Hormone Assays in Biological Fluids (Methods in Molecular Biology, 1065) 1627036156, 9781627036153

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Methods in Molecular Biology 1065

Michael J. Wheeler Editor

Hormone Assays in Biological Fluids Second Edition

METHODS

IN

M O L E C U L A R B I O LO G Y ™

Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes: http://www.springer.com/series/7651

Hormone Assays in Biological Fluids Second Edition

Edited by

Michael J. Wheeler Consultant Clinical Scientist, The Old Dairy, Exmouth, UK

Editor Michael J. Wheeler Consultant Clinical Scientist The Old Dairy Exmouth, UK

ISSN 1064-3745 ISSN 1940-6029 (electronic) ISBN 978-1-62703-615-3 ISBN 978-1-62703-616-0 (eBook) DOI 10.1007/978-1-62703-616-0 Springer New York Heidelberg Dordrecht London Library of Congress Control Number: 2013945870 © Springer Science+Business Media New York 2013 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher’s location, in its current version, and permission for use must always be obtained from Springer. Permissions for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution under the respective Copyright Law. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Humana Press is a brand of Springer Springer is part of Springer Science+Business Media (www.springer.com)

Dedication This book is dedicated to the memory of my friend and colleague Morley Hutchinson, an enthusiastic and respected scientist and teacher who is greatly missed.

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Preface Endocrinology is a fascinating field of work, and the measurement of hormones is an essential element in clinical and biological investigations. Hormone measurement is required for the diagnosis of a wide range of clinical conditions as well as for monitoring the effectiveness of treatment. It also provides an insight into the body’s response to a wide range of physiological stimuli, such as heat, cold, and toxins, in both humans and animals. The number of hormone requests, in the clinical field in particular, has risen exponentially, and now thousands of requests may be received by a laboratory each year. Initially hormones were measured in specialized laboratories which had the equipment, facilities and expertise to develop suitable methods. Nonspecialized laboratories were able to offer hormone determination, as commercial companies developed diagnostic kits for hormones. Even so these procedures were very labor-intensive and required a large number of staff if a wide range of hormone assays was going to be carried out. Today, most routine clinical laboratories in both the developed and the developing world have automatic robotic systems. In its simplest form, this requires a sample to be placed on a machine, together with calibrators and quality control samples, and after a period of analysis a result is generated. In the highly sophisticated systems, automatic machines are linked to a track system. A single sample can be analyzed for clinical chemistry, serology, and hematology as well as endocrinology. Such systems are capable of handling large numbers of samples and producing thousands of results in a day. The simplest solution for researchers working with human material, who require hormones to be measured, is to approach their local clinical laboratory for analysis. However, not all clinical assays give the sensitivity, e.g., testosterone concentrations in women and children, or the specificity, e.g., free thyroxine, required by the researcher. In these cases separate assays may have to be set up. In some cases a commercial kit can be sufficient but sometimes a manual assay may be needed. For those working with animals commercial kits may not be available. Although steroids circulating in different animal species are generally the same as humans, the proteins are not. Antisera raised to human protein hormones do not recognize or recognize poorly the same protein hormones in animals. In these cases separate hormone assays have to be established. In addition, hormone research progresses at a considerable pace and new hormones, or substances with hormone-like activity, are being investigated both in humans and animals. New assays have to be developed in such cases. However, the techniques required to do this are the same as used to establish human hormone assays. Although the authors of the chapters contained within this book come from a clinical background the principles and techniques they present are readily transferable to animal species. It is for these groups who are unable to utilize automated assays or require to set up their own assays that this book will find most use. The first few chapters give an overview of the common techniques now used in the measurement of hormones. Tandem mass spectrometry is a relatively new technique to the routine laboratory and has a potentially wide application.

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Gas chromatography-mass spectrometry is used in specialized laboratories for some routine investigations and is an important method in establishing reference preparations for the steroid hormones. The subsequent chapters give details of methods for a wide range of hormones. It has not been possible to cover all hormones. In some cases, such as pregnancy screening, a number of automated systems are used and advice from a local screening laboratory would be more appropriate. Nevertheless the techniques and principles explained within Hormone Methods in Biological Fluids, Second Edition are transferable to a wide range of substances across species. Where reference ranges are quoted, these are only given for guidance and are appropriate only for humans. It is always recommended that the assayist establish their own reference ranges which would be applicable to the method used and population studied. This second edition has drawn heavily on the first but has been expanded and brought up-to-date. I am grateful to the many authors who have contributed to its production and their patience with me as I have harried them, particularly in the later stages. I am also grateful to the late Morley Hutchinson who invited me to get involved in the first edition. He is sorely missed and I hope he would be pleased with this second edition of a book which he launched. Exmouth, UK

Michael J. Wheeler

Contents Preface.............................................................................................................................. Contributors ....................................................................................................................

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1 A Short History of Hormone Measurement...................................................... Michael J. Wheeler 2 Immunoassay Techniques ................................................................................. Michael J. Wheeler 3 Introduction to Gas Chromatography-Mass Spectrometry................................. Alberto Sánchez-Guijo, Michaela F. Hartmann, and Stefan A. Wudy 4 Tandem Mass Spectrometry in Hormone Measurement.................................... Helen P. Field 5 Methods for the Investigation of Thyroid Function........................................... Alireza Morovat 6 The Measurement of LH, FSH, and Prolactin................................................... Michael J. Wheeler 7 Assays for GH, IGF-I, and IGF Binding Protein-3............................................ Nishan Guha 8 Measurement of Arginine Vasopressin............................................................... Nadia El-Farhan, David Hampton, and Michael Penney 9 The Measurement of Anti-Müllerian Hormone (AMH).................................... Michael J. Wheeler 10 Measurement of Gut Hormones in Plasma........................................................ Steve Bloom, Mohammad Ghatei, and Paul Bech 11 Measurement of Melatonin and 6-Sulphatoxymelatonin.................................... Benita Middleton 12 Measurement of Glucocorticoids in Biological Fluids........................................ Laura Owen 13 The Measurement of Androgens....................................................................... Helen P. Field and Michael J. Wheeler 14 Measurement of Aldosterone in Blood.............................................................. Sophie C. Barnes 15 Measurement of Plasma Renin Activity.............................................................. Sophie C. Barnes

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7 27 45 75 105 117 129 141 147 171 201 211 227 235

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16 Measurement of Vitamin D .............................................................................. Loretta Ford 17 Urinary Steroid Profiling.................................................................................. Norman F. Taylor 18 Internal Quality Control................................................................................... Edward Kearney 19 External Quality Assessment Schemes for Immunoassays................................... Catharine Sturgeon Index ...............................................................................................................................

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Contributors SOPHIE C. BARNES • Imperial College Healthcare NHS Trust, Charing Cross Hospital, London, UK STEVE BLOOM • Division of Diabetes, Endocrinology and Metabolism, Imperial College Healthcare NHS Trust, London, UK PAUL BECH • Division of Diabetes, Endocrinology and Metabolism, Imperial College Healthcare NHS Trust, London, UK NADIA EL-FARHAN • Department of Clinical Biochemistry, Royal Gwent Hospital, Newport, South Wales, UK HELEN P. FIELD • Department of Specialist Laboratory Medicine, St. James’s University Hospital, Leeds, UK LORETTA FORD • Department of Clinical Biochemistry, City Hospital, Birmingham, UK MOHAMMAD GHATEI • Division of Diabetes, Endocrinology and Metabolism, Imperial College Healthcare NHS Trust, London, UK NISHAN GUHA • Department of Clinical Biochemistry, John Radcliffe Hospital, Oxford, UK DAVID HAMPTON • Department of Clinical Biochemistry, Royal Gwent Hospital, Newport, South Wales, UK MICHAELA F. HARTMANN • Laboratory for Translational Hormone Analytics in Paediatric Endocrinology, Steroid Research & Mass Spectrometry Unit, Division of Paediatric Endocrinology & Diabetology, Justus Liebig University, Giessen, Germany EDWARD KEARNEY • Department of Clinical Chemistry, Queen Elizabeth the Queen Mother Hospital, Margate, Kent, UK BENITA MIDDLETON • Chronobiology Group, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, UK ALIREZA MOROVAT • Department of Clinical Biochemistry, Oxford University Hospitals, Oxford, UK LAURA OWEN • Biochemistry Department, University Hospital of South Manchester, Manchester, UK MICHAEL PENNEY • Department of Clinical Biochemistry, Royal Gwent Hospital, Newport, South Wales, UK ALBERTO SÁNCHEZ-GUIJO • Laboratory for Translational Hormone Analytics in Paediatric Endocrinology, Steroid Research & Mass Spectrometry Unit, Division of Paediatric Endocrinology & Diabetology, Justus Liebig University, Giessen, Germany CATHARINE STURGEON • UKNEQAS (Edinburgh), Department of Laboratory Medicine, Royal Infirmary of Edinburgh, Edinburgh, UK NORMAN F. TAYLOR • Department of Clinical Biochemistry, King’s College Hospital, London, UK MICHAEL J. WHEELER • Consultant Clinical Scientist, The Old Dairy, Exmouth, UK STEFAN A. WUDY • Division of Paediatric Endocrinology & Diabetology, Laboratory for Translational Hormone Analytics in Paediatric Endocrinology, Steroid Research & Mass Spectrometry Unit, Justus Liebig University, Giessen, Germany

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Chapter 1 A Short History of Hormone Measurement Michael J. Wheeler Abstract Huge changes have occurred in the measurement of hormones over the last 50 years or so. Methods have become simplified, sensitivity has increased manyfold, and automation has allowed the analysis of large number of specimens in a single day. The most significant steps in the history of hormone measurement were the development of radioimmunoassay and later the production of monoclonal antibodies. There has also been increased commercialization, the technique has been applied to an ever-increasing range of substances, and radioactive measurement has been replaced with colorimetric, fluorescent, and chemiluminescent end-points. However, all these changes have not been without their problems. Collaboration between laboratories has seen standardization of reagents and methods, the development of reference methods, and the setting up of external quality assurance schemes. All these have led to improved sensitivity, precision, and reliability. More recently tandem mass spectrometry has brought further improvements in the measurement of certain hormones. Although many hormones are now measured by automated systems there is still a place for manual assays whether developed in-house or by using a commercial kit. Key words Radioimmunoassay, Immunometric assay, Tandem mass spectrometry, Automation, Monoclonal antibodies’ External quality assurance schemes

It now seems slightly surreal that 45 years ago I was slaughtering mice and rats in order to measure FSH and LH in urine. Bioassays were the only methods available for measuring these hormones and urine provided a large source of material. Collections of urine over 24–48 h were extracted using several absorption and purification steps. LH was determined by the ovarian ascorbic acid depletion method of Parlow [1]. This required 7-day priming of immature rats with hCG before injection of urine extract. FSH was frequently measured by the increase in the uterine weight of immature mice following injection of purified urine extract [2]. This assay was nonspecific as the amount of LH present influenced the result [3]. A more precise method was the ovarian weight augmentation assay of immature mice or rats [4] where urine extract and hCG were injected over 3 days. The hCG was given to overcome LH crossreaction. Commonly four animals were used at two dose levels in order to achieve reasonable precision. The results were quantified by comparing with the results of injected reference preparation. Michael J. Wheeler (ed.), Hormone Assays in Biological Fluids: Second Edition, Methods in Molecular Biology, vol. 1065, DOI 10.1007/978-1-62703-616-0_1, © Springer Science+Business Media New York 2013

