10 µl each of test solution (1), reference solution (2), reference solution (4) and reference solution (5). Continue the chromatography for 1.5 times the retention time of the principal peak in the chromatogram obtained with test solution (1). When the chromatograms are recorded in the prescribed conditions, the substances are eluted in the following sequence: thymine, zidovudine and impurity B. Limits: In the chromatogram obtained with test solution (1): The area of any peak corresponding to thymine is not greater than the area of the peak in the chromatogram obtained with reference solution (2) (2%). The area of any peak corresponding to impurity B is not greater than the area of the corresponding peak in the chromatogram obtained with reference solution (4) (1%). The area of any other peak, apart from the principal peak, is not greater than the area of the peak in the chromatogram

obtained with reference solution (5) (0.5%). The sum of the peak areas of all impurities, apart from the principal peak, is not greater than 6 times the area of the peak obtained with reference solution (5) (3.0%). Disregard any peak with an area less than 10% the area of the peak in the chromatogram obtained with reference solution (5). Note: Impurity A: 1-[(2R,5S)-5-(hydroxymethyl)-2,5-dihydrofuran-2yl)-5-methylpyrimidine-2,4(1H,3H)-dione. Impurity B: 1-(3-chloro-2,3-dideoxy-β-D-erythro-pentofuranosyl)5-methylpyrimidine-2,4(1H,3H)-dione. Impurity C: 5-methylpyrimidine-2,4(1H,3H)-dione (thymine).

Heavy metals Not more than 20 ppm (Appendix 9.4.8, method 4). 1.0 g complies with the limit test for heavy metals, method 4. Prepare the standard using 2 ml of lead standard solution (10 ppm Pb) R. Loss on drying Not more than 1.0% (Appendix 9.6). (1.000 g; 100 °C to 105 °C). Sulfated ash Not more than 0.25% (Appendix 9.9, method 2). Determined on 1.0 g. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol - water (20 : 80). Test solution (1): Dissolve 50.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase, and mix. Test solution (2): Dilute 10.0 ml of test solution (1) to 50.0 ml with the mobile phase, and mix. Reference solution (1): Dissolve 10.0 mg of zidovudine RS in the mobile phase and dilute to 50.0 ml with the mobile phase, and mix. Reference solution (2): Dissolve 10.0 mg of thymine R in methanol R and dilute to 50.0 ml with the same solvent, and mix. Dilute 5.0 ml of the solution to 50.0 ml with the mobile phase, and mix. Reference solution (3): Dissolve 5.0 mg of zidovudine impurity B RS in 25.0 ml of reference solution (1) and dilute to 50.0 ml with the mobile phase, and mix. Reference solution (4): Dilute 5.0 ml of reference solution (3) to 50.0 ml with the mobile phase, and mix. Reference solution (5): Dilute 0.25 ml of test solution (1) to 50.0 ml with the mobile phase, and mix. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 265 nm. Flow rate: 1.2 ml/min. Volume of injection: 10 µl. 1019

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Procedure: Equilibrate the column with the mobile phase at a flow rate of 1.2 ml per minute for about 45 minutes. Inject reference solution (3). Adjust the sensitivity of the detector so that the height of the principal peaks in the chromatogram obtained is not less that 70% of the full scale of the recorder. The test is not valid unless the resolution between the peaks due to zidovudine and impurity B is at least 1.0. Inject separately test solution (2) and reference solution (1). Adjust the sensitivity of the detector so that the height of the peaks in the chromatograms obtained is not less than 50% of the full scale of the recorder. Calculate the content of C10H13N5O4 in the substance to be examined, using the areas of the principal peaks obtained with test solution (2), reference solution (1), and the declared content of C10H13N5O4 in zidovudine RS.

Storage Store in an airtight container, protected from light. Action and use Antiviral. Preparations Tablets, oral solution. Zidovudine and lamivudine tablets. ZIDOVUDINE ORAL SOLUTION Zidovudini solutionum peroralum Zidovudine oral solution is a solution containing zidovudine in a suitable vehicle. The oral solution complies with the requirements under “Solutions” (Appendix 1.3) and with the following requirements:

Content of zidovudine, C10H13N5O4, 90.0% to 110.0% of the stated amount. Identification: A. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Dichloromethane - methanol - glacial acetic acid (90 : 10 : 3). Reference solution: Prepare a solution of zidovudine RS having a concentration of about 1 mg zidovudine per ml methanol R. Test solution: Dilute a volume of the solution to be examined containing 20 mg of zidovudine to 20 ml with methanol R, filter if necessary. Procedure: Apply separately 5 µl of each solution to the plate. Develop over a path of about three-quarters of the plate. Remove the plate and allow to dry in air and examine under ultraviolet light at 254 nm. In the chromatogram obtained with the test solution, the principal spot is similar in colour, shape and Rf value to that in the chromatogram obtained with the reference solution. 1020

B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution is similar to that of the zidovudine peak in the chromatogram obtained with the reference solution.

pH (Appendix 6.2) 3.0 to 4.0. Related substances Examine by liquid chromatography (Appendix 5.3) with the mobile phase and chromatographic system as described in the Assay. Test solution: Dilute the solution to be examined with the mobile phase to obtain a solution having a concentration of about 0.2 mg of zidovudine per ml. Reference solution (1): Dilute 1.0 ml of the test solution to 200.0 ml with the mobile phase. Reference solution (2): Dissolve 1 mg of thymine RS (zidovudine impurity C), in 10 ml of the mobile phase. Transfer 1.0 ml of this solution into a 10-ml volumetric flask containing 5 mg of zidovudine RS, dissolve and dilute to volume with the mobile phase. Placebo solution (prepare if available): Dissolve all excipients (other than any parahydroxy benzoates) with a suitable solvent (the solvent used in the formulation) to obtain a solution having the same concentration as the solution to be examined. Dilute the obtained solution to obtain a solution having the same concentration as the test solution. Inject the test solution, the reference solutions and the placebo solution. Record the chromatograms for 4 times the retention time of zidovudine. For preparation containing parahydroxybenzoate, record the chromatograms for the 8 times the retention time of zidovudine to elute all these components. In the chromatogram obtained with reference solution (2), the relative retention time with reference to zidovudine (retention time about 12 min) are as follows: about 0.3 for impurity C (thymine); about 0.4 for impurity A (stavudine) and about 1.2 for impurity B. The resolution factor between impurity C and zidovudine peaks is not less than 5.0; between zidovudine and impurity B peaks is not less than 2.0; the symmetry factor of zidovudine peak is not more than 2.0. Limits: Comply to the following requirement A and B. In the case insufficient information of excipients or any peak in the chromatogram obtained with the placebo solution having the same retention time as any peak in the chromatogram obtained with reference solution (2), or interference by excipients has been demonstrated, applies only the requirement A. A. In the chromatogram obtained with the test solution, the peak area of impurity C after multiple with the correction factor 0.6, is not more than 6 times the area of the principal peak in the chromatogram obtained with reference solution (1) (3.0%).

ZIDOVUDINE TABLETS

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B. In the chromatogram obtained with the test solution, the area of any secondary peak is not more than twice the area of the principal peak in the chromatogram obtained with reference solution 1 (1.0%) and not more than one secondary peak having the area greater than that of the principal peak in the chromatogram obtained with reference solution (1) (0.5%), the sum of the area of impurity C peak (after multiple with the correction factor 0.6) and all other secondary peaks is not more than 12 times the area of the principal peak in the chromatogram obtained with reference solution (1) (6.0%). Disregard any peak having the same retention time as that of any peak in the chromatogram obtained with the placebo solution, any peak having the relative retention time with reference to lamivudine greater than 2.0 (corresponding to parahydroxybenzoate) and any peak having the area less than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (1) (0.1%).

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol - buffer solution pH 5.3 (20 : 80). Buffer solution pH 5.3: 0.045 M sodium acetate solution, adjusted the pH to 5.3 with glacial acetic acid R. Reference solution: Dissolve a quantity of zidovudine RS in the mobile phase to obtain a solution having a concentration of about 0.2 mg per ml. Test solution: Determine the density of the solution to be examined (Appendix 6.5). Weigh accurately a quantity of the solution to be examined containing about 20 mg of zidovudine and transfer into a 100-ml volumetric flask. Dilute to volume with the mobile phase, mix well, filter. Resolution solution: Dissolve 2 mg of thymine RS in 10 ml of methanol R. Transfer 1.0 ml of the obtained solution into a 50-ml volumetric flask and dilute to volume with the test solution. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 265 nm. Flow rate: 1.2 ml/min. Volume of injection: 10 µl. Procedure: System suitability: Inject the resolution solution, in the chromatogram obtained, the relative retention time of thymine peak with reference to zidovudine (retention time about 12 min) is 0.3. The resolution factor between the thymine and zidovudine peaks is not less than 5.0; the symmetry factor of the zidovudine peak is not more than 2.0. Inject the reference solution and the test solution. Calculate the content of zidovudine, C10H13N5O4, in the solution using the peak areas in the chromatogram obtained with the reference solution, the test solution, the density of the solution to be examined and the declared content of C10H13N5O4 in zidovudine RS.

Storage Store in a well-closed container, in a cool place, protected from light. Action and use Antiviral. Usual strength 50 mg/5ml. ZIDOVUDINE TABLETS Tabellae Zidovudini Zidovudine tablets contain zidovudine. They may be coated. The tablets comply with the requirements stated under “Tablets” (Appendix 1.20) and with the following requirements.

Content of zidovudine, C10H13N5O4, 90.0% to 110.0% of the stated amount. Identification A. Examine by ultraviolet and visible absorption spectrophotometry (Appendix 4.1). Solvent mixture: Methanol - water (75 : 25). Reference solution: A 15 µg/ml solution of zidovudine RS in the solvent mixture. Test solution: Transfer an accurately weighed quantity of the powdered tablets (coating layer removed if necessary) containing the equivalent of 150 mg of zidovudine to a 100 ml volumetric flask. Add 70 ml of the solvent mixture. Dissolve by sonicating for 5 minutes, dilute to vomlume with the solvent mixture and mix. Filter or contrifuge. Dilute 1.0 ml of the filtrate or clear supernatant to 100.0 ml with the solvent mixture. Measure the absorbance (Appendix 4.1) of the test solution and the reference solution in the range 200 nm to 400 nm, in a 1 cm cell, using the solvent mixture as a blank. The absorption spectrum of the test solution is concordant with that of the reference solution. B. In the Assay, the retention time of the principal peak in the chromatogram obtained with the test solution corresponds to that of zidovudine peak in the chromatogram obtained with the reference solution. Dissolution (Appendix 11.4) Apparatus: Paddle. Medium: 900 ml of water. Rotation speed: 50 rpm. Time: 30 min. Procedure: Examine by liquid chromatography (Appendix 5.3). Mobile phase, reference solution and chromatographic system: Prepare as directed in the Assay. 1021

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Test solution: After the specified time, withdraw a sample of the medium, filter, discard the first portion of the filtrate. Dilute a volume of the filtrate with water to obtain a solution having a concentration of 0.12 mg/ml of zidovudine. Tolerance: Not less than 75% (Q) of the stated amount of zidovudine, C10H13N5O4, is dissolved in 30 minutes.

Related substances Examine by liquid chromatography (Appendix 5.3). Mobile phase, reference solution, test solution, chromatographic system and procedure: prepare as directed in the Assay. Calculate the percentage of each impurity using the following expression: 100(1/F)(Si/Ss) (Cs/Ci) In which: F: Relative response factor, equal to 1.7 for zidovudine related compound C and 1.0 for all other impurities. Si: The peak response for each impurity obtained from the test solution. Ss: The peak response for zidovudine obtained from the reference solution. Cs: Concentration of zidovudine in the reference solution (mg/ml). Ci: Concentration of zidovudine in the test solution (mg/ml). Limits: Zidovudine related compound C: Not more than 1.5%. Other individual impurities: Not more than 0.2%. Total impurities: Not more than 2.0%. Note: Zidovudine related compound B: 1-(3-cloro-2,3-dideoxy-β-Derythro-pentofuranosyl)-5-methylpyrimidine-2,4(1H,3H)-dion. Zidovudine related compound C: 5-methylpyrimidine2,4(1H,3H)-dion (thymine).

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Dissolve 3.0 g of sodium acetate R and 1.3 g of sodium octanesulfonate R in 900 ml of water. Add 90 ml of methanol R and 40 ml of acetonitrile R, mix. Adjust to pH 5.3 with glacial acetic acid R. Filter and degas. Make adjustments if necessary. Zidovudine related compound B reference solution (stock solution): Dissolve an accurately weighed quantity of zidovudine related compound B RS in methanol R to obtain a solution containing 0.1 mg/ml. Zidovudine related compound C reference solution (stock solution): Dissolve an accurately weighed quantity of zidovudine related compound C RS in methanol R to obtain a solution containing 0.2 mg/ml. Reference solution: Transfer an accurately weighed quantity of about 30 mg of zidovudine RS to a 250 ml volumetric flask, add 3 ml of methanol R, shake to dissolve. Add 2.5 ml of zidovudine related compound B reference solution and 5.0 ml of zidovudine related compound C reference solution, mix and dilute to volume with 1022

water. This solution contains 0.12 mg/ml of zidovudine, 0.001 mg/ml of zidovudine related compound B and 0.004 mg/ml of zidovudine related compound C. Test solution: Weigh 20 tablets (with the coating layer removed if necessary), calculate the average mass and powder finely. Transfer an accurately weighed quantity of the powdered tablets containing the equivalent of about 300 mg of zidovudine to a 100 ml volumetric flask, add 10 ml of water, shake to disperse the powder tablets, add 30 ml of methanol R and sonicate for 10 minutes. Dilute to volume with water, mix and filter. Dilute 4.0 ml of the filtrate to 100.0 ml with water. Chromatographic system: A column (15 cm × 4.6 mm) packed with stationary phase C. Detector: A spectrophotometer set at 265 nm. Flow rate: 1.3 ml/min. Volume of injection: 20 μl. Procedure: System suitability: Inject the reference solution, the relative retention times are 0.17 for zidovudine related compound C, 1.0 for zidovudine and 1.2 for zidovudine related compound B. The resolution factor between the peaks due to zidovudine and zidovudine related compound B is at least 2.5. The tailing factor of the zindovudine peak is not more than 2.0. The relative standard deviation of the peak areas due to zindovudine peak for 6 replicate injections is not more than 2.0%. Inject the test solution. Calculate the content of zindovudine, C10H13N5O4, in the tablets using the peak areas in the chromatograms obtained with the test solution and the reference solution and the declared content of C10H13N5O4 in zindovudine RS.

Storage Store in an airtight container, in a cool and dry place, protected from light. Action and use Antiviral. Usual strength 200 mg; 300 mg. ZINC OXIDE Zinci oxidum ZnO



M. 81.39

Zinc oxide contains not less than 99.0% and not more than 100.5% of ZnO, calculated with reference to the ignited substance.

Characters A soft, white or faintly yellowish-white, amorphous powder. Absorb moisture and carbon dioxide when expose to the air.

ZINC OXIDE OINTMENT

VP V

Practically insoluble in water and in ethanol (96%), dissolves in dilute mineral acids and in solution of alkali hydroxides and in dilute ammonia.

Identification A. It becomes yellow when strongly heated; the yellow colour disappears on cooling. B. Dissolve 0.1 g of the substance to be examined in 1.5 ml of dilute hydrochloric acid R and dilute to 5 ml with water. The solution gives the reaction characteristic of zinc (Appendix 8.1). Alkalinity Shake 1.0 g of the substance to be examined with 10 ml of boiling water. Add two drops of phenolphthalein solution R and filter. If the filtrate is red, not more than 0.3 ml of 0.1 N hydrochloric acid VS is required to discolourize the solution. Carbonates and substances insoluble in acids Dissolve 1.0 g of the substance to be examined in 15 ml of dilute hydrochloric acid R. It dissolves without effervescence and the solution is not more opalescent than reference suspension II (Appendix 9.2) and is colourless (Appendix 9.3, method 2). Arsenic Not more than 5 ppm (Appendix 9.4.2). Take 0.2 g of the substance to be examined and carry out the method A for arsenic. Cadmium Not more than 10 ppm. Examine by atomic absorption spectrometry (Appendix 4.4, method 2). Test solution: Dissolve 2.0 g of the substance to be examined in 14 ml of a mixture of equal volumes of water and cadmium- and lead-free nitric acid R, boil for 1 min, cool and dilute to 100.0 ml with water. Reference solutions: Prepare the reference solutions using cadmium standard solution (1000 ppm Cd) R and diluting with a 3.5% (v/v) solution of cadmium- and lead-free nitric acid R. Measure the absorbance at 228.8 nm using a cadmium hollow-cathode lamp as source of radiation and an airacetylene or an air-propane flame. Iron Not more than 0.02% (Appendix 9.4.13). Dissolve 50 mg of the substance to be examined in 1 ml of dilute hydrochloric acid R and dilute to 10 ml with water. The solution complies with the limit test for iron. Use in this test 0.5 ml of mercaptoacetic acid R. Lead Not more than 50 ppm. Examine by atomic absorption spectrometry (Appendix 4.4, method 2).

Test solution: Dissolve 5.0 g of the substance to be examined in 24 ml of a mixture of equal volumes of water and cadmium- and lead-free nitric acid R, boil for 1 min, cool and dilute to 100.0 ml with water. Reference solutions: Prepare the reference solutions using lead standard solution (1000 ppm Pb) R and diluting with a 3.5% (v/v) solution of cadmium- and lead-free nitric acid R. Measure the absorbance at 283.3 nm using a lead hollowcathode lamp as source of radiation and an air-acetylene flame. Depending on the apparatus, the line at 217.0 nm or 283.3 nm may be used.

Loss on ignition Not more than 1.0%. (1.00 g, at 500 °C to constant weight). Assay Dissolve 0.150 g of the substance to be examined in 10 ml of dilute acetic acid R. Titrate with 0.1 M trilon B VS following the complexometric titration of zinc (Appendix 10.5). 1 ml of 0.1 M trilon B VS is equivalent to 8.14 mg of ZnO. Storage Store in a well-closed container. Action and use Mild astringent. Preparations Cream, ointment. ZINC OXIDE OINTMENT Unguentum Zinci oxidi Zinc oxide ointment intended for extranal use contains zinc oxide. Zinc oxide should be powdered finely and passed a sieve No.125 before the preparation of the ointment. The ointment complies with the requirements stated under Topical semi-solid preparations (Appendix 1.12) and with the following requirements.

Content of zinc oxide, ZnO, 90.0% to 110.0% of the stated amount. Characters White ointment. Identification Transfer a quantity of the ointment, equivalent to about 50 mg of zinc oxide, to a crucible, melt by heating gently, and continue to heat, raising gradually the temperature until the mass is completely charred. Ignite until the residue becomes yellow and turns white on cooling. To the residue add 10 ml of water and 5 ml of 10% hydrochloric acid R, mix well and filter. To the filtrate add 2 to 3 drops 1023

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of a 10% solution of potassium ferrocyanide R, a white precipitate is formed.

Calcium, magnesium and other foreign inorganic substances Transfer 2 g of the ointment to a crucible, melt by heating gently, and continue to heat, raising gradually the temperature until the mass is completely charred. Ignite until the residue becomes homogeneously yellow. To the residue add 6 ml of 10% hydrochloric acid R, and heat on a water bath for 10 to 15 minutes. The solution is clear and colourless. Dilute the solution to 10 ml with water, and add 10% ammonia solution R until the first formed precipitate dissolves. Add 2 ml of a 3.5% solution of ammonium oxalate R and 2 ml of a 12% solution of disodium hydrogen phosphate R. The solution is clear and colourless or becomes slightly turbid within 5 minutes. Assay Transfer an accurately weighed quantity of the ointment, equivalent to about 75 mg of zinc oxide, to a crucible, melt by heating gently, and continue to heat, raising gradually the temperature until the mass is completely charred. Ignite until the residue becomes homogeneously yellow, and allow to cool. Dissolve the residue in 10 ml of 1 M sulphuric acid R, heating if necessary. Transfer the solution to a suitable conical flask. Wash the crucible with small portions of water and transfer the washings to the flask, to obtained about 50 ml of the solution. Adjust the pH of the obtained solution to 6.0 to 7.0 with 10% ammonia solution R, add 10 ml of ammonia buffer pH 10.0 R and 1 ml of eriochrome black T R, and titrate with 0.05 M trilon B VS. 1 ml of 0.05 M trilon B VS is equivalent to 4.069 mg of ZnO. Storage Store in an airtight container at cool place.

M. 287.5

Zinc sulfate contains not less than 99.0% and not more than 104.0% of ZnSO4,7 H2O.

Characters White, crystalline powder or colourless, transparent crystals, odourless, efflorescent in dry air. Very soluble in water, freely soluble in glycerol, practically insoluble in ethanol (96%). Identification Solution S: Dissolve 2.5 g of the substance to be examined 1024

pH pH of solution S is 4.4 to 5.6 (Appendix 6.2). Chlorides Not more than 0.03% (Appendix 9.4.5). Take 3.3 ml of solution S, dilute to 15 ml with water and carry out the limit test for chlorides. Iron Not more than 0.01% (Appendix 9.4.13). Take 2 ml of solution S and dilute to 10 ml with water. The solution complies with the limit test for iron. Use in this test 0.5 ml of mercaptoacetic acid R. Assay Dissolve 0.200 g of the substance to be examined in 5 ml of dilute acetic acid R. Titrate with 0.1 M sodium edetate VS following the complexometric titration of zinc (Appendix 10.5). 1 ml of 0.1 M sodium edetate VS is equivalent to 28.75 mg of ZnSO4,7H2O. Storage Store in a well-closed container, in a cool place. Action and use Astringent, antiseptic.

ZINC SULFATE EYE DROPS Collyrium Zinci sulfatis

ZINC SULFATE Zinci sulfas

Appearance of solution Solution S is clear (Appendix 9.2) and colourless (Appendix 9.3, method 2).

Preparation Eye drops.

Usual strength 1%.

ZnSO4,7H2O

in carbon dioxide-free water R and dilute to 50 ml with the same solvent. Solution S give the reactions of zinc and sulfates (Appendix 8.1).

Zinc sulfate eye drops are a sterile solution of zinc sulfate heptahydrate in purified water. The eye drops comply with the requirements stated under “Eye preparations” (Appendix 1.14) and with the following requirements.

Content of zinc sulfate heptahydrate, ZnSO4,7H2O, 95.0% to 105.0% of the stated amount. Characters A clear, colourless solution.

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ZINC SULFATE EYE DROPS

Identification The eye drops yield the reactions characteristic of zinc salts and the reactions characteristic of sulfates (Appendix 8.1). pH 4.5 to 5.5 (Appendix 6.2). Assay To an accurately measured volume of the eye drops containing about 25 mg of zinc sulfate add 50 ml of water and 10 ml of ammonia buffer pH 10.0 R. Titrate with 0.01 M Trilon B VS using eriochrome black T solution R as indicator. 1 ml of 0.01 M Trilon B VS is equivalent to 2.875 mg of ZnSO4,7H2O. Storage Store in a airtight container at dry and cool place, protected from light. Usual strength 0.5%.

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Monographs IMMUNOLOGICAL PRODUCTS

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IMMUNOSERA

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IMMUNOSERA FOR HUMAN USE Immunosera ad usum humanum Immunosera for human use are preparations containing immunoglobulins that have the power of specifically neutralizing the antigen.

Production Immunosera are obtained from immunized healthy animals or humans. Purification of immunosera is carried out to obtain the immune substance with specified content of globulin and to remove unnecessary proteins. Suitable preservatives may be incorporated into preparations. In freeze-dried immunosera, the residual moisture is not more than 3% (m/m). Liquid immunosera are colorless or very faintly yellow, free from turbidity. Freeze-dried immunosera are freely soluble in water to form colorless or very faintly yellow solutions that show the same characteristics as the corresponding liquid preparations. Quality control pH 6.0 to 7.0 (Appendix 15.33). Foreign proteins Only protein from the declared animal species or human is shown to be present (Appendix 15.10). Total protein Not more than 170 g per liter (Appendix 15.18). Albumin When examined immunoelectrophoretically, purified immunosera show not more than a trace of albumin.

Inject intraperitoneally with a quantity equivalent to a human dose but not more than 1 ml into each white Swiss mouse and not more than 5 ml into each guinea pig (Appendix 15.11). Criteria: After an observation period of at least 7 days, none of the animals shows any decrease of body mass and any pathological signs. Pyrogens Inject into each rabbit a dose that varies from 0.5 ml to 10 ml of immunoserum per kg of body mass according to the product to be examined and is prescribed in the individual immunoserum (Appendix 15.12). Criteria: The initial temperature should be within the range from 38 °C to 39.8 °C; the temperature difference between two successive readings is not greater than 0.2 °C; the initial temperature of rabbits does not differ from one another by more than 1 °C; the highest temperature recorded for the rabbit in the 3 hours after the injection is the value to be determined which is not more than 0.6 °C for each rabbit and the summed values of three rabbits does not exceed 1.3 °C (Appendix 15.12).

Storage and expiry date Immunosera are kept under cold conditions, at 2 - 8 °C. Liquid immunosera must not be allowed to freeze. The expiry date is calculated from the day of potency testing, the duration depends on each manufacturer and should be validated by the National Control Authority. Labelling The information on the label, box and leaflet must comply with the current regulations.

Antimicrobial preservatives Thimerosal: Not more than 0.02% (Appendix 15.29). Phenol: Not more than 0.25% (Appendix 15.28).

DIPHTHERIA ANTITOXIN Immunoserum diphthericum Antidiphtheria serum

Sterility Determined by using either direct inoculation method or membrane filtration method (Appendix 15.7). Criteria: No evidence of bacterial or fungal growth is found on appropriate media after 14 days, at 30 °C - 35 °C for bacteria detection and at 20 °C - 25 °C for fungi detection.

Diphtheria antitoxin is a preparation containing antitoxic globulins that have the power of specifically neutralizing the toxin formed by Corynebacterium diphtheria.

Potency Carry out the test for potency as prescribed in individual immunosera and express the result in International Units (IU) per milliliter. The International Units are defined by the World Health Organization. Criteria: The criterion of potency is prescribed in individual immunosera. General safety Carry out the test for general safety using 5 healthy white Swiss mice, each weighing 17 g - 22 g and 2 healthy guinea pigs, each weighing 250 g - 350 g.

Identification It specifically neutralizes the toxin formed by C. diphtheria, rendering it harmless susceptible animals. Tests It complies with the tests prescribed in “Immunosera for human use” monograph and with the following requirement. Potency The potency of diphtheria antitoxin is expressed in international units per milliliter (IU/ml). Not less than 500 IU/ml. The potency of diphtheria antitoxin is determined by neutralizing method in rabbit skin or in guinea pigs weighing 250 g to 350 g (Appendix 15.15). 1029

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IMMUNOSERA

Storage Store at 2 °C to 8 °C, avoid being freeze. Labelling See “Immunosera for human use” monograph. HUMAN HEPATITIS B IMMUNOGLOBULIN Immunoglobulinum humanum hepatitidis B Human Hepatitis B Immunoglobulin is sterile liquid or freeze-dried preparation containing immunoglobulin, mainly immunoglobulin G against hepatitis B surface antitgen (HBsAg).

Production Human Hepatitis B Immunoglobulin is obtained from plasma by selected and/or immunized donors having antibodies against hepatitis B surface antigen. Immunoglobulin must be extracted to obtain the regulated quantity of immunoglobulin and remove unnecessary proteins. The preservative may be added to the final product. For the freeze-dried immunoglobulin, the residual moisture is not greater than 3% (w/w). Property Liquid hepatitis B immunoglobulin is colorless or light yellow, no residue. The liquid may be turbid during storage. Freeze-dried hepatitis B immunoglobulin is white or light yellow powder, solid type. Quality control Identification (Appendix 15.10) The identification of Hepatitis B immunoglobulin is tested by immunoelectrophoresis or immunodiffusion method. The purpose of the test is sure that the hepatitis B immunoglobulin has only anti-HBsAg antibody and human protein. Immunodiffusion method technique The technique is based on the principle of free doublediffusion between antigens and antibodies from separating wells on 1% agarose gel and making the precipitation line by specific reaction. Immunoelectrophoresis Based on an electric field in agarose gel, the negatively charged antigen from wells at cathode and the positively charged antigen from wells at anode move contrariwise and react to make a visible precipitation line. Acceptance criteria Hepatitis B immunoglobulin is verified if there is the precipitation line between sample and corresponding human antibody. Potency The potency of Hepatitis B immunoglobulin is determined by comparing the antibody titre of the immunoglobulin to be examined with that of a reference calibrated in International 1030

Units, using a suitable immunoassay and expressing by International Units (IU) of 1 ml or using other method approved by National Control Laboratory (NCL). Acceptance criteria: For intramuscular preparation: The titre of Hepatitis B immunoglobulin is not less than 100 IU/ml and not less than 80% and not more than 125% of the labeled potency (P = 0.95). For intravenous preparation: The titre of Hepatitis B immunoglobulin is not less than 50 IU/ml and not less than 80% and not more than 125% of the labeled potency (P = 0.95). pH (Appendix 15.33) 6.4 - 7.2. Total protein (Appendix 15.34) Total protein is 100 - 180 g/l and not less than 90% and not more than 110% of the stated amount. Sterility (Appendix 15.7) Carry out by direct innoculation method or membrane filtration method. Specification: There are no bacteria and fungi growing on the suitable media after 14 days of incubation at 30 °C - 35 °C (for bacteria) and 20 °C - 25 °C (for fungi). General safety (Appendix 15.11) Use 5 mice, each weighing 17 g - 22 g and 2 guinea pigs, each weighing 250 g - 350 g. Inject intraperitoneally with 1 human dose and not more than 1 ml for each mouse and not more than 5 ml for each guinea pig. Specification: After at least 7 days of observation, all of the mice and guinea pigs stay healthy, increase weight and no pathological signals. Pyrogens (Appendix 15.12) Inject with dose from 0.5 ml to 10 ml per 1 kg of rabbit weight based on the requirement of immunoglobulin type. The initial temperature of the rabbits is from 38 °C to 39.8 °C, the difference between 2 measurements is not greater than 0.2 °C; the initial temperature among rabbits is not greater than 1 °C; measure the highest temperature during 3h after injection and the value is not greater than 0.6 °C for each rabbit and the temperature total of 3 rabbits is not greater than 1.3 °C.

Package Comply with GMP and the current regulations. Storage For the liquid preparation: store in colorless vial, protect from light. Do not freeze the liquid. For the freeze-dried preparation: store in colorless vial, vacuum or inert gas, protect from light. Storage temperature: 2 °C - 8 °C Labelling The information on the label, container, and leaflet must comply with the current regulations, especially information

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about: name, potency, manufacturing date, expiry date, and storage condition.

Expiry date Expiry date should be defined on the day starting to test the final valid titre. The announcements of the expiry date and storage temperature are based on the result of the stability studies and approved by the NCL. HUMAN NORMAL IMMUNOGLOBULIN Immunoglobulinum humanum normale Human immunoglobulin is a liquid or freeze-dried preparation containing immunoglobulins mainly immunoglobulin G (IgG) from human normal plasma. The liquid immunoglobulin is a colorless or pale-yellow to light-brown transparent solution Freeze-dried immunoglobulin is a white or slightly yellow powder or solid, friable mass. Non-specific human normal immunoglobulin is prescribed for treatment of hepatitis A, hepatitis B, measles, rubella… or in case the shortage of globulin gamma, immune suppression, infection but antibiotic resistance.

Production Human normal immunoglobulin is prepared from pooled material from at least 1000 donors. The source plasma may be derived directly from human blood of from freezed plasma containing not less than 0.1 IU/ml of diphtheria or tetanus antitoxin or 0.25 IU/ml of measles antibody. Immunoglobulin G fraction is prepared by the column chromatography method that has been found not to deteriorate antibodies and to be safe in respect of the transmission of hepatitis and any other microorganism. The concentration of immunoglobulin shall be 50g/l consisting different types of antibodies and at least two of them (one antibacterial and one for antiviral) shall be shown more than 3 times that in the initial pooled material. Any antimicrobial preservative or stabilizer used shall have been shown to have no deleterious effect on the potency of antibodies in the final product. Identification Identification of the origin of immunoglobulin: Human normal immunoglobulin is confirmed by precipitation technique using specific antiserum to human plasma proteins. Acceptance criteria: The tested sample of human normal immunoglobulin shall be shown positive reaction with antiserum to normal human serum and negative reaction with animal antisera. Identification of immunoglobulin G component (IgG) Using antiserum to normal human serum, compare normal human serum and the preparation to be examined by immune-electrophoresis technique.

Acceptance criteria: The main component of the preparation to be examined corresponds to the IgG component of normal human serum.

Potency The potency of diphtheria antitoxin and tetanus antitoxin The potency of diphtheria antitoxin: Appendix 15.15. The potency of tetanus antitoxin: Appendix 15.16. Acceptance criteria: The diphtheria antitoxin content is no less than 2 IU/ml. Potency of measles antibody The potency of measles antibody of test sample is determined by neutralization on Vero cells and expressed in international units (IU) per ml. Materials Test sample: Human normal immunoglobulin. International Standard. Fetal bovine serum (FBS). Vero cell line. Measles challenge virus for neutralization. Medium 199 containing 2% of FBS. A 0.25% solution of trysin. Microwell plates (96 wells, flat-bottomed). Procedure Use two plates for one test. The first plate Dilute the test sample and the reference preparation to 5 IU/ml in 199 medium. Perform serial twofold dilution from the diluted solution (5 IU/ml) of the test sample and and the reference preparation to obtain dilution of 2-1; 2-2; 2-3; …. to 2-12. Prepare measles virus to 100 CCID50/50 μl suspension (cell culture infectious dose) in 199 medium. Dispense 50 μl of each dilution from 2-6 to 2-12 of the test sample and and the reference preparation by previously mapped locations on a plate. Dispense 50 μl of the measles challenge suspension The plate is incubated at 37 °C, 5% of CO2 for 60 - 90 min. Prepare Vero cell suspension containing 2 × 105 cells/ml Dispense 100 μl of the cell suspension to each well. The plate is incubated at 37 °C, 5% of CO2 for 7 - 9 days. Observe CPE under microscope. The second plate The second plate is a control plate for a determination of CCID50/50 μl used by neutralization of the potency test of measles antibody. Dilute the freezed stock measles virus (stored at -35 °C) to 100 CCID50/50 μl. This suspension is signed as 100. Dilute the suspension 100 to 10-1; 10-2; 10-3 and 10-4. Dispense 50 μl of each virus suspension into each row. The plate is incubated at 37 °C, 5% of CO2 for 60 - 90 minutes. Dispend 100 μl of the cell suspension containing 2 × 105 cells/ml to each well. The plate is incubated at 37 °C, 5% of CO2 for 7 - 9 days. Observe CPE under microscope. 1031

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Calculate amount of CCID50/50 μl for neutralization by using Karber formula: Log CCID50 = - log C - [(sum of % CPE in each dilution/100 – 0.5) × log d] Where: CCID50: Cell culture infectious dose causing cytopathic effect of 50% suitable cells. C: The highest viral dose causing 100% CPE. % CPE: The rate between amount of wells having CPE and amount of wells used in each dilution. d: Dilution factor. Acceptance criteria: The challenge virus dose shall be in the range of 31-316 CCID50. Calculate effective dose 50% (ED50) Use Karber or Reed-muench formula: Log ED50 = - log C - [(sum of % ED50 in each dilution/100 – 0.5) × log d] Where: ED50: Effective dose that protects 50% of the cells. C: The lowest dilution of the test sample of the reference preparation where 100% cells are protected. % ED50: The rate between the amount of protected well and amount of wells used in each dilution. d: Dilution factor. Calculation of the potency The potency of measles antibody is calculated by formula: HG = (ED50 of the reference preparation/ED50 of the test sample) × 5 (IU) Acceptance criteria: The potency of measles antibody is not less than 5 IU/ml.

Immunoglobulin G content (IgG) Immunoglobulin G is determined by electrophoresis technique (Appendix 5.6). Acceptance criteria: No less than 90% of the total proteins. Solubility The freeze-dried preparation dissolves completely within 20 minutes at 20 - 25 °C after reconstitution as instructions stated on the label. Abnormal toxicity It complies with the test for abnormal toxicity (Appendix 15.11). Pyrogens Appendix 15.12. Sterility test It complies with the test for sterility (Appendix 15.7). Total nitrogen Appendix 15.18. Thimerosal Not more than 0.012% (Appendix 15.29). 1032

pH 6.4 - 7.2 (Appendix 15.33). Filling and storage Depend on each manufacturer, in a colorless glass container, protected from light. Labelling and package The information on the label, box and leaflet complies with the current regulations. INTERFERON ALPHA - 2 Interferoni alfa 2 Interferon alpha-2 is a liquid or a freeze-dried preparation containing human recombinant interferon alpha-2. Liquid interferon alpha-2 is a colorless transparent solution. Freeze-dried interferon alpha-2 looks like a white spongy cake and change into a clear liquid quickly after reconstitution. Interferon alpha-2 is used in the treatment of certain viral infections, in inhibition of cancer cell proliferation, and in reconciling immune system.

Production Interferon alpha-2 is produced by a method based on recombinant DNA (rDNA) technology: human interferon alpha-2 gene is isolated from human lymphocytes and inserted into an expression vector; then, this recombinant vector is transformed in a suitable bacterial strain which fermented in a specific media for biomass. The recombinant proteins are isolated and purified by biochemical techniques. The preparation is filled and freeze-dried with variable interferon alpha-2 content depending on use purpose. Identification Interferon alpha-2 is identified by a decrease of antiviral activity after neutralization it with a corresponding monoclonal antibody to human interferon alpha-2. Materials Interferon alpha-2 to be tested. Monoclonal antibody to human interferon α-2. Cell line MDBK (Madin Darby Bovin Kidney). Vesicular Stomatitis Virus (VSV) for infecting cell culture already titrated. Minimum Essential Medium (MEM) containing Fetal bovine serum (FBS). A 0.25% solution of trypsin. Sterile 96-well microtiter plate (flat-bottomed). Procedure The first day: Dilute monoclonal antibody to human interferon alpha-2 200 IU/ml solution with 2% FBS MEM under an aseptic condition

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Mix up 3 to 5 vials of interferon alpha-2 sample and dilute under an aseptic condition to approximately 200 IU/ml solution with 2% FBS MEM (depend on stated content). Mix 1 ml of diluted interferon alpha-2 sample and 1 ml of diluted monoclonal antibody solution. Incubate the mixture solution at 37 °C for 60 min. Dispense 100 µl of 5% FBS MEM in wells of 96-well plate. Add 100 µl of diluted sample to well A1. Serially dilute by transferring 100 µl from well A1 to well A12, and discard 100 µl from well A12. Add 100 µl of neutralized solution between sample and monoclonal antibody to human interferon alpha-2 to well B1. Dilute two-fold by transferring 100 µl from well B1 to well B12, and discard 100 µl from well B12. Dispense 100 µl of MDBK cell suspension containing 6.105 to 8.105 cells/ml to each well. The plate is sealed with polyethylene sheet and incubated at 37 °C, 5% CO2 for 18 to 24 hours. The second day: Observe the growth of MDBK cells in the control wells under the reverse microscope. The cell control exhibits a monolayer. Discard the supernatant from all wells of the plate. Dilute the vesicular stomatitis virus (VSV) to 100 - 200 CCID50/ml suspension with 2% FBS MEM. Dispense 100 µl of VSV suspension to each well. The plate is sealed with polyethylene sheet and incubated at 37 °C, 5% CO2 for 18 to 24 hours. The third day: Observe the damage of infected MDBK cells under the reverse microscope: The cytopathic effect in the virus control wells must be more than 90%. Stain the plate: add 50 µl of staining solution containing crystal violet into each well for 10 minutes. Discard staining solution and wash plate with water. The plate is dried at the room temperature. Add 100 µl of 2-methoxyethanol to each well of the dried test plate with continuous gentle shaking for 10 minutes. The absorbance is determined in a micro plate reader at 540 nm (Appendix 4.1). Acceptance criteria Compare absorbance value of the sample wells with that of the neutralized solution wells: The absorbance value of the neutralized solution wells must be lower than the sample wells.

Potency The potency of interferon alpha-2 is determined by the antiviral activity titration on MDBK cell culture. The potency of interferon alpha-2 to be tested is calculated by International standard of recombinant interferon alpha-2. Materials International standard of interferon α-2. Interferon alpha-2 to be examined. Monoclonal antibody to human interferon α-2.

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Cell line MDBK (Madin Darby Bovine Kidney). Vesicular Stomatitis Virus (VSV) for infecting cell culture already titrated. Minimum Essential Medium (MEM) containing Fetal bovine serum (FBS). Sterile 96-well microtiter plate. Procedure The first day: Dilute the international standard of human interferon alpha-2 under an aseptic condition to 100 IU/ml solution with 2% FBS MEM. Mix up 3 to 5 vials of interferon alpha-2 sample and dilute under an aseptic condition to approximately 100 IU/ml solution with 2% FBS MEM (depend on stated content). Dispense 100 µl of 5% FBS MEM in wells of 96 well plate. Add 100 µl of each diluted sample and international standard solution to each well of the first column (standard: row B1, C1, D1, E1, F1, G1, H1). Perform 1:2 serial dilutions with 100 µl each up to column 12 and discard 100 µl from column 12. Add 100 µl of MDBK cell suspension containing 6.105 to 8.105 cells/ml to each well. The plate is sealed with polyethylene sheet and incubated at 37 °C, 5% CO2 for 18 to 24 hours. The second day: Observe the growth of MDBK cells in the cell control wells (A1 to A6) under the reverse microscope. The cell control exhibits a monolayer. Discard the supernatant from all wells of the plate. Dilute the vesicular stomatitis virus (VSV) to 100 - 200 CCID50/ml suspension with 2% FBS MEM. Dispense 100 µl of VSV suspension to each well except cell control wells. The plate is sealed with polyethylene sheet and incubated at 37 °C, 5% CO2 for 18 to 24 hours. The third day: Observe the damage of infected MDBK cells under the reverse microscope: The cytopathic effect in the virus control wells (A7 to A12) must be more than 90%. Stain the plate: Add 50 µl of crystal violet solution into each well for 10 minutes. Discard crystal violet solution and wash plate with water. Allow the plate to dry at the room temperature. Add 100 µl of 2-methoxyethanol to each well of the dried test plate with continuous gentle shaking for 10 min. The absorbance is determined in a microplate reader at 540 nm (Appendix 4.1). Calculate Calculate A50 using the following formula: A50 = (Ac + Av)/2 Where: Ac: Average absorbance of the cell control. Av: Average absorbance of the viral control. A50: The absorbance of the interferon where 50% CPE has occured 1033

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IMMUNOSERA

Determine the well number of the standard and the interferon sample where 50% CPE has occurred using the following formula: N = n + (An – A50)/(An – An+1) Where: N: The well number of the standard or sample where 50% CPE has occurred. n: The maximum well number larger than A50 value. An: The absorbance of the number “n”. An+1: The absorbance of the number “n+1”. Calculate the potency using the following formula: Potency (IU/ml) = 100 × dilution factor × 2(Nsam – Nref) Where: Nsam: The well number of the sample where 50% CPE has occurred. Nref: the well number of the standard where 50% CPE has occurred. Acceptance criteria The potency of the test sample shall be 70% to 150% for the value stated on the label.

Abnormal toxicity The abnormal toxicity test is carried out with a dose of 15M IU/5 ml for each guinea pig and 1M IU/0.5 ml for each mouse (Appendix 15.11). Pyrogens Interferon alpha sample is diluted to 600 000 IU/ml with reconstituting physiological saline (Appendix 15.12). Sterility It complies with the test for sterility (Appendix 15.7). Residual moisture (for freeze-dried preparation) Not more than 3% (Appendix 15.35). pH 7.0 ± 0.5 (Appendix 15.33). Filling and storage Preparation shall be filled with different content in each vial depending on a manufacturer: 3 M/vial, 4.5 M/vial, 6 M/vial, 90 μg/vial, 180 μg/vial, enclosing reconstituting solution. Store at 2 - 8 °C, protected from light. Labelling and package The information on the label, box and leaflet must comply with the current regulations.

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ANTIRABIES SERUM Serum antirabicum

Definition Antirabies serum is a preparation containing immunoglobulins that have the power of specifically neutralizing the rabies virus. Identification Antirabies serum specifically neutralizes rabies virus, rendering them harmless to susceptible animals. Tests It complies with the tests prescribed in “Immunosera for human use” monograph and with the following requirements: Extraneous protein Absence of extraneous protein in final products. Potency The potency of the antirabies serum is expressed in international unit per milliliter (IU/ml). Criteria: Not less than 150 (IU/ml). It is determined by neutralizing method on mice (Appendix 15.17). The reaction bases on the neutralization of a fixed amount of rabies virus with different dilutions of antirabies serum.

Storage Store at 2 °C to 8 °C, avoid being freeze dried. Labelling See “Immunosera for human use” monograph. TETANUS ANTITOXIN Immunoserum tetanicum ad usum humanum Antitetanus serum Tetanus antitoxin is a preparation containing antitoxic globulins that have the power of specifically neutralizing the toxin formed by Clostridium tetani.

Identification It specifically neutralizes the toxin formed by Cl. tetani, rendering it harmless to susceptible animals. Tests It complies with the tests prescribed in “Immunosera for human use” monograph and with the following requirements: Potency The potency of the tetanus antitoxin is expressed in international unit per milliliter (IU/ml). Criteria: Not less than 1000 IU/ml when intended for prophylactic use. Not less than 3000 IU/ml when intended for therapeutic use. The international units of antitoxin per milliliter are determined by the neutralization method on mice (Appendix 15.16).

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Storage Store at 2 °C to 8 °C. Tetanus antitoxin should not be allowed to freeze. Labelling See “Immunosera for human use” monograph. ANTIVENOM SERA Immunoserum contra venena Antivenom sera are a preparation containing immunoglobulin that have power of specifically neutralizing the corresponding snake venom. Antivenom sera are colorless or slightly yellowish transparent liquid preparation. It may be in freeze-dried form, accompanies with the diluent. Antivenom sera are indicated for the treatment of patients with snake bites

Production Antivenom sera are produced from the plasma of healthy, aged 4 to 7 years old, weighed about 200 kg, immunized horses with specific venom and purified, concentrated by Pope method using pepsin. The preservative is merthiolate 1/10000. The bulk material is sterilely filtrated through a 0.2-μm membrane, diluted as appropriate and then dispensed into vials. Identification The antivenom sera are identified by the potency test. Potency The potency test of antivenom sera is carried out by neutralization method between venom and a specific antivenom serum. The test is performed on Swiss mice for determination a quantity of LD50 per ml of the serum to be examined. Method Materials Antivenom serum to be examined Purified venom Swiss mice weighing 18 - 20 g Physiological saline solution Procedure * Determination of LD50 of venom Preparation of stock venom solution: Weigh 10 mg of venom and dissolve in 10.0 ml of physiological saline solution to obtain a 1000 μg/ml solution of venom (store at 2 - 8 °C). Preparation of test venom solution: Dilute the stock venom solution with physiological saline solution to obtain a 250 μg/ml or 500 μg/ml solution of venom (depends on venom toxicity). Dilute the test venom solution to five suitable logarithmic serial dilutions at concentration interval of 1.1 or 1.4 times so that the highest concentration has shown mortality rate

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of 100% and the lowest has shown survival rate of 100% (mix well after dilution and allow to stand at 37 °C for 30 minutes, protected from light). Inject intravenously via the tail vein 0.5 ml into each mouse. Use 6 mice for each dilution. Read results after 24 hours to 48 hours. Calculate the result using Spearman-Karber formula: Log LD50 = X0 + d/2 x (Σ ri/n) Where: X0: Log of the lowest concentration of venom at which 100% of mice are dead. d: Log of dilution factor. ri: Number of dead mice of each dilution. n: Number of injected mice of each dilution. LD50 = AntilogLD50 *Determination of potency Preparation of venom solution containing 20 LD50/ml: Dilute the stock venom solution (1000 μg/ml, stored at 2 - 8 °C) to obtain a 20 LD50/ml solution with physiological saline solution. Preparation of antivenom serum to be examined: The sample (undiluted or diluted twofold, threefold … if the sample has concentration higher than 200 LD50) is chosen for at least 5 levels of volume differing from each to another 1.1 or 1.4 times so that the highest level can protect 100% of mice and the lowest level causes 100% of mice mortality after neutralization with a fixed amount of venom. Neutralization Add into each of the series of tubes 2.5 ml of venom solution containing 20 LD50, a chosen variable volume of the serum to be examined and then dilute to 5.0 ml with physiological saline solution. Shake gently and incubate the tubes at 37 °C for 30 minutes. Use 6 mice for each dilution. Read results after 24 to 48 hours. Calculate the effective dose 50% (ED50) using SpearmanKarber formula: Log ED50 = X0 + d/2 – d × (Σri /n) Where: X0: Log of the concentration of the antivenom serum to be examined at which 100% of mice are protected. d: log of dilution factor. ri: Number of the survived mice at each neutralized solution. n: Number of the injected mice at each neutralized solution. ED50 = AntilogED50 * Determination an amount of LD50 in the venom solution for neutralization (control group) Dilute the test venom solution containing theoretically 20LD50 to 1/20; 1/14; 1/10; 1/7 and 1/5 solutions in sterile saline (if determination of LD50 using the dilution factor d = 1.4) or 1/12.1; 1/11; 1/10; 1/9.1; 1/8.3 (if determination of LD50 using the dilution factor d = 1.1). Mix gently and incubate these tubes at 37 °C for 30 min, protected from light. Inject intravenously via the tail vein 0.5 ml into each 1035

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mouse. Use 6 mice for each dilution. Read results after 24 to 48 hours. Calculate neutralizing LD50 (T) as the same as the determination LD50 of venom. Log LD50 = - [X0 + d/2 - d × (Σ ri/n)] X0: Log of the lowest concentration of venom at which 100% of mice are dead. d: Log of dilution factor. ri: Number of dead mice of each dilution. n: Number of injected mice of each dilution. * Determination potency (or amount LD50 in 1 ml of the antivenom serum sample) by formula: Potency = (T-1)/ED50 Where: T: Amount of LD50 in 1 injected dose ED50: Effective dose 50% Acceptance criteria The potency test is not valid unless T value is in range of 4 to 6. The potency of the antivenom serum to be examined is not less than 1000 LD50 per vial.

Abnormal toxicity It complies with the test for abnormal toxicity (Appendix 15.11). Pyrogens It complies with the test for pyrogens (Appendix 15.12). Sterility test It complies with the test for sterility (Appendix 15.7). Total nitrogen Not more than 15% (Appendix 15.18). Thimerosal Not more than 0.01% (Appendix 15.29). Sodium chloride 0.85% - 0.90% (Appendix 15.26). pH 6 - 7 (Appendix 15.33). Filling and storage Fill from 2 ml to 5 ml per vial or freeze-dried form depending on manufacturers. Store at 2 °C - 8 °C, protected from light.

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VACCINES

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VACCINES FOR HUMAN USE Vaccina ad usum humanum Vaccines for human use are preparations containing antigenic substances capable of inducing a specific and active immunity in man against infecting organisms. Vaccines are prepared from bacteria, Rickettsia or viruses. Vaccines for human use may contain (1) organisms inactivated but retain adequate immunogenic properties; (2) living organisms that are naturally avirulent or (3) antigens extracted from the organisms.

Production The methods of production for each type of vaccines are described in the following sections or in individual monographs; these methods are designed to maintain adequate immunogenic properties, to render the preparation harmless and to prevent contamination with extraneous agent. In the production process suitable additives may be incorporated but penicillin is not used at any stage of production. To increase adequate immunogenic properties of certain vaccines, suitable additives may be used as included in individual monographs. Suitable preservatives may be used as specified for certain inactivated vaccines. The final lot is prepared by aseptically distributing the final bulk into sterile ampoules (or vials) which are closed so as to exclude contamination. Final lots of certain vaccines are prepared by freezedrying. Freeze-dried vaccines are only reconstituted right before use. Bacterial vaccines Bacterial vaccines are prepared from suitable bacteria grown in media under suitable culture conditions. The inactivated bacterial vaccines contain inactivated bacterial or antigenic substances that maintain adequate immunogenic properties. The live bacterial vaccines are prepared from attenuated strain whilst retaining specific immunogenic properties. The concentration of inactivated bacteria in vaccines is expressed in terms of International Units of Opacity or, where appropriate, is determined by direct cell count or, for living bacteria, by viable count. Bacterial toxoids Bacterial toxoids are prepared from toxins by eliminating their toxicity whilst retaining adequate immunogenic properties and the toxoids do not revert to toxin. Purified and adsorbed toxoids may form white or yellowish sediment at the bottom of the container, which will be dispersed in the vaccine liquids after shaking. Viral vaccines Viral vaccines are prepared using a seed lot system from viruses grown in animals, in avian embryos or in suitable cell cultures. They are liquids or may be freeze-dried.

Viral vaccines are often prepared from attenuated specific virus strains. Viral vaccines may be colored if they contain a pH indicator such as phenol red. The inactivated viral vaccines are treated by physical or chemical method. Combined vaccines Combined vaccines are the combination of 2 or several different vaccines.

Chemical content in vaccines Phenol For vaccines containing phenol, the content of it should be determined as described in Appendix 15.28. Where phenol has been used in the preparation of the vaccine, not more than 2.5 g/L is present in the final product, unless otherwise stated. Formaldehyde For vaccines containing formaldehyde, the content of it should be determined as described in Appendix 15.25. Where formaldehyde has been used in the preparation of the vaccine, not more than 0.2 g/L of free formaldehyde is present in the final product, unless otherwise stated. Aluminium For vaccines containing an aluminium component (Al3+), the content of it should be determined as described in Appendix 15.27. Where an aluminium adsorbent has been used in the preparation of the vaccine, not more than 1.25 mg of aluminium (Al+3) is present in single human dose, unless otherwise stated.

Storage Store protected from light. Unless otherwise stated, the storage temperature is 2 - 8 °C; liquid adsorbed vaccines must not be allowed to freeze. Expiry date The expiry date is calculated from the beginning of the assay for the vaccines stored in the prescribed conditions. The expiry date of vaccines is prescribed in individual monographs. Labelling The information on the label, box and leaflet must comply with the current regulations. BCG VACCINE Vaccinum BCG cryodessicatum Freeze-dried BCG vaccine is a preparation of live bacteria derived from a culture of the bacillus of Calmette and Guerin, intended for intradermal injection and protection against tuberculosis. BCG vaccine is produced in freezedried form and reconstituted with a suitable diluent immediately before use. 1037

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VACCINES

Production Production of BCG vaccine is based on a seed-lot system. The strain is chosen for and maintained to preserve its stability. The production method shall have been shown to yield consistently BCG products with the capacity to induce sensitivity to tuberculin and to protect against tuberculosis. Seed lots must be safe for man and laboratory animals. The strain 1173P2 - Pasteur Paris is used to produce BCG vaccine in Vietnam. The strain is cultured in Sauton-Potato medium and then passed into liquid Sauton medium. After harvesting, a homogenous suspension is prepared and then freeze-dried. The product is prepared from cultures which are separated from the master seed lot by as few subcultures as possible and in any case not more than 8 subcultures. Seed lots The strain used to establish the master seed lot is chosen for and maintained to preserve its stability, its capacity to induce sensitivity to tuberculin in man and guinea pigs and its absence of pathogenicity for man and laboratory animals. BCG for immunotherapy must be protected from sunlight. The procedure for production must be designed so that all stages of manufacture, testing and storage are protected from sunlight and ultraviolet light. Quality control of final bulk Sterility The final bulk complies with the test for sterility (Appendix 15.7). Virulent mycobacteria Only final bulk that has been stored at 4 °C for not more than 72 hours after harvesting may be used for the test. The test shall be performed in the final bulk or final lot (if necessary) (Appendix 15.9). BCG bacterial concentration Determine the total bacterial concentration in BCG final bulk by a suitable method, either directly determining the mass of the micro-organism or indirectly by measuring the optical density that has been calibrated in relation to the mass of the micro-organisms. Viability test Carry out the test as described in Appendix 15.1.

Quality control of final lot Characteristic appearance and suspension formation White, dry, loose and non-shrunken powder. When mixed with physiological saline it becomes a homogenous suspension within 1 minute. Opacity test The optical density (OD) of the BCG vaccine suspension (1 mg/ml) should not be more than 0.5. Measured by spectrophotometry at 490 nm with the green filter (Appendix 4.1). 1038

Dispersion test The vaccine complies with the test for dispersion (Appendix 15.3). Vacuum The vaccine complies with the test for vacuum (Appendix 15.2). Residual moisture Not more than 3% (Appendix 15.35). Sterility BCG vaccine complies with the test for sterility (Appendix 15.7). Identification BCG vaccine is identified by microscopic examination of the bacilli in stained smears demonstrating their acid-fast property and by the characteristic appearance of colonies grown on solid medium. General safety The general safety test for BCG vaccine is performed only in mice: 5 mice, each weighing 17 - 22 g. Each mouse is given a subcutaneous injection of 1 mg BCG vaccine per 1 ml. Observe and weigh each mouse for 7 days. If any of the animals shows losing weight or dies within 7 days of observation, repeat the test on twofold number of mice and samples. Acceptance criteria: The vaccine complies with the test if all of 5 mice survive and remain healthy, gain weight, without significant signs of toxicity after the observation period of seven days. Specific safety (absence of virulent mycobacteria) The test shall be performed in the final bulk or final lot (if necessary) (Appendix 15.9). Dermal reactivity The vaccine is injected intradermally into at least four guinea pigs of the same sex (use only non-pregnant females) that are negative to tuberculin test and each weighing 250 - 400 g. The injections consist of 0.1 ml of reconstituted vaccine and 0.1 ml of vaccine dilutions: 1/1; 1/10 and 1/100 (of both reference and testing vaccine). The lesions formed at the site of the injection are observed weekly for four weeks. After four weeks, the tuberculin test is performed by injecting intradermally into each guinea pig 5TU per 0.1ml. Read the results of the skin reaction after 24 hours. Acceptance criteria: The vaccine complies with the test if the reaction it produces is not markedly different from that produced by the reference vaccine. Viability Determine the number of viable units of reconstituted vaccine by viable count on Lowenstein-Jensen medium. The number of viable units shall be within the range of 1.106 - 6.106 CFU/mg of BCG vaccine (Appendix 15.1).

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Thermal stability Maintain the vaccine at 37 °C and 4 °C for 28 days. Determine the number of viable units in the incubated vaccine and in refrigerated vaccine. The vaccine complies with the test if the number of viable units in the incubated vaccine is not less than 20% of that in refrigerated vaccine (Appendix 15.1).

Storage and expiry date Freeze-dried BCG vaccine is stored at 2 - 8 °C and protected from sunlight. The expiry date is at least 24 months. After reconstitution, the BCG vaccine is kept at 2 - 8 °C, protected from sunlight and should be used within four hours. Labelling The information on the label and leaflet must comply with the current regulations. ORAL INACTIVATED CHOLERA VACCINE Vaccinum cholerae perorale inactivatum

Definition Oral inactivated cholera vaccine (mORCVAX) is a biological product derived from cholera bacterial whole cell which is inactivated by formaldehyde or heat. Each 1.5 ml of oral cholera vaccine contains: Vibrio cholerae O1, El Tor, Phil. 6973 (inactivated by formaldehyde): 600 E.U.LPS. Vibrio cholerae O139, 4260B (inactivated by formaldehyde): 600 E.U.LPS. Vibrio cholerae O1, Cairo 50 (inactivated by heat): 300 E.U.LPS. Vibrio cholerae O1, Cairo 50 (inactivated by formaldehyde): 300 E.U.LPS. Vibrio cholerae O1, Cairo 48 (inactivated by heat): 300 E.U.LPS. Preservative: Thimerosal. Production Working Seed lots Working seed lots (V.cholerae with El Tor biological type, Inaba serum type (strain Phil. 6973); V. cholerae with new O139 biological type (strain 4260B); V. cholerae with classic biological type, Ogawa serum type (strain Cairo 50); V. cholerae with classic biological type, Inaba serum type (strain 48) used for production of oral cholera vaccine must be approved by National Control Authority. Identity and purity of seed lots: determine V. cholerae bacteria only (with above biological types): is a commashaped, slightly curved gram-negative enteric bacteria. In the blood agar: produces convex, smooth, round colonies. V. cholerae grows well on thiosulfate-citrate-bile-sucrose agar (TCBS), on which it produces specific yellow colonies. It ferments saccharose and mannose but can not with arabinose.

Quality control of working seed lots by agglutination reaction: Remark points on glass slides. Dispense 20 µl of the cholera antiserum (Polyvalent, Inaba, Ogawa, O139) and physiological saline (K) on the remarked points. Dispense 20 µl of the cholera bacterial suspension into each of above antiserum. Thoroughly mix and read results. Acceptable criteria: Strain

Polyvalent Inaba Ogawa O139 K

V. cholerae O1, El Tor, Phil. 6973

+

+

-

-

-

V. cholerae O139, 4260B

-

-

-

+

-

V. cholerae O1, Cairo 48

+

+

-

-

-

V. cholerae O1, Cairo 50

+

-

+

-

-

Quality control of working seed lots in TCBS agar: Thoroughly shake the tubes containing cholera bacterial suspension, dispense 0.1 ml of it into TCBS agar plates, cultivate on TCBS agar plates by a sticker. Incubate this agar plates at (35 ± 1.0) °C in 48 hours. Observe the growth of bacterial colonies and the change of the medium color at 24 hours and 48 hours. Read result after 48 hours. Acceptance criteria: Bacterial colonies are clear, the color of TCBS medium changed from green to yellow. Quality control of working seed lots for chemico-biological characteristic: Thoroughly shake the tubes containing cholera bacterial suspension, cultivate 0.1 ml of it into every medium tube (containing Arabinose, Mannose, Saccharose). Incubate medium tubes at temperature (35 ± 1.0) °C in 48 hours, observe color change of the medium. Read result after 48 hours. Positive (+): Color of the medium changes to yellow. Negative (-): Color of the medium is same as original. Acceptance criteria: Chemico-biological characteristic of all cholera strains: Arabinose (-), Mannose (+), Saccharose (+). Production (1) Cholera bacteria are cultivated in a suitable medium. Determine the concentration of the cholera bacteria after fermentation. The cholera bacteria are inactivated by formaldehyde or heat depending on each strain. After inactivation, collect the cholera bacteria by centrifugation or tangential flow filtration (TFF).

Quality control of bulk vaccine Sterility The vaccine complies with the test for sterility (Appendix 15.7). Quality control after inactivation Process method: Shake thoroughly the tubes containing inactivated cholera bacterial suspension, cultivate 0.5 ml into agar plate (with 15% horse blood), make smoothly. 1039

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Each sample is cultivated in 2 blood agar plates. The agar plates are incubated in an incubator at (35 ± 1.0) °C in 48 hours. Observe the growth of cholera bacteria in 24 h and 48 h. Acceptance criteria: No cholera bacteria growth in blood agar. Determine bacteria concentration (by standard optical density test) Process method: as described in instructions of the standard optical density set supplied with manufacturers. For example: Mix thoroughly the cholera stock solution by vortex. Take 100 µl of the solution into test-tube of optical density set, gradually dispense (x) ml of the diluent (PBS 0.01 M pH 7.2 or physiological saline) until the optical density of test-tube equal to standard tube No. 10. Calculate the cholera bacteria density as below: - Convert to 1 ml: (0.1 ml of the stock + x ml of the diluent) × 10 = X ml - Density of the sample = X ml × (a × 109 bacteria/ml) (where: a × 109 is equivalent to standard tube No. 10 of standard optical density set supplied with manufacturers). LPS content Use ELISA method to determine the content of each LPS in the bulk vaccine.

Quality control of final bulk vaccine Sterility The vaccine complies with the test for sterility (Appendix 15.7).

Quality control of final product Physical appearance The physical appearance of every lot of the product is visually examined. After shaking, it produces a homogeneous and brownish suspension. Sterility The vaccine complies with the test for sterility (Appendix 15.7). pH 6.8 to 7.4 (Appendix 15.33). Total protein Not more than 5% (Appendix 15.18). Formaldehyde Not more than 0.02% (Appendix 15.25). Thimerosal Not more than 0.02% (Appendix 15.29). Potency The test is performed in rabbits that are negative with antibodies against to cholera bacteria. Rabbits weighing 2 kg to 2.2 kg are used for the test of each cholera vaccine lot. Dilute oral cholera vaccine with PBS 0.01M (pH = 7,2) or physiological saline for in order to gather concentration of the suspension is 4 billion bacteria/ml. Immunize the 1040

rabits by injecting to muscle area of hind leg of rabbits, 1 ml of diluted vaccine for each rabbit. Inject 03 doses for each rabbit, the duration is 1 week (on the days 0, 7th and 14th). After vaccination, take care and observe the rabbits every day. On the day 24th, take blood and separate serum in sterility condition. Perform the agglutination test in tubes between rabbit serum and specific antigen of cholera bacteria. Serum of each rabbit is dilluted 2-fold: 1/20, 1/40, 1/80, 1/160, 1/320 và 1/640. Antigen of cholera bacteria containing 3 strains 569B, Cairo 50 và Phil. 6973 is determined cholera bacteria concentration and diluted until the concentration is 1 billion bacteria/ml. Conduct agglutination reaction between specific antigen and dilluted rabbit serum. Read result after 48 hours. Result: The titration is determined at lowest dilution which still happens agglutination reation. Acceptance criteria: Potency of oral cholera vaccine meet the requirements if imean titration of each antigen ≤ 1/80.

Packaging and storage Inactivated oral cholera vaccine is filled in neutral glass vials. Store at 2 °C to 8 °C, do not frozen. Expiry date Shelf life of vaccine is studied from potency stability of 3 consecutive lots (produced from 3 separated lots of the bulk vaccine). Vaccine lots must be stored as mentioned in leaflet during study period. Shelf life of oral cholera vaccine is not more than 2 years since manufacturing date. Labelling Follow the requirements of the current regulations. Administration Oral cholera vaccine is for oral administration only. Dosage: 1.5 ml/person. Schedule: Duration between 2 doses is 14 days. ADSORBED DIPHTHERIA VACCINE Vaccinum diphtheriae adsorbatum Adsorbed Diphtheria Vaccine is prepared from diphtheria toxin which is treated by formaldehyde and heat to render it non-toxic without destroying its immunogenic potency. The toxoid contains not less than 1500 Lf per milligram of protein nitrogen and is adsorbed onto a suitable adjuvant, either hydrated aluminium phosphate or aluminium hydroxide in a 0.85% solution of sodium chloride. A suitable preservative can be added.

Production Strain of Corynebacterium diphtheriae used in production of diphtheria toxoid must comply with the regulations of defined seed-lot system grown in a suitable liquid medium.

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At the end of cultivation, the purity of each culture is tested and single contaminated harvest is discarded. The toxin content (Lf/ml) is checked by flocculation test. Single harvests may be pooled and detoxified with formaldehyde and purified by a suitable method. The specific toxicity, toxic reversibility and antigenic purity of purified toxoid are tested before preparation of the final product vaccine.