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Problems included limited purity of the reference preparation, cross-reaction of the hormones, and variation in the sensitivity of the animals to the hormone [5]. Obviously the number of clinical samples that could be measured in a week was few and, in hindsight, the results gave only a reflection of the physiological and pathological changes that occur [6]. The most significant advance in the history of hormone measurement was the development of radioimmunoassay by Yalow and Berson [7] for which they received the Nobel prize and Ekins [8]. A radioactive signal in the form of iodine-131 and later iodine-125 provided measurement of small amounts of protein hormone. An antibody to the protein provided specificity. Later, enzyme, fluorescent, and chemiluminescent signals replaced radioactivity overcoming the hazards and problems of waste disposal associated with the latter. Chapter 2 provides more detail of immunoassay techniques and detailed reviews are provided by refs. [9, 10]. There were major problems with the early radioimmunoassay methods. Non-specificity was common to both steroid and peptide hormone assays. Protein hormone methods suffered from lack of standardization and many lacked sufficient sensitivity to measure concentrations in the low physiological range or the hypo-secretory state. Comparison of results from different assays for the polypeptide hormones showed that values could differ by as much as ten times. The main reason was the use of different reference preparations. This led to the standardization of reference preparations which greatly improved the situation. However, large differences still remained. Further investigations showed that antisera had different specificities by cross-reacting with similar hormones by different amounts and causing falsely high results. Gradually more specific antibodies were developed improving agreement further. At this stage a radioimmunoassay for LH, FSH, and other protein hormones would take 4 days which was shortened by a day with the introduction of polyethylene glycol-accelerated precipitation of the final second antibody stage. The workload was relatively small and so it was common to run all samples in duplicate at two dilutions. This was also the age of innocence with many of us iodinating hormones in the open laboratory! For many protein hormones sensitivity remained a problem and it was not possible to establish a lower limit for the reference range or measure sub-physiological levels. Even when lower limits were established this was later found to be due to “noise,” e.g., TSH. The next major step in the development of immunoassay was the production of monoclonal antibodies by Kohler and Milstein [11] which, in turn, led to the development of specific, sensitive immunometric assays. Hence there was a move from competitive assays for protein hormones to reagent excess assays which reduced the time of assay even further to about 24 h. These features are the essence of modern assays where computerization,

A Short History

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precise engineering, and a much greater end-point signal, such as chemiluminescence, have led to assays that can be completed in a very short period of time. Specific assays were developed for steroid hormones over 50 years ago but were restricted initially to urine because of the large volume of material that could be collected and extracted in order to achieve sufficient sensitivity. Steroids were extracted with solvents, purified with chromatography, and then measured by spectrophotometry or fluorimetry. Purification was carried out by paper [12], column [13], and gas liquid chromatography [14, 15]. Methods were hazardous, complex, and very lengthy. As well as flammable and carcinogenic solvents other hazardous chemicals, such as concentrated sulfuric acid, were employed by some common methods [16]. Steroids were also measured in serum and plasma at an early stage using protein-binding methods [17]. These early methods employing C14-labeled steroids used a lot of blood and were not very sensitive or specific. Gradually these methods were replaced with radioimmunoassay methods which were more sensitive and used smaller volumes of blood. Lengthy chromatographic steps were replaced by simple solvent extraction. The steroids were unable to illicit an immune response in animals but could be made to do so by attaching to large proteins such as albumin and keyhole limpet hemocyanin (see Chapter 4). The introduction of radioactive iodine directly into the steroid molecule, to provide a tracer, destroyed the latter, so a substance like tyrosine or histamine was attached via a reactive position on the steroid and then iodinated [18]. The simplified technique was applicable to a wide range of steroids as well as drugs and small molecules. These assays also had pitfalls such as contamination of solvents and cross-reaction with similar steroids. Like protein hormones all the common steroids may be measured now on modern automated analyzers. An overview of the methods for the extraction, purification, and measurement of steroids is provided in ref. 9. As with the protein hormones, radioactivity was replaced with enzyme, fluorometric, and chemiluminescent labels, the latter now being the most common. The diagnostics industry began to develop simple kits for the measurement of a wide range of hormones and so many laboratories were able to offer hormone determinations to their clinicians. Although methods were relatively simple they still needed skilled technical staff. The assay performance of some laboratories was less than good. With training courses and the introduction of External Quality Assessment Schemes (see Chapter 19) laboratories rapidly improved. However, results from laboratories could still differ significantly. Kit comparisons have been carried out over many years examining specificity, sensitivity, and reliability of commercial kits [19, 20]. The Department of Health in the UK set up laboratories in the 1990s to carry out kit evaluations and comparisons to give

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guidance to laboratories in the choice of commercial kits. This work was later extended to automated systems as they became a common tool in the routine clinical laboratory [21]. Despite all the training and standardization of reference preparations significant differences between the commercial methods for any one analyte still remain. This can be due to differences in the cross-reaction of the antisera or the medium used to prepare the calibrators. Although companies try and mimic serum or urine matrices a number of additional chemicals are added to improve stability and overcome bacterial growth. They are often, therefore, a poor imitation of serum and urine and their matrix may cause deviation from the true result. To address this problem reference methods, using gas chromatography–mass spectrometry (see Chapter 3) and more recently tandem mass spectrometry (see Chapter 4), have been established to provide a definitive value for comparison. This has been easier to do for the steroids which circulate in the body as a distinct form than for the protein hormones. The latter may circulate in more than one form due to loss of one or two amino acids (GH), variation in glycosylation (glycoprotein hormones), and conjugated as macroforms (prolactin) (see Chapters 7 and 8). These different forms cross-react by different amounts with the various antisera but to give a definitive result on any one form would not be helpful as some forms are biologically active. There have been times when commercial pressures, such as following the market leader, have led to better agreement at the expense of accuracy. Automation has enabled a large number of samples and a wide range of analytes to be analyzed in a relatively short period of time. The measurement of a hormone may take only 15 min with several hormones being measured almost simultaneously. Track systems deliver samples to the analyzer and can even store the samples after analysis. This reduces technical intervention but knowledge of the limitations of assays is very important for accurate interpretation of results. Limited sensitivity and interference from other substances can invalidate results and it is important for researchers, who have little or no experience of measuring hormones, to seek help from the skilled technologist. Where there are few research samples to be analyzed it is probably not worth the time and effort for the researcher to establish their own assays. It is more economical to ask the local laboratory, carrying out routine hormone measurements, to analyze the samples. Where a large number of samples are to be analyzed over a long period of time it may be worth the researcher to set up their own assay as it may be difficult for the routine laboratory to accommodate such large numbers. Specific requirements of increased sensitivity or specificity may need a separate assay to be developed. In addition the researcher may be trying to measure a new substance, whether in humans, other animal species, or other medium, for which no assay exists.

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Chapters 6–17 give details of hormone methods that can be set up in the laboratory. Automated methods are not described although such methods are available for nearly all the hormones covered. Some chapters describe noncommercial methods but where a commercial kit is described it should be viewed as an example of one of several manual kits and automated methods may also be available. In the case of anti-müllerian hormone there is only one method currently available and that is a commercial product. Some omissions may be found, e.g., pregnancy screening, where the methods are all automated or have many established commercial kits. It is still incumbent on the assayist to examine the literature more widely or to seek expert advice before choosing a particular manual method to ensure that it is appropriate to their studies. Whatever method a person uses the quality of the assay can only be assured by the use of internal quality control (IQC) samples (see Chapter 18). These must be of sufficient number and at a concentration relevant to the study to be of use. Chapter 18 gives details of how to set up IQC for an assay and the subsequent interpretation of the results collected. IQC is essential to give confidence in the results produced. External quality assurance schemes (EQAS) (see Chapter 19) provide additional information on the quality of an assay. As these schemes distribute samples typically on a monthly basis for the most common hormones it is not worth joining a scheme if only one or two assays are carried out. However, it is still worth trying to obtain information on the agreement between methods from scheme organizers so that methods with poorer precision or greater bias may be avoided. Confidence in results can be established by asking another laboratory to analyze a few samples and comparing the results. Where an assay is to be used over an extended period of time participation in EQAS is an important adjunct to quality measures of an assay. EQAS provides a comparison of results (a) between methods (b) between users of the same method and (c) where repeat samples are sent out, assay precision. EQAS have been set up in many countries and welcome overseas participation. The chapters of this book illustrate a variety of methods used for a range of hormones. Across this book techniques covering radioimmunoassay to tandem mass spectrometry are described. The techniques described for any one hormone may be applicable to other hormones. For instance, a tandem mass spectrometry method is described for testosterone (see Chapter 12) but this technique is equally applicable to other hormones and methods have been published. In other words chapters additional to the hormone of interest may provide a more appropriate method for the investigator. Nevertheless it is hoped that this book will give the reader a good overview of hormone methods and the quality measures that should be adopted.

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References 1. Parlow AF (1961) In: Albert A (ed) Human pituitary gonadotrophins. Thomas, Springfield, Illinois. P 300 2. Vangilse HA, Nass CAG, Kassenaar AAH (1956) The assay of urinary gonadotrophins by ultra-filtration and the mouse uterine weight bioassay. Acta Endocrinol (Copenh) 21:19–31 3. Butt WR, Lynch SS, Robinson W (1975) Assays of gonadotrophins and their application. Proc R Soc Med 68:71–73 4. Steelman SL, Pohley FM (1953) Assay of the follicle stimulating hormone based on the augmentation of human chorionic gonadotropin. Endocrinology 53:606–616 5. Parlow AF, Reichert LE (1963) Biological assay of luteinizing hormone (LH, ICSH) by the ovarian hyperemia method of Ellis: An evaluation. Endocrinology 72:955–961 6. Fukushima M, Stevens VC, Gantt CL et al (1964) Urinary FSH and LH excretion during the normal menstrual cycle. J Clin Endocrinol Metab 24:205–213 7. Yalow RS, Berson SA (1960) Immunoassay of endogenous plasma insulin in men. J Clin Endocrinol Metab 89:1157–1175 8. Ekins RP (1960) The estimation of thyroxine in human plasma by an electrophoretic technique. Clin Chim Acta 5:452–459 9. Wild D (2013) The immunoassay handbook. Elsevier, Oxford 10. Wheeler MJ, Barnard G (2010) Immunoassay of steroids. In: Makin HLJ, Gower DB (eds) Steroid analysis, 2nd edn. Springer, London, pp 283–327 11. Kohler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of pre-defined specificity. Nature 256:495–497

12. Bush IE (1954) The possibilities and limitations of paper chromatography as a method of steroid analysis. Recent Prog Horm Res 9:321–335 13. Berthou F, Picart D, Bardou L et al (1976) Separation of C-19 and C-21 dihydroxysteroids by open-hole tubular glass columns and lipophilic gel chromatography. J Chromatogr 118:135–155 14. Vandenheuval WJA, Sweeley CC, Horning EC (1960) Separation of steroids by gas chromatography. J Am Chem Soc 82:3481 15. Bailey E, Fenoughty M, Wheeler MJ (1973) The group estimation of urinary 11-oxy-17hydroxy-corticosteroids using gas-liquid chromatography. Biochem Med 7:1–13 16. Mattingly D (1962) A simple fluorimetric method for the estimation of free 11-hydroxycorticosteroids in human plasma. J Clin Pathol 15:374–379 17. Vermeulen A, Vandeweg M, Verdonck L (1969) Cortisol and testosterone determination with protein-binding methods. Z Clin Chem Clin Biochem 7:111 18. Nars PW, Hunter WM (1973) Method for labelling oestradiol-17beta with radioiodine for radioimmunoassays. J Endocrinol 57: R47–R48 19. Wheeler MJ, Shaikh M, Jennings RD (1986) An evaluation of 13 commercial kits for the measurement of testosterone in serum and plasma. Ann Clin Biochem 23:303–309 20. Lamph SA, Wheeler MJ, Halloran SR (2005) GMEC evaluation of seven oestradiol assays. Clin Chim Acta 355:S274–S274 21. Wheeler MJ (2001) Automated immunoassay analysers. Ann Clin Biochem 38:217–229