Bulk purified toxoid The strain for production is of high toxicity, well-known origin and history. The diphtheria toxin-containing culture medium is separated aseptically from the bacteria mass as soon as possible. The toxin content (Lf per milliliter) is determined by flocculation test to monitor consistency of production. Diphtheria toxoid is purified to remove components likely to cause adverse reactions in human. The purified toxin is detoxified with formaldehyde by a suitable method that avoids destruction of the immunogenic potency of the toxoid and toxic reversion. Alternatively, purification may be carried out after detoxification. Only bulk purified toxoid that complies with the following requirements may be used in the preparation of the final bulk vaccine. Sterility Carry out the test for sterility of the bulk purified diphtheria toxoid using 10 ml for each medium (Appendix 15.7). Specific toxicity and irreversibility of diphtheria toxoid Test of specific toxicity: Inject subcutaneously 1 ml of the purified toxoid containing 500 Lf per ml into each of 5 healthy guinea pigs, each weighing 250 - 350 g, that have not previously been treated with any material that will interfere with the test. Observe and investigate the animal mass within 6 weeks after administration. The animals that die during the observation period are to be examined by autopsy for signs of diphtheria toxaemia (red-swollen suprarenal gland). The purified toxoid complies with the requirement of specific safety if within 6 weeks of injection, the experimental guinea pigs show no signs of diphtheria toxaemia and at least 80% of guinea pigs survive. Test of irreversibility of diphtheria toxoid: The purified diphtheria toxoid is tested for the compliance with the absence of reversibility of toxoid. Use the same buffer solution as for the final bulk vaccine, without adsorbent, to prepare a suspension of bulk purified toxoid containing 35 Lf per ml. Divide the suspension into 2 equal parts. Incubate 1 part at (5 ± 3) °C and the other at 37 °C for 6 weeks. Inject subcutaneously 5 ml of each part into each of 5 healthy guinea pigs, each weighing 250 - 350 g, that has not previously been treated with any material that will interfere with the test. The bulk purified toxoid complies with the requirement if both groups of guinea pigs remain healthy and no animal shows signs of diphtheria toxaemia within 6 weeks of observation.

Antigenic purity The purified diphtheria toxoid is tested for antigenic purity by determining the antigenic content in units of Lf per mg of protein nitrogen. Determine the antigenic content by comparing with the reference toxoid calibrated by an international standard of diphtheria toxoid for the flocculation test or an equivalent national reference toxoid. The purified diphtheria toxoid complies with the requirement if it contains not less than 1500 Lf per mg of protein nitrogen.

Quality control of final bulk vaccine The final bulk vaccine is prepared by adsorption of a suitable quantity of bulk purified toxoid onto a mineral carrier such as hydrated aluminium phosphate or aluminium hydroxide. Suitable antimicrobial preservatives may be added. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lots. Antimicrobial preservative Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The amount is not less than 85% and not more than 115% of the stated amount (Appendix 15.29). Sterility Carry out the test for sterility of the final bulk vaccine using 10 ml for each medium (Appendix 15.7).

Quality control of final lot Identification An identification test is performed on at least one labelled container from each final lot by flocculation test as described in Appendix 15.19. Sterility The vaccine is not contaminated by bacteria and fungi (Appendix 15.7). Potency Carry out the test as described in Appendix 15.23. Acceptance criteria: The potency of diphtheria vaccine for children complies with the requirement if it contains not less than 30 IU per human single dose. For the tests using 3 dilutions, limits of the 95% confidence intervals of the estimate are within 50 - 200%, or the lower limit of the 95% confidence interval is ≥ 30 IU per single human dose. General safety Inject intraperitoneally one human dose into each of 5 healthy mice, each weighing 17 - 22 g, that have not previously been treated with any material that will interfere with the test; inject intraperitoneally at least one human dose (not more than 1 ml) into each of 2 healthy guinea pigs, each weighing 250 - 350 g, that has not previously been treated with any material that will interfere with the test. The final product complies with test of general safety (without abnormal toxicity) if all the experimental animals remain healthy, gain weight and show no signs of intoxication during seven days of observation. 1041

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Antimicrobial preservative The content of thimerosal is not less than 85% and not more than 115% of the amount stated on the label (Appendix 15.29). Aluminium Not more than 1.25 mg of aluminium per human single dose (Appendix 15.27). pH 6.0 - 7.0 (Appendix 15.33). Visual examination Each final vaccine lot after manufacturing is visually examined to ensure that the vaccines in the final closed containers before release for use comply with the requirements, showing no abnormal signs of visualization such as extraneous agents in the vaccine containers or tightness of closures and ensure their integrity. In the process of testing, any vaccine containers that do not comply with the requirement are discarded.

Storage and expiry date If kept under correct storage conditions, the vaccine can maintain its potency for 3 years from the date of potency testing. Labelling The information on the label, box and leaflet must comply with the requirements of the current regulations. DIPHTHERIA AND TETANUS VACCINE (ADSORBED) FOR ADULTS AND ADOLESCENTS Vaccinum diphtheriae et tetani ad usum adulti et adulescentis adsorbatum Adsorbed diphtheria and tetanus vaccine is a combined preparation of diphtheria and tetanus toxoids. The diphtheria and tetanus toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae or Clostridium tetani. After that, the toxins are treated by formaldehyde and heat to render them non-toxic without destroying its immunogenic potency. The toxoids are adsorbed onto a suitable adjuvant such as hydratedaluminium phosphate or aluminium hydroxide.

Production Bulk purified diphtheria toxoid The strain used in preparing diphtheria toxin is managed in a defined seed-lot system. A highly toxinogenic strain of Corynebacterium diphtheriae with known origin and history is grown in a suitable liquid medium. At the end of cultivation, the purity of its culture is tested and single contaminated cultures are discarded. The toxin-containing culture medium is separated aseptically from the bacterium mass. The toxin content (Lf per milliliter) is checked to monitor consistency of production. 1042

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Single harvests may be pooled, detoxified and purified. Purification may be carried out before or after detoxification. The toxin is purified to remove components likely to cause adverse reactions in humans. The purified toxin is detoxified with formaldehyde by a suitable method (usually by heating) that avoids destruction of the immunogenicity of the toxoid and reversion of the toxoid to toxin. Only bulk purified toxoid that complies with the prescribed requirements may be used in the preparation of the final bulk vaccine. Sterility Carry out the test for sterility using at least 10 ml for each medium. Specific safety (absence of toxin) Inject subcutaneously 1 ml of the purified toxoid containing at least 500 Lf per ml into each of 5 healthy guinea pigs, each weighing 250 - 350 g, that have not previously been treated with any material that will interfere with the test. Observe the guinea pigs within 42 days of the injection. If no animal shows signs of or dies from diphtheria toxaemia, the vaccine complies with the test. If more than one guinea pig dies from non-specific causes, repeat the test once; if more than one guinea pig dies in the second test, the vaccine does not comply with the test. Irreversibility of diphtheria toxoid Use the same buffer solution as for the final bulk vaccine to prepare a solution of bulk purified toxoid containing the same concentration of diphtheria toxoid as the final vaccine. Divide the suspension into 2 equal parts. Maintain 1 part at (5 ± 3) °C and the other at 37 °C for 6 weeks. Inject subcutaneously 5 ml of each part into each of 5 healthy guinea pigs, each weighing 250 - 350 g. Observe the animals within 6 weeks. The bulk purified diphtheria toxoid complies with the requirement if the results of the tests do not show any presence of diphtheria toxin. Antigenic purity Not less than 1,500 Lf per mg of protein nitrogen. Bulk purified tetanus toxoid The strain used in preparing tetanus toxin is managed in a defined seed-lot system. A highly toxinogenic strain of Clostridium tetani with known origin and history is grown in a suitable liquid medium. At the end of cultivation, the purity of its culture is tested and single contaminated cultures are discarded. The toxin-containing culture medium is separated aseptically from the bacterium mass. The toxin content (Lf per milliliter) is checked to monitor consistency of production. Single harvests may be pooled, detoxified and purified. Purification may be carried out before or after detoxification. The toxin is purified to remove components likely to cause adverse reactions in humans. The purified toxin is detoxified with formaldehyde by a suitable method that avoids destruction of the immunogenic potency of the toxoid and reversion of the toxoid to toxin, particularly on exposure to heat.

VP V

Only bulk purified tetanus toxoid that complies with the prescribed requirements may be used in the preparation of the final bulk vaccine. Sterility Carry out the test for sterility, using at least 10 ml for each medium (Appendix 15.7). Specific safety (absence of toxin) Inject subcutaneously 1 ml of the purified toxoid containing at least 500 Lf per ml into each of 5 healthy guinea pigs, each weighing 250 - 350 g, that have not previously been treated with any material that will interfere with the test. Observe the guinea pigs within 21 days of the injection. If no animal shows signs of paralysis or dies from tetanus toxaemia, the vaccine complies with the test. If more than one guinea pig dies from non-specific causes, repeat the test once; if more than one guinea pig dies or is paralysed in the second test, the vaccine does not comply with the test. Irreversibility of tetanus toxoid Use the same buffer solution as for the final bulk vaccine to prepare a solution of bulk purified toxoid containing the same concentration of tetanus toxoid as the final vaccine. Divide the suspension into 2 equal parts. Maintain 1 part at (5 ± 3) °C and the other at 37 °C for 6 weeks. Inject subcutaneously 5 ml of each part into each of 5 healthy guinea pigs, each weighing 250 - 350 g Observe the animals within 21 days. The bulk purified tetanus toxoid complies with the requirement if the results of the tests do not show any presence of tetanus toxin. Antigenic purity Not less than 1,000 Lf per mg of protein nitrogen.

Quality control of final bulk The final bulk vaccine is prepared by adsorption of suitable quantities of bulk purified diphtheria toxoid and tetanus toxoid onto a mineral carrier such as hydrated aluminium phosphate or aluminium hydroxide. Suitable antimicrobial preservative (usually use thimerosal) is added. Phenol is not used as it causes adverse effects on antigenic activity. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative Determine the amount of antimicrobial preservative in the final bulk by a suitable method. The amount is not less than 85% and not more than 115% of the intended amount (Appendix 15.29). Sterility Carry out the test for sterility of the final bulk, using 10 ml of the final bulk (Appendix 15.7). Potency Carry out the test for potency on the final bulk or the final lot if necessary (Appendices 15.22 and 15.23). Specific safety Inject subcutaneously at least 5 times the single human dose stated on the label into each of at least 5 healthy guinea pigs, each weighing 250 - 350 g, that have not

VACCINES

previously been treated with any material that will interfere with the test. Observe the experimental animals within 42 days of the injection. The animals that die during the observation period are to be examined by autopsy. The vaccine complies with the requirement if the experimental guinea pigs show no signs of tetanus toxaemia (tetanus paralysis or tetanus symptoms) and during the test period not less than 80% of the guinea pigs survive.

Quality control of final lot The final lot is prepared by aseptically distributing the final bulk into sterile ampoules (or vials) which are closed so as to exclude contamination. Only the final lots that comply with the requirements of tests recommended by WHO and VN standards may be released for use. Visual examination Each final vaccine lot after manufacturing is visually examined to ensure the vaccine in the final closed containers before release for use comply with the requirements, showing no abnormal signs of visualization. In the process of testing, any vaccine containers that do not comply with the requirement are discarded. Identification Identify the diphtheria and tetanus toxoids by flocculation test or by immunodiffusion. Potency For diphtheria component: Carry out the test as described in Appendix 15.23. The lower limit of the 95% confidence intervals of the estimated potency is not less than 2 IU per single human dose. For tetanus component: Carry out the test as described in Appendix 15.22. The lower limit of the 95% confidence intervals of the estimated potency is not less than 20 IU per single human dose. Sterility The vaccine complies with the requirement of sterility test, not contaminated by bacteria and fungi (Appendix 15.7). General safety Carry out the test as prescribed in Appendix 15.11. pH 6.0 - 7.0 (Appendix 15.33). Thimerosal The content of thimerosal is not less than 85% and not greater than 115% of the amount stated on the label (Appendix 15.29). Aluminium Not greater than 1.25 mg of aluminium per single human dose (Appendix 15.27). Content of sodium chloride Carry out the test as prescribed in Appendix 15.26. Residual formaldehyde Carry out the test as prescribed in Appendix 15.25.

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Storage and expiry date The expiry date is accepted by the National Control Authority and fixed, relying on the data of stability study of the vaccine on at least 3 consecutive lots (produced from separate final bulk). Stored at 2 - 8 °C, the vaccine can maintain its potency for 3 years from the date of the last potency testing. The vaccine must not be allowed to freeze.

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Labelling The information on the label, box and leaflet must comply with the current regulations.

Test of irreversibility of diphtheria toxoid Use the same buffer solution as for the final bulk vaccine, without adsorbent, to prepare a solution of bulk purified toxoid so as to contain the same toxoid content as that of the final lot (35 Lf per ml). Divide the solution into 2 equal parts. Maintain 1 part at (5 ± 3) °C and the other at 37 °C for 6 weeks. Inject subcutaneously 5 ml of each part into each of 5 healthy guinea pigs, each weighing 250 - 350 g. The purified toxoid complies with the requirement if the experimental guinea pigs remain healthy, normally gain body mass and no animal shows signs of diphtheria toxaemia within 6 weeks of observation.

ADSORBED DIPHTHERIA, TETANUS AND PERTUSSIS VACCINE (DTwP) Vaccinum diphtheriae, tetani et pertussis adsorbatum

Antigenic purity The purified diphtheria toxoid complies with the requirement if it contains not less than 1,500 Lf per mg of protein nitrogen.

Adsorbed diphtheria, tetanus and pertussis vaccine is a combined preparation of purified adsorbed diphtheria and tetanus toxoid, to which adsorbed whole cell pertussis vaccine is added.

Production of diphtheria toxoid Production is based on a seed-lot system. The strain of Corynebacterium diphtheriae used in preparing diphtheria toxoid is managed in a defined seed-lot system and grown in a suitable liquid medium. At the end of cultivation, the purity of each culture is tested and single contaminated cultures are discarded. The toxin content (Lf per milliliter) is checked by flocculation test. Single harvests may be pooled and detoxified with formaldehyde and purified by a suitable method. The sterility, specific toxicity, irreversibility and antigenic purity of the purified toxoid are tested before preparation of the final bulk vaccine. Sterility Carry out the test for sterility of the purified diphtheria toxoid in the thioglycolate medium and soybean-casein medium, for each medium using 10 ml of the purified diphtheria toxoid. Carry out the test and apply the acceptance criteria as prescribed in Appendix 15.7. Test of specific toxicity Inject subcutaneously 1 ml of the purified diphtheria toxoid containing at least 500 Lf per ml into each of 5 healthy guinea pigs, each weighing 250 - 350 g, that have not previously been treated with any material that will interfere with the test. Observe the animals for 42 days after administration. If within the period of observation any of the animals shows signs of or dies from diphtheria toxaemia, the purified diphtheria toxoid does not comply with the test. If more than one animal dies from nonspecific causes during the period of observation, repeat the test once. If more than one animal dies in the second test, the purified diphtheria toxoid does not comply with the test. 1044

Production of tetanus toxoid The tetanus vaccine is produced on the basis of a defined seed-lot system. The strain of Clostridium tetani used in preparing tetanus toxoid is managed in a defined seedlot system and grown in a suitable medium. At the end of cultivation, the purity of each culture is tested and single contaminated cultures are discarded. The toxin content (Lf per milliliter) is checked by flocculation test. Single harvests may be pooled and detoxified with formaldehyde and heat; then purified by a suitable method. The toxin content (Lf per ml) of the bulk purified tetanus toxoid is prescribed depending on the primary formula of each manufacturer but it contains not more than 25 Lf per single human dose if more than 1 dose is used to induce immunity with a primary vaccination schedule. The sterility, specific toxicity, irreversibility and antigenic purity of the purified toxoid are tested before preparation of the final bulk vaccine. Sterility Carry out the test for sterility of the purified tetanus toxoid in the thioglycolate medium and soybean-casein medium, for each medium using 10 ml of the purified tetanus toxoid. Carry out the test and apply the acceptance criteria as prescribed in Appendix 15.7. Test of specific toxicity Inject subcutaneously 1 ml of the purified tetanus toxoid containing at least 500 Lf per ml into each of 5 healthy guinea pigs, each weighing 250 - 350 g, that have not previously been treated with any material that will interfere with the test. Observe the animals for 21 days after administration. If within the period of observation any of the animals shows signs of or dies from tetanus toxaemia, the purified tetanus toxoid does not comply with the test. If more than one animal dies from non-specific causes during the period of observation, repeat the test once. If more than one animal dies in the second test, the purified tetanus toxoid does not comply with the test.

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Test of irreversibility of tetanus toxoid Using the same buffer solution as for the final bulk vaccine, without adsorbent, prepare a solution of bulk purified toxoid so as to contain the same toxoid content as that of the final lot (12.5 Lf per ml). Divide the solution into 2 equal parts. Maintain 1 part at (5 ± 3) °C and the other at 37 °C for 6 weeks. Inject subcutaneously 5 ml of each part into each of 5 healthy guinea pigs, each weighing 250 - 350 g. The purified tetanus toxoid complies with the requirement if the experimental guinea pigs remain healthy, normally gain body mass and no animal shows signs of tetanus toxaemia within 3 weeks of observation. Antigenic purity The purified tetanus toxoid complies with the requirement if it contains not less than 1,000 Lf per mg of protein nitrogen.

Production of inactivated pertussis suspension The pertussis suspension is produced relying on a seed-lot system. Strains of Bordetella pertussis used in preparing pertussis vaccine are managed in a defined seed-lot system and grown in a suitable medium. Strains, culture medium and cultivation method are chosen in such a way that agglutinogens 1, 2 and 3 are present in the final vaccine. Each strain is grown for 24 - 72 hours in a liquid medium or on a solid medium. Human blood or blood products are not used in any culture media. The medium used in the final cultivation stage does not contain blood or blood products. The bacteria are harvested, washed to remove substances derived from the medium and suspended in a sterile physiological saline solution to obtain a concentrated pertussis suspension. Test for purity of single harvests The purity of each single harvest is tested by microscopic examination on stained smears or by growing in a suitable culture medium. Only single harvests that are not contaminated with extraneous substances may be used in the preparation of the final bulk vaccine. Test for opacity The opacity of the concentrated pertussis suspension is determined (not later than 2 weeks after harvest and before being introduced into any subsequent preparing process) by comparison with the international reference preparation of opacity or by measuring the optical density at the wave length 560 nm. The concentration of the suspension is used as the basis of calculation for subsequent stages in vaccine preparation. The B. pertussis concentration of the final bulk vaccine (combined vaccine) does not exceed that corresponding to 20 International Opacity Units (IOU) per single human dose. If two or more strains of B.pertussis are used, the composition of consecutive lots shall be consistent with

VACCINES

respect to the proportion of each strain as measured in opacity units. The proportion of B. pertussis strains must have been registered on file and accepted by the National Control Authority. Test for viability The B. pertussis bacteria in the suspension are killed and detoxified in controlled conditions by a method together with the killing and detoxifying chemicals that have been approved by the National Control Authority. B. pertussis bacteria in the suspension are killed by heating for a suitable period, freedom from live B. pertussis is tested using Bordet-Gengou medium. After inactivation, the suspension is maintained at (5 ± 3) °C for a necessary period to diminish its toxicity. Only single pertussis suspension lots that comply with the requirements of sterility, freedom from live B. pertussis (test of viability), identification, growth and agglutination of the bacteria (identified with the seed-lot) may be used in the preparation of the final bulk vaccine. The production method is validated to demonstrate that the product, if tested, would comply with the following requirements.

Quality control of final bulk The final bulk vaccine is prepared by adsorption of suitable quantities of bulk purified diphtheria toxoid and tetanus toxoid onto a mineral carrier such as hydrated aluminium phosphate or aluminium hydroxide and admixture of an appropriate quantity of a suspension of inactivated B. pertussis; the resulting mixture is approximately isotonic with blood. The B. pertussis concentration of the final bulk vaccine does not exceed that corresponding to the opacity of 20 IU per single human dose. If 2 or more strains of B. pertussis are used, the composition of consecutive lots of the final bulk vaccine shall be consistent with respect to the proportion of each strain and the total opacity units do not exceed 20 IU per single human dose. The content of antimicrobial preservative in adsorbed diphtheria, tetanus and pertussis vaccine does not affect the immunogenic activity of the diphtheria and tetanus toxoids and the B. pertussis vaccine, and does not cause adverse effects on the users. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative Determine the amount of antimicrobial preservative in the final bulk by a suitable method. The amount is not less than 85% and not more than 115% of the intended amount (Appendix 15.29). Sterility Carry out the test for sterility of the final bulk in the thioglycolate medium and Soybean-casein medium, for each medium using 10 ml of the vaccine (Appendix 15.7).

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Potency Carry out the test for potency as described in Appendices 15.22; 15.23; and 15.24. Specific toxicity Specific toxicity of the DTwP vaccine is tested on the final bulk or the final lot if necessary (Appendix 15.4). Specific toxicity of the diphtheria and tetanus components Inject subcutaneously at least 5 times of the single human dose stated on the label into each of 5 healthy guinea pigs, each weighing 250 - 350 g. Observe daily the experimental animals. The vaccine complies with the requirement if within 6 weeks of the injection, the experimental guinea pigs show neither signs of diphtheria toxaemia nor signs of tetanus paralysis and during the test period not less than 80% of the guinea pigs survive. The animals that die during the observation period are to be examined by autopsy for signs of diphtheria toxaemia (red-swollen suprarenal gland). Specific toxicity of the pertussis component Use not fewer than 20 mice each weighing 14 - 16 g for the vaccine group and for the saline control. Use mice of the same sex or distribute males and females equally between the groups. Inject each mouse of the vaccine group intraperitoneally with 0.5 ml, containing a quantity of the vaccine equivalent to not less than half the single human dose. Inject each mouse of the control group with 0.5 ml of a sterile physiological saline solution (preferably containing the same amount of antimicrobial preservative as that injected with the vaccine). Weigh the groups of mice 72 hours and 7 days after the injection. The vaccine complies with the test if it complies with the 3 following criteria: At the end of 72 hours the total mass of the group of vaccinated mice is not less than that preceding the injection. At the end of 7 days the average increase in mass per vaccinated mouse is not less than 60 percent of that per control mouse. Not more than 5% of the vaccinated mice die during the test.

Quality control of final lot Identification Diphtheria, tetanus and whole cell pertussis components in adsorbed DTwP vaccine are segregated by adding sufficient sodium citrate (C6H5O7Na3,2H2O) to give a 5% solution and maintain at 37 oC for 48 hours. Centrifuge for 15 minutes at 2,000 rpm. Use the clear supernatant liquid to identify the diphtheria and tetanus components by flocculation test. Reserve the precipitate for identification of the pertussis component by glide agglutination test with antisera specific to B. pertussis (Appendix 15.19). Sterility The final lot vaccine complies with the test for sterility (Appendix 15.7). General safety Carry out the test on healthy mice and guinea pigs. Inject 1046

intraperitoneally half a human dose into each of 5 healthy mice, each weighing 17 - 22 g; inject intraperitoneally one human dose (not more than 1 ml) into each of 2 healthy guinea pigs, each weighing 250 - 350 g. The vaccine complies with test of general safety (without abnormal toxicity) if all the experimental animals remain healthy, gain weight and show no signs of intoxication during seven days of observation. Potency Carry out the test as described in Appendices 15.22; 15.23; and 15.24 (if potency test has not been carried out on final bulk). Physical and chemical characteristics Visual examination Each final vaccine lot after manufacturing is visually examined. The vaccine suspension is separated into 2 layers: the upper layer is a clear, colorless or yellowish solution, the lower layer is a greyish-white precipitate settled at the bottom. Physical characteristics The volume of vaccine in the container: The labeled volume +10%. Discard ratio: For multi-dose vaccines: Not more than 3%. For single-dose vaccines: Not more than 5%. Unfrozen (carry out the test only after storage or transport on cold chain system, the test is not involved in the manufacturer specifications of product release): After shaking, the sedimentation speed of the vaccine to be examined is much slower than that of the positive control and the vaccine suspension does not show any formation of particles or hovering floccules or aggregated clots. Preservative The content of thimerosal is not less than 0.005% and not more than 0.02% (Appendix 15.29). Adjuvant content Not more than 1.25 mg of Al+++ per single human dose (Appendix 15.27). pH 6.0 - 7.0 (Appendix 15.33). Free formaldehyde Not more than 0.02% (Appendix 15.25).

Storage and expiry date Stored at 2 - 8 °C, the vaccine can maintain its potency for 30 months from the date of potency testing. The manufacturer should give recommendations of storage and transport conditions to ensure its potency until the expiry date as registered and labeled. The adsorbed DTwP vaccine is stored in such a way as not to be allowed to freeze. The expiry date is accepted by the National Control Authority and fixed, relying on the data of stability study

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of the vaccine and does not exceed 30 months from the end of potency testing (calculated from the day of vaccination on the experimental animals).

Labelling The information included in label, box and leaflet must comply with the current regulations. DIPHTHERIA, TETANUS AND PERTUSSIS (ACELLULAR, COMPONENT) VACCINE (ADSORBED) Vaccinum diphtheriae, tetani et pertussis sine cellulis ex elementis praeparatum adsorbatum Diphtheria, tetanus and pertussis (acellular, component) vaccine (adsorbed) is a combined vaccine composed of: diphtheria toxoid; tetanus toxoid; individually purified antigenic components of Bordetella pertussis; adsorbed with aluminium salt such as aluminium hydroxide or hydrated aluminium phosphate. When shake thoroughly, vaccine makes a homogenous solution. PRODUCTION

Diphtheria toxoid and tetanus toxoid production See “Diphtheria toxoid production” and “Tetanus toxoid production” of “Diphtheria, tetanus and pertussis (whole cell) vaccine (adsorbed) - DTwP” section. Production of the pertussis component Working seed lot Use Bordetella pertussis phage I for the production. Bordetella pertussis strain for vaccine production must comply with the regulation of a master seed lot. If using a DNA changed Bordetella pertussis strain, the DNA changed sequence must be described the properties clearly and completely. The property of the working seed lot is the same as of the master seed lot; the working seed lot may be stored at liquid nitrogen or freeze-dried. Culture media: Do not use any media containing blood, blood-product of human to inoculate Pertussis strain for pertussis vaccine production including a final stage. The purity of the culture media has to be verified. Protective antigenic components are purified by chemicals methods such as: Repeated cycles of ammonium sulfate precipitation, sucrose density gradient centrifugation. After the purity, the antigens are detoxified by formaldehyde or a suitable method to inhibit their toxicity. Control of bacterial purity: Inoculation individual cultures in appropriate media. Gram - stain sample of all individual cultures. If a contamination is found, the culture must be discarded. Sterility test: No growth of bacteria and fungi (Appendix 15.7).

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Characterization of antigens The stability of individual antigens must be validated with the following requirements during the production. After validation, the tests are not required for all production batches. Adenylate cyclase: Not greater than 500 ng/single human dose. Windpipe - cell toxin: Not greater than 2 pmol/single human dose. Residual necrosis toxin: Inject intradermally 3 mice (no wean of breeding), 0.1 ml of each mouse, with quantity of antigen equivalent to 1 single dose for human. Observe for 48 h. The test is not valid unless the absence of necrosis reaction is verified. Pertussis toxin: Chinese hamster ovary (CHO) cellclustering effect and haemagglutination as in vitro methods; lymphocytosis-promoting activity, histaminesensitizing activity and insulin secretory activity as in vivo methods. Specification: The toxin shows ADP-ribosyl transferase activity using transducin as the adsorbent. Filamentous haemagglutinin: Use the hemagglutinin method or agglutination inhibition method. Specification: Haemagglutination and inhibition by a specific antibody. Pertactin, fimbrial-2 and fimbrial-3 antigens: Use suitable immunoassay. Specification: Reactivity with specific antibodies. Pertussis toxoid: Consider that the inhibition ability of mouse antibody with the toxicity of pertussis toxin is detected or not. Specification: The toxoid induces in animals the production of antibodies capable of inhibiting all the properties of pertussis toxin. Purity of antigens Before detoxification: The purity of individual or copurified antigens is verified by appropriate methods such as: SDS-PAGE, liquid chromatography or other suitable method. In case where 2 or more antigens are co-purified, the proportion of each antigen must be detected by suitable method such as SDS-PAGE, HPLC, electrophoresis on non-denaturing gel or densitometry, and proved that the effect of vaccine is shown in the clinical trials. Endotoxin level: Endotoxin level is less than 100 IU/single human dose. Use Limulus amoebocyte test (LAL) or other equivalent test (Appendix 13.2). Sterility: The vaccine complies with the test for sterility (Appendix 15.7). Detoxification: PT, FHA (Filamentous hemagglutinin), Pertactin are detoxified by formaldehyde using a validated suitable method (heating method normally) to ensure to not revert to biologically active pertussis toxin upon storage. Other components, fimbrial-2 and fimbrial-3 are proved that there is no link between them and other toxins. After detoxification: control residual activity of pertussis toxin (PT): the amount of residual activity of PT should be determined by method of a sufficiently sensitive test such as the agglutination of CHO cell. By adequate dilution of 1047

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the test solution, the total of amount of residual activity PT from all pertussis antigens should not exceed that found in vaccine lot shown to be safe in clinical trials. Antigen content: By immunochemical method, nitrogenprotein method or other suitable methods. Specification: the proportion of antigen to nitrogen-protein should not exceed the determined limit. Sterility: Each bath of the purified antigen must be performed the sterility test (Appendix 15.7). Specification: No growth of bacteria and fungi after 14 days of inoculation. CONTROL OF FINAL BULK

The final bulk is prepared by adsorption of suitable quantities of bulk purified diphtheria toxoid, tetanus toxoid, and pertussis toxoid components separately or together onto hydrated aluminium phosphate, aluminium hydroxyde, suitable amount of excipients and preservatives are added so as to gain the safety and effect.

Residual toxin content (free formaldehyde) Residual formaldehyde content is not greater than 0.2 g/l (Appendix 15.25). Residual glutaraldehyde content is not greater than 0.1 g/l. Preservative Determine the amount of antimicrobial preservative by a suitable chemical or microbiological method. The amount is not less than 85% and not greater than 115% of the intended content (Appendix 15.29). If the antibiotic is used, the content of antibiotic is not less than the content minimum shown the storage effect and not greater than 115% of the intended content (Appendix 13.9). Aluminium Not greater than 1.25 mg per single human dose (Appendix 15.27). Calcium Not greater than 1.3 mg per single human dose. Sterility The final bulk complies with the test for sterility (Appendix 15.7). Potency Carry out the potency of diphtheria and tetanus component as Appendix 15.22 and 15.23. Immunological activity of pertussis component Make immune for mice, and then titrate antibody by ELISA method. Specification: Many calculating software may be applied. If use SoftMaxPro software, anti-PT antiserum: Not less than 38 EU/ml, anti-FHA antiserum: Not less than 125 EU/ml, anti-PRN antiserum: not less than 9400 EU/ml. Confident limit (P = 0.95), the potency is not less than 50% and not greater 200% for relative potency of each pertussis antigen. 1048

The statistically valid assay shows that the slope is valid, and no deviation of dose immune curve.

Specificity test For diphtheria and tetanus components, see Appendix 15.4. Reversion to toxicity and residual activity of pertussis toxin test Test the sensitivity of pertussis toxin with histamine. 2 methods: the lethal rate after 24h of injection of histamine challenge dose and the temperature measurement of mice after injection of histamine dose. For the 1st method, when make immune by 1 vaccine human dose for each mouse, no mice die at the histamine challenge dose 8 mg/ml of histamine dihydrochloride or 4 mg/ml of histamine base. Total of mice per each challenge dose is 10. For 2nd method, the PT content of residual activity or reversion to toxicity is not greater than 0.4 HSU/ml (HSU: histamine sensitization unit). CONTROL OF FINAL LOT

Inspection Inspect visually each container, suspension dividing 2 layers: the supernatant is transparent, colorless or light yellow; the residue is gray-white. Shake gently, 2 layers make a homogenous solution quickly. No abnormal subject. Identification For tetanus, diphtheria component identification, see Appendix 15.19. For acellular pertussis identification: Double immunodiffusion method. There is the specific interaction of reference antibodies with pertussis antigens of vaccine Sterility The final lot complies with the test for sterility (Appendix 15.7). Aluminium Not greater than 1.25 mg per single human dose (Appendix 15.27). Calcium Not greater than 1.3 mg per single human dose. Thimerosal 0.005% - 0.02% (Appendix 15.29). Residual formaldehyde Not greater than 0.02% (Appendix 15.25). pH 6.0 - 7.0 (Appendix 15.33). General safety Carry out the test on healthy mice and guinea pig. Inject intraperitoneally 5 mice, each weighing 18 - 22 g, and 2 guinea pigs, each weighing 250 - 350 g.

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Injection dose: for mice: A single human dose but not greater than 1 ml per each mouse, for guinea pig: 10 human doses but not greater than 5 ml. Vaccine is passed for unexpected toxicity if all animals are healthy, increase the weight after 7 days of observation and no poisonous signal.

Bacterial endotoxins Less than 100 EU per single human dose. Use Limulus amoebocyte test (LAL) or other equivalent test (Appendix 13.2). Reversion to toxicity and residual activity of pertussis toxin test See the control of final bulk. Diphtheria, tetanus potency Cary out this test on the final lot if not carry out on the final bulk or if necessary (Appendix 15.22 and 15.23). Immunological activity of pertussis component Make immune for mice, and then titrate antibody by ELISA method. Specification: many calculating software may be applied. If using SoftMaxPro software, anti-PT antiserum is not less than 38 EU/ml, anti-FHA antiserum is not less than 125 EU/ml, anti-PRN antiserum is not less than 9400 EU/ml. For confidential limit (P = 0.95), the potency is not less than 50% and not greater 200% for relative potency of each pertussis antigen. The statistically validity shows that the slope is valid, and no deviation of dose immune curve. Storage and expiry date Store at 2 °C - 8 °C, the potency may be maintained at least 2 years. The manufacturer should recommend conditions of storage and transport to ensure that the vaccine satisfies the potency requirements until the expiry date stated on the label. The vaccine must not be frozen. The expiry date should be defined on the basis of shelflife supported by the stability studies and approved by the national regulatory authority. Labelling The information on the label, container, and leaflet must comply with the current regulations. DIPHTHERIA - TETANUS - PERTUSSIS HEPATITIS B - HEAMOPHILUS INFLUENZA TYPE b (DTwP - HeB- Hib) COMBINED VACCINE. Vaccinum diphtheriae, tetani, pertussis, hepatitidis B et haemophili stirpis b coniugatum adsorbatum Diphtheria - Tetanus - Pertussis - Hepatitis B - Heamophilus Influeanzae type b (DTwP – Heb - Hib) combined vaccine

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is the mixture of Diphtheria toxoid, Tetanus toxoid, whole cell Pertussis vaccine, recombinant Hepatitis B surface antigen (HBsAg), conjugated Heamophilus Influeanzae type b antigens which is adsorbed on to aluminum. Here, aluminum is considered as an adjuvant.

Diphtheria toxoid production Diphtheria vaccine is produced base on a seed-lot system. Corynebacterium diphtheriae strain used in production complies with the requirements for seed-lot system, and it is cultured in liquid Lingood medium. At the final stage of this culture, carry out the test for bacterial purity to discard single harvests in case of contamination. Determine diphtheria toxin content (Lf/ml) by using flocculation test. Single harvests can be mixed to detoxify by using formaldehyde and purified by appropriate methods. Purified diphtheria toxoid shall be tested for sterility, specific toxicity, reversion to toxicity, antigenic purity of antigen before preparing the final bulk. Sterility Carry out the test for sterility in fluid Thioglycolate medium and Soybean-casein-digest medium, using 10 ml of inoculum diphtheria toxoid for each medium. The method and criteria for this test complies with Appendix 15.7. Specific toxicity test Use 5 guinea pigs, weighing 250 g - 350 g per guinea pig, healthy and haven’t been used previously for experimental purposes. Inject subcutaneously 1 ml of a dilution of purified toxoid containing at least 500 Lf of toxoid for each pig. Observe the animals during 42 days after injection. If any of the animals shows signs of or dies from diphtheria toxaemia, the vaccine does not comply with the test. If more than 1 animal dies from non-specific causes, repeat the test once; if more than 1 animal dies in the second test, the vaccine does not comply with the test. Reversion for toxicity test The bulk purified toxoid shall be diluted in the same buffer as use in the final bulk vaccine, except for the presence of adjuvant in order to obtain the same concentration (35 Lf/ml). Diluted toxoid have been stored at 37 °C for six weeks, similar dilutions of toxoid held at (5 ± 3) °C during the same period. Reversion of toxicity in diphtheria toxoid can be tested by injecting each of dilutions subcutaneously into five healthy guinea pigs weighing from 250 g to 350 g per guinea pig. Purified diphtheria toxoid shall pass the reversion toxicity test if all animals are healthy, increase their body weight, and none shows any symptom of specific diphtheria toxin during six weeks of test. Diphtheria antigen purity Concentration of purified diphtheria toxoid is not less than 1500 Lf per mg of protein nitrogen. 1049

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Tetanus toxoid production Tetanus vaccine is produced base on a seed lot system. Clostridium tetani strain used complies with the requirements for seed lot system, and it is cultured in appropriate medium. At the final stage of this culture, carry out the test for bacterial purity to discard single harvests in case of contamination. Determine tetanus toxin content (Lf/ml) by using flocculation test. Determine tetanus toxicity by using Minimum Lethal Dose test (MLD). Single harvests can be mixed to detoxification by using formaldehyde, temperature, and it is purified by appropriate method. Antigen concentration (Lf/ml) in the final bulk depend on origin formula of manufactures but it contains not less than 25 Lf per single human dose when use more than one dose in basic vaccination schedule. Purified tetanus toxoid shall be tested for sterility, specific toxicity, reversion to toxicity, antigenic purity of antigen, concentration of antigen (Lf/ml) before preparing the final bulk. Sterility Carry out the test for sterility in fluid Thioglycolate medium and Soybean-casein-digest medium, using 10 ml of inoculum diphtheria toxoid for each medium. The method and criteria for this test complies with Appendix 15.7. Specific toxicity test Use 5 guinea pigs, weighing 250 g - 350 g per guinea pig, healthy and haven’t been used previously for experimental purposes. Inject subcutaneously of 1 ml of a dilution of purified toxoid containing at least 500 Lf of toxoid. Observe the animal during 21 days after injection. If any of the animals shows signs of or dies from tetanus toxin, the vaccine does not comply with the test. If more than 1 animal dies from non-specific causes, repeat the test once; if more than 1 animal dies in the second test, the vaccine does not comply with the test. Reversion for toxicity test The bulk purified toxoid shall be diluted in the same buffer as use in the final bulk vaccine, except for the presence of adjuvant in order to obtain the same concentration (≤ 20 Lf/ml). Diluted toxoid have been stored at 37 °C for six weeks, similar dilutions of toxoid held at (5 ± 3) °C during the same period. Reversion of toxicity in tetanus toxoid can be tested by injecting each of dilutions subcutaneously into five healthy guinea pigs weighing from 250 g to 350 g per guinea pig. Purified tetanus toxoid shall pass the reversion toxicity test if all animals are healthy, increase their body weight, and none shows any symptom of specific diphtheria toxin during three weeks of test. Tetanus antigen purity Concentration of purified tetanus toxoid is not less than 1000 Lf per mg of protein nitrogen. 1050

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Inactivated whole cell pertussis suspension production Inactivated whole cell pertussis suspension is produced base on a seed lot system. Bordetella pertussis strain used complies with the requirements for seed-lot system; it is cultured in appropriate medium. The appropriate strain, medium and method for culture shall be chosen in such a way that the final pertussis vaccine includes agglutinogens 1, 2 and 3. Each strain is cultured in liquid or solid medium for 24 hours to 72 hours. Don’t use human blood or blood products in culture media in any stage of pertussis strain culture to vaccine production. At the final stage of this culture, have to use culture media without human blood or blood products. After culture, residual contamination present in medium shall be removed by washing the harvested bacteria. Add saline solution to harvested bacteria to make condensed pertussis suspension. Control bacteria purity of single harvests Samples of single harvests taken before killing shall be tested for purity by microscopic examination of stained smears or by inoculation into appropriate culture media. Single harvests shall not be used for the final bulk if contamination has occurred at any stage in their production. Control of opacity Either use International opacity reference or measurement OD (optical density) value of condensed pertussis suspension at wavelength of 560 nm (the opacity of each harvest shall be measured not later than two weeks after harvesting and before the bacteria suspension has been subjected to any next process) to determine density of condensed pertussis suspension. This value is used as a baseline to calculate when preparing the final bulk. Pertussis component in DTwP - HeB - Hib vaccine does not exceed 15 IOU (International Opacity Unit) per a single human dose. The proportion of B. pertussis strains doesn’t change when mixing vaccine; it is written clearly in the document and approved by the national control authority. Survival bacteria The bacteria shall be killed and detoxified by a method approved by the national control authority. If chemicals are used for this purpose, they shall be approved by the national control authority. B. pertussis bacteria can be killed by temperature in an appropriate period of time. Survival of the bacteria is tested on Border - Gengou. After inactivation, B. pertussis suspension is held at (5 ± 3) °C to reduce its toxicity. Single B. pertussis suspension lot is not used to prepare final bulk when it doesn’t meet criteria for sterility, complete inactivation (test for survival), identification, growth and agglutination as these in original strains.

Recombinant Hepatitis B surface antigen (HBsAg) production Recombinant Hepatitis B vaccine has been produced by using HBsAg derived from yeast (Saccharomyces

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cerevisiae, Pichia pastoris, Hansenula polymorpha), or from CHO (Chinese Hamster Ovary), or from other appropriate cell lines which is transformed a plasmid that has HBsAg encodes gene. This HBsAg derived by lysing yeast. It is purified by biological, chemical and physical technique. Recombinant Hepatitis B vaccine may contain the S gene product (essential protein) or products of the S/pre-S2 combination (medium protein) or products of the S/pre-S1 and pre-S2 combination (big protein). Protein content Determine protein content in vaccine by Lowry method (appendix 15.34) Identification and HBsAg content The HBsAg content shall be determine by a suitable method include radioimmuno assay (RIA), enzymlinked immunosorbent assay (ELISA), single radial immunodiffusion, immunosorbent assay. Sample is compared with international reference standard or national reference standard. Purity antigen It is determined by liquid chromatography method (Appendix 5.3). Lipid content Not more than 100 µg/100 µg protein (Appendix 15.40). Cesi chloride (CsCl) content Not more than 5 µg/20 µg protein (Appendix 15.41). Tween 20 Not more than 50 µg/100 µg protein (Appendix 15.6). Polysaccharide Not exceed 10 µg/100 µg protein (Appendix 15.38). This test is performed at the final bulk. Thimerosal Not more than 0.012% (Appendix 15.6). Sterility It complies with the test for sterility (Appendix 15.7).

Conjugated Hib antigen production Haemophilus influenzae type b vaccine is the chemical vaccine prepared from synthesis oligosaccharide that likes capsular polysaccharide of bacteria from nature. Oligosaccharide covalently linked to tetanus toxoid as carrier protein. In a single human dose, PRP-T antigen content is 12 µg. Hib Identification Carry out identification test by using latex agglutination base on specific reaction between antibody and antigen. Either use biological test kit with suitable requirements for Hib identification test, perform test follow the guideline of manufacture, or use a suitable immuno-chemical method. Acceptance criteria: Agglutination occurs; it is visible to naked eyes.

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Total polysaccharide content (Polyribosyl ribitol phosphate = PRP) Determine total polysaccharide content by using Orcinol method (appendix 15.42) or liquid chromatography method. Acceptance criteria: At least 80% of total saccharide content on the label. Free Saccharide content Not more than 25.0% (≤ 25.0%). Determine saccharide free content by using Orcinol method (appendix 15.42) or liquid chromatography method. Immunogenicity test Biological materials F1 female rabbit (1.8-2.2 kg) is chosen, transported and taken care in the normal condition. Positive control: Hib vaccine. Prepare solutions: 20×PBS solution contains: 2.74 M sodium chloride R, 0.054 M potassium chloride R, 0.16 M disodium hydrogen phosphate R and 0.028 M potassium dihydrogen phosphate R. Weigh 320 g of sodium chloride R, 8 g of potassium chloride R, 46 g of disodium hydrogen phosphate R, 8 g of potassium dihydrogen phosphate R. Dissolve in 1500 ml of distilled water R. Adjust to pH 7.2 to 7.3 with either hydrochloride acid R or 2 M sodium hydroxide R. Transfer whole solution into a 2-liter volumetric flask; add distilled water R to volume. This solution have 20 times at concentration if compare with working solution. Store this solution at room temperature, use within one month. PBS solution: Dilute 20 × PBS solution 20 times by distilled water as following: Transfer 100 ml of 20 × PBS solution into 2 liter volumetric flask, add distilled water R to volume, shake well. If necessary, adjust to pH 7.2 to 7.3 with either hydrochloride acid R or 2 M sodium hydroxide R. Store this solution at 4 °C for 15 days. Procedure This procedure is carried out in parallel for the control vaccine. Vaccine dose is 10 µg/0.5ml. Inoculate 5 rabbits by received sample, at the vaccine dose of 10 µg/0.5ml. If the vaccine has higher concentrations, dilute with PBS solution and use the sterile materials. If the vaccine has lower concentration, inoculate with as much volume as necessary to reach the dose of 10 µg, but not more than 2.5 ml per one rabbit. Inoculate on day 0 and day 14th. Route of inoculation: subcutaneous. Transfer animals to the animal quarantine area; observe animals during 28 days after inculcating. Twenty-one days after the first dose, take not less than 5 ml of blood of each rabbit in 15 ml corning tube. Centrifuge each of the blood samples at 3000 rpm for 20 minutes at room temperature. 1051

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Extract carefully the serum with a pipette (the serum of each tube must not be mixed) and pass it through reaction vials. If the test for anti-Hib antibodies assay is not done right now, store the serum at -20 °C for later assay. Determine the content of antibodies in the serum after centrifugation stage by means of an ELISA system for the determination of anti-Hib antibodies. Equipments ELISA reader, at a wavelength of 492 nm. Bio safety cabinet. Washing ELISA plate machine. Multichannel ELISA meter. Materials A 96-well microtitre plate. Humid chamber (take a tight metallic or plastic box and recover with a filter paper damped with distilled water). Reagents Tween 20. BSA Fraction V. OPD. Anti-rabbit peroxidase conjugate. Negative control: Anti-Hib serum. Positive control: Anti-Hib rabbit serum. Hib oligosaccharide conjugate with HSA (coating) (HbOHSA). Prepare solutions 20× PBS solution: Prepared as above. Citrate phosphate buffer solution contains 0.048 M citric acid R, 0.1 M disodium hydrogen phosphate R. Washing solution: Containing 0.027 M potassium chloride R, 0.137 M sodium chloride R, 0.008 M disodium hydrogen phosphate, 0.0014 M dipotassium hydrogen phosphate R, 0.05% Tween 20, is prepared as below: Add 2.5 ml of Tween 20 to 5 liters of PBS solution. Keep at room temperature, use within 7 days. Tween 20 is diluted to 1/4: Mix 10 ml of Tween 20 with 30 ml of distilled water. Keep at room temperature, use within 1 month. Stop solution: Weigh 10 g of sodium metabisulfit R, dissolve in 840 ml of distilled water, and add slowly 160 ml of sulfuric acid R. Mix well. Put it into ice box. Carry out this procedure in safety cabinet, wear gloves. Store this solution for one month. Blocking solution (Solution of 1% BSA): Prepare this solution before use. Weigh 0.2 g of BSA; dissolve in 20 ml of distilled water. Diluent solution for ELISA anti-Hib: Weigh 1 g of BSA, 0.372 g of sodium edetate R, add 1.2 ml of Tween 20 is diluted to 1/4 solution, Dissolve whole components in 80 ml of PBS solution. Adjust to pH 7.2 to 7.3 with either hydrochloride acid R or 2 N sodium hydroxide R. Add PBS solution up to 100 ml. Store this solution at 2 - 8 °C, use within one month. Procedure: Prepare the positive control (Anti-Hib rabbit serum) and 1052

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the negative control (Anti-Hib serum) as below: Dilution at 1/50 with the diluent solution for ELISA anti - Hib. Discard the remaining materials. Note: Keep all in humid chamber. After the wash step, if not use immediately, put the plate (with the face upward) in humid chamber for not more than 15 minutes. Dilute the coating HbO-HSA for a final concentration of 1 µg/ml of HbO- HSA in PBS for a final volume of 11 ml. This final volume is enough for a 96-well plate. Incubate the plate at 37 °C for 90 minutes or the whole night at 4 °C. Wash the plate three times with the washing solution. Add 100 µl of blocking solution per well. Incubate the plate at 37 °C for 30 minutes. Wash the plate three times with the washing solution. Design the plate The volume of samples and controls to adds is 100µl per well. Add four replicates of the positive control and negative control of the ELISA system in the 1st, 2nd columns from the line A to H. The samples are added from the 3rd column, from the line A to H every serum to evaluate in duplicate. Incubate the plate 90 minutes at room temperature. Wash the plate three times with the washing solution. Dilute the anti-rabbit peroxidase conjugate in the diluent buffer of sample at the dilution declared by the supplier of the reagent and add 100 µl of the dilution to the whole plate. Incubate the plate at 37 °C for one hour. Wash the plate three times with the washing solution. Prepare the substrate solution as follows: Weigh 0.1 g of OPD and dissolve into 25 ml of phosphate-citrate buffer, dissolve in the darkness and add 10 ml of hydrogen peroxide, mix well and add 100 µl per well to the whole plate. Incubate the plate at room temperature for 5 min in the darkness. Stop the reaction by adding 50 µl of the stop solution to each well. Read the plate at a wavelength of 492 nm (Multiscan). Analyze and interpret the results The test is valid when OD value of positive control and negative control is in the range which is established previously. If more than 50% of OD values fail, repeat ELISA test. Calculate the cutoff value of the trial to establish the standard for declaring the positiveness or negativeness of the serum using the following formula: Cutoff value = Mean of the baseline + 2 SD Where: SD: Standard deviations. All OD values that are equal or more than the cutoff value are positive. All OD values that are less than the cutoff value are negative. Calculate the responded percentage.

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The percentage of responded animals = total of responded animals × 100/ total of inoculated animals. Acceptance criterion of test The test is invalid unless the percentage (%) of responded animals is not less than 50% (≥ 50%) in control group. Acceptance criterion of the sample The sample passes the test when the percentage of responded animals is not less than 50% (≥ 50%). The production method is validated to demonstrate that the product, if tested, would comply with the following criteria.

Quality control of bulk vaccine The bulk vaccine is prepared and mixed by using a suitable quantity of diphtheria toxoid, tetanus toxoid, hepatitis B surface antigen previously adsorbed on aluminum phosphate and add an appropriate quantity of inactivated B.pertussis suspension. Finally, mix with Hib antigen adsorbed on to aluminum phosphate. The mixture is suitable about physiological like blood when inject it to human. Opacity of B. pertussis in the final bulk does not exceed 15 IOU per single human dose. If use two or more than two of B. pertussis strains, total opacity unit of all B. pertussis strains is consistent in lots of vaccine and not more the 15 IOU per a single human dose. Preservative content in adsorbed DTPw - HeB - Hib combined vaccine have been show not to have any deleterious on the immunogenicity of tetanus toxoid, diphtheria toxoid, pertussis vaccine, Hib antigen, HeB antigen and it doesn’t cause any unexpected adverse reactions in human. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Preservative Determine preservative content in the final bulk vaccine by using an appropriate method (appendix 15.29). Preservative content must be from 85% to 115% of preservative content stated on the label. Sterility Carry out the test for sterility by using fluid thioglycollate medium and Soybean-casein-digest medium. Use 10 ml of inoculum tetanus toxoid for each medium (appendix 15.7). Potency test Carry out the potency test according to Appendix 15.22, Appendix 15.23, and Appendix 15.24. Potency test of recombinant hepatitis B vaccine is carried out in parallel for the sample and national reference standard vaccine. Use in vivo method to determine antibody titer or in vitro method to determine HBsAg content in vaccine. In vivo potency test Animal White mice BALB/C or ICR strain, healthy, and come from the same pack of mice, aged at about from 5 weeks to 6 weeks, and in the same sex preferably.

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Relative potency Dilute sample and national reference standard vaccine with 2-fold with 5 dilutions. The diluent is saline containing aluminum hydroxide (not more than 500 µg of aluminum hydroxide per ml). Inject intraperitoneally 1 ml of each dilution per mouse. Inject for at least 15 mice per each dilution. All of immunized mice are taken care from 28 days to 30 days in the same condition. Anaesthetize mice and take blood from their heart. Centrifuge each of the blood samples at 3000 rpm at 4 - 8 °C. Extract the serum; store at -20 °C. Determine anti-antibody HBsAg titer by enzym-linked immunosorbent assay (ELISA) by using trade kit. Calculate result by Probit Analysis program (WHO). The test is valid if: For both the sample and national reference standard vaccine, the ED50 lies between the smallest and largest does given to animals. Statistical analysis shows to be satisfactory in linearity and parallel. The limit of the confidence interval is in range from 33% to 300%. Criteria for in vivo potency test Relative potency (P = 95%) is not less than 1. In vitro potency test Determine HBsAg antigen content by ELISA technique. Materials 96-well plates. Hepanostika HbsAg Uni-Form II kit (Biomerieux). Reagents BSA (bovine serum albumin). HBV surface antigen, conjugated. Prepare solutions PBS solution: as described in “immunogenicity test”. Buffer solution (0.2% of BSA, PBS, 0.05% of Tween 20): Dissolve 0.2 g of BSA in 100 ml of PBS solution, add 0.2 ml of Tween 20. Store this solution at 4 °C. Prepare this solution before use and discard the remaining. Procedure Dilute sample and reference in order to its OD value in the range of 0.8 to 1.2. Prepare three replicas separately this solution of sample and reference. Make 4 dilutions at 1:2. Prepare three replicas maximum and minimum control (Notes: Minimum control as negative control, it is buffer solution. Maximum control is the first solution having concentration higher than that in the reference). HBsAg content determination must follow the guideline of test kit. All solutions, controls, reagents, biologicals used in test must be equivalent to sample and suitable with ELISA technique. All coating steps have to follow in guideline of test kit. Design the plate depending on analyst and sample size but should be anticipated. 1053

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Result analysis: Base on Parlin program, use “logarit” to convert with P = 95%. Criteria for test The test is valid if: Statistical analysis shows to be satisfactory in linearity, parallel and deviation between dilutions. If the test is invalid, must do retest. Criteria: Relative potency (P=95%) is not less than 0.65. Polysaccharide Not more than 10 µg/100 µg protein (appendix 15.38). Total polysaccharide content (Polyribosyl ribitol phosphate = PRP) Determine total polysaccharide content by using Orcinol method (Appendix 15.42) or liquid chromatography method. Acceptance criteria: Not less than 80% of total saccharide content stated on the label. Free saccharide content Not more than 25.0% (≤ 25.0%) Determine free saccharide content using Orcinol method (Appendix 15.42) or liquid chromatography method. Hib immunogenicity test (the same with “Hib antigen production” part) This procedure is carried out in parallel for the control vaccine. Vaccine dose is 10 µg/0.5ml. Inoculate on day 0 and day 14th. Route of inoculation: abdomen subcutaneous Inoculate 5 rabbits by received sample, at the vaccine dose of 10 µg/0.5 ml. Transfer animals to the animal quarantine area; observe animals during 28 days after inoculation. Twenty-one days after the first dose, take not less than 5 ml of blood of each rabbit in a 15 ml corning tube. Determine the content of anti-Hib antibodies using ELISA. Acceptance criteria: ≥ 50% of seroconversion. Specific toxicity Specific toxicity of DTPw-HeB-Hib combined vaccine is tested in final bulk or in final product if necessary (Appendix15.4). For diphtheria toxoid and tetanus toxoid Use 5 guinea pigs, weighing 250 g - 350 g per guinea pig. Give a subcutaneous injection the amount of vaccine equivalent to at least 5 human doses for each guinea pig. Observe the animals every day. Vaccine meets specific toxicity requirement if no guinea pig shows any signs of tetanic paralysis or symptoms of diphtheria intoxication and at least 80% of the animals survive for 6 weeks after injection. Animals that die must be autopsied and examined for symptoms of diphtheria intoxication (red adrenals). For pertussis toxicity Use at least 20 white mice, each weighing 14 - 16 g. The mice used for the test vaccine(s) and the control group of 1054

mice should be of the same sex (if both sexes are used, they should be equally distributed in all groups). Each mouse is given an intraperitoneal injection of 0.5 ml of suspension containing at least a half of a single human dose. Control group of mice is injected with 0.5 ml of physiological saline (containing the same amount of preservative as the injected solution into the test mice). The total weigh of each group of mice is determined 72 h and seven days after injection. Vaccine is considered to be satisfactory if it complies with 3 following criteria: At the end of 72 h, total weight of the group of the vaccinated mice is not less than that before injection. At the end of seven days, the average weight gain per mouse is not less than 60% of that of the control group of mice. Not more than 5% of the total number of injected mice die.

Quality control of final product Identification of diphtheria - tetanus - pertussis Deabsorption of diphtheria - tetanus - whole cell pertussis component in adsorbed DTPw - HeB - Hib vaccine by adding sodium citrate (C6H5O7Na3,2H2O) at concentration of 5%, store at 37 °C for 48h. Centrifuge this solution at 2000 rpm for 15 minutes. The supernatant part is used for diphtheria and tetanus toxoid identity test by using flocculation reaction, sediment is used for pertussis indentity test by agglutination reaction of the organisms with specific antipertussis serum (Appendix 15.19). Hib identification (the same with “Hib antigen production” part) Method: Use Latex agglutination test or a suitable immunochemical method. Criteria: Positive with specific antiserum. HBsAg identification The HBsAg content shall be determine by a suitable method include radioimmuno assay (RIA), enzymlinked immunosorbent assay (ELISA), single radial immunodiffusion, immunosorbent assay. Sample is compared with international reference standard or national reference standard. Sterility The final product complies with the test for sterility (Appendix 15.7). Innocuity Use healthy white mice and guinea pig for this test. Inject by the intraperitoneal route of half a human dose into each of five mice (each weighing 17 g - 22 g) and a human dose into each of two guinea pigs (each weighing 250 - 350 g), but not more than 1 ml. Vaccine is considered innocuous if whole animals survive, stay healthy and increase their body weight for at least 7 days without showing significant signs of toxicity. Potency Carry out this test for DTPw - HeB - Hib vaccine at final

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product when it has not done at bulk or when it is necessary (Appendix 15.22, 15.23, 15.24, 15.43).

Each vial is closed by a rubber cap and rounded by an aluminum ring.

Characteristics Appearance Each container in each final lot must be inspected visually, vaccine suspension is divided into 2 layes, the upper layer is transparent, colorless or yellowish, sediment layer is greywhite at the bottom of container. Become a homogenous suspension quickly after shaking lightly, lack of particles. Physical The volume of each container: Tolerance +10% calculated with reference to the volume stated on the label. Percentage of vaccine that is discarded. For multi-dose vaccine: Not more than 3%. For single-dose vaccine: Not more than 5%. The vaccine is not be frozen (this criterion is checked only in vaccine storage process or vaccine transport process with cold chain, it does not apply for lot release of vaccine). Sedimentation speed in the sample must be much more slowly than positive control. Vaccine suspension doesn’t become to seed shape or like snow-flower pendently or curdle after shaking.

Labelling The information stated on the label, the carton box and the leaflet must comply with the current regulations.

Preservative Thimerosal content must be in the range from 0.005% to 0.02% (Appendix 15.29). Adsorbent substance Al3+ content in DTPw-HeB-Hib vaccine must be from 0.55 mg/ml to 0.77 mg/ml. Aluminum phosphate content is from 2.5 mg/ml to 3.5 mg/ml. (appendix 15.27).

pH 6.0 - 7.0 (Appendix 15.33). Residual formaldehyde Not more than 0.02% (Appendix 15.25). Storage and expiry date Store vaccine at 2 - 8 °C, vaccine maintains its potency for 2.5 years. The manufacturer must recommend conditions of storage and transport adsorbed DTPw-HeB-Hib vaccine that ensures that the vaccine satisfies the potency requirements until the expiry date stated on the label. Adsorbed DTPwHeB-Hib vaccine is not being frozen during the storage process. The expiry date must be fixed with the approval of the national control authority based on the stability studies referred to in and not be more than 2.5 years after the date of the last satisfactory potency test (the date on which the animals were immunized with the vaccine). Packaging 10 vials equivalent to 100 doses in a box (Each vial contains 5 ml equivalent to 10 doses).

HEPATITIS A VACCINE (live, attenuated) Vaccinum Hepatitidis A vivum Hepatitis A vaccine (live, attenuated) is a freeze dried vaccine, is produced from a suitable attenuated strain of hepatitis A virus, cultured on human diploid cells. The vaccine is reconstituted immediately with an accompanying diluent solution to give a clear homogenous liquid.

Production Virus strain Complies with the following criteria: Have full document and information on the origin of the virus. Achieve the standards set for the virus used to produce live, attenuated vaccine. Show ability to produce a safe and effective vaccine though clinical trials Production strains are produced based on the virus seed lot system approved by competent authority. Production strains are stored at minus 60 °C (-60 °C). Cells substrate Including the human diploid cells, continuous cell lines or primary cells were full characteristics and approved by the competent authority. Cell culture media used in production Must be validated to ensure the productivity and efficiency of vaccine production. All the ingredients added to the culture medium must be sterile and safe. Production process Hepatitis A virus is usually cultured in the human diploid cell line or other continuous cell lines, using an appropriate culture medium. Virus suspension is harvested, pooled, purified, added preservatives, filtrated, diluted, freeze-dried by approved procedures. During production, all processes are tested from the source material, such as virus strain, cell substrate, culture media, fetal bovine serum, trypsin to dissociate cells, pH indicators, antibiotics, adjuvants. In case of use of primary monkey kidney cells, carry out the specific tests to control the virus in monkeys including: SIV, SV 40, hepatitis B virus and tuberculosis. Reference vaccines: Must comply with and meet the general criteria for reference standard used in biological products and medicine applications in human.

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Quality control on single harvests Virus titre: Determined by approved methods. Titre should be within the approved limits. Sterility: The vaccine complies with the test for sterility (Appendix 15.7). Mycoplasma Determined by approved methods. Absence of Mycoplasma (Appendix 15.36).

determine viral titers of hepatitis A vaccine sample using the ELISA method. - Hepatitis A virus titre of the vaccine stored at 2 - 8 °C must not be under 6.5lg CCID50/dose; - Virus titre of incubated temperature stability vaccine must not under 6.5 lgCCID50 /dose and shall not have decreased by more than 0.5 lgCCID50/dose compared with a titer of vaccine at 2 - 8oC.

Quality control on bulk vaccine

Residual BSA content Determined by a commercial ELISA kit. BSA content must be within the approved limit.

Virus titre: Determined by approved methods. Titre should be within the approved limits. Sterility: The vaccine complies with the test for sterility (Appendix 15.7). Mycoplasma Absence of Mycoplasma (Appendix 15.36).

Quality control on final bulk vaccine Sterility The vaccine complies with the test for sterility (Appendix 15.7). Determined on 10  ml of a final bulk vaccine for each culture medium.

Quality control on final vaccine

Gentamicin Determined by commercial ELISA kit. Gentamicin content in vaccine must not exceed 50 ng/a single human dose. Sterility The vaccine complies with the test for sterility (Appendix 15.7). General safety Meet the requirements on general safety of mice and guinea pigs. All of the mice must be healthy, gain weight after 7 days of observation (Appendix 15.11).

Storage Stored at 2 °C to 8 °C, protected from light.

Identification Determined by ELISA or PCR methods. The potency test can also serve to identify.

Labelling The information on the label, box, and leaflet must comply with the current regulations.

Appearance Must meet specifications by the manufacturer's registration that has been approved.

HEPATITIS AVACCINE (INACTIVATED, ADSORBED) Vaccinum hepatitidis A inactivatum adsorbatum

Residual moisture Not more than 3.0% (Appendix 15.35). Chloroform Not more than 0.006% or not more than 60 μg/a single dose for human. Potency and thermal stability Carry out titration and thermal stability test (vaccines kept at 37 °C for 3 days) in parallel by infecting human diploid cell (MRC5 or KMB17) and then quantify virus by the ELISA method. Use commercial kits or kits of the manufacturer (in-house). Mix 5 reconstituted vaccine vials well, then dilute 10-fold by Hanks medium. Then add 1 ml diluted vaccine in each cell flask (25 cm2), use at least 4 dilutions, each dilution infects 4 cell flasks. Use 4 cell flasks as control cell; inoculate 1 ml of Hank’s medium for each flask. Incubate in 2 hours at 37 °C. Add 6 - 8 ml maintain medium containing 2% of FBS into each flask and incubate cell flaks at (36 ± 0.5) °C in an incubator. Check and replace maintain medium every week. After 25 - 28 days, harvest cells and purify, 1056

Hepatitis A vaccine (inactivated, adsorbed) is a preparation consisting of a suitable strain of hepatitis A virus grown in cell cultures, inactivated by a validated method and often adsorbed on aluminium hydroxide or aluminium phosphate.

Production Virus seed Complies with the following criteria: Have full document and information on the origin of the virus Achieve the standards set for the virus used to produce inactivated vaccines Show ability to produce a safe and effective vaccine though clinical trials Working seeds are produced based on the original seed lot system approved by the competent authority. Production strains are stored at below minus 60 °C (-60 °C). Cell substrate Including the human diploid cells, continuous cell lines or primary cells were full characterize and approved by the competent authority.

VP V

Cell culture media used in the production Must be validated to ensure the productivity and efficiency of vaccine production. All the ingredients added to the culture medium must be sterile and safe fetal bovine serum, trypsin, pH indicator antibiotic. Production process Hepatitis A virus is usually cultured in the human diploid cell line (MRC5 cells or KMB17) or other continuous cell lines, using an appropriate culture medium. Virus suspension is harvested, pooled, purified, inactivated, added preservatives, filtrated, diluted, added adjuvants and filled by approved procedures. During production, all processes are tested from the source material, such as virus strain, cell substrate, culture media, fetal bovine serum, trypsin, pH indicators, antibiotics, chemical inactivation, preservatives... In case of using primary monkey kidney cells, carry out the specific tests to control virus in monkeys including: SIV, SV 40, hepatitis B virus and tuberculosis.

Final bulk vaccine Sterility The final bulk vaccine complies with the test for sterility (Appendix 15.7). Antimicrobial preservative Where applicable, determine the amount of antimicrobial preservative by a suitable chemical or physico-chemical method. The amount is not less than 85% and not greater than 115% of the amount stated on the label. Aluminum content If using aluminum hydroxide or aluminum phosphate hydrate as the adsorbent, the maximum concentration of 1.25 mg Al+3 in a single human dose (Appendix 15.27). Residual formaldehyde Not more than 0.2 g/L (Appendix 15.25).

Final lot Provided that the tests for residual formaldehyde content (where applicable), antimicrobial preservative content (where applicable) or the tests on animals have been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Identification Must contain Hepatitis A virus antigen. Determined by ELISA method, using specific antibody or in vivo test. Appearance When shaking inactivated hepatitis A vaccine, obtain a turbid white suspension. Sterility The final lot vaccine complies with the test for sterility (Appendix 15.7).

VACCINES

pH Must be within the limit by the approved manufacturer's registration (Appendix 15.33 General safety Hepatitis A vaccine inactivated adsorbed is safe in mice and guinea pigs. All of the test animals must be healthy, gain weight after 7 days of observation (Appendix 15.11). Pyrogens Hepatitis A vaccine (inactivated, adsorbed) must meet the requirements of pyrogens in rabbits. The total temperature increase of 3 rabbits not more than 1.3 °C (Appendix 15.12). Endotoxins Meet the specification by the approved manufacturer's registration (Appendix 13.2). Potency Carry out the potency test of hepatitis A vaccine (inactivated, adsorbed) and the reference vaccine in parallel. May be determined by the method of immunization in mice to determine the antibody titer obtained (in vivo test) or using specific antibodies to quantify directly the amount of antigens contained in the vaccine (in vitro test). The in vivo test: Experiment animals: Healthy albino mice (strain depending on the process of production) and from the same flock, about 5 weeks old, and at the same sex as the best choice. Determine the relative potency: Tested vaccines and reference vaccines are diluted at least 3 dilutions. The diluent is physiological saline (0.9% solution of sodium chloride) containing types of adjuvants used for the tested vaccine. Intraperitoneal injection of 1 ml per mouse, use at least 10 mice for each dilution. Use a group of control mice injected the diluent. All of the mice after immunization were brought up from 28 to 32 days in the same conditions. Perform anesthesia, take blood from heart. Blood samples were centrifuged at 3000 rpm for 10 min at 4 °C to 8 °C, the serum is separated and stored separately for each mouse at -20 °C. Determine specific antibody titers of hepatitis A virus antibodies by ELISA with good quality commercial kit. Results are calculated by Probit Analysis Program (WHO) Test is valid if: - ED50 of both the vaccine to be examined and the reference vaccine lie between the largest and smallest doses given to the mice. - The statistical analysis shows no significant deviations from linearity or parallelism of the dose-response curves. - The confidence limits (P = 0.95) are not less than 33% and not more than 300% of the estimated potency. Specification for in vivo test: The correlation potency (p = 95%) is not less than 1 compared with the reference vaccine.