Chapter 2 Immunoassay Techniques Michael J. Wheeler Abstract No other development has had such a major impact on the measurement of hormones as immunoassay. Reagents and assay kits can now be bought commercially but not for the more esoteric or new hormones. This chapter explains the basics of the immunoassay reaction and gives simple methods for immunoassays and immunometric assays and for the production of reagents for both antigenic and hapten hormones. Alternative methods are given for the preparation of labeled hormones as well as several possible separation procedures. The methods described here have been previously used in a wide range of assays and have stood the test of time. They will allow the production of usable immunoassays in a relatively short period of time. Key words Immunoassay, Antibodies, Tracers, Separation techniques, Peptides, Protein hormones, Steroids

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Introduction The first immunoassays were described by Yalow and Berson [1] and Ekins [2] for the measurement of insulin and thyroxine, respectively. As they say, the rest is history. There are now hundreds of immunoassays for scores of analytes including hormones, tumor markers, bone markers, drugs, antibodies, proteins, viruses and cardiac markers covering the fields of endocrinology, oncology, hematology, toxicology, serology, infectious diseases, et cetera. Developments in antibody and label production, and automation, have resulted in highly specific and sensitive assays. This chapter provides an overview of immunoassay technology and some general methods for the production of antibodies, labeled analytes, and solid-phase separation, but assays specific to different analytes will be found in the assay-specific chapters. The basic requirements of an immunoassay are given in Table 1.

Michael J. Wheeler (ed.), Hormone Assays in Biological Fluids: Second Edition, Methods in Molecular Biology, vol. 1065, DOI 10.1007/978-1-62703-616-0_2, © Springer Science+Business Media New York 2013

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Table 1 Basic requirements of an immunoassay An antibody to the analyte to be measured A labeled form of the analyte (competitive immunoassay) Or a labeled second antibody to the analyte (reagent excess immunoassay) A separation system to separate antibody-bound tracer from unbound tracer

Both polyclonal and monoclonal antibodies are employed in current immunoassays as will be explained in Subheading 1.1. Labels attached to analytes and antibodies may be radioactive, usually iodine-125 [3] (radioimmunoassay and immunoradiometric assays), enzymes such as alkaline phosphatase and horseradish peroxidase [4] (enzyme immunoassay or immunometric assay, or enzyme-linked immunosorbent assay [ELISA]), chemiluminescent [5] (e.g., acridinium ester), fluorescent [6] (e.g., fluorscein), or an earth chelate such as europium or terbium [7, 8] (time-resolved fluorescent assays). All the nonradioactive labels are used in different commercial-automated immunoassays. Manual assays using radioactive or nonradioactive labels are available for a great number of different analytes and the only expensive equipment required is for measuring the end-point. Because γ-counters and colorimeteric plate readers have been generally available, in-house assays have tended to use either radioactive or enzyme labels. Both types of label are also available commercially for many hormones and are fairly easy to use and each has its own advantages and disadvantages. The main advantage of a radioactive label is that the assay is relatively free from interference and counting can be delayed for several days if necessary. Enzyme labels remove the hazard of radiation and radioactive contamination but can be affected by inferring substances that can inhibit enzyme activity or affect the colorimetric reading. Manual assays are useful for independent studies where a significant number of samples will be analyzed, whereas automated assays are more convenient for routine diagnostic laboratories that carry out a large number of determinations of a defined repertoire of analytes from week to week. Raising antibodies is fairly simple but lengthy, whereas producing labels, especially the majority of nonradioactive labels, can be rather complex, so it is much easier to set up an assay if the antibody and label or tracer are available commercially. The basic immunoassay reaction is as follows: Antibody (Ab) + Antigen (Ag)  Antibody. Antigen (Ab. Ag) An antibody specific to the endogenous antigen (analyte) in the sample binds to the antigen after a period of incubation. In order to quantify this reaction a labeled form of the antigen is

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added that competes for a limited number of antibody-binding sites with the endogenous antigen: Ab + Ag + Ag *  Ab.Ag + Ab.Ag * In this system, the amount of labeled antigen bound to antibody is inversely proportional to the amount of endogenous antigen in the sample. In order to measure the amount of labeled antigen that is bound, the bound moiety must be separated from the unbound reagents. Greater assay precision is achieved with the most specific separation system. Hormone immunometric assays use labeled antibodies in a “sandwich” assay. The antibodies are either both monoclonal or one is monoclonal and the other polyclonal: Ab1 + Ag ® Ab1.Ag Ab1.Ag + Ab2 * ® Ab1.Ag.Ab2 * The systems that have been developed for general diagnostics use a capture antibody that is usually bound to a solid phase such as the reaction tube or microtiter well surface or to a particle, such as a plastic bead or cellulose. Sandwich assays use excess reagents because the assay depends on occupancy of binding sites rather than competition for the binding sites. This assay design can produce fast, sensitive, and highly specific assays since two epitopes on the hormone have to be recognized by the two antibodies. 1.1

Antibodies

Immunoassay techniques have been made possible by the unique characteristics of the mammalian immune system. These are the following: 1. A foreign substance (antigen) entering the body stimulates the immune system to produce antibodies against that substance. 2. Antibodies recognize specific and sometimes unique characteristics of the antigen. 3. Once antibodies have been produced there is an enhanced production of antibody following a second challenge with the antigen. Antibodies used in immunoassays fall into two groups, polyclonal and monoclonal antibodies. Polyclonal antibodies may be produced in mammals such as rabbits or sheep. When a foreign substance enters the body, it stimulates the immune system to produce antibodies to the substance making it attract macrophages that will attack the foreign substance. Using this natural reaction, an analyte in as pure a form as possible is injected into the animal stimulating the production of antibodies.

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In the case of many proteins (hormones, tumor markers), this may involve the injection of the protein without modification because the protein of one species is usually different enough from the same substance in the host to cause an immune reaction. Some substances such as drugs and steroids are too small to produce an antigenic reaction and natural steroids are the same as those circulating in the target animal. These latter substances, referred to as haptens, can be made antigenic by attaching them to a protein such as albumin or keyhole limpet hemocyanin. The conjugated molecule is larger and, as a hybrid substance, is now seen as a foreign substance by the host animal which will produce antibodies to it. A number of different routes have been used to inject the antigen into animals [9] but ethical restrictions have led to subcutaneous injection being the first and preferred method of administration. Monoclonal antibodies (MAbs) use the same characteristics of the immune system but provide unique specificity by recognizing only one epitope on an antigen. The method for the production of MAbs was developed by Kohler and Milstein in 1975 [10] and led to the development of very specific and sensitive sandwich or twosite immunoassays. The first step is to produce a polyclonal antiserum in a mouse or a rat. The lymphocytes are harvested and fused with a myeloma cell line. The cells are then cultured in a medium containing hypoxanthine, aminopterin, and thymidine (HAT medium). Aminopterin blocks the main biochemical pathway for DNA synthesis, whereas hypoxanthine and thymidine provide substrate for an alternative salvage pathway. The myeloma cells lack the salvage pathway and die and the lymphocytes are short-lived and also die after about 7 days. The remaining cells are the hybrid cells where the alternative biochemical pathway for DNA and RNA synthesis has been inherited from the original lymphocytes and immortality has been inherited from the myeloma cells. The surviving cells are diluted, plated out, and grown in microtiter plates. The medium is tested for antibody, and where present, the individual clones can then be plated into microtiter plates and grown further. These cell lines will be from a single original hybrid cell and will produce one type of antibody recognizing a single epitope on the antigen. These wells are further screened to assess antibody quality and the chosen wells can be investigated further or cryopreserved for later investigation. Full details of monoclonal preparation and cryopreservation may be found in ref. 11. 1.2 Labeled Reagents (Haptens, Proteins, and Antibodies) or Tracer

The ideal tracer will be a homogeneous preparation that has the same affinity between antibody and antigen as the unlabeled substance. Competitive immunoassays are generally used for small molecules such as drugs and steroids. In such assays, a labeled form of the substance is prepared. Although steroids can be labeled directly with radioactive iodine, the product has poor affinity for the antibody and is unstable. Steroids and other small molecules

Immunoassay Techniques

11

are usually labeled by first producing a reactive bridge to which the label (iodinated amine, enzyme, and others) can be attached. The most common intermediate products are carboxymethyloximes and hemisuccinates. The former can be attached through a carboxyl group, whereas the latter is usually linked through a hydroxyl group. Enzymes and fluorescent and chemiluminescent substances can then be attached through this bridging structure. In the case of iodinated tracers, histamine or tyramine is iodinated first and then attached to the bridge structure. The chemical bridge increases the distance between the substance and the attached label, thus reducing inhibition of antibody binding. Peptides and proteins can be labeled by direct methods because of the availability of reactive tyrosine groups in most of them. The most common methods for labelling with iodine-125 are as follows: (1) chloramine T (N-chloro-ptoluenesulfonamide), (2) lactoperoxidase, (3) iodogen, and (4) the Bolton–Hunter reagent (5-[p-hydroxyphenyl]propionic acid N-hydoxysuccinimide ester). Enzyme-, luminescent-, and chemiluminescent-labeled hormones may be prepared in a similar way to iodinated tracers with only slight modifications in some instances [12]. These methods were developed in the early days of immunoassay and their simplicity and robustness have withstood the test of time. Little detail of the methods used is given in current texts and one has to go back to early publications to search these out [13, 14]. A common method of labelling proteins with an enzyme is with glutaraldehyde. There are a large number of variations of the original method [15–18]. One method is described here which uses dialysis to purify the products. Fuller reviews of immunoassay development and techniques may be found in two recent references [19, 20].

2

Materials

2.1 Preparation of Steroid–Albumin Conjugate

1. Dioxane. 2. Histamine. 3. 1 M Hydrochloric acid. 4. Isobutylchloroformate. 5. 1 M Sodium hydroxide. 6. Steroid-3-conjugate, e.g., testosterone, cortisol, and other steroids with a 3 carboxyl group. 7. Tri-n-butylamine.

2.2 Polyclonal Antibody Production

1. Freund’s adjuvant. 2. Saline: 9 g sodium chloride in 1 L distilled water. 3. Steroid conjugate, hapten conjugate, or any antigen.

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Michael J. Wheeler

2.3 Production of Labeled Antigen or Antibody (Tracers)

1. Chloramine T.

2.3.1 Iodination Using Chloramine T Oxidation

4. 0.5 M Phosphate buffer, pH 7.4: Dissolve 57 g disodium hydrogen orthophosphate and 13.4 g potassium dihydrogen orthophosphate into 800 mL distilled water and check pH. Adjust pH if necessary with 0.1 M NaOH or 0.1 M HCl and make up to 1 L.

2. 0.1 M Hydrochloric acid. 3. Peptide/protein.

5. 0.05 M Phosphate buffer, pH 7.4: Make up 100 mL 0.5 M phosphate buffer to 1 L with distilled water. 6. 0.1 M Sodium hydroxide. 7. Sodium iodide. 8. Sodium [125] iodine. 9. Sodium metabisulfite. 2.3.2 Iodination Using Lactoperoxidase Oxidation

1. Hydrogen peroxide. 2. Lactoperoxidase. 3. Prolactin (National Institute for Biological Standards and Control, Potters Bar, UK). 4. 0.5 M Phosphate buffer, pH 7.4 (see Subheading 2.3.1, item 4). 5. 0.05 M Phosphate buffer, pH 7.4 (see Subheading 2.3.1, item 5). 6. Sodium [125] iodine.

2.3.3 Iodination of Steroids and Other Haptens

1. Chloramine T.

Iodination of Histamine

4. Sodium [125] iodine.

2. Histamine. 3. 0.5 M Phosphate buffer, pH 7.4 (see Subheading 2.3.1, item 4). 5. Sodium metabisulfite.

Formation of a Mixed Anhydride

1. Dioxane. 2. Testosterone-3(O-carboxymethyl)oxime. 3. Tri-n-butylamine. 4. Isobutylchloroformate.

Conjugation

1. 0.01 M Hydrochloric acid: Prepared as 10 % dilution from 0.1 M solution. 2. Mixed anhydride prepared from Subheading 3.3.3.2. 3. 0.05 M Phosphate buffer, pH 7.4 (see Subheading 2.3.1, item 5). 4. Sodium hydroxide. 5. 0.01 M Sodium hydroxide. 6. Toluene.

Immunoassay Techniques Separation and Purification of Iodinated Product by TLC

13

1. Ethanol. 2. Glacial acetic acid. 3. Methanol. 4. Silica gel TLC plates ultraviolet absorbing at 254 nm. 5. Toluene.

Separation and Purification of Iodinated Product by HPLC

1. Acetonitrile. 2. High-pressure liquid chromatography (HPLC) column 0.5 cm × 10 cm C18-ODS column. 3. Methanol. 4. Sodium acetate.

2.3.4 Conjugation of Proteins with Enzymes

2.3.5 Glutaraldehyde Conjugation of Enzyme Label to Protein

One of the commonest methods of labelling proteins with an enzyme is to use glutaraldehyde. There have been numerous modifications. The original method required two lengthy dialysis steps plus purification on gel columns. Modifications have tried to simplify the technique and two methods are described linking horseradish peroxidase to a protein. Other enzymes could be used. The amount of purification will depend on the concentration of the measured analyte and the sensitivity required. The purer the enzyme label the greater the sensitivity that should be achieved. 1. Enzyme to be conjugated. 2. Phosphate buffer: 0.1 M, pH 6.8. 3. Phosphate-buffered saline (PBS): 0.05 M, pH 7.4. 4. Glutaraldehyde solution. 5. Dialysis tubing. 6. Bovine serum albumin (BSA). 7. Carbonate/bicarbonate buffer: 1 M, pH 9.5. 8. Lysine: 0.2 M. 9. Ammonium sulfate: Saturated solution.

2.4 Separation Methods 2.4.1 Coating of Microtiter Plate Wells and the Inside of Polystyrene Tubes for Solid-Phase Assays

1. Carbonate buffer, pH 10.0: 13 mM sodium bicarbonate, 6.4 mM sodium carbonate. 2. PBS 0.05 M, pH 7.2 with 0.1 % BSA.

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Michael J. Wheeler

2.4.2 Separation of Antibody-Bound and Unbound Antigen by a Second Antibody Method

1. Donkey anti-rabbit immunoglobulin serum. 2. PBS 0.05 M, pH 7.2 with 0.1 % BSA. 3. Polyethylene glycol 6000. 4. Rabbit serum.

2.4.3 Antigen Separation of Tritiated Tracers Using Dextran-Coated Charcoal Preparation of Dextran-Coated Charcoal Separation with Dextran-Coated Charcoal

1. Charcoal. 2. Dextran. 3. Dextran-coated charcoal: 0.05 g Dextran + 0.5 g charcoal in 100 mL 0.05 M phosphate buffer, pH 7.4. 1. Dextran-coated charcoal: 0.05 g Dextran + 0.5 g charcoal in 100 mL 0.05 M phosphate buffer, pH 7.4. 2. Scintillation fluid: Optiphase Hisafe 3. 3. Scintillation vials.

3

Methods

3.1 Preparation of Steroid–Albumin Conjugate

1. Place a stoppered conical glass test tube in water at 10 °C. 2. Add 5–10 mg steroid 3-carboxymethyloxime, 15 μL tri(m) butylamine, and 300 μL dioxane. 3. Shake gently to dissolve steroid. 4. Add 5 μL isobutylchloroformate. 5. React for 20 min, shaking periodically. 6. To a small (25 mL) conical flask, add 20 mg human albumin in 2 mL distilled water:dioxane (1:1). 7. Place the flask on ice and cool to 8 °C. 8. Add the reactants in the conical test tube to the conical flask and stir for 4 h (see Note 1). (After 1 h add 25 μL 1 M sodium hydroxide.) 9. At the end of 4 h dialyze overnight against distilled water. 10. Precipitate the protein conjugate by slowly adding very small drops of 1 M HCl (see Note 2). 11. Stand overnight at 4 °C. 12. Centrifuge, pour off supernatant, and freeze-dry. 13. Confirmation of conjugate can be carried out spectrophotometrically.

3.2 Polyclonal Antibody Production

There are a number of different methods that have been used to produce polyclonal antibodies. Subcutaneous, intradermal, intralymphatic, intrasplenic, and intramuscular injections and injections into the footpad [11] have been used. In the United Kingdom, regulating bodies tend to give permission for subcutaneous injection only unless there are very good reasons for trying a different route. The following procedures have been used in this laboratory

Antibody Titre

Immunoassay Techniques

Primary Injection

15

Secondary Injection

Time

Fig. 1 A diagram showing the trend in antibody titer for a hormone antiserum raised in a rabbit

to prepare a number of steroid and peptide antibodies, but modifications (given in the notes) may be required to meet local licensing requirements for animal work. 1. Dissolve 500 μg to 1 mg steroid conjugate (100 μg peptide) in 1 mL saline (see Note 3). 2. Add 1 mL Freund’s complete adjuvant (see Note 4). 3. Mix on a vortex mixer or by some other means to produce a stable emulsion. 4. Inject subcutaneously along the back of the rabbit into about 12 sites (see Note 5). 5. Repeat this procedure after 4 weeks substituting Freund’s incomplete adjuvant for the Freund’s complete adjuvant. 6. Take a small amount of blood from an ear vein after a further 4 weeks for testing (see Note 6). A diagrammatic graph is shown in Fig. 1 of typical antibody production measured as titer. Antibody has to be tested for titer (the number of antigen-binding sites available), binding affinity (how tightly antigen is bound), and specificity. As this chapter is restricted to reagent and assay development, the reader is referred to two general immunoassay books that fully describe the assessment of immunoassay performance [20, 21]. Polyclonal antibodies can be produced in amounts that are far in excess of the needs of the individual researcher. For example, 1 mL of a modest antibody that can be used at a dilution of 1/10,000 will, using 100 L per tube, be sufficient for 100,000 tubes and would last almost 4 years if one were to carry out an assay of 100 tubes every working day of the year. It is relatively easy to collect up to 10 mL of blood by ear bleed from a rabbit or about

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Michael J. Wheeler

60 mL by exsanguination. Nevertheless, this production is an insufficient amount for commercial requirements. Polyclonal antibodies can also suffer from lack of specificity because antibodies to all the epitopes on an antigen will be present. Some of these epitopes will be common to other similar antigens and will result in cross-reaction with these other antigens. An example is polyclonal antibodies to luteinizing hormone that cross-react with chorionic gonadotropin, follicle-stimulating hormone, and thyroidstimulating hormone owing to the presence of a common α subunit. The degree of cross-reaction and the binding affinity is unpredictable because this will vary between the animals used to raise the antibody as well as between different bleeds from the same animal. 3.3 Production of Labeled Antigen or Antibody (Tracers) 3.3.1 Iodination Using Chloramine T Oxidation

The iodination reaction may be carried out in a small (LP3) polystyrene tube. After each solution is added, the reactants should be mixed by gently flicking the end of the tube. 1. Add 10 μL iodine-125 (37 MBq), 10 μL 0.5 M phosphate buffer, pH 7.2 (see Notes 1 and 8), 2 μg peptide in 10 μL 0.05 M phosphate buffer, pH 7.2, and 10 μL chloramine T in 0.05 M phosphate buffer (1 mg/mL). 2. Mix for 10 s. 3. Add 10 μL sodium metabisulfite in 0.05 M phosphate buffer (4 mg/mL) and 200 μL potassium iodide in 0.05 M phosphate buffer (10 %). 4. Separate the iodinated peptide from free iodide by running down a column of Sephadex. Where there is only one form of peptide a 1 cm × 10 cm column of Sephadex 50 or Sephadex 100 may be used. Where the peptide exists as or breaks down during the labelling process to other isoforms, longer columns are required using Sephadex 200. Figure 2a shows the elution profile of radioactivity from 0.5 cm × 10 cm column of Sephadex G100 after chloramine iodination of LH.

3.3.2 Iodination Using Lactoperoxidase Oxidation

Chloramine T is a powerful oxidizing agent and may cause degradation of the protein molecule. Therefore, a more gentle procedure may be required. Hydrogen peroxidase may be used as oxidizing agent for the iodination of prolactin. Again, the reaction may be carried out in a small (LP3) polystyrene tube, mixing after the addition of each solution. 1. Add 10 L 0.5 M phosphate buffer, pH 7.2, 5 μL iodine-125 sodium (18.5 MBq), 2 μg prolactin in 10 μL 0.05 M phosphate buffer, pH 7.2, 10 μL lactoperoxidase (1 mg/mL in 0.05 M PBS, pH 7.2), and 10 μL hydrogen peroxide solution (1:12,000 dilution of 30 % solution in distilled water) (see Notes 7 and 8). 2. Cap the reaction tube, mix gently, and incubate for 5 min at room temperature. 3. Add 10 μL hydrogen peroxide solution and incubate for a further 5 min.