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Aluminum content If using aluminum hydroxide or aluminum phosphate hydrate as an adsorbent, the maximum concentration of 1.25 mg Al+3 in a single human dose (Appendix 15.27). Residual formaldehyde Not more than 0.2 g/L (Appendix 15.25). Preservative Where applicable, determine the amount of preservative by a suitable chemical or physico-chemical method. The amount is not less than 85% and not greater than 115% of the amount stated on the label. Protein content Determined by appropriate methods, often using Lowry method. Protein content must be within the allowable limit (Appendix 15.34).

Storage, expiry date Stored at 2 °C to 8 °C, protected from light and avoided freezing vaccines. The statement on expiry date, storage temperature based on the research results about the stability of the vaccine and should be approved by the competent authority. Labelling The information on the label, box, and leaflet must comply with the current regulations. Usage and dosage According to the leaflet (approved). HEPATITIS A VACCINE (INACTIVATED, VIROSOME) Vaccinum hepatitidis A inactivatum virosomale Hepatitis A vaccine (inactivated, virosome) is a suspension of a suitable strain of hepatitis A virus grown in cell cultures and inactivated by an approved method and then mixed with suitable ratio of virosome, use phospholipids as adjuvants. Virosomes composed of influenza proteins of a strain approved for the particular product.

Production The production method is validated to demonstrate that the product, if tested, would comply with the test for abnormal toxicity for immunosera and vaccines for human use (Appendix 15.11). The production process consists of three stages. Stage 1: Production of hepatitis A vaccine (inactivated). Process for production of vaccines for hepatitis A inactivated are described in the monograph for hepatitis A vaccine (inactivated, adsorbed) and must comply with all the requirements for the production and testing vaccines as described in Production part in hepatitis A vaccine (inactivated, adsorbed) monograph. 1058

Phase 2: Production of virosomes Virosome is envelope part of a flu virus that has been approved for a specific product, containing hemagglutinin surface antigen. The production process of these virosomes as same as that of inactivated influenza vaccine (split format), include the steps: a suitable hepatitis A virus strain is propagated in fertilized eggs, incubated and then harvest the allantoic fluids; purified virus; inactivated; disrupted. Collect the viral envelope parts containing glycoprotein of the influenza virus (including hemagglutinin antigens). Then add the appropriate phospholipids such as lecithin, kephalin ... then remove the substances used in the process of purification by adsorption chromatography or a suitable method to obtain virosomes. The production process of virosomes must comply with all the requirements for manufacturing and testing as described in Production part of the inactivated influenza vaccine. Stage 3: Mix to final bulk vaccine The final bulk vaccine is prepared by adding virosomes to inactivated hepatitis  A viruses to yield an approved hepatitis A antigen:haemagglutinin ratio. During production, all processes are tested from the source material, such as virus strain, cell seed, cell substrate, culture media, fetal bovine serum, trypsin, pH indicators, antibiotics, chemical for inactivation, and preservatives... vaccines must complies with the requirements stated in Vaccines for human use monograph and the following requirements.

Quality control on final bulk vaccine Protein content Determined by a suitable method, often using Lowry method (Appendix 15.34). Protein content shall comply with the approved limit. Phospholipids content Determined by a suitable method, often using the immunochemical or physicochemical method. The phospholipids content shall comply with the limit approved for the particular product. Haemagglutinin identification and content Determined by a suitable method, often using single radial immunodiffusion method (SRD). The haemagglutinin content shall with the limit approved for the particular product. The quantitative test is used as identification test. HVA antigen identification and content Determined by a suitable immunochemical method, often using ELISA method. The amount of HAV antigen shall comply with the limit approved. The quantitative test is used as identification test. Ratio of hepatitis A antigen to haemagglutinin Must comply with the approved limit. Ovalbumin content Determined by a suitable immunochemical method, often using ELISA method. Not more than 1 µg of ovalbumin per single human dose.

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Residual BSA Determined by a suitable immunochemical method, often using ELISA method. BSA content must comply with the registered limit by the manufacturer, but not more than 50 ng/ml. Sterility The final bulk vaccine shall comply with the test for sterility (Appendix 15.7). Antimicrobial preservative Where applicable, determine the amount of antimicrobial preservative by a suitable chemical or physico-chemical method. The amount is not less than 85% and not greater than 115% of the amount stated on the label. Residual chemicals Chemical substances used during the production process must be tested according to approved methods. The residual chemical content complies with the limit approved for the particular product. Residual formaldehyde Not more than 0.2 g/L (Appendix 15.25).

Quality control on final lot Provided that the tests for residual formaldehyde content (where applicable), antimicrobial preservative content (where applicable), residual BSA, Ovalbumin or the tests on animals have been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. HAV antigen identification Vaccines contain antigens to hepatitis A virus were identified by ELISA method, using specific antibodies or by in vivo test. Haemagglutinin identification and content Determined by a suitable method, often using a single radial immunodiffusion method (SRD). The haemagglutinin content must be within range of 12 - 25 μg/ml. The quantitative test is used as identification test. Appearance Meet specification by the manufacturer's registration that has been approved. Mycoplasma Determined by an appropriate method. Absence of Mycoplasma (Appendix 15.36). Sterility The final lot vaccine complies with the test for sterility (Appendix 15.7). pH 6.3 to 7.3 (Appendix 15.33). Volume Not less than the volume stated on the label (Appendix 11.1). General safety Meet the requirements of general safety on mice and guinea pigs. All of the test animals must be healthy, gain weight after 7 days of observation (Appendix 15.11).

Bacterial endotoxin Not more than 4 EU/ml or 2 EU/a single human dose (Appendix 13.2). Phospholipids content Determined by a suitable method, often using the immunochemical or physicochemical method. The phospholipid content complies with the limit approved for the particular product. Formaldehyde Not more than 0.2 g/L (Appendix 15.25). Virosome size The size of virosome-HVA complies with the limit approved for the particular product. Antimicrobial preservative Where applicable, determine the amount of antimicrobial preservative by a suitable chemical or physico-chemical method. The amount is not less than 85% and not greater than 115% of the amount stated on the label. Potency Determine the potency in parallel for hepatitis A vaccine (inactivated, virosome) and the reference vaccine by a suitable immunochemical method, often using ELISA method on biological diagnostic kits or ELISA process of manufacturer (in-house method). Use HAV antibody to determine the amount of HVA antigen in the vaccine to be examined base on reference vaccine. HAV antigen content complies with the approved limit.

Storage Stored at 2 °C to 8 °C, protected from light and avoided freezing vaccines. Labelling The information on the label, box, and leaflet must comply with the current regulations. The label states the biological origin of the cells used for the preparation of the vaccine, influenza envelope made from eggs, avoid freezing the vaccine, shake well before use. HEPATITIS A (INACTIVATED) AND HEPATITIS B (rDNA) VACCINE (ADSORBED) Vaccinum Hepatitidis A inactivatum et Hepatitidis B (ADNr) adsorbatum Hepatitis A (inactivated) and hepatitis B (rDNA) vaccine (adsorbed) is a suspension consisting of a suitable strain of hepatitis A virus, grown in cell cultures and inactivated by a validated method, and of hepatitis B vaccine recombinant (produced from HBsAg, a component protein of hepatitis B virus obtained by recombinant DNA technology), the antigens are adsorbed on aluminium hydroxide or hydrated aluminium phosphate.

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Production Produce separately each vaccine then blended together to make Hepatitis A (inactivated) and hepatitis B (rDNA) vaccine (adsorbed). The two components are prepared as described in the monographs on Hepatitis A vaccine (inactivated, adsorbed) and hepatitis B vaccine (rDNA, adsorbed) and comply with the requirements prescribed therein. Reference vaccine: The reference preparation is part of a representative batch shown to be at least as immunogenic in animals as a batch that, in clinical studies in young healthy adults, produced not less than 95% seroconversion, corresponding to a level of neutralizing antibody recognized to be protective, after a full-course primary immunisation. For hepatitis A, an antibody level not less than 20 mIU/ml is recognized as being protective. For hepatitis B, an antibody level not less than 10 mIU/ml against HBsAg is recognized as being protective.

The free HAV antigen content complies with the approved limits.

Quality control on final bulk vaccine

Residual formaldehyde Not more than 0.2 g/L (Appendix 15.25).

Sterility The final bulk vaccine complies with the test for sterility (Appendix 15.7). Antimicrobial preservative Where applicable, determine the amount of antimicrobial preservative by a suitable chemical or physico-chemical method. The amount is not less than 85% and not greater than 115% of the intended amount. 2-phenoxyethanol content Determined by the liquid chromatography method. The content of 2-phenoxyethanol in vaccines complies with the allowed limit.

Quality control on final lot Provided that the tests for residual formaldehyde content (where applicable), antimicrobial preservative content (where applicable), free HBsAg antigen content, free HAV antigen content (not adsorbed) or the tests on animals have been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Identification The vaccine must contain hepatitis A virus antigen and hepatitis B surface antigen HBsAg) that are identified by ELISA method (see Notes bellow), using specific antibodies or by in vivo tests. Description Turbid liquid, colorless; after shaking create opaque white suspension. Sterility The final lot vaccine complies with the test for sterility (Appendix 15.7).

Free HAV antigen content (not adsorbed) Determined by ELISA method (see Notes below). 1060

Free HBsAg antigen content (not adsorbed) Determined by ELISA method (see Notes below). The free HBsAg antigen content complies with the allowed limits. pH 5.8 to 6.6 (Appendix 15.33). Volume Not less than the volume stated on the label (Appendix 11.1). Aluminum content If using aluminum hydroxide or aluminum phosphate hydrate as the adsorbent, the maximum concentration of 1.25 mg Al+3 in a single human dose (Appendix 15.27). Free aluminum content Must be within the allowed limits.

Antimicrobial preservative Where applicable, determine the amount of antimicrobial preservative by a suitable chemical or physico-chemical method. The amount is not less than 85% and not greater than 115% of the amount stated on the label. General safety Hepatitis A vaccine inactivated adsorbed must be safe in mice and guinea pigs. All of the test animals must be healthy, gain weight after 7 days of observation (Appendix 15.11). Bacterial endotoxins Not more than 2 EU per single human dose. Potency test Potency of hepatitis A (inactivated, adsorbed) and hepatitis B vaccine (recombinant, adsorbed) is conducted independently by the suitable methods and procedures for each vaccine; each vaccine must be tested in parallel with the standard vaccine. Potency can be determined by the method of immunization in mice to determine antibody titers obtained (in vivo test) or using specific antibodies to quantify directly concentration of each antigen contained in the vaccine (in vitro test) by the commercial kits or process of the manufacturer (in-house method). The processes and the steps may be changed according to the guidance of each method, each kit.

Storage Stored at 2 °C to 8 °C, protected from light and avoided freezing vaccines. Labelling The information on the label, box, and leaflet must comply with the current regulations.

VP V Notes:

Quantify and identify for HAV component in hepatitis A (inactivated, adsorbed) and hepatitis B vaccine (recombinant, adsorbed) Principle HAV content is determined by ELISA method. Sample and reference are diluted to some concentrations then added into plates (attached HAV antibody). Complex antibodyantigen was detected by anti-HAV antibody conjugated to peroxidase enzymes. Then add the substrate (TMB). Color intensity is proportional to the concentration of antigen in the sample. Procedure: Prepare plate: Human anti-HAV IgG is diluted by PBSTween solution then coated into 96-well plates. Incubate plates overnight at 25 °C, then wash and dry the plates. Sealed, stored at 2 °C to 8 °C and use within 1 week. Prepare test sample: Test vaccine, standard vaccine, and internal control (adsorbed aluminum) are desorbed by adding EDTA solution (supplemented with disodium hydro phosphate-Tween 20 and gelatin) or sodium citrate supplemented with BSA. Procedure as follows: - Centrifuge the sample at 6000 r/min for 10 min to remove the supernatant and then add the desorption solution. Shake well, and then incubate at 37 °C. Centrifuge as above, collect the supernatant. - Repeat 2nd and 3rd extraction and the 4th extraction incubated overnight. - Mix obtained the supernatant after the extraction times with PBS (without Ca2+ and Mg2+) supplemented with Tween 20 and gelatin. The desorbed samples and reference HVA (unabsorbed) are diluted at least 6 dilutions. - Add diluted samples into the plate coated with human anti-HAV IgG, 2 wells/dilution; add the diluent in a column for blank. Incubate for 90 min at 37 °C. Add anti- HAV antibody conjugated to peroxidase. Incubate for 90 min at 37 °C. - Add TMB supplemented with H2O2 30% in acetate buffer into the plate. Incubate at room temperature, protected from light. - Add 50 µl 1 N sulfuric acid to stop the reaction. - Measure OD at 450/620 nm wavelength. Calculate results Results are calculated by Paralelline or Excel software. The correlation coefficient (R2) linear line of standard calculated by Excel software should be greater than 95%. HAV antigen content in the sample is calculated from at least 3 dilutions in a row of the dilutions used in the test. Procedure of assay and identification test for HBsAg in Hepatitis A (inactivated, adsorbed) and hepatitis B vaccine (recombinant, adsorbed) by the indirect ELISA method. Principle: HBsAg content in test vaccine is quantified by indirect ELISA method. The vaccine is incubated for the amount

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of human polyclonal antibodies. Then the excess of antibodies are determined by incubation with HBsAg standard and then detected by human IgG antibody conjugated peroxidase. Procedure: - Vaccine samples are diluted in a solution of PBS containing 0.1% BSA and 0.1% (V/v) Tween 20, then mixed with anti-HBs polyclonal antibodies and incubated overnight at room temperature. Then centrifuged, and transfer the supernatant into the HBsAg coated plate, incubate overnight at 2 - 8 °C with reference recombinant HBsAg in 0.05 M carbonate buffer solution (pH 9.6) to determine the amount of excess antibodies. The amount of excess antibody is determined by using an anti-human antibody attached peroxidase. Incubate at 37 °C for 1 h. Rinse, and then add substrate solution (TMB). Add reaction stop solution (1 N sulfuric acid). Measure OD at 450/620 nm wavelength. Color density is proportional with HBsAg content in the sample. (*) Prepare 0.05 M carbonate buffer solution (pH 9.6): Solution 1: Dissolve 2.1 g of sodium hydrocarbonate R in 500 ml of water. Solution 2: Dissolve 2.65 g of sodium carbonate R in 500 ml of water. Store the solutions in sealed containers, at 25 °C or in the refrigerator, use within 3 months. Before use, mix 70 ml solution 1 and 30 ml solution 2 to obtain 0.05 M carbonate buffer solution (pH 9.6). Stored the resulting solution in a sealed container, at room temperature, use within 1 month. Calculate results: Results are calculated by Paralelline or Excel software. The correlation coefficient (R2) linear line of standard calculated by Excel software should be greater than 95%. HBsAg antigen content in the sample is calculated from at least 3 dilutions in a row of the dilutions used in the test. Acceptance criteria: HAV antigen content is not less than 60% content stated on the label. HBsAg antigen content must be from 21 to 37 micrograms of HBsAg per ml. RECOMBINANT HEPATITIS B VACCINE (rDNA) Vaccinum Hepatitidis B recombinatum (rDNA) Recombinant hepatitis B vaccine (rDNA) is a preparation of hepatitis B surface antigen (HBsAg), a protein of hepatitis B virus; the antigen may be adsorbed on a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate. The antigen is produced by recombinant DNA method.

Production Recombinant hepatitis B vaccine (rDNA) is produced by the expression of the viral gene coding for HBsAg in yeast (Saccharomyces cerevisiae, Pichia pastoris, Hansenula 1061

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polymorpha) or mammalian cell (CHO - Chinese Hamster Ovary) cells or other suitable cell lines carrying coded HBsAg which has been transformed into a plasmid. This hepatitis B surface antigen (HBsAg) expressed in yeast cells is purified by physico-chemical techniques. The recombinant vaccine may contain the product of the S gene (small protein), a combination of the S gene and pre-S2 gene products (middle protein) or a combination of S gene, pre-S2 gene and pre-S1 gene products (large protein).

Quality control of bulk vaccine Total protein Determined by Lowry method (Appendix 15.34). Identification and HBsAg content Determined by suitable immunochemical methods such as radio-immuno assay (RIA), enzym-linked immunosorbent assay (ELISA), immunoblot or single radial diffusion. Vaccine sample is compared with the standard vaccine. Antigenic purity Determined by liquid chromatography (Appendix 5.3) or other suitable methods such as SDS-PAGE (Appendix 15.39). Acceptance criteria: Not less than 95% of the total protein. Lipid Not more than 100 µg/100 µg protein (Appendix 15.40.) Cesi cloride (CsCl) Not more than 5 µg /20 µg protein (Appendix 15.41). Tween 20 Not more than 50 µg /100 µg protein (Appendix 15.42). Polysaccharide Not more than 10 µg /100 µg protein (Appendix 15.38).

Quality control of final bulk vaccine Thimerosal Not more than 0.012% (Appendix 15.29). Sterility The vaccine complies with the test for sterility (Appendix 15.7).

Quality control of final lot Appearance Once shaken the vaccine obtains a slightly opaque, white suspension. pH 5.4 to 7.4 (Appendix 5.33). Total protein Determined by Lowry method. The content is not more than 40 µg/ml (Appendix 15.34).

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Aluminium Not more than 1.25 mg per single human dose (Appendix 15.27). Free formaldehyde Not more than 0.01% (Appendix 15.25). Thimerosal Not more than 0.012% (Appendix 15.29). Sterility The vaccine complies with the test for sterility (Appendix 15.7). General safety The vaccine is safety when injected into mice and guinea pigs. Mice and guinea pigs must be healthy and increase their weight within 7 days of observation (Appendix 15.11). Pyrogens The vaccine is safety when injected into rabbits. Total temperature disparity of 03 rabbits should not be more than 1.3 °C (Appendix 15.12). Potency The potency of the vaccine is determined by comparing vaccine sample and the standard vaccine; can use animals (in vivo method) to determine antibody titre or by labor immune techniques (in vitro method) to measure HBsAg content of the vaccine. Potency test in animals (in vivo) Animals Mice BALB/C or ICR, are healthy and from the same stock, about 5 - 6 weeks old, preferable same sex. Relative potency The standard reference and tested vaccine sample are diluted into 5 levels of 2 fold dilution. The diluent is sterile physiologic saline solution containing aluminium hydroxide (aluminium hydroxide content is not more than 500 µg/ml). Inject intraperitoneally 1 ml of each dilution level into mice. Each dilution level is injected to at least 15 mice. All the mice are fed from 28 to 32 days after injection in the same condition. Anaesthetizing and taking blood from their heart. Blood samples are centrifuged with speed of 3000 rpm at 4 - 8 °C, then separate serum from the blood sample. The serum samples should be stored at -20  °C. HBsAg Antibody titre is determined by ELISA technique with commercial diagnostic kit. The result is analyzed by statistic software. The assay is valid if: ED50 of both National Standard Reference and tested vaccine sample are in the interval of the highest and lowest dose. Both linearity and parallel should meet requirements. Confidence limit of relative potency should be from 33% to 300%. Acceptance criteria: Relative potency (P = 95%) is not less than 1.

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Potency test in vitro Vaccine sample and Standard Reference are diluted in parallel into suitable levels of 2 fold dilution by PBS 0.1 M. ELISA technique is performed with commercial diagnostic kit. HBsAg content of vaccine sample which is compared with standard reference determined by monoclonal anti-HBsAg antibody. Analyze the result by excel, Paralelline Program, or other programs. Acceptance criteria: Comply with the requirements registered by manufacturer.

Packaging Hepatitis B vaccine is presented in neutral glass vial; each vial of 1 ml containing 20 µg; or 10 µg; or 5 µg depending on each manufacturer. Stability For stability of vaccine, data of at least 3 continuously vaccine lots studied in storage condition are required (manufactured from different 3 bulk lots) under storage condition stated on the vial stamp. Storage, expiry Hepatitis B vaccine should be stored at 2 °C to 8 °C. Do not freeze. Discard if vaccine has been frozen. In above storage condition, shelf-life of recombinant hepatitis B vaccine is 36 months. Labelling Information on the label, box and leaflet must comply with the current regulations. Dosage and administration Administration: Inject intramuscularly in the deltoid region in adults (in the anterolateral thigh in neonates is prefer). Dosage, schedule: Manufacturer’s recommendation. Booster dose after 5 years should be applied with all age ranges. HAEMOPHILUS INFLUENZAE TYPE b CONJUGATE VACCINE Vaccinum haemophili stirpis b coniugatum Haemophilus influenzae type b conjugate vaccine (Hib) is a preparation of capsular polysaccharide from H. influenzae type b covalently linked to a carrier protein. Polysaccharide capsule is a linear polymer contained units of 3-β-D-ribofuranosyl (1→1)-D-ribitol-5-phosphat (PRP). Carrier protein is diphtheria toxoid or tetanus toxoid or non-toxin mutant diphtheria toxin - CRM 197 or outer membrane protein complex of Neisseria meningitidis group B.

Production Working seed The strain of H. influenzae type b used in preparing H. influenzae type b conjugate vaccine must be approved by the National control authority. The strain must have specific polysaccharide capsule and be capable of producing type b polysaccharide, make the immune response and be safe with the human. To be approved for production of Hib vaccine, there is only strain having properties as following: in optimum medium, the strain has typical shape of H. influenzae: small rod shape, aerobic, Gram-negative; immobile, non-spore, non-hemolysis, glucose fermenting, lactose, sucrose, mannose non-fermenting and positive reaction with catalase and indole. Seed lot system The master seed has the detail document containing its history, physiological, biochemical characteristics, serology and genetic properties. Cultures derived from the working seed lot have the same characteristics as cultures derived from the master seed lot and meet the requirements as master seed lot. Cultures and single harvests The growth of H. influenzae type b has to consistency by monitoring the growth rate, pH and yield of polysaccharide. The H. influenzae type b should be inoculated into a liquid medium that does not contain high-molecular-weight polysaccharides; if the medium contains blood-product, assurance of the absence of blood product is proved after the purity. The purity of culture after inactivation has to be verified by a suitable method that includes inoculation on to appropriate plating media to verify shape-characteristics, Gram staining and agglutination with the specific serum. Control of purified polysaccharide The culture is inactivated. PRP isolated from the culture is purified by a suitable method. Each purified polysaccharide lot should be tested for purity. The purified polysaccharide that complies with the following requirements may be used in the preparation of the bulk conjugate. Identification Apply an immunological method or 1H (or 13C) NMR spectroscopy; PRP is positive. Molecular size distribution: Apply gel filtration chromatography method to determine the molecular size distribution of polysaccharide. The distribution constant K0 is determined using the main peak of polysaccharide in the chromatogram met by the manufacture specification and approved for each kind of Hib conjugate vaccine. High performance liquid chromatography is also applied for this purpose. Moisture content: Apply a suitable method approved by the national control authority and shown to be consistent within agreed limits. 1063

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Polysaccharide composition: Carry out as the approved method. The quantity of the polysaccharide can be estimated by measuring the ribose content (Bial method). The ribose content should be not less than 32.0% of the polysaccharide dry weight. Phosphorus content: Apply Chen method or another suitable validated method. The phosphorus content should be between 6.8% and 9.0% calculated on the dry weight. Protein impurity: Not more than 1.0% calculated with reference to the dried polysaccharide (determined by Lowry method). Nucleic acid: Not more than 1.0% calculated with reference to the dried polysaccharide and determined by measuring the absorbance at 260 nm wavelength or another validated method. Endotoxin content: Not more than 10.0 EU/µg polysaccharide (Appendix 13.2). Control of carrier protein Carrier protein is used to improve immune response at B and T cells. Strain to be used for the production of the carrier protein should have a clear document containing its history, physiological and biochemical characteristics. The strain shows stable growth by monitoring the growth rate, pH of the medium and yield. Protein carrier produced from a suitable strain, culture should be tested for purity, the culture is inactivated and purified by a suitable method. Only protein carrier that complies with the following requirements may be used in the preparation of bulk conjugate: Diphtheria and tetanus toxoid: The content is not less than 1500 Lf/mg protein. CRM197: The content of purified protein is not less than 90.0%. Outer-membrane protein complex of Neisseria meningitidis group B: The content of protein is not more than 8.0% of lipopolysaccharide by weight. Pyrogen It complies with the test for pyrogens (Appendix 15.12). Inject intravenously via rabbit ear vein with 0.25 µg/kg rabbit body weight.

Quality control of bulk conjugate The bulk conjugate that complies with the following requirements may be used in the preparation of the final bulk: Residual reagents The conjugate purification procedure should remove residual reagents, for example: cyanide should be confirmed by a suitable validated method, complies with the manufacturer specifications for each kind of Hib conjugate vaccine. 1064

Conjugation markers Determined by the number of PRP repeating units or total PRP content; complies with the manufacturer specifications for each kind of Hib conjugate vaccine. Residual reactive functional groups Carry out a suitable validated method; meet the manufacture specifications for each kind of Hib conjugate vaccine. Content of PRP Carry out a suitable validated method; complies with the manufacturer specifications for each kind of Hib conjugate vaccine. Conjugated and free PRP content Amount of free PRP is less than 40.0% that depending on each kind of Hib conjugate vaccine. Protein content Carry out a suitable validated method. PRP-to-protein ration The ratio should be within the range approved by manufacturer for each kind of Hib conjugate vaccine. Molecular size distribution Carry out a suitable validated method, for example on gel Sepharose CL-4B, complies with the manufacturer specifications for each kind of Hib conjugate vaccine. Sterility Complies with the test for sterility (Appendix 15.7). Specific toxicity of carrier protein in the conjugate The bulk conjugate should be tested for the absence of specific toxicity of the carrier protein (if the carrier protein is tetanus toxoid or diphtheria toxoid).

Final bulk Adjuvants, preservatives of stabilizers are added with a suitable quanity to the bulk conjugate to make final bulk. Only a final bulk vaccine that complies with the following requirement may be used in the preparation of the final product. Sterility The final bulk complies with the test for sterility (Appendix 15.7).

Final product Only the final product that complies with the following requirements is approved of lot release. Identification Use an appropriate immunological test, using specific antibodies for the purified polysaccharide (Ouchterlony double immunodiffusion or ELISA). Sterility The final product complies with the test for sterility (Appendix 15.7).

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PRP content Tolerance ± 20.0% of the PRP content stated on the label. Use colorimetric or chromatographic method. Residual moisture Not more than 3.0% (Appendix 15.35). Pyrogens The final product complies with the test for pyrogens. Inject intravenously via rabbit ear vein ranging from 0.025 µg to 1.0 µg of PRP per 1 kg of rabbit body weight (Appendix 15.12). Adjuvant The amount of aluminium: Not more than 1.25 mg per single human dose (Appendix 15.27). The amount of calcium: Not more than 1.3 mg per single human dose. Carry out a suitable validated method. Preservative Carry out a suitable validated method, the specification is approved by the national control authority. General safety Each final lot is passed for general safety if all mice and guinea pigs are alive and gain weight after 7 days of observation (Appendix 15.11). pH For the liquid product: pH is within the range of values that found to be safe and effective in clinical trials and stability studies (Appendix 15.33). For the freeze-dried product: Measure pH after reconstitution by a suitable solvent. Inspection Inspect visually, vaccine showing abnormalities such as improper sealing, lack of integrity, abnormal particles.

Storage, expiry date The storage temperature is range from 2 °C to 8 °C. The announcement of the expiry date, storage condition is based on the stability studies and approved by the national control authority. Labelling The information on the label, box, and leaflet must comply with the current regulations and specially indicate the information of PRP content, name, and carrier protein content of one single human dose. INFLUENZA VACCINE (INACTIVATED) Vaccinum Influenzae inactivatum Influenza vaccine inactivated is a transparent sterile suspension, containing one or more sterile strains of influenza virus, type A or B or a mixture of both types, has been inactivated by the appropriate method.

Influenza vaccine can be in 4 forms: - The suspension of viral particles is inactivated by appropriate methods. - The suspension was treated by physical and chemical methods that the virus particle disrupts completely or partially. - The suspension has been treated so that only contains the majority of the haemagglutinin and neuraminidase antigens (subunit vaccines). - The suspension of inactivated influenza virus particles, splitted virus particles or the subunit composition with the adjuvants. Products must comply with the following requirements:

Production Strain for production According to the annual recommendations of the World Health Organization and approved by the national accrediting authority. Currently, it is common to use high-yielding strains of surface antigens. The origins and passage history of virus strains shall be approved by the competent authority. Egg for production If vaccines are produced in embryonated eggs, the eggs must be selected from healthy block, no disease and monitored by methods approved by the competent authority of animal health. Cell for production Cell line for influenza vaccine production must be based on the suitable cell banking system and are allowed to use for the production of biological products for human use. The National Control Laboratory must approve master cell bank and establish the maximum passage number of cell banks. Cell culture medium Serum used for cell culture is not infected bacteria, fungi, Mycoplasma, and other viruses. Penicillin and other beta-lactam antibiotics are not used in any stage of any production. The other antibiotics can be used, but must be approved by the National Control Laboratory. Single harvests For production, the virus of each strain is grown in the allantoic cavity of fertilized hens’ eggs from healthy flocks. After incubation at the appropriate temperature, the allantoic fluids are harvested. For vaccines produced from mammalian cells, viruses of each strain to be developed on the cell line approved for vaccine production. The single harvest batches of the same strain are combined to form a monovalent pooled harvest. Monovalent viral suspensions from the cell are not mixed with Monovalent viral suspensions from eggs.

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The purity of vaccines produced from cell To monitor the stability of the purity, the monovalent batch suspensions harvested from mammalian cells have been tested concentration ratio of HA (hemagglutinin) and total protein. This rate must be within the limit approved by the National Control Laboratory. For virus growth in continuous cell lineages, single harvest must be tested for DNA residues. DNA content must be less than 10 ng/dose in human. This test may be omitted with the approval of the national Control Laboratory if the production process has validated as achieving and maintaining this standard.

Test on final bulk vaccine The final bulk vaccine is produced by blending and diluting the monovalent pooled harvests of each strain. Only a preservative or adjuvant solution approved by National Control Laboratory is added in the final bulk vaccine. These substances must not affect the safety and efficacy of the product. Vaccine production to pandemic should only contain 1 strain. Potency test This test may be omitted if it has been carried out on each batch of the final lot. Test for identify and determine the HA antigen content of influenza vaccine in single radial immunodiffusion technique (SRD, SRID). Principle: Influenza antigen diffusion in agar containing corresponding antibodies, at region containing suitable antibody-antigen content, the precipitate ring will be formed. The size of this ring is proportional to the concentration of antigen. Base on the ring sizes of the standard antigen are made in the same time, calculate concentration of antigen in the tested sample. Tested sample and standard antigen are treated with the suitable detergent solution, and then diluted with PBS at the appropriate dilutions. A volume of sample and standard are added to the antigen wells of agar plates contained a suitable amount of antibody. Keep this agar plates at 20 °C for at least 18 h. Antigens will diffuse around and combine with antibodies to make up the precipitate circle. Dye and measure the diameter of the rings. Compare to the rings made by standard antigens to calculate results. Procedure Prepare the SRD agar molds: An agar mold will use 13 ml of agarose. Melt agarose enough by microwave. Then incubate at 60 ° C for 15 min. Prepare glass plates; write a code on the top left corner of the glass. Wipe the surface of plate glass by melting agarose. Put the mold on plate glass. Sealed on site mold with melted agarose and keep for 5 min. Add an amount of standard antibodies in the agarose and then poured into the molds above. Keep room temperature for 30 min, and then punch holes (4 mm diameter). 1066

Preparation of antigen: Reconstitute lyophilized antigen standard with 1 ml of distilled water and allow it to stand for 5 min before use. Dilute standard antigen with PBS (-) to obtain a final concentration of 30 mcg HA/ml according to the leaflet. Mix 450 µl of standard antigen and tested vaccine with 50 µl of 10% Zwittergent and keep into the humid box for 30 min at room temperature. Example: Dilutions by the PBS (-) solution may be done by the following table: Table 1 - Dilutions by the PBS (-) solution Diluted content 1.0 0.75 0.5 0.25

Volume (µl ) Treated antigen PBS (-) with Zwittergent 200 150 100 50

0 50 100 150

Tested antigen and incubation conditions: Petite 20 µl each antigen content into the corresponding well. Put plate glass into the humid box and incubate at 20 °C for at least 18 h. Treatment of plate glass: Wash agar plates (gel) under running water, place the filter papers on the surface of the gel carefully to remove all bubbles. Squeeze the gel for 30 min by plate glass weighing 600 g. Dry the gel in the incubator at 37 ° C. Stain gel with a coomassie blue solution. Destain gel with a solution and dry. Measure the diameter of diffusion ring in two perpendicular directions, and calculate results by dedicated Paranell software ("BioAssay assist" or EDQM Combistats software, ...). First, assess SRD test to be valid or not based on statistical analysis. If the test is valid, the amount of HA vaccine will be calculated by comparing the data with standard antigen. Evaluation results: The potency of each type of virus in the test sample shall not be less than 15 µg HA/dose. The test is valid if: The precipitation rings are clear. The diameters of the precipitation rings are in range from 5.0 mm to 14 mm if measured with the naked eye. The confidence limits (P = 0.95) are not less than 80% and not more than 120% of the estimated haemagglutinin antigen content. Sterility It complies with the test for sterility (Appendix 15.7). If preservatives were added to vaccines, use an appropriate method to prevent interference with sterility test. Total protein content Total protein content must not exceed 6 times the concentration of HA virus strains in the vaccine. However,

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in any case does not exceed 100 µg protein /1 strain /1 single dose in humans, and no more than 300 µg protein/ total of viral antigens/1 single human dose (Appendix 15.34). Ovalbumin Ovalbumin content must not exceed 1 µg/dose in humans. Ovalbumin concentrations should be determined by appropriate methods. May be ovalbumin quantitative based on the principle of "sandwich ELISA". Ovalbumin monoclonal antibodies are fixed on the plate; samples and conjugate were incubated on the plates in the same time. The color reaction by adding substrate containing TMB and H2O2. Stop reaction by adding sulfuric acid. Read the reaction on the colorimeter. Biological standard: Ovalbumin kit (Serazym). Sample: Influenza vaccines 2 ml/sample. Procedure: Serazym Ovalbumin ELISA Kit may be used within 2 months after opening, stored at 4 °C to 8 °C. Put kits at room temperature before use. Dilute of ovalbumin sample to obtain solutions containing from 1 ng to 20 ng of ovalbumin per ml. Add 200 µl of the standard, diluted samples into each well. Add 100 µl of HRP conjugate into each well, mix well. Incubate at room temperature for 60 min. Remove all solution in the wells. Add 300 µl of washing solution into all wells, keep 5 seconds and then pour out the solution in wells, perform repeat this step 4 times . Add 100 µl of substrate to all wells; incubate 15 min at room temperature. Add 100 µl of reaction stop solution to all wells and read the results by the ELISA reader at 450 nm in 30 min after reaction stop. Calculate the result by “Calculation ovalbumin” software. Test is valid if: The mean absorbance of Standard 1 (20 ng/ml) is ≥ 1.50. The mean absorbance of Standard 6 (0.625 ng/ml) is ≤ 0.50. 5.0 ng/ml ≤ mean absorbance of Ovalbumin control ≤ 10.0 ng/ml. OD value of the diluted samples should be within the limit of standard sample (from 0.625 ng/ml to 20 ng/ml). CV of all diluted samples must not exceed 15% (≤ 15%). Adjuvant content If an adjuvant is added to the vaccine, its concentration must be determined by the approved method by the National Regulatory Authority. Content must be enough to ensure the highest clinical effectiveness of the product. The formula of adjuvant and antigens must be consistent and stable throughout the production process.

Test on final lot Identification Identification test is carried out on at least one vial from each batch of finished product (final lot) by the appropriate

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approved method by the national accrediting authority. Using single radial immunodiffusion technique (SRD, SRID) as follows: Principle: A suitable amount of sample (antigen) is added to the wells of agar plates SRD (with a suitable amount of flu antibodies). The form of the diffusion specific ring in agar allows to identify the corresponding influenza antigens. Procedure: The same as SRD test is presented at the potency test for final bulk vaccine. Evaluate result: Vaccines identification test is satisfactory if test results have precipitated ring. If the first test was not passed, have to do the second test If the second test still did not form diffuse ring the vaccine lot is not satisfactory. Identification of haemagglutination antigen in vaccines is determined by immunological methods such as diffusion immune, inhibitory antigens, or using specific immune corresponding sera. Potency test HA antigen content is determined by single radial immunodiffusion technique as presented at the potency test for final bulk vaccine above. Accepted standards: Vaccines should contain at least 15 µg HA/dose/each produced strain. For pandemic vaccines, it may contain different content HA by the World Health Organization recommendations. General safety According to Appendix 15.11. Inject intraperitoneal 1 dose of influenza vaccine for each of 5 healthy mice, weighing 17 - 22 g/mouse and not use for any purpose before; injected intraperitoneal 10 doses of human for each of two healthy guinea pigs, weighing 250 g to 350 g/mouse and not use for any purpose before. Vaccine meet the requirement for general safety test, abnormal toxicity if the whole experiment mice are healthy, gain weight and show no toxic signs in 7 days of observation. Sterility The vaccine complies with the test for sterility (Appendix 15.7). Total protein content Total protein content must not exceed 6 times the concentration of HA virus strains in the vaccine. However, in any case does not exceed 100 µg of protein/1 strain/ 1 single human dose, and no more than 300 µg protein/total of viral antigens/1 single human dose (Appendix 15.34). Bacterial endotoxin Not more than 100 EU/human dose. Endotoxin content must be determined by suitable methods (Appendix 13.2). Endotoxin content from cell-derived vaccines may be lower than from egg-derived. 1067

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Requirements The sensitivity of the positive control is in range from ½λ - 2λ to valid test. If the sensitivity of the labeled lysate was 0.125 EU/ml, the sensitivity of laboratory fact can range from 0.06 to 0.25 EU/ml. If formed gel of lysate phenomenon is not clear, have to do the retest.

Expiry date, For pandemic vaccines: specifies the particular dose (example as 2 doses).

Formaldehyde Not more than 0.2 g/L (Appendix 15.25).

Definition Japanese Encephalitis Vaccine is a sterile preparation consisting of a suspension derived from mouse brain infected with Japanese encephalitis virus strain (Nakayama or Beijing strain). The production process includes the steps of purification by protamine sulfate, ammonium sulfate, and ultra-centrifugation in sucrose solution. The virus suspension is highly purified and maintains its antigenic nature for induction of optimal immune response. The purified antigen is inactivated by formaldehyde and thimerosal is added as preservative.

Antimicrobial preservative Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The amount is not less than the minium concentration that causes effect and not greater than 115% of the intended amount (Appendix 15.29). Appearance Each of the vaccine lot after production must be checked to ensure that sensory vaccine vials or syringes before released must comply the requirements, no abnormal signs such as a foreign substance in the vaccine vials/syringes, not sealed cover or and/or not ensure integrity. During the test, if there is any vaccine syringes or vials that do not meet the requirements must be removed.

Packing and storage Inactivated influenza vaccine is packed in neutral glass vials or syringes with volume of 0.5 ml or 0.25 ml per vial and must be stored at temperatures between 2 °C to 8 °C. If used in other storage conditions, the storage conditions must be fully verified and approved by National Control Laboratory. Expiry date Expiry date must be approved by the National Control Laboratory. Generally expiry date is not exceeding 1 year from the date of manufacture, because the virus strain may not be suitable for the following year. Indications, dose Subcutaneous injection. Often is deltoid region. Dose: From 6 months to 35 months old: 0.25 ml/dose; over 36 months: 0.5 ml/dose. Labelling The information on the label, box, and leaflet must comply with the current regulations. Specifically as follows: The vaccine has been prepared on eggs or mammalian cells, The strain or strains of influenza virus used in the preparation of the vaccine, The haemagglutinin content in micrograms per virus strain per dose, Name and maximum content of any antibiotic presenting in vaccine, Recommended temperature for the storage and transportation of vaccines, 1068

JAPANESE ENCEPHALITIS VACCINE Vaccinum Encephalitidis japonicae

Quality control of final bulk vaccine Sterility The final bulk vaccine complies with the test for sterility (Appendix 15.7). Virus titer The titer of Japanese encephalitis virus is determined by ELISA technique. The titer serves as the monitoring indicator for production consistency.

Quality control of final lot Visual examination Each final vaccine lot after manufacturing is visually examined to ensure the filled containers showing no abnormal signs such as extraneous agents in the vaccine containers or tightness of closures and ensure their integrity. The vaccine is colorless and transparent. Identification The vaccine should induce neutralizing antibody, which is able to neutralize the specific virus, when the vaccine is injected to guinea pigs or mice. pH 6.8 - 7.4 (Appendix 15.33). Total protein The total protein content is determined by the method of Lowry (Appendix 15.34). The protein content is not more than 80 µg/ml. Preservative The thimerosal content is not more than 0.12 mg/ml (Appendix 15.29). Formaldehyde Not more than 0.1 mg/ml (Appendix 15.25).

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Pyrogens The vaccine shall be safe in pyrogen test. The summed response in injected rabbits is not higher than 1.3 °C (Appendix 15.12). Sterility The vaccine complies with the test for sterility (Appendix 15.7). General safety The vaccine complies with the general safety test on guinea pigs and mice. The experimental animals remain healthy and normally gain body mass after 7 days of observation (Appendix 15.11). Specific safety The vaccine complies with the specific safety test on mice. The experimental animals remain healthy and normally gain body mass after 14 days of observation. The specific safety of Japanese encephalitis vaccine is tested by determining the presence of survival virus after inactivation. Animal: Use at least 10 mice about 4 weeks old, weighing about 12-14 grams. The healthy mice without any abnormal signs and with a normal increase in body mass during a quarantine period of at least 3 days are selected just before testing. All animals are kept and observed in standard conditions. Procedure: The undiluted test sample is injected intracerebrally at a dose of 0.03 ml into each mouse. Observation and criteria for interpretation: All animals are observed and weighed before injection and daily after injection. The animals are observed for 14 days after the day of injection. The experimental animals remain healthy, gain body mass and do not show any abnormal signs during the observation period. If one animal is paralyzed or dies, repeat the test once with double number of animals and vaccine samples. In the second test if one animal is paralyzed or dies, the vaccine is rejected, as it does not comply with the test. Potency Perform simultaneous tests of potency on the vaccine to be examined and on the reference preparation. Prepare 3 dilutions of the vaccine to be examine an of the reference preparation such that to make 1/16, 1/32, 1/64 serial dilutions in 0.75 M phosphate buffer solution pH 7.4 containing 0.02% of gelatin. Inject intraperitoneally each solution in two doses of 0.5 ml at an interval of 7 days into each of 16 mice. All animals are kept and observed for a period of 2 weeks. After that, each animal is bled in aseptic condition. The separated blood is kept at 4oC overnight. The serum separation is made individually and equal part of each serum (0.1 ml) is taken to make a serum mixture for each dilution. The serum is diluted in 1/10 and kept at 56 °C for 30 minutes to inactivate serum complement and then stored at -20 °C. The potency is determined by titrating the neutralizing antibody on BHK21 cells.

The result is calculated by the formula of reducing 50% of the necrotic plaque. The titer of neutralizing antibody of the vaccine is not less than that of the reference preparation.

Packaging, storage and expiry date The vaccine is bottled in neutral glass vials of 5.5 ml/vial and 1 ml/vial. The vaccine is stored at 2 - 8 °C, protected from direct light and must not be allowed to freeze. When stored under the prescribed conditions, the vaccine has expiry date of 24 months from the date of manufacturing. Labelling The information on the label, box and leaflet must comply with the current regulations. Dosage, administration and schedule Intramuscular injection into the deltoid muscle. Under 36 months of age: 0.5 ml/dose. Over 36 months of age: 1 ml/dose. Vaccination schedule: 2 doses at an interval of 1 - 2 weeks. Booster dose: after 1 year and then every 3 years. MEASLES VACCINE (LIVE) Vaccinum morbillorum vivum Measles vaccine (live) is a freeze-dried preparation of an attenuated strain of measles virus grown on suitable cell cultures. The vaccine complies with the following requirements.

Production Working seed virus: The production of vaccine is based on a seed-lot system. The attenuated working seed virus used for production is approved by the National Control Laboratory and stored at a temperature below -60 °C. Working cells: Use working cells from cell-bank or from hen-embryo cells derived from a chicken flock free from specified pathogens (SPF) that are approved by the National Control Authority. Production process and quality control: The strain of measles virus is grown on hen-embryo cells or on cells from cell-bank using the suitable medium. The virus suspension is harvested, pooled, supplemented with preservative, filtered, diluted and freeze-dried by a validated process. Quality control should be carried out during manufacturing process at different stages: source materials (hen-eggs, medium used for production, calf serum, trypsin,…), cells used for production, single harvests, pooled vaccine lot, final bulk vaccine. Appearance The vaccine includes 01 vial of freeze-dried product and 01 vial of reconstituting liquid.

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Quality control of final product Identification Carry out the suitable methods such as micro-neutralization method and immunofluorescence method. Criteria: This is measles virus. Sterility The vaccine complies with the test for sterility (Appendix 15.7). Mycoplasmas Absence of Mycoplasmas (Appendix 15.47). Potency and thermal stability Titrate the vaccine for effective virus and test the thermal stability by a micro-neutralization on Vero cell or plaque forming unit (PFU) method on verocell. Inject the diluted vaccine (10-1; 10-2; 10-3;...) on to a 1-layer vero cell (6 well plates), inculate at (37 ± 1)°C for 60 min, first coat of agar (without red), incubate at (37 ± 1)°C, 5% CO2 for 7 days, second coat of agar (with red), incubate at (37 ± 1)°C, 5% CO2 for 3 days, count numbers of PFU that are not stained on red background, calculate potency per 0.5 ml. Determination of titer: The measles vaccine is reconstituted and prepared in serial tenfold-dilutions from 10-1 to 10-5 with medium DMEM supplemented with 2% calf serum. Add 0.1 ml of each vaccine dilution to all wells, 10 wells for each dilution. Then add 0.1 ml of Vero cell suspension (150,000 cells per ml) to each well, incubate at 36 °C for 9 days. The result is calculated by formula Karber. Final product complies with the potency test if the titer is not less than 1×103 CCID50/0.5 ml. Determination of thermal stability: Maintain samples of the freeze-dried measles vaccine at 37 °C for 1 week, the virus concentration of the heated vaccine is not more than 1.0 log10 lower than that of the vaccine stored at 4 °C. General safety The measles vaccine complies with the test for general safety on guinea pigs and mice. The experimental animals remain healthy and normally gain body mass after 7 days of observation (Appendix 15.11). Residual moisture The residual moisture of the vaccine is not more than 3% (Appendix 15.35). Residual albumin Not more than 50 ng per ml.

Storage, expiry The measles vaccine is stored at 2 - 8 °C, protected from light. When stored under the prescribed conditions, the measles vaccine may be expected to retain its potency for 24 months from the date of the last potency testing.

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Labelling The information on the label, box and leaflet must comply with the current regulations. Dosage, route of administration Use 0.5 ml per dose by subcutaneous injection. MEASLES, MUMPS, AND RUBELLA VACCINE  MMR VACCINE (LIVE) Vaccinum morbillorum, parotitidis et rubella vivum Measles, mumps, and rubella vaccine is a freeze-dried preparation of suitable live-attenuated strains of measles virus, mumps virus and rubella virus. The vaccine is reconstituted immediately before use to give a clear liquid that colored owing to the presence of a pH indicator. The vaccine complies with the following requirements:

Production Working seed-lot system Prepare the working seed-lot system derived from the attenuated master seed-lot system and is approved by the national control authority. Cell seed Use cell seed from cell-bank of human, animal or other origin; no specific pathogen free agents (SPF) and approved by the national control authority. The cells are stored frozen at -70 °C or in liquid nitrogen. Production and production control The embryo cells or cells from cell bank are used for the propagation of measles virus, mumps virus and rubella virus in suitable culture. Each virus is harvested, pooled together, added the suitable stabilizer, filtered, diluted as appropriate, capped and freeze-dried according to the approved procedures. During the production, all of the steps are controlled such as source materials (egg, cell culture medium, bovine albumin serum, trypsin...); cells using for single harvests, pooling steps, bulk, final bulk... are controlled extraneous agents, virus titer, and sterility. Single harvests Each component single harvest of measles, mumps, and rubella has to ensure that: Virus fluids shall be harvested by a method approved by the national control authority. A single harvest may be a combination of several consecutive harvests from one production cell culture. Single harvests are partly or fully stabilized and stored at or below -60 °C until pooling. No antibiotics shall be added at the time of harvesting. Each component single harvest of measles, mumps, and rubella complies with the test for sterility (Appendix 15.7), test for Mycoplasma by an approved method (Appendix 15.36) and virus titration by cell culture titration.

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Control of final bulk Virus harvests for each component are pooled and clarified to remove cells. Only add the preservatives, adjuvants or the diluent (reconstituted solution) that have been approved by the national control authority to the final bulk and cause no harm to vaccine safety and effectiveness. A suitable stabilizer may be added and for each component the pooled harvests diluted as appropriate. Suitable quantities of the pooled harvest for each component are mixed, and may be diluted if necessary. The final bulk vaccine is sealed and freeze-dried. The final bulk vaccine is ensured that there is no antibiotic. Sterility The final bulk vaccine complies with the test for sterility (Appendix 15.7).

Quality control of final lot Identification Use a neutralizing method: use specific antibodies to neutralize each virus of measles, mumps, and rubella in vaccine. Use Vero cell (for measles, mumps virus), RK-13 cell (for rubella virus). Dilute antigens and antibodies with sufficient concentration, use a suitable procedure to neutralize and infect on cell. The vaccine contains quite measles, mumps, and rubella virus if there is no infected cell of the virus infected wells. Other method such as RT-PCR may be used (use the specific gene for each virus). Potency Use a neutralizing method (neutralizes any 2 components to determine the others) on determined cell or other method approved by NCL. Acceptance criteria: Titre of measles component ≥ lg103CCID50/dose. Titre of mumps component ≥ lg103.7CCID50/dose. Titre of rubella component ≥ lg103CCID50/dose. (Manufacturer dose is 0.5 ml or 0.7 ml). Use at least 3 vials in potency test for each component. The test is valid if: the titre difference of at least 3 vaccine vials (or more) is not 0.5 logCCID50. (Potency units may be CCID50, PFU, TCID50…. and approved by NCL). Thermal stability Determined on MMR vaccine stored at 37 °C for 7 days. Compare to that stored at 2 - 8 °C. Acceptance criteria: The vaccine is passed if the titre of each component is as following: Titre of measles component ≥ lg103CCID50 /dose. Titre of mumps component ≥ lg103.7CCID50/dose. Titre of rubella component ≥ lg103CCID50/dose. the titre difference between the heated vaccine and the vaccine stored at 2 °C - 8 °C is not less than 1 lg.

General safety Carry out on mice and guinea pigs. These animals are healthy and increase weight after 7 days of observation (Appendix 15.11). Sterility Absence of bacteria and fungi (Appendix 15.7). Residual moisture Not more than 3.0% (Appendix 15.35). Inspection No abnormal subject in vaccine. Reconstitute the vaccine with the diluent; the resulting solution is clear liquid and colored owing to the manufacturer specification. Mycoplasma No Mycoplasma grows on suitable media after incubation (Appendix 15.36). PCR method may be used. Ovalbumin Ovalbumin content is less than 1 µg/dose (0.5 ml). Use a chemical-immunological method. BSA BSA content is less than 50 ng/a single human dose. Use a chemical-immunological method.

Storage Stored at 2 °C - 8 °C, protected from light. Expiry date Approved by NCL. Labelling The information of label, container, and leaflet must comply with the current regulations. Normal containing: The strains of virus used in the preparation The type and origin of the cells used for the preparation The minimum virus concentration for each component Residual antibiotic, preservatives: name and content (if any) Volume of the diluent used for reconstitution as manufacturer recommendation Store and reconstitute the vaccine protected from light The vaccine should not to be administered to pregnant women and women intend to be pregnant within two months Clear information about: how long the freeze-dried vaccine takes to dissolve completely and the expiration date for reconstituted vaccine. Appearance of the vaccine from the beginning to the expiration date for reconstituted vaccine. Dosage and administration Subcutaneous injection, dose of 0.5 ml or 0.7 ml. Diluent specification The volume of the diluent is greater than dosage (0.5 ml or 0.7 ml), clear, and no abnormal subject. There is document approved by NCL. 1071

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MENINGOCOCCAL POLYSACCHARIDE VACCINE Vaccinum meningococcale polysaccharidicum Meninigococcal polysaccharide vaccine contains only polysaccharide or conjugate vaccine type, containing purified specific polysaccharide group A, C, Y or W135 covalently linked to a carrier protein. Meningococcal polysaccharide vaccine is a sterile preparation of purified polysaccharides obtained from one or more suitable strains of Neisseria meningitidis group A, group C, group Y and group W135 that are covalently linked to carrier-protein (tetanus toxoid, diphtheria toxoid, diphtheria protein CRM197).

International name “Vaccinum meningococcale polysaccharidicum” and following by A, C, Y or W135 group. The name translated to native language is suitable to the international name. Production Production of the meningococcal polysaccharides is based on a seed-lot system. The production method shall have been shown to yield consistently meningococcal polysaccharide vaccines of satisfactory immunogenicity and safety in man. The production method is validated to demonstrate that the product, if tested, would comply with the test for abnormal toxicity for immunosera and vaccines for human use Quality control of final product A final bulk is prepared by mixing a conjugate bulk with an absorbent (where applicable), Suitable stabilizers and preservatives may be added as appropriate as long as the final product meets the result of safety test and potency test equivalent to the vaccine passed for the clinical trial. Appearance Each final lot must be visible with the naked eye for minimum package unit to ensure that no abnormal particles, the cover and cap are integrity. During testing, remove the vial not to comply with the specification. Identification Apply an immunological method; use a purified specific anti-polysaccharide antiserum. Sterility The final lot vaccine complies with the test for sterility (Appendix 15.7). Saccharide content Saccharide content of vaccine is quantified and the result complies with the limit approved by National Control Laboratory. The different manufacturer has a different formula of conjugate vaccine so that the saccharide content may be different. The method is specific for the product containing determination method of phosphorus such as colorimetric method, chromatography, HPAEC-PAD. The 1072

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immunological method such as nephelometry or inhibition ELISA may be used. Specification: The content of each polysaccharide is in the range from 70% to 130% of the labeled content. Conjugate saccharide and free saccharide content Hydrophobic-interaction  chromatography method precipitate with ice, with anti-protein carrier antiserum, ultracentrifugation, gel filtration, ultrafiltration. The free saccharide content can also be determined by a chemical method, immunological method or HPAEC after dialysis. Distribution of molecular size The specific size of the conjugate polysaccharide-protein is determined by gel filtration on Sepharose-4B or HPSEC (High Pressure Size Exclusion Chrotomagraphy) with the appropriate column. Residual moisture For freeze-dried vaccine, control residual moisture by the method approved by NCL. The test is carried out with the rate 1/1000 vials, not more than 10 vials and not less than 5 vials. The average content of the residual moisture is not more than 2.5% and no vial is more than or equal 3.0% (Appendix 15.35). Pyrogens The pyrogens are controled by rabbit intravenous injection method with the following dose: Monovalent vaccine: 0.025 µg of polysaccharide/1ml/1 kg of rabbit weight. Multivalent vaccine: Vaccine containing 2-antigen group: 0.05 µg of polysaccharide/1ml/1 kg of rabbit weight. Vaccine containing 3-antigen group: 0.075 µg of polysaccharide/1ml/1 kg of rabbit weight. Vaccine containing 4-antigen group: 0.10 µg of polysaccharide/1ml/1 kg of rabbit weight. Total of temperature difference of 3 rabbits is not more than 1.3 °C (Appendix 15.27). Adsorbent content Where applicable, determine the amount of adsorbent by a method approved by NCL. If aluminium is used as an adsorbent, the amount of aluminium is not greater than 1.25 mg per single human dose (Appendix 15.27). Preservatives The different manufacturers may use different preservatives for the vaccine and the preservative content is determined by a method approved by NCL. If thimerosal is used as a preservative, the amount of thimerosal is not greater than 0.012% (Appendix 15.29). General safety To ensure the safety when testing on mice and guinea pigs, use 2 guinea pigs weighing approximately 350 g/guinea pig. Inject intraperitoneally into each guinea pig 500 µg of the polysaccharide.

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Use 5 mice weighing 17-25 g/mouse. Inject intraperitoneally into each mouse 100 µg of the polysaccharide. The animals increase their weight, no significant symptoms for 7-day observation (Appendix 15.11). pH (Appendix 15.33) For liquid vaccine, pH is in the range of the pH value of the vaccine lot passed the clinical trial. For freeze-dried vaccine, pH test must be controlled after reconstitution with the solvent. Package Base on the manufacturer and comply with the registered document approved by National Regulatory Authority.

Storage Store the vaccine (liquid or freeze-dried) at 2 - 8 °C. Expiry date Base on the manufacturer and comply with the registered document approved by National Regulatory Authority. Labelling Comply with the current regulations. Need to state: The group of polysaccharides (A, C, Y or W135) present in the vaccine and micrograms of polysaccharide per single human dose. MUMPS VACCINE, LIVE Vaccinum parotitidis vivum Mumps vaccine (live) is a freeze-dried preparation of a suitable attenuated strain of mumps virus. The vaccine is reconstituted immediately before use, as stated on the label, to give a clear liquid that may be colored owing to the presence of a pH indicator. The vaccine complies with the following requirements:

Production Master seed lot The production of the vaccine bases on a virus seed-lot system and a cell-bank, and be approved by national control authority. The strain of mumps virus used shall be identified by historical records that include information on the origin of the strain and its subsequent manipulation. Master seed-lot are stored at temperatures below -20 °C if freeze-dried, or below -60 °C if not freeze-dried and during storage period, temperatures are monitored. Only seed lot that complies with the following requirements may be used for virus propagation. Identification The master and working seed-lots are identified as mumps virus by serum neutralization in cell culture, using specific antibodies or RT-PCR method. Virus concentration The virus concentration of the master and working seedlot is determined to ensure consistency of production.

VACCINES

Extraneous agents The working seed lot complies with the requirement for seed lots, without extraneous agents. Neurovirulence The working seed-lot complies with the test for neurovirulence of live virus vaccine. Macaca and Cercopithecus monkeys are suitable for the test. Cells used for vaccine virus Use the cell-bank or the cell from embryonated eggs that are free from specified pathogens and approved by National Control Authority. Serum used in cell-culture medium Serum used for the propagation shall be tested to demonstrate that complies with the test for sterility (absence of bacteria, fungi, and Mycoplasma) and absence of virus. Human serum shall not be used. Production control Strain of virus growth on the embryonated eggs or cell bank, medium suitable for production shall be used. The virus harvest is mixed and added preservatives, filtrated, diluted, sealed and lyophilized by approved procedures. For those test that require prior neutralization of mumps virus, the antiserum used shall be not human, simian, or avian origin. The immunizing antigen used for the preparation of the antiserum shall be produced in cell cultures from a species different to that used for the production of vaccine. Such cell cultures shall be free from extraneous microbial agent that might elicit antibodies inhibitory to growth of extraneous agents present in the mumps virus pool. The cell used for vaccine shall be tested extraneous agents, sterility... All production as cell bank, culture cell in the vaccine production shall be done in the sterility environment, which does not contain another cell. Animal serum may be used in culture. Serum and trypsin used for cell dilution and medium shall not be free from extraneous agents. The cell culture medium may contain a pH indicator such as phenol red and suitable antibiotics at the lowest effective concentration.

Single harvest Only a single harvest that complies with the following requirements may be used in the preparation of the final bulk vaccine. Identification The single harvest contains virus that is identified as mumps virus by serum neutralization in cell culture, using specific antibodies or RT-PCR method. Virus concentration The virus concentration in the single harvest is determined to monitor consistency of production and to determine the dilution to be used for the final bulk vaccine. Extraneous agents: The single harvest complies with the tests for extraneous agents (free from extraneous agents). 1073

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Control cells and eggs If human diploid cells are used for production, the control cells comply with a test for identification; the control cells and the control eggs comply with the tests for extraneous agents. Mumps vaccine (live, attenuated) may be added suitable stabilizers, and lyophilized, it is single vaccine or combine vaccine (with measles vaccine and rubella vaccine).

Quality control of final bulk Single harvests that comply with the above tests are pooled and clarified to remove cells. A suitable stabilizer may be added and the pooled harvests diluted as appropriate. Only a final bulk vaccine that complies with the following requirement may be used in the preparation of the final lot. Sterility Each final bulk shall be tested in appropriate medium. Absence of bacteria, fungi, and Mycoplasma.

Quality control of final vaccine Identification Use a suitable method approved by the national control authority. Identification using specific antiserum Specific antiserum shall be suitable with mumps vaccine. Then the Vero cell shall be effective. The test always has negative and positive samples, read the results after two weeks. Accepted standards: The vaccine complies with this test if the Vero cell is not infective as the same as the negative sample. Procedure: Day 1: Culture the cell on the 6-well plate or 24-well plate. Amount of the cell is about 1×105 - 1.5×105 cell per ml. Day 2: Neutralize the vaccine with specific antiserum. Dilute antibody at different dilutions Reconstitute the vaccine Mix antibody with antigen, the volume is ratio 1 : 1 (neutralization reaction). Incubate the neutralized suspension (antigen and antibody) and unneutralized suspension (including the reconstituted vaccine at concentration of 10-1, 10-2, 10-3, 10-4, 10-5, and antibody diluted as appropriate) in a suitable thermal cycle. Note: Thermal cycle as follow may be apply: In the waterbath at 37 °C for 30 min, 23 °C for 30 min, 4 °C for more than 18 h (≥ 18 h). Day 3: Infect the cells: Drop the neutralized and unneutralized suspension on plates. There are cell-reference wells, vaccine-reference wells at different concentration and antibody-reference wells diluted as appropriate. Incubate in the incubator at 37 °C, containing 5% of CO2. Observe daily, read results from 4th day to 7th day) The test is valid if: In reference cells, cells are normal growing Concentration dilutions of vaccine have many CPE 1074

Identification using RT-PCR method Use specific primer for mumps virus. Primer has detail information. The tests have samples and the negative. Accepted standards: The vaccine complies with this test if the production of RT-PCR is the same information of primer. Notes: The primer may be used: Right: 5’-AGT AGT GTC GAT GAT CTC AT-3’, Left: 5’-GCT CAA GCC TTG ATC ATT GA-3’. Size: 674 bp. Thermal cycle/time: 50 °C/30 min; 95 °C/time (following the guidance of RT-PCR kits) 94 °C/1 min 55 °C/1 min 40 cycles; 72 °C/1 min 72 °C/10 min 4 °C/ until electrophoresis.

Titration Titer of mumps vaccine is determined by cell culture infectious doses on 50% of the Vero cell (CCID50). Materials: The mumps vaccine to be examined: 03 vials, with the diluents supplied with the vaccine. Reference vaccine: 1 vial. Two 75-cm2 flasks of Vero cell growing well on a monolayer. Chemicals: PBS (Phosphate buffered Saline) Trysin 0.25% EDTA MEM or M199 containing 2% FBS Method: Carry out this test in sterile BSC class II in sterile area. Three containers from the final freeze-dried vaccine lot shall tested titer. The geometric mean infectious virus titre of three vials. Dilute the sample and standard. Ex: Dilute as in Table 1. Table 1 - Example for the dilution of the vaccine Volume of diluent Volume of vaccine Dilution added (ml) dilution (ml) -1.0 10 (A) 2.7 0.3 -1.5 10 (B) 2.16 1.0 (from A) -2.0 10 (C) 2.16 1.0 (from B) -2.5 10 (D) 2.16 1.0 (from C) -3.0 10 (E) 2.16 1.0 (from D) -3.5 10 (F) 2.16 1.0 (from E) -4.0 10 (G) 2.16 1.0 (from F) -4.5 10 (H) 2.16 1.0 (from G) -5.0 10 (K) 2.16 1.0 (from H)

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Add the media as described in the above table into glass penicillin. Reconstitute vaccines, shake well; to 0.3 ml, add to the first dilution level of 10-1.0, mix well; to 1 ml, add to the dilution level of 10-1.5, to continue the process till the last dilution level. Remember to change tip after each dilution level. After diluting, add each dilution in 10 wells in a row, each well of 100 µl. Start from the lowest dilution to the highest dilution, if the process is reversed, the tip should be changed after each dilution level. Add the media into the last 2 rows, each well of 100 µl. Repeat the above process with the remaining vials, and standard vaccine. After diluting, put the plates in the fridge (2 °C - 8 °C). Prepare the cell suspension Cell concentration is 2×105 cells per ml. Calculate enough of volume Vero cell then into them on the tray. Add 100 µl of the Vero cell suspension to all wells of all plates. Wrap the plate in Adhesive film. Incubate the plates at 36 °C in incubator containing 5% of CO2. Read the results from 7th day to 9th day. Read and calculate results: Results shall be read by microscopy. These the cells are damage which is called the cell infected mumps virus. Calculate the results using the Karber or Reed-muench formula: Log CCID50 = Negative log of the lowest dilution used – (sum % CPE in each dilution/100-0.5)× log dilution factor Evaluation of results: The negative have cells, which shall be growing normal. The titre of working reference preparation should be within 100.5 of the established titre, which is based on the geometric mean titre of all valid assay for the working reference preparation performed during the previous period. The titre should also be within the confidence limits of the test as establishes before. The variation between the two samples of the vaccine under test should be not more than 100.5. It should also be within the confidence limit of the test as established before. The cytopathic effect should decrease with increasing dilution. Dilution should include 0% and 100% cytopathic effect readings. Acceptance criteria: The titer of the assayed vial should be at least 103.0 per human dose. Virus titer of mumps vaccine was determined not less than on the label

Acceptance criteria: The virus concentration of the heated vaccine at 37 °C is not more than 1.0 log lower than that of the unheated vaccine at 5 ± 3 °C. The minimum quantity is generally considered to be log103 CCID50/0.5 ml (single dose for human)

Thermal stability Keep the vaccine in the dry state at 37 °C for 7 days. Determine the virus concentration as described under Assay in parallel for the heated vaccine and for vaccine stored at the temperature recommended for storage (5 ± 3 °C).

Administration Subcutaneously injection, 0.5 ml/dose or 0.7 ml/dose (accordance with the manufacturer regulation).

General safety (Appendix 15.11) Each final lot shall be tests for the absence of abnormal toxicity in mice and guinea pigs by appropriate tests approved by national control authority. Mice and guinea pigs must be healthy and gain weight after 7 days of observation. Sterility It complies with the test for sterility (Appendix 15.7). Residual moisture Not more than 3.0% (Appendix 15.35). Appearance It complies with the requirements of the manufacturer. Mycoplasma Absence of Mycoplasma ((Appendix 15.36). Note: PCR method may be used to identify Mycoplasma on the mumps vaccine. Ovalbumin If the mumps component is produced in chick embryos, the vaccine contains not more than 1 µg of ovalbumin per single human dose, determined by a suitable immunochemical method. Residual albumin Not more than 50 ng of albumin per single human dose. Determined by a suitable immunochemical method. Storage Store at temperature about 2 °C to 8 °C and protected from light.

Labelling The label complies with the current regulations and contains the following information: The strain of virus used for the preparation of the vaccine; That the vaccine has been prepared in chick embryos or the type and origin of cells used for the preparation of the vaccine; The minimum virus concentration; Residual antibiotic content; the preservatives and their content (where applicable); Protected from light That contact between the vaccine and disinfectants is to be avoided.

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HUMAN PAPILLOMAVIRUS VACCINE (RECOMBINATION) Vaccinum papillomaviri humani (ADNr) Human Papillomavirus vaccine (HPV) is preparation of purified virus-like particles (VLPs) but not infectious, highpurity, the capsid protein (L1) of one or more types of HPV. HPV antigens were prepared by recombinant DNA technology. Depending on the manufacturer, purified VLPs can be adsorbed on the adjuvant aluminum hydroxide combined and 3-0-desacyl-4'-monophosphorylated lipid A (MPL) or aluminum hydroxyphosphate sulfate. PRODUCTION

HPV vaccine is produced by the expression of the viral genes encoding for the capsid proteins in yeast cells or in an insect cell/Baculovirus expression vector system and then collected and purified VLPs. The HPV vaccine production is based on the cell-bank system and seed-lot system. Reference vaccine: HPV reference vaccine is prepared from a vaccine batch or one of vaccine batches that has been studied clinically and shown to be effectively protected. Reference vaccine need to achieve stability requirements.

Evaluate characterization of VLPs Characterization of VLPs must be evaluated in the process of vaccine production. The techniques are often used to evaluate the characterizations of VLPs including determine the composition of protein as SDS-PAGE (sodium dodecyl sulfate Polyacrylamide gel electrophoresis), Western Blot, mass spectrometry, peptide mapping and/or terminal amino acid sequence analysis. Morphological characteristics of the VLPs and degree of aggregation are determined to confirm the presence of the conformational epitopes that are essential for creating effective vaccines. In addition, the protein, lipid, nucleic acid and carbohydrate components should also be determined in the vaccine manufacturing process. Cell banks and seed lots Vaccine production in yeast cells The use of any cell line must been based on the cell bank systems. Master/original and working cell banks must to meet the requirements of identity, purity; growth characteristic and stability shall be used for production. Gene homogeneity must be studied for the master and working cells. A full description of biological characteristics of the host cell and expression vectors is given. The physiological methods used to in the production process to promote and control the expression of the cloned gene in the host cell. The characteristics of the recombinant strain produced (host cell combination with expression vector system) must be fully described, demonstrated no spontaneous 1076

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elements and information about genetic homogeneity of the working and master cell bank. Need full description of the biological characteristics of host cells and the expression vector. Vaccine production in insect cell/Baculovirus expression vector system In insect cells: cell banks that meet the requirements of identity, purity, growth characteristic, stability, extraneous agents shall be used for production. These characteristics can evaluated at the appropriate stage in the production process. If the insect cells used to produce VLPs, the use of insect cells must rely on a system of master cell bank. In addition, master and working cell bank if multiplied/ passaged in next time or beyond the allowed limit, must be test ability born tumors in animal; the presence of the retroviruses and other virus in arthropods. Recombinant baculovirus: The use of the recombinant baculovirus vector is based on a seed-lot system with a defined number of passages between the original virus and the master and the working seed-lots, as approved by the competent authorities. The recombinant baculovirus expression vector contains the coding sequence of the HPV L1 antigen. Using molecular biology techniques combined with some other tests to determine the structure of the fractional expression in recombinant protein purification and confirmed the quality and stability of the L1 antigen. Recombinant Baculovirus should be monitored during vaccine production, including genetic information, such as the origin, identity, structure, composition and genetic characteristics. Baculovirus recombinant original strain is produced in large quantities and stored at appropriate temperatures. Only a seed lot that complies with the following requirementsmay be used for virus propagation Identification The master and working seed lots are identified by an appropriate method such as nucleic acid amplification techniques (NAT). Virus content The virus concentration of the master and working seed lots is determined to monitor consistency of production. Extraneous agents The working seed lot complies with the requirements for seed lots and control cells. Special attention is given to Spiroplasma spp. and insect-borne viruses, in particular insect-borne potential human pathogens (example: Arboviruses). Sterility Baculovirus recombinant strains have been tested with a bacterial infection, fungal and Mycoplasma using appropriate test (Appendix 15.7).

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Mycobacteria Baculovirus recombinant strains will be tested for Mycobacterium spp.

pay special attention to insect-borne viruses in cell banks and master seed virus strain that capable of causing human disease (example: Arbovirus).

Control cells use for production cell See the test for control cells below.

Control cells In cell culture process/production Baculovirus expression system, control cells have meet requirements for identity and exogenous agents. Need to pay particular attention insect-borne viruses in cell banks and master seed virus strain that capable of causing human disease.

Propagation and harvest The process of cell culture and Baculovirus master seed strain must be done under completely aspeptic conditions and without any other type of cell being cultured. In the production area for culture cells/Baculovirus expression vector systems, a stored virus intermediate culture that complies with the 5 following tests may be used for virus propagation. Identification Determined by identifying HPV types and using immunoassay with specific antibodies or molecular biological techniques such as genome amplification techniques. Sterility Each intermediate culture medium containing virus must be tested and meet the requirements for sterility, using 10 ml for each medium (Appendix 15.7). Virus concentration The virus concentration of each stored baculovirus intermediate culture is determined by a suitable method such as plaque assay or NAT, to ensure consistency in the production process. Extraneous agents All mediated culture medium containing the virus must comply with the test for extraneous agents. Control cells The control cells of the production cell culture from which each stored virus intermediate is derived comply with a test for identity and with the requirements for extraneous agents

Single harvest Only a single harvest that complies with the following requirements may be used in the preparation of the purified monovalent antigen. Identification Determined by identifying HPV types and using immunoassay or molecular biological techniques such as PCR or hybridization. Identification test can replace by a part of the test checking the purity of the antigen. Sterility Each single harvest must be tested and met the requirements for sterility (Appendix 15.7). Extraneous agents All single harvest must be tested for extraneous agents during cell culture process/production Baculovirus expression vector system. As mentioned above, need to

Purified monovalent antigen Only the purified monovalent antigen that complies with the following requirements may be used in the preparation of the final bulk. Provided that the following tests have been carried out with satisfactory results on the “Adsorbed monovalent antigen” stage, it may be omitted on the final lot. Total protein The total protein content is determined by a suitable evaluated method. The protein content must be within the limits approved for product (Appendix 15.34). Antigen content and identification The quantity and specificity of each antigen type is determined by a suitable immunochemical method such as radio-immunoassay (RIA), enzyme-linked immunosorbent assay (ELISA). Preferably using a monoclonal antibody directed against a protective epitope or single radial diffusion. The antigen/protein ratio may be determined and is within the limits approved for the particular product. Antigenic purity The purity of each purified monovalent antigen is determined by a suitable method, such as SDS-PAGE with quantification by densitometry analysis, the limit of detection being 1% of impurities or better with respect to total protein. A reference preparation is used to validate each test. The protein purity is calculated as the ratio of the L1 protein-related bands relative to the total protein bands, expressed as a percentage. For the genotypes included in the vaccine, the value calculated for purity complies with the limits approved for the particular product. Percent of intact L1 monomer The antigenic purity assay serves also to assess the integrity of the L1 monomer. The percent intact L1 monomer is the ratio of the intact L1 monomer to the total protein, expressed as a percentage. VLP size and structure The size and structure of the VLPs is established and monitored by a suitable method such as dynamic light scattering. The size is within the limits approved for the particular product.

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Composition The protein, lipid, nucleic acid and carbohydrate contents are determined, where applicable. Residual DNA Not more than 10 ng of DNA in a quantity of purified antigen equivalent to a single human dose of vaccine, determined by a highly sensitive method. Residual host-cell proteins The residual host-cell protein content is determined by a suitable method. The content is within the limits approved. Chemicals used for disruption and purification The chemicals used for purification or/and other stages of production are determined by a suitable method. The content is within the limits approved (for the particular products). Albumin If animal serum is used in cultured insect cells or mammalian cells for vaccine production, the concentration of albumin residues have been identified. Sterility Each purified monovalent antigen must be tested and meet the requirements for sterility, carried out using 10 ml for each medium (Appendix 15.7).

Adsorbed monovalent antigen The purified monovalent antigens may be adsorbed onto a mineral vehicle such as an aluminum salt. Only an adsorbed monovalent antigen that complies with the following requirements may be used in the preparation of the final bulk vaccine. Sterility Each absorbed monovalent antigen must be tested and meet the requirements for sterility, carried out using 10 ml for each medium (Appendix 15.7). Bacterial endotoxins Each adsorbed monovalent antigen is tested for bacterial endotoxins (Appendix 13.2). The content is within the limits approved for the particular product. Antigen content and identification Each antigen type is identified by a suitable immunochemical method such as radio-immunoassay (RIA), enzyme-linked immunosorbent assay (ELISA). Preferably using a monoclonal antibody directed against a protective epitope or single radial diffusion. The antigen/ protein ratio is within the limits approved for the particular product. The concentration of the adsorbent Each adsorbed monovalent antigen batch is tested for the content of adsorbent. The content is within the limits approved for the particular product.

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CONTROL ON THE FINAL BULK VACCINE

The final bulk vaccine is prepared directly from each purified monovalent antigen HPV type or adsorbed purified monovalent antigen HPV type. An antimicrobial preservative, a mineral vehicle such as an aluminum salt and the adjuvant 3-O-desacyl-4-monophosphoryl lipid A or aluminum  hydroxyphosphate sulfate may be included in the formulation of the final bulk. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot.

Antimicrobial preservative The amount of antimicrobial preservative (where applicable) is determined by a suitable method. The total amount is not less than 85% and not greater than 115% of the intended content. Sterility The final bulk vaccine complies with the test for sterility, carried out using 10 ml for each batch (Appendix 15.7). Adsorbent If aluminum is used as an absorbent, aluminum content is within the limits approved for particular product. Degree of adsorption The degree of adsorption of each antigen (adsorption capacity completely) of vaccine antigens in the semi-final must be assessed. However, it may be omitted if the stability of the process has been proved or if it has been carried out with satisfactory results on the final bulk vaccine. Potency (The assay is performed by an in vivo test or an in vitro test) The potency of HPV vaccine is carried out in parallel with the reference vaccine and internal control. There may use the method of immunization in mice to determine the antibody titer obtained (in vivo test) or methods using specific antibodies carry out ​​in the laboratory to quantify directly the amount antigens contained in the vaccine (in vitro test). CONTROL ON THE FINAL LOT

Only vaccines to meet requirements of the following tests may be used in humans The test for antimicrobial preservative content (where applicable) may be omitted if it has been carried out with satisfactory results on the final bulk vaccine. In addition, the in vivo assay may be omitted, too if it has been carried out with satisfactory results on the final bulk vaccine.

Closed volume and appearance/description Volume: Test at least 3 vials for sealed and tight. Volume of each vaccine vial is not less than the volume stated on the label and in the registration document, ensure sealed, snugly. Description: Vaccines have no foreign material, morphology and color as registered. Observe with naked eye of product form and color.

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Identification The different HPV types in the vaccine are identified by a suitable immunochemical method (usually by ELISA using specific antibodies or by in vivo tests). The potency test may include/serve the identification test. Sterility The vaccine complies with the test for sterility (Appendix 15.7). pH Must be within the approved limits (Appendix 15.33). Antimicrobial preservative The amount of antimicrobial preservative (where applicable) is determined by a suitable method. The total amount is not less than 85% and not greater than 115% of the intended content (Appendix 15.29). Pyrogens (Appendix 15.12) Each lot final must comply with the test for pyrogens in rabbits. It is possible to carry out the test endotoxin testing if appropriate conditions. Particularly vaccines using MPL adjuvant is this test should be checked. This test was carried out until prove the safety and stability of production. Bacterial endotoxins (Appendix 13.2). Not more than 5 EU per single human dose. If the adjuvant prevents the determination of endotoxin, a suitable inprocess test is carried out. Adsorbent (Appendix 15.27) If vaccines containing the adsorbent, adsorbent content must be within the limits approved. Degree of adsorption The degree of adsorption of each antigen (adsorption capacity completely) of vaccine antigens is assessed. However, it may be omitted in tests for release if it has demonstrated the stability of the product. Adjuvants (Where applicable MPL: 3-O-desacyl-4’monophosphoryl lipid A) Determined by gas chromatography. Accepted standard: From 30 µg/dose to 50 µg/dose. General safety Each batch of finished vaccine must be tested for abnormal toxicity (which may be called the non-specific toxicity) with general safety test (Appendix 15.11). This test may be omitted in tests for release of vaccine if the potency test is carried out by the method of in vivo. In vitro potency assay In vitro potency assay of the HPV vaccine was determined by ELISA method, according to the "sandwich" two stages. The VLP antigen (L1) in combination with anti-HPV

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antibodies polyclonal rabbit IgG (antibodies or specific HPV type) was mounted on plastic plates (antibody 1). Complex antigen - antibody 1 further mounted mouse antibody, specific VLP (antibody 2) and conjugate. The development color is attached with complex 1 antibody - antigen - antibody 2. Antigen contents were calculated based on the color density of wells containing samples vaccines. ELISA was performed with the supernatants after centrifugation of vaccine to get not get antigens not absorbed. Procedures in vitro effects of each type of HPV antigens are similar and are done separately. The potency and identification for Cervarix vaccine (in vitro) Materials: Immunoplate Maxisorp F96 microplate (Nunc or equivalent) Cervarix vaccine HPV 16 and HPV 18 standard HPV 16 and HPV 18 internal control (IC) Anti-HPV rabbit polyclonal antibodies of type 16 and type 18 (antibody 1- capture antibody) Anti-HPV 16 and Anti-HPV 18 mouse monoclonal antibodies (antibody 2- “primary antibody”). Biotinylated mouse anti-Ig antibodies (antibody 3) Streptavidin-biotin-peroxidase complex Procedure: Preparation of coating plate: The immunoplate 96 wells are coated rabbit polyclonal antibody diluted HPV antibodies in PBS solution, incubated plates 4 °C overnight, washed and saturated with PBS-casein solution at 37 °C for 1 h. Procedure for test vaccines: Centrifuge the standard vaccine, IC and Cervarix vaccine at 13,000 rpm for 10 min to separate the supernatants whole. Dilute fold-2 the supernatants that vaccine antigen concentrations in the lowest phase of approximately 250 ng/ml for HPV-16 and 2000 ng/ml for HPV-18. Then dilute fold-2 from 20 to 2-10 to the potency test. The identity test just needs one dilution of 20. The diluted vaccine samples were added in plates, 3 wells for each dilution, incubated at 37 °C for 2 h, shaking slightly. Wash plates and add mouse monoclonal antibodies to HPV at all wells after washing, incubated at 37 °C for 1 h. Wash plates and add mouse antibody conjugate - Biotin solution and incubated at 37 °C for 1 h. Wash plates and add the streptavidin-biotin-peroxidase complex, incubated at 37 °C for 30 min. Wash plates and add the TMB color solution, incubated for 15 min at room temperature, protected from light. Add solution to stop the reaction. Measure optical density at 450/620 nm. Acceptance criteria Identification test: + Test is not valid unless: The mean OD of the blanks must be less than 2.0 + HPV antigen content of each type in the test sample was considered positive when the mean OD of tested sample 1079

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greater than the OD value corresponds to asymptotic lines under of standard samples. Potency test: Calculate the results in Excel software. The correlation coefficient (R2) of the standard samples must be greater than 95%. Antigen content of each type in the tested sample was calculated at least three dilutions in a row of the 10 continuous dilutions used in the reaction. Accepted standards for potency test: Type 16: The relative potency of tested vaccines and internal control sample must be from 0.78 to 1.45. Type 18: The relative potency of tested vaccines and internal control sample must be from 0.79 to 1.47. The potency and identification for Gardasil vaccine (in vitro) Materials: Immunoplate Maxisorp F96 microplate (Nunc or equivalent) Gardasil vaccine (HPV-6, 1, 16, 18) Capture antibody (HPV-6,11,16,18) Monoclonal antibodies specific HPV type 6, 11, 16, and 18. Goat anti-mouse IgG2b-HRP antibody. Horseradish peroxidase (HPR-conjugated antibody). Tetramethylbenzidine (TMB). Procedure: Preparation of coating plate: The immunoplate 96 wells are coated rabbit monoclonal specific HPV antibodies in PBS solution, incubated plates at 2-8 °C overnight, washed and saturated with PBS solution containing 1% BSA, and 0.05% Tween 20 at 22-28 °C from 1 h to 6 h. Procedure for test vaccines: Centrifuge the standard vaccine, IC and Cervarix vaccine at 650 rpm/10 min to separate the supernatants whole. Dilute 2-fold the supernatants with PBS solution containing 1% BSA, and 0.05% Tween 20 that vaccine antigen concentrations in the lowest phase of approximately 2 µg/ml. Then dilute 3-fold from 30 to 3-10 to the potency test. The identity test just needs one dilution is 30. The diluted vaccine samples were added in coated plates, Citrate buffer solution was added to the wells immediately after to eliminate the adsorbent in the vaccine, the plates incubated at temperatures from 22 °C to 28 °C overnight and shake. Then wash the plates and add mouse monoclonal antibody to all wells. Incubate for 1 h at temperatures from 22 °C to 28 °C. Wash the plates and add conjugate solution, incubated for 1 h at temperatures from 22 °C to 28 °C. Wash and add undiluted TMB solution. Wait for 5 min and then add a solution to stop the reaction. Measure optical density at 450 nm. Calculate a result by the Pro program SoftMax or Excel software. Acceptance criteria: Content of each type must be within the limits that manufacturers approved registration. 1080

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HPV total content must be not greater than 500 IU/ml (≤ 500 IU/ml). Potency test (in vivo) (for bivalent vaccene) Experiment animal: white mice (BALB/C strain or a suitable approved strain) healthy and from the same flock, about 6 to 8 weeks old, and the best is the same sex. Preparation of tested vaccine: Tested Vaccine and reference Vaccine are diluted from 2-fold to 5-fold dilutions so that the concentration of antigen diluted sample finally reached about 0.56 mcg/0.5 ml. The solution for dilution is physiological saline (0.9% solution of sodium chloride) contain more types of adjuvant used for the vaccine production. Intraperitoneal injection of the 0.5 ml/mouse, each dilution injects least 10 mice. All of the mice after immunization were brought up from 21 days to 22 days in the same conditions. Perform of anesthesia, take blood mice heart. Blood samples were centrifuged at 3000 rpm for 10 min at 4 °C to 8 °C, the serum separated and stored separately for each mouse blood samples at -20 °C. Determine specific antibody titers of HPV antibodies by enzyme immunoassay (ELISA) with good quality commercial kit. Results were calculated by Probit Analysis Program (WHO PRO). Test is not valid unless: ED50 of tested vaccine and standard vaccine were in the range of the largest and smallest doses. The statistical analysis shows no significant deviation from linearity or parallelism. The confidence limits (P = 0.95) are within the limit approved for the particular product. Acceptance criterion: The correlation potency (p = 95%) is not less than 0.5 compared with reference vaccine.

Package HPV vaccines are packed in neutral glass vials or prefilled syringe (type I glass) with piton has rubber bottom (butyl rubber), with or without needles. Each vial/syringe containing 1 single dose of 0.5 ml suspension. Carton box can contain one or more vials/syringe single dose depending on the manufacturer. Storage Store at temperatures 2 °C to 8 °C, protected from light and avoid freezing vaccines. Labelling The information on the label, box, and leaflet must comply with the current regulations Administration Route of administration: Intramuscular injection in the deltoid region. Dose, injection schedule: According to the specific instructions of the registered manufacturers.

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PNEUMOCOCCAL POLYSACCHARIDE CONJUGATE VACCINE (ADSORBED) Vaccinum pneumococcale polysaccharidicum coniugatum adsorbatum Pneumococcal polysaccharide conjugate vaccine (adsorbed) is a sterile suspension of purified capsular polysaccharide obtained from S. pneumoniae serotypes (abbreviated to pneumococcal antigens); each antigen covalently links to a carrier protein. The vaccine is adsorbed on a suitable adjuvant or adsorbent such as aluminum phosphate... Liquid or freeze-dried vaccine is complied with the following requirements:

Production The production method shall have been shown to yield consistently S. pneumoniae conjugate vaccines of adequate safety and immunogenicity in man. The production of polysaccharides and of the carrier(s) is based on a seed-lot system. The production method is validated to demonstrate that the product, if tested, would comply with the test for abnormal toxicity for immunosera and vaccines for human use. Master seed and working seed The bacterial strains used for master seed lots shall be identified by historical records that include information on their origin and the tests used to characterize the strain (physiological, biochemical, serology and genetic characteristics...). S. pneumoniae serotype strains for pneumococcal vaccine production are approved by National Control Laboratory (NCL). Each strain must be shown to be capable of producing polysaccharide of the appropriate serotypes, of adequate safety and immunogenicity in man. Cultures derived from the working seed lot have the same characteristics as cultures of the master seed lot. Positive Gram staining, immobility, non-spore forming, microscopically appear typical of S. pneumonia, grow at 37 °C, have smooth alpha-haemolytic colonies; be lysed in the bile solubility test, ferment inulin, be sensitive to optochin, give a positive Quellung reaction. Cultures and harvests The cultures of seed lot should be free from ingredients that will form a precipitate upon purification of the polysaccharide. The purity of culture before separation is tested by suitable method that includes inoculation on to appropriate plating media. If the contamination is found, the culture is removed. Polysaccharide purity Each strain of S. pneumoniae serotypes is individually grown in a liquid medium that does not contain highmolecular-mass polysaccharides; if any ingredient of the medium contains blood-group substances; the process is

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validated to demonstrate that after the purification step they are no longer detectable. After inactivation, polysaccharide is isolated and purified by a suitable method such as fractional precipitation, chromatography, enzyme treatment and ultrafiltration. The purified polysaccharides complies with the following specifications may be used in the preparation of the bulk conjugate. Identification: Apply an immunological method or 1H (or 13 C) Nuclear magnetic resonance (NMR) spectroscopy; each pneumococcal antigen is verified. Polysaccharide composition: should be based on the dry weight of the polysaccharide or the percentage of total nitrogen, phosphorus, uronic acid, hexosamine, methyl pentose and O-acetyl groups that are determined by colorimetric method or high-performance-anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The specification is approved by NRA. Moisture content: If the purified polysaccharide is to be stored as a lyophilized powder, the moisture content should be determined and must comply with the limit approved by NRA. Protein impurity: Apply Lowry method, not more than 3.0% by weight of polysaccharide. Nucleic impurity: Not more than 2.0% by weight of polysaccharide as determined by absorbance of nucleic acid at 260 nm length or by another validated method. Pyrogens: It complies with the test for pyrogens (Appendix 15.12). Carry out the test in rabbits. Bacterial endotoxin: Carry out the test as described in Appendix 13.2. Less than 0.5 EU of endotoxin per microgram of polysaccharide. Molecular size: use gel filtration chromatography or highperformance size-exclusion chromatography (HPSEC). The distribution constant (KD) is determined by measuring the molecular size distribution of the polysaccharide at the main peak of the elution curve; meets manufacturer specification approved by NRA. Modify polysaccharide Before or during a chemical modification, polysaccharide preparations are partially depolymerized. The actived polysaccharide that complies with the following requirements may be used in the preparation of the conjugation. Molecular size: Use gel filtration chromatography method or other validated method. The molecular size of each pneumococcal antigen is in the specification approved by NRA. Carrier protein Carrier protein is used to improve immune response at B and T cells. Strain to be used for the production of the carrier protein has its history, origin of strain, physiological, biochemical characteristics, safety. The growth of strain has to consistency by monitoring the growth rate, pH and 1081

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yield. The purity of culture should be tested; the culture is inactivated and purified by a suitable method. Normally, carrier protein is tetanus toxoid, diphtheria toxoid, CRM 197 protein, protein D – outer-surface protein of non-typable Haemophilus influenza. Only a carrier protein that complies with the following requirements may be used in the preparation of the conjugate bulk: Identifycation: Use a serological method, positive reaction. Sterility: It complies with the test for sterility (Appendix 15.7). Endotoxin: Endotoxin content is not more than 1.0 EU/µg protein. Protein content: Determined by a validated suitable method, the content is not less than 90% of total protein content. Monovalent bulk The conjugate is obtained by the covalent binding of activated polysaccharide to the carrier protein by different methods depending on manufacturer. The conjugate purification procedure is designed to remove residual reagents used for the conjugation by suitable method. Only a bulk that complies with the following requirements may be used in the preparation of the final bulk. Identification: Apply for a serological method, positive to each pneumococcal antigen. Residual reagent: Use of chromatography mass spectrometry or other validated method; no residual reagents are verified. Polysaccharide - protein ratio: 0.3 - 3.0 for each pneumococcal antigen. Determine each polysaccharide content and protein content or directly measure the ratio by 1H NMR spectroscopy or HPSEC or other validated method. Conjugated and unbound (free) polysaccharide: Meet the manufacturer specification approved by NRA. Use a validated suitable method such as HPAEC. Protein content: Meet the manufacturer specification for each antigen. Use a validated appropriate method such as HPLC, capillary electrophoresis. Sterility: It complies with the test for sterility (Appendix 15.7) Specific toxicity of carrier protein: If carrier protein is tetanus toxoid, diphtheria toxoid; use a validated suitable method; absence of specific toxicity of the carrier protein. Endotoxin: Use LAL test, the endotoxin content is not more than 0.75 EU/µg polysaccharide.

Final bulk Adjuvants and preservatives are added with the appropriate monovalent bulk of each pneumococcal antigen to make the final bulk. The final bulk that complies with the following requirements may be used in the preparation of the final product: Sterility: It complies with the test for sterility (Appendix 15.7). 1082

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Final product Identification: Apply for a serological method, positive to each pneumococcal antigen. Sterility: It complies with the test for sterility (Appendix 15.7). Polysaccharide content: Determine the polysaccharide content of each pneumococcal antigen and total polysaccharide by an immunological method (rate nephelometry or ELISA inhibition,...) or other validated suitable method; the content is in range of ±30.0% of labeled polysaccharide content. Residual content: If the vaccine is freeze-dried, the residual moisture content is not more than 3% (Appendix 15.35). Endotoxins (Appendix 13.2): Meet the specification approved by National Regulatory Authority (NRA). Aluminum: not more than 1.25 mg/1 single human dose (Appendix 15.27). Preservative content: Use a suitable validated method that approved by NRA. General safety: The vaccine complies with the requirement for abnormal toxicity if all mice survive and increase their weight after 7 days of observation (Appendix 15.11). pH: For liquid vaccine: pH specification is within the range of values shown to be safe and effective in clinical trials and in stability studies (Appendix 15.33). For lyophilized vaccine: pH is measured after reconstitution with the appropriate diluent. pH specification is within the range of values shown to be safe and effective in clinical trials and in stability studies (Appendix 15.33). Inspection: Inspect visually vaccine; there is no abnormality such as improper sealing, lack of integrity, particles... Package It complies with the GMP requirements and current regulations. Storage, expiry date Store in an airtight container, at 2 °C - 8 °C. The expiry date is based on the stability of the vaccine and approved by NRA. Labelling The information on the label, box, and leaflet must comply with the current regulations and indicates the information of content of each polysaccharide, the name and content of protein carrier of single human dose, the name and amount of adjuvant, the temperature recommended during storage and transport (shake before use, not freeze). PNEUMOCOCCAL POLYSACCHARIDE VACCINE Vaccinum pneumococcale polysaccharidicum

Definition Pneumococcal polysaccharide vaccine consists of a mixture of the equal parts of 23 purified capsular polysaccharide antigens prepared from S. pneumoniae strain (abbreviated

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to pneumococcal antigens): 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17A or 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F. The vaccine is a clear, colorless liquid.

Production Master seed and working seed Production of the vaccine is based on a seed-lot system for each type. S. pneumoniae serotype strains for pneumococcal vaccine production are approved by National Control Laboratory (NCL). Each strain must be shown the capability of producing polysaccharide of the appropriate serotypes with consistent yield, of adequate safety and immunogenicity in man. Each master seed lot must have its document (history, identification of the strain’s characteristics (physiological, biochemical, serology and genetic characteristics). Cultures derived from the working seed lot have the same characteristics as cultures of the master seed lot. Positive Gram staining, immobility, non-spore forming, microscopically appear typical of S. pneumonia, grow at 37 °C, have smooth alpha-haemolytic colonies; be lysed in the bile solubility test, ferment inulin, be sensitive to optochin, give a positive Quellung reaction. Cultures and harvests The cultures of seed lot should be free from ingredients that will form a precipitate upon purification of polysaccharide, from blood-group substances or high molecular mass. The purity of culture before separation is tested by suitable method that includes inoculation on to appropriate plating media. If the contamination is found, the culture is removed. Monovalent bulk After inactivation by phenol, polysaccharides are isolated and purified by a suitable method such as fractional precipitation, enzymatic digestion and ultrafiltration, washed, and dried in a vacuum to a residual moisture content shown to be favorable to the stability off the polysaccharide and stored at a suitable condition. Only the purified polysaccharides that complies with the following specifications may be used in the preparation of the final bulk Identification: Apply an immunological method (double immunodiffusion or immune-electrophoresis; each pneumococcal antigen is verified. Polysaccharide composition: Base on the dry weight of the polysaccharide or the percentage of total nitrogen, phosphorus, uronic acid, hexosamine, and methyl pentose and O-acetyl groups that are determined by colorimetric method or high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The specification is approved by NRA. Protein impurity: Not more than 3.0% by weight of polysaccharide. Determined by Lowry method.

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Nucleic acid impurity: Not more than 2.0% by weight of polysaccharide. Determined by measuring the absorbance of nucleic acid at 260 nm or by another validated methods. Molecular size: Use gel filtration chromatography or high-performance size-exclusion chromatography. The distribution constant (KD) is determined by measuring the molecular size distribution of polysaccharide at the main peak of the elution curve; meets manufacturer specification approved by NRA. Sterility: It complies with the test for sterility (Appendix 15.7) Specificity: No across reaction occurs when the antigens are tested against all the antisera specific for the other polysaccharides of the vaccines. Final bulk Final bulk is prepared by mixing different types of polysaccharide powder in sterile condition. Disperse homogeneous mixture in an appropriate isotonic solution to obtain a single human dose containing 25 μg of each polysaccharide type. Only the final bulk that complies with the following requirement may be used in the preparation of the final product: Sterility: It complies with the test for sterility (Appendix 15.7).

Final product Identification: Positive to each pneumococcal antigen. Apply for immunological method. Sterility: It complies with the test for sterility (Appendix 15.7) Polysaccharide content: Determine the polysaccharide content of each pneumococcal antigen and total polysaccharide by immunological assays (rate nephelometry or ELISA inhibition...) or other suitable validated method; the content is within a tolerance of ±30.0% of labeled polysaccharide content. Preservative: Antimicrobial preservative: determine by validated suitable method; the content is in the range from 85% to 115% of labeled content. Phenol: determine by validated suitable method; the content is not more than 2.5 g/l. Pyrogens: Inject intravenously via rabbit’s ear with 2.5 μg of each pneumococcal antigen/ml/kg; comply the pyrogens (Appendix 15.12). General safety: The vaccine complies with the requirement for abnormal toxicity if all mice survive and increase their weight after 7 days of observation (Appendix 15.11). pH: 4.5 - 7.4 (Appendix 15.33). Inspection: Inspect visually vaccine; there is no abnormalities such as improper sealing, lack of integrity, particles... Package Comply with the GMP requirements and current regulations. 1083

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Storage, expiry date Store in an airtight container, at 2 °C - 8 °C. The expiry date base on the stability of the vaccine and approved by NRA. Labelling The information on the label, box, and leaflet must comply with the current regulations and indicates the information of name, content of each polysaccharide and the total content of polysaccharide. INACTIVATED POLIOMYELITIS VACCINE (IPV) Vaccinum poliomyelitidis inactivatum Inactivated poliomyelitis vaccine is a sterile suspension of suitable strains of human poliovirus types 1, 2 and 3 grown in suitable cell cultures and concentrated, purified and inactivated by a validated method. The vaccine is used to induce a specific and active immunity in man to prevent poliomyelitis caused by polioviruses.

Production Working seed virus The production of vaccine is based on a seed-lot system. The working seed virus, wild or attenuated, should not have undergone more than 2 passages from the master seed-lot by a method that has been approved by the NICVB. Working seed virus is stored at a temperature below -60 °C. Working cells Working cells used for vaccine production and derived from primary monkey kidney cells comply with the WHO requirements. The animal serum may be used for propagation of the cells. Monkeys for vaccine production Monkeys used for vaccine production must comply with the requirements of WHO and NICVB. Production process and quality control The vaccine production process complies with the processes of WHO. The working seed virus is propagated on primary monkey kidney cells or Vero cells and grown in a suitable medium. After harvested, the virus suspension is triply frozen and thawed and then centrifuged to remove cellular debris, filtered through a bacteria-retentive filter, then introduced into the processes of concentration and purification of antigen; the following step is virus inactivation by formaldehyde and formaldehyde neutralization. Finally, the virus suspension is processed to produce inactivated vaccine, tripe vaccine. Quality control tests should be carried out during manufacturing process at different stages: source materials (cells, culture media…), single harvest, final bulk after ultra-centrifugation and before inactivation and after inactivation…, in order to check extraneous agents, to determine successively the vaccine titer before inactivation, the content of D-antigen, the virus purity and the residual live polio virus after inactivation. 1084

Only a final product that complies with the requirements stated under Vaccine for human use, production and with the following requirements may be released for use.

Quality control of final product Visual examination The inactivated poliomyelitis vaccine is a clear liquid. Identification Carry out the test on at least 1 vital. The test for Potency such as the determination of D-antigen is accepted as Identification test. Sterility The vaccine complies with the test for sterility (Appendix 15.7). General safety The vaccine complies with the general safety test on guinea pigs and mice. The experimental animals remain healthy and normally gain body mass after 7 days of observation (Appendix 15.11). Potency Determine the concentration of D-antigen For the inactivated poliomyelitis vaccine from wild strains: 40 D-antigen units for type 1; 8 D-antigen units for type 3. For the inactivated poliomyelitis vaccine from Sabin virus without adjuvants: 30 D-antigen units for type 1, 100 D-antigen units for type 2, 100 D-antigen units for type 3. for the inactivated poliomyelitis vaccine from Sabin strains with aluminm: 3 D-antigen units for type 1; 10 D-antigen units for type 2, 10 D-antigen units for type 3. Total protein 50 μg of protein per human dose (Appendix 15.34). Bacterial endotoxins Not more than 0.25 EU/ml. Free formaldehyde Not more than 0.12 mg per ml (Appendix 15.25). pH 6.8 - 7.5 (Appendix 15.33). Storage The vaccine is stored at 2 - 8 °C, protected from light and must not be allowed to freeze.

Labelling The information on the label, box and leaflet must comply with the current regulations. Expiry date When stored under the prescribed conditions, the vaccine has expiry date of 12 months from the date of the last potency testing. Dosage, route of administration Use 0.5 ml per dose by subcutaneous injection.

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POLIOMYELITIS VACCINE, LIVE (ORAL) Vaccinum Poliomyelitidis perorale Oral Poliomyelitis Vaccine is a preparation of approved strains of live attenuated poliovirus (Sabin strains) types 1, 2 and 3 grown in in-vitro cultures of suitable cells, containing anyone of the three types of Sabin strains induce specific immune active in man to prevent poliomyelitis caused by Polioviruses. Oral Poliomyelitis Vaccine could contain one, two or all three types of virus. The vaccine is a clear, pink liquid.

Production Seed virus The production of vaccine is based on a virus seed-lot system. The virus for production shall not have undergone more than 2 passages from the master seed lot. Unless otherwise justified and authorized, the attenuated working seed virus is validated by the National Control Authority. The working seed virus is stored at a temperature below -60 °C. Monkey and working cells Monkey and working cells used for vaccine production should comply with WHO requirements. Approved animal serum may be used in the media for propagation of the cells, but the medium for cell maintaining during virus multiplication should not contain protein. Phenol red and antibiotics may be used in cell culture medium provided that they are of acceptable nature and concentration. Production process and quality control Vaccine production process should comply with WHO requirements. Quality control tests should be carried out during manufacturing process at different stages: source materials (monkey, cells, culture media...); single harvest, monovalent pooled harvest, final bulk, final lot..., in order to determine bacterial and fungal contamination; mycoplasmas; extraneous viruses and genetic consistency of polioviruses (Rct/40, D-marker; neurovirulence...). Only a final product that complies with the requirements stated under Vaccine for human use, Production and the following requirements may be released for use.

Quality control of final product Visual examination Each final vaccine lot after manufacturing is visually examined to ensure the vaccine in the final closed containers before release and complies with the requirements, showing no abnormal signs of visualization such as extraneous agents in the vaccine containers or tightness of closures and ensuring their integrity. In the process of testing, any vaccine containers that do not comply with the requirement are discarded. Identification When neutralized with type-specific antiserum, virus can not cause cytopathic effects on sensitive cells.

VACCINES

Sterility It complies with the test for sterility. Absence of bacteria, fungi (Appendix 15.7). Potency Determine the titer by infecting sensitive cells with virus. Estimate criteria: The estimated virus titer per single human dose (0.1 ml) must be not less than:106.0 infectious virus units (CCID50) for type 1,105.0 infectious virus units (CCID50) for type 2,105.5 infectious virus units (CCID50) for type 3.Carry out the method as described in Appendix 15.21. Thermal stability The titer loss of exposed vaccine vial (incubated at 37 °C for 48 hours) is not greater than 0.5 log10 infectious units per human dose in comparison with the unexposed vaccine. Physical characteristics and chemical contents The vaccine complies with WHO requirements.

Storage, expiry The vaccine should be kept continuously at -20 °C. After defrosting, the vaccine should be kept at 2 - 8 °C and use within 6 months. Keep out of light and avoid repeated defrosting. The expiry date is not more than 2 years when stored continuously at -20 °C. Labelling The information on the label, box and leaflet must comply with the current regulations. RABIES VACCINE FOR HUMAN USE Vaccinum Rabiei ex cellulis ad usum humanum Rabies vaccine for human use prepared in cell culture is a freeze-dried preparation of a suitable strain of fixed rabies virus grown in cell cultures and inactivated by a validated method. The vaccine is reconstituted immediately before use as stated on the label to give a clear liquid that may be colored owing to the presence of a pH indicator. Rabies vaccine prepared in cell culture must comply with the general requirements of vaccine for human use.

Production The production of the vaccine is based on a virus seedlot system and, if a cell line is used for virus propagation, a cell-bank system. Production method shall have been shown to yield consistently vaccines that comply with the requirements for immunogenicity, safety and stability. Substrate for virus propagation The virus is propagated in a human diploid cell, chick-embryo cell, Vero cell or Chinese Hamster Ovary (CHO) cell. Seed lots The strain of rabies virus used shall be identified by historical records that include information on the origin of the strain and its subsequent manipulation. 1085

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Working seed lot are prepared by not more than 5 passages from the master seed lot and only a working seed lot that complies with the following tests may be used for vaccine production. Identification: Each working seed lot is identified as rabies virus using specific antibodies. Virus concentration: The virus concentration of each working seed lot is determined by an injection into brain of mice or cell culture method using immunofluorescence, to ensure consistency of production. Sterility: Each working seed lot complies with the test for sterility (Appendix 15.7).

Virus propagation and harvest All processing of the cell bank and subsequent cell cultures is done under aseptic conditions in an area where no other cells are handled. Approved animal (but not human) serum may be used in the media, but the final medium for maintaining cell growth during virus multiplication does not contain animal serum; the media may contain human albumin. Serum and trypsin used in the preparation of cell suspensions and media are shown to be free from extraneous agents. The cell culture media may contain a pH indicator such as phenol red and approved antibiotics at the lowest effective concentration. About 10% of the cell cultures employed for vaccine production is set aside as uninfected cell cultures (control cells). The virus suspension is harvested on one or more occasions during incubation. Multiple harvests from the same production cell culture may be pooled and considered as a single harvest. Only a single harvest that complies with the following requirements may be used in the preparation of the inactivated viral harvest. Identification: Each working seed lot contains virus that is identified as rabies virus using specific antibodies. Virus concentration: The titre of infective virus in cell cultures is determined by some method: Inoculation in mice brain, cell culture method using immunofluorescence, or ELISA. The titre is used to monitor consistency of production. Control cells: The control cells of the production cell culture comply with a test for identification and without presence of extraneous agents. Purification and inactivation The virus harvest may be concentrated and/or purified by a suitable methods; the virus harvest is inactivated by a validated method at a fixed, well-defined stage of the production process. The inactivation method shall have been shown to be capable of inactivating rabies virus without destruction of the immunogenic activity. If betapropiolactone is used, the concentration shall at no time exceed 1 : 3500. Only an inactivated viral suspension that complies with the following requirements may be used in the preparation of the final bulk vaccine. 1086

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Specific safety Examine for the presence of infectious rabies virus in the sample after inactivation by specific safety test in mice. The test is passed if the mice should be healthy and increase their weight after 14 days of observation. Method: Animals: At least 10 mice of 4 weeks old, each weighing 11 g to 13 g. The mice must be healthy, without any sign of illness and increase their weight during at least 5 days of quarantine period preceding the test. Procedure: Inject intracerebrally 0.03 ml undiluted sample into each mouse. Observation and interpretation of results: all tested animals must be observed and assessed daily their weight before and after injection. Observe the animals for 14 days. During observation time, the animals must be healthy, increase their weight and none of animals show abnormal signs.

Final bulk vaccine The final bulk vaccine is prepared from one or more inactivated viral suspension. An approved stabilizer may be added to maintain the activity of the product during and after freeze-drying. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Glycoprotein content Determine the glycoprotein content by a suitable immunochemical method, for example, single-radial immunodiffusion, enzyme-linked immunosorbent assay or an antibody-binding test. The content is within the limits approved for the particular product of each manufacturer. Sterility The final bulk vaccine complies with the test for sterility (Appendix 15.7).

Final lot The final lot is distributed aseptically into sterile containers and free-dried to a moisture content shown to be favorable to the stability of the vaccine. The containers are then closed so as to avoid contamination and the introduction of moisture. Only a final lot that complies with following requirements may be used for release. Identification The vaccine is shown to contain rabies virus antigen by a suitable immunochemical method using specific antibodies, preferably monoclonal. Specific safety Determine presence of infectious rabies virus in samples after inactivation by a specific safety test in mice. In the case if the inactivated virus suspension passes the test, it may no need to carry out the test in final lot.

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Sterility It complies with the test for sterility (Appendix 15.7). Pyrogens The vaccine must comply with the test for pyrogens. The test is performed by injecting into each rabbit a single human dose of the vaccine diluted to 10 times its volume (Appendix 15.12). General safety The vaccine complies with the test for general safety on guinea pigs and mice. The experimental animals remain healthy and normally gain body mass after 7 days of observation (Appendix 15.11). Residual moisture Appendix 15.35. Potency It is required for all batches of the vaccine final lot. Use N.I.H method (Appendix 15.31). The test is valid if: ED50 of both the reference preparation and vaccine to be examined lies between the largest and smallest doses. The statistical analysis shows no significant deviations from linearity and parallelism. The confidence limits of the potency are not less than 25% and not more than 400% of the estimated potency. Acceptance criteria: LD50 of challenge virus strain (CVS) must be within 10 - 100 LD50/0.03ml. The potency of vaccine to be tested is not less than 2.5 IU per human dose. Preparation of the challenge virus strain (CVS) Preparation: Inoculate mice intracerebrally with CVS, each mouse weighing 11 - 13 g. When the mice show signs of rabies, but before they die, euthanize them, and then collect the brains. The brains are ground and diluted with a suitable diluent to obtain a final concentration of 20%. Distribute the suspension in small volumes in ampoules, seal and store at a temperature below -60 °C. Titration: Thaw one ampoule of the CVS suspension and make serial dilutions in a suitable diluent. Allocate each dilution to a group of 10 mice and inject intracerebrally into each mouse 0.03 ml of the dilution allocated to its group. Observe the mice for 14 days. Calculate the LD50 of the CVS following Reed - Muench formulation using the number in each group that, only after 5th days, died or developed signs of rabies. Absorbent Where applicable, determine the amount of the absorbent by an approved method. Acceptance criteria: Comply with the current regulations.

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Preservative Where applicable, determine the amount of preservative by an approved method. Acceptance criteria: Comply with the current regulations.

Packaging, storage Packaging: Includes one vial of freeze-dried vaccine and one vial of diluent. Pack the freeze-dried vaccine in the neutral glass vials Store the Rabies vaccine at 2 - 8 °C. Labeling The information on the label, box and leaflet must comply with the current regulations. Indication, posology Take all the diluent and pour into freeze-dried vaccine vials, carefully shake until the vaccine completely thaws. The vaccine should be reconstituted immediately before use. Immediately discard syringes and needles after use. Inject intramuscularly into delta area, preferably thigh muscle in children. Do not inject into loose subcutaneous areas of the body. The vaccination schedule depends on each subject. There are 03 main groups: Prophylactic dose for high risk subjects (expose with rabies virus): Inject 03 main doses on days: 0, 7th, 28th. Booster dose: after 1 year. Inject a booster dose every 5 years. Vaccination schedule on day 28th can be replaced by vaccination on day 21st. Inject into people who are in doubt or surely be infected with rabies virus (to be bitten by dog): * If the people were bitten by dog do not have immunity against to rabies virus: Dosage of 0.5 ml, same between adult and children. Inject 5 doses: on days 0, 3rd, 7th, 14th, 28th of the month. In case the subject has many bites or scratches, saliva..., it is necessary to use immunoglobulin immediately on day 0 with dosage of: Human rabies immunoglobulin: 20 IU/1 kg. Human horse immunoglobulin: 40 IU/1 kg. Vaccine should be injected into the site which opposite to immunoglobulin vaccinated site. In severe cases, for example a large bite or a bite near to center neural system should be vaccinated by 2 doses in the same day 0 depending on each specific case. * If the people were bitten by dog have immunity against to rabies virus: In case vaccination within 5 years: continue to inject 02 doses on days 0 and 3rd. In case vaccination over 5 years but not enough doses following schedule: should apply with schedule which is used for subjects has not immunity against to rabies virus. 1087

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VACCINES

ROTAVIRUS VACCINE (LIVE ATTENUATED, ORAL) Vaccinum Rotaviri vivum perorale Rotavirus vaccine - live attenuated (oral) contains alive attenuated rotavirus is a preparation of liquid or freeze-dried type. The freeze-dried preparation after reconstitution with the diluent gives a colored liquid owing to the presence of a pH indicator.

Production Working seed lot The production of vaccine is based on a virus seed lot system. The number of passage from a master seed lot to virus lot used to prepare the final vaccine is less than the number of passage from a master seed lot to virus lot used to prepare the clinical trial vaccine if there is no other regulation and approved by National Control Laboratory (NCL). The seed lot is stored at below -60 °C if not freezedried and below -20 °C if freeze-dried. Working cell bank A cell bank prepared vaccine production complies with the WHO requirements. Animal serum can be used in cell culture medium but there is no protein in the cell maintaining medium for virus propagation. The medium may contain red-phenol and appropriate antibiotics with appropriate content. Production process and control The production process must comply with the WHO requirements. Need to perform tests during process at the stages: staring materials (cell, culture medium...); single harvest, bulk before centrifugation and before filtration; bulk after filtration; final vaccine. The vaccine has to comply with the requirements of human vaccine and release if satisfactory the production and control requirements and below requirements. Virus propagation and single harvest Cell and culture are performed in the sterile condition. There are no extraneous agents in serum and trypsin. Animal serum is used in cell propagation and maintenance media; for the virus propagation, there is no serum in the culture media. The medium may contain red-phenol and appropriate antibiotics with the appropriate content. The optimum culture medium does not contain antibiotic. Store the intermediate virus during the culture at below -60 °C. Infect virus, control identity, sterility, and virus concentration. The virus suspensions are harvested. The single harvest that complies with the requirements may be used for purity process. Need to control identity, sterility, virus concentration and extraneous agents. Monovalent harvest purity and control The purified monovalent harvest is prepared from a single harvest or a pooled monovalent harvest. The single harvest is filtered to remove cell debris. Only a purified monovalent 1088

harvest that complies with the requirements may be used in the preparation of the vaccine bulk. Sterility: It complies with the test for sterility (Appendix 15.7). Virus concentration: determine by cell-culture method and PCR method. Residual DNA: maximum 100 µg DNA per single human dose (for viruses grown in continuous cells line).

Quality control of final bulk vaccine The final bulk vaccine is prepared from one or more purified monovalent harvests, containing more than one virus type. Suitable stabilizer may be added. Sterility: It complies with the test for sterility (Appendix 15.7). Quality control of final lot Inspection Each final lot is tested inspection for minimum package unit to ensure that no abnormal particles; integrity of the cover and cap are. During testing, remove the vial not to comply with the specification. Identification The vaccine is shown to contain rotavirus stated on the label, for multivalent rotavirus vaccine, use the suitable method for each type identity. The method such as plaque neutralization and immunofocus assays are suitable to identify the presence of rotavirus using rotavirus- specific polyclonal antiserum. PCR may also be appropriate. Sterility: It complies with the test for sterility (Appendix 15.7). pH The pH of the final lot is tested in a pool of final containers to get an appropriate volume limit for pH measurement. In case of freeze-dried vaccine, pH is measured after reconstitution of vaccine with the diluent. pH is in the range of registered specification. Residual moisture The residual moisture of the freeze-dried vaccine is not greater than 3% (Appendix 15.35). Potency (virus concentration) The virus concentration is determined individually for at least 3 final containers. An immunofocus or plaque assay may be used in MA-104, Vero or other sensitive cells to determine virus concentration. Result is recorded as FFU/dose or PFU/ml. A cell culture infectious dose assay may also be used to determine virus concentration. Result is recorded as CCID50/dose. Freeze-dried vaccine is reconstituted with the diluent to determine virus concentration. Titre limit of vaccine dose is established by the manufacturer after being shown to be safe and effective in human clinical trials. Specification for virus concentration is essentially specify the minimum titre guaranteed to be

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contained in one human dose and approved by National Regulatory Authority (NRA). The virus concentration is equivalent to and greater than the labeled concentration. Recommendation: For freeze-dried vaccine, the virus concentration is at least 106.0CCID50/dose. PFU, FFU unit can be used. Stability test Determine virus titre for using does when storing vaccine at regulated temperature and elevated temperature. The maximum allowable loss of titre during the accelerated stability test is confirmed on the basis of the manufacturer’s experience and approved by NRA. For a multivalent vaccine, if there is no significant difference in the virus loss between serotypes, the loss is based upon total virus concentration. Recommendation: For freeze-dried vaccine, the titre of vaccine stored at 37 °C for 7 days is not less than 0.5logCCID50 compared to the titre of vaccine stored at 2 - 8 °C and also the titre of vaccine is at least 106.0CCID50/dose. PFU, FFU unit can be used.

Storage Store at 2 - 8 °C. Expiry date Approved by National Regulatory Authority. Labeling The information of label, box, and leaflet must comply with the current regulations. Diluent The diluent complies with the GMP requirements and approved by NRA. RUBELLA VACCINE Vaccinum Rubellae vivum Rubella vaccine (live) is a freeze dried preparation of a suitable attenuated strain of mumps virus. The vaccine is reconstituted immediately before use, as stated on the label, to give a clear liquid that may be colored owing to the presence of a pH indicator. Vaccine must comply with the following requirements:

Production Master seed lot The production of vaccine bases on a virus seed-lot system and a cell-bank, and approved by national control authority. The strain of Rubella virus used in the production of mumps vaccine shall be identify by historical records that include information on the origin of the train, its method of attenuation and passage level at which attenuation was demonstrated by clinical evaluation. Store master seed lot at temperatures below -20 °C if freeze-dried, or bellow

VACCINES

-60 °C if not freeze-dried and control the temperatures during storage. Only seed lot that complies with following requirements may be used for virus propagation. Identification The master and working seed lots are identified as rubella virus by serum neutralization in cell culture, using specific antibodies or RT-PCR method. Virus concentration The virus concentration of the master and working seed lot is determined to ensure consistency of production Extraneous agents The working seed lot complies with the requirement for seed lots, without the extraneous. Neurovirulence The working seed lot complies with the test for neurovirulence of live virus vaccine. Macaca and Cercopithecus monkeys are suitable for the test. Cells used for vaccine virus Cell used for vaccine which is cell bank or origin of cell is embryonated eggs and which shall be tested to pathogens by national control authority. Serum used in cell-culture medium Serum used for the propagation of cell for mumps vaccine production shall be tested to demonstrate freedom from bacteria, fungi and Mycoplasma as freedom virus. Human serum shall not be use. Production control Strains of virus grow on the embryonated eggs or cell bank systems; use a suitable medium for production shall be used. Harvest of virus is mixed and added preservatives, filtrated, diluted, sealed and lyophilized by an appropriate laboratory test. For those test that require prior neutralization of mumps virus, the antiserum used shall be not human, simian, or avian origin. The immunizing antigen used for the preparation of the antiserum shall be produced in cell cultures from a species different to that used for the production of vaccine. Such cell cultures shall be free from extraneous microbial agent that might elicit antibodies inhibitory to growth of extraneous agents present in the rubella virus pool. The cell used for vaccine shall be tested extraneous agents, sterility...... All production as cell bank, culture cell in the vaccine production shall be done in the sterility environment which does not have another cell. Serum and trypsin used for cell dilution and medium shall not be freedom extraneous agents. The cell culture medium may contain a pH indicator such phenol red and suitable antibiotics at the lowest effective concentration.

Single harvests Only a single harvest that complies with the following requirements may be used in the preparation of the final bulk vaccine.

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Identification The single harvests are identified as rubella virus by serum neutralization in cell culture, using specific antibodies or RT-PCR method. Virus concentration The virus concentration of each single harvest or pool of harvest must be to ensure consistency of production and to determine the dilution to be used for the final bulk vaccine. Extraneous agents The single harvest complies with the tests for extraneous agent (freedom extraneous agent). Control cells and eggs If using human diploid cells in production vaccine, they shall be test for identification, the control cell and eggs shall be tested freedom extraneous agents. Rubella vaccine live, attenuated add suitable stabilizer then lyophilized, it is single vaccine or combine vaccine.

Quality control of final bulk Single harvests that comply with the above tests are pooled and clarified to remove cells. A suitable stabilizer may be added and the pooled harvests diluted as appropriate. Only a final bulk vaccine that complies with the following requirement may be used in the preparation of the final lot. Sterility The final bulk complies with the test for sterility (Appendix 15.7).

Quality control of final vaccine Identification Use suitable methods approved by the national control authority. Use specific antiserum Use specific antibody to neutralize rubella vaccine. Use Rubella virus- antibody suspension to infect on RK13 cell. The test has the negative and positive. Read the results after two weeks. Acceptance criterion: Vaccine is passed if the RK-13 cell is not infective as the same negative. Procedure: Day 1: The cell is culture on the 6-well plate or 24-well plate. Amount of the cell is used about 1×105 - 1.5×105 cell/ml. Day 2: Dilute antiserum at different level dilution Reconstitute the vaccine Mix antibody with antigen; the volume is ratio 1:1 (neutralization reaction) Incubate neutralized suspension (including antigen and antibody) and unneutralized suspension (the vaccine at concentration of 10-1, 10-2, 10-3, 10-4, 10-5 and antibody at suitable dilutions) in an appropriate thermal cycle. 1090

VP V Note: Thermal cycle as following: in the water-bath 37 °C for 30 min, 23 °C for 30 min, 4 °C for more than 18h (≥ 18 h) may be used.

Day 3: Solution has antigen combined antibody shall be infected on the RK-13 cell. Vaccine shall be infected the Vero cell. Methods have negative and positive. Incubate in the incubator at 32 °C, containing 5% of CO2. Observe daily, read results from day 4 to day 7. The test is valid if: The cell is normal growing. Concentration dilution of vaccine have many CPE. RT-PCR method Use specific primer for rubella virus. Primer has information. The tests have negative and positive. Acceptance criterion: Rubella vaccine is passed if the production of RT-PCR is the same information of primer. Disclaimer: The primer may be used as following: Forward primer 5’-CAA CAC GCC GCA CGG ACA AC-3’. Reverse primer 5’-CCA CAA GCC GCG AGC AGT CA-3’. Size: 185 bp. Thermal cycle/time 50 °C/30 min; 95 °C/time (following the guidance of RT-PCR kits) 94 °C/1 min, 60 °C/1 min, 35 cycles; 72 °C/1 min 72 °C/10 min; 4 °C/ until electrophoresis.

Potency Principle: Titer of rubella vaccine is determined by cell culture infectious doses on the RK-13 cell (CCID50) Materials: 03 vials of freeze-dried rubella vaccine and the diluents supplied with the vaccine. Reference rubella vaccine: 1 vial Two of 75 cm2 flasks of RK-13 cell growing well on a monolayer. Chemicals: PBS (Phosphate buffered Saline) Trysin containing 0.25% EDTA. MEM containing 2% FBS Method: Cary out the test in sterile BSC class II, in sterile area. Three containers from the final freeze-dried vaccine lot shall tested titer. The geometric mean infectious virus titre of three vials. Dilute the sample and standard from 10-5 to 10-1

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Table 1 - Example for dilution Dilution

Volume of diluent added (ml)

Volume of vaccine dilution (ml)

10-1.0(A)

2.7

0.3

10

(B)

2.16

1.0 (from A)

10

(C)

2.16

1.0 (from B)

(D)

2.16

1.0 (from C)

(E)

2.16

1.0 (from D)

10

(F)

2.16

1.0 (from E)

10

(G)

2.16

1.0 (from F)

10

(H)

2.16

1.0 (from G)

-1.5 -2.0

10

-2.5

10

-3.0

-3.5

-4.0 -4.5

10 ( K) 2.16 1.0 (from H) Add the media as described in the above table into glass penicilin. Reconstitute vaccines, shake well; to 0.3 ml, add to the first dilution level of 10-1.0, mix well; to 1 ml, add to the dilution level of 10-1.5, to continue the process till the last dilution level. Remember to change tip after each dilution level. After diluting, add each dilution in 10 wells in a row, each well of 100 µl. Start from the lowest dilution to the highest dilution, if the process is reversed, the tip should be changed after each dilution level. Add the media into the last 2 rows, each well of 100 µl. Repeat the above process with the remaining vials, and standard vaccine. After diluting, put the plates in the fridge (2 °C - 8 °C). Prepare the cell suspension - Cell concentration is 1×105 to 2×105 cells per ml. - Add 100 µl of the Vero cell supension to all wells of all plates. - Incubate all plates at 32 °C for 12 days. - Read the results from 10th day to 12th day. Read and calculate the result: Firstly, observe the positive control wells by inverse microscope to distinguish easily cells that are damaged by viral infection from normal RK-13 cells. Signs of a damaged cell consist of creating giant multi-nucleus cells, plaque; a well having the signs is considered as a damaged well. Calculate the viral titer by Karber or Reed - Muench equation: Log CCID50 = L – d(S – 0.5) Where: L: Log of the lowest dilution in the test; D: Log of dilution coefficient; S: Total % of infected wells. The test is valid if: Negative control wells: Cells growth normally, there is no growth of bacteria or fungi; Potency of reference vaccine within 0.5 Log10 of the established value; Potency variation of 3 sample vials is not more than 0.5 Log10; -5.0

The cytopathic effect decreases consecutively with increasing dilution; Used dilutions have cytopathic effect rate of 0% to 100%. Acceptance criteria: The rubella viral titer of vaccine is not less than the titer stated on the vial label. The lowest titer stated on the table does not less than Lg 103.0 CCID50 per human dose. Thermal stability Determine the viral titer in parallel for vaccine incubated at 370C for 7 days and vaccine stored at (5±3)0C. Acceptance criteria: The viral titer of the heated vaccine is not more than 1 Lg CCID50 lower than that of unheated vaccine. The minimum viral titer is not less than Log 103.0 CCID50/0.5 ml (one human dose). General safety (Appendix 15.11). Each final lot must be safe in mice and guinea pigs. Acceptance criterion: Mice and guinea pigs must stay healthy and gain weight after 7 days of observation. Sterility It complies with the test for sterility (Appendix 15.7) Residual moisture Not more than 3.0% (Appendix 15.35). Appearance It complies with the registered regulations of the manufacturer. Mycoplasma Absence of Mycoplasma ((Appendix 15.36). Note: May be used direct culture method or PCR to identify Mycoplasma on the rubella vaccine. Albumin Not more than 50 ng/dose of albumin per single human dose. Determined by a suitable immunochemical method.

Storage Store at 2 °C to 8 °C and protected from light. Labelling The information of label, container, and leaflet must comply with the current regulations. Normal containing: The strains of virus used in the preparation The type and origin of the cells used for the preparation The minimum virus concentration for each component Residual antibiotic, preservatives: name and content (if any) That contact with disinfectants is to be avoided. Note: a statement concerning the photosensitivity of the vaccine, cautioning that both lyophilized and reconstituted vaccine should be protected from light. The vaccine should not to be administered to pregnant women and women intend to be pregnant within two months

Dosage and administration Subcutaneous injection, dose of 0.5 ml.

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ADSORBED TETANUS VACCINE Vaccinum tetani adsorbatum Adsorbed Tetanus Vaccine is prepared from tetanus toxin which is treated by formaldehyde and heat to render it non-toxic without destroying its immunogenic potency. The toxoid contains not less than 1,000 Lf per milligram of protein nitrogen and is adsorbed onto a suitable adjuvant, either hydrated aluminium phosphate or aluminium hydroxide in an isotonic solution of sodium chloride. A suitable antimicrobial preservative can be added.

Production Strain of Clostridium tetani used in preparing tetanus toxoid is managed in a defined seed-lot system and grown in a suitable liquid medium. At the end of cultivation, the purity of each culture is tested and single contaminated cultures are discarded. The toxin content (Lf per milliliter) is checked by flocculation test. Single harvests may be pooled and detoxified with formaldehyde and heat and then purified by a suitable method. Lf amount in final bulk vaccine is not greater than 25 Lf per human single dose if use more than one dose for primary immunization is required. The amount of preservative should not be harmful to toxoid and should not cause reactions in human. Bulk purified toxoid The strain of Clostridium tetani for production is of high toxicity, known origin and history. The toxin-containing culture medium is separated aseptically from the bacterium mass. The toxin content (Lf per milliliter) is checked by flocculation test to monitor the consistency of production. The toxin is purified to remove components likely to cause adverse reactions in humans. The purified toxin is detoxified by a suitable method that avoids destruction of the immunogenic potency of the toxoid and reversion of the toxoid to toxin. Alternatively, purification may be carried out after detoxification. Only bulk purified toxoid that complies with the following requirements may be used in the preparation of the final bulk vaccine. Sterility Carry out the test for sterility of the bulk purified tetanus toxoid using 10 ml of each medium (Appendix 15.7). Specific toxicity and irreversibility of tetanus toxoid Test of specific toxicity: Inject subcutaneously 1 ml of the purified toxoid containing 500 Lf per ml into each of 5 healthy guinea pigs, each weighing 250 - 350 g, that have not previously been treated with any material that will interfere with the test. Weekly observe and investigate the animal mass, signs of paralysis caused by tetanus within 6 weeks after administration. The animals that die during the observation period are to be examined by autopsy. The purified toxoid complies with the requirement of specific 1092

safety if within 3 weeks of the injection, the experimental guinea pigs show neither signs of tetanus paralysis nor tetanus symptoms and during the test period not less than 80% of guinea pigs survive. Test of irreversibility of tetanus toxoid: The purified tetanus toxoid is tested for the compliance with the absence of reversibility of toxoid. Using the same buffer solution as for the final bulk vaccine, without adsorbent, prepare a suspension of bulk purified toxoid containing 12.5 Lf per ml. Divide the suspension into 2 equal parts. Maintain 1 part at (5 ± 3) °C and the other at 37 °C for 6 weeks. Inject subcutaneously 5 ml of each part into each of 5 healthy guinea pigs, each weighing 250 - 350 g, that has not previously been treated with any material that will interfere with the test. The bulk purified toxoid complies with the requirement if both groups of guinea pigs remain healthy and no animal shows signs of tetanus symptoms within 3 weeks of observation. Antigenic purity The purified tetanus toxoid is tested for antigenic purity by determining the antigenic content in units of Lf per mg of protein nitrogen. Determine the antigenic content by comparing with the reference toxoid calibrated by an international standard of tetanus toxoid for the flocculation test or an equivalent national reference toxoid. The purified tetanus toxoid complies with the requirement if it contains not less than 1,000 Lf per mg of protein nitrogen.

Quality control of final bulk vaccine The final bulk vaccine is prepared by adsorption of a suitable quantity of bulk purified toxoid onto a mineral carrier such as hydrated aluminium phosphate or aluminium hydroxide. Suitable antimicrobial preservatives may be added. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The amount is not less than 85% and not more than 115% of the intended amount (Appendix 15.29). Sterility Carry out the test for sterility of the final bulk vaccine using 10 ml for each medium. The final bulk vaccine is not contaminated by bacteria and fungi (Appendix 15.7).

Quality control of final lot Identification Identity test is performed on at least one container from each final lot by flocculation test as described in Appendix 15.19. Sterility Absence of bacteria and fungi (Appendix 15.7).

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Potency Carry out the test as described in Appendix 15.23. Criteria of acceptance: The potency of the adsorbed single tetanus vaccine complies with the requirement if it contains not less than 40 IU per human single dose. For the tests using 3 dilutions, limits of the 95% confidence intervals of the estimate are within 50 - 200%. General safety Carry out the test as described in Appendix 15.11. Antimicrobial preservative The content of thimerosal is not less than 85% and not greater than 115% of the amount stated on the label (Appendix 15.29). Adjuvant content Adjuvant content is not greater than 1.25 mg of aluminium per human single dose (Appendix 15.27). pH 6.0 - 7.0 (Appendix 15.33). Visual examination Each final vaccine lot after manufacturing is visually examined to ensure the vaccine in the final closed containers before release for use comply with the requirements, showing no abnormal signs of visualization such as extraneous agents in the vaccine containers or tightness of closures and ensure their integrity. In the process of testing, any vaccine containers that do not comply with the requirement are discarded.