Immunoassay Techniques

a

17

Radioactivity Profile from a 0.5 cm x 10 cm Sephadex G100 column following iodination of LH

120

Radioactivity (cps x 1000)

Macroprolactin 100 80 60

Prolactin Monomer

40 20 0

0

b

2

4

6

8 10 12 Fraction number

14

18

20

Profile from a G200 column of radioiodine after the iodination of prolactin 1600

Free Iodide

Prolactin Monomer

1400

Radio active counts

16

1200 Prolactin Dimer

1000 Prolactin Aggregate

800 600 400 200 0 0

10

20

30 Fraction number

40

50

60

Fig. 2 (a) Radioactivity profile from a 0.5 cm × 10 cm Sephadex G100 column following iodation of LH. (b) Profile from a G200 column of radioiodine after the iodination of prolactin

4. Separate the iodinated products (monomer, dimer, aggregate, and iodinate enzyme) on a 1.5 cm × 500 cm Sephadex G200 column. A separation profile is shown in Fig. 2b (see Note 9). 3.3.3 Iodination of Steroids and Other Haptens

The addition of a radioactive iodine atom to a hapten is more complex. Although there have been a number of published methods for the direct incorporation of an iodine molecule into the structure of a hapten, such as a steroid, these have not been very successful. This is because the iodine molecule is one-third of the size of the whole hapten and causes disruption to its structure. This makes the molecule unstable as well as sometimes not being recognized by the antibody. Therefore, the procedure with haptens is to

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Michael J. Wheeler

label histamine or tyrosine and then attach it to the hapten via a chemical bridge. The following method describes the iodination procedure we have used for many years for the iodination of testosterone, cortisol, and other steroids having a 3 carboxyl group and is based on the method of Nars and Hunter [22]. Iodination of Histamine

The reaction should be carried out in a glass-stoppered conical pyrex tube behind appropriate shielding. After the addition of each solution, the reactants should be mixed by gently flicking the bottom of the tube. 1. Add 10 μL histamine (2.2 g) in 0.5 M PBS, pH 7.2, 10 μL sodium iodide-125 (37 MBq), and 10 μL chloramine T (5 mg/mL water). 2. Incubate for 30 s at room temperature. 3. Add 10 μL sodium metabisulfite (30 mg/mL).

Formation of a Mixed Anhydride

The reaction should be carried out in a glass-stoppered conical pyrex tube behind appropriate shielding. After the addition of each solution, the reactants should be mixed by gently flicking the bottom of the tube. 1. Add 100 μL testosterone-3(O-carboxymethyl) oxime, 10 μL tri-n-butylamine in dioxane (1:5), and 10 μL isobutylchloroformate in dioxane (1:10). 2. Incubate at 10 °C for 20 min with frequent mixing. 3. Add 3.45 mL dioxane and place the tube in ice.

Conjugation

1. Add 50 μL of the mixed anhydride solution to the iodination mixture followed by 10 μL 0.1 M NaOH. 2. Incubate in ice for 1 h with occasional mixing. 3. After 1 h, add 10 μL 0.1 M NaOH. 4. Incubate in ice for a further hour with occasional mixing. 5. Add 1 mL 0.1 M HCl followed by 1.0 mL toluene. 6. Mix for 1 min on vortex mixer. 7. Remove and discard the solvent layer. 8. Add 1.0 mL 0.1 M NaOH and 1.0 mL 0.5 M sodium phosphate buffer, pH 7.2. 9. Add 0.5 mL toluene (see Note 10) and carefully mix on a vortex mixer for 1 min to extract the iodinated steroid conjugate. 10. Remove the toluene layer into a clean conical glass tube. 11. Repeat last step and combine the toluene extracts.

Immunoassay Techniques Separation and Purification of Iodinated Product by TLC

19

1. Reduce the volume of the extract to approx 250 μL under nitrogen or air. 2. Add a solvent mixture of toluene:methanol:glacial acetic acid (75:24:1) to a TLC chromatography tank. 3. Draw an origin line on a 5 mm × 20 cm silica gel plate. 4. Carefully streak the reduced extract along the origin, using about 20 μL at a time, and dry with an air or a nitrogen stream. 5. After the TLC tank has equilibrated for about 1 h place the TLC plate in the tank. 6. When the solvent front has reached to about 1 cm from the top of the plate, remove the plate, mark the solvent front, and air-dry. 7. Place the dry TLC plate on top of an X-ray film for about 30 min (see Note 11). 8. Develop the X-ray film and locate radioactive band on TLC plate. 9. Scrape off the silica gel carefully onto a large no. 1 Whatman filter paper that has been folded down the middle. 10. Fold the filter paper and tip the silica gel into a stoppered conical tube. 11. Add 5 mL ethanol and mix for 1 min. 12. Place the tube in a lead container and allow to settle in a suitable refrigerator overnight. Most steroids made in this way are stable for several months. 13. The next day the radioactivity in the solution can be assessed by counting a small aliquot in a γ-counter in order to determine the specific activity and the concentration required for an assay. Small amounts can then be taken, dried down, and buffer added to give a working solution for radioimmunoassay (RIA).

Separation and Purification of Iodinated Steroid by HPLC

1. Take the toluene extract to dryness under nitrogen. 2. Add 100 μL column solvent mixture (see Note 12). 3. Run on a lead-shielded 0.5 cm × 10 cm C18-ODS reverse-phase column using the same solvent mixture as the mobile phase. 4. Collect 1 mL fractions. 5. Count the fractions in a γ-counter and, if necessary, pool the fractions in the first radioactive peak that indicates the iodinated hapten. 6. Take about 10 μL and count the radioactivity to determine the specific activity and the amount to be used in an assay.

3.3.4 Glutaraldehyde Conjugation of Enzyme Labels to Protein

1. Prepare a 1.25 % solution glutaraldehyde. 2. Add 10 mg enzyme. 3. Incubate overnight at room temperature. 4. Dialyze against PBS at 4 °C.

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Michael J. Wheeler

5. Prepare a 5 mg/mL solution of protein in PBS. 6. Add 1 mL to the dialysate. 7. Add 50 μL carbonate/bicarbonate buffer. 8. Incubate overnight at 4 °C. 9. Add 50 μL 0.2 M lysine 10. Incubate at room temperature for 2 h. 11. Dialyze against PBS at 4 °C overnight. 12. Add an equal volume of saturated ammonium sulfate to the dialysate. 13. Leave for 30 min at 4 °C. 14. Centrifuge at 4,000 × g for 20 min. 15. Dissolve the precipitate in 1 mL saline. 16. Dialyze against PBS overnight. 17. Store conjugate solution at 4 °C for up to 3 months. 3.4 Separation Methods

3.4.1 Coating of Microtiter Plate Wells and the Inside of Polystyrene Tubes for Solid-Phase Assays

Once the antigen and antibody have reacted, the mixture contains both bound and unbound antigen and antibody. In order to determine the amount of antigen bound to the antibody it is necessary to separate the bound and unbound moieties [23]. Nonspecific methods, such as adding ethanol or ammonium sulfate solution, have been used but these methods give rise to high nonspecific binding that reduces the sensitivity of the assay. More specific methods are preferred for routine methods. Antibodies can be attached to solid surfaces, such as a coating on the bottom of the polystyrene reaction tubes or microtiter plates, plastic beads, or on cellulose particles. Commercial methods frequently used magnetized particles. The advantage of the latter is that these are held in suspension during the reaction providing shorter incubation times and can be quickly pulled out of the mixture by applying a magnetic force either at the bottom or the sides of the reaction tube. The solution can then be aspirated and the particles thoroughly washed. During the wash step the magnet is moved away from the tube during addition of the wash solution and reapplied so that the solution can be aspirated. Several wash steps may be used to reduce the nonspecific binding to a minimum. A method for coating the inside of a polystyrene reaction tube, microtiter well, plastic particles, or beads is described. 1. Add antibody diluted in carbonate buffer pH 10.0 (see Note 13) to the microtiter plate wells and incubate at 4 °C overnight. 2. Aspirate the liquid, add 0.1 % BSA–0.05 M PBS pH 7.2, and then leave at room temperature for 1 h. 3. Aspirate to dryness before washing with 0.05 M phosphate buffer pH 7.2.

Immunoassay Techniques

21

4. Aspirate to dryness. 5. Place the microtiter plate in a plastic bag, preferably with a small amount of silica gel, and store in a refrigerator for no more than 24 h. 3.4.2 Separation of Antibody-Bound and Unbound Antigen by a Second Antibody Method

Another simple separation method is the double-antibody method. After the initial reaction, a solution is added containing a second antibody to the first antibody. The solution also contains nonspecific serum of the animal species in which the first antibody was raised. For example, if the method uses a rabbit antihormone antibody, the separation solution contains an antiserum to rabbit immunoglobulins plus rabbit serum. The anti-rabbit immunoglobulin antibody will bind to the rabbit antihormone antibody and to rabbit immunoglobulins in the rabbit serum. As the second antibody can bind more than one first antibody molecule a protein matrix forms that precipitates out. This usually takes about 12 h but the precipitation can be speeded up by adding the second antibody and rabbit serum in an 8 % solution of polyethylene glycol 6000. Precipitation is almost instantaneous but is probably best left for about 20 min. The precipitate is centrifuged down. In the case of iodine tracers the supernatant is aspirated and the amount of radioactivity in the precipitate is determined in a gamma scintillation counter. For enzyme tracers the precipitate can be resuspended in a substrate solution and the color determined. When a second antibody method is used the amounts of second antibody and animal serum have to be optimized to give maximum formation of the protein matrix. Poor matrix and hence poor precipitation occur if either the antigen or the second antibody is in excess. Optimum conditions may be determined in the following way and are described for a simple RIA using a rabbit polyclonal antiserum to a hormone. 1. Prepare solutions of anti-rabbit immunoglobulin antiserum in 8 % polyethylene glycol (PEG) solution: typically 1/10, 1/25, 1/50, and 1/100. 2. Prepare solutions of nonimmune rabbit serum in 8 % PEG solution: typically 1/100, 1/250, 1/500, and 1/1,000. 3. Label 32 reaction tubes and set up as in Fig. 3. 4. Add 100 μL assay buffer (0.1 % BSA in PBS 0.05 M, pH 7.2) and 100 μL tracer solution to give 10,000 cpm and 100 μL antihormone antibody to all tubes. 5. Add 100 μL tracer solution to two tubes, cap, and put to one side (total count tubes). 6. Mix and incubate overnight at 4 °C. 7. Add 250 μL each rabbit serum dilution to two columns of tubes as shown in Fig. 3.

22

Michael J. Wheeler Second antibody dilution 1/5

1/5

1/10 1/10 1/20 1/20 1/40 1/40 1/50 1/50 1/100 1/100

1/25

Non-immune serum

1/50 1/100 1/200 1/400 1/800 1/1000 1/1500

Fig. 3 Arrangement of tubes and dispensing for optimizing the second antibody and nonimmune animal serum

8. Add 250 μL each second antibody solution across each row as shown in Fig. 3. 9. Mix the tubes and incubate for 20 min. 10. Centrifuge for 30 min at 1,500 × g. 11. Decant or aspirate the supernatant. 12. Count the radioactivity in the precipitates and calculate the percentage binding of tracer. 13. Determine which tubes contain the maximum amount of radioactivity and note the dilutions of second antibody and rabbit serum used. If this was rabbit serum at a dilution of 1/500 and second antibody at 1/25, then for the routine assay 500 μL PEG solution, containing rabbit serum at 1/1,000 and second antibody at 1/50, would be added to each tube at the separation stage. Once the optimum conditions have been found further experiments can then be carried out on first antibody and tracer concentrations and first incubation times in order to optimize the assay for sensitivity and precision. Therefore, in the development of any immunoassay it is important to have optimized the separation step first. 3.4.3 Antigen Separation of Tritiated Tracers Using Dextran-Coated Charcoal

Some steroid assays use tritiated tracers. These may be bought commercially and obviate the need to prepare iodinated tracers that require special facilities. Tritiated tracers may also be available for other haptens.

Immunoassay Techniques Preparation of Dextran-Coated Charcoal

23

This may be carried out in a large glass or plastic beaker. 1. Wash the charcoal to remove fines by adding distilled water (about five times the volume of charcoal) to several grams of charcoal. 2. Allow to settle for 60 min. 3. Aspirate most of the water from the top of the charcoal. 4. Repeat step 1 at least five more times. 5. On the final occasion, aspirate the water from the top of the charcoal as completely as possible and dry the charcoal in an oven at 100 °C. 6. To 100 mL assay buffer (see Note 14) add 0.5 g charcoal and 0.05 g dextran. 7. Store the reagent at 4 °C and always add to the assay at 4 °C while keeping the charcoal in suspension by gentle mixing on a magnetic stirrer.