Storage and expiry date Adsorbed tetanus vaccine is stored at 2 - 8 °C. If kept under correct storage conditions, the vaccine can maintain its potency for 3 years from the date of potency testing. Labelling The information on the label, box and leaflet must comply with the current regulations. TYPHOID VACCINE (ORAL) Vaccinum febris typhoidi perorale vivum

Definition Typhoid vaccine (live, oral, and attenuated) is a freezedried preparation of Salmonella typhi strain Ty 21a grown in a suitable medium. The vaccine is presented in gelatinous capsules with the content of 1×109 - 5×109 live S. typhi strain Ty 21a per capsule. Production Working seed lots The mutational strain of S. typhi Ty 21a has been shown satisfactory with respect to safety and efficacy on humans. The main characteristic of the strain is the defect of uridine diphosphate-galactose-4-epimerase enzyme. The activities

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of galactopermease, galactokinase and galactose-1phosphate uridyl-transferase are reduced by 50% to 90%. Whatever the growth conditions, the strain does not contain Vi antigen. The strain agglutinates to anti-O:9 antiserum only if grown in medium containing galactose. It contains the flagellar H:d antigen and does not produce hydrogen sulphide on Kligler iron agar. The strain is non-virulent for mice. Cells of strain Ty 21a lyse if grown in the presence of 1% of galactose. The vaccine is prepared using a seed-lot system. Production of master seed lots: From a single colony, the culture is performed on a suitable medium (BHI medium is suitable), that is free from galactose, and incubated at a temperature suitable for optimum development. When the culture attains consistent phase, the bacterial broth is harvested, centrifuged to obtain the bacterial precipitate and distributed into tubes, then prepared into suspension with suitable concentration and freeze-dried in such a way that each tube containing at least 109 units of live strain. The tubes of freeze-dried master seed lot are stored at 5 ± 3 °C. Production of working seed lots: Working seed lot is produced from a master seed lot tube. The steps of cultivation, harvesting, centrifugation to obtain the bacterial precipitate, preparation and freeze-drying are successively processed in the same order as the master seed lot. Each tube of freeze-dried working seed lot contains not less than 109 units of live strain. The tubes of freeze-dried working seed lot are stored at 5 ± 3 °C. Tests of working seed lots Identification and purity Only bacterium Ty 21a is identified as a mobile, gramnegative bacillus. The strain is grown through 3 subcultures in the medium containing or free from galactose, agglutinates to H:d antiserum and does not agglutinate to Vi antiserum. On the contrary, the colonies agglutinate to 0:9 antiserum only if grown in medium containing galactose (1 g per liter). When grown on Endo agar medium and incubated at 37 °C for 7 days, the colony growth has the medium colour, cellular lysis and gradually becomes transparent. The galactose-fermenting colonies shall not occur at any time in the period of 7 days of incubating at 37 °C. When grown in the indicator medium containing galactose incubated at 37 °C for 48 hours, the colonies appear to be concave, surrounded with greyish-blue zones and dark coloured in the middle until they die and lyse in the same manner as strain Ty 21a on the agar medium containing galactose. When grown on Kligler iron agar medium, the colonies that are not blackened prove to produce no hydrogen sulphide. Produce lysis to the bacterium in the medium containing galactose: By shaken culture in BHI medium (with the presence of 1% of galactose) at 37 °C, the bacterium lyses within 1 hour. 1093

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Test of toxicity in mice The strain grown in BHI medium at 37 °C for 6 hours is used to inject intraperitoneally into each of 5 healthy mice, each weighing 18 - 22 g. The bacterial suspension containing at least 5 × 107 bacteria shall not kill the experimental mice during 7 days of observation. Determination of enzymes (including those contained in the process of galactose fermenting) Experimentation shows that the enzyme activity of strain Ty 21a is weaker than that of strain Ty 2. In comparison with the enzyme activity of strain Ty 2 (considered as 100 per cent), the enzyme activity of strain Ty 21a with respect to epimerase, galactokinase, galactose-1-phosphate uridyl-transferase and galactose-permease is 0%, 5 - 20%, 25 - 50% and 40 - 50%, respectively. Adsorption and intracellular distribution of galactose 14C After 7 hours of incubating at 30 °C, not less than 90% of galactose in the medium (1 g per liter) is reduced due to the bacterial strain (determined by measuring 14C on the cells). The testing method also shows that about 75% of galactose settles down on the bacterial cell walls. Characteristic of lipopolysaccharide (LPS) Lipopolyaccharide extracted from cell walls of S. typhi strain Ty 21a, grown in BHI medium containing galactose (1 g per liter) at 30oC for 7 hours shall be hydrolyzed in a 1% solution of acetic acid and tested for polysaccharide. Examine by size-exclusion chromatography using Sephadex G50; S. typhi bacteria of both colonies, smooth and rough, contain LPS in the ratio corresponding to the toxic strain of S. typhi Ty 2. Moreover, the distribution of keto-deoxy-octonate (KDO), galactose, glucose and rhamnose of LPS in strain Ty 2 and strain Ty 21a is corresponding with respect to proportion. Requirements for production process Culture media for working seed lots do not cause toxic and allergic reactions in man. Working seed lots are grown and incubated at a suitable temperature for a period of time sufficient for an early consistent phase and harvested. The number of subcultures from working seed lot to final fermenting shall not exceed four. Before centrifugation of fermenting products: Taking samples for identification of strain Ty 21a, testing viability on BHI agar medium and incubating at 37 °C for 36 hours. After centrifugation, preparing the biomasses of single harvests into consistent suspension (or letting single harvests be) and maintaining in freezing condition until freeze-dried.

Quality control of final bulk Identification The samples in powder form shall be reconstituted and tested for identification as prescribed under “Identification and purity”.

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Viability Determine the viability by the mass of the micro-organisms. The ratio of the count of viable units after freeze-drying to that before is not less than 10%. Criteria of viability: Not less than 40 × 109 viable units of S. typhi 21 a per gram. Residual moisture (Appendix 15.35). Final bulk shall be tested for residual moisture and the result should not exceed the limits prescribed by the National Control Authority. The test for residual moisture may be performed either by Karl Fischer method or semi-micro method. When determined by Karl Fischer method (Appendix 10.3) the residual moisture in final bulk should be less than 3%. When determined by semi-micro method the residual moisture in final bulk should range from 1.5% to 4.0%.

Quality control of final product Take a quantity from the final bulk that has been completely homogenized and determined the number of viable units, if it contains not less than 2-5 × 109 viable units it shall be filled in 1 gelatinous capsule. The filling process should ensure that the content of each capsule is a human dose and must be not less than 2 × 109 viable units. The sample for quality control of final product shall be taken from capsules of each final lot and used to perform the following tests. Identification Carry out the test for identification with 3 capsules for each sample by the method prescribed under “Identification and purity”. Viability Carry out the test for viability using 5 capsules for each sample. The viability obtained from the sample is considered as the potency of the attenuated, live, oral typhoid vaccine Ty 21a. The test for viability of each final vaccine lot shall be performed in parallel on 2 samples and the result is the average value of viability of the 2 samples. Procedure of testing viability for each sample: Place the powder contents of 5 capsules of each sample in each flask that has contained a few sterile glass beads; add 20 ml of sterile physiological saline solution. Shake the above sample-containing flask, using an oscillating-agitator at the speed of 200 rpm and keeping it in a cold room at 4 °C for 30 minutes. From the obtained suspension, prepare serial dilutions in physiological saline solution and set appropriate dilution level depending on the number of viable units contained in each vaccine capsule. Apply 0.1 ml of the diluted vaccine suspension (of appropriate dilution) onto each of at least 5 BHI agar dishes and incubate at 35 - 37 °C for 30 - 36 hours or more. Count the number of colonies on the medium dishes and calculate the average number of colonies of each sample. From this

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result, calculate the average number of colonies for the 2 samples tested in parallel. For example, the vaccine suspension after being oscillatingshaken in cold room as described above shall be prepared in serial tenfold dilutions (to 10-6) in sterile physiological saline solution. Inoculate 0.1 ml of the vaccine suspension diluted to 10-6 onto each of at least 5 BHI agar dishes. Calculate the average viability of the stock solution using the following expression: X = C × 20 × 106 Where: X is the viable unit. C is the average number of colonies of the 2 samples. From the above result, calculate the number of viable units (Y) contained in one capsule of oral typhoid vaccine Ty 21a using the following expression: Y = X/5 Acceptance criteria: The vaccine contains 2 × 109 - 5 × 109 viable units of S. typhi Ty 21a per gelatinous capsule. Contamination Carry out the test using at least 3 vaccine capsules for each sample. Use suitable selective media for checking and finding the presence of such pathogenic bacteria as Salmonella (except for strain Ty 21a), Shigella, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Vibrio parahaemolyticus. Acceptance criteria: The vaccine complies with the requirement if the above pathogenic bacteria are not found in the vaccine. The number of contaminating nonpathogenic live bacteria per dosage unit (1 capsule) is not more than 2 × 102 bacteria and 20 fungi. General safety The test for general safety is carried out on each final lot of oral typhoid vaccine Ty 21a by injecting intraperitoneally 0.01 of human single dose of vaccine into each of 5 mice, each weighing 18 - 22 g; and by administering orally 1 human dose to each of 3 guinea pigs, each weighing 250 - 350 g. Mice are observed within 7 days after the injection, guinea pigs are observed within 14 days after the administration. Acceptance criteria: No experimental animal shows any signs of infection during the period of observation. Stability The vaccine preparation is tested for stability by a method that has been approved by the National Control Authority. Stability of the vaccine is usually estimated by the viability of the vaccine. Acceptance criteria: The viability of the vaccine is not less than 2 × 109 viable units of S. typhi Ty 21a per gelatin capsule during the expiry date that has been submitted by the manufacturer. Visual examination Each final vaccine lot after manufacturing is visually examined. The final vaccine lot is discarded if the vaccine to

be examined shows any abnormal signs that do not comply with the requirement

Packaging, storage and expiry date Packaging and labeling As label cannot be printed on vaccine capsules, the label on blister packs, cartons and leaflets with instructions for use shall provide adequate information on general regulations and particularly state the minimum number of viable units per vaccine capsule and that the capsule is for oral use only. Storage The manufacturer shall give recommended conditions for storage and transport to ensure that the vaccine preparations when stored under suitable conditions shall comply with the specifications until the last date of expiry. The vaccine capsules are usually stored in dry and dark places at 5 ± 3 oC. Expiry date The vaccine may be expected to retain its potency for not less than 18 months from the date of manufacturing. The expiry date may be extended if the data have been shown that the vaccine product has a longer stability. TYPHOID VI POLYSACCHARIDE VACCINE Vaccinum Vi polysaccharide Typhoidi

Definition Typhoid Vi polysaccharide vaccine is a preparation of purified capsular Vi polysaccharide obtained from Salmonella typhi Ty 2 strain or some other suitable strain that has the capacity to produce Vi polysaccharide. Capsular Vi polysaccharide consists of partly 3-O-acetylated repeated units of 2-acetylamino-2-deoxyD-galactopyranuronic acid with ∝- (1→4) linkages. Production Working seed lot The strain of Salmonella typhi used in the preparation of Vi polysaccharide vaccine should be approved by the National Control Authority. The strain should be shown to yield consistently Vi polysaccharide of adequate immunogenicity and safety in man. The production method is validated to demonstrate that the product, if tested, would comply with the test for general safety for immunosera and vaccines for human use. The strain of S. typhi Ty 2 has shown to be suitable for preparing typhoid Vi polysaccharide vaccine. Only a strain that has the following characteristics may be used in the preparation of the vaccine: stained smears from a culture are typical of S. typhi; the culture medium has fermented glucose but without produced gas; colonies on agar are oxidase-negative; a suspension of the culture agglutinates specifically with a suitable Vi antiserum or colonies form haloes on an agar plate containing a specific antiserum. 1095

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Production of master seed lot and working seed lot The strain of S. typhi used for the master seed lot shall be identified by historical records that include information on its origin and by its biochemical and serological characteristics. Cultures from the working seed lot shall have the same characteristics as the strain that was used to prepare the master seed lot. Culture and harvest The growth of S. typhi strain shall have shown to be consistent by monitoring the growing rate of bacteria, culture pH and yield of Vi polysaccharide. The temperature for growing is usually at 35 - 37 °C. The working seed lot is cultured on a solid medium, incubated at 35 - 37 °C for 12 - 18 hours; the inoculum obtained is transferred to a liquid medium which is used to inoculate the final medium. The liquid medium and the final medium are semi-synthetic, free from substances that are precipitated by cetrimonium bromide and do not contain blood group substances or high-molecular-mass polysaccharides, unless it has been demonstrated that they are removed by the purification process. The bacterial purity of the culture is verified by microscopic examination (at a magnification of x1,000) of stained smears and by inoculation into suitable solid media. The culture is then inactivated at the beginning of the stationary phase by the addition of formaldehyde. Bacterial cells are eliminated by centrifugation; the polysaccharide is precipitated from the culture medium by addition of cetrimonium bromide (hexadecyltrimethylammonium bromide). The precipitate is harvested and stored at -20 °C before purification. Purify Vi polysaccharide The polysaccharide is purified by precipitating with cetrimonium bromide complex, the using suitable procedures to eliminate successively nucleic acids, proteins and lipopolysaccharides. The polysaccharide is precipitated as the calcium salt in the presence of ethanol R and dried at 2 - 8 °C; the obtained powder is purified Vi polysaccharide. Residual moisture is determined by thermogravimetry and used to calculate the results of the chemical tests shown below with reference to the dried substance. Only a purified Vi polysaccharide that complies with the following requirements may be used in the preparation of the final bulk. Protein: Not more than 10 mg per gram of polysaccharide. Nucleic acids: Not more than 20 mg per gram of polysaccharide. O-Acetyl groups: Not more than 2 mmol per gram of polysaccharide. Molecular size: Examine by size-exclusion chromatography using cross-linked agarose for chromatography R. Use a column 0.9 m long and 16 mm in internal diameter equilibrated with a solvent having an ionic strength of 0.2 mol per kg and a pH of 7.0 - 7.5. Apply about 5 mg 1096

of polysaccharide in a volume of 1 ml to the column and elute at 20 ml per hour. Collect fractions of about 2.5 ml. Determine the point of corresponding to KD = 0.25 and make 2 pools consisting of fractions eluted before and after this point. Determine O-acetyl groups on the 2 pools. Not less than 50 per cent of the polysaccharide is found in the pool containing fractions eluted before KD = 0.25. Identification: Carry out an identification test using a suitable immunochemical method. Bacterial endotoxins: Not more than 150 EU per microgram of polysaccharide.

Quality control of final bulk vaccine Final bulk vaccine is prepared from one or more batches of purified Vi polysaccharide. If the final bulk vaccine is used to distribute into multi-dose preparations, it needs to add antimicrobial preservatives and to be dissolved in a suitable solvent so that the volume corresponding to 1 single human dose contains 25 μg of polysaccharide. The preservative content in final bulk vaccine should not make any alterations to Vi polysaccharide and other components of the vaccine. The final bulk vaccine is prepared (in aseptic conditions) in a solution free from pyrogens and aseptically filtered through a filter membrane. Only a final bulk vaccine that complies with the following tests may be used in the preparation of the final lot. Sterility The final bulk vaccine complies with the test for sterility (Appendix 15.7). Thimerosal The content of thimerosal in vaccine complies with the requirements prescribed in Appendix 15.29. Content of Vi polysaccharide Determine the content of Vi polysaccharide in final bulk vaccine using a suitable immunochemical titration that has been approved by the National Control Authority (Appendix 15.37). Identification Use the serological test relying on immuno-precipitation.

Quality control of final product The following tests are performed on each final lot. Visual examination The vaccine is a clear, colorless liquid. By usual examination the vaccine shows no abnormal signs such as extraneous particles in the vaccine containers, loose closures, bad welding… Identification Carry out an identification test using a suitable immunochemical method. Determination of Vi polysaccharide content Take at random the samples, at least 3 vials (or ampoules). Determine the content of Vi polysaccharide by a suitable

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immunochemical method, using a reference polysaccharide (Appendix 15.37). Sterility The final product complies with the test for sterility (Appendix 15.7). Pyrogens Carry out the test for pyrogens on each final lot by injecting intravenously into rabbit auricular the typhoid Vi polysaccharide vaccine containing 25 ng of Vi polysaccharide per ml (1 ml per kg of rabbit body mass). General safety Each final lot of typhoid Vi polysaccharide complies with the test for general safety if all the experimental animals survive and do not lose weight during at least 7 days of observation. Preservatives Thimerosal: Determine the content of thimerosal as prescribed in Appendix 15.29. Phenol: If phenol has been used in the preparation of the vaccine, the concentration is not mare than 2.5 g per liter (Appendix 15.28). Residual moisture (applied to freeze-dried vaccine) The applied method should have been approved by the National Control Authority. The residual moisture is usually determined by Karl Fischer method. Samples are taken at random in the ratio of 1 to 1,000 vials, but the number of samples is not more than 10 vials and not less than 5 vials (Appendix 15.35). Acceptance criteria: Mean residual moisture is not more than 2.5%, and there is no vial having residual moisture being equal or more than 3.0%, pH 7.0 ± 0.5 (Appendix 15.33). Stability study Stability study is performed to determine the expiry date of the typhoid Vi polysaccharide vaccine. The study may be based on the determination of O-Acetyl content and molecular size on at least 3 final vaccine lots (these lots must be produced from individual intermediates of purified Vi polysaccharide). The final vaccine lots used for the study throughout the period of studying should be maintained under the prescribed conditions as stated in the instructions for use. Acceptance criteria: Molecular size of each final vaccine lot is not less than 50 per cent of the Vi polysaccharide found from the column eluted before KD = 0.25. Stability tests should have beer approved by the National control Authority.

Packaging Packaging and labeling must comply with the requirements for GMP.

Storage, expiry date The information about expiry date and storage bases on stability studies and be approved by National control Authority. The vaccine is stored at 2 - 8 °C. Not more than 3 years from the date of filling into the final containers. Labelling The information on the label, box and leaflet must comply with the current regulations. VARICELLA VACCINE (LIVE) Vaccinum Varicella vivum Varicella vaccine (live) is a freeze-dried preparation of live attenuated varicella virus strain grown in human diploid cell cultures. The vaccine is reconstituted immediately before use to give a clear liquid that may be colored owing to the presence of a pH indicator. The vaccine complies with the following requirements.

Production Vaccine production is based on virus seed lot system and a cell bank system. The production method has shown to yield consistently live varicella vaccine of adequate immunogenicity and safety in man. The virus in the final vaccine shall not have been passaged in cell culture more than 38 passages from the original isolated virus. Virus seed lot The varicella virus strain is identified as Oka strain by historical records that include information on the origin of the strain, live attenuated method and its subsequent manipulation and show to be attenuated and effective in the clinical. The seed lots are prepared in the same kind of cells as those used for the production of the final vaccine. The seed lot is stored at below -20 °C if freeze-dried or below -60 °C if not freeze-dried. Only a virus seed lot that complies with the following requirements may is used for virus production. Identification The master and working seed lot are identified as human herpes virus 3 by serum neutralization in cell culture using specific antibodies. Virus concentration The virus concentration of the master and working seed lot is determined as regulation to ensure the stability of production. Extraneous agents The working seed lot complies with the requirements for seed lot of live virus vaccine. Selection of cell culture The cell line for production of varicella vaccine is based on cell bank system. The human diploid cells are stored at -70 °C or liquid nitrogen. The cell bank system and the maximum passage are approved by and registered with the national control authority (NRA). 1097

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Cell-culture medium Serum used for the propagation of cells is free of bacteria, fungi and Mycoplasma and other virus, extraneous agents. Penicillin and other beta-lactam are not used at any production stages. Other antibiotics may be used at production stages but approved by national control laboratory (NCL). Single harvest Before strain virus inoculation, the cells are examined for degeneration caused by infective agents. If such examination shows evidence of the presence in a cell culture of any adventitious agent, the whole group of culture concerned is not used for vaccine production. After virus inoculation, the cells are incubated at suitable temperature. If animal serum is used in the growth or maintenance medium for the cell cultures, the serum is removed from the cultures either before or after inoculation with the seed virus but before harvest. The cell culture is rinsed and the growth medium replaced with serum - free maintenance medium. The infected cells from a single harvest will be washed and pooled. The cell suspension is disrupted by sonication or another appropriate method.

Virus pool The virus pool is prepared from one or several single harvests that satisfy the requirements. The virus pool is tested for sterility. Sample that not tested immediately is stored at or below -60 °C. The virus pool is clarified by a suitable method to remove maximum cell debris. The quantity of live virus is determined by titration method in cell against reference standard – live varicella vaccine. Quality control of final bulk The final bulk is prepared from one or more clarified virus pool. Only preservatives, adjuvant or diluent approved by NRA are added to the bulk. The substances do not effect on the safety and efficacy of the vaccine. Sterility It complies with the test for sterility (Appendix 15.7). If the vaccine contains preservatives, carry out a suitable method to prevent agents influencing the sterility test.

Quality control of final product Inspection Each final lot is inspected visually to ensure that the vial or ampoule passes for inspection before release such as: no abnormal particles, cap or cover is tight and integrity. If the vial or ampoule is not passed, discard it. Identification Perform at least 1 vial of final product by a suitable method approved by NCL. Antigen of vaccine is identified by seroneutralization in cell culture with specific antiserum. 1098

Potency (virus content) Use method having high precision to determine the virus content. Use reference standard of varicella vaccine to validate the procedure. Specification: Not less than 2 × 103 PFU per single human dose. The thermal stability test is considered by NCL as an element in establishing consistency of production. General safety Pass for general safety test (Appendix 15.11). Sterility It complies with the test for sterility (Appendix 15.7). Residual moisture Not more than 2% (Appendix 15.35). The residual moisture is determined by a suitable method approved by NCL. Residual animal serum proteins If using serum in the cell-culture system, the residual amount of serum albumin is less than 50 ng per single human dose.

Storage Varicella vaccine is stored at 2 - 8 °C if freeze-dried and single dose. If stored at different condition, need to be validated by a suitable method approved by NCL. Expiry date Approved by National Regulatory Authority. Labelling The information of label, box, and leaflet must comply with the current regulations; need to have information such as: Virus strain for vaccine production; Cell name; The minimum virus content; Time for reconstitution, time for using vaccine after reconstitution; Recommendation for pregnancy; Avoid disinfectant.

DIAGNOSTIC PREPARATION

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TUBERCULIN PPD Tuberculini deviratum proteinosum purificatum

The two nonsensitised guinea pigs in control group shall be shown negative reaction to the test sample of tuberculin.

Tuberculin purified protein derivative (tuberculin PPD) is a preparation containing the active substance causing specific cutaneous reaction to tuberculosis obtained from culture filtrate and lysate of Mycobacterium tuberculosis. Freeze-dried tuberculin PPD is off-white, spongy, and odorless; the diluted preparation may be a freeze-dried powder which upon dissolution gives a colorless. Using freeze-dried tuberculin PPD is one of the methods used to detect tuberculosis cases or used to evaluate late hypersensitivity reactions to tuberculosis after injecting of BCG vaccine.

Potency The potency of tuberculin PPD is determined by comparing the reactions produced by the intracutaneous injection of different doses of the preparation to be examined into sensitized guinea pigs with the reaction produced by known concentrations of the reference preparation.

Production The strain for production shall be inoculated onto suitable culture medium. After 6 weeks, the developed strain shall be harvested, killed by steam at 100 °C for 60 minutes and then filtered to remove bacteria. The active fraction of the filtrate, consisting mainly of protein, is isolated by precipitation with ammonium sulfate, washed and redissolved in a suitable buffered solution. Phenol is used as preservative. The final bulk shall be dispensed sterilely in final containers and freeze-dried. Identification The test shall be conducted by the intradermal injection in sensitized guinea pigs previously sensitized with BCG vaccine. Method Materials Freeze-dried BCG vaccine 1mg/ml. Use four healthy guinea pigs, weighing 350 - 400g with negative reaction to tuberculin, separate equally into a test group and a control group. The test group shall be sensitized with BCG vaccine. Sensitization Freeze-dried BCG vaccine shall be reconstituted and diluted to 0.5 mg/ml suspension. Inject intradermally 0.4 ml into each white guinea pig at four different sites (0.1 ml per one site). Observe the animals for 5 - 6 weeks after injection. Inject tuberculin The test sample of tuberculin shall be reconstituted to 50 IU/ml solution. Inject intradermally 0.1 ml (equivalent to 5IU) into both animal groups. Reading results After 24 h of injection, measure the diameter of local reactions at the injection site. The reaction shall be considered as a positive to tuberculin if the diameter of the lesion is equal or more than 6 mm. Criteria for judgment The two sensitised guinea pigs in test group shall be shown positive reaction to the test sample of tuberculin.

Method Materials Test samples of tuberculin Reference tuberculin Six guinea pigs: healthy, white or pall-colored guinea pigs, of the same sex, and weighing 350 - 400 g. Sensitization Freeze-dried BCG vaccine shall be reconstituted and diluted to 0.5 mg/ml suspension. Inject subcutaneously 0.4 ml into each white guinea pig at four different sites (0.1 ml per one site). The guinea pigs shall be observed for 5 -6 weeks after injection and used for the potency test. Procedure Prepare dilution of the preparation to be examined and of the reference preparation using phosphate-buffered saline to 3 or 4 different doses such that the produced lesions have a diameter of not more than 25 mm for the highest dose and not less than 6 mm for the lowest dose. Inject intracutaneously 0.1 ml of each dose of the test or the reference preparation at six sites of the flanks of animals. The site of injection shall be selected by the Latin square method. Measure the diameters of the lesions 24 to 48 hours later. The reaction shall be considered positive if the lesions have diameters more than 6 mm and negative if less than 6 mm. The potency of test tuberculin is calculated by the parallel line program of WHO. Acceptance criteria The estimated potency is not less than 80% and not more than 125% of the stated potency.

Abnormal toxicity It complies with the test for abnormal toxicity (Appendix 15.11). Sterility It complies with the test for sterility (Appendix 15.7). Phenol content Not more than 5.5 g/l (Appendix 15.28). pH 6.5 - 7.5 (Appendix 15.33). Administration and dose Intracutaneous injection at dose of 5 IU/0.1 ml.

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Filling and storage Depend on each manufacturer, normally consist 3 types below: A vial contains 15 IU, equivalent to 3 doses. A vial contains 50 IU, equivalent to 10 doses. A vial contains 100 IU, equivalent to 20 doses. Store at 2 - 8 °C, protected from light. Labelling and package The information on the label, box and leaflet must comply with the current regulations.

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Monographs MATERIA MEDICA

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ARILLUS LONGAN

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ALOE Lô hội (Nhựa) Aloes is the dried concentrated juice obtained mainly from the leaves of Aloe vera L. and Aloe ferox Mill, (Fam. Asphodelaceae).

Description Irregular pieces, varying in size; externally blackishbrown, lustrous, easily broken, fracture glassy. Odour, slightly uncomfortable; taste, very bitter. Identification A. Place 0.5 g of the powder in a 250 ml conical flask, add 50 ml of water, shake thoroughly for 5 minutes, filter. Using the filtrate (solution A) for the tests below: Place 5 ml of solution A in a test tube, add 0.2 g of sodium tetraborate R, heat to dissolve. To 1 ml of the obtained solution, add 30 ml of water, mix well. Examine under ultraviolet light (366 nm), a bright yellow fluorescence is shown. Place 2 ml of solution A in a test tube, add 2 ml of saturated bromine water R, a yellow precipitate is formed. B. Place 0.1 g of the powder in a 100 ml conical flask, add 5 ml of a 3% solution of ferric chloride R and 5 ml of a 10% solution of hydrochloric acid R, shake thoroughly, heat on a water bath for 10 minutes, allow to cool, add 15 ml of ether R, shake well for 1 minute. To the ether extract, add 5 ml of a 10% solution of ammonia R, shake, a violet - pink colour is produced in the ammonia layer. C. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel G Mobile phase: Water - ethyl acetate - methanol (13:100:17). Test solution: To 0.5 g of the powder, add 20 ml of methanol R, boil on a water bath. Shake for several minutes and filter. Reference solution: Dissolve 25 mg of barbaloin in methanol R and dilute to 10 ml with the same solvent. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 10 cm, remove the plate and allow to dry in air. Spray with a 10% solution of potassium hydroxide in methanol R. Examine under ultra-violet light at 366 nm. The chromatogram obtained with the test solution has no violet fluorescent spot, has a fluorescent spot corresponding in colour and position to the fluorescent spot due to barbaloin in the chromatogram obtained with the reference solution.

water previously warmed to about 60 °C, mix well. Add further 75 ml of water, and heat in a water bath at 60 °C for 30 minutes, with occasional shaking. Allow to cool, filter into a 1000 ml volumetric flask, wash the conical flask and filter paper with 20 ml of water, combine the washings into the volumetric flask, add water to volume, mix well. Transfer accurately 10 ml of the obtained solution to a 100 ml spherical flask containing 1 ml of a 60% solution of ferric chloride R and 6 ml of hydrochloric acid R, heat under a reflux condensor in a water bath for 4 hours. Allow to cool, then transfer the solution to a separator, wash the spherical flask successively with 4 ml of water, 4 ml of 1N sodium hydroxide R and 4 ml of water, combine the washings into the separator. Extract with 3 quantities, each of 20 ml, of ether R. Transfer the combined ether extract into another separotor and wash with 2 quantities, each of 10 ml, of water. Transfer the ether layer to a 100 ml volumetric flask, add ether R to volume, mix well. Evaporate carefully 20.0 ml of the obtained ether solution, on a water bath to dryness, add 10.0 ml of a 0.5% solution of magnesium acetate in methanol to dissolve. Measure the absorbance at 512 nm (Appendix 4.1), using methanol R as the blank. The content of hydroxyanthracen is calculated as barbaloin by the following expression: (A×19.6) X%= m Where: A: Absorbance at 512 nm. m: Weight of the powder being examined, calculated on the dried basis (g). X: Content of hydroxyanthracen, calculated as barbaloin. It contains not less than 18.0% of hydroxyanthracen, calculated as barbaloin (C21H22O9) which reference to the dried drug.

Preliminary processing Take Aloe leaves, press to collect the leaf juice, concentrate it to dryness. Storage Preserve in well closed containers; store in dry and cool places. ARILLUS LONGAN Long nhãn

Loss on drying Not more than 12.0% (Appendix 9.6, 1.000 g, 105 °C, 4 h).

Sun-or heat-dried arils of Dimocarpus longan Lour. (Fam. Sapindaceae).

Total ash Not more than 4.0% (Appendix 9.8).

Description Dried arils, irregular in thickness, broken longitudinally, usually agglutinated, yellowish brown or brown, translucent, one surface shrunken and the other lustrous, with longitudinally fine wrinkles, 1.5 cm long, 2 - 4 cm wide, about 1 mm thick. Texture soft and smooth, tough, unsticky to fingerss. Odour, slightly aromatic, taste, sweet.

Assay Moisten about 0.4 g of the powder (passed through a sieve with a pore size of 0.18 mm), accurately weighed, with 2 ml of methanol R in a 250 ml conical flask, add 5 ml of

1103

VP V

ARILLUS MOMORDICAE COCHINCHINENSIS

Transverse section Outer epidermis consists of one row of sub-squared cells. Inner epidermis consists of one row of slightly thick-walled cells, covered with slightly thick cuticle. Between the outer epidermis, and inner epidermis there are parenchymatous cells, large, thin-wall, distorted shape, up to 310 µm in diameter, that are arranged in numerous rows. Loss on drying Not more than 15.0% (Appendix 9.6, 1 g, 100 °C, 4 h). Foreign matter Dark-brown slices: Not more than 5% (Appendix 12.11). Total ash Not more than 4.0% (Appendix 9.8) Extractives Not less than 70.0%, calculated with reference to the dried drug. Carry out the hot extraction method (Appendix 12.10), using water as the solvent. Preliminary processing In summer and autumn, collect ripe fruits, thick and draind arils, dry in strong sunlight or at a temperature of 40 - 50 °C until hearing a clop when shaking dry fruits, remove shells and nutlets, take out wrinkled yellow arils, dry at 50 - 60 °C, until they become unsticky to fingers (water content of the crude drug, under 15.0%). Pay attention to hygienic conditions during aril taking off and drying. Possibly soak bunches of longan fruits in boiling water, for 1 - 2 minutes, before drying in the sun or at a low temperature. Storage Preserve in well closed containers with damp proof lining; store in dry, cool and ventilated places, protected from mould, weevils, damp, decolorization and acidification. ARILLUS MOMORDICAE COCHINCHINENSIS Gấc (Áo hạt) Sun- or heat-dried seed coats from seeds of ripe fruits of Momordica cochinchinensis (Lour.) Spreng. (Fam. Cucurbitaceae).

Description Membranes about 1 mm thick, 2 - 3 cm long, 2 - 2.5 cm wide, orangish-red, surface wrinkled. Texture dry and fragile, easily fragmentally broken; odour, slightly acrid; taste, weak. Powder Orangish-red. Parenchymatous fragments consisting of polygonal cells, relatively regular in size, wall slightly thickened, arranged evenly and closely. Nunmerous 1104

orange oil droplets. Scattered with blackish-brown masses of substance.

Identification Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Cyclohexane - ethylic ether (4 : 1). Test solution: To 1 g of the coarse powder, add 10 ml of petroleum ether (40 - 60 °C) R, macerate for 1 hour, filter, evaporate the filtrate to dryness. Dissolve the residue in 1 ml of petroleum ether (40 - 60 °C) R. Reference solution: Dissolve β-carotene in petroleum ether (40 - 60  °C) R to obtain a solution containing 0.1 mg per ml. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 12 cm and remove the plate, dry in air. Examine in visible light, the principal spot in the chromatogram obtained with the test solution corresponds in colour and position to the spot due to β-carotene in the chromatogram obtained with the reference solution. Water Not more than 10.0% (Appendix 12.13). Determined on 10 g of the coarse powder. Foreign matter Not more than 1.0% (Appendix 12.11). Broken matter Pass through a sieve with a pore size of 4.0 mm: Not more than 5.0% (Appendix 12.12). Total ash Not more than 10.0% (Appendix 9.8). Extractives Weigh accurately about 10 g of the powder, put into a 250 ml ground-glass stoppered conical flask, add 100 ml of petroleum ether (40 - 60  °C) R, heat under a reflux condenser on a warm water bath for 30 minutes. Decant the extract and filter. Repeat the extraction twice, each of 50 ml of petroleum ether (40 - 60 °C) R. Combine all the filtrates to a tared cup, evaporate on a water bath to dryness. Allow to cool in a desiccator. Weigh and calculate the percentage content of the residue in the drug. It contains not less than 8.0% of oil residue, caculated with reference to the dried drug. Preliminary processing Take out the seeds from the ripe fruits, dry under the sun or steam well until they are less sticky then peel the arils, dry or heat at temperature of 40 - 60 °C to dryness. The aril is used for making oil of Momordica cochinchinensis. Storage Store in a cool and dry place.

VP V

BENZOINUM Cánh kiến trắng, An tức hương Dried aromatic resin, collected from the trunk of Styrax tonkinensis (Pierre) Craib ex Hardw., (Fam. Styracaceae).

Description Detached small irregular pieces, somewhat flattened, sometimes stuck into masses; externally orange yellow with waxy lustre (naturally injured resin); or irregularly cylindrical, flat pieces, externally greyish-white to yellowish-white (artificially incised resin). Texture, fragile, easily fractured; fracture, even, white, gradually becoming yellowish-brown or reddish-brown on long storage. Soften and melt on heating. Odour, vanilla characteristic. Taste, slightly pungent, with a sensation of sand when chewing. Identification A. Heat gently 0.25 g of the drug in a dry test tube, an irritant aromatic odour and numerous prismatic sublimates adhering to the tube wall are produced. B. To 0.1 g of the crude drug in a test tube, add 5 ml of ethanol (96%) R, stir well and filter. To the filtrate, add 0.5 ml of ferric chloride solution in ethanol R, a light green colour is produced in the solution, then changes to yellowish-green. Loss on drying Not more than 2.0%. Weigh accurately about 2.0 g of the coarse powder, dry to constant mass over sulphuric acid R in a desiccator (Appendix 9.6). Ethanol-insoluble matter Not more than 2.0%. Extract 2.5 g, accurately weighed, of the fine powder in a Soxhlet’s extractor with ethanol (96%) R until all of ethanol-soluble matter is extracted. Discard the ethanol extract, dry the residue to constant mass at 100 °C, weigh the dried residue mass. Total ash Not more than 0.50% (Appendix 9.8) Sulphated ash Not more than 2.0%, using 1.0 g (Appendix 9.9). Assay Weigh accurately about 1.5 g of the powder (passed through a sieve No. 355), put into a ground-glassstoppered conical flask, add 25 ml of 0.5 M ethanolic potassium hydroxide R, heat under a reflux condenser on a water bath for 1.5 hours. Evaporate the extract to dryness on a water bath and disperse the residue in 50 ml of hot water. Allow to cool, add 150 ml of water and 50 ml of a 5% solution of magnesium sulphate R, shake well, and allow to stand for 10 minutes. Filter, wash the residue with 20 ml of water. Combine the filtrate and washings, acidify

BOMBYX BOTRYTICATUS

with hydrochloric acid R, then transfer to a separator and extract successively with 50, 40, 30 and 30 ml of ether R. Extract the combined ether extract with 20, 20, 10, 10 and 10 ml of a 5% solution of sodium carbonate R, successively; washing each aqueous extract with 20 ml of ether R. Combine the aqueous extracts, acidify with hydrochloric acid R, and extract again successively with 30, 20, 10 and 10 ml of ether R. Combine the ether extracts and transfer to a tared conical flask, evaporate the ether with a current of air and rotate the flask to disperse evenly the residue on the inner wall of the flask. Dry the flask to constant mass over sulphuric acid in a desiccator, weigh accurately. The mass of residue represents the content of total balsamic acid in the sample. Calculate the percentage content of total balsamic acid in the dried basis (subtracted the content of ethanol-insoluble matter). It contains not less than 30.0% of total balsamic acid, calculated on the basis of ethanol soluble extractives.

Preliminary processing The resin is collected from the trunks of Styrax tonkinensis which are injured naturally or incised in summer and autumn, and dried in the shade to dryness. Storage Preserve in well closed containers; store in cool and dry places. BOMBYX BOTRYTICATUS Tằm vôi, Bạch cương tàm, Cương tàm Sun-or heat-dried body of the 4th - 5th stage larva of Bombyx mori L., (Fam. Bombycidae) died of infection (or artificial infection) of Botrytis bassiana Bals. (Fam. Mucedinaceae), Fungi imperfecti (Deuteromycotina).

Description Somewhat cylindrical, usually curved and shrunken, 2 - 5 cm long and 0.5 - 0.7 cm in diameter. External surface, greyish-yellow, covered with white powdery aerial mycelia and conidia. The whole body, distinctly segmental. The head, relatively round with two eyes at two sides; 8 pairs of false legs present at two sides of abdomen; caudal portion somewhat dichotomic. Texture, hard and fragile, easily broken; fracture, even and lustrous; peripheral layer, white; middle part with 4 bright brown or bright black rings of silk glands. Odour, slightly stinking; taste, slightly salty. Powder Greyish-brown or dark greyish-brown in colour. Examine under microscope: The mycelia almost colourless, slightly curved or arch-like, with brown or dark brown spiral filaments. Outer surface, epidermis with reticulated shrunken striations and small pointed projections risen from the striations; with rounded setae pits, margin of the 1105

VP V

BULBUS ALLII SATIVI

pits yellow. Setae, yellow or yellowish-brown; external surface, smooth; walls, slightly thick. Tissues contain the indigested mulberry leaf containing prismatic crystals of calcium oxalate.

Loss on drying Not more than 11.0% (Appendix 9.6, 1 g, 105 °C, 4 h). Foreign matter Not more than 0.5% (Appendix 12.11). Total ash Not more than 7.0% (Appendix 9.8). Preliminary processing In spring and autumn select intact hardened dead silkworms that the outer and inner parts are white, because of infection with micro-fungus Botrytis brassiana Bals., wash clean, dry under the sun (cover with gauze) or at a low temperature (40 °C - 50 °C) to dryness. Processing Eliminate foreign matter, wash clean, dry in the sun or at a low temperature. Bombyx Botryticatus (stir-baked): Spread the bran in a pan, bake until it smokes, add clean and dry Bombyx Botryticatus, stir-bake until the crude drug surface turns yellow, sift out the bran and cool. Use 1 kg of bran per 10 kg of Bombyx Botryticatus. Storage Store in dry places, protected from weevils. BULBUS ALLII SATIVI Tỏi Sun-dried compound bulbs of Allium sativum L., (Fam. Alliaceae).

Description Sub-globular compound bulbs 3 - 5 cm in diameter, consisting of 8 - 20 cloves, surrounded by 2 - 5 layers of thin white scale leaves formed from sheaths of former leaves, adhering to a flattened rounded base (corm). Cloves ovoid, 3- or 4-sided, apex acute, base truncate. Each clove covered with several layers of white scale leaves and a pinkish-white epidermal layer easily exfoliated from the inner hard part. The cloves layered around a long core rising from the centre of the base, 1 - 3 mm in diameter. The inner hard part of cloves, watery. Odour, aromatic; taste, pungent and lasting. Transverse section Detached scale leaves examine under microscope showing parallel layers of spiral vessels. Outermost scale leaves consisting of epidermal cells, elongated, thick-walled. Each cell containing a prismatic crystal with 20 - 50 µm 1106

in diameter or stomata possibly visible depending on its position. Leaf flesh cells, thin-walled, disorderly arranged. The inner epidermis consisting of thin-walled cells. Thick compound bulbs containing thin-walled epidermis, leaf flesh layer consisting of parenchymal cells varying in shape and spiral conductive vessels arranged in lines.

Total ash Not more than 5.0% (Appendix 9.8). Preliminary processing Collect the drug in late winter to early spring, when the leaves turn withering, lift the bulbs, shake soil and sand off, bind in bundle and hang to dry. Processing Eleminate old coats, wash clean, use fresh bulbs, pound or macerate in alcohol. Storage Store in dry and cool places. BULBUS ELEUTHERINIS SUBAPHYLLAE Sâm đại hành, Sâm cau, Tỏi lào, Hành lào Sun- or heat-dried bulbs of Eleutherine subaphylla Gagnep., (Fam. Iridaceae).

Description Bulbs rounded like onions or elongated, 1 - 2 cm in diameter at the largest place, 4 - 5 cm long. Externally with some layers of dry scales, brown on the upper part, red on the lower part; inner layers, bright red like blood. Transverse cut surface, pale red, alternating with white concentric rings. Bulbs bearing some dry rootlets, 1 - 3 cm long. Transverse section Transverse section of succulent scales: Outer epidermis consisting of one row of rectangular cells, arranged evenly. Parenchyma containing abundant starch granules and stick-shaped crystals of calcium oxalate. Double collateral phloem-xylem bundles ellipsoid, lying in the middle of parenchyma, phloem coating both ends, xylem in the centre, xylem vessels few, arranged disorderly. Inner epidermis consisting of one row of rectangular cells, thinner than outer epidermis. Powder Pink in colour. Taste, slightly bitter, then sweet. Examine under microscope: Polymorph starch granules, abundant, 1.6 - 40 µm in size; hilum, distinct on several granules. Crystals of calcium oxalate, stick-shaped, with acute ends similar to pencilends or with truncate ends. Vessel fragments, fragments of parenchyma containing starch granules, fragments of outer epidermis and inner epidermis visible.

BULBUS LILII

VP V

Water Not more than 10.0% (Appendix 12.13). Using 10 g of slivered drug. Foreign matter Not more than 1% (Appendix 12.11). Total ash Not more than 5.0% (Appendix 9.8). Preliminary processing Collect the drug from over one-year-old plants. When the plants turn withering, lift the bulbs, remove roots and leaves, wash clean; cut vertically into slices; dry in the sun or at a low temperature (under 60 °C) to dryness; use whole slices or pulverize them before use. If the drug is not used yet, after lifting the bulbs, shake soil off and let the root layers and outer coats be intact. The fresh bulbs being scale by scale separated will be buried in moistened sand to keep them dry slowly. Storage Store dry bulbs in dry and cool places, protected from mould and weevils. Store fresh bulbs in moistened sand or in cool places BULBUS FRITILLARIAE Bối mẫu, Xuyên bối mẫu Sun- or heat-dried bulbs of Fritillaria cirrhosa D. Don, Fritillaria unibracteata Hsiao et K.C. Hsia, Fritillaria przewalskii Maxim. or Fritillaria delavayi Franch., (Fam. Liliaceae). According to different characters, the drug derived from the former three species are known as “Tung boi”, “Thanh boi” and “Lo boi”, respectively.

Description Tung boi: Bulbs conical or spherical, 0.3 - 0.8 cm high, 0.3 - 0.9 cm in diameter. Externally, ivory-white. Outer scale leaves, two, varying considerably in size, the larger embracing the smaller, the uncovered part appearing crescent, commonly known as “Hoai trung bao nguyet” (holding the moon in the arms). Bulb apexes, closed; buds, subcylindrical and slightly tapering, with 1 - 2 small scales inside, obtuse or slightly acute apexes, even and slightly concave bases, with a grayish-brown disk at central part, remains of fibrous roots occasionally found. Texture, hard and fragile; fracture, white, starchy. Taste, slightly bitter. Thanh boi: Bulbs, nearly oblate, 0.4 - 1.4 cm high, 0.4 - 1.6 cm in diameter. Outer scale leaves, two, almost uniform in size, embracing each other. Apexes, open, with buds and 2 - 3 small scales inside and remains of slender cylindrical stem. Lo boi: Bulbs, long conical, 0.7 - 2.5 cm high, 0.5 - 2.5 cm in diameter. Externally, ivory-white or brownish-yellow, sometimes brown-maculate. Outer scale leaves, two,

almost uniform in size. Apexes, open, somewhat tapering; base, slightly acute or relatively obtuse.

Powder Ivory-white in colour. Odour characteristic; taste slightly bitter. Examine under microscope: Tung boi and Thanh boi: Starch granules, fairly abundant, broadly ovoid, long spheroidal or irregularly spheroidal, some with uneven or slightly branch-like edges, 5 - 64 µm in diameter, hilum short slit-shaped, pointed, V-shaped or U-shaped, and faint striations visible. Epidermal cells, subrectangular, uneven, with sinuous anticlinal walls; rounded or oblate anomocytic stomata occasionally found. Spiral vessels, 5 - 26 µm in diameter. Lo boi: Starch granules, broadly ovoid, conchoidal, reniform or ellipsoidal, up to 60 µm in diameter; hilum, V-shaped, stellate or pointed, striations distinct. Spiral and reticulate vessels, up to 64 µm in diameter. Loss on drying Not more than 12.0% (Appendix 9.6, 1 g, 105 °C, 5 h). Total ash Not more than 5.0% (Appendix 9.8). Foreign matter Not more than 0.5% (Appendix 12.11). Broken matter Pass through a sieve with a pore size of 3.15 mm: Not more than 5.0% (Appendix 12.12). Extratives Not less than 9.0%, calculated with reference to the dried drug. Carry out the hot extraction method (Appendix 12.10), using ethanol (50%) R as the solvent. Preliminary processing The drug is collected in summer and autumn; bulbs are lifted, removed from rootlets and coarse barks, washed clean, dried in the sun or at a low temperature. Storage Preserve in well closed containers (buckets or jars), store in dry places, protected from mould and weevils. BULBUS LILII Bách hợp Processed, dried bulb scales of Lilium brownii F. E. Brown var. viridulum Baker or Lilium pumilum DC., (Fam. Liliaceae).

Description Scales, elongated elliptical, 2 - 5 cm long, 1 - 2 cm wide, the middle part 1.3 - 4 mm thick. Externally ivory white, brownish-yellow or purplish, with longitudinal white 1107

VP V

CACUMEN PLATYCLADI

veins (vascular bundles). Apex acute, base relatively even, margins thin, non-serrated, slightly curved inwards. Texture, hard and tough. Fracture even, horn-like lustrous. Odourless; taste, slightly bitter.

Transverse section Cut surface, elongated elliptical. Outer epidermis consisting of very even cells, internally polygonal cells filled with starch, and scattered with vascular bundles. Powder Pale yellow in colour, with epidermal fragments and parenchymatous fragments containing starch. Starch granules ovate, hilum stellate or orbiculate, distinctly striated. Fragments of reticulate vessels. Identification Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel G. Mobile phase: Use the upper layer of a mixture of petroleum ether (60 - 90 °C) - ethyl acetate - formic acid (15 : 5 : 1). Test solution: Place 1 g of the powder in a conical flask, add 10 ml of methanol R, shake or sonicate for 20 minutes, filter. Concentrate the filtrate to about 1 ml. Reference drug solution: Prepare a solution of 1 g of Bulbus Lilii reference drug in the same manner as described under Test solution. Procedure: Apply separately to the plate 10 µl of each solution. After developing and removal of the plate, dry in air, spray with a 10% solution of phosphomolybdic acid in ethanol R, heat the plate at 105 °C untill the spots appear. Examine under visible light. The spots in the chromatogram obtained with the test solution correspond in colour and position to the spots in the chromatogram obtained with the reference drug solution. Loss on drying Not more than 12.0% (Appendix 9.6, 1 g, 105 °C, 4 h). Total ash Not more than 3.0% (Appendix 9.8). Extratives Not less than 18.0% , calculated with reference to the dried drug. Carry out the cold extraction method (Appendix 12.10), using water as the solvent. Preliminary processing When the weather is dry, lift the bulbs, wash clean, remove soil and sand, dry slightly until dryish, then separate scale by scale, and dry under the sun. Processing Bulbus Lilii (honeyed): To the clean Bulbus Lilii, add honey and a small quantity of boiling water, stir regularly until infused, put into a clean pan, stir bake with gentle 1108

heat until no longer sticky to fingers, take out and cool; using 5 kg of refined honey per 100 kg of Bulbus Lilii.

Storage Store in ventilated dry places. CACUMEN PLATYCLADI Trắc bách diệp, Trắc bá Sun- or heat-dried twig and leaf of Platycladus orientalis (L.) Franco, (Fam. Cupressaceae).

Description Usually branched, twigs flattened. Leaves small, scaleshaped, decussate, close to stems, dark green or yellowishgreen. Texture fragile, easily broken. Odour delicately aromatic; taste bitter, astringent and slightly pungent. Powder Yellowish-green in colour. Examine under microscope: Upper epidermal cells of leaves rectangular with slightly thickened walls. Lower epidermal cells subsquare, with numerous stomata, hollowed; subsidiary cells relatively large, dumbbell-shaped in side view. Parenchymatous cells containing oil droplets. Fibres slender, about 18 µm in diameter. Bordered pitted tracheids sometimes present. Identification Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel H, using a 0.2 - 0.5% sulution of carboxymethylcellulose to prepare slurry. Mobile phase: Cyclohexane - ethyl acetate - formic acid (9 : 1 : 0.5). Test solution: To 3 g of the powder, add 50 ml of ethanol (70%) R and 3 ml of hydrochloric acid R, heat under a relux condenser for 3 hours, filter, use the filtrate as the test solution. Reference substance solution: Dissolve quercetin RS in ethanol (95%) R to obtain a solution containing 0.5 mg per ml. Procedure: Apply separately to the plate 5 µl of the test solution and 1 µl of the reference substance solution. After developing over a path about 12 cm and removal of the plate. Allow the plate to dry in air, spray with a 1% solution of aluminium chloride in ethanol R and examine in ultraviolet light (366 nm). The fluorescent spot in the chromatogram obtained with the test solution corresponds in colour and position to the spot due to quercetin in the chromatogram obtained with the reference substance solution. Water Not more than 11.0% (Appendix 12.13). Determined on 10 g of the coarse powder. Foreign matter Not more than 6.0% (Appendix 12.11).

CALYX KAKI

VP V

Total ash Not more than 10.0% (Appendix 9.8).

CALYX KAKI Thị đế, Tai hồng

Acid-insoluble ash Not more than 3.0% (Appendix 9.7)

Sun- or heat-dried persistent calyx of Diospyros kaki L. f., (Fam. Ebenaceae).

Extractives Not less than 15.0%, calculated with reference to the dried drug. Carry out the hot extraction method (Appendix 12.10), using the ethanol (95%) R as the solvent.

Description Calyx round flattened, 1.5 - 2.5 cm in diameter. Relatively thick in the middle, slightly prominent, with a rounded scar of fruit fallen off and somewhat thin at the edge, 4-lobeb, the lobes frequently reflexed, easily broken. A fruit stalk or a pitted scar of fruit stalk remained at the base. Outer surface, yellowish-brown or reddish-brown; inner surface, yellowish-brown, densely covered with fine pubescences. Texture hard and fragile. Odourless; taste astringent.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: A mixture of methanol - 0.01 M potassium dihydrogen phosphate solution - glacial acetic acid (40 : 60 : 1.5). Test solution: Place 0.5 g of the powder, accurately weighed, in a ground-glass stoppered conical flask, add 20.0 ml of methanol R, weigh. Sonicate for 30 minutes, allow to cool, weigh again and compensate the loss of weight with methanol R, shake well and filter. Reference solution: Dissolve quercitroside RS, accurately weighed, in methanol R to produce a solution containing 50 µg per ml. Chromatographic system: A column (0.25 m × 4.6 mm) packed with stationary phase C (5 µm) (RP18 column is suitable). Detector: A spectrophotometer set at 254 nm. Flow rate: 1 - 2 ml/minute. Volume of injection: 10 µl. Procedure: Inject the reference solution, record the chromatogram. The number of theoretical plates of the column is not less than 1500, calculated for the peak due to quercitroside. Inject the reference solution and the test solution separately. Calculate the percentage content of quercitroside in the drug using the areas of quercitroside peaks in the chromatograms obtained with the test solution and the reference solution and the declared content of C21H20O11 in quercitrosid RS. It contains not less than 0.1% of quercitroside (C21H20O11), calculated with reference to the dried drug. Preliminary processing Collect the drug in summer and autumn; cut and take twigs and leaves, dry in the shade Processing Cacumen Platycladi: Eliminate hard twigs and foreign matter and use the drug. Cacumen Platycladi (carbonized): Take the clean Cacumen Platycladi, place it in a hot pan and stir bake at a high temperature until the surface becomes burned brown and the inner part burned yellow (stir baking for natural preservation). Storage Store in a dry and cool place and preserve in well covered containers.

Identification Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Toluen saturated with water - methyl formate - formic acid (5 : 4 : 1). Test solution: To 2 g of the powder, add 10 ml of ethanol (70%) R, and warmly steep for 2 hours, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of methanol R. Reference substance solution: Dissolve gallic acid RS in methanol R to obtain a solution containing 0.5 mg per ml. Reference drug solution: Alternatively, prepare a solution of 2 g of Calyx kali reference drug in the same manner as described under Test solution. Procedure: Apply separately to the plate 5 µl of each solution. After developing and removal of the plate. Allow the plate to dry in air, spray with a 1.0% solution of ferric chloride in ethanol R. The chromatogram obtained with the test solution must have a spot corresponding in colour and position to the spot of gallic acid in the chromatogram obtained with the reference substance solution or the spots in the chromatogram obtained with the test solution correspond in colour and position to the spots in the chromatogram obtained with the reference drug solution Loss on drying Not more than 12.0% (Appendix 9.6, 1 g, 105 °C, 4 h). Foreign matter Not more than 1.% (Appendix 12.11). Preliminary processing Collect the ripe fruits of Diospyros kaki in autumn and winter, and take off Calyx kaki or collect Calyx kaki from eaten fruits; wash clean and dry in the sun. Processing Eliminate foreign matter, wash clean, remove the stalks; dry or break to pieces and dry. Storage Store in a ventilated, cool and dry place, protected from mould and weevils. 1109

VP V

CARAPAX ET PLASTRUM TESTUDINIS

CARAPAX ET PLASTRUM TESTUDINIS Qui giáp và qui bản, Mai rùa và yếm rùa

CARAPAX TRIONYCIS Miết giáp, Mai ba ba

Sun-dried carapaces and plastrons of Chinemys reveesii (Gray), (Fam. Emydidae).

Sun- or heat-dried shells of Trionyx sinensis Wiege - mann, (Fam. Trionychidae).

Description Carapaces and plastrons being connected by bony bridges. Carapaces are slightly longer than plastrons. Carapaces narrow elliptical, arciform, 7.5 - 22 cm long, 6 -18 cm wide, the anterior part slightly narrower than the posterior part; the outer surface, brown or blackish-brown, with a cervical scute at the anterior part and 5 vertebral scutes along the mid line of the black. Four costal scutes symmetrical at both sides of carapace, 11 marginal scutes at each side of margin. Two pygal scutes (đồn giáp) in the posterior part. Plastrons, plate-like, subelliptical, diagonal claviform or long rectangular, 5.5 - 17 cm wide, 6.4 - 21 cm long; the outer surface, pale yellowish-brown to brown, with 12 horny scutes, each with purplish-brown radiated veins; the inner surface, yellowish-white to greyish-white, with blood stains or remaining flesh. After scraping and cleaning, 9 pieces of bony plates visible, the serrated connecting edges inlaid each other. The anterior end, obtuse or truncate, the posterior end with an isoscele triangular notch, wing-like bony bridge, curved upwards obliquely at both sides. Texture is hard. Odour is slightly stinking; taste is slightly salty.

Description Turtle shells elliptical or ovoid , with convexes; dorsal surface, 10 - 15 cm long, 9 - 14 cm wide. Outer surface blackish-brown or dark green, slightly lustrous, with fine reticulate wrinkles, greyish-yellow or greyish-white spots and a longitudinal ridge in the middle of the back. Cervical vertebrae curved inwards, 8 ribs arranged on each side of the vertebrae, stretching towards the margin. Texture, hard. Odour, slightly stinking; taste, salty.

Preliminary processing Capture the animals all the year round, but usually in autumn and winter. After capturing, kill the tortoises, collect carapaces and plastrons, remove the remaining flesh, and dry; this is known as “huyet ban” (bloody drug). Or after capturing, scald the tortoises with boiling water, collect carapaces and plastrons, remove the remaining flesh and dry, this is known as “Thang ban” (boiled drug). “huyet ban” drug is glossy, with remaining skin, and sometimes with remaining blood stains. “Thang ban” drug has darker colour, with traces of detached skin, the inner surface is greyish-white or yellowish, and not glossy. Processing Carapax et plastrum Testudinis: Steam for 45 minutes, take out and put in hot water, remove the remaining skin and flesh immediately, then wash clean and dry under the sun. Carapax et plastrum Testudinis (processed with vinegar): put clean sand in a pan, stir bake the sand at a high temperature to dryness, put Carapax et plastrum Testudinis in sand and stir bake until the colour of the surface turns yellowish. Take out, remove sand, soak quickly in vinegar and dry. Use 2 litres of vinegar per 10 kg of Carapax et plastrum Testudinis; break to slivers before use. Storage Store in cool and dry places, protected from weevils. 1110

Loss on drying Not more than 5.0% (Appendix 9.6, 1 g, 105 °C, 5 h). Foreign matter Not more than 1.0% (Appendix 12.11). Extractives Not less than 5.0%, calculated with reference to the dried drug. Carry out the hot extraction method (Appendix 12.10), using ethanol (50%)R as the solvent. Preliminary processing Collect turtles all the year round, but mostly in autumn and winter, kill and take out carapaces, put in boiling water and boil for 1 - 2 hours, until the hard skin of carapace can be detached. Pick up carapaces, remove all the remaining flesh, wash clean, dry under the sun or at a low temperature. Processing Carapax Trionycis: Steam dry carapaces in a pot for 45 minutes, take out and put in hot water, remove the remaining skin and flesh by a hard brush immediately, wash clean and dry under the sun. Carapax Trionycis (processed with vinegar): Put the clean sand in a pan, stir bake at a high temperature until the sand loose, put clean carapaces in the pan and stir bake until the surface becomes yellowish, take out, remove the sand, soak quickly in vinegar and dry; break to slivers before use. Per 10 kg of Carapax Trionycis, use 2 litres of vinegar. Storage Store in dry places, protected from weevils and insects, occasionally dry the crude drug again. CAULIS COSCINII FENESTRATI Vàng đắng (Thân) Sun-or heat-dried stems of Coscinium fenestratum (Gaertn) Colebr., Syn. Menispermum fenestratum Gaertn., (Fam. Menispermaceae).

VP V

Description Stems cylindrical, over 2 cm in diameter, nearly straight or slightly curved, sometimes with large swollen knobs. Externally dirty yellow or brown, sometimes full of patched, with numerous longitudinal shallow wrinkles, sometimes bearing rounded scars of small branches. Transverse cut surface exposing a thin bark layer and a thick wood part occupying 4/5 of the stem diameter, straw yellow, with radial rays and spotted with dots (xylem vessels). Texture hard, difficult to break. Odourless; taste is bitter. Transverse section Cork cells, flattened rectangular, arranged evenly. Cortical parenchyma scattered with numerous masses of sclerenchymatous cells, thick-walled, pit canals present. Fibre masses occurring outside, separated from phloem by a ring of sclerenchyma. The outer sclerenchymatous ring, continuous, surrounding the top of crescent phloem masses. Cambium visible. Secondary wood, xylem vessels large rounded, wood parenchymatous cells with thick walls. Medullary rays, broad. The inner sclerenchymatous ring, disinterrupted, consisting of cells with thick walls and concentric striations. Pith scattered with single sclerenchymatous cells. Powder Fragments of reticulate, dotted vessels. Sclerenchymatous cells, numerous, bright yellow, fusiform, polygonal or rectangular with thickened walls, lumina broad or narrow. Fibres thick-walled, with pit canals distinct or absent. Starch granules bell-shaped, single or double, triple compound, 8 - 10 µm in diameter. Crystals of calcium oxalate, minute bacilliform, about 8 µm long, scattered in parenchymatous. Prismalic crystals of calcium oxalate sometimes visible in lumina of sclerenchymatous cells. Identification A. Macerate 0.10 g of the powder in 10 ml of water in a test tube for about 2 hours, filter. Transfer 2 ml of the filtrate to another test tube, add 1 ml of sulphuric acid R, cool and add slowly 1 ml of saturated bromine solution R along the wall of the tube; a dark red ring is produced at the junction of the two layers. B. Macerate 0.10 g of the powder in 1 ml of ethanol (90%) R in a test tube for 10 - 15 minutes. Apply 1 - 2 drops of the ethanol extract to a slide, heat gently to nearly dryness, add 1 drop of hydrochloric acid R, cover with a coverslip allow to stand for 5 - 10 minutes, then examine under a microscope: Bunched or separate yellow needle-shaped crystals are observed. C. Examine the powder under ultraviolet light (366 nm), a light yellow fluorescence is shown. D. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel GF254. Mobile phase: n-Butanol - acetic acid - water (7 : 1 : 2). Test solution: To 0.10 g of the powder, add 5 ml of ethanol

CAULIS COSCINII FENESTRATI

(90%) R, heat under a reflux condenser on a water bath for 15 minutes. Filter, use the filtrate as the test solution. Reference substance solution: A 0.1% solution of berberine chloride RS in ethanol (90%) R. Procedure: Apply separately to the plate 20 µl of each solution. After developing and removal of the plate, dry in air, spray with Dragendorff reagent R. The chromatogram obtained with the test solution must have a spot corresponding in colour (orange red) and position to the spot due to berberine in the chromatogram obtained with the reference substance solution.

Loss on drying Not more than 13.0% (Appendix 9.6, 1 g, 100 °C, 4 h). Total ash Not more than 6.0% (Appendix 9.8). Foreign matter (Appendix 12.11) Discoloured drug: Not more than 2.0%, Stems below 2 cm in diameter: Not more than 2.0%. Other foreign matter: Not more than 1.0%. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: A mixture of acetonitrile - 0.1% solution of phosphoric acid (50 : 50). Add 0.1 g of dodecylsulfonate sodium R into each of 100 ml of the obtained mixture. Reference solution: Dissolve berberine chloride RS in mobile phase to produce a solution containing about 0.1 mg per ml. Test solution: Place 0.1 g of the powder (passed through a sieve No 710), accurately weighed, in a 100 ml volumetric flask, add 80 ml of mobile phase, sonicate for 40 minutes, allow to cool, add mobile phase to volume. Shake well, filter. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 μm). Detector: A spectrophotometer set at 265 nm. Flow rate: 1.0 - 1.5 ml per minute. Procedure: Inject 5 µl of the reference solution; the column efficiency determined on the berberine chloride peak is not less than 4000 theoretical plates. Inject separately 5 - 20 µl of the test solution and 5 µl of the reference solution. Adjust the volume of injection so that the area of the peak due to berberine in the chromatogram obtained with test solution and the reference solution is equal. Use the areas of the peak due to berberine in the chromatograms obtained with the test solution and the reference solution, and the declared content of C20H18NO4Cl in berberine chloride RS to calculate the content of berberine in the drug. It contains not less than 1.5% of berberine chloride (C20H18NO4Cl), calculated with reference to the dried drug. 1111

CAULIS CUM FOLIUM LONICERAE

Preliminary processing The drug is collected all the year round, scrape bark, cut into 10 - 13 cm segments, dry under the sun or at a low temperature (50 °C - 60 °C). Processing Wash clean, cut into thin slices, dry under the sun or at a low temperature (50 °C - 60 °C). The processed drug can be used as powder and to extract. Storage Store in cool and dry places, protected from mould and weevils. CAULIS CUM FOLIUM LONICERAE Kim ngân (cuộng) Sun-or heart-dried branches and leaves of Lonicera japonica Thunb and other species of the same branch such as L.dasystyla Rehd.; L. confusa DC. and L.cambodiana Pierre ex Danguy (Fam. Caprifoliaceae).

Description Segments of stem, cylindrical, 2 - 5 cm long, 0.2 - 0.5 cm in diameter; outer surface, light brown to dark brown; inner surface, light yellow; core, spongy or hollow. Leaves, dry, entire, ovate, opposite, 3 - 5 cm long, with short petioles, both sufaces pubescent. Odour, slightly aromatic; taste, slightly bitter. Transverse section Stems: Cut surface is subsquare. Epidermis consisting of a single layer of small cells, arranged regularly, bearing numerous non-glandular hairs. Collenchyma composed of 2 - 3 row of thick-walled cells present beneath the epidermal layer. Cortical parenchyma containing thinwalled, rounded cells. Irregular cells, with thick walls, lignified, arranged in a ring lying on the inner side of cortical parenchyma. Phloem forming continuous rings. Rows of xylem vessels grouped into bundles. Pith parenchyma consisting of roundish polygonal cells with lignified walls, the nearer inwards the cells lying, the lager their size. Old branches usually containing lacunaee in the centre. Leaves: Midribs: Upper and lower epidermis consisting of one layer of cells arranged evenly, cuticularized outer wall bearing numerous non-glandular hairs. Adjacent to upper and lower epidermis lying collenchyma which composed of 3 - 4 rows of roundish polygonal cells with angles thick walls. Parenchyma composed of polygonal cells, large sized, thinwalled, bearing scattered urchin-form crystals of calcium oxalate. Phloem-xylem bundles lying in the midddle of midrib. Phloem arranged in a ring bordering xylem. Lamina: Upper and lower epidermis consisting of one row of cells cuticularized on the outerside, bearing 1112

VP V

scattered non-glandular hairs. Palisade tissue consisting of rectangular cells arranged perpendicularly to upper epidermis.

Powder Pale brown powder, slightly aromatic, slightly bitter. Unicellular non-glandular hairs, thick-walled, smooth, with slightly swollen base. Epidermal fragments bearing non-glandular hairs. Conveying vessels composed of several kinds scalariform, dotted, reticulate and spiral. Starch granules, single, double or triple compounds. Crystals of cacium oxalate, urchin-form. Parenchymal fragments of lamina present. Epidermal fragments bearing stomata. Identification A. Place 10 g of the powder in a 100 ml conical flask, add 20 ml of ethanol (90%) R. Shake well, heat in a waterbath for 15 minutes, filter. Concentrate the filtrate on a water-bath to about 5 ml. Transfer 1 ml of the filtrate to a test tube, add 2 - 3 drops of hydrochloric acid R and a few powder of magnesium R or zinc R. The colour of the solution turns from yellow to orange to red. B. Place 1 g of the powder in a test tube, add 10 ml of water, shake gently for 5 minutess, filter. Transfer the filtrate to two test tube, 2 ml to each. To one test tubes, add 2 - 3 drops of a 10% solution of sodium hydroxide R. The yellow colour of the solution in this test tube is more intense than that in the test tube without adding sodium hydroxide solution. C. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel 60F254. Mobile phase: Butyl acetate - formic acid - water (7 : 2.5 : 2.5) Test solution: To 1 g of the powder add 20 ml of methanol R, sonicate for 20 minutes, cool, filter. Evaporate the filtrate on a water bath to dryness. Dissolve the residue in 1 ml of ethanol R. Reference substance solution: Dissolve chlorogenic acid RS in ethanol R to produce a solution containing 1 mg/ml. Reference drug solution: Alternatively, prepare a solution of 1 g of the powder of caulis cum folium Lonicerae reference drug in the same manner as described under Test solution. Procedure: Apply separately to the plate 10 μl of each solution. After developing and removal of the plate, allow to dry in air. Examine under ultraviolet light at 366 nm. The spot in the chromatogram obtained with the test solution corresponds in colour and position to the spot due to chlorogenic acid in the chromatogram obtained with the reference substance solution or the spots in the chromatogram obtained with the test solution correspond in colour and position to the spots in the chromatogram obtained with the reference drug solution. D. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel 60F254.