Separation with Dextran-Coated Charcoal

1. 500 μL dextran-coated charcoal is added to each tube. 2. The reaction tubes are incubated at 4 °C for 15 min (see Note 15). 3. The supernatant is poured into a scintillation vial and scintillation fluid added. 4. The radioactivity in each tube is counted in a beta scintillation counter. This is a very brief account of the methods used in immunoassay development, but it provides sufficient information for setting up a basic immunoassay. However, further development work is required to achieve an assay that fulfils the need for a sensitive and precise method. Development includes investigating different volumes of reagent in the assay system, the concentration of reagent constituents, the need for a wash step, the temperature for the reaction, and the length of incubation times. There have been several books written on immunoassay techniques over the years that will give many references although the older books give more detail of the methods [13, 14].

4

Notes 1. The method adopted to keep the reactants at the right temperature and to mix continuously was to place the flask at 45 °C in a beaker of iced water. A small flea could be placed in the liquid and the whole apparatus placed on a magnetic mixer. Extra ice was added to the iced water at intervals to keep the reactants cool.

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Michael J. Wheeler

2. This step has to be carried out with great patience and care as the end-point, when the conjugate precipitates, is very sudden. If too much acid is added the precipitate goes back into solution. The pH is just under 7.0. The precipitate is usually fairly fluffy in appearance, but if a fine precipitate forms it is better to continue, but retain the supernatant after centrifugation for further manipulation. 3. Smaller volumes can be taken. We use a total volume of about 500 μL when we carry out intradermal injections. In this case the back of the animal has to be shaved first. 4. In the United Kingdom, the use of incomplete adjuvant is preferred by the regulatory body. 5. For intradermal injection we inject at about 30 sites along the back. 6. Some researchers advocate taking some blood after the first 4 weeks to check for antibody production before the second injection. This is to save injecting precious material into an animal that is not producing antibodies. We have found this unnecessary. 7. Sodium iodide I125 is provided in alkaline solution. 8. The higher molarity buffer neutralizes this solution before analyte is added. The 30 % stock solution should be stored at 4 °C for no longer than 1 year. 9. A number of different procedures have been published. Coupling the lactoperoxidase to cellulose particles allows the iodinated enzyme to be removed by centrifugation. Iodogen, available commercially, is also a gentle oxidizer, but we have found that it is not very efficient for amounts of less than 10 g protein. Some proteins are deficient in tyrosine and histidine, which are iodinated in the above reactions, and so cannot be iodinated by the methods described previously. Bolton and Hunter developed a method using 3-(p-hydroxyphenyl) propionic acid N-hydroxysuccinimide ester that is iodinated with chloramine T. The ester is added to a solution containing the polypeptide and combines with lysine residues or the N-terminal. 10. Toluene may be used for the less polar steroids such as testosterone, but ethyl acetate is used for polar steroids such as cortisol. 11. To position the TLC plate after the X-ray plate has been developed, punch two holes with a pin through the paper envelope of the X-ray holder, one hole at each of the two bottom corners, and one hole at the top center of the plate. This allows a small amount of light through that which produces three black spots when the plate is developed. 12. For testosterone we use isocratic separation using acetonitrile :water 1:1, and for progesterone acetonitrile:sodium acetate 1:4.

Immunoassay Techniques

25

13. The antibody dilution used in this laboratory has been between 1/2,000 and 1/10,000 found by experimentation. 14. The concentrations and solutions described here have been used in many assays by many centers, but variations occur. Some researchers have described assays where water is used in place of buffer and others have not used dextran. 15. These are typical conditions to reduce stripping of the antigen from the antibody. This occurs if the reactants are kept too long in the presence of the charcoal. Optimum conditions may be found by incubating the charcoal suspension in a blank tube (tracer only) and a zero tube (tracer plus antibody solution) for increasing periods of time. The counts in the blank will increase as stripping occurs. References 1. Yalow RS, Berson SA (1959) Assay of plasma insulin in human subjects by immunological methods. Nature 184:1648–1649 2. Ekins RP (1960) The estimation of thyroxine in human plasma by an electrophoretic technique. Clin Chim Acta 5:453–459 3. Rezaieyazdi Z, Hesamifard A (2006) Correlation between serum prolactin and lupus activity. Rheumatol Int 26:1036–1039 4. Shimizu A, Ohkubo M, Hamaguchi M (2012) Development of non-competitive enzymelinked immunosorbent assays for mummichog Fundulus heteroclitus gonadotrophins— examining seasonal variations in plasma FSH and LH levels in both sexes. Gen Comp Endocrinol 178:463–469 5. Sekiguchi S, Kohno H, Yasukawa K et al (2011) Chemiluminescent enzyme immunoassay for measuring leptin. Biosci Biotechnol Biochem 75:752–756 6. Sun M, Du L, Gao S et al (2010) Determination of 17-beta-estradiol by fluorescence immunoassay with streptavidin-conjugated quantum dots as label. Steroids 75:400–403 7. Johnsen AH, Assaad FN, Rehfeld JF (2011) Competitive solid-phase immunoassay of gastrin in serum using time-resolved fluorometry. Scand J Clin Lab Invest 71:216–220 8. Hoferi M, Krist S, Buchbauer G (2005) Adaptation of DELFIA(TM) cortisol kit for determination of salivary cortisol concentration. Arch Pharm 338:493–497 9. Harlow E, Lane D (eds) (1988) Antibodies: a laboratory manual. Cold Harbor Spring Laboratory, New York, NY 10. Kohler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495–497 11. Zola H (ed) (2000) Monoclonal antibodies. BIOS Scientific Publishers Ltd, Oxford

12. Allman BL, Short F, James VHT (1981) Fluoroimmunoassay of progesterone in human serum or plasma. Clin Chem 27:1176–1179 13. Kirkam KE, Hunter WM (eds) (1971) Radioimmunoassay methods. Churchill Livingstone, Edinburgh 14. Hunter WM, Corrie JET (eds) (1983) Immunoassays for clinical chemistry. Churchill Livingstone, Edinburgh 15. Avrameas S (1969) Coupling of enzymes to proteins with glutaraldehyde. Immunochemistry 69:43–52 16. Avrameas S, Ternynck T (1974) Peroxidase labelled antibody and Fab conjugates with enhanced cellular penetration. Immunochemistry 8:1175–1179 17. Sheng J-W, He M, Shi H-C et al (2006) A comprehensive immunoassay for the detection of microcystins in waters based on polyclonal antibodies. Anal Chim Acta 572:309–315 18. Ray S, Chowdry P, Das N et al (2010) Development of an efficient and simple method for conjugation of laccase to immunoglobulin and its characterization by enzyme immunoassay. J Immunoassay Immunochem 31:217–232 19. Wheeler MJ, Barnard G (2010) Immunoassay of steroids. In: Makin HJL, Gower DB (eds) Steroid analysis, 2nd edn. Springer, London, pp 283–327 20. Wild D (ed) (2013) The immunoassay handbook. Nature Publishing Group, London 21. Price CP, Newman DJ (eds) (1997) Principles and practice of immunoassay. Stockton, New York 22. Nars PW, Hunter WM (1973) A method for labelling oestradiol-17β with radioiodine for radioimmunoassay. J Endocrinol 57:157–158 23. Newman DJ, Price CP (1997) Separation techniques. In: Price CP, Newman DJ (eds) Principles and Practice of immunoassay, 2nd edn. Stockton Press: New York

Chapter 3 Introduction to Gas Chromatography-Mass Spectrometry Alberto Sánchez-Guijo, Michaela F. Hartmann, and Stefan A. Wudy Abstract Gas chromatography-mass spectrometry (GC-MS) is a technique of pivotal importance for the analysis of hormones in biological fluids. In consequence, it has gained relevance in clinical and endocrinological laboratories, providing reference analytical methods. This chapter offers a general description of its principles, and a real example for GC-MS profiling of plasma steroids. Key words Gas chromatography, Mass spectrometry, Derivatization, Isotopic labeling, Steroids, Plasma

1  Introduction Gas chromatography-mass spectrometry (GC-MS) is a powerful analytical technique which has maintained a prominent position as a gold standard in several research fields during the last 40 years. GC-MS plays a critical role in hormone analysis too, in which selectivity and quantification are crucial requirements. Different analytical techniques have emerged ever since the development of GC-MS and, although they have been successfully applied to the study of hormonal components in biological fluids (e.g., liquid chromatography-mass spectrometry), they cannot compete with GC-MS in terms of reproducibility, chromatographic separation, or sensitivity. GC-MS combines the extraordinary resolving power of gas chromatography, which facilitates the separation of structurally related molecules, with the high specificity offered by mass spectrometry detection. In this chapter, we describe the principles of this important technique and the components of a typical GC-MS instrument, with a particular focus on its application to hormone analysis.

Michael J. Wheeler (ed.), Hormone Assays in Biological Fluids: Second Edition, Methods in Molecular Biology, vol. 1065, DOI 10.1007/978-1-62703-616-0_3, © Springer Science+Business Media New York 2013

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2  Gas Chromatography 2.1  Principle

Gas chromatography (GC) is the term that encompasses all the chromatographic methods in which the mobile phase is a gas. GC is divided into two categories: gas–liquid chromatography (GLC) and gas–solid chromatography (GSC). In GLC the stationary phase is a liquid and in GSC a solid. GLC is an extensively used analytical technique and the only one with applications in hormone analysis. Gas chromatographic separation stems from the distribution of the components of a mixture between the stationary and the mobile phase along the chromatographic column. The particular chemical properties of each component in the mixture result in a specific interaction with the stationary phase. Compounds with a higher affinity for the stationary phase stay longer in the column than the rest. The mobile phase acts as a carrier gas and it has no interaction with the sample constituents. When the separation is successful, the constituents of the mixture are eluted at different times (retention times) from the column and then they are finally represented as different bands in a chromatogram. In Fig. 1, a mixture of substances with different chemical properties is separated using a chromatographic method. In most cases, the samples analyzed by GC are liquids. An effective GC separation can only happen when the analyte is both volatile and stable at the operating temperature, so it can properly interact with the liquid stationary phase and be carried by the mobile phase. Hormones usually do not fit these requirements and a ­chemical derivatization step is most often needed after sample preparation. Derivatization is a chemical modification of the molecule, so its