VP V

Mobile phase: Chloroform - methanol - water (65 : 35 : 10). Shake well, use the lower layer. Test solution: To 1 g of the powder add 10 ml of methanol (50%) R, sonicate for 30 minutes, cool, filter. Reference drug solution: Prepare a solution of 1 g of the powder of caulis cum folium Lonicerae reference drug in the same manner as described under Test solution. Procedure: Apply separately to the plate 5 μl of each solution. After developing and removal of the plate, allow to dry in air. Spray with a 10% solution of sulfuric acid in ethanol R. Examine under visible light. The spots in the chromatogram obtained with the test solution correspond in colour and position to the spots in the chromatogram obtained with the reference drug solution.

Loss on drying Not more than 13.0% (Appendix 9.6, 1 g, 85 °C, 4 h). Total ash Not more than 9.0% (Appendix 9.8). Foreign matter Not more than 0.5% (Appendix 12.11). Extractives Not less than 12.0%, calculated with reference to the dried drug. Carry out the hot extraction method (Appendix 12.10), using ethanol (50%) R as the solvent. Preliminary processing Collect branches bearing leaves, remove foreign matter, cut into 2 - 5 cm long segments, dry in the shade or at a low temperature. Storage Store in dry and cool places, protected from mould and weevils CAULIS ET FOLIUM GYMNEMATIS SYLVESTRIS Dây thìa canh Sun- or heat-dried stems and leaves of Gymnema sylvestre (Retz.) R. Br. ex Schult., (Fam. Asclepiadaceae).

Description The intact drug, not cut into segments forming liana, upto 6 m long, 3 mm in diameter. Normally, cut into segments of 1.5 - 3 cm long. Becomes green after drying. Blade, oval opposite or elongated oval, 6 - 7 cm long, 2.5 - 5 cm wide, pointed apex with tip, consisting of 4 - 6 couples of lateral veins, shown clearly on lower surface, wrinkled after drying; petiole, 5 - 8 cm long. Transverse section Leaves Midrib: Upper surface slightly prominent. Lower epidermis consisting of one layer of round cells, sometimes

CAULIS ET FOLIUM GYMNEMATIS SYLVESTRIS

bearing multicellular non-glandular hairs; 3 - 5 lower collenchyma layers, thick-wall, located closely to the epidermis; phloem-xylem bundles consisting of internal wood vessels, phloem arch surrounding medulla, located in the middle of the main midrib; parenchyma consisting of ovoid, thin-walled cells, crystals of calcium oxalate in urchin form scattered in the midrib; upper collenchyma consisting of 3 - 4 layers of cells, structure similar to lower collenchymas; upper epidermis consisting of one layer of rectangular cells. Lamina: Upper and lower epidermis consisting of one layer of rectangle cells, arranged evenly, bearing multicellular non-glandular hairs. Palisade tissue composed of 2 - 3 rows of rectangular cells, arranged perpendicularly to the upper epidermis, covered one-fifths the thickness of the lamina. Spongy tissue located in mesophyll, composed of rounded cells, arranged irregularly and leave small openings. Phloem-xylem bundles of lateral veins and spiral vessels located closely to spongy and the palisade tissues. Stems: Transverse cut surface, round. Cork layer consisting of 2 - 3 rows of rectangle cells, arranged in concentric rows, numerous big shell holes. Young stems, epidermics layer consisting of one row of rectangular cells, arranged evenly in rows, with numerous multicellular non-glandular hairs; cortical parenchyma compose of numerous layers of ovoid, thin-walled cells; fibre bundles, arranged adjoiningly to each other or in concentrated masses around the stem; phloem-xylem composed of the phloem around medulla, surounded inner wood vessels; secondary xylem arranged in a circle, open small medullary rays; parenchyma layer, inner pith.

Powder Green in colour; odour mild aromatic. Multicellular nonglandular hairs; parenchymatous fragments; epidermis fragments bearing stomata; stomata cells; cork fragments consisting of thin-walled cells; crystals of calcium oxalate in urchin form; numerous vessel fragments: spiral, line and coin vessels; fibres and fibre bundles. Identification A. To 1 g of the powder in a big test tube, add 5 ml of water, boil slightly, filter while hot. Transfer the filtrate to another big test tube, add 10 ml of water. Shake vigorously and vertically for 2 minutes, a foam column about 4 cm in height is produced, persisting for at least 15 minutes. B. To 2 g of the powder in a big test tube, add 10 ml of ethanol (96%) R, heat at about 800C for 10 minutes, filter immediately. Evaporate the filtrate to dryness. Add 1 ml of chloroform R to the residue, shake to dissolve. Add 1 ml of sulfuric acid R, shake well. A red colour is produced. Loss on drying Not more than 13.0% (Appendix 9.6, 1 g, 85 °C, 3 hours).

1113

CAULIS ET RADIX FIBRAUREAE

Total ash Not more than 10.0% (Appendix 9.8). Foreign matter Not more than 1.0% (Appendix 12.11). Extractives Not less than 25.0%, calculated with reference to the dried drug. Carry out the hot extraction method (Appendix 12.10), using water as the solvent. Preliminary processing The drug is collected all the year round, dried under the sun or at a low temperature, cut into slices, 1.5 - 3 cm long. Stirbake with gentle heat until its colour turns yellow before use. Storage Store in a dry place, protected from moisture. CAULIS ET RADIX FIBRAUREAE Hoàng đằng (Thân và rễ) Sun-or heat-dried stems and roots of Fibraurea recisa Pierre and Fibraurea tinctoria Lour., (Fam. Menispermaceae).

Description Segments of stem and root straight or slightly curved cylinder, 10 - 30 cm long, 0.6 - 3 cm in diameter. Externally brown with several longitudinal wrinkles and scars of petioles (stem segments) or scars of rootlets (root segments). Transverse cut surface yellow, showing 3 distinct parts: cortex narrow, wood with medullary rays in spoke-like arrangement, pith in the centre rounded and narrow. Texture hard, difficult to break. Taste bitter. Transverse section Stems and roots of both species are similar in structure. Stems: Remains of cork consisting of numerous rows of rectangular cells, arranged evenly. Cortical parenchyma composed of thin-walled cells subrounded, ovoid or rectangular, scattered with sclerenchymatous cells, thick-walled, lumina large, striations numerous and distinct. Crystals of calcium oxalate cubic, rectangular or fusiform, occurring in or near sclerenchymatous cells. Sclerenchymatous ring continuous, sinuous, protruding and depresssed along side phloem-xylem bundles, consisting of thick-walled cells, lumina large, crystals of calcium oxalate also scattered. Phloem bundles lying adjacently to the sclerenchymatous ring, divided by narrow medullary rays, consisting of 2 - 3 rows of rectangular cells. Xylem bundles composed of numerous large wood vessels, separated by medullary rays, also scattered with some sclerenchymatous cells. Pith parenchyma consisting of rounded or polygonal cells. Roots: Remains of cork consisting of rectangular, thickwalled cells. Outer cambium composed of one row of 1114

VP V

thin-walled cells, arranged evenly. Cortical parenchyma composed of thin-walled cells. Sclerenchymatous ring continuous, consisting of thick-walled and lignified cells, with distinct striations, scatered with numerous cubic or fusiform crystals of calcium oxalate. Secondary phloem and xylem divided into 2 or 3 fan-wings. Each wing divided by broad medullary rays into several small parts.

Powder Yellow in colour. Examine under microscope: Sclerenchymatous cells rectangular, fusiform or subrounded, yellow. Fragments of dotted and scalariform vessels visible. Parenchymatous fragments containing starchy cells. Crystals of calcium oxalate cubic or parallelepiped. Starch granules rounded, campanulate or ovoid, several granules double compound, hilum distinct, 10 - 23 µm in diameter. Identification A. Examine a slice of the drug under ultraviolet light (366 nm), a bright yellow fluorescence is shown. B. Shake vigorously 0.1 g of the powder with 3 - 4 ml of a 1% solution of sulphuric acid R, filter. Transfer 2 ml of the filtrate to a test tube, add slowly 0.5 ml of chlorine water R along the tube wall, a red colour is formed at the interface. C. Macerate 0.2 g of the powder in 2 ml of ethanol (90%) R for 1 hour, filter. Apply 1 drop of the filtrate to a slide, add 1 drop of a 32% solution of nitric acid R. After 5 - 10 minutes, examine the slide under a microscope, yellow needle-shaped crystals are observed. D. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Toluene - ethyl acetate - isopropanol methanol - ammonia (6 : 3 : 1.5 : 1.5 : 0.5) Test solution: To 0.1 g of the powder, add 5 ml of ethanol (90%) R, boil on a water bath for 2 - 3 minutes and filter, use the filtrate as the test solution. Reference substance solution: Dissolve palmatine chloride RS in ethanol (90%) R to produce a solution containing 0.1 mg per ml. Reference drug solution: Prepare a solution of 0.1 g of the powder of Caulis et Radix Fibraureae reference drug in the same manner as described under Test solution. Procedure: Apply separately to the plate 20 µl of each solution. Develope in a chamber pre-saturated with ammonia vapour. After removal of the plate, dry at room temparature. Examine under ultra-violet light (366 nm). The chromatogram obtained with the test solution shows at least two yellow fluorescent spots, of which, one spot corresponds in colour and position to the spot due to palmatine chloride in the chromatogram obtained with the reference substance solution. Spray with Dragendorff reagent R. The chromatogram obtained with the test solution must have a spot which corresponds in position and orange-red colour to the spot due to palmatine chloride in the chromatogram obtained with the reference substance solution and must have spots corresponding in colour and

VP V

position to the spots in the chromatogram obtained with the reference drug solution.

Loss on drying Not more than 14.0% (Appendix 9.6, 1 g, 105 °C, 5 h). Total ash Not more than 8.0% (Appendix 9.8). Foreign matter Not more than 1.0% (Appendix 12.11). Extractives Not less than 17.0%, calculated with reference to the dried drug. Carry out the hot extraction method (Appendix 12.10), using a 1% solution of hydrochloric acid in methanol R as the solvent. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - 0.4% solution of phosphoric acid (32 : 68). Test solution: Weigh accurately 0.6 g of the powder (passed through a sieve No. 355) into a ground-glassstoppered conical flask, add 100.0 ml of a 1% solution of hydrochloric acid in methanol R, stopper the flask, weigh and allow to stand overnight, then heat on a water bath for 1 hour, allow to cool, weigh again and compensate for the loss of solvent with a 1% solution of hydrochloric acid in methanol R. Mix well and filter. Dilute 2.0 ml of the filtrate to 10.0 ml with a 1% solution of hydrochloric acid in methanol R and mix well. Filter through a membrane filter (0.45 μm). Use the filtrate as the test solution. Reference solution: Dissolve palmatin chloride RS, weighed accurately, in a 1% solution of hydrochloric acid in methanol R to produce a solution containing about 30 µg per ml. Chromatographic system: A column (25 cm × 4 mm) packed with stationary phase C (5 μm). Detector: A spectrophotometer set at 345 nm. Column temperature: 40 °C. Flow rate: 1 ml/min Volume of injection: 5 μl. Procedure: Inject the reference solution. The number of theoretical plates of the column is not less than 5000, calculated for the peak due to palmatin chloride. Inject separately the test solution and the reference solution. Use peak areas in the chromatograms obtained with the test solution and the reference solution, and the declared content of C21H22NO4Cl in palmatin chloride RS to calculate the percentage content of palmatin chloride (C21H22NO4Cl) in the drug. It contains not less than 2.0% of palmatin chloride (C21H22NO4Cl), calculated with reference to the dried drug.

CAULIS SPATHOLOBI SUBERECTI

Preliminary processing Roots and stems of Fibraurea are washed clean; scraped to remove the cork layer, cut into segments and dried under the sun or at a low temperature. Storage Store in dry places, protected from mould and weevils. CAULIS PERILLAE FRUTESCENSIS Tía tô (Thân) Sun-or heat-dried stems of Perilla frutescens (L.) Britt., (Fam. Lamiaceae).

Description Stems square, four angles obtuse, varying in size, 0.5 - 1.5 cm in diameter. Externally purplish-brown or dark purple, four sides with longitudinal furrows and fine longitudinal striations, nodes slightly swollen with opposite branch scars and leaf scars. Texture light and hard; fracture lobed. Slices 2 - 5 mm thick, usually long bevelled fusiform; wood yellowish-white; medullary rays fine and dense, radial; pith white and lax. Odour mildly aromatic; taste insipid. Water Not more than 12.0% (Appendix 12.13). Pesticide residue Benzene hexachloride (BHC): Not more than 0.2 ppm (Appendix 12.17). Preliminary processing In autumn, after fruit is ripe, cut the aerial parts of the plant, remove twigs and leaves and foreign matter, dry, or cut into sections or slices, then dry in the sun. Processing For unsliced stems, eliminate foreign matter, soak in water quickly, take out, wrap up thoroughly to soften, cut into sections or thick slices and dry in the sun. Storage Store in dry and cool places. CAULIS SPATHOLOBI SUBERECTI Kê huyết đằng (Thân), Huyết đằng

Sun-or heat-dried bevelled slices of climbing stems of Spatholobus suberectus Dunn (Fam. Fabaceae). Description Large, long, cylindrical stems or irregularly elliptical bevelled slices, 0.3 - 0.8 cm thick. Cork greyish-brown, sometimes greyish-white spots visible and appearing reddish-brown when the cork exfoliated. Transverse 1115

VP V

CAULIS TINOSPORAE SINENSIS

section showing reddish-brown or brown wood, expoessing numerous pores of vessels; phloem with resinous secretion, reddish-brown to blackish - brown, arranged alternately with wood forming 3 - 8 eccentric and crescent rings; pith inclined to one side. Texture dry and hard. Taste astringent.

Total ash Not more than 4.0% (Appendix 9.8).

Transverse section Transverse cut surface: Cork consisting of several layers of cells, containing brownish-red contents. Cortex relatively narrow, having groups of stone cells with lumen filled with brownish-red contents; parenchymatous cells containing prismatic crystals of calcium oxalate. Abnormal vascular bundles arranged by phloem alternating with xylem in several whorls. The outermost of phloem being a layer of sclerenchymatous cells, consisting of stone cells and fibre bundles; most rays pressed; secretory cells abundant, filled with brownish-red contents, usually several to 10 or more cells arranged tangentially into layers. Fibre bundles relatively abundant, non-lignified or slightly lignified, surrounded by cells containing prismatic crystals of calcium oxalate forming crystal fibres; the walls of crystal cells lignified and thickened; groups of stone cells scattered. Xylem rays sometimes containing brownish-red contents; most vessels single, subrounded, up to 400 µm in diameter, arranged sparsely; bundles of wood fibres also forming crystal fibres. Some wood parenchymatous cells containing reddish-brown contents.

Extractives Not less than 8.0%, calculated with reference to the dried drug. Carry out the hot extraction method (Appendix 12.10), using ethanol (96%) R as the solvent.

Identification Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Chloroform - methanol (30 : 1). Test solution: To 1 g of the coarse powder, add 100 ml of ethanol (96%) R, heat under a reflux condenser for 1 hour, filter, evaporate the filtrate to dryness. Dissolve the residue in 2 ml of methanol R, add 1 g of silica gel R, stir well, expel the solvent. Transfer to a column (1.0 cm in inner diameter) packed with 2 g of silica gel (75 - 150 µm in particle size). Pre-elute with 30 ml of petroleum ether (60 °C 90  °C) R. Elute with 40 ml of chloroform R, evaporate the chloroform eluate to dryness, dissolve the residue in 0.5 ml of chloroform R, as the test solution. Reference drug solution: Extract 1 g of the powder of caulis Spatholobi reference drug in the same manner as described under Test solution. Procedure: Apply separately 5 - 10 µl of each solution to the plate. After developing and removal of the plate, allow to dry in air. Examine under ultraviolet light at 254 nm. The fluorescent spots in the chromatogram obtained with the test solution correspond in colour and position to the fluorescent spots in the chromatogram obtained with the reference drug solution.

Sliced, sun- or heat-dried stems of Tinospora sinensis (Lour.) Merr. (Fam. Menispermaceae).

Loss on drying Not more than 13.0% (Appendix 9.6, 1 g, 105 °C, 5 h).

1116

Acid-insoluble ash Not more than 0.6% (Appendix 9.7).

Preliminary processing Collect the drug in autumn and winter, cut climbing stems, remove branches and leaves, cut into slices and dry in the sun. Processing For the crude drug of long cylindrical form, eliminate foreign matter, wash clean, soak in water and wrap up thoroughly to soften, cut into slices, and dry in the sun. Storage Store in cool and dry places, protected from mould and weevils. CAULIS TINOSPORAE SINENSIS Dây đau xương (Thân)

Description Stem slices dried, varying in thickness, usually 0.3 - 0.5 cm thick, 0.5 - 2 cm in diameter. Externally greyish brown or greyish green. Cork layer thin, shrunken when dried, easily stripped off. Lenticels numerous, prominent on the outer surface. Transverse cut surface ivory white or pale yellow. Cortical parenchyma thin. Wood part broad, spreading like wheel-spokes, medullary rays distinct. Pith in the middle small, rounded. Transverse section Older stems bearing a not very thick cork layer, with prominent lenticels. Cortical parenchyma less developed, sporadically scattered with large cells containing resin. Cortical parenchyma of young stems marked with fibrous masses, in older stems the cortical parenchyma scattered with small sclerenchymatous groups accompanied by rectangular or rhombic crystals of calcium oxalate. Outside the phloem-xylem mass, a ring of sclerenchyma; this ring continuous in young stems, but divided into arches covering each phloem-xylem bundle in older stems. Phloem-xylem separated by medullary rays. A mass of thin-walled cells existing between phloem-xylem bundle and sclerenchymatous arch. Phloem composed of thinwalled cells arranged radially. Cambium winding through phloem-xylem bundles. Secondary wood consisting of

CERA FLAVA

VP V

large vessels scattered in wood parenchyma. Medullary rays broad in older stems, narrow in younger stems, cells elongated in radial direction. Pith parenchyma intermingled with small sclerenchymatous masses containing crystals of calcium oxalate. Numerous starch granules left on transverse section.

Powder Grey in colour. Taste slightly bitter. Starch granules vary in form frequently ovoid. Crystals of calcium oxalate cubic, urchin-form. Sclerenchymatous cells in different shapes thick wall, distinct pit canals. Fragments coveying vessels dotted, recticulate. Identification To 3 g of the dried powder add 1 ml of a 10% solution of ammonia R, mix well. Add 25 ml of chloroform R, shake for 10 minutes, then macerate for 1 hour, filter. Transfer the filtrate to a separating funnel, extract with 5 ml of a 10% solution of sulfuric acid R. Divide the acidic layer into 3 test tubes. Test tube 1: Add 2 drops of Mayer reagent R, a milky white precipitate is produced. Test tube 2: Add 2-3 drops of Bouchardat reagent R, a brownish-red precipitate is produced. Test tube 3: Add 2-3 drops of a 1% solution of picric acid, a yellow precipitate is produced. Loss on drying Not more than 14.0% (Appendix 9.6, 1 g, 105 °C, 4 h). Foreign matter (Appendix 12.11). Black and rotten stems: Not more than 0.5%. Other foreign matters: Not more than 1.0%. Broken matter Pass through a sieve with a pore zise of 4 mm: Not more than 5.0% (Appendix 12.12). Preliminary processing Collect the crude drug year-round, eliminate foreign matter, grade according to size, cut into thin slices and dry in the sun or at low temperature. Storage Preserve in a dry place, protected from mould and weevils. CERA ALBA Sáp ong trắng White beeswax is obtained by bleaching yellow beeswax.

Description Irregular masses, varying in size, opaque white. Texture compact, harder and more fragile than yellow beeswax, honey-odourless, tasteless, insoluble in water, soluble in ethanol (96%) and hot ether.

Relative density About 0.96 at 20 °C (Appendix 6.5). Melting point 62 - 69 °C (Appendix 6.7). Fat, fatty acid, resin, soap Requirement and method are specified in “Cera flava”. Acid value 17 - 24. Carry out the method as described under “Cera flava”. Saponification value 80 - 100. Carry out the method as described under “Cera flava”. Storage Store in a cool and ventilated place, protected from high temperature. CERA FLAVA Sáp ong vàng The wax collected from the honeycomb of Apis cerana Fabr. or Apis mellifera L. or other Apis species, (Fam. Apidae).

Description Irregular pieces or lumps, varying in size, yellow or pale brown. Softened and twisted when rubbed. Texture becoming more fragile at lower temperatures. Odour with a tint of honey; tasteless. Insoluble in water, partly soluble in hot ethanol (96%); soluble in hot ether, in chloroform, fatty oil and volatile oil. Relative density About 0.960 - 0.966 at 20 °C (Appendix 6.5). Melting point 62 - 66 °C (Appendix 6.7). Fat, fatty acid, resin, soap To 2 g of the substance being examined in a 250 ml beaker, add 70 ml of 3.5 N sodium hydroxide R, boil gently for 30 minutes and compensate the water lost while heating, cool at room temperature for about 2 hours. The liquid must be clear or little translucent. Filter through a plug of glass wool, acidify the filtrate with hydrochloric acid R, the filtrate must not be opaque or precipitated. Acid value 17 - 24 (Appendix 7.2). Weigh accurately about 3 g of the substance being examined, put into a 250 ml conical flask, add 50 ml of ethanol R having been neutralised by 0.1 N potassium hydroxide R, heat until the substance is completely 1117

COLLA CORNUS CERVI

dissolved, shake well, add 0.5 ml of phenolphtalein R and titrate with 0.1 N potassium hydroxide in ethanol VS until a pale pink colour which persists for 30 seconds is obtained. Calculate the acid value from the expression: n × 5.61 Acid value = Amount of sample (g) Where: n is volume (ml) of 0.1 N ethanolic potassium hydroxide VS required.

Saponification value 80 - 100 (Appendix 7.7). Test sample: Weigh accurately about 3 g of the substance being examined, put into a 250 ml ground stoppered conical flask, add 25.0 ml of 0.5 N potassium hydroxide in ethanol VS and 50 ml of ethanol (96%) R, boil under a reflux condenser in a water bath for 4 hours (the obtained mixture will not be opaque when dilute with water), allow to cool, dilute with 25 ml of freshly boiled and allow to cool water. Add 5 drops of phenolphthalein solution R. Titrate with 0.5 N hydrochloric acid VS until the solution becomes colourless. Carry out a blank titration at the same time under the same condition. Calculate the saponification value from the expression: (b - a) × 28.05 Saponification value = Amount of sample (g) where: a: volume (ml) of 0.5 N hydrochloric acid VS required in the sample titration. b: volume (ml) of 0.5 N hydrochloric acid VS required in the blank titration. Storage Store in a dry and cool place, protected from high temperature. COLLA CORNUS CERVI Cao gạc Hươu, Cao Ban long, Lộc giác giao The drug is a solid glue prepared from deerhorn by decoction and concentration.

Procedure Cut the deerhorn into pieces, soak in cold water and rinse until clear (the washing is clear). Decoct with water several times, filter, combine the filtrates (or add a small quantity of fine powder of alum), allow to stand and filter. Concentrate the glutinous filtrate (or add a quantity of rice wine, crystal sugar, soybean oil) to thick glue, allow to cool and congeal, cut into pieces, and dry in the air. Description Square flat pieces, 3 - 4 cm long and 0.6 cm thick, yellowish-brown or reddish-brown, translucent, some with a yellowish-white foam layer in the upper part; texture 1118

VP V

fragile, easily broken; fracture bright; taste slightly sweet.

Water Not more than 15.0%. Weigh accurately about 1 g of drug (cut into pieces), dissolve in 2 ml of hot water, evaporate on a water bath to dryness, keep the thickness is not over than 2 - 3 mm, carry out the method for determination of water (Appendix 9.6, 105 °C, 5 hours). Total ash Not more than 1.0% (Appendix 9.8), determined on 1.0 g. Heavy metals Not more than 30 ppm. Carry out the limit test for heavy metals (Appendix 9.4.8, method 3), using the residue obtained in the test for Total ash. Prepare the reference solution using 3.0 ml of lead standard solution (10 ppm Pb) R. Arsenic Not more than 3 ppm. To 1 g of drug (cut into pieces), add 1 g of calcium hydroxide R and a small quantity of water, stir well. Evaporate to dryness, ignite gently at first and then incinerate at 500 - 600 °C until an almost white residue is obtained. Allow to cool, dissolve the residue in 5 ml of hydrochloric acid R and 2 ml of water, carry out the limit test for arsenic (Appendix 9.4.2, method A). Water-insoluble matter Not less than 2.0%. Weigh accurately about 1.0 g of drug (cut into pieces), add 10 ml of water and heat to dissolve. Transfer to a centrifuge tube, previously dried at 105 °C to constant mass. Weigh and centrifuge, discard the oil slick on tube wall and the supernatant layer. Add warm water to volume along the tube wall, stir well and centrifuge, discard the oil slick on tube wall and the supernatant layer. Repeat the process of washing with warm water three times. Heat the centrifuge tube at 105 °C for 2 hours, allow to cool in a desiccator for 30 minutes, weigh and calculate in percentage of waterinsoluble matters. Microbial contamination Acceptance criteria of provision for oral dosage forms containing raw materials of natural (Appendix 13.6). Assay Weigh accurately about 0.05 g of drug (cut into pieces), carry out the method for determination of nitrogen in organic compounds (Appendix 10.9, method 1). It contains not less than 10.0% of the total nitrogen calculated with reference to the dried drug. Storage Preserve in closed containers, store in a cool and dry place.

CORNU CERVI

VP V

CONCHA OSTREAE Mẫu lệ, vỏ hàu, vỏ hà

CONCRETIO SILICAE BAMBUSAE Thiên trúc hoàng, Phấn nứa

Sun-dried shells of several oysters such as Ostrea gigas Thunberg, Ostrea rivularis Gould or Ostrea talienwhanensis Crosse, (Fam. Ostreidae).

Dried masses of secretion in stems of Bambusa textilis McClure or Schizostachyum chinense Rendle, (Fam. Poaceae).

Description Shell of Ostrea gigas: In elongated pieces, consisting of two halves, dorsal and ventral ribs almost parallel, 10 - 50 cm long, 4 - 15 cm thick. The left shell is larger than right one; the right shell relatively small; scales hard, thick, arranged in even laminations or striated layers. Outer surface smooth or with several depressions, pale purple, greyish-white or yellowish-brown; inner surface porcelain white. Both sides of umbo without denticles. The left shell very deeply depressed with scales rougher and bigger than those of the right shell. Attachment surface of umbo small. Texture hard, heavy; fracture stratiform, white. Odourless; taste slightly salty. Shell of Ostrea rivularis: Shells usually 15 - 25 cm long, ovoid or triangular, the left shell larger than the right one, the right shell more even. Outer surface of the right shell slightly uneven, grey, purple, brown, yellow, with concentric scales. Young scales thin, fragile; old scales many-layered; inner surface white; the edge sometimes pale purple. Shell of Ostrea talienwhanensis: Triangular, dorsal and ventral edges V-shaped. Outer surface of the right shell pale yellow, with thin concentric scales undulating up and down; the inner surface shining white. Concentric scales of the left shell thick, hard, with distinct ribs radiating from umbo; inner surface concaved in shape of a box; hinge surface small.

Description Irregular pieces, varying in size and shape, greyishblue, slight yellow, greyish-white or white, transparent and somewhat lustrous. Texture hard, difficult to break, strongly hygroscopic. Odourless, with tongue-sticky feeling on tasting. Identification Mineralize completely about 1 g of the drug, dissolve the residue in water, filter. To 2 ml of the filtrate, add 2 ml of a 10% solution of ammonium molybdate R (dissolve 10 g of ammonium molybdate R in water, add sufficient water to produce 100 ml), shake well. Add 1 ml of a 8% solution of ferrous sulphate R, a blackish-brown is produced, then turns to a persistent blue after 5 minutes. Loss on drying Not more than 10.0% (Appendix 9.6, 1 g, 105 °C, 5 h). Foreign matter Not more than 0.5% (Appendix 12.11). Volume ratio Place gently 10 g of the powder (passed through a sieve No. 250) to a measuring cylinder, the volume is not less than 35 ml.

Loss on drying Not more than 5.0% (Appendix 9.6, 1 g, 105 °C, 4 h).

Water absorbing capacity To 5 g of the powder, add 50 ml of water. Allow to stand for a moment (about 3 minutes), filter through moistened filter paper, the filtrate is not more than 44 ml.

Foreign matter Not more than 1.0% (Appendix 12.11).

Total ash Not less than 80.0% ( Appendix 9.8).

Preliminary processing Possibly collect the oysters all the year round, remove the soft part, take and wash clean the shell, dry in the sun or at a low temperature.

Preliminary processing The drug is collected in autumn and winter. Take milky or transparent white masses of secretion in burnt bamboo section, removed foreign matter, dry in the sun or at a low temperature.

Processing Dry Concha Ostreae: Wash clean, then dry and grind into powder, or bake and grind into powder before use. Concha Ostreae (calcined): Take clean Concha Ostreae and place on a burning charcoal stove; calcinate until the drug turns whitish ashy and spongy, take out, and cool, grind it finely. Storage Store in dry places.

Storage Preserve in well closed containers; store in dry and cool places. CORNU CERVI Lộc giác, gạc hươu The drug is the old ossified antler or antler base which fallen off after the pilose antler is cut, of Cervus Nippon Temminck (Fam.Cervidae) 1119

VP V

CORNU CERVI DEGELATINATUM

Commonly known as “Gạc hươu sao” or “Mai hoa lộc giác” (the ossified antler of Hươu sao) and “Gốc gạc hươu rụng” or “Lộc giác thoát bàn” (the antler base).

Description Gạc hươu sao: Usually divided into 3 - 4 branches, 30 - 60 cm in length, 2.5 - 5 cm in diameter, the two sides symmetrical. The lateral branches mostly stretched toward two sides. The first branch is near the antler base, known as “Trân châu bàn” (pearl plate) and the second one relatively apart from the first one. The primary branch (the main branch) is divided two small branches. Externally yellowish-brown or greyish-brown, the apex of branch is greyish-white and glabrous, the middle and the lower parts usually contain knobs as known “cốt đinh” (bony nails), longitudinally arranged, discontinuous. Below of the base is prominent pearl plate as known “Trân châu bàn”. Texture hard, cross-section: out side is white, the center is grey, sparse small pores; Taste salty. Gốc gạc hươu rụng (Lộc giác thoát bàn): In helmet or flattened helmet, 3 - 6 cm in diameter. Pearl plate is 4.5 - 6.5 cm in diameter and 1.5 - 4 cm high. Externally greyish-brown or greyish- yellowish- brown, lustrous; the middle part with honeycomb-like pores; bottom surface flat, honeycomb-like, mostly yellowish-white or yellowish-brown. The margin of the pearl plate usually contains sparse small pores. The upper surface is slightly flat or irregular semi spheroidal. Texture hard; outer part of the section bony greyish-white; the central part is white. Taste slightly salty. Extractives Not less than 17.0%. Weigh accurately about 4 g of the coarse powder into a beaker, add 90 ml of water, heat the mixture to boiling and maintain gentle boiling for 1 hour (compensate for the loss of solvent with water), filter while hot, wash the residue with 10 ml of hot water, filter. Combine the filtrate and washing to a 100-ml volumetric flask, dilute with water to volume, shake well. Introduce 25.0 ml of this solution into a previously dried and weighed beaker, evaporate in a water-bath to dryness, heat the residue for 3 hours at 105 oC, allow to cool for 30 minutes in a desiccator, weigh. Calculate percentage content of the extractive. Preliminary processing Collect ossified antler in spring or antler base fallen in spring after the pilose antler was cut in the last year. Processing The drug is washed clean, sawed into sections, soaked in warm water, took out, cut into slices and dried in shade or pulverized into a coarse powder. Storage Store in a cool and dry place.

1120

CORNU CERVI DEGELATINATUM Lộc giác sương The ossified antler is decocted to eliminate the gelatin, sun- or heat- dried. The product is pulverized or crushed to produce white powder.

Description Long cylindrical or irregular pieces, and varying in size. The surface is white and powdery, often with longitudinal ridges, occasionally with grey or greyish-brown small dots. Light, texture crisp, the outer layer of fracture relatively dense, white or greyish-white, the inner layer is greyishbrown or greyish-yellow, with honeycomb-like pores. Hygrosocpical. Taste weak, sticky to teeth on chewing. Loss on drying Not more than 8.0% (Appendix 9.6, 2 g, 105 °C, 5 h). Preliminary processing The ossified antler is decocted to eliminate the gelatin. The product is taken out and dried. Processing Dried drug is broken to pieces before using. Storage Store in a dry place, protected from moisture, and preserve in closed containers. CORNU CERVI PANTOTRICHUM Lộc nhung, Nhung hươu Young unossified hairy antlers of male Cervus nippon Temminck, (Fam. Cervidae).

Description Pilose antler known as “sika deer pilose antlers”: cylindrical, branched. Antlers with one side branch commonly known as “double branches”. Main or big branches, 17 - 20 cm long; cut surface, 4 - 5 cm in diameter. Side branches arising at about 1 cm from the cut surface known as “side branches”, 9 - 15 cm long, slightly smaller than main branches in diameter. Outer skin reddishbrown or brown, usually lustrous, densely covered with brownish-yellow or reddish-yellow soft hairs, relatively thick at the upper end and sparse at the lower end, with a greyish-black vein at the base between the main and side branches, the skin and hairs sticking closely to each other. Cut surface yellowish-white, without bony element in outer part, densely perforated in central part. Texture light; odour slightly fishy; taste slightly salty. Antlers with two side branches commonly known as “triple branches”, main branches 23 - 33 cm long, smaller than those of “double branches” in diameter, slightly curved and flattened, tip slightly pointed, lower part usually bearing longitudinal ridged veins and lumps. Skin reddish-yellow, soft hairs relatively sparse and thick.

VP V

Identification A. To about 0.1 g of the powder, add 4 ml of water, heat for 15 minutes, allow to cool and filter. To 1 ml of the filtrate, add 3 drops of ninhydrin solution R, mix well, boil for several minutes, a bluish-violet colour is produced. To 1 ml of the filtrate, add 2 drops of a 10% solution of sodium hydroxide R, mix well, and add a 0.5% solution of cupric sulphate R dropwise, a bluish-violet colour is produced. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silical gel G. Mobile phase: n-Butanol - glacial acetic acid - water (3 : 1 : 1) Test solution: To 0.4 g of the powder, add 5 ml of ethanol (70%) R, sonicate for 15 minutes, filter, and use the filtrate as the test solution. Reference drug solution: Prepare a solution of 0.4 g of Cornu Cervi pantotrichum reference drug in the same manner as described under Test solution. Reference substance solution: Dissolve glycine RS in ethanol (70%) R to produce a solution containing 2 mg per ml. Procedure: Apply separately to the plate 8 µl of the test and drug reference solutions and 1 µl of the reference substance solution. Develope over a path of 12 - 13 cm and remove the plate, allow to dry in air, spray with a 2% solution of ninhydrin in acetone R and heat at 105 °C until the spots appear. Examine under visible light. The spots in the chromatogram obtained with the test solution correspond in colour and position to the spots in the chromatogram obtained with the reference drug solution and in which must have a spot corresponding in colour and position to the spot due to glycine in the chromatogram obtained with the reference substance solution. Preliminary processing Pilose antlers are usually collected in spring, saw the antlers, then wire the margin, hang over a burning charcoal stove, spray with moderate hot water, frequently turn over for gradually drying to make pilose antlers unbroken. Dry continuously for 3 - 4 days and nights to dryness, alternatively dry to plastic dryness, cut into slices and stir bake with gentle heat to dryness. Processing Cornu cervi pantotrichum (sliced): Take dried pilose antlers, burn a way soft hairs, scrape clean, wrap with clothing bands, pour hot ethanol into the small holes on the cut surface until softened thorougly or steam after being softened with ethanol, cut into round, thin slices, press to flatten the slices, and dry at a low temperature. Cornu cervi pantotrichum (powdered): Take dried pilose antlers, burn away soft hairs, scrape clean, cut into slivers and pulverize.

CORTEX ACANTHOPANACIS GRACILISTYLI

Storage Store in dry places, preserved in well closed with a desiccant inside containers, protected from weevils. CORTEX ACANTHOPANACIS GRACILISTYLI Ngũ gia bì hương (Vỏ rễ, Vỏ thân) Sun- or heat-dried root and stem bark of Acanthopanax gracilistylus W. W. Smith, (Fam. Araliaceae).

Description Bark fragments roll channel shape, various size, usually 3 to 7 cm in length, 0.3 to 1 cm in width, about 1 mm to 2 mm in thickness. Outer is thin cork, pale brown. The surface of fracture is irregular. Texture light, crispy, slightly porous. Slightly aromatic. Transverse section Stem bark transverse section is rectangular, from outer to inner consisting of: epidermis layer turns to cork containing many rectangular cells arranged into concentric and radial row. Nearly epidermis layer is collenchymas layer, structured by ovoid or oval, thick-walled cells. Cortial parenchyma consisting of thin-walled, polygonal cells; in cortical parenchyma, urchin-form crystal of calcium oxalate scattered. Large phloem, with cracked outside, medullary rays radial large with 1 to 5 rows of cells; secretory tubes clearly, surrounded by 4 to 11 secretory cells. Powder Colour pale yellow. The corks consisting of rectangular or polygonal, thick-walled, yellowish brown cells. Parenchyma structured by polygonal, big, thin-walled cells. A lot of sclerenchyma cells with rectangular shape, nearly round or polygonal, not branches, light yellow, thick-walled, scattered or together to large clusters. Sclerenchyma cells: rectangular, 40 µm to 110 µm in length, 30 µm to 50 µm in width, on wall with clearly horizontal grooves; Sclerenchyma cells are nearly round with 30 µm to 40 µm in size. Fibers scattered or together into bundles, thick-walled, narrow cavity. Urchin-form crystal of calcium oxalate with 10 µm to 38 µm in diameter. Dotted vessels. Identification A. To 5 g of the powder, add 20 ml of ethanol (96%) R, heat under a reflux condenser on a water bath for 15 minutes, cool, filter. Transfer 1 ml of the filtrate to a test tube, add 5 drops of acetic anhydride R, add carefully and gradually along the wall of the test tube 0.5 ml of sulfuric acid R. A reddish-brown colour is produced at the interface. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel 60F254. Mobile phase: Chloroform - methanol - water (7.5 : 2.5 : 0.5 ). Test solution: To 1 g of the powder, add 10 ml of a mixture 1121

VP V

CORTEX ACANTHOPANACIS TRIFOLIATI

of methanol - water (4 : 1), sonicate for 15 minutes, filter, use the filtrate as the test solution. Reference drug solution: Prepare a solution of 1 g of the powder of cortex Acanthopanacis gracilistyli reference drug in the same manner as described under Test solution. Reference substance solution: Alternatively, dissolve siringin RS in methanol R to obtain a solution containing 0.1 mg per ml. Procedure: Apply separately 10 µl of each solution to the plate. After the development and removal of the plate, dry in air, spay with a 10% solution of sulfuric acid in ethanol R, heat at 105 °C for 5 minutes. Examine under visible light or under ultraviolet light at 366 nm. The spots in the chromatogram obtained with the test solution correspond in colour and position to the spots in the chromatogram obtained with the reference drug solution or the chromatogram obtained with the test solution must have a spot that coressponds in colour and position to the spot due to siringin in the chromatogram obtained with the reference substance solution.

Loss on drying Not more than 13.0% (Appendix 9.6, 1 g, 105 °C, 5 h). Total ash  Not more than 12.0% (Appendix 9.8), determined on 1 g. Forgein matter Not more than 1% (Appendix12.11). Extractives Not less than 3.5%, calculated with reference to the dried drug. Carry out the hot extraction method (Appendix 12.10), using ethanol (96%) R as solvent. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Acetonitrile R. Mobile phase B: A 0.05M solution of sodium dihydrogen phosphate. Reference solution: Dissolve siringin RS in methanol R to obtain a solution containing 0.02 mg per ml. Test solution: Weigh accurately about 0.5 g of the powder (passed through a sieve No. 250) and put into a 100 ml-conical flask, add accurately 50 ml of a mixture of methanol - water (70 : 30), weigh, sonicate for 30 minutes, cool, weigh again and compensate for the loss of solvent with a mixture of methanol - water (70 : 30), filter through a paper filter, then pass through a 0.45-µm membrance filter. Chromatographic system: A column (25 cm × 4 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 265 nm. Flow rate: 0.5 ml per minute. Volume of injection: 10 µl. Procedure: Use the gradient elution described as follows: 1122

Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 50

10 → 30

90 → 70

50 - 60

30 → 100

70 → 0

Inject the reference solution. Maximum relative standard deviation of 2.0% after 6 injections calculated for the peak due to siringin. Inject separately the reference solution and the test solution. Use the peak areas due to siringin in the chromatogram obtained with the test solution, the reference solution and the declared content of C17H24O9 in siringin RS to calculate the percentage content of siringin in drug. It contains not less than 0.15% siringin (C17H24O9), calculated with reference to the dried drug.

Processing Peel the bark of stem or big branches in summer and autumn; scrape off the outer layer, dry under the sun or heat under 60 °C to dryness. Before use, wash clean, drain, cut into segments of 5 cm to 7 cm in length, dry under the sun or heat at 60 °C. For collecting the root bark, after digging up root, wash clean, peel the bark, scrape off the outer layer, cut into segments of 5 cm to 7 cm in length, dry under the sun or at heat at 60 °C. Storage Preserve in well moisture-proof containers, store in a dry and cool place, prevent from mold, fungi. CORTEX ACANTHOPANACIS TRIFOLIATI Ngũ gia bì gai (Vỏ rễ, vỏ thân) Sun- or heat-dried root and stem barks of Acanthopanax trifoliatus (L.) Merr., (Fam. Araliaceae).

Description Bark pieces semi-tubularly rolled, 10 - 20 cm long, 0.5 - 1 cm wide, about 1 - 3 mm thick. Outer surface containing thin, pale brownish-yellow cork; several places ragged, exposing the inner dark brown layer. Transverse cut surface uneven. Texture light, fragile and slightly spongy. Odour mildly aromatic. Transverse section Cork cells rectangular, occurring in several layers, superimposed into even radial lines. Pericambium consisting of one layer of rectangular cells. Parenchyma composed of distorted, thin-walled cells. Cortical parenchyma scattered with secretory tubes and urchinform crystals of calcium oxalate. Sclerenchymatous fibres arranged into sparse masses in an interrupted ring between parenchyma and phloem. Phloem part thick, with radial medullary rays. Cambium visible.

CORTEX CINNAMOMI

VP V

Powder Sclerenchymatous cells numerous, rectangular or polygonal, pale yellow in colour, walls very thick, pit canals distinct, singly or grouped into masses. Fibres with thick walls, pit canals distinct. Cells of cork fragments polygonal, with thick walls, pale yellow. Parenchymatous cells polygonal, thin - walled, filled with abundant, fine, polygonal starch granules, single or compound. Urchinform crystals of calcium oxalate, 12 - 40 µm in diameter. Identification Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: n-Butanol - ethanol - ammonia solution (7 : 2 : 5). (Ammonia solution: Mix 1 volume of ammonia R with 3 volumes of water). Test solution: Macerate 0.5 g of the powder in 5 ml of ethanol (80%) R, heat on a water bath for 15 minutes, and filter. Reference substance solution: Dissolve oleanolic acid RS in chloroform R to produce a solution containing 0.5 mg per ml. Procedure: Apply separately to the plate 20 µl of the test solution and 10 µl of the reference substance solution. After developing and removal of the plate, allow to dry at room temperature, spray with a 1% solution of vanillin R in a mixture of methanol R and phosphoric acid R (1 : 1). Heat the plate at 120 °C for 5 minutes. Examine under visible light. The chromatogram obtained with the test solution shows 3 violet spots, of which, 1 spot corresponds in colour and position to the spot due to oleanolic acid in the chromatogram obtained with the reference substance solution. Water Not more than 12.0% (Appendix 12.13), determined on 10 g of the slivered drug. Total ash Not more than 6.5% (Appendix 9.8). Acid-insoluble ash Not more than 4.0% (Appendix 9.7). Foreign matter Not more than 1.0% (Appendix 12.11). Preliminary processing Root barks and stem barks are collected in summer and autumn, wrapped up to produce aromatic smell, dried in the shade, in ventilated places, or at a low temperature (50 °C) to dryness. Storage Store in dry and cool places.

CORTEX CINNAMOMI Quế (Vỏ thân, vỏ cành) Sun-dried stem or branch barks of Cinnamomum cassia Presl. or some other Cinnamomum species (Cinnamomum zeylanicum Blume, Cinnamomum loureirii Nees.) (Fam, Lauraceae).

Description Cinnamomi cassia: Bark pieces usually tubularly rolled, 5 - 50 cm long, 1.5 - 10 cm wide, 1 - 8 mm thick. Outer surface brown to greyish-brown, bearing lenticels and leaf stalk remains (will not been seen in bark bearing thick cork). Inner surface reddish-brown to dark brown, smooth or slightly rough. Texture hard and fragile, easily broken; fracture uneven, fibrous. A transverse cut surface showing 2 layers: Outer layer brownish-yellow and slightly rough; inner layer reddish-brown, with short fibres; a yellowishbrown ring occurring between the two layers. Odour aromatic; taste hot, sweet and slightly pungent. Cinnamomi zeylanicum, bark usually thiner than that of C.cassia; inner layer, yellowish - brown; odour, slightly aromatic. Cinnamomi loureirii, cork layer brown. Outermost layer possibly scraped and leaving a reddish-brown or dark brown layer. Odour strongly aromatic. Texture fragile, easy to break. Fracture fibrous. Tranverse section Outermost cork containing several layers of fairly thickwalled cells, ragged or exfoliated at some places. Cortical parenchyma consisting of thin-walled cells, polygonal or flatened rectangular, containing minute starch granules. Parenchyma scattered with essential oil secretory cells, mucillage cells and fine needle crystals of calcium oxalate. Adjacently to cork layer, occurring sclerenchymatous cells, single or grouped into masses in cluster arrangement, polygonal or tapered, with evenly thick walls. Several sclerenchymatours cells thick-walled, in U-shape. Closely to phloem occurring a sclerenchymatous ring nearly continuous with thick-walled cells and narrow lumina. Secondary phloem abundantly developed, consisting of thin-walled cells alternated with several fibres which changed from cells, square or subrounded sectional, thick walled, lying sparsely. Secondary phloem divided into clusters by medullary rays which consisting of 1 - 5 rows of cells and extending very often up to cortical parenchyma, containing needlecrystals of calcium oxalate. Phloem also scattered with essential oil secretory cells, mucillage cells, cells containing starch granules as in cortical parenchyma. Powder Powder brownish - yellow or dark brown; odour aromatic; taste hot slightly sweet. Numerous pale yellow fibres, 200 - 400 µm long, 20 - 50 µm in diameter, thick - walled, lumen narrow. Fragments of sclerenchymatous cells in 2 types: one with oval or 1123

VP V

CORTEX EUCOMMIAE

rectangular cells, thick - walled, lumen large or narrow, pit canals distinct; the other consisting of U-shaped thick walled cells, lumen narrower, pit canals distinct. Sclerenchymatous cells often singly or aggregated into masses, 60 - 120 µm long, 30 - 50 µm wide. Fragments of parenchyma with thin - walled cells, containing starch granules. Starch granules fine, subrounded or polygonal, 6 - 15 µm in diameter, single or double, triple compound. Needle crystals of calcium oxalate often broken into short portions. Cork fragments brownish-yellow, consisting of thick walled polygonal cells.

Identification Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: n-Hexane - chloroform - ethyl acetate (4 : 1 : 1). Test solution: Shake 2.0 g of the powder (passed through a sieve No 250) with 10 ml of ether R for 3 minutes, filter. Reference substance solution: Dissolve cinnamic aldehyde RS in ether R to produce a solution containing 1 mg per ml. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 12 cm, remove the plate, dry in air, spray with 2,4-dinitrophenylhydrazine solution R. Examine under visible light. The chromatogram obtained with the test solution shows 5 orange spots, one of which corresponds in colour and position to the spot due to cinnamic aldehyde in the chromatogram obtained with the reference substance solution. Water Not more than 14.0% (Appendix 12.13), determined on 10 g of the coarse powder. Total ash Not more than 5.0% (Appendix 9.8). Foreign matter Not more than 1.0% (Appendix 12.11). Assay Carry out the method described under “Determination of volatile oil in herbal drugs” (Appendix 12.7). Weigh accurately about 20 g of the powder (passed through a sieve No 355), put into a 500 ml round bottom flask of the apparatus for volatile oil determination, add 200 ml of 0.1 N hydrochloric acid R. Transfer 0.5 ml of xylene R to the graduated tube of the apparatus, distill for 3 hours at the rate of 2.5 - 3.5 ml per minute. It contains not less than 1.0% of volatile oil, calculated with reference to the dried drug. Preliminary processing Collect the drug in april - may and september - october, choose the cinnamon trees of over 5 years old (the most perennial tree is the best one) to strip the bark. Before stripping cassia bark, tie bamboo tapes around the stem and large branches to divide them into sections, at a 1124

distance of 40 - 50 cm long, to cut the bark evenly. Use a sharp and pointed knife to cut transversely to half stems or half branches, then cut longitudinally each section of bark. Each time stripping, take only the bark of one half side, leave the other half side regenerating. After that, strip the barks off with a thin and pointed neohouzeaua knife, sort them in categories. Take care when stripping off the barks do not let bark remain in wood. The large thick cassia barks are wrapped up, soaked for a day in water, washed clean and left until water is drained off, then placed in a bamboo basket, lined and covered with a 5 cm layer of banana leaves, for wrapping up for 3 days in summer or 7 days in winter. The barks are turned upside down every day to keep even hot temperature. After that the barks are taken out and soaked in water for 1 hour. Pick up the barks from water and place on a neohouzeaua wattle. Press and flatten the barks with another wattle placed over them, then expose the barks in a dry and cool place until dryish. At last, the cinnamon bark is submitted to shaping. Each cinnamon strip is tied around a straight cylindrical bamboo tube. During shaping time, the cinnamon strip is untied and wiped inside, twice a day, and tied around the bamboo tube again until the strip is dry. The wrapping up and drying time of cinnamon bark lasts about 15 - 16 days in summer or one month in rainy season and sometimes still longer.

Processing Eliminate foreign matter, remove subereous layers, grind into powder to make pills or powder; for combined decoction, grind the bark in the decoction for oral administration Storage Preserve in well closed containers, store in dry and cool places. To protect from flavour loss of cinnamon bark, smooth beeswax on the two ends of cinnamon strips, then wrap in polyethylene bags and preserve in well closed buckets, store in dry and cool places. CORTEX EUCOMMIAE Đỗ trọng (Vỏ thân) Sun- or heat-dried stem barks of Eucommia ulmoides Oliv., (Fam. Eucommiaceae).

Description Flat pieces or the two edges slightly incurved, irregular varying in size, 0.2 - 0.7 cm thick, ash-grey. Outer surface scabrous, with numerous longitudinal wrinkles and branchlet traces. Inner surface dark purple, smooth. Texture fragile, easily broken; fracture linked up by fine, dense, silvery and elastic threads of gutta-percha. Taste slightly bitter.

VP V

CORTEX EUCOMMIAE

Transverse section Under a microscope, a transverse section, from outermost to innermost reveals: Cork thick, cracked at some places, consisting of flattened suberized cells arranged in concentric rings and radial rows. Cortical parenchyma consisting of numerous rows of cells, outer cells usually pressed flat. Sclerenchyma scattered in masses in cortical parenchyma and even in phloem. Secondary phloem thick, with fibrous masses. Several cells containing guttapercha scattered in all cortical parenchyma, phloem and sclerenchyma. Medullary rays with 2 - 3 rows of cells, running tortuously, sinuously from cambium to cortical parenchyma. Innermost being cambium.

of 3/4 plate and removal of the plate, dry in air. Spray with a mixture of sulphuric acid R and water R (20 : 80) and heat at 120 oC until the spots appear. Examine under visible light. The chromatogram obtained with the test solution exhibits a spot corresponding in colour and position to the spot due to pinoresinol diglucoside in the chromatogram obtained with the reference substance solution or the spots in the chromatogram obtained with the test solution correspond in colour and position to the spots in the chromatogram obtained with the reference drug solution.

Powder Powder greyish-brown; odourless; taste slightly bitter. Under a microscope: Cork fragments consisting of polygonal cells with unevenly thickened, lignified and finely pitted walls. Cork fragments consisting of rectangular cells with wall thickened on three sides and relative thin on one side, pit canals distinct. Numerous gutta-percha threads long, thin, tortuous, concentrated into opaque white masses or stretching like strings, the surface granular. Sclerenchymatous fragments consisting of yellow, elongated or oval cells, lumina narrow; pit canals distinct. Stone cells numerous, mostly in groups, rectangular to sub-rounded, elongated-rectangular or irregular, thick-walled, some containing resinous masses.

Foreign matter Not more than 1.0% (Appendix 12.11).

Identification A. Macerate 1 g of the powder in 10 ml of chloroform R for 2 h, filter. Evaporate the filtrate to dryness, add 1 ml of ethanol (96%) R, allow to stand for about 5 min. An elastic film is produced. B. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel GF254 . Mobile phase: Dichloromethane - methanol - formic acid (3 : 1 : 0.1). Test solution: To 2.5 g of the powder, add 30 ml of methanol R, sonicate for 30 min. Filter and evaporate the filtrate to dryness under reduced pressure in a rotary evaporator. Dissolve the residue in 20 ml of water. Transfer the aqueous solution to a separating funnel. Extract with 50 ml of dichloromethane R and discard the dichloromethane layer. Extract the aqueous layer with 50 mL of n-butanol R. Evaporate the n-butanol extract to dryness under reduced pressure. Dissolve the residue in 1 ml of methanol R. Reference substance solution: Dissolve pinoresinol diglucoside RS in methanol to produce a solution containing 1 mg/ml. Reference drug solution: Alternatively, prepare a solution of 2.5 g of Cortex Eucommiae reference drug in the same manner as described under Test solution. Procedure: Apply separately to the plate 5 µl of each solution. After developing the chromatogram over a path

Loss on drying Not more than 10.0% (Appendix 9.6, 1 g, 70 °C, 5 h).

Total ash Not more than 8.5% (Appendix 9.8). Acid-insoluble ash Not more than 6.0% (Appendix 9.7). Extractives Not less than 11.0% , calcualated with reference to dried drug. Carry out the hot extraction method (Appendix 12.10), using ethanol (75%) R as the solvent. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Water. Mobile phase B: Acetonitrile R. Test solution: Transfer about 0.4 g of  the fine powder (passed through a sieve with a pore size of 0.850 mm), accurately weighed, to a 100-ml stopper conical flask, add 10.0 ml of ethanol (50%) R, stopper. Sonicate for 30 min. Centifuge for 5 min. Filter through a membrane filter of 0.45-µm and transfer the filtrate to a 25-ml volumetric flask. Repeat the extraction once more. Combine the filtrate into the 25 ml volumetric flask and dilute to volume with ethanol (50%) R. Mix well and filter through a membrane filter of 0.45-µm. Reference solution: Dissolve pinoresinol diglucoside RS in methanol R to produce a solution containing 1 mg/ml. Dilute this solution in methanol R to obtain reference solutions containing with each ml about 1, 10, 50, 100, and 200 μg of pinoresinol diglucoside. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm) (RP18 column is suitable). Detector: A spectrophotometer set at 228 nm. Flow rate: 1.0 ml/min. Volume of injection: 5 µl. Procedure: Use gradient elution as follows:

1125

VP V

CORTEX HOLARRHENAE

Time (min) 0 - 20

Mobile phase A Mobile phase B (% v/v) (% v/v) 90 → 80

10 → 20

Elution

CORTEX HOLARRHENAE Mộc hoa trắng, Mực hoa trắng, Thừng mực lá to

Linear gradient

Sun- or heat-dried barks of Holarrhena pubescens (Buch. - Ham.) Wall. ex G. Don., Syn. Holarrhena antidysenteria (Roxb. ex Flem.) A. DC., (Fam. Apocynaceae).

System suitability: Perform five replicate injections, each of 5 μL of pinoresinol diglucoside reference solution (50 μg/ml). The relative standard diviation of the peak areas of pinoresinol diglucoside should not be more than 5.0%; the retention time of pinoresinol diglucoside peak should not be more than 2.0%; the column efficiency, determined on pinoresinol diglucoside peak, should not be less than 50000 theoretical plates. The resolution between pinoresinol diglucoside peak and the adjacent peak in the chromatogram obtained with the test solution should not be less than 1.5. Calibration curve: Inject a series of pinoresinol diglucoside reference solution and record the chromatograms. Plot the peak areas of pinoresinol diglucoside against the corresponding concentrations of pinoresinol diglucoside reference solution. Inject the test solution and record the chromatogram. Identify pinoresinol diglucoside peak in the chromatogram obtained with the test solution by comparing its retention time with that in the chromatogram obtained with the reference solution. The retention times of pinoresinol diglucoside peaks from the two chromatograms should not differ by more than 5.0%. Calculate the percentage content of pinoresinol diglucoside in the sample using the chromatograms obtained with the test solution, the calibration curve established and the declared content of C32H42O16 in pinoresinol diglucoside RS. It contains not less than 0.10% of pinoresinol diglucoside (C32H42O16), calculated with reference to the dried drug. Preliminary processing The crude drug is collected from april to june, the bark is stripped off, removed from the coarse outer layer, piled up until the inner surface becomes blackish-purplish-brown and dried in the sun. Processing Cortex Eucommiae: Scrape off the remains of coarse outer layer, wash, cut into pieces or slivers with remaining guttapercha threads and dry. Use raw drug or processed drug. Cortex Eucommiae (processed with salt): soak the pieces of Cortex Eucommiae with remaining gutta - percha threads in salt solution for 2 h (use 30 g of salt in 200 ml of water per kg of Cortex Eucommiae); stir-bake until the gutta-percha threads broken off, or until the outer surface charred. When breaking the bark, the elasticity of guttapercha threads is weaker than that before baking. Taste slightly salty. Storage Store in ventilated and dry places. 1126

Description Bark pieces slightly curved, varying in length, 0.2 - 1.5 cm thick. Outer surface dark brown, scattered with grayishwhite patches, densely nodulate, easily exfoliated. Inner surface smooth, pale yellow or brownish-yellow. Texture easily broken; fracture rough, exposing numerous distinct superposed layers. Transverse cut surface showing thin parenchyma, dark red-brown; thick phloem, pale yellow, arranged in many gravelly layers. Odourless; taste very bitter. Transverse section Cork layer consisting of 3 - 5 rows of rectangular cells. Cortical parenchyma dark red-brown. Secondary phloem layer heavily thick, alternating with sclerenchymatous masses which arranged in numerous strata with latex vessels. Numerous cubic crystals of calcium oxalate occurring adjacently to each fiber mass. Medullar ray composed of 1 - 2 rows of thin-walled cells, radially elongated. Cambium lying innermost. Powder Parenchymatous fragments consisting of thinwalled, polygonal cells. Cork fragments pale brown. Sclerenchymatous cells arranged separately or in groups, pale yellow, polygonal, thick-walled, with large lumina and distinct pit canals. Crystals of calcium oxalate parallelepiped, about 40 µm long, 30 µm wide. Vascular fragments visible. Starch granules ovate-oblong, with distinct hila. Identification A. Moisten 1 g of the powder with ammonia R, add 10 ml of chloroform R, shake, tightly covered, macerate for 12 hours. Filter, transfer the filtrate to a separating funnel, add 5 ml of 1M hydrochloric acid R, shake well. Collect the acid layer and divide into three test tubes: Tube 1: Add 2 - 3 drops of Mayer reagent R, a white precipitate is produced. Tube 2: Add 2 - 3 drops of Dragendorff reagent R, a reddish orange precipitate is appeared. Tube 3: Add 2 - 3 drops of Bouchardat reagent R, a brown precipitate is produced. B. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel G. Mobile phase: Chloroform - methanol - 10% solution of ammonium hydroxide (50 : 9 : 1). Test solution: Moisten 5 g of the powder with ammonia R, tightly covered, macerate for 12 hours, transfer to a ground-glass stoppered conical flask, add 20 ml chloroform R, heat under a reflux condenser on a warm water bath for

VP V

15 minutes, allow to cool, filter. Evaporate the filtrate on a water bath to dryness. Dissolve the residue in 1 ml of chloroform R. Reference substance solution: Dissolve conessine RS in methanol R to produce a solution containting 1 mg/ml. Reference drug solution: Alternatively, prepare a solution of 5 g of Cortex Holarrhenae reference drug in the same manner as described under Test solution. Procedure: Apply separately to the plate 10 µl of each solution. After developing and removal of the plate, dry in air, spray with Dragendorff reagent R. The spots in the chromatogram obtained with the test solution correspond in colour and postion to the spots obtained with the reference drug solution or the chromatogram obtained with test solution must have a spot corresponding in colour and position to the spot due to conessine in the chromatogram obtained with the reference substance solution.

Loss on drying Not more than 13.0% (Appendix 9.6, 1g, 105 °C, 4 h). Total ash Not more than 9.0% (Appendix 9.8). Hydrochloric acid-insoluble ash Not more than 5.5% (Appendix 9.7). Heavy metal Not more than: 10 ppm of Pb, 2 ppm of Cd, 0.5 ppm of Hg, 3 ppm of As (Appendix 9.4.11). Assay Weigh accurately about 5 g of the powder (passed through a sieve No 355), moisten with 5 ml of 2 M sodium hydroxide R, allow to stand for 1 hour, spread at an airy area to dry. Extract alkaloids with mixture of ethanol - chloroform (1 : 3) in a Soxhlet’s extractor until alkaloid is compeletely extracted (check with Mayer reagent R). Shake the obtained extractive with 2 N hydrochloric acid R, each of 20, 20, 10, 10 and 10 ml. Combine the acid extracts, alkalize slowly to pH 9 - 10 with ammonia R. Extract the alkaline mixture with chloroform R five times, each of 20, 20, 10, 10 and 10 ml. Before the last extraction, add 1 ml of 2 M sodium hydroxide R to aqueous layer. Transfer 10 ml of distilled water to the separating funnel, wash separately the chloroform extracts, each twice. Combine the chloroform extracts, add 20.0 ml of 0.1 N sulfuric acid VS, shake carefully for 5 minutes. Transfer the acid layer to a conical flask, wash the chloroform layer with distilled water twice, each of 10 ml, combine the washings and transfer to above conical flask. Add 3 drops of a mixture of indicator solution (see the note) and titrate with 0.1 N sodium hydroxide VS until the colour of solution changes to light blue. Prepare a blank sample as following: To 20 ml of 0.1N sulfuric acid VS, add 20 ml of water and 3 drops of the

CORTEX MAGNOLIAE OFFICINALIS

mixture of indicator solution, titrate with 0.1N sodium hydroxide VS until the colour of solution changes to light blue. 1 ml of 0.1 N sulfuric acid VS is equivalent to 0.017829 g of alkaloid. Calculate the percentage content (m/m) of total alkaloid from the expression: (n’ – n) × 1.7829 P Where: n’: volume of 0.1 N sodium hydroxide VS used in blank, in ml, n: volume of 0.1 N sodium hydroxide VS used in sample, in ml, P: weight of drug, in g (which subtracted the water content). Drug contains not less than 1.0% of alkaloid, based on conessine (C24H10O2) and calculated with reference to the dried drug.

Mixture of indicator solution: Mix 13 ml of methylene blue solution (dissolve 0.15 g of methylene blue R in 100 ml of ethanol R) and methyl red solution (dissolve 0.04 g of methyl red R in 70 ml of ethanol R and 25 ml of water) to produce 100 ml.

Preliminary processing Collect the drug in dry weather, strip off and take stem barks, dry in the sun or at a low temperature. Storage Store in dry, cool and ventilated places, protected from mould and weevils. CORTEX MAGNOLIAE OFFICINALIS Hậu phác (Vỏ) Sun- or heat-dried barks of stems, roots and branches of Magnolia officinalis Rehd. et Wils. or Magnolia officinalis Rehd. et Wils var. biloba Rehd. et Wils. (Fam. Magnoliaceae).

Description Stem barks: Dry barks, singly or double tubularly rolled, 30 - 35 cm long, 0.2 - 0.7 cm thick, usually known as “ống hậu phác” (Magnolia tube). The end near the root spread out like a bell, 13 - 25 cm long, 0.3 - 0.8 cm thick, commonly known as “Hoa đồng phác”. Outer surface greyish-brown, rough, sometimes scaly, easily exfoliated, with distinct elliptical lenticels and longitudinal wrinkles. Appearing yellowish-brown when the coarse outer layer scraped off. Inner surface purplish- brown or dark purplishbrown, relatively smooth, with fine longitudinal striations, exhibiting distinct oily traces on scratching. Texture hard, difficult to break. Fracture scabrous, granular, greyishbrown in outer layer, purplish-brown or brown in inner layer, oily, sometimes small bright spots visible. Odour, aromatic; taste pungent, slightly bitter. 1127

VP V

CORTEX MORI ALBAE RADICIS

Root barks (Căn phác): Tubularly rolled or irregularly pieced, some twisted like chicken intestines, known as “Kê trường phác”. Texture hard, easily broken; fracture fibrous. Branch barks (Chi phác): Tubularly rolled, 10 - 20 cm long, 0.1 - 0.2 cm thick. Texture fragile, easily broken; fracture fibrous.

Transverse section Cork layer consisting of over 10 rows of cells, sometimes rhytidome visible. The outer side of cortex showing a ring of sclerenchymatous cells and the inner side scattered with numerous oil cells and groups of sclerenchymatous cells. Phloem rays consisting of 1 - 3 rows of large cells; fibres mostly arranged in bundles gathering in pericyclic part; oil cells scattered. Powder Brown in colour. Fibres numerous, 15 - 32 µm in diameter, walls strongly thickened, sometimes undulate or serrated at one side, lignified, pit canals indistinct. Sclerenchymatous cells square, elliptical, ovoid or irregularly branched, 11 - 65 µm in diameter, sometimes with distinct striations. Oil cells, elliptical or subrounded, 50 - 85 µm in diameter, containing yellowish-brown oily contents. Cork fragment consisting of rectangular cells, thick-walled, brownishyellow. Identification Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Cyclohexane - ethyl acetate - acetone (9 : 1 : 0.5). Test solution: To 0.5 g of the powder, add 5 ml of methanol R, shake for 30 minutes, filter. Reference substance solution: Prepare a 0.1% solution of magnolol RS and honokiol RS in methanol R. Reference drug solution: Alternatively, prepare a solution of 0.5 g of Cortex Magnolia officinalis reference drug in the same manner as described under Test solution. Procedure: Apply separately to the plate 5 µl of each solution. After developing and removal of the plate, dry in air, spray with a 10% solution of sulphuric acid in ethanol R. Heat at 100 °C for 10 minutes. Examine under visible light. The spots in the chromatogram obtained with the test solution correspond in colour and position to the spots in the chromatogram obtained with the reference drug solution or the chromatogram obtained with test solution must have two spots corresponding in colour and position to the spots due to magnolol and honokiol in the chromatogram obtained with the reference substance solution. Water Not more than 15.0% (Appendix 12.13). Determined on 10 g of the drug cut to the small pieces. 1128

Foreign matter (Appendix 12.11) Cork bark: Not more than 2.0%, Other foreign matter: Not more than 1.0%. Total ash Not more than 6.0% (Appendix 9.8). Extractives Not less than 5.0%, calculated with referece to the dried drug. Carry out the cold extraction method (Appendix 12.10), using ethanol (70%) R as the solvent. Preliminary processing The drug is collected from april to june; root barks, stem barks and branch barks are stripped off. Root and branch barks are dried in the shade. Stem barks are put in boiling water and boiled quickly. Then taken out and piled up to sweat in a wet place until the inner surface becomes purplish-brown or brown, steamed to be softened, taken out and rolled into a pipe, then dried. Processing Cortex Magnoliae officinalis (sliced): Scrape off the cork outer layer, wash clean, soften thoroughly, cut into slices and dry in the sun. Cortex Magnoliae officinalis (processed with ginger): Crush the fresh clean ginger to pasty, press to get juice. Add some water to the residue, and press once more, mix well the juices. Add ginger juice to the sliced Cortex Magnoliae officinalis, mix well until the drug is absorbed completely. Stir-bake with gentle heat to dryness, slices curved, fracture fibrous and purplish-brown. Use 10 kg of fresh ginger or 3 kg of dried ginger per 100 kg of Cortex Magnoliae officinalis. Storage Store in a dry and cool place, preserve in well-closed containers, protected from flavour loss. CORTEX MORI ALBAE RADICIS Dâu (Vỏ rễ), Tang bạch bì, Vỏ rễ dâu Scraped clear of cork then sun - or heat - dried root barks of Morus alba L., (Fam. Moraceae).

Description Root barks tubular, semi - tubular rolled with two margins incurved or flattened or twisted pieced, varying in length and wide, 1 - 4 mm thick. Outer surface white or pale yellow, relatively smooth; some yellow or brownishyellow remains of scaly bark. Inner surface pale yellow or greyish-yellow, with fine longitudinal striations. Texture light and tough, strongly fibrous, uneasily broken transversely, but easily stripped longitudinally into small bands. Odour mild; taste slightly sweet.

CORTEX OROXYLI

VP V

Transverse section Large phloem containing 2 - 4 layers of cells. Laticiferous tubes scattered. Fibres scattered singly or grouped in bundles, walls unlignified or slightly lignified. Parenchymatous cells containing starch granules, sometimes containing prismatic crystals of calcium oxalate. Groups of sclerenchymatous cells intermingled with stone cells, scattered in old root bark; most of these cells containing prismatic crystals of calcium oxalate. Powder Pale greyish-yellow in colour, odour slightly aromatic. fibres numerous, mostly broken, 13 - 26 µm in diameter, thick-walled, unlignified or slightly lignified. Cubic crystals of calcium oxalate, 11 - 32 µm in diameter. Stone cells subrounded, rectangular or irregular, 22 - 52 µm in diameter, relatively or extremely thick-walled, with distinct pits and pit-canals, sometimes containing cubic crystals of calcium oxalate. Starch granules numerous, subrounded, 4 - 16 µm in diameter scattered or grouped in bundles. Remains of cork fragments yellow, containing polygonal cells. Identification A. Heat under a reflux condenser 1 g of the powder in 20 ml of n-hexane R on a water bath for 15 minutes, filter. Evaporate the filtrate to dryness, dissolve the residue in 10 ml of chloroform R. Transfer 0.5 ml of the chloroform solution to a test tube, add 0.5 ml of acetic anhydride R, slowly and carefully add 0.5 ml of sulphuric acid R to make two layers. A reddish-brown colour develops at the zone of contact. B. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel 60F254. Mobile phase: Acetic acid. Test solution: Place 2 g of the powder in a conical flask, add 20 ml of the saturated solution of sodium carbonate R, sonicate for 20 minutes, filter. Adjust the pH of the filtrate to 1 - 2 with dilute hydrochloric acid R. Allow to stand for 30 min and filter. Extract with two 10 ml quantities of ethyl acetate R. Combine the ethyl acetate extracts and evaporate to dryness. Dissolve the residue in 1 ml of methanol R. Reference drug solution: Prepare a solution of 2 g of the powder of Cortex Mori alba radicis reference drug in the same manner as described under Test solution. Procedure: Apply separately to the plate 5 µl of each solution. Develope over a path of 10 cm, remove of the plate, dry in air. Examine under ultraviolet light (366 nm). Two fluorescent spots in the chromatogram obtained with the test solution correspond in position and colour to the spots in the chromatogram obtained with the reference drug solution. Loss on drying Not more than 12.0% (Appendix 9.6, 1 g, 105 °C, 5 hours).

Total ash Not more than 9.0% (Appendix 9.8). Foreign matter Not more than 1.0% (Appendix 12.11). Preliminary processing The drug is collected in late autumn when leaves are falling off and in early spring before germination. Roots are lifted, washed clean, removed from the yellowishbrown cork, cut longitudinally. Ivory white root barks are stripped off, washed clean; dried under the sun or at a low temperature. Processing Dried Cortex Mori: Wash clean, wrap up until softish, strip into fibres, dry in the sun or at a low temperature. Honey Cortex Mori (processed with honey): Add the slivers of Cortex Mori to refined honey, mix carefully; wrap up until honey is absorbed; then put them into a pot and stir bake with gentle heat until become yellow and no more sticky to fingers, take out and cool; using 2 kg of refined honey per 10 kg of mulberry root bark. Storage Store in a ventilated and dry place, protect from mould and weevils. CORTEX OROXYLI Núc nác (Vỏ thân) Sun- or heat-dried stem barks of Oroxylon indicum (L.) Vent., (Fam. Bignoniaceae).

Description Barks tubularly or semi-tubularly rolled, 0.6 - 1.3 cm thick, varying in length. Outer surface pale brownish-yellow, shrunken, with numerous longitudinal and transversal striations. Inner surface smooth, greyish-yellow or greenish-yellow. A thin cork layer visible on transverse fracture. Cortical parenchyma gritty as full of grit; innermost occurring a layer of fibers easy to longitudinally exfoliate. Transverse section Cork layer extremely thick, consisting of 30 - 40 rows of rectangular cells in tangential and relatively even arrangement, deeply dehiscent and ragged at numerous places. Cortical parenchyma consisting of thin-walled cells, with tangential and slight elongation, scattered with numerous sclerenchymatous masses. Secondary phloem extremely thick, branched by medullar rays. Phloem cells thin-walled, regularly arranged and flattened in tangential rings. Numerous fibrous masses with lignified and thin-walled cells, distinct, stratified in phloem. Medullar rays large, composed of 3 - 5 rows of rectangular cells, in radial arrangement, running 1129

VP V

CORTEX PERIPLOCAE

from cambium to parenchyma and needle-shaped crystals of calcium oxalate scattered throughout cortical parenchyma and phloem. Cambium visible.

Powder Yellow in colour; taste bitter. Examine under a microscope: Numerous fibers long, pale yellow, thickwalled, walls sometimes evenly protruding, distinct pit canals. Sclerenchymatous cells yellow, polygonal, slightly thick-walled, with large lumina and distinct pit canals. Abundant needle-shaped crystals of calcium oxalate, both ends sharply tapered or square, 2 - 4 µm in diameter, sparsely occurring or grouped into bundles. Cork fragments consisting of thin-walled, polygonal cells. Parenchymatous fragments polygonal, usually containing needle-shaped crystals of calcium oxalate. Identification Place 0.5 g of the coarse powder in a test tube, add 5 ml of ethanol R, shake. Heat in a water-bath for 5 - 10 minutes, filter. Use the filtrate to perform the following reactions: To 1 ml of the filtrate, add 3 drops of hydrochloric acid R and a small quantity of magnesium powder R, an orange yellow colour is produced. To 1 ml of the filtrate, add 1 drop of a 5% solution of ferric chloride R, a brownish-green or blackish-green colour is produced. To 1 ml of the filtrate, gently add 0.5 ml of sulphuric acid R along the tube wall, 2 layers are produced. In the lower layer, a brown colour appears and more distinct on standing. Los on drying Not more than 14.0% (Appendix 9.6, 1 g, 100 °C, 4 hours).

CORTEX PERIPLOCAE Hương gia bì (Vỏ rễ) Sun- or heat-dried root barks of Periploca sepium Bge. (Fam. Asclepiadaceae).

Description Bark pieces tubular or semi-tubular, usually tubularly rolled, 0.5 - 3 mm thick, 3 - 17 cm or more long. Outer surface brownish-yellow, scabrous, with irregular longitudinal fissures, easily exfoliated. Texture light and fragile, easy to break. Odour characteristically pungent. Transverse section Cork layer consisting of numerous rows of rectangular cells, regularly arranged in concentric rings and radial rows. Cortical parenchyma composed of thin-walled cells, polygonal, disorderly arranged, bearing scattered cubic crystals of calcium oxalate. Secondary phloem bundles occupying about 2/3 of the transverse section. Medullary rays consisting of 2 - 3 rows of cells. Parenchyma scattered with phloem containing sacs of aromatic contents. Powder Brown powder, aromatic, bitter. Examine under a microscope: cortical fragments, dark brown, numerous; parenchymatous fragments containing starch granules, parallelepiped crystals of calcium oxalate, 30 - 40 µm. Starch granules composed mainly of small single granules, subrounded or polygonal. Sleroid polygonal, thick-walled, arranged separately or grouped into masses. Vascular fragments usually consisting of dotted vessels.

Processing Eliminate foreign matter, scrape cortical layers, wash clean, cut into slices 2 - 5 cm long, dry in the sun or stirbake with gentle heat until the drug surface turns yellow.

Identification Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Toluene - ethyl acetate - methanol (9 : 2 : 0.5). Test solution: To 1.0 g of the powder, add 5 ml of methanol R, shake for 3 minutes, filter. Reference drug solution: Prepare a solution of 1 g of the powder of Cortex Periplocae reference drug in the same manner as described under Test solution. Procedure: Apply separately to the plate 10 µl of each solution. After developing and removal of the plate, dry in air. Spray with a 1% vanillin in sulphuric acid R. Heat the plate at 105 °C for 3 - 5 minutes. Examine under visible light. The spots in the chromatogram obtained with the test solution correspond in colour and position to the spots in the chromatogram obtained with the reference drug solution.

Storage Store in a dry and ventilated place.

Water Not more than 13.0% (Appendix 12.13).

Foreign matter Not more than 1.0% (Appendix 12.11). Extractives Not less than 10.0%, calculated with reference to the dried drug. Carry out the hot extraction method (Appendix 12.10), using ethanol (96%) R as the solvent. Preliminary processing Collect the drug all the year round, whittle the bark part from their plants, dry under the sun or at a low temperature.

Foreign matter Not more than 1.0% (Appendix 12.11).

1130

VP V

Preliminary processing Collect the root barks, wash clean, dry under the sun or at a low temperature. Storage Store in a cool and dry place, preserve in well closed containers. CORTEX PHELLODENDRI Hoàng bá (Vỏ thân, vỏ cành) Sun- or heat-dried stem and branch barks (removed from cork) of Phellodendron chinense Schneid. or Phellodendron amurense Rupr., (Fam. Rutaceae).

Description Stem barks brownish-yellow, 0.3 - 0.5 cm thick, 20 - 40 cm long, 3 - 6 cm wide. Outer surface bearing remains of greyish-brown cork, scabrously sunken spots and longitudinal furrows. Inner surface pale brown, with fine long longitudinal ridges. Fracture uneven; texture hard and light, straw yellow. Branch barks 0.15 - 0.20 cm thick, long pieces, tubularly rolled. Outer surface having greyish-brown coarse bark, exhibiting dark brown cork when scaling off, spotted with numerous scars of lenticels. Inner surface pale brown, with fine longitudinal wrinkles. Texture brittle, easily broken; fracture scabrous, showing straw yelow parenchyma. Transverse section Remains of cork very thin, consisting of some rows of flattened rectangular cells. Cortical parenchyma occupying one third of stem bark thickness, composed of thinwalled cells, scattered with several fibrous masses and fusiform crystals of calcium oxalate. Secondary phloem thick, occupying two thirds of stem bark thickness, with numerous fibrous masses inside, thick walls, lumina narrow; adjacently being fusiform crystals of calcium oxalate. Medullary rays consisting of 2 - 4 rows of rectangular cells, thin-walled, arranged tortuously and radially. Powder Bright yellow, odourless; taste very bitter. Examine under a microscope: Fibrous masses abundant, containing prismatic crystals of calcium oxalate, some brownish - yellow or bright yellow, some separately arranged, thick-walled. Fragments of cortical parenchyma with subrounded cells. Fragments of cork (remaining) consisting of rectangular cells, walls slightly sinuous, brownish-yellow. Under ultra-violet light, both transverse section and powder giving bright yellow fluorescence. Identification A. To 0.2 g of the powder, add 3 ml of water, heat gently, filter. To 2 ml of the filtrate, add 1 ml of a 1% solution of

CORTEX PHELLODENDRI

sulphuric acid R, add gradually chlorine water R. Allow to stand for 10 minutes. A dark red ring is produced at the zone of contact. B. To 0.2 g of the powder, add 1 ml of ethanol (90%) R, heat gently for some minutes, filter. Apply 1 - 2 drops of the filtrate to a slide, gently heat on a spirit fire to nearly dried, add 1 drop of a 25% solution of nitric acid R or 1 drop of hydrochloric acid R, cover with a lamina. After 20 minutes, examine under a microscope: Bright yellow needle-shaped crystals are observed. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: n-Butanol - acetic acid - water (7 : 1 : 2). Test solution: To 0.1 g of the powder, add 5 ml of methanol R, heat gently in a water bath for some minutes, filter, use the filtrate as the test solution. Reference substance solution: Dissolve berberine chloride RS in methanol R to obtain a solution containing 1 mg/ml. Procedure: Apply separately to the plate 20 µl of each solution. After developing and removal of the plate, allow to dry at room temprature. Examine under ultraviolet light at 366 nm. The bright yellow fluorescent spot in the chromatogram obtained with the test solution corresponds in colour and position to the spot due to berberine chloride in the chromatogram obtained with the reference substance solution. D. Identify P. amurense and P. chinensis. Examine the chromatograms obtained under Assay. P. amurense: The chromatogram obtained with test solution has two principal peaks coressponding in retention time to the peaks due to berberine chloride and palmatine chloride in the chromatogram obtained with reference solution. P. chinensis: The chromatogram obtained with the test solution has only one principal peak coressponding in retention time to the peak due to berberine chloride in the chromatogram obtained with the reference solution and has not any peak in the chromatogram obtained with the test solution corresponding in retention time to the peak due to palmatine chloride in the chromatogram obtained the with reference solution.

Loss on drying Not more than 13.0% (Appendix 9.6, 1 g, 105 °C, 5 h). Foreign matter Not more than 1.0% (Appendix 12.11). Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase A: A 0.1% solution of trifluoacetic acid. Mobile phase B: Acetonitrile R. Reference solution: Dissolve berberine chloride RS and palmatine chloride RS in methanol R to produce a series of reference solutions containing 1, 10, 100, 200 and 400 mg/L of each reference substance. 1131

VP V

CORTEX RADICIS LYCII

Test solution: Weigh accurately 0.2 g of the powder (passed through a sieve No 355), put it into a 50 ml volumetric flask, add 10 ml of methanol R and weigh. Sonicate the mixture for 30 min and weigh again. Compensate the weight loss with methanol R. Mix and centrifuge, decant the clear solution. Filter through a membrane filter of 0.45 µm. Chromatographic system: A column (30 cm × 3.9 mm) packed with stationary phase C (4 μm). Detection: A spectrophotometer at 346 nm. Flow rate: 0.8 ml per minute. Volume injection: 5 μl. Procedure: Use gradient elution as follows: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0-20

90 → 10

10 → 90

Perform at least five replicate injections each reference standard solution of berberine chloride and palmatine chloride (200 mg/L). Record the chromatograms. Elution order: palmatine chloride peak, berberine chloride peak. System suitability: The relative standard diviation of the peak areas of berberine chloride and palmatine chloride should be not more than 5.0%; the relative standard diviation of the retention times of berberine chloride peak and palmatine chloride peak should be not more than 2.0%; the column efficiency determined from berberine chloride peak and palmatine chloride peak should be not less than 30000 theoretical plates. Inject a series of the reference solutions and record the chromatograms. Plot the peak areas of berberine chloride and palmatine chloride against the corresponding concentrations of the mixed berberine chloride and palmatine chloride. Inject the test solution and record the chromatogram. Identify peak due to berberine chloride and peak due to palmatine chloride in the chromatogram of the test solution by comparing their retention times with those in the chromatogram of the reference solution. Use the areas of the peaks in each of the chromatograms obtained with the test solution and the reference solution, and the declared content of C20H18NO4Cl in berberine chloride RS to calculate the content of berberine chloride in the drug. It contains not less than 0.33% of berberine chloride (C20H18NO4Cl) for P. amurense and 2.5% of berberine chloride (C20H18NO4Cl) for P. chinensis, calculated with reference to the dried drug.

Preliminary processing Stem and branch barks are collected, scraped clean of cork, cut into pieces, dried in the sun or at 50 °C. Storage Store in ventilated, dry and cool places, protected from mould and weevils. 1132

CORTEX RADICIS LYCII Địa cốt bì Sun- or heat-dried root barks of Lycium chinense Mill. or Lycium barbarum L., (Fam. Solanaceae).

Description Root barks small, semi-tubularly or tubularly rolled, 3 - 10 cm long, 0.5 - 1.5 cm wide, 1 - 3 mm thick. Outer surface greyish-yellow to brownish-yellow, scabrous, with irregular longitudinal fissures, easily exfoliated. Inner surface pale yellow to greyish-yellow, relatively smooth, with fine longitudinal wrinkles. Texture light and fragile easy to break; fracture uneven; outer layers brownishyellow; inner layers gryish-white. Odour mild; taste slightly sweet, then biter. Transverse section Cork consisting of 4 - 10 or more rows of cells. Cortical parenchymatous cells containing sandy crystals of calcium oxalate, aggregated in masses with numerous starch granules. Most bundles of phloem rays separated by one row of large cells, fibres singly scattered, or two to several grouped into bundles. Identification Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel G. Mobile phase: Toluene - acetone - formic acid (10 : 1 : 0.1). Test solution: Place 1.5 g of the powder in a conical flask, add 15 ml of methanol R, sonicate for 30 minutes, filter. Evaporate the filtrate on a water bath to dryness. Dissolve the residue in 1 ml of methanol R. Reference drug solution: Prepare a solution of 1.5 g of Cortex Radicis Lycii reference drug in the same manner as described under Test solution. Procedure: Apply separately to the plate 5 µl of each solution. After developing and removal of the plate, dry in air. Examine under ultraviolet light (366 nm). The spots in the chromatogram obtained with the test solution correspond in colour and position to the spots in the chromatogram obtained with the reference drug solution Total ash Not more than 11.0% (Appendix 9.8). Loss on drying Not more than 11.0% (Appendix 9.6, 1 g, 105 °C, 5 h). Broken matter Fragments under 1.5 cm: Not more than 2.0% (Appendix 12.12). Preliminary processing The drug is collected in early spring and late autumn; roots are lifted, washed clean, root barks are stripped off and dried in the sun or at a low temperature to dryness, or roots are washed clean, cut into sections of 6 - 12 cm and slit with

VP V

a knife up to the wood; then steamed, root barks are peeled off, gathered and dried in the sun or at a low temperature.

Processing Eliminate foreign matter and remains of wood, wash clean, dry and cut into sections. Storage Store in a dry place. CORTEX RADICIS PAEONIAE SUFFRUTICOSAE Mẫu đơn bì (Vỏ rễ) Sun-dried root barks of Paeonia suffruticosa Andr., (Fam. Paeoniaceae).

Description Root barks tubular or semitubular, longitudinally fissured, margins usually incurved or opened, 5 - 20 cm long, 0.5 - 1.2 cm in diameter, 0.1 - 0.4 cm thick. Outer surface brown or brownish - yellow, bearing numerous transverse lenticels and scars of rootlets, the exposed layer where cork scaling off appearing pink. Inner surface ashy-yellow or pale brown, with distinct fine longitudinal striations, usually showing bright small crystals. Texture hard, fragile, easily broken. Fracture nearly even, starchy, pale pink. Odour characteristically aromatic; taste slightly bitter and astringent.

CORTEX SCHEFFLERAE HEPTAPHYLLAE

and filter. Evaporate the filtrate to dryness on a water-bath, and dissolve the residue in 2 ml of acetone R. Reference substance solution: Dissolve paeonol RS in acetone R to produce a solution containing 5 mg per ml. Reference drug solution: Alternatively, prepare a solution of 1 g of Cortex Paeoniae suffruticosae reference drug in the same manner as described under Test solution. Procedure: Apply separately to the plate 10 µl of each solution. After developing and removal of the plate, dry it in air. Spray with a 5% solution of ferric chloride in ethanol R. Examine under visible light. The spots in the chromatogram obtained with the test solution correspond in colour and position to the spots in the chromatogram obtained with the reference drug solution. Or the chromatogram obtained with the test solution has a spot coressponding in colour and position to the spot due to paeonol in the chromatogram obtained with the reference substance solution.

Water Not more than 13.0% (Appendix 12.13). Foreign matter (Appendix 12.11) Wood matter: Not more than 5.0%. Other foreign matter: Not more than 1.0%. Total ash Not more than 5.0% (Appendix 9.8).

Powder Pale reddish-brown in colour. Starch granules fairly abundant; single granules rounded or polygonal, 3 - 16 µm in diameter, hilum dotted, cleft or V-shaped, compound granules of 2 - 6 components. Clusters of calcium oxalate crystals 9 - 45 µm in diameter, sometimes crystalcontaining cells adjoining each other, arranged in clusters, bundles of crystals, at times one cell containing several bundles of calcium oxalate. Cork cells rectangular, slightly thick-walled, pale red.

Assay Steam distill 0.2 g of the powder (passed through a sieve No. 355), accurately weighed, until about 450 ml of the distillate are collected, adjust the volume to 500 ml with water and shake well. Measure the absorbance of the solution at 274 nm and calculate the content of paeonol, taking 862 as the value of A (1%, 1 cm) of paeonol at 274 nm. It contains not less than 1.2% of paeonol (C9H10O3), calculated with reference to the dried drug.

Identification A. Shake 0.15 g of the powder with 25 ml of ethanol R for several minutes and filter. Dilute 1 ml of the filtrate to 25 ml with ethanol R. Measure the absorbance of the obtained solution (Appendix 4.1). The light absorption exhibits a maximum at (274 ± 1) nm. B. Shake 0.5 g of the powder with 5 ml of ether ethylic R for 10 minutes and filter. Evaporate the filtrate to dryness on a water-bath, dissolve the residue in 3 ml of ethanol R, add 1 drop of a 5% solution of ferric chloride R, a reddishpurple colour is produced. C. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel G. Mobile phase: Cyclohexane - ethyl acetate (3 : 1). Test solution: To 1 g of the powder, add 10 ml of ether ethylic R, shake vigorously, allow to stand for 10 minutes

Preliminary processing Collect the drug in autumn, lift the roots, remove rootlets and soil, wash clean, strip off and take soft barks, cut into slices and dry in the sun. Storage Store in dry and cool places, preserve in well closed containers. CORTEX SCHEFFLERAE HEPTAPHYLLAE Ngũ gia bì chân chim (Vỏ thân, Vỏ cành) Sun- or heat-dried stem and branch barks of Schefflera heptaphylla (L.) Frodin, (Fam. Araliaceae).

Description Bark pieces slightly curved semi-tubularly roolled, 20 - 50 cm long, 3 - 10 cm wide, 0.3 - 1 cm thick. After 1133

VP V

CORTEX STRYCHNI WALLICHIANAE

removal of cork, externally appearing pale brown, with pale whitish-grey punctate traces. Transverse cut surface showing outer sandy layers and inner soft fibrous, easily longitudinally split layers. Texture light and fragile. Odour mildly aromatic; taste slightly bitter.

Transverse section Remaining cork consisting of about 10 rows of flattened rectangular cells, slightly thick - walled, superimposed into even radial rays. Pericambium consisting of one layer of flattened rectangutar cells, arranged evenly. Sclerenchymatous cells flattened rectangular or polygonal, grouped in masses, walls heavily thick - walls, lumen narrow, pit canals distinct, arranged adjacently to Pericambium in continuous ring. Cortical parenchyma containing narrow, tangentially elongated, thin-walled cells and scattered with secretory tubes. Secondary phloem ring, thick accupying 2/3 of stem bark thickness; phloem cells thin-walled. Phloem fibres arranged into masses, alternating in several layers in phloem. Fibre cells rounded thick-walled. Crystals of calcium oxalate occurring beside fibre masses. Medullary rays narrow, consisting of 3 rows of cells running radially through secondary phloem. Powder Sclerenchymatous cells numerous rectangular or polygonal, pale yellow, walls strongly thick-walled, pit canals distinct, singly or grouped into masses. Fibres with thickened walls and distinct pit canals. Cork fragments consisting of rectangular cells, arranged evenly, thickwalls. Parenchymatous fragments composed of polygonal, thin - walled cells. Crystals of calcium oxalate rectangular or cubic, about 40 µm wide. Starch granules fine, 4 µm in diameter, sometimes up to 16 µm. Identification A. To 5 g of the powder, add 20 ml of ethanol (96%) R, boil, shake, cool, and filter. To 1 ml of the filtrate in a test tube, add 5 drops of anhydride acetic R, slowly and carefully add 0.5 ml of sulphuric acid R along the tube wall, a brownish-red ring developes at the junction of the two layers. B. Examine by thin - layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Chloroform - methanol - water (65 : 35 : 10), shake thoroughly, using the lower layer. Test solution: To about 2 g of the powder, add 25 ml of a mixture of methanol R and water (4 : 1), heat under a reflux condenser on a water bath for 30 minutes, filter. Evaporate the filtrate on a water bath to dryness. Dissolve the residue in 2 ml of methanol R. Reference drug solution: Prepare a solution of 2 g of Cortex Schefflerae heptaphyllae reference drug in the same manner as described under Test solution. 1134

Procedure: Apply separately to the plate 10 µl of each solution. After developing and removal of the plate, allow to dry in air. Spray with a 1% solution of vanillin in phosphoric acid (50%) R. Heat the plate at 120 °C for 5 minutes. Examine under visible light. The spots in the chromatogram obtained with the test solution correspond in colour and position to the spots in the chromatogram obtained with the reference drug solution.

Water Not more than 12.0% (Appendix 12.13). Total ash Not more than 4.5% (Appendix 9.8). Foreign matter Not more than 1.0% (Appendix 12.11). Preliminary processing Collect the stem and branch barks all the year round, mainly in spring and autumn, when the weather is dry, strip the bark off the tree according to specified size, wash clean, remove remains of wood and the outer cork, dry in the shade, then wrap up the barks with leaves of banana for 7 days, and occasionally turn upside-down sometimes until giving out aroma, take the barks out, dry in the shade or at a low temperature (50 °C - 60 °C) until dryness. Processing Wash clean the barks, cut into short segments, steam to soften thoroughly, cut into pieces and dry under the sun. Storage Store in dry and cool places, protected from mould and weevils. CORTEX STRYCHNI WALLICHIANAE Hoàng nàn (Vỏ thân, Vỏ cành) Sun-or heat-dried stem or branch barks of Strychnos wallichiana Steud. ex DC., (Fam. Loganiaceae).

Description Bark pieces thick, usually semi-tubularly or tubularly rolled. Outer surface containing numerous greyish-brown nodules. Inner surface blackish-brown, with numerous fine longitudinal striations; texture fragile, easily broken; fracture uneven; taste very bitter. Transverse section Cortical layer consisting of numerous rows of rectangular cells arranged in concentric rings and radial rows, ragged and exfoliated at some places. Cortical parenchyma consisting of thin-walled cells, outer cells usually pressed flat having numerous crystal masses of calcium oxalate. In parenchyma occurring a continuous sclerenchymatous ring which consisting of lignified thick-walled cells, narrow

VP V

lumina. Secondary phloem abundantly developed, divided into conical bundles by medullar rays. Medullar rays composed of 1 - 3 rows of cells running through phloem and broadening sometimes up to cortical parenchyma.

Powder Dark brown in colour; taste very bitter. Examine under a microscope. Brownish-yellow cortical fragments consisting of thick-walled polygonal; parenchymatous fragments containing thin-walled cells, polygonal or subrounded, bearing crystal masses of calcium oxalate. Numerous sclerenchymatous cells with heavy thick walls and narrow lumina, pit canals distinct. Crystal masses of calcium oxalate abundant, 25 - 35 µm long, 10 - 24 µm wide. Identification A. Moisten 1 g of the powder with 1 ml of ammonia R, add 10 ml of chloroform R and shake for 5 minutes. Filter the chloroform layer through a paper filter containing anhydrous sodium sulphate R. Divide the filtrate into 2 equal parts, transfer to 2 cups and evaporate on a water bath to dryness. Dissolve the residue in the first cup with 2 - 3 drops of sulphuric acid R, add some crystals of potassium dichromate R, tilt the cup, some violet streaks are produced and quickly decolorized (strychnine). Add 2 - 3 drops of nitric acid R to the residue in the second cup, an orange-red colour develops (brucine). B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Toluene - acetone - ethanol - ammonia (4 : 5 : 0.6 : 0.4). Test solution: Place 0.5 g of the powder in a groundglass stoppered conical flask, add 5 ml of a mixture of chloroform - ethanol (10 : 1) and 0.5 ml of ammonia R. Stopper the flask and shake for 5 minutes, allow to stand for 1 hour with occasional shaking, filter. Reference substance solution: Dissolve strychnine RS and brucine RS in chloroform R to produce a solution containing 2 mg of each per ml. Procedure: Apply separately to the plate 10 µl of each solution. After developing and removal of the plate, allow to dry in air and spray with Dragendorff reagent R. Examine under visible light. The chromatogram obtained with the test solution shows several spots, two of which correspond in colour and position to the spots due to strychnine and brucine in the chromatogram obtained with the reference substance solution. Loss on drying Not more than 12.0% (Appendix 9.6, 1 g, 105 °C, 5 h). Total ash Not more than 17.0% (Appendix 9.8). Foreign matter Not more than 1.0% (Appendix 12.11).

CORTEX TERMINALIAE NIGROVENULOSAE

Assay Weigh accurately about 2 g of the powder (passed through a sieve No 355), put into a 250 ml ground-glass stoppered conical flask. Add 2.0 ml of ammonia R and 100 ml of a mixture of ether ethylic - chloroform (1 : 1). Stopper the flask, allow to stand for 24 hours with occasional shaking. Allow the layers to separate, quickly filter and collect 50 ml of the supernatant layer through a folding paper filter placed in a filtering funnel with cover, carry out the filtration in a cool place. Carry out the alkaloid extraction by using three successive quantities (20, 20 and 10 ml) of a 4.0% solution of hydrochloric acid R by shaking. Combine the extracts, adjust to pH 8 - 9 with ammonia R (about 2 ml) and continue the alkaloid extraction with three quantities (50, 30 and 20 ml) of a mixture of ether ethylic - chloroform (1 : 1). Combine the extracts and evaporate to expel the solvent completely. Dry the residue at 100 ºC to constant mass, weigh the residue. Calculate the content of total alkaloids (X) in the drug using the following expression: m×10 000×2 X (%) = p×(100 – a) Where: m: mass of the residue (g) p: mass of the drug powder being examined (g) a: water content (%) The drug contains not less than 2.0% of total alkaloids, calculated with reference to the dried drug.

Preliminary processing The drug is collected in dry weather, barks are stripped off, dried in the sun or at a low temperature. Before use, macerate the drug in water, or in the rice washing for 12 - 24 hours. Take out and wash clean. Scrape and remove the yellow cortical layer and black inner layer. Continue to macerate in the rice washing for 3 days and nights. Replace the rice washing every day. Wash clean, dry under the sun or at a low temperature. Cut into small slices. Soak the slices with peanut or sesame oil, stir-bake to yellow. Storage Preserve in well-closed containers, store in a dry place. CORTEX TERMINALIAE NIGROVENULOSAE Chiêu liêu (vỏ thân), Chiêu liêu nghệ Sun-or heat-dried stem barks of Terminalia nigrovenulosa Pierre ex. Laness., (Fam. Combretaceae).

Description Stem bark pieces 40 - 50 cm long, 5 - 10 cm or more wide, about 0.8 - 1.5 cm thick. Outer surface cortical layer white to pale yellow, scabrous, relatively thin in comparison with bark. Inner surface fairly smooth, all dark red. Fracture bristly; texture heavy and compact. Odour mild; taste slightly acrid. 1135

VP V

EMBRYO NELUMBINIS NUCIFERAE

Transverse section Cortical layer thick, composed of rectangular cells with thick walls in concentric and radial arrangement, ragged and exfoliated at some places. Pericambium consisting of one row of rectangular cells fairly regularly arranged. Cortical parenchyma containing thin-walled rectangular cells, numerous masses of sclerenchymatous cells, starch granules and crystals of calcium oxalate. Sclerenchymatous cells usually grouped in rectangular or polygonal masses, with heavy thick walls, pit canals distinct. Crystals of calcium oxalate urchin-form varying in size. Secondary phloem fairly thick, composed of thin-walled cells. Phloem divided into numerous strata (stratified phloem) by grouped phloem fibers. Strata of phloem alternating with strata of cells which filled with urchin-form crystals of calcium oxalate. Numerous medullary rays separating strata into small compartments. Powder Pinkish-red in colour; odour mild; taste slightly acrid. Examine under a microscope. Cortical fragments consisting of rectangular cells, thick-walled, in fairly regular arrangement. Parenchymatous fragments containing polygonal or rectangular cells, full of starch and urchinform crystals of calcium oxalate. Crystals of calcium oxalate abundant, varying in size from 10 to 40 µm. Parenchymatous fragments containing colour contents (orange-red, brown-red…). Coloured masses (orange-yellow, orangered, brown-red…). Numerous sclerenchymatous cells rectangular or polygonal, pale yellow, heavy thick-walled, pit canals distinct, usually grouped in masses. Fibres lying separately or grouped in bundles. Starch granules abundant, varying in form, pills scalariform or stelliform, 2 - 5 µm sometimes up to 10 µm in size. Identification A. To 1 g of the powder, add 30 ml of water, heat in a boiling water-bath for 15 minutes. Filter through one or two paper filters if necessary (solution A). Reaction 1: Transfer separately 2 ml of solution A to each of 2 test tubes. Test tube 1: Add 1 - 2 drops of sodium chloride-gelatin solution R, a milky white precipitate is produced. Test tube 2: Add 1 - 2 drops of a 1.3% solution of ferric chloride R, the solution colour turns moss green. Reaction 2: Transfer separately 2 ml of solution A to each of 2 test tubes. Test tube 1: Add 3 drops of Stiasny reagent R, heat in a water-bath for 10 minutes, a curdy brick-red precipitate is produced. Test tube 2: Add 1 - 2 drops of bromine water R, an ivory precipitate is produced. B. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel 60GF254. Mobile phase: Chloroform - ethyl acetate - formic acid (5 : 4 : 1). 1136

Test solution: Place 2 g of the powder in a 250 ml conical flask, add 20 ml of mobile phase, sonicate for 30 minutes, filter. Evaporate the filtrate on a water-bath to dryness. Dissolve the residue in 2 ml of mobile phase. Reference drug solution: Prepare a solution of 2 g of the powder of Cortex Terminaliae nigrovenulosae reference drug in the same manner as described under Test solution. Procedure: Apply separately to the plate 5 µl of each solution. After developing and removal of the plate, dry in air, spray with a 2% solution of vanillin in sulphuric acid R. Heat the plate at 110 °C until the spots appear. Examine under visible light. The spots in the chromatogram obtained with the test solution correspond in colour and position to the spots in the chromatogram obtained with the reference drug solution.

Loss on drying Not more than 12.0% (Appendix 9.6, 1 g, 100 °C, 5 h). Total ash Not more than 14.0% (Appendix 9.8). Hydrocloric acid-insoluble ash Not more than 2.0% (Appendix 9.7). Foreign matter Not more than 1.0% (Appendix 12.11). Extractives Not less than 25.0%, calculated with reference to the dried drug. Carry out the hot extraction method (Appendix 12.10), using ethanol (96%) R as the solvent. Preliminary processing Collect the crude drug in autumn, take stem barks from big plants, dry under the sun or at a low temperature. Storage Preserve in well closed containers, store in a dry place. EMBRYO NELUMBINIS NUCIFERAE Sen (Cây mầm), Liên tâm Lotus embryo is the sun-or heat-dried embryo collected from seeds of Nelumbo nucifera Gaertn., (Fam. Nelumbonaceae).

Description Lotus embryo about 1 cm long, about 1 mm wide; the upper part being plumule, dark green, consisting of 4 young folded leaflets; the lower part consisting of radicle and hypocotyl, cylindrical, pale yellow; transverse cut surface showing numerous spaces (observed under a magnifying glass). Transverse section Epidermis consisting of one row of evenly arranged cells. Cells of parenchyma rounded, thin-walled. Phloem-xylem

VP V

bundles abundant, gradually increasing in size towards the inner ring. Each phloem-xylem bundle consisting of phloem outside and xylem inside. Numerous large lacunae arranged in a ring between the 2 innermost phloem-xylem rings.

Powder Dark green in colour; taste bitter. Examine under a microscope. Fragments of parenchyma consisting of numerous chlorophyll-containing cells. Starch granules spheroid or ovoid, 4 - 6 µm in diameter, sometimes up to 15 µm. Epidermal fragments visible. Identification A. To about 1 g of the powder, add 20 ml of ethanol (90%) R, heat on a water bath for 5 minutes, then filter through a plug of absorbent cotton. Evaporate the filtrate to dryness. Dissolve the residue in 5 ml of a 5% solution of sulphuric acid R and filter. Divide the filtrate into 4 test tubes: Test tube 1: add 2 drops of Mayer reagent R, a white precipitate is produced. Test tube 2: add 2 drops of Bouchardat reagent R, a brown precipitate is produced. Test tube 3: add 2 drops of Dragendorff reagent R, a red precipitate is produced. Test tube 4: add 2 drops of picric acid solution R, a yellow precipitate is produced. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Chloroform - methanol - ammonia (50 : 9 : 1). Test solution: Moisten about 1 g of the powder with 1 ml of ammonia R. Add 10 ml of chloroform R, allow to stand for 20 minutes, occasionally shake, then filter through a plug of absorbent cotton. Transfer the filtrate to a small separating funnel, shake with 10 ml of a 5% solution of sulphuric acid R. Separate the acid layer out. Make it alkaline with ammonia R and shake with chloroform R. Evaporate the chloroform extract on a water bath to about 0.5 ml. Reference substance solution: Dissolve nuciferin RS in chloroform R to produce a solution containing 1 mg per ml. Reference drug solution: Alternatively, prepare a solution of 1 g of Embryo Nelumbinis reference drug in the same manner as described under Test solution. Procedure: Apply separately to the plate 20 µl of each solution. After developing and removal of the plate, dry in air, spray with Dragendorff reagent R. Examine under visible light. The chromatogram obtained with the test solution shows at least 5 orange-red spots, of which, one spot corresponds in colour and position to the spot due to nuciferin in the chromatogram obtained with the reference substance solution or the spots in the chromatogram obtained with the test solution correspond in colour and position to the spots in the chromatogram obtained with the reference drug solution. Loss on drying Not more than 12.0% (Appendix 9.6, 1 g, 105 °C, 4 h).

ENDOTHELIUM CORNEUM GIGERIAE GALLI

Total ash Not more than 5.0% (Appendix 9.8). Hydrochloric acid-insoluble ash Not more than 0.3% (Appendix 9.7). Foreign matter Not more than 0.5% (Appendix 12.11). Heavy metals Not more than 10 ppm of Pb, 1 ppm of Cd, 0.4 ppm of Hg. 1 ppm of As ( Appendix 9.4.11). Assay Carry out the method described under Assay of Folium Nelumbinis monograph. It contains not less than 1.0% of total alkaloids, calculated as nuciferin and with reference to the dried drug. Preliminary processing Gather over ripe achenes from the fruit torus, take off their external hard pericarps, soak in water, wrap up to soften, remove red membranes, take out plumules, dry under the sun or at a low temperature (40 °C - 50 °C) to dryness. Storage Store in a dry, ventilated and cool place. ENDOTHELIUM CORNEUM GIGERIAE GALLI Kê nội kim, Màng mề gà Sun-or heat-dried inner wall of the gizzard of Gallus gallus domesticus Brisson, (Fam. Phasianidae).

Description Gizzards nearly entire or in rolled dry slices, about 2 mm in thickness. Externally yellow, yellowish-green or yellowish-brown, thin and translucent, with prominent streak-shaped winkles. Texture fragile, easily broken; fracture horny lustrous. Odour slightly stinking; taste slightly bitter. Loss on drying Not more than 15.0% (Appendix 9.6, 1 g, 105 °C, 4 h). Foreign matter Not more than 1.0% (Appendix 12.11). Total ash Not more than 2.0% (Appendix 9.8). Acid-insoluble ash Not more than 1.0% (Appendix 9.7). Extractives Not less than 7.5%, calculated with reference to the dried drug. Carry out the hot extraction method (Appendix 12.10), using ethanol (50%) R as the solvent. 1137

VP V

FLOS CARTHAMI TINCTORII

Preliminary processing After the chicken is killed, the gizzard is collected; the inner wall of the gizzard, still warm, is peeled off immediately, washed clean, dried under the sun or at a low temperature to dryness. Processing Endothelium Corneum Gigeriae Galli (stir-baked): Stirbake the clean Endothelium Corneum Gigeriae Galli in a sand-bath until it inflates, take out and cool. Externally dark yellowish-brown to charring yellow, fine bubble-shaped; broken when bend gently; fracture lustrous. Endothelium Corneum Gigeriae Galli (processed with vinegar): Stir-bake the clean Endothelium Corneum Gigeriae Galli until it inflates, spray with vinegar, take out and dry in the sun or at a low temperature to dryness. Use 15 liters of vinegar per 100 kg of Endothelium Corneum Gigeriae Galli. Storage Preserve in well closed containers, stored in a dry place, protect from mould and weevils. FLOS CARTHAMI TINCTORII Hồng hoa (hoa) Sun- or heat-dried flowers of Carthamus tinctorius L., (Fam. Asteraceae).

Description Flowers 1 - 2 cm long, externally reddish-yellow or red. Corolla, tubular, slender, 5-lobed at the apex, narrowly belt-shaped lobes, 5 - 8 cm long. Stamens 5, anthers attached to tube-walls, yellow. Stigma cylindrical, slightly 2-cleft, protruding from corolla lobes. Texture soft; odour slightly aromatic; taste slightly bitter. Powder Orange yellow in colour. Fragments of corolla, filament and stigma frequently visible. Long tubular secretory cells present, accompanied by vessels, up to 66 μm in diameter, containing yellowish-brown to reddish-brown secretion. Outer walls of terminal epidermal cells of corolla lobes projecting to be tomentellate. Upper epidermal cells of stigma and style differentiated into conical unicellular hairs, acuminate or slightly obtuse at apex. Pollen grains spherical, 60 - 70 μm in diameter, with 3 germinal pores, exine with dentate-spinula. Prismatic crystal of calcium oxalate occurring. Parenchymatous fragments consisting of rectangular cells. Terminal fragments of corolla consisting of numerous cells, imbricated. Filament cells thin-walled, rectangular. Fragments of reticulate and scalariform vessels visible.

1138

Identification A. Macerate 1 g of the powder in 10 ml of ethanol (50%) R for about 10 min. Transfer the supernatant liquid into a beaker, hang a band of filter paper, of which the lower end is well dipped into the solution. After 5 minutes, remove the band then dipped into the water and lifted up immediately. The upper band is yellowish, the lower band is reddish. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel H. Mobile phase: Ethyl acetate - formic acid - water methanol (7 : 2 : 3 : 0.4). Test solution: To 0.5 g of the powder, add 5 ml of acetone (80%) R, stopper tightly, shake constantly for 15 min and filter, use the filtrate as the test solution. Reference drug solution: Prepare a solution of 0.5 g of the powder of Flos Carthami tinctorii reference drug in the same manner as described under Test solution. Procedure: Apply separately to the plate 5 μl of each of solution. After developing and removal of the plate, dry in air. Examine under visible light. The spots in the chromatogram obtained with the test solution correspond in colour and position to those in the chromatogram obtained with the reference drug solution. C. Absorbance Yellow pigment: Dry the drug in a desiccator over silica gel for 24 h and grind into fine powder. Macerate 0.1 g of the powder, accurately weighed, with 150 ml of water in a conical flask for 1 h with shaking and filter through a sintered glass funnel (No 3) into a 500 ml volumetric flask. Wash the funnel and residue with successive quantities of water until the washing is colourless, add sufficient water to volume and mix well. The measured absorbance of the solution at 401 nm (Appendix 4.1) is not less than 0.40. Red pigment: Macerate warmly about 0.25 g of the fine powder (obtained from section yellow pigment), accurately weighed, with 50 ml of acetone (80%) R in a conical flask at 50 °C on a water bath for 90 min, cool, filter through a sintered glass funnel (No 3) into a 100 ml volumetric flask, wash the residue with 25 ml of a acetone (80%) R in portions. Transfer the washings into the volumetric flask, add sufficient acetone (80%) R to volume and mix well. The measured absorbance of the solution at 518 nm (Appendix 4.1) is not less than 0.20. Water Not more than 13.0% (Appendix 12.13). Foreign matter (Appendix 12.11) Blackish-brown flowers: Not more than 0.5%. Others: Not more than 2.0%. Total ash Not more than 15.0% (Appendix 9.8).

VP V

Acid-insoluble ash Not more than 5.0% (Appendix 9.7). Extractives Not less than 30.0%, calculated with reference to the dried drug. Carry out the cold extraction method (Appendix 12.10), using water as the solvent. Assay Hydroxysafflor yellow A Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol - acetonitrile - 0.7% solution of phosphoric acid (26 : 2 : 72). Test solution: Weigh accurately 0.4 g of powder (passed through a sieve No.355), put into a stoppered conical flask, add accurately 50 ml of methanol (25%) R, weigh and sonicate for 40 min, allow to cool and weigh again. Replenish the loss of mass with methanol (25%) R, mix well, filter through a membrane filter (0.45 μm) and use the filtrate as the test solution. Reference solution: Weigh accurately a quantity of hydroxysafflor yellow A RS, add methanol (25%) R to produce a reference solution containing about 0.13 mg per ml. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 μm). Detector: A spectrophotometer set at 403 nm. Flow rate: 1 ml/min. Volume of injection: 10 μl. Procedure: Inject the reference solution and record the chromatograms. The number of theoretical plates of the column is not less than 3000, determined on the peak of hydroxysafflor yellow A. Inject the reference solution and the test solution. Calculate the content of hydroxysafflor yellow A in the crude drug using the areas of hydroxysafflor yellow A peaks in the chromatograms obtained with the test solution, the reference solutions and the declared content of C27H32O16 in hydroxysafflor yellow A RS. It contains not less than 1.0% of hydroxysafflor yellow A (C27H32O16), calculate with reference to the dried drug. Kaempferol Examine by liquid chromatography (Appendix 5.3). Mobile phase: Methanol - 0.4% solution of phosphoric acid (52 : 48). Test solution: Weigh accurately 0.5 g of powder (passed through a sieve), put into a stoppered conical flask, add accurately 25 ml of methanol R, weigh. Heat under a reflux condenser for 40 min, allow to cool and weigh again. Replenish the loss of mass with methanol R, mix well, filter. Measure accurately 15 ml of the sussessive

FLOS CHRYSANTHEMI INDICI

filtrate to a flat bottom flask, add 5 ml of a 17% solution of hydrochloric acid R, mix well and heat to hydrolysis on a water bath for 30 min, cool immediately. Transfer to a 25 ml volumetric flask, dilute with methanol R to volume, mix well. Reference solution: Weigh accurately a quantity of kaempferol RS, add methanol R to produce a reference solution containing about 9 µg per ml. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 μm). Detector: A spectrophotometer set at 367 nm. Flow rate: 1 ml/min. Volume of injection: 10 μl. Procedure: Inject the reference solution and record the chromatograms. The number of theoretical plates of the column is not less than 3000, determined on the peak of kaempferol. Inject the reference solution and test solution. Calculate the content of kaempferol in the crude drug using the areas of kaempferol peaks in the chromatograms obtained with the test solution, the reference solution and the percentage of C15H10O6 in kaempferol RS. It contains not less than 0.05% of kaempferol (C15H10O6), calculated with reference to the dried drug.

Preliminary processing Collect the drug in summer, pluck the flowers when their colour turns from yellow to red, dry in the shade, wellventilated, or in soft sunlight to dryness. Storage Store in dry and cool places, protected from moisture, mould and weevils. FLOS CHRYSANTHEMI INDICI Cúc hoa vàng, Cam cúc, Kim cúc Sun- or heat-dried and processed capitula (frequently known as flowers) of Chrysanthemum indicum L., (Fam. Asteraceae).

Description Capitula brownish-yellow, sometimes with adhering peduncles, 0.5 - 1.2 cm in diameter. Involucre consists of 4 - 5 rows of bracts; outer bracts grayish-green or pale brown, very pale and scarious between margins. Flowers of two kinds: ligulate florets, one whorl, monosexual, irregular outside; the other tubular florets numerous, regular, pentamerous, bisexual inside. Texture light. Odour aromatic; taste bitter. Powder Yellow in colour; odour aromatic. Examine under a microscope: Petal fragments, yellow, bearing thin-walled cells, wrinkled. Bract fragments having long, thin- and 1139

VP V

FLOS CLEISTOCALYSIS OPERCULATI

thick-walled cells with porous canals distinct. Pollen grains urchin-form, yellow in colour. Non-glandular hairs broken into pieces. Stigma fragments bearing round-headed cells, piled up each other in layers, long cells protruding at the end of the stigma.

Identification A. To 3.0 g of the powder, add 20 ml of ethanol (96%) R, heat to boiling under a reflux condenser in a water-bath for 30 min and filter (solution A). To 2 ml of solution A, add a small quantity of magnesium powder R and 3 - 4 drops of hydrochloric acid R, heat, a red colour is produced. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Ethyl acetate - formic acid - water (8 : 1 : 1). Test solution: Evaporate 10 ml of solution A to dryness. Dissolve the residue in 20 ml of hot water, filter and transfer the filtrate to a separating funnel and extract with two 10 ml quantities of ethyl acetate R. Evaporate the combined ethyl acetate extracts to dryness. Dissolve the residue in 1 ml of ethanol R, use it as the test solution. Reference drug solution: Prepare a solution of 3.0 g of Flos Chrysanthemi indici reference drug in the same manner as described under Test solution. Procedure: Apply separately to the plate 5 µl of each solution. After developing the chromatogram over a path of 15 cm and removal of the plate, dry in air. Put the plate in a chamber pre-saturated with the vapour of ammonia R. Examine under visible light. The spots in the chromatogram obtained with test solution (6 spots, of which, there are 4 brownish-yellow spots and 2 greenishyellow spots) correspond in colour and position to those in the chromatogram obtained with reference drug solution. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel 60F254. Mobile phase: Ethyl acetate - 2-butanol - formic acid water (25 : 3 : 1 : 1). Test solution: To 1 g of the powder, add 20 ml of methanol R, sonicate for 10 min and filter. Evaporate the filtrate in a water-bath to dryness. Dissolve the residue in 1 ml of ethanol R, use it as the test solution. Reference drug solution: Prepare a solution of 1 g of Flos Chrysanthemi indici reference drug in the same manner as described under Test solution. Procedure: Apply separately to the plate 10 µl of each solution. After developing and removal of the plate, dry in air. Spray with a 5% solution iron (III) chloride in ethanol R. Examine in daylight. The spots in the chromatogram obtained with test solution correspond in colour and position to those in the chromatogram obtained with reference drug solution. Water Not more than 13.0% (Appendix 12.13). Determined on 10 g of the slivered drug. 1140

Total ash Not more than 9.0% (Appendix 9.8). Broken matter Pass through a sieve with a pore size of 4 mm: Not more than 2.0% (Appendix 12.12). Heavy metals Not more than 10 ppm of Pb, 0.5 ppm of Cd, 0.5 ppm of Hg, 1 ppm of As (Appendix 9.4.11). Extractives Not less than 30.0%, calculated with reference to the dried drug. Carry out the hot extraction method (Appendix 12.10), using ethanol (50%) R as the solvent. Preliminary processing In autumn and winter, when the weather is dry, the flowers are picked up, fumigated with sulfur thoroughly, pressed tightly overnight until the running out liquid has black colour, take out and dry under the sun or at temparature of 40 °C - 50 °C to dryness. Storage Store in dry places, periodically fumigate with sulfur. FLOS CLEISTOCALYSIS OPERCULATI Vối (Nụ hoa) Sun- or slightly heat-dried flower buds of Cleistocalyx operculatus (Roxb.) Merr. et Perry (Fam. Myrtaceae).

Description Flower buds oval, two acute ends, 4 - 6 mm long, 2 - 3 mm wide, brownish-yellow. Calyx campanulate, grey, 1/3 - 1/2 in length of the bud, the upper part 4-shallow lobed. Small flower pale bluish-white when fresh; and pale yellowishgrey, distinct aroma after processing. Taste slightly acrid. Powder Colour pale yellow, non-glandular hair, parenchymatous fragment consist of polygonal cells. Pollen grains spherical, 3-lacunae. Petal fragment, spiral vessel, vascular fragment. Identification A. To 2 g of the powder, add 20 ml of ethanol (90%) R, boiled 2 minutes on a water-bath, filter. The filtrate (solution A) is used for the following reactions and as the test solution in Identification B. To 2 ml of solution A, add 5 drops of hydrochloric acid R and a small quantity of magnesium R powder, the solution changes from yellow to pink after some minutes. To 1 ml of solution A, add 3 drops of a 5% solution of ferric chloride R, shake slightly, a blackish-green colour is produced.

FLOS DATURAE METELIS

VP V

To 1 ml of solution A, add 2 - 3 drops of a 10% solution of sodium hydroxide R, an yellow precipitate is produced, the precipitate is dissolved in excess of a 10% solution of sodium hydroxide R. B. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel GF254. Mobile phase: Toluene - ethyl acetate - formic acid (6 : 4 : 1) Test solution: Evaporate 5 ml of solution A on a water-bath to about 1 ml. Reference drug solution: Prepare a solution of 3 g of Flos Cleistocalysis operculati reference drug in the same maner as described under Test solution. Procedure: Apply separately 10 µl of each solution to the plate. After the development and removal the plate, dry in air, examine under ultraviolet light at 254 nm, the blue fluorescent spots in the chromatogram obtained with the test solution correspond colour and position to the blue fluorescent spots obtained with the reference drug solution. Spray with a 10% solution of sulfuric acid in ethanol R, heat at 100 °C until reddish-violet spots appear and examine under visible light. The spots in the chromatogram obtained with the test solution correspond colour and position to the spots obtained with the reference drug solution.

Loss on drying Not more than 10.0% (Appendix 9.6, 1 g, 70 °C, 4 h). Total ash Not more than 10.0% (Appendix 9.8). Foreign matter (Appendix 12.11) Blooming flowers: Not more than 10%. Other foreign matter: Not more than 3%. Extractives It contains not less than 15.0%, calculated with reference to the dried drug. Carry out the hot extraction method (Appendix 12.10), using ethanol (70%) R as the solvent. Preliminary processing Collect the flower-bud branches, macerate in water for 20 - 30 minutes. Put out, dry, pluck the buds. Dry under the sun until the content of water is about 50%. Percolate for 0.5 - 1 hour to pale yellow and aromatic buds. Dry in the sun until the buds are pale greyish-yellow, distinct aromatic. Storage Preserve in a well-closed containers, store in a cool and dry place, protect from mould.

FLOS DATURAE METELIS Cà độc dược (Hoa) Sun- or heat-dried flowers of Datura metel L., (Fam. Solanaceae).

Description Dry flowers usually crumpled, strip-shaped. Big buds 3 - 5 cm long; opening flowers 7 - 12 cm long. Calyx tubular, two fifths in length of corolla, grayish-green or grayish-yellow; apex 5-lobed, with 5 longitudinal veins at the base; surface slightly fine pubescent. Corolla trumpetshaped, pale yellow or brownish-yellow; apex 5-lobed; lobes short, acuminulate with 3 distinct longitudinal veins below the acumination, slightly sunken between two lobes. Stamens 5, filaments adhering close to corolla tube, three quarters in length of corolla; styli rode-shaped. Texture of heat-dried samples soft and flexible; of sun-dried samples fragile. Odour mild; taste slightly bitter. Powder Pale yellow in colour; taste slightly bitter. Pollen grains subspherical or oblong, 39 - 42 µm in diameter, tricolporatestructured, surfaced with branching veinlets which forming reticulated veination at both poles. Non-glandular hairs of calyx 1 - 3 celled, with warts on the walls. Glandular hairs with a 1 - 5 celled head and a 1 - 5 celled stalk. Nonglandular hairs on the edge of corolla lobe, 1 - 10 celled, with slightly warty prominences on the walls. Non-glandular hairs on the base of filaments thick, 1 - 5 celled, up to 128 µm in diameter at the base, apex obtusely rounded. Sandy, prismatic crystals and clusters of calcium oxalate occurring in the cells of corolla and calyx. Identification Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Ethyl acetate - methanol - ammonia (17 : 2 : 1). Test solution: To 1 g of the powder add 1 ml of ammonia R, mix well, add 25 ml of chloroform R and stir well. Allow to stand overnight, filter. Evaporate the filtrate to dryness. Dissolve the residue in 1 ml of chloroform R. Reference substance solution: Dissolve atropine sulfate RS and scopolamine hydrobromide RS in methanol R to obtain a solution containing 4 mg of each per ml. Reference drug solution: Alternatively, prepare a solution of 1 g of the powder of Flos Daturae metelis reference drug in the same manner as described under Test solution. Procedure: Apply separately to the plate 10 μl of each solution. After the development and removal of the plate, allow to dry in air. Spray with Dragendorff reagent R. Examine under visible light. The chromatogram obtained with the test solution shows several spots, two of which, correspond in colour and position to the spots due to atropine and scopolamine in the chromatogram obtained with the reference substance solution or the spots in the 1141

VP V

FLOS ERIOCAULI

chromatogram obtained with the test solution correspond in colour and position to the spots in the chromatogram obtained with the reference drug solution.

Storage Store in a dry place, protect from mould and weevils.

Loss on drying Not more than 12.0% (Appendix 9.6, 1 g, 85 °C, 4 h).

FLOS ERIOCAULI Cốc tinh thảo, Cỏ dùi trống

Total ash Not more than 9.0% (Appendix 9.8).

Sun-dried or slightly heat-dried capitula with peduncles of Eriocaulon sexangulare L. or Eriocaulon buergerianum Koern., (Fam. Eriocaulaceae).

Foreign matter Not more than 1.0% (Appendix 12.11). Assay Place about 10 g, accurately weighed, of the fine powder previously dried for 4 hours at 60 °C, in a Soxhlet’s extractor and moisten with a quantity of a mixture of ethanol ammonia - ether (5 : 4 : 10). Allow to stand for 12 hours on a water bath until the alkaloids are extracted completely (Appendix 12.3, using Dragendorff reagent R). Evaporate the extract on a water bath to remove ether, add 25 ml of 0.5 N sulphuric acid R and continue to evaporate the ether completely. Allow to slightly warm, filter through a plug of absorbent cotton wool, transfer the filtrate to a separator. Wash the residue with 5 ml of 0.5 N sulphuric acid R and then with two 5 ml quantities of water. Combine the washings with the filtrate in the separator, and extract successively with 10, 5, 5 ml of chloroform R until the chloroform layer becomes colourless. Combine the chloroform solutions, extract with 10 ml of 0.1 N sulphuric acid R. Discard the chloroform layer, combine the acid solutions and neutralize with ammonia R and add 2 ml in excess. Extract immediately and successively with 20, 15, 15, 10, 5 ml of chloroform R until the alkaloids are extracted completely. Filter the chloroform solutions through a funnel containing a layer of anhydrous sodium sulphate R. Wash the funnel with two 4 ml quantities of chloroform R. Combine the chloroform extracts and washings and evaporate the solvent on a water bath. Add 3 ml of neutral ethanol R to dissolve the residue, evaporate to dryness and heat for further l5 minutes. Dissolve the residue in 2 ml of chloroform R by heating gently, add accurately 20 ml of 0.02 N sulphuric acid VS and heat on a water bath to remove the chloroform. Cool to room temperature, add 2 - 3 drops of methyl red R, titrate with 0.02 N sodium hydroxide VS to yellow colour. Each ml of 0.02 N sulphuric acid VS is equivalent to 6.068 mg of C17H21NO4. The drug, dried at 60 °C for 4 hours, contains not less than 0.30% of alkaloids, calculated as scopolamine (C17H21NO4). Preliminary processing The flowers are collected from april to november, at the beginning of flowering time, dried in the sun or at a low temperature. 1142

Description Capitula flattened rounded, 4 - 5 mm in diameter (about 6 mm, as for Eriocaulon sexangulare L.). Bracts densely arranged in numerous layers at the flower base, pale yellowish-green, lustrous, densely pubescent at the upper margin. Upper surface of capitula grayish-white. After rubbing, numerous black anthers and many fine greenyellow unripe fruit, visible. Peduncles slender, varying in length, rarely less than 1 mm in diameter, pale greenishyellow, bearing numerous twisted ridges on the surface. Texture soft, difficult to break; odourless; taste, insipid. Powder Yellowish-green in colour. Fine hairs with punctate surface. Non-glandular hairs large and long, 2 - 4 celled. Fragments of epidermal cells of capitula peduncles long, flattened; fragments of epidermal cells bearing stomata containing rectangular satellite cells. Pericarpic cells subpolygonal in surface view; walls with thickened prominent plicae, necklace-shaped. Pollen grains subrounded, with narrow lumen. Identification Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Toluene - acetone (10 : 0.6). Test solution: Place 1 g of the powder in a conical flask, add 30 ml of ethanol (96%) R, sonicate for 30 minutes, filter. Evaporate the filtrate to dryness, dissolve the residue in 1 ml of ethanol (96%) R. Reference drug solution: Prepare a solution of 1 g of the powder of Flos Eriocauli reference drug in the same manner as described under Test solution. Procedure: Apply separately to the plate 5 µl of each solution. After developing and removal of the plate, allow to dry in air. Examine under ultraviolet light at 366 nm. The fluorescent spots in the chromatogram obtained with the test solution correspond in colour and position to the fluorescent spots in the chromatogram obtained with the reference drug solution. Loss on drying Not more than 13.0% (Appendix 9.6, 1 g, 105 °C, 4 h). Preliminary processing The drug is collected in autumn, pick up capitula with

FLOS LONICERAE

VP V

peduncles and dry under the sun or at a low temperature (50 - 60 °C) to dryness.

Storage Store in dry places. FLOS LONICERAE Kim ngân (Hoa) Sun- or heat-dried flower opening flowers of Lonicera other species like Lonicera confusa DC. and Lonicera Caprifoliaceae).

buds mixed with several japonica Thunb. and some dasystyla Rehd., Lonicera cambodiana Pierre, (Fam.

Description Flower buds tubular, slightly curved, 1 - 5 cm long, large head, 0.2 - 0.5 cm in diameter. Externally yellow to brown, densely pubescent. Lower part of corolla tube having 5 small, green sepals. Five stamens and 1 style appearing when the bud head pressed strongly. Odour mildly aromatic; taste slightly bitter. Opening flowers 2 - 5 cm long, corolla divided into 2 lips rolling upwards. Upper lip 4- lobed; lower one entire. Stamens and style frequently exserted. Powder Pale brownish-yellow in colour; odour mildly aromatic. Pollen grains spheroidal, 53 - 62 μm in diameter, yellow, 3 germinal pores distinct, surfaced with sparse spinula. Glandular hairs of two types: one with claviform head composed of 20 - 30 cells and the other with spherical head of about 10 cells. Unicellular non-glandular hairs also of two types: one with thick walls, smooth or with small raised dots, the other with thin walls, raised dots very prominent. Corolla fragments bearing glandular and non-glandular hairs. Identification A. To 5 g of the powder in a 100 ml conical flash, add 20 ml of ethanol (90%) R, shake well, heat on a waterbath for 15 min, filter. Evaporate the filtrate on a water bath to about 5 ml. To 1 ml of the filtrate in a test tube, add 2 - 3 drops of hydrochloric acid R and a small quantity of magnesium powder R or zinc powder R, the solution changes from yellow colour to orange then red colour. B. To 1 g of the powdered herbal drug in a test tube, add 10 ml of water, gently shake for 5 min, filter. Transfer the filtrate to 2 test tubes, 2 ml each tube. To test tube 1, add 2 - 3 drops of 10% sodium hydroxide solution R, the obtained solution is yellower than the solution in test tube 2. C. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel 60 F254. Mobile phase: Buthyl acetate - formic acid - water (7 : 2.5 : 2.5).

Test solution: To 0.2 g of the powder, add 10 ml of methanol R. Sonicate for about 20 min, with frequent shaking, allow to cool then filter. Evaporate the filtrate on a water-bath to dryness. Dissolve the residue in 2 ml of ethanol R. Reference substance solution: Prepare a 1 mg/ml solution of chlorogenic acid RS in ethanol R. Reference drug solution: Alternatively, prepare a solution of 0.2 g of Flos Lonicerae reference drug in the same manner as described under Test solution. Procedure: Apply separately to the plate 10 µl of each solution. After developing and removal of the plate, dry in air and examine in ultra-violet light at 366 nm. The chromatogram obatained with the test solution exhibits a spot corresponding in colour and position to that in the chromatogram obtained with the reference substance solution or the spots in the chromatogram obtained with the test solution correspond in colour and position to the spots in the chromatogram obtained with the reference drug solution.

Loss on drying Not more than 12.0% (Appendix 9.6, 1 g, 85 °C, 4 h). Total ash Not more than 9.0% (Appendix 9.8). Hydrochloric acid-insoluble ash Not more than 1.5% (Appendix 9.7). Foreign matter (Appendix 12.11). Stems and leaves: Not more than 2%, Others: Not more than 0.5%. Blooming flowers Not more than 10% (weigh the blooming flowers selected from 100 g of Flos Lonicerae, calculate the blooming flower rate). Extractives Not less than 29.0%, calculated with reference to the dried drug. Carry out the hot extraction method (Appendix 12.10), using ethanol (96%) R as the solvent. Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - 0.4% solution of phosphoric acid (13 : 87). Reference solution: Prepare a 0.15 mg/ml solution of chlorogenic acid RS in methanol (50%) R. Test solution: Transfer about 0.5 g of the powder (passed through a sieve No 355), accurately weighed, to a 100-ml stopper conical flask, add 50.0 ml of methanol (50%) R, stopper tightly, weigh. Sonicate for 30 min, with frequent shaking, allow to cool, weigh again and replenish the loss of weight with methanol (50%) R, mix well and filter through a membrane filter of 0.45-µm. 1143

VP V

FLOS PLUMERIAE RUBRAE

Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 327 nm. Flow rate: 1.0 ml/min. Volume of injection: 10 µl. Procedure: Inject the reference solution and record the chromatograms. The column efficiency, determined on the peak due to chlorogenic acid, is not less than 1000 theoretical plates. Inject the reference solution, the test solution and record the chromatograms. Calculate the percentage content of chlorogenic acid (C16H18O9) in the drug using the areas of chlorogenic acid peaks in the chromatograms obtained with the test solution, the reference solution and the declared content of chlorogenic acid RS. It contains not less than 1.5% of chlorogenic acid (C16H18O9), calculated with reference to the dried drug.

Preliminary processing Pluck flower buds mixed with a few flowers; remove foreign matter, dry in the shade or at a low temperature to dryness. Storage Store in cool and dry places, protected from weevils and insects. FLOS PLUMERIAE RUBRAE Đại (Hoa), Bông sứ, Hoa sứ trắng Sun- or slightly heat-dried flowers of Plumeria rubra L. var. acutifolia (Aiton) Woodson, (Fam. Apocynaceae).