Fig. 1 Chromatographic separation principle

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29

Fig. 2 MO-TMS combined derivative conversion of testosterone

chromatographic properties are improved (stability, volatility, better separation, peak shapes, and detector response). When the hormone undergoes derivatization, it is converted into a new compound, termed “derivative.” The ideal derivatization reagent provides clean, predictable, high-yield, and easy-to-perform reactions. The derivative must permit direct fragmentation in the mass spectrometer. This process offers additional structural information about the reactive groups that were affected during the modification. Typical derivatization reactions include esterification, alkylation, acylation, or silylation, the latter being the most popular in hormone research due to its ease of preparation [1]. The transformation of hydroxyl groups into trimethylsilyl (TMS) derivatives can be achieved with several reagents; dichloromethylsilane, trimethylchlorosilane, bis-trimethylsilylacetamide (BSA), bistrimethylsilyltrifluoroacetamide (BSTFA), or trimethylsilylimid­ azole (TSIM). Other useful derivatization reagents are tertiary butyldimethylsilyl chloride (TBDMS-Cl) or heptafluorobutyric acid anhydride (HFBA). Oxo groups are normally transformed into methyl oximes (MO) in hormone analysis. The reagent used in this case is methyloxime hydrochloride. The biggest disadvantage associated with the formation of oxime derivatives is that in some cases the process generates syn and anti isomers, which can be separated during the GC run. Combination of both oxo and hydroxyl group derivatization procedures makes it possible to obtain simultaneous methyl oxime— TMS derivatives (see Fig. 2 for a combined derivatization example). The carboxyl groups, which are also present in some hormones, are normally transformed into methyl esters for GC analyses. The high temperatures needed to maintain volatilization of the analytes have a strong influence on their eluting time, being an additional factor to be considered. 2.2  Carrier Gas

The carrier gas, the mobile phase in gas chromatography, must fulfill certain characteristics. It should be inert, avoiding undesirable reactions with the analytes or with the stationary phase, pure

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enough, easy to obtain, affordable, and compatible with the detection method. Unlike the analytes, the carrier gas needs a high diffusion coefficient. Temperature affects the efficiency of the separation of the carrier gas, as it changes its viscosity. Ideally, the ratio viscosity– diffusion coefficient should be as low as possible [2]. Hydrogen presents better properties than helium and nitrogen, and it provides great chromatographic efficiency and faster analysis, but still the most common carrier gas for GC-MS analysis is helium. 2.3  Injection Ports

The injection port is the system that enables the introduction of the sample into the chromatographic column. This process must be carried out in a quick and quantitative fashion. The injectors for capillary columns include split, splitless, on-column, and programmed-­temperature vaporization (PTV). The split injection port emerged as the size of the chromatographic columns decreased. The syringes used in older packed columns were not applicable to capillary columns, and the volume of sample needed had to be reduced as well. In the split configuration, the sample is evaporated and mixed with the carrier gas, and only a fraction of this mixture is injected into the column while the rest is purged through a waste port. This strategy avoids the capillary column overload, facilitating quick sample injection. The biggest restriction of this technique is the high sample concentrations required. Splitless inlet solves this problem using almost the same instrumentation as the split inlet. The purge valve remains closed during the injection time (20–60 s), so all the vaporized sample is pushed to enter the column. Possible overloads and high peak widths do not take place because of a set of effects: solvent focusing, temperature focusing, and stationary phase focusing. The most important one is the solvent effect (solvent focusing); low-­polarity solvents saturate the head of the column until the purge is opened. Then the increase in temperature and the evaporation of the solvent concentrate the analytes into narrow bands. This configuration is typically used for samples with very low concentration. Figure 3 shows the scheme for the split and splitless injection systems. The so-called cold injectors are the on-column and PTV inlets which allow the injection of high volumes of sample. In the on-­ column configuration the sample is not vaporized as it enters the column as a liquid. Vaporization is achieved with a temperature programming of the column. This port is simple and practical, with excellent quantitative results. In the PTV port, the injection occurs without vaporization too, although not directly into the column but into a chamber similar to the split/splitless inlet. After the injection, the chamber is heated to about 300 °C during 30 s, and the sample reaches the column.

2.4  Columns

GC columns contain the stationary phase. Capillary columns offer a much better peak resolution than the older packed columns and

Introduction to GCMS

31

Fig. 3 Split and splitless injection ports

hence packed technology has been almost completely superseded by capillary chromatography. Capillary columns, also known as open tubular columns (OTC), have a small-diameter tubing ranging from about 0.1 to 1 mm and can have a length up to 60 m. The flow rate of the mobile phase is most often in the range between 0.5 and 15 ml/min, and the column head pressure is typically set at 4 bar, or higher (up to 6 bar) for longer columns. They are composed of a tube (generally fused silica tube) coated with a protecting material such as polyamide. The stationary phase is applied to the inside wall in the wall-coated open tubular columns (WCOAT). This stationary phase, also known as liquid phase, liquid substrate, or solvent, is a thin layer coating the inside part of the column without chemical bonding, with a thickness of 0.1–1 μm [2]. The principal characteristics of this liquid are low volatility at working conditions, thermal stability, inertness, and capacity to permit the partitioning of the sample. The immobilization of the liquid phase is accomplished by cross-linking the stationary phase or by chemical bonding. Gas chromatography users have access to different stationary phases, to achieve a better separation of the components of their specific sample mixture. Nevertheless, the most used stationary phases in capillary chromatography are the polysiloxanes. The polysiloxane structure can be modulated, including different functional groups, to modify the polarity of the solvent. The hormone analysis based on GC uses a limited number of liquid phases, among which polysiloxanes are the most important ones (dimethylsiloxane, etc.). Chromatographic separation is determined by the affinity of the analyte for the solvent with extreme influence of the temperature and of its physicochemical nature. The most relevant

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criteria for the selection of the column should be based on the stationary phase characteristics. The factors to consider are polarity, selectivity, formation of hydrogen bonds, dipole–dipole energy, and dispersion forces. The choice of the right liquid phase is not an easy task, and the best way to get good results is to look into the bibliographic resources. Other important factors like the column length and diameter mainly affect the retention times of the analyte or the separation efficiency. The retention time cannot be reported as an absolute value because it is affected by different experimental conditions and would not be comparable with other experiments. To overcome this problem, the retention time of the hormone is usually calculated as a methylene unit (MU) or as a Kovats retention index (RI or I). In the methylene unit, the retention time of an n-alkane corresponds to the number of carbons (or methylene units) that it contains. For instance, n-decane equals 10 MU. In a similar way, the Kovats retention indices assign, in the alkane structure, a value of 100 times the carbon number, so one RI is equal to 100 MU. In the same example, n-decane equals to 1,000 RI. The MU or the RI values for n-alkanes are independent of the chromatographic conditions. The calculation of these values for any other compound requires a calibration plot of a spiked mixture of alkanes added to the analytes. The plot represents the adjusted retention times in logarithmic scale versus the MU or the RI. The MU/RI values for the hormones are obtained by extrapolation and in this case they are not independent of the experimental chromatographic conditions (stationary phase, column temperature, etc.), which always have to be specified [2].

3  Interface Gas chromatography ideally achieves a correct separation of the components of a sample which have to be immediately identified. There are several available detectors that can be coupled with GC, e.g., flame ionization detector or mass spectrometers. Some detectors have applications just for certain type of molecules, like the thermionic specific detector (nitrogen phosphorus detector), which is used for the selective detection of molecules with nitrogen or phosphorus in their structure as for example pesticides. A mass spectrometer (MS) is an instrument that detects ions formed from the molecules of interest, separating and identifying each one of them according to its mass-to-charge ratio (m/z). Nowadays, MS detectors are widely used for analytical purposes. Hormone analysis is not an exception and MS has become a routine technique in this field [3, 4]. Mass spectrometers require, in most of the cases, a high vacuum to avoid undesirable collisions of the ions with air molecules.

Introduction to GCMS

33

Fig. 4 GC-MS unit

To facilitate this process, large volumes of the carrier gas should not enter the MS, this being a problem in older packed columns. This issue was overcome with the introduction of capillary columns which use reduced gas flows. The existing high-capacity vacuum systems can work with a flow of about 1–2 ml/min, which permits direct coupling of the GC to the MS. However, the interface between both instruments needs to meet certain conditions: (a) the temperature must be maintained across the interface, up to the ion source to prevent condensations or peak broadening, (b) the interface volume should be small so that retention times do not suffer alterations and (c) the interface’s material must be inert and resistant. GC-MS belongs to the “hyphenated” or “hybrid” analytical techniques, as it combines a chromatographic separation with a spectrometric detection method. A representation of the GC-MS unit is shown in Fig. 4.

4  Mass Spectrometry 4.1  Ionization

The molecules eluted from the GC column are generally neutral compounds carried by the mobile gas phase. The MS unit detects those analytes as ions and therefore they have to be ionized. This process takes place in the ion source. When the process uses

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Alberto Sánchez-Guijo et al.

Fig. 5 EI ionization source

electrons to impact the molecules, it is known as electron ionization (EI) but if the fragmentation is achieved through collisions with charged ions, we refer to chemical ionization (CI). High-energy electrons in EI can be obtained from a heated filament (cathode) and they interact with the analytes to produce molecular ions by displacement of an electron of the substance, or negative ions (see Fig. 5). The system uses high temperatures in order to prevent sample condensation complications. In this process, some molecules experience reproducible fragmentation processes. In fact, EI causes rather extensive molecular fragmentation, thus providing detailed structural information. Most of the available mass spectra libraries are based on this technique. Chemical ionization needs a gas reagent, usually methane, isobutene, or ammonia, which is ionized by electron impact. The collision of these ions with the analyte generally produces protonated analyte ions. This is the positive chemical ionization (PCI), the most common type of CI. Chemical ionization is the preferred ionization method for quantification purposes as its ionization characteristics compared to EI are better. It produces fewer fragmentations and therefore a better signal. On the other hand, EI is, as aforementioned, still widely used for qualitative studies and library searching. 4.2  Mass Analyzers

The mass analyzer filters the ions, generated in the ionization process, according to their mass-to-charge ratio, in most of the cases applying a magnetic or an electric field.

4.2.1  Magnetic Sector Analyzer

It was the first analyzer to be introduced and it provides high mass determination resolution. It is an expensive analyzer and for this reason it is mostly used in very specific experiments that require high accuracy. It normally includes an electric focusing component, so the mass analysis is based on the response of the ions to an electromagnetic field (double-focusing magnetic sector analyzer).

Introduction to GCMS

35

Fig. 6 Principle of quadrupole analyzer

The m/z ratio in this case depends on the magnetic field strength applied (H), the radius of the trajectory of the ion during its path in the analyzer (r), and the inverse of the accelerating voltage (V):

m / z  H 2 ·r 2 / 2 V . When the magnetic field strength is changed a specific range of masses is brought into focus so that the analyte of interest can be selected.

4.2.2  Quadrupole Analyzer

This is the most common type of analyzer. It consists of four cylindrical rods arranged at the corners of a square (see Fig. 6). The quadrupoles are electrodes that make it possible to maintain an electrostatic field (direct current, DC) and a radio-frequency field (RF). By adjusting the DC and RF values an ion coming from the ion source can pass through the rods with a stable trajectory and reach the detector (the green ion in Fig. 6). An increase in these values permits the detection of ions with higher m/z. Those ions describing unstable trajectories cannot reach the detector as they are expelled from the rods (red ion in Fig. 6). A quadrupole analyzer can scan a mass range from 2 up to 4,000 Da. It is fast, easy to operate, and relatively inexpensive compared to other analyzers.

4.2.3  Ion Trap Analyzer

The ion trap analyzer is capable of confining the ions using the same principle as the quadrupole analyzer. Accordingly, they are considered as a quadrupole three-dimensional version. In a similar way, the adjustment of the RF results in stable concentric orbitals of those ions with a certain value of m/z. They require elevated pressures compared to the other analyzers. To maintain this pressure a gas is used, generally helium. The purpose of this buffer gas is to increase the stability of the orbits that the ions describe. The increase of the RF ejects the ions with lower mass-to-charge ratio to the detector as they become more unstable. Ion trap analyzers are a comparably cheap option with excellent sensitivity and they can accomplish tandem mass analysis as it is described in Subheading 4.3.2.