Description Flowers with 5 thin petals, externally white, internally lemon at lower part, 4 - 5 cm long. Dried flowers earthbrown, very light, tortuous; petals sometimes twisting each other. Odour mildly aromatic. Powder Brown in colour; odour aromatic; taste slightly sweet. Examine under a microscope: Pollen grains spheroidal, about 25 µm in diameter, pale yellow, with 3- germinal pores distinct. Fragments of petal epidermis consisting of sinuous thin-walled cells. Corolla containing unicellular non-glandular hairs which having finely dotted surface. Epidermal fragments of calyx composed of polygonal thin-walled cells, scattered with spiral vascular bundles. Identification Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Petroleum ether - ethyl acetate - formic acid (7.5 : 2.5 : 0.1). Test solution: To the dried residue obtained under Assay B, moisten with 5 ml of a 25% solution of ammonia, 1144

stopper tightly, allow to stand for 30 minutes. Extract with chloroform R in a Soxhlet’s extractor for 2 hours (from boiling solvent). Distil the extract to recover the solvent to dryness. Dissolve the residue with 10 ml of a 10% solution of sulphuric acid R, filter, adjust pH of the filtrate with a 10% solution of ammonia R to pH 10 - 11, extract with 25 ml of chloroform R by shaking. Decant and filter the chloroform extract through anhydrous sodium sulphate R. Distil to recover the solvent to dryness. Dissolve the residue in 1 ml of chloroform R. Reference substance solution: Dissolve ajmalin RS in chloroform R to obtain a solution containing 0.1 mg per ml. Reference drug solution: Alternatively, prepare a solution of the residue obtained from 5 g of Flos Plumeriae rubrae reference drug that extracted with petroleum ether (boiling range 60 °C - 90 °C) or with n - hexane R (see Assay B), in same manner as described under Test solution. Procedure: Apply separately to the plate 10 µl of each solution. After developing and removal of the plate, dry it in air, spray with Dragendorff reagent R. Examine under visible light. The chromatogram obtained with the test solution shows several spots, one of which corresponds in colour and position to the spot due to ajmalin in the chromatogram obtained with the reference substance solution or the spots in the chromatogram obtained with the test solution correspond in colour and position to the spots in the chromatogram obtained with the reference drug solution.

Loss on drying Not more than 15.0% (Appendix 9.6, 1 g, 85 °C, 4 h). Foreign matter Blackened flowers: Not more than 0.5% (Appendix 12.11). Total ash Not more than 7.0% (Appendix 9.8). Acid-insoluble ash Not more than 1.5% (Appendix 9.7). Assay A. Total alkaloids: Weigh accurately about 5 g of the powder (passed through a sieve No. 355), determine the content of water in the drug at the same time. Moisten the powder with 5 ml of a 25% solution of ammonia R, stopper tightly and allow to stand for 30 minutes. Extract with chloroform R in a Soxhlets extractor for 2 hours as from boiling solvent. Distil the extract to recover the solvent to dryness. Dissolve the residue with 10 ml of a 10% solution of sulphuric acid R, filter. Wash the Soxhlet extractor and the paper filter with 10 ml of a 1% solution of sulphuric acid R, continue to wash with 5 ml of water. Combine the filtrates and washings, adjust to pH 10 - 11 with a 10% solution of ammonia R, extract successively with four quantities (25, 20, 15, 10 ml) of chloroform R. Decant and filter the chloroform extract through anhydrous sodium

FLOS SAMBUCI JAVANICAE

VP V

sulphate R into a previously weighed flask, wash the paper filter and anhydrous sodium sulphate with 10 ml of chloroform R. Combine the filtrates and washings. Distil to recover the solvent to residue. Dry the residue at 80 °C to constant mass and weigh. Calculate the content of total alkaloids of the drug from the expression: a × 10000 X% = b × (100 − c)

(1)

Where: a: mass of the extracted residue (g), b: mass of the powder to be examined (g), c: content of water (%). It contains from 0.08 - 0.13% of total alkaloids calculated with reference to the dried drug. B. n-Hexane-soluble extractives or petroleum ether (60 °C - 90 °C) - soluble extractives: Weigh accurately about 5 g of the powder (passed through a sieve No.355), determine the content of water in the drug at the same time. Extract with n-hexane R or petroleum ether (60 - 90 °C) R in a Soxhlet’s extractor for 2 hours (from boiling solvent). Filter the extract to a previously dried and weighed flask. Distil the solvent to dryness. Dry the residue at 50 °C to dryness, then keep it in a desiccator to constant mass and weigh accurately. Calculate the content of extractives from the expression (1). It contains not less than 5.0% of the extractives calculated with reference to the dried drug.

Preliminary processing The drug is collected from may to august, opening flowers are picked and dried under the sun or at 40 °C - 50 °C to dryness. Storage Store in a dry and cool place. FLOS SAMBUCI JAVANICAE Cơm cháy (Hoa) Sun- or heat- dried flowers of Sambucus javanica Blume., (Fam. Sambucaceae).

Description Dried flowers pale yellow to brownish-yellow; odour specially pungent; 1.5 - 2.5 mm in diameter. Five corollas wheel shape, sharp-tipped. Five stamens arrange alternatively. Anther with 2 cells, cracks longitudinally, directs outside. Pistil composed of 3 carpels, divided ovary into 3 cells, the each cell contains one ovule. Some flowers may not reproduce and turn to cup-shaped gland forms, 1 - 2 mm in diameter.

Powder Brownish-yellow in colour, specialy pungent odour, tasteless. Parenchyma fragment of petal consisting of polygonal cells, arranged evenly, tissue fragment bearing stomatas and multicellular grandular hairs. Yellow, cylindrical pollen grains with 3 zonocolporates, outside cortical layer, rough, single or mass compound. Fragments of spiral and pitted vessels. Identification To 10 g of the powder in a filter paper bag, transfer to a Soxhlet's extractor, extract with petroleum ether R 30 - 60 °C) until the extract is colourless. Remove the extract and dry the residue. Transfer the residue to a conical flask, add 30 ml of ethanol (90%) R, heat on a water bath for 15 minutes, filter immediately. The filtrate complies with the following tests: A. Apply 1 drop of the filtrate to a filter paper, allow to dry, expose to ammonia vapour, the initial yellow colour is darkened. B. To 2 ml of the filtrate in a test-tube, add 2 - 3 drops of a 10% solution of sodium hydroxide R, a yellow precipitate is produced, add 1 ml of distilled water R, the precipitate is dissolved. C. To 2 ml of the filtrate in a test-tube, add a small amount of magnesium powder R and 3 - 4 drops of hydrochloric acid R, heat on a water bath, a pink colour is produced. Loss on drying Not more than 12.0% (Appendix 9.6, 1 g, 85 °C, 4 hours). Total ash Not more than 9.0% (Appendix 9.8). Foreign matter Not more than 1% (Appendix 12.11). Extractives Not less than 25.0%, calculated with reference to the dried material. Carry out the hot extraction method (Appendix 12.10), using distilled water R as the solvent. Preliminary processing The flowers are collected immediately after blooming. Dry under the sun or at 50 - 60 °C. Stir-bake at low temperature before use. Storage Preserve in well closed containers. Store in a dry place, protected from weevils and insects.

1145

VP V

FLOS STYPHNOLOBII JAPONICI IMMATURUS

FLOS STYPHNOLOBII JAPONICI IMMATURUS Flos Sophorae japonicae immaturus Hòe (Nụ hoa) Sun- or heat-dried flower buds of Styphnolobium japonicum (L.) Schott; Syn. Sophora japonica L., (Fam. Fabaceae).

The yellow spot in the chromatogram obtained with the test solution corresponds in position and colour to the spot due to rutin in the chromatogram obtained with the reference substance solution (Rf value is about 0.5 - 0.54).

Loss on drying Not more than 11.0% (Appendix 9.6, 2 g, 105 °C, 5 h).

Description Flower buds ovoid, with slender and short pedicels, one end slightly acute, 3 - 6 mm long, 1 - 2 mm wide, greyish yellow. Calyx campanulate, greyish yellow, 1/2 to 2/3 in length of the bud, upper part 5-shallow lobed. Big buds 4 - 10 mm long, 2 - 4 mm in diameter. Petals of unopened flowers yellow. Odour aromatic; taste slightly bitter.

Total ash Not more than 10.0% (Appendix 9.8).

Powder Pollen grains numerous, spherical, 16 µm in diameter, tricolporate, surfaced with reticulate striae. Multicellular non-glandular hairs 2 - 4 celled; head cells long and acuteoblong, base cells short. Epidermal fragments of petals consisting of polygonal, finely striated cells, arranged closely. Epidermal fragments of calyx composed of polygonal cells, bearing anomocytic stomata and nonglandular hairs. Fragments of spiral vessels also found.

Assay Standard solution: Weigh accurately about 0.2 g of rutin RS, previously dried to constant mass in vacuum, and put into a 100 ml volumetric flask, dissolve in 70 ml of methanol R on a warm water bath, cool, add methanol R to volume, mix well. Pipet 10.0 ml of this solution into another 100 ml volumetric flask, add water to volume, mix well (the solution contains 0.2 mg of rutin per ml). Setting up the standard curve: Pipet 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0 ml of the standard solution, place separately each in a 25 ml volumetric flask, add water to get 6.0 ml of solution in each flask, add 1 ml of a 5% solution of sodium nitrite R, mix well, allow to stand for 6 minutes. Add 1 ml of a 10% solution aluminium nitrate R, mix well, allow to stand for 6 minutes. Add 10 ml of a 10% solution of sodium hydroxide R, add sufficient water to volume, mix well, allow to stand for 15 minutes. Measure the absorbance of the obtained solutions at the wave-length of 500 nm (Appendix 4.1). Draw the standard curve with the absorbances on the vertical axis and concentrations on the horizontal axis. Test solution: Weigh accurately about 1 g of the coarse powder, previously dried at 60 °C for 6 hours, transfer to a Soxhlet’s extractor. Add 120 ml of ether R, extract until the ether extract is colourless, cool, and discard the ether extract. Add 90 ml of methanol R, extract until the methanol extract is colourless. Transfer the methanol extract to a 100 ml volumetric flask, wash the extractor with small quantities of methanol R for several times, transfer the washings to the volumetric flask, add sufficient methanol R to volume, mix well. Pipet 10.0 ml of the methanol solution into a 100 ml volumetric flask, add sufficient water to volume, mix well. Pipet accurately 3 ml of this diluted solution into a 25 ml volumetric flask, add 3 ml of water and 1 ml of a 5% solution of sodium nitrite R, mix well, allow to stand for 6 minutes. Add 1 ml of a 10% solution of aluminium nitrate R, mix well, allow to stand for 6 minutes. Add 10 ml of a 10% solution of sodium hydroxide R, dilute with water to volume, mix well, allow to stand for 15 minutes. Measure the absorbance of the obtained solution at the wave-length of 500 nm (Appendix

Identification A. To 0.5 g of the powder, add 10 ml of ethanol R, boil for 3 minutes, cool, and filter. Use the filtrate (solution A) to perform the following reactions: Dilute 2 ml of solution A with 10 ml of ethanol 90% R. The obtained solution is divided into 3 test tubes: Test tube 1: Add 5 drops of hydrochloric acid R and a small quantity of magnesium powder R. The colour of the solution changes from pale yellow to pink then reddishviolet. Test tube 2: Add 2 drops of a 20% solution of sodium hydroxide R, a yellowish-orange precipitate is produced and dissolved in the excess reagent. Test tube 3: Add 2 drops of a 5% solution of ferric chloride R, the solution turns mossy green colour. B. Apply 2 - 3 drops of solution A to a piece of filter paper, allow to dry, examine under ultra-violet light at 366 nm a brownish-yellow fluorescent spot is observed. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: n-Butanol - acetic acid - water (4 : 1 : 5). Test solution: Solution A. Reference substance solution: Dissolve rutin RS in ethanol (90%) R to produce a solution containing 1 mg per ml. Procedure: Apply separately to the plate 20 µl of each solution. After developing and removal of the plate, dry in air. Examine under ultra violet light at 366 nm. The brown fluorescent spot in the chromatogram obtained with the test solution corresponds in colour and position to the spot due to rutin in the chromatogram obtained with the reference substance solution. Expose the plate to ammonia vapour. 1146

Foreign matter (Appendix 12.11) Blooming flowers: Not more than 10.0%. Dark colour flowers: Not more than 1.0%. Other parts: Not more than 2.0%.

VP V

4.1). Calculate the mass (μg) of rutin in the test solution from the concentration read on the standard curve and determine the percentage of rutin in the crude drug. It contains not less than 20.0% of rutin (C27H30O16), calculated with reference to the dried drug.

Preliminary processing Usually in the morning, when the weather is dry, pluck panicles of flower buds, pull off buds from panicles, remove all the other parts of the plant, dry the buds in the sun or at a low temperature to dryness. Storage Store in dry places, protected from mould and weevils. FLOS SYZYGII AROMATICI Đinh hương (Nụ hoa) Sun- or heat-dried flower buds of Syzygium aromaticum (L.) Merill et Perry, (Fam. Myrtaceae).

Description Flower bud similar to a nail, dark brown, consisting of a cylindrical inferior ovary with 10 - 12 mm long, 2 - 3 mm in diameter, and a spherical head with 4 - 6 mm in diameter. The lower part of ovary sometimes bearing a short remain of pedicel, the upper part bearing 4 thick triangular sepals, arranged crossly opposite. The spherical head consisting of 4 unopened petals, arranged adjacently to each other. After removing petals, numerous stamens visible with a short, straight style in the centre. Transverse section Ovarium elliptical or rounded. Epidermis sinuous, consisting of one row of cells, covered with thick cuticle, scattered with stomata. Parenchyma: Outer layers composed of thin-walled cells, usually compressed; containing 2 to 3 rings of ovoid, oil secretory vesicles. Inner layers composed of polygonal cells, phloem-xylem bundles abundant, xylem in the middle, surrounded by phloem, fibres or fibrous masses occurring outside phloem. Spongy tissue: Cells thin-walled, arranged in succession, forming reticulation and large lacunae. Stele part: A few rows of parenchymatous cells surrounding outside, containing abundant urchin-form crystals of calcium oxalate. Phloem-xylem bundles in continuous rings with xylem inside and phloem outside. Parenchyma in the innermost part containing numerous urchin-form crystals of calcium oxalate. Powder Dark brown in colour. Odour, pungently aromatic; taste, hot. Examine under a microscope. Parenchymatous fragments of ovary containing large spherical oil secretory vesicles, 80 - 100 µm in diameter. Epidermal cells with stomata. Fibres short, thick-walled, narrowly luminal,

FLOS TUSSILAGINIS FARFARAE

occurring singly or in groups of 2 - 3 fibres. Pollen grains triangular, pale yellow, 15 - 20 µm in diameter. Petal fragments consisting of numerous thin-walled cells. Urchin-form crystals of calcium oxalate abundant, occurring inside the cells or separately outside. Fragments of spiral vessels occurring separately or in bundles; cells of sclerenchyma also found.

Water Not more than 13.0% (Appendix 12.13). Foreign matter (Appendix 12.11) Blooming flowers and peduncles: Not more than 5.0%, Others: Not more than 1%. Total ash Not more than 7.0% (Appendix 9.8). Assay Carry out the method for Determination of essential oils in herbal drugs (Appendix 12.7) Weigh accurately about 5 g of the coarse powder, place it in a 250 ml round bottom flask, add 100 ml of water. Distil for 4 hours, use 0.50 ml of xylene R. It contains not less than 15.0% of volatile oil, calculated with reference to the dried drug. Preliminary processing Flower buds are collected when turning to deep red, removed from foreign matter and flower stalks and dried under the sun or at a low temperature. Storage Preserve in well closed containers, store in dry and cool places, protected from light. FLOS TUSSILAGINIS FARFARAE Khoản đông hoa Sun- or heat-dried thyrse buds of Tussilago farfara L., (Fam. Asteraceae).

Description Thyrse buds long clavate headed, usually solitary or 2 - 3 accreted on one branch, 2 - 2.5 cm long, upper part broader and lower part tapering. Thyrse peduncle beaks having numerous scaly bracts. Outer surface of bracts purplish-red or pale red, inner surface densely covered with white flocky hairs. Odour aromatic; taste slightly bitter and pungent. Powder Pale brownish-yellow in colour, coarse, stuck to big pieces from pubescence. Examine under a microscope: Numerous epidermal fragments of petals containing big polygonal cells which scattered with brownish-yellow pigments. Numerous epidermal fragments of bracts containing polygonal cells and stomata, several cells 1147

VP V

FOLIUM AMPELOPSIS

containing brownish-red pigments. Numerous unicellular non-glandular hairs ulotrichous, white flocky. Pollen grains spherical, yellow, with spined primine. Wood vessels orange-red. Stigma fragments also found.

Identification A. To 1 g of the powder, add 10 ml of ethanol (96%) R, heat to boiling, shake well and filter. To 1 ml of the filtrate, add 1 drop of a 5% solution of ferric chloride R, a blackishgrey colour and precipitate is produced. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Cyclohexane - ethyl acetate (9.5 : 0.5). Test solution: To 2 g of the powder, add 2 ml of petroleum ether (40 - 60 °C) R. Soak with occasional shaking for 1 hour, filter. Allow the filtrate to evaporate naturally to dryness. Dissolve the residue in 1 ml of chloroform R. Reference drug solution: Prepare a solution of 2 g of the powder of Flos Tussilaginis farfarae reference drug in the same manner as described under Test solution. Procedure: Apply separately to the plate 20 μl of each solution. After developing and removal of the plate, allow to dry in air or at a low temperature. Examine under ultraviolet light at 366 nm, then spray with vanillin solution in sulphuric acid R. Heat the plate at 105 °C until the spots appear. The spots in the chromatogram obtained with the test solution correspond in colour and position to the spots in the chromatogram obtained with the reference drug solution. Water Not more than 14.0% (Appendix 12.13). Foreign matter Blackened flower buds: Not more than 0.5% (Appendix 12.11). Extractives Not less than 20.0%, calculated with reference to the dried drug. Carry out the hot extraction method (Appendix 12.10), using ethanol (96%) R as the solvent. Preliminary processing Flower buds are collected in december or cold season, removed from flower stalks and soil, then dried in the shade. Processing Flos Tussilaginus farfarae: Eliminate foreign matter and remained flower stalks. Flos Tussilaginis farfarae (processed with honey): Add honey and a small quantity of boiling water to clean Flos Tussilaginus farfarae, mix well, wrap up until honey is thoroughly absorbed, then stir-bake with gentle heat until no more sticky to fingers; take out and cool. Use 2.5 kg of honey per 10 kg of Flos Tussilaginis farfarae. 1148

Storage Store in a dry and cool place; protect from mould and moth. FOLIUM AMPELOPSIS Chè dây Sun- or heat-dried leaves of Ampelopsis cantoniensis (Hook. et Arn.) Planch., (Fam. Vitaceae.)

Description Dried leaflets usually crumpled, ovate or lanceolate when stretched, 2.5 - 7.5 cm long; base obtuse or subrounded; apex acute; margins, slightly denticular. Upper surface greyish-green, full of white patches like mouldy; lower surface lighter in colour. Leaf peduncles smooth, 3 - 12 mm long. Texture light, fragile easily broken to shreds; odour aromatic; taste, bitter then a bit sweet. Transverse section Leaf veins: Midribs heavily protruding on upper surface (protrusion height may be as thick as leaf-blade), less protruding on lower surface. Upper and lower epidermis of leaf veins consisting of one row of cells, small, even, arranged continuously and regularly, bearing short unicellular non-glandular hairs. Collenchyma composed of cells thick-walled, varying in size, arranged in some rows lying adjacently to epidermal layer. Phloem-xylem bundles of midrib consisting of small bundles separately arranged; one bundle found on the upper side of the centre with centripetal xylem; other bundles forming an arc on the lower side with centripetal xylem. A medullar parenchyma area found among phloem-xylem bundles, composed of thin-walled cells. Lamina: Upper and lower epidermis of leaf-blade containing small cells, arranged in a continuous and regular layer. Palisade tissue consisting of one row of cells, arranged vertically and regularly beneath the upper epidermal layer. Lamina scattered with fragments of spiral vessels, urchinform crystals of calcium oxalate; bundles of needle-shaped crystals of calcium oxalate lying in thin-walled lumina. Powder Greyish-green in colour. Examine under a microscope. Fragments of leaf-blade possibly bearing vascular fragments, epidermal fragments containing stomata, unicellular non-glandular hairs. Numerous needle-shaped crystals of calcium oxalate scattered or grouped into bundles. Fragments of dotted vessels, fragments of spiral vessel, urchin-form crystals of calcium oxalate, 30 - 35 µm in diameter, also found. Identification A. To 0.5 g of the powder, add 10 ml of ethanol (90%) R. Heat in a water-bath for 3 minutes, filter. Pipet the filtrate (solution A) into 2 test-tubes, each of 1 ml, to perform the following reactions:

FOLIUM ARDISIAE

VP V

Test-tube 1: Add 5 drops of hydrochloric acid R and a small quantily of magnesium R powder, allow to stand for some minutes, the solution changes gradually from light yellow to red. Test tube 2: Add 2 drops of a 5% solution of ferric chloride R, the solution turns to blue. Apply 2 - 3 drops of solution A on a filter paper, allow to dry, expose the filter paper above the mouth of an opened bottle of concentrated ammonia R, the yellow colour becomes much more intense. B. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel G. Mobile phase: Toluene - ethyl acetate - formic acid (5 : 6 : 1.5). Test solution: Solution A. Reference substance solution: Dissolve separately myricetin RS and dihydromyricetin RS in methanol R to produce a solution containing 2 mg per ml. Alternatively, prepare a solution of 0.5 g of the powder of Folium Ampelopsis reference drug in the same manner as described under Test solution. Procedure: Apply separately to the plate 8 µl of each solution. After developing and removal of the plate, allow to dry in air at room temperature, spray with a mixture of a 10% solution of boric acid R and a 10% solution of oxalic acid R (2 : 1) and dry the plate at 100 °C until the spots appear clearly. Examine under visible light. The spots in the chromatogram obtained with the test solution correspond in colour and positions to the spots in the chromatogram obtained with the reference substance solution.

to expel completely the chloroform from the drug residue. Transfer the filtering paper bag into a Soxhlet's extractor, add 120 ml of methanol R, boil in a water bath until 1 ml of the extract has no colour on addition of 1 drop of a 10% solution of sodium hydroxide R. Evaporate the methanol extract to partial dryness. Allow to cool, transfer to a 50 ml volumetric flask, add sufficient methanol R to volume, shake well. Measure accurately 2 ml of the obtained solution and apply to a column packed with activated stationary phase C. Elute with methanol (50%) R and a flow rate of 15 drops per minute, collect about 17 ml of the eluate, add sufficient methanol (50%) R to 20.0 ml. Shake well, filter through a 0.45 μm filtering membrane. Chromatographic system: A column (25 cm × 4 mm) packed with stationary phase C (5 μm) (Lichrosorb RP 18). Detector: A spectrophotometer set at 260 nm. Flow rate: 1 ml per minute. Volume of injection: 20 μl. Procedure: Inject separately the test solution and the reference solution. Use the peak areas due to dihydromyricetin in the chromatograms obtained with the test solution and the reference solution, and the declared content of C15H12O8 in dihydromyricetin RS to calculate the content of dihydromyricetin (C15H12O8) in the drug. It contains not less than 18.0% of dihydromyricetin (C15H12O8), calculated with reference to the dried drug.

Loss on drying Not more than 13.0% (Appendix 9.6, 1 g, 100 °C, 4 h).

Storage Store in dry and ventilated places.

Total ash Not more than 8.0% (Appendix 9.8, method 2), determined on 1 g.

FOLIUM ARDISIAE Khôi (Lá)

Foreign matter Not more than 1.0% (Appendix 12.11).

Sun- or heat- dried leaves of Ardiasia sylvestris Pitard.,(Fam. Myrsinaceae).

Heavy metal Not more than 3 ppm of Pb, 1.0 ppm of Cd, 0.5 ppm of Hg, 2.0 ppm of As (Appendix 9.4.11).

Description Leaf is oblong-lanceolate shape, 25 - 35 cm in length, 6 - 10 cm in width; petiole 1.5 - 3 cm in length; fine leaf blades, equally and continuously serated; green upper surface, violet lower surface with a lot of tiny spots, two surfaces have smooth hairs as velvet.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile - 0.01 M phosphoric acid (25 : 75). Reference solution: Dissolve dihydromyricetin RS in methanol R to produce a solution having known concentration of about 2 mg per ml. Test solution: Macerate about 0.5 g of the powder (passed through a sieve No. 355), accurately weighed, in a filtering paper bag, with 50 ml of chloroform R for 12 hours. Discard the chloroform extract. Evaporate on a water bath

Preliminary processing Leaves are collected and removed from mined, old and withered leaves, then dried under the sun to dryness.

Transverse section Midrib: upper concave, lower convex. Epidermal cells bear multicellular non-gradular hairs and glandular hair with unicellular base and multicellular apex. Epidermal cells arranged evenly in a single row. Collenchymas including thick wall cells are located closely to the epidermis. Parenchyma including thin walled, round cells with glandular pipes scattering about. Phloem-xylem 1149

VP V

FOLIUM ARTEMISIAE ANNUAE

bundles arranged closely in an arch: in each of the bundles, there are a phloem bundle out and a xylem one in-side. Medullary parenchyma consist big, thin-walled round cells, with glandular pipes scattering about. Lamina: under the epidermis, a row of parenchymatous palisade tissue and spongy parenchyma consisting of thinwalled, small cells irregularly arranged.

Powder Grayish-brown powder, odor, slight sweet. Many multicellular hairs; glandular hair with unicellular base and multicellular apex; parenchymatous fragment, stomata, a lot of spiral vessel, calcium oxalate crystals in urchin form. Identification A. To 3 g of the powder add 20 ml of water, boil in 2 - 3 minutes, filter, and the filtrate complies with the following tests: To 1 ml of the filtrate, add 2 drops of a 5% solution of ferric chloride R, a dark-green colour is produced. To 1 ml of the filtrate, add 2 drops of a 1% solution of gelatin R, a white cotton precipitate is produced. B. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel GF254. Mobile phase: Ethyl acetate - chloroform - formic acid (3 : 3 : 1). Test solution: To 3 g of the powder, transfer to a Soxhlet's extractor, extract with petroleum ether (40 °C - 60 °C) R until the extract is colourless. Discard the petroleum ether extract, the residue is extracted with 30 ml of ethyl acetate R in 2 hours, collect the extract, then distill to recover the solvent until obtaining dry residue. Dissolve the residue in 1 ml of methanol R. Reference drug solution: Prepare a solution of 3 g of the powder of Folium Ardisiae reference drug in the same manner as described under Test solution. Procedure: Apply separately to the plate 10 µl of each solution. After the development and removal of plate, allow to dry in room temperature. Spay with a 10% solution of sulfuric acid in ethanol R. Heat the plate at 105 °C until spots are visible. Examine under visible light. The spots in the chromatogram obtained with test solution correspond in position and colour to spots in the chromatogram obtained with reference drug solution. Loss on drying Not more than 12.0% (Appendix 9.6, 2 g, 100 °C, 5 hours). Total ash Not more than 20.0% (Appendix 9.8) Foreign matter Not having soil, rock, gravel (Appendix 12.11) Heavy metals Not more than 2.0 ppm of Pb, 0.5 ppm of Cd, 0.5 ppm of Hg, 1.0 ppm of As (Appendix 9.4.11). 1150

Extractives Not less than 10.0%, calculated with reference to the dried drug. Carry out the hot extraction method (Appendix 12.10), using ethanol (96%) R as solvent. Preliminary processing After collecting, wash clean, dry under sun. When using, cut into sections 3 - 5 cm in length and stir-bake at low temperature. Storage Store in a dry, cool and ventilates place, protected from mould. Note: Do not use the leaves with two sufaces of green colour.

FOLIUM ARTEMISIAE ANNUAE Thanh cao hoa vàng, thanh hao hoa vàng (lá) Sun- or heat- dried folium of Artemisia annua L. (Fam. Asteraceae).

Description Leaves brownish-yellow or dark brown, fragile, easy to crush, odour horrible, taste bitter. Sometime mixed with twig or bud. Transverse section Midrib: Upper and lower epidermis consist of one layer of regular cells, waxy cuticle thin, bearing glandular hair and non- glandular hair. Collenchyma closed to the upper and lower epidermis. Parenchyma consisting wrinkled and thin-walled cells. Xylem- phloem bundles located at center of vein include: two sclerenchymatous arc face each other consist of polygonal and thick-walled cells. Phloem arc be adjacent to lower sclerenchymatous arc. Xylem bundles fusiform, consisting xylem vessel arranged in rows, between phloem arc and upper sclerenchymatous arc. Lamina: the upper and lower epidermis consist of one layer of regular cell, bearing glandular hair and nonglandular hair. Lower epidermis consisting stomata. Palisade mesophyll located at upper and lower. Small xylem- phloem bundles arranged in spongy mesophyll. Powder Epidermis fragment consist of thin-walled cells and stomata. Two types of non- glandular hair, one T-shaped, with fusiform unicellular at apex and multiple cell stalk consisting 2-3 cells; each othermulticellular, cells arranged in row and small head. Glandular hair, 2 cells at head and 1-2 rows of cell at stalk. Spiral vessel, scalariform vessel, parenchymatous fragment. Identification Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel G. Mobile phase: Toluene - ethyl acetate (95 : 5).

FOLIUM CALOTROPIS

VP V

Test solution: Weigh 1 g of the powder, add 30 ml of petroleum (30 - 60 oC) R, heat under a reflux condenser on a water-bath for 5 - 10 min. Cool, filter, evaporate the filtrate on a water-bath to dryness. Dissolve the residue in 1 ml of chloroform R, add 9 ml of ethanol R, shake well and filter. Reference substance solution: A 0.1% solution of artemisinin RS in ethanol R. Procedure: Apply separately 10 μl of each solution to the plate. Develop over a path of 10 cm, remove the plate, allow to dry in air at the room temperature. Spray a solution of 0.025 g of 4-dimethylaminobenzaldehyde R in a mixture of 5.0 ml of acetic acid R and 5.0 ml of a 10% solution of phosphoric acid R, heat the plate at 110 °C for 5 min. Examine in daylight. The chromatogram obtained with the test solution show a purplish-green spot corresponding in colour and position to the spot in the chromatogram obtained with the reference substance solution.

will have ivory colour. Mark 0.5 - 0.7 cm above and below zone 1 to 4. Scrape the marked regions separately. Scrape zone 5 as a blank. Add 1 ml of ethanol R to each of the scraped silica gel, shake well and then add 9 ml of 0.05 N sodium hydroxide R, shake well, keep at 50 °C in incubator for 30 min, cool for 15 min, filter. Determine the absorbance of the filtrate at the wavelength of 292 nm (Appendix 4.1). The absorbance of each solution is an average value of two measures. Use the absorbances of the reference solution, the test solution and concentration of artemisinin in the reference solution to calculate content of artemisinin in drug. It contains not less than 0.7% of artemisinin (C15H22O5), calculated with reference to the dried drug.

Loss on drying Not more than 13.0% (Appendix 9.6, 1 g, 85 °C, 5 h).

Preliminary processing The drug is collected just before flowering stage, best in the summer. Collect the aerial part, dry under the sun, shake or beat for fallen leaves. Discard trunks and branches, leaves are dried under the sun or at low temperate.

Total ash Not more than 6.0% (Appendix 9.8).

Storage Preserve at a dry place, protected from weevil and mould.

Foreign mater (Appendix 12.11) Twig or bud: Not more than 10.0% Other: Not more than 2.0%

FOLIUM CALOTROPIS Lá hen, Bồng bồng, Nam tỳ bà

Assay Examine by ultraviolet and visible absorption spectrophotometry (Appendix 4.1) and thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel G. Mobile phase: n-Hexane - ethyl acetate (70 : 30). Test solution: Weigh accurately about 1 g of the powder (passed through a sieve No 355), put into a Soxhlet’s extractor, add 50 ml of petroleum ether (30 °C - 60 °C) R. Heat on a water bath for 6 hours. Evaporate the petroleum ether extract to dryness, dissolve the residue in 1 ml of chloroform R and transfer to a 10 ml volumetric flask with ethanol R and add ethanol R to volume. Filter through a filter paper, discard the first 0.5 ml - 1 ml of the filtrate and use 2 ml of the filtrate as the test solution. Reference solution: A 0.1% solution of artemisinin RS in ethanol R (use within a day after preparation). A silica gel G plate (20 cm × 20 cm) is activated in 110 °C for 2 hours and divided into 5 zones with note from 1 to 5. Apply 0.1 ml of the test solution on zone 1, 2 and 0.1 ml of the reference solution on zone 3, 4 and 0.1 ml of ethanol R on zone 5 (as a blank) as lines, each 2 cm long, 0.3 cm wide. After the development over a path of 18 cm and removal of the plate, dry in air. Spray with water to determine the areas containing artemisinin. The zones containing artemisinin

Sun- or heat-dried leaves of Calotropis gigentea (L.) Dryand. ex Ait. f., (Fam. Asclepiadaceae).

Description Leaves short petioled about 0.5 cm long; blades large and elongated, 12 - 20 cm long, 5 - 10 cm wide; both surfaces bearing white hairs, more abundant on the lower. Veins distinctly conspicuous on lower surface; midrib large, containing a big gland lying near the petiole and surrounded by reddish-brown hairs, slightly bristle and coarse. Transverse section Midrib: Upper surface aplanate; lower surface prominent. Upper and lower epidermis consisting of one layer of small cells, arranged evenly, bearing multi-cellular nonglandular hairs (more abundant on the lower). Collenchyma occurring below epidermis, consisting of 2 - 3 layers of rounded, thick-walled cells. Adjacently, lying parenchyma with larger cells, ovoid or polygonal, thin-walled, varying in size; latex vessels scattered in parenchyma or wood vessels. The phloem-xylem bundle of midrib composed of a xylem arch with wood vessels arranged in rows, and a surrounding ring. Crystals of calcium oxalate urchin-form, 0.03 - 0.04 mm in diameter, scattered in parenchymatous cells. Lamina: Upper and lower epidermis consisting of one layer of evenly arranged small cells, scattered with 1151

VP V

FOLIUM CATHARANTHI ROSEI

stomata. Above epidermis occurring multi-cellular nonglandular hairs. Below upper epidermis lying palisade tissue containing 3 - 4 layers of rectangular cells, arranged perpendicularly to leaf surface. Parenchyma consisting of closely arranged, thin-walled cells, with uncovered intercellular spaces. Lamina scattered with spiral vessels and phloem-xylem bundles, phloem outside and xylem inside.

Powder Leaf powder slightly green; taste bitter and slightly austere. Examine under a microscope: Epidermis fragments containing numerous stomata and multicellular non-glandular hairs. Plenty of non-glandular hairs multi-cellular, thin-walled, transparent, visible. Spiral vascular fragments found abundantly and striped vascular fragments sparsely. Crystals of calcium oxalate urchinform, scattered. Identification A. Transfer about 20 g of the coarse powder to a 250 ml conical flask. Add 70 ml of a 10% solution of sulfuric acid R. Boil for 10 minutes. Filter, transfer the filtrate to a 100 ml separating funnel. Alkalize to pH 10 with a 25% solution of ammonia R. Extract alkaloid with three quantities, each of 10 ml, of chloroform R by shaking. Combine the chloroform extracts, shake with three quantities, each of 5 ml, of a 10% solution of sulfuric acid R. Evaporate the extracts to remain about 3 ml, transfer to three test tubes, each of 1 ml. Tube 1: Add 2 drops of Dragendorff reagent R, a yellowish orange precipitate is appeared. Tube 2: Add 2 drops of Bouchardat reagent R, a brown precipitate is produced. Tube 3: Add 2 drops of Mayer reagent R, a white precipitate is produced. B. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel G. Mobile phase: Chloroform - methanol - ammonia (50 : 9 : 1). Test solution: Take about 5 g of the powder, extract with petroleum ether R in a Soxhlet’s extractor to remove chlorophyl (discard the ether extract). Spread the residue to evaporate the solvent, moisten with a 10% solution of amomia R, add 30 ml of chloroform R, heat under a relux condenser on a water bath for 15 min, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of ethanol R. Reference drug solution: Prepare a solution of 5 g of powder of Folium Calotropis reference drug in the same manner as described under Test solution. Procedure: Apply separately to the plate 20 µl of each solution. After developing, removal of the plate, dry in air, spray with vanillin - sulfuric acid solution R. Heat at 105 °C until spots appear. Examine under visible light. The spots in the chromatogram obtained with the test solution correspond in position and colour to the spots obtained with the reference drug solution. 1152

Total ash Not more than 12.0% (Appendix 9.8). Acid-insoluble ash Not more than 2.0% (Appendix 9.7). Preliminary processing Collect the drug in september to november. Wipe white pollen clean from leaf lower surface and dry under the sun to dryness. Before using, wash clean, allow the water to drain off, cut into filaments, stir-bake with gentle heat or stir-bake with honey to yellowing. Storage Store in a cool and dry place. FOLIUM CATHARANTHI ROSEI Dừa cạn (Lá) Sun- or heat-dried leaves of Catharanthus roseus (L.) G. Don (Fam. Apocynaceae).

Description Leaves entire, long elliptical, greyish green or pale green; apex slightly acuminate; base narrowly oblong; leaf blades 3.5 - 5 cm long, 1.5 - 3 cm wide. Nerves pinnate, conspicuous on lower side. Petioles 0.3 - 0.7 cm long. Odour pungent; taste bitter. Transverse section Upper and lower epidermis consisting of one row of evenly arranged, rectangular cells; bearing two kinds of non-glandular hairs: One long, multicellular, 2- to 5-celled (usually 2-celled), with short base cells and long pointed head cells; and the other short, unicellular. Midrib: Angular collenchymatous masses occurring under the layer of upper epidermal cells. Parenchyma consisting of thin-walled cells, irregular in size, among these cells occurring triangular intercellular spaces. Phloem-xylem bundles in double-piled arch arrangement in the middle of veins, consisting of small-celled phloem masses, in 2-arch arrangement surrounding the xylem arch. Wood vessels regularly arranged. Lamina consisting of one row of evenly arranged palisade cells and spongy parenchyma with small cells, thin-walled, irregularly arranged. Powder Epidermal fragments bearing stomata and multicellular non-glandular hairs, a few unicellular. Stomata having 3 subsidiary cells varying in shape, usually with one smaller cell. Fragments of veins consisting of rectangular, thinwalled cells. Non-glandular hairs scattered, 2- to 5- celled, with spray-like dots on the surface. Fragments of palisade parenchyma, spongy parenchyma visible. Fragments of scalariform vessels and reticulate vessels present.

VP V

Identification Place 3 g of the powder in a conical flask, moisten with 2 ml of ammonia R, add 30 ml of chloroform R, allow to stand for 30 minutes, occasionally shake, filter. Transfer the filtrate to a separator, shake with 5 ml of a 10% solution of sulphuric acid R for 2 - 5 minutes. Allow the layers to separate. Divide the acid layer into 4 test tubes, each tube containing 0.5 ml. Test tube 1: Add 2 drops of Mayer reagent R, a white precipitate is produced. Test tube 2: Add 2 drops of Dragendorff reagent R, a orange-red precipitate is produced. Test tube 3: Add 2 drops of Bouchardat reagent R, a brown precipitate is produced. Test tube 4: Add 2 drops of picric acid solution R, a yellow precipitate is produced. Loss on drying Not more than 12.0% (Appendix 9.6, 1 g, 85 °C, 4 h). Total ash Not more than 3.0% (Appendix 9.8). Broken matter Pass through a sieve with a pore size of 4 mm: Not more than 4.0% (Appendix 12.12). Foreign matter (Appendix 12.11) Inorganic matter: Not more than 0.5%. Other parts: Not more than 3.0%. Black burned leaves: Not more than 1.0%. Assay Weigh accurately about 15 g of the powder (passed through a sieve No. 355) and put into a 250 ml groundglass stoppered conical flask, moisten well with 5 ml of ammonia R, add 150.0 ml of chloroform R, shake vigorously, allow to stand overnight, filter. Shake 100 ml of the filtrate (equivalent to about 10 g of the powder) with four quantities, each of 10 ml of a 10% solution of sulphuric acid` R. Combine the acid extracts, adjust to pH 10 with ammonia R, extract with four successive quantities (15, 15, 15, 10 ml) of chloroform R. Combine the acid extract, adjust again to pH 11 - 12 with ammonia R, extract again with four successive quantities (15, 15, 15, 10 ml) of chloroform R. Combine the chloroform extracts, filter through anhydrous sodium sulphate R, wash anhydrous sodium sulphate R with 10 ml of chloroform R, combine the chloroform extracts and the washing, distil to recover the solvent then transfer the extract to a previously weighed flask. Evaporate the extract to dryness. Dry the residue in a desiccator containing silica gel to constant mass and weigh accurately. Calculate the content of total alkaloids in drug. It contains not less than 0.7% of total alkaloids, calculated with reference to the dried drug.

FOLIUM CLEISTOCALYSIS OPERCULATI

Preliminary processing Leaves are collected in plants, in forthcoming flowering time; dry under the sun or at a low temperature to dryness. Storage Store in a dry and cool place, protect from mould. FOLIUM CLEISTOCALYSIS OPERCULATI Vối (Lá) Sun- or heat- dried leaves of Cleistocalyx operculatus (Roxb.) Merr. et Pery (Fam. Myrtaceae).

Description Leaves thick, oval or ellipse, 8 - 15 cm long, 5 - 7 cm wide; base round; apex acute. Two sides of leaf have different colour, the upper surface greyish-brown, the lower surface pale green. Odourless; taste slightly acrid. Transverse section Midrib: Upper and lower epidermis consisting of a row ofevenly arranged small cells; with thick cuticle outside. Parenchymatous fragments consist of round, thin-walled cells; irregular dimension. In cortical parenchyma, oil vesicles arranged adjcacently to epidermis. Sclerenchymatous ring is almost constantly surrounded phloem-xylem bundles. Phloem-xylem bundles arc, amphicribal. Crystals of calcium oxalate in phloem, urchinform, scattered. Xylem consists of xylem vessels, 1 - 2 bundles lying peculiarly near main arc-form wood vessel. Lamina: Upper and lower epidermis consisting of one row of evenly arranged small cells, with thick cuticle outside. Palisade parenchyma containing a row of cells, arranged perpendicularly to the upper epidermis. Parenchyma included round, thin-walled cells, irregular dimension. Dermis included 2 - 3 layers of small cells lying next to the lower epidermis. Powder Colour brown; taste acrid. Parenchymatous fragment, oil vesicle, spiral vascular fragment, fibers with scattered or arranged in bundle, crystals of urchin-form calcium oxalate; sclerenchymatous cells polygonal, thick-walled cell. Identification A. To 3 g of the powder, add 30 ml of ethanol (90%) R, boil on a water bath for 3 minutes, filter, using the filtrate (solution A) to carry out the following tests: To 2 ml of solution A, add 3 drops of a 5% solution of ferric chloride R, shake gently, the solution turns blackishgreen. To 1 drop of solution A to a piece of filter paper, dry in air, the spot is pale yellow, expose the spot to ammonia vapour by putting the paper on a opened boltle containing concentrated ammonia R, the spot turns dark yellow. 1153

VP V

FOLIUM CLERODENDRI CHINENSE

To 1 ml of solution A, add 3 drops of a 10% solution of sodium hydroxide R, the brownish-yellow precipitate is produced. B. Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel GF254. Mobile phase: Toluene - ethyl acetate - formic acid (6 : 4 : 1). Test solution: Evaporate 5 ml of solution A on a water bath to about 1 ml. Reference drug solution: Prepare a solution of 3 g of Folium Cleistocalysis operculati reference drug in the same manner as described under Identification A. Procedure: Apply separately 10 µl of each solution to the plate. After the development and removal of the plate, allow to dry in air. Examine under ultraviolet light at 254 nm and then spray with a 10% solution of sulfuric acid in ethanol R, heat at 100 °C until spots appear and examine under visible light. The spots in the chromatogram obtained with the test solution correspond in colour and position to the spots in the chromatogram obtained with the reference drug solution.

Loss on drying Not more than 8.0% (Appendix 9.6, 1 g, 70 °C, 4 h). Total ash Not more than 9.0% (Appendix 9.8). Foreign matter Not more than 2% (Appendix 12.11). Extractives Not less than 1.0%, calculated with reference to the dried drug. Weigh accurately about 10 g of the powder, put it into a Soxhlet’s extractor, extract with ethanol (90%) R for 4 hours. Evaporate the ethanol extract on a water bath to dryness. Dissolve the residue 3 times, each with 20 ml of hot water, filter. Combine the filtrates, extract with 5 quantities, each of 20 ml of ethyl acetate R. Combine the ethyl acetat extracts, distil to recuperate the solvent, transfer to a priviously weighed beaker, evaporate on a water bath to dryness, dry at 60 °C to constant mass. Calculate the content of the extractives. Preliminary processing Collect the leaves, eliminate foreign matter, dry under the sun or heat. Storage Store in well-closed containers at a cool and dry place, protect from mould. Processing Collect the mature leaf, macerate in water for 20 - 30 minutes. Put the leaf out, dry until the content of water is about 50%. Percolate from 0.5 - 1 hour to pale yellow and aromatic leaf. Dry under the sun. Dried drug is pale greyish-yellow, distinct aroma. Cut into 3 - 5 mm threads 1154

in perpendicular direction with the leaf or pulverize to coarse powder. FOLIUM CLERODENDRI CHINENSE Bạch đồng nữ (Cành mang lá) Sun- or heat- dried leaves of Clerodendrum chinense var. simplex (Mold.) S.L. Chen (Fam. Verbenaceae) Synonyms: Clerodendrum philippinum Schauer var. simplex Mold.; Clerodendrum fragrans Schauer in DC

Description Branches bear leaves, sometime bear flowers. Young branches are nearly square, contain milk white pubescences; old branches are nearly glabrous, brownish-green. Leaves simple, opposite, blade wide ovate, about 20 cm long and 8 cm wide, slightly cordate at the base, acuminate at the apex, margin denticulate; the upper surface rough, greyish-green; the lower surface contains pubescences and round small glandules, side veins 4 - 5 couples, 3 veins from the base; petiole 3 - 10 cm long, pubescent. Corymb inflorescence at the apex of branches, thick and 5 - 10 cm wide, pedicel covered with pubescences. Flower solitary. Bract lanceolate, greyish-green, 1 - 2 cm long, has veins. Calyx cone- shaped and darkish-brown, 1.5 - 2.5 cm long, externally pubescents and glandulars; 5 lobes elongate, 10 - 16 mm long. Corollas slightly crumpled, brownishyellow, tubular; include: corolla tube 2 - 2.5 cm long, smooth and 5 oval lobes, 8 - 10 mm long. Four stamens, higher than corolla; filaments attached on the corolla tube. Styles are as long as filaments, divided two lobes at the apex. Transverse section Midrib: The upper surface slightly prominent and the lower surface clearly prominent. Upper and lower epidermis each consists of one layer of round small cells, arranged regularly, bear non-glandular hair containing 4 - 5 cells and glandular hair. Cluster of collenchymatous cells closed to upper epidermis; collenchymas closed to lower epidermis consists of 4 - 5 layers of ovoid and thickwalled cells. Many phloem-xylem bundles arranged in a big arc, phloem-xylem bundles are collateral. Parenchyma consists of round or polygonal and thin-walled cells. Lamina: Epidermis is similar to those of vein, containing many non-glandular hairs. Palisade merophyll consists of one layer of rectangular cells, arranged closely together and perpendicularly to upper epidermis. Powder Colour greyish-brown; Examine under a microscope: Cork consists some layers of rectangular or polygonal cells; parenchyma containing nearly round starch granules; epidermis consist of thin-walled cells, oval stomata; fibres with thickened walls and in bundles; in side view, glandular hair is round at the head and thin at the stalk,

FOLIUM CRATOXYLI PRUNIFLORI

VP V

it is round from the top; non-glandular hair is unicellular or multicellular, entire or broken. Collenchymatous cells with thickened walls contain cubic crystal of calcium oxalate; pitted vessel or scalariform vessel.

Identification A. Use the Test solution obtained in identification B to perform the following reactions: To 1 ml of the filtrate, add 5 drops of acid hydrochloric R and a small quantity of magnesium powder R, shake gently, a pinkish-red colour is produced. To 1 ml of the filtrate, add 2 drops of a 20% solution of sodium hydroxide R, an orange precipitate is produced, the precipitate is dissolved in the excess reagent. To 1 ml of the filtrate, add 1 ml of ethanol (90%) R, shake gently, add 3 drops of a 5% solution of ferric chloride R, the solution changes from pale green to blackish-green. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Ethyl acetate - formic acid - water (8 : 1 : 1). Test solution: Weigh 10 g of the powder and place it in a 250-ml conical flask, add 100 ml of ethanol (90%) R, shake gently and heat under a reflux condenser for 30 minutes in a water-bath. Filter and evaporate the filtrate on a water-bath to dryness. Add 10 ml of n-hexane R to the residue, stir well and discard the solvent, repeat this washing once. Dissolve the residue in 5 ml of ethanol (90%) R by gentle heating. Filter, use the filtrate as the test solution. Reference drug solution: Prepare a solution of 10 g of Folium Clerodendri chinense reference drug in the same manner as described under Test solution. Procedure: Apply separately 2 μl of each solution to the plate. After the development and removal of the plate, dry in air. Examine the plate under ultraviolet light at 366 nm and then expose to ammonia R vapour until the spots become visible, examine under visible light. The spots in the chromatogram obtained with the test solution correspond in colour and position to the spots in the chromatogram obtained with the reference drug solution. Loss on drying Not more than 12.0% (Appendix 9.6, 1 g, 105 °C, 4 h). Total ash Not more than 12.0% (Appendix 9.8). Foreign matter Not more than 1% (Appendix 12.11). Assay Weigh accurately about 5 g of the powder (passed through a sieve No. 355) and place it in a conical flask, add 50 ml of chloroform R, macerate and shake for 30 minutes, filter. Discard the chloroform extract, allow the residue to dry. Add 50 ml of methanol R to the residue, sonicate for 30 minutes, decant the liquid and repeat the extraction

twice, combine the methanol extracts, evaporate the solvent under reduced pressure to dryness. Dissolve the residue in 10 ml of hot water by heating for 15 minutes in a waterbath at 60 °C, and stiring well. Immediately filter through cotton, and then through a twisted filter paper to obtain the hot water extract. Repeat this procedure for five more times, each with 5 ml of hot water. Combine the filtrates, allow to cool, transfer to a separating funnel, extract for five times, each with 50 ml of ethyl acetate R. Combine the ethyl acetate extracts, evaporate the solvent to about 20 ml under reduced pressure, transfer the concentrated extract to a previously weighed beaker, evaporate the solvent on a water-bath to dryness, and heat the residue at 60 °C to constant mass. Allow to cool in a desiccator for 30 minutes and weigh immediately. Calculate the content of the obtained residue. It contains not less than 0.5% of the residue, calculated with reference to the dried drug.

Preliminary processing The drug is collected all year round, preferably during flowering period in summer, cut leaf branches, washed clean, and dried. Processing Choose mature leaves, remove petioles, cut into small sections and dry under the sun. Storage Preserve in well-closed containers, store in a dry place. FOLIUM CRATOXYLI PRUNIFLORI Ngành ngạnh (Lá), Đỏ ngọn, Co kín lang Sun- or heat- dried leaves of Cratoxylum pruniflorum Kurtz (Fam. Hypericaceae)

Description Leaves lanceolate or oval, 6 - 11cm long, 2.5 - 3.5 cm wide, pubescences at the upper surface are more than the lower surface, petiole short. Transverse section Midrib: The upper surface is concave, the lower surface is convex. Both upper and lower epidermis consists of one row of thick-walled cells, containing multi-cellular non-glandular hairs. Parenchyma consists of polygonal and thin-walled cells. Phloem-xylem is arranged in an arc closed to parenchyma. The phloem-xylem bundles are surrounded by fibres bundles which are arranged in an arc. Many urchin-formed crystals of calcium oxalate scattered in parenchyma. Lamina: Both upper and lower epidermis consists of one row of cells, containing multi-cellular non-glandular hairs. Palisade tissue consists of one layer of rectangular cells, arranged perpendicularly with epidermis. 1155

VP V

FOLIUM CRINI ASIATICI

Powder Colour yellowish-brown; odour aromatic; taste slightly sour and acrid. Examine under a microscope: Epidermis consists of rectangular cells, bearing non-glandular hairs, and containing paracytic stomata arranged singly. Multicellular non-glandular hairs scattered. Fibres long, walls thick, grouped in bundles. Epidermis consists of polygonal and thin-walled cells. Urchin-formed crystals of calcium oxalate; spiral vessel and scalariform vessel. Identification A. To 10 g of the powder, add 30 ml of ether R, heat under a reflux condenser for 5 minutes on a water-bath, filter and discard the ether extract. The residue is dried at the room temperature, add 30 ml of ethanol R to the residue, boil for 10 minutes on a water-bath, filer. The filtrate is carried out the following reactions: Place 1 ml of the filtrate in a test tube, add a small quantity of magnesium R powder, 3 drops of hydrochloric acid R, heat gently, the solution changes from yellow to redishbrown. Place 1 ml of the filtrate in a test tube, add 3 drops of a 5% solution of ferric chloride R, a dark green colour is produced. To 1 ml of the filtrate, add 3 drops of a 10% solution of sodium hydroxide R, a yellow precipitate is produced. Add 1 ml of water, the precipitate is dissolved and the yellow colour is more intensive. B. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel GF254. Mobile phase: Toluene - ethyl acetate - formic acid (5 : 4 : 1). Test solution: Place 10 g of the powder in a filter-paper bag, extract in Soxhlet’s extractor with 50 ml of n-hexane R until the extract is colourless. Discard the hexane extract, evaporate the remained solvent in residue to dryness, add 50 ml of methanol R, extract for 1 hour in Soxhlet’s extractor. Evaporate the methanol extract and recover the solvent to dryness. Dissolve completely the residue in 20 ml of water by heating in a water-bath, filter. The filtrate is extracted for three times, each with 20 ml of ethyl acetate R. Combine the ethyl acetate extract, evaporate and recover the solvent to dryness. Dissolve the residue in 5 ml of 0.1 M sodium hydroxide R, filter. Adjust to pH 3 with 0.1 M hydrochloride acid R, filter to obtain the precipitate. The precipitate is dried at room temperature and then dissolved in 10 ml of methanol R. Reference drug solution: Prepare a solution of 10 g of the semi-croase powder of Folium Cratoxyli pruniflori reference drug in the same manner as described under Test solution. Procedure: Apply separately 10 μl of each solution to the plate. After the development and removal of the plate, allow to dry in air at the room temperature. Expose the plate to ammonia R vapour until the spots appear and examine under visible light. The spots in the chromatogram obtained with the test solution (6 yellow to brownish1156

yellow spots, two of them are most intense) correspond in colour and position to the spots in the chromatogram obtained with the reference drug solution.

Loss on drying Not more than 12.0% (Appendix 9.6, 1 g, 105 °C, 4 h). Total ash Not more than 5.0% (Appendix 9.8). Broken matter Passed through a sieve (nominal pore size 4 mm): Not more than 3.0% (Appendix 12.12). Foreign matter Not more than 1% (Appendix 12.11). Extractives Not less than 20.0%, calculated with reference to the dried drug. Carry out the hot extraction method (Appendix 12.10), using ethanol (96%) R as the solvent. Preliminary processing The leaves are collected all year round, wrapped up, and dried under the sun or heat. Storage Preserve in a cool and dry place, protect from mould. FOLIUM CRINI ASIATICI Náng hoa trắng (Lá) Sun- or heat-dried leaves of Crinum asiaticum L. (Fam. Amaryllidaceae).

Description Dried leave pale brown or pale yellow in colour, slightly thin, light, central of leaves is thick, as thin as reaching to margin, many veins parallel to midrib. At the torn leaves having a lot of white silks. Natural leavesare oblong lanceolate, 70 - 120 cm in length, maybe longer, width about 10 cm to 18 cm. Powder Colour light brown, odourless, slightly bitter. Examine under a microscope: epidermis fragment bearing stomata, stomata arranged in anomocytic. A lot of needle-formed calcium oxalate crystals, both heads acute, about 100 - 110 µm long, 2 - 3 µm in diameter, single or gathered to groups. Epidermis fragment of leaves composed of rhombus or polygonal cells consiting of urchin-formed calcium oxalate crystals. Many spiral vessels. Identification A. To about 3 g of the powder, add 2 ml of amonia R, mix well. Add 20 ml of cloroform R, shake for 30 minutes, allow to stand for 1 hour. Filter, transfer the filtrate to a

FOLIUM CRINI LATIFOLII

VP V

separating funnel. Add 10 ml of a 10% solution of sulfuric acid R, shake well, allow the layers to separate and decant the supernatant layer, divide into 3 test tubes. Test tube 1: Add a drop of Mayer reagent R, a white precipitate is produced. Test tube 2: Add a drop of Dragendorff reagent R, an yellowish orange precipitate is produced. Test tube 3: Add a drop of Bouchardat reagent R, a brown precipitate is produced. B.Examine by thin-layer chromatography (Appendix 5.4) Coating substance: Silica gel 60F254. Mobile phase: Chloroform - methanol - ammonia (70 : 10 : 1). Test solution: To 1 g of the drug (cut into small pieces), add 10 ml of ethanol R, sonicate for 20 minutes, filter, using the filtrate as the test solution. Reference drug solution: Prepare a solution of 1 g of Folium Crini asiatici reference drug in the same manner as decribed under Test solution. Reference substance solution: Dissolve lycorine RS in ethanol R to produce a solution containing 0.025%. Procedure: Apply separately 10 µl of each solution to the plate. After the development and removal of the plate, allow to dry in air. Examine under ultraviolet light at 366 nm or spray with Dragendorff reagent R then with a 10% solution of sulfuric acid R. The spots in the chromatogram obtained with the test solution correspond in colour and position to the spots in the chromatogram obtained with the reference drug solution and the chromatogram obtained with the test solution must have a spot that corresponds in colour and position to the spot due to lycorine in the chromatogram obtained with the reference substance solution.

Loss on drying Not more than 12.0% (Appendix 9.6, 1 g, 105 °C, 5 h).

extracts, evaporate on a water bath to dryness. Dissolve the residue in 0.1 M hydroclorid acid R and transfer to a 25 ml volumetric flask, add 0.1 M hydrochloric acid R to volume, mix well, filter through the paper filter then pass through a 0.45 µm membrane filter. Chromatographic system: A column (25 cm × 4.6 mm) packed with stationary phase C (5 µm). Detector: A spectrophotometer set at 290 nm. Flow rate: 1 ml per minute, adjusted if necessary. Volume of injection: 10 µl. Procedure: Using gradient elution described as below: Time (min)

Mobile phase A (% v/v)

Mobile phase B (% v/v)

0 - 10 10 - 11 11 - 21 21 - 22 22 - 30

10 10 → 40 40 40 → 10 10

90 90 → 60 60 60 → 90 90

Inject the reference solution. Maximum relative standard deviation of 2.0% after 6 replicate injections, calculated for the peak due to lycorine. Inject separately the reference solution, the test solution. Use peak areas in the chromatograms obtained with the test solution, the reference solution and the declared content of C16H17NO4 in lycorine RS to calculate the content of lycorine (C16H17NO4) in the drug. It contains not less than 0.4% lycorine (C16H17NO4), calculated with reference to the dried drug.

Total ash Not more than 12.0% (Appendix 9.8), determined on 1 g.

Preliminary processing Collect the mature leaf, eliminate yellow leaves, cut into segments of 2 - 5 cm in length, dry in the shade or heat at 40 °C to 50 °C to dryness.

Forgein matter Not more than 1% (Appendix 12.11).

Storage Store in a dry and cool place.

Assay Examine by liquid chromatography (Appendix 5.3). Mobile phase A: Acetonitrile R. Mobile phase B: 0.02 M potassium dihydrogen phosphate solution - triethylamine (100 : 0.3) adjusted to pH 3 with phosphoric acid R. Reference solution: Weigh accurately and dissolve lycorine RS in 0.1 M hydrochloric acid R to obtain a solution containing 0.15 mg per ml Test solution: Weigh accurately about 1 g of the powder (passed through a sieve No. 250) and place in a 100-ml ground-glass-stoppered conical flask, moisten with 1 ml of ammonia R, stoppered, allow to stand for 15 minutes. Add 50 ml of ethanol R, sonicate for 30 minutes, cool, decant the supernatant liquid. Repeat the extraction with 4 quantities, each of 30 ml of ethanol R. Combine the liquid

FOLIUM CRINI LATIFOLII Trinh nữ hoàng cung (lá) Sun- or heat-dried leaves of Crinum latifolium L., (Fam. Amaryllidaceae).

Description Leaf linear, strap - shaped, 30 - 50 cm long, 3 - 8 cm wide, thin, light, thickened in the center, as attenuate as the edge, wavy edges, apex acute or obtuse, base flattened. Lamina yellowish - brown or pale brown, with numerous small veins parallel to midrip. Texture tough, surface linked up by fine, numerous, white threads. Odour slight sour, characteristic; taste slightly bitter.

1157

VP V

FOLIUM CRINI LATIFOLII

Transverse section Upper and lower epidermis consisting of small square cells, cutinized thickened - walls, arranged evenly in row, scattered with stomata. Palisade tissue indistinct. In the lamina, each subsidiary vein is formed from a phloem-xylem bundle with 2 - 3 xylem vessels, those with phloem arranged in rows perpendicularly to the surface of the epidermis. Spongy tissue alternate phloem - xylem bundles, clusters of fibre frequently occurring above the spongy tissue. Parenchyma consisting of thin-walled cells, irregular in sizes. Structure of the midrib is similar to the subsidiary but the phloem-xylem bundle is larger and located in the protuberance, closely to the lower epidermis, a mass of collenchyma is visible. Powder Pale brown powder, characteristic odour, slightly bitter taste. Fragments of epidermis bearing stomata. Fragments of epidermis bearing convex cutin. Numerous fragments of spiral vessels, single or in cross lines, scalariform vessels; fragments of vessels arranged particularly sparse. Needle crystals of calcium oxalate, frequently broken, single or grouped into clusters. Identification A. To 2 g of the powder, add 20 ml of ethanol (96%) R, boil on a water-bath for 5 min, filter. Transfer the filtrate to 2 test tubes, each of 1 ml, carry out the following reactions: Test tube (1): Add some drops of a 5% solution of sodium hydroxide R. The colour of the solution changes from pale yellow to dark orange. Test tube (2): Add 0.5 ml of hydrochloric acid R, add a little powder of magnesium R, a red colour is produced. B. To 2 g of the powder, moisten with ammonia R, add 30 ml of chloroform R, ultrasonicate for 15 minutes, filter. Evaporate the filtrate on a water-bath to dryness. Dissolve the residue in 10 ml of 0.1 N hydrochloric acid R, filter. Transfer the filtrate to 3 test tubes, each of 2 ml, carry out the following reactions: Test tube (1): Add 1 drop of Mayer reagent R, a white precipitate is produced. Test tube (2): Add 1 drop of Bouchardat reagent R, a brown precipitate is produced. Test tube (3): Add 1 drop of Dragendorff reagent R, an orange-red precipitate is produced. C. Examine by thin-layer chromatography (Appendix 5.4). Coating substance: Silica gel G. Mobile phase: Chloroform - methanol - ammonia (7 : 1 : 0.05). Test solution: To 4 g of the coarse powder, add 50 ml of ethanol (50%) R, heat under a reflux condenser for 1 hour, filer. Evaporate the filtrate on a water-bath to dryness. Dissolve the residue in 10 ml of water. Alkalize the solution with ammonia R to pH 10 - 11, shake with 30 ml of chloroform R. Decant the chloroform extract, evaporate on a water-bath to dryness. Dissolve the residue in 1 ml of a mixture of chloroform - methanol (9 : 1). 1158

Reference substance solution: Dissolve crinamidine RS in a mixture of chloroform - methanol (9 : 1) to produce a solution containing 0.5 mg per ml. Reference drug solution: Extract 4 g of the coarse powder of Folium Crini latifolii (reference drug) in the same manner as described under Test solution. Procedure: Apply separately to the plate 10 µl of each solution. Develop over a path of 12 cm, remove the plate, allow to dry in air, spray with Dragendorff reagent R. The spots in the chromatogram obtained with the test solution correspond in colour and position to the spots in the chromatogram obtained with the reference drug solution and one of the spots in the chromatogram obtained with the test solution corresponds in colour and position to the spot due to crinamidine in the chromatogram obtained with the reference substance solution. D. In the test for Assay B, the principal peak in the chromatogram obtained with the test solution is similar in retention time to crinamidine peak in the chromatogram obtained with the reference solution.

Loss on drying Not more than 12.0% (Appendix 9.6, 1 g, 100 °C, 5 hours). Total ash Not more than 12.0% (Appendix 9.8). Foreign matter Not more than 0.5% (Appendix 12.11). Assay A. Total alkaloids Weigh accurately about 10 g of the coarse powder (previously determimed water), put into a 250 ml stopper conical flask, add 100 ml of ethanol (96%) R previously acidified with a 5% solution of acetic acid R. Boil under a reflux condenser for 30 minutes, filter through cotton. Continue the extraction in the same manner as described above until extracts no longer react with the reagent of alkaloids (no precipitate with Mayer reagent R). Combine the ethanol extracts, evaporate on a water-bath to dryness. Dissolve the residue in 40 ml of a 5% solution of sufuric acid R, divided into 3 times, by ultrasonicating for 5 minutes, filter through filter paper into a separator. Wash the filter paper with 10 ml of a 5% solution of sulfuric acid R. Combine the washing with the acid extracts into above separator. Alkalize the solution with ammonia R to pH 10 and shake with 5 quantities, each of 30 ml of chloroform R. Combine the chloroform extracts into other separator, shake gently with 50 ml of water. Discard the water layer. Decant the chloroform layer, dry over anhydrous sodium sulfate R, decant the chloroform solution into a 250 ml conical flask. Wash the anhydrous sodium sulfate with 3 quantities, each of 5 ml of chloroform R. Combine the washing into above 250 ml conical flask, evaporate to dryness. Dissolve the residue

FOLIUM CYNARAE SCOLYMI

VP V

in 1 ml of ethanol (96%) R, add accurately 10.0 ml of 0.02 N hydrochloric acid VS, ultrasonicate for 10 minutes, add 2 drops of methyl red solution R, titrate excess acid with 0.02 N sodium hydroxide VS until the colour of the solution changes from red to yellow. Carry out a blank. Each ml of 0.02 N sodium hydroxide VS is equivalent to 0.005746 g of lycorine (C16H17NO4, M. 287.32). Calculate the content (X%) of total alkaloids by using the following expression:

X (%) =

(V − v) × 0.005746 ×100 m

In which: V: The volume of 0.02 N sodium hydroxide VS used for the blank, in ml. v: The volume of 0.02 N sodium hydroxide VS used for the test solution, in ml. m: The weight of the powder taken to prepare the test solution (excepted from water), in g. It contains not less than 0.2% of total alkaloids calculated as lycorine (C16H17NO4) with reference to the dried drug. B. Crinamidine Examine by liquid chromatography (Appendix 5.3). Mobile phase: Acetonitrile R and phosphate buffer pH 3.0 as described in gradient elution program. Phosphate buffer pH 3.0: Dissolve 13.6 g of potassium dihydrogen phosphate R in 800 ml of water, add 3 ml of triethylamine R, mix well, adjust to pH 3.0 with p­ hosphoric acid R, add sufficient water to produce 1000 ml. Test solution: Weigh accurately about 2.5 g of the powder (passed through a sieve No. 355), put into a 100 ml stoppered conical flask, moisten with ammonia R, stopper tightly, allow for 1 hour. Add 50 ml of a mixture of chloroform - methanol (3 : 1), ultrasonicate for 30 minutes, allow to stand to settle, filter through filter paper. Residue is extracted in the same manner as described above 4 times more, each of 40 ml of a mixture of chloroform - methanol (3 : 1). Combine the extracs, evaporate on a water-bath to dryness. Use 0.1 M hydrochloric acid R to dissolve and transfer all the residue to a 20 ml volumetric flask, add 0.1 M