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4.2.4  Time-of-Flight Analyzer

Time-of-flight (TOF) analysis is based on the measurement of the time that the accelerated ion requires to reach the detector from the ion source. Every ion receives the same kinetic energy, but their different m/z results in different velocities and therefore different times. Those ions with higher m/z arrive later than those with lower ratios. Probably the most remarkable feature of this analyzer is its excellent mass accuracy. They are a real alternative to the magnetic sector analyzers and their popularity in GC-MS is increasing.

4.3  Ion Detection and Data Handling

The detector transforms the small currents of the ions into signals that are amplified and can be more easily interpreted as a mass spectrum. The detector in GC-MS is almost always the electron multiplier. When the ions reach the detector, they collide with different series of dynodes that amplify their signals. These signals can be stored in a computer and studied subsequently. A photomultiplier works under the same principles but the currents from the ions are converted into photons.

4.3.1  Detector

4.3.2  Scanning Techniques

The final representations that summarize the information about the experiment are the mass spectrum and the mass chromatogram. The mass spectrum is a plot of the mass-to-charge ratio (x-axis) versus the intensity of the signal. The mass chromatogram is more informative as it includes the time and the intensity of the signal and each of its points corresponds to the mass spectrum at a particular time. There are different preferred MS configurations to solve specific analytical problems. The most general scanning method is the full mass range scanning which provides a chromatogram that encompasses all the currents from all the ions in a defined range (usually from 50 to 500 m/z). This chromatogram is known as the “total ion current” (TIC) chromatogram. Each point in the chromatogram includes the MS information recorded at that specific time. This scanning method gathers all the information from all the ions that reach the detector and are included in the chosen range, even from those that may interfere with the analytes in the experiment. Nevertheless, it is still useful as a first approach to get qualitative information from the available spectral libraries. An example of a TIC is shown in Fig. 7. Commercial libraries include thousands of mass spectra and they can be used to study the GC-MS analysis results. The comparison between the experimental unknown mass spectrum and the database spectra is effected by a program that uses a matching algorithm. These programs are capable of searching thousands of mass spectra in a few seconds to give the most probable match. Extracted ion chromatograms (EIC) permit the selection of an m/z value from the TIC and they represent the chromatographic peaks corresponding to that signal.

Fig. 7 TIC (upper panel) and example of mass spectrum (lower panel, testosterone) of the methyloxime-­trimethylsilylether derivatives of a standard solution of urinary steroids

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Alberto Sánchez-Guijo et al.

The selected ion monitoring (SIM) mode increases the sensitivity of the detection, as it focuses on recording one or two specific m/z typical of a certain analyte, thus producing a SIM chromatogram. When several different m/z are selected, the method is termed “multiple-ion monitoring mode” (MIM). The chromatograms obtained from these approaches are more useful for quantification studies, as the sensitivity of the detection is increased. Gas chromatography-tandem mass spectrometry (GC-MS/ MS) consists basically of an analysis performed with two or more different mass-to-charge ratio measurements from the same ion. In the most used configuration, the ion of interest (precursor or parent ion) is detected in a first quadrupole and then it is fragmented in a second quadrupole, which acts as a collision cell containing a collision inert gas. The fragment (daughter or fragment ion) is later detected in a third quadrupole analyzer. The advantage of this configuration is the increased selectivity. Other configurations are possible, too. Ion traps allow additional fragmentations of the fragments (MSn) with just one analyzer. After the parent ion is selected and the other ions expelled from the ion trap, fragmentation takes place through collision-induced dissociation, a process that includes excitation of the parent ion and collision with the collision gas. Additional fragmented products can be analyzed from their previous product ions which provide a very selective tool to distinguish chemically related molecules. GC-MS/MS chromatograms are selected reaction monitoring (SRM) chromatograms, in a similar way to the SIM but with the additional fragmentation to achieve a selective detection. SRM can also be named as multiple reaction monitoring (MRM). 4.3.3  Quantitative Mass Spectrometry

Internal standards (IS) are another important tool in hormone quantitative analysis. An internal standard is a substance added to the samples in a known and constant concentration. The IS prevents instrument variations and compensates for possible errors and losses during the analytical process, minimizing matrix effects and increasing the precision and accuracy of the analysis. Instead of using the intensity of the signal provided by the analyte (peak area, peak height), when adding internal standard, the concentration is plotted against the response ratio of the compound to the internal standard, usually the area ratios. The ideal internal standard should be as similar as possible, in terms of physicochemical properties, to the analyte so that they can both experience the same process. It should be stable and inert, and the final calibration plots should be linear. An internal standard can be an analogue of the hormone or a natural or a synthetic isomer, but the best choices are stable isotope-labeled compounds. Labeled molecules have the same chemical properties as their unlabeled analogs, but their physical properties differ. Thus, losses during sample preparation can be compensated. In their structure some

Introduction to GCMS

39

Fig. 8 Structure of [1α,2α -2H2] cortisol

atoms are substituted by some of their stable isotopes [5]. In GC-MS, two or three atoms of deuterium or carbon-13 are ideal for labeling the molecule [1]. With this replacement, the isotope-­ labeled internal standard maintains the original characteristics of its unlabeled analog, but its molecular weight is increased. For example, if in the structure three atoms of hydrogen are exchanged with deuterium atoms, the new mass-to-charge ratio will be approximately (m + 3)/z. As long as a mass spectrometer can analyze simultaneously different masses, the co-elution of the compound and its isotope-labeled internal standard in the chromatographic run is not a problem for distinguishing both species. As a prerequisite, the deuterium-labeled standards should be of high isotopic purity to avoid overlapping with the corresponding analyte. Furthermore, they should also be stable, that is, no loss of deuterium should be observed during sample preparation. As an example, the structure of twofold deuterium-labeled cortisol is depicted in Fig. 8. The method that includes stable isotope-labeled hormones as internal standards is known as isotope dilution mass spectrometry (IDMS), and it is the best choice whenever the stable isotope-­ labeled hormones are available [6, 7]. Several methods based on IDMS are considered reference methods in hormone quantification and they are used as “gold standards” in medical diagnosis.

5  Example of ID-GC-MS Analysis of Hormones: Profiling Plasma Steroids In our laboratory, we are currently able to simultaneously determine up to 11 diagnostically important steroid hormones. The method has undergone constant expansion since its earliest days. 5.1  Sample Workup

1. Into 0.2–1.0 ml of plasma a cocktail is added of internal standards, containing [16,16,17-2H3] testosterone (d3-T), [7,7-2H2] 4-androstenedione (d2-4A), [16,16,17-2H3] 5α-androstanediol3α,17β (d3-AD), [7,7-2H2] dehydroepiandrosterone 2 (d2-DHEA), [16,16,17- H3] dihydrotestosterone (d3-DHT), [11,11,12,12-2H4] 17-hydroxyprogesterone (d4-17OHP),

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Alberto Sánchez-Guijo et al.

[2,2,4,4,21,21,21-2H7] 17-­ hydroxypregnenolone (d7-17PE), [1α,2α-2H2] 11-­ deoxycortisol (d2-S), [2,2,4,6,6,17α,21,21,212 H9] progesterone (d9-Prog), [2,2,4,6,6,17α,21,21-2H8] corticosterone (d8-B), and [1α,2α-2H2] cortisol (d2-F) in amounts similar to the expected values. 2. The samples are allowed to equilibrate. 3. After extraction, the combined organic extracts are dried and thereafter purified by gel chromatography on Sephadex LH-20 mini columns (50 mm × 5 mm ID). Cyclohexane/ethanol (90:10 v/v) is used to elute all steroids except cortisol. Chloroform/cyclohexane/methanol (100:80:15 v/v/v) is used for elution of cortisol. 4. For derivatization, HFBA is used. 5. Gas chromatography is performed on an Optima® 1-MS capillary column (25 m × 0.2 mm I.D., df 0.1 μm, Macherey-Nagel, Düren, Germany) housed in an Agilent 6890N GC with an Agilent 7683B Series injector (split/splitless automatic liquid sampler) coupled to Agilent 5975 inert XL Mass Selective Detector. Helium is used as carrier gas at 1.0 ml/min. The injector temperature is 270 °C and the initial column temperature is set at 80 °C. The steroids of interest elute at a rate of 3 °C/min until the column temperature reaches 250 °C. 6. Quantification is performed in the SIM mode using the peak area ratio between analyte and internal standard. The following m/z ratios are measured for the analytes and their corresponding internal standards: m/z 680.4/683.4 for T/d3-T, m/z 482.3/484.3 for 4A/d2-4A, m/z 470.3/473.3 for AD/ d3-AD, m/z 270.2/272.2 for DHEA/d2-DHEA, m/z 414.3/417.3 for DHT/d3-DHT, m/z 465.4/469.4 for 17OHP/d4-17OHP, m/z 467.4/471.4 for 17PE/d4-17PE, m/z 465.2/467.2 for S/d2-S, 720.4/726.4 for B/d8-B, m/z 510.3/518.3 for P ­ rog/d9-Prog, and m/z 489.3/491.3 for F/d2-F. 5.2  Method Validation

In order to assess linearity of our method, 6-point calibration plots with amounts of analytes between 0.1 and 10 ng/ml for T, AD, 17PE, and S, 5-point calibration plots with amounts of analytes between 0.1 and 5 ng/ml for 4A and DHT, 5-point calibration plots with amounts of analytes between 0.5 and 25 ng/ml for DHEA, 5-point calibration plots with amounts of analytes between 2 and 200 ng/ml for Prog and B, and 6-point calibration plots with amounts of analytes between 20 and 800 ng/ml for F are prepared. To each sample, a cocktail containing the internal standards in fixed amounts (for d3-T, d2-4A, d3-AD, d3-DHT, d4-17OHP, d4-17PE: 1 ng/ml; for d2-S: 1.2 ng/ml; for d2-DHEA: 5 ng/ml; for d9-Prog, d8-B: 20 ng/ml; and for d2-F:

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200 ng/ml) is added. All calibration plots show good linearity with coefficients of determination above 0.99. The accuracy of steroid measurement is determined by adding various amounts of our analytes to aliquots of pooled human plasma (see Table 1). A good agreement between the values found and the amount added is observed. The relative errors are between 0.06 % (Prog, spike level 1) and 14.17 % (DHT, spike level 1). The assay shows good reproducibility for intra-assay (withinday) as well as inter-assay (between-day) precision (see Table 2).

Table 1 Accuracy of the GC-MS method for profiling plasma steroids T

4A

AD

DHEA

DHT

0.79

n.d.

Mean

0.52

0.57

CV

8.16

5.91

Spiked

1.00

1.00

1.00

5.00

Exp. m

1.52

1.57

1.00

Mean

1.51

1.59

CV

2.23

Rel. err

S

B

Prog

F

0.40

0.64

0.99

3.77

8.45

6.82

6.28

5.69

3.97

1.83

1.00

1.00

1.00

1.00

20.00

10.00

100.0

5.79

1.00

1.40

1.64

1.99

23.77

18.45

123.0

1.05

6.02

1.14

1.42

1.59

2.27

25.34

18.46

128.6

3.67

2.16

0.47

6.41

4.58

3.58

5.03

12.02

2.97

3.91

−0.66

1.28

4.83

3.91

14.17

1.19

−2.96 13.97

6.61

0.06

4.54

Spiked

4.00

4.00

4.00

20.00

4.00

4.00

4.00

4.00

80.00

40.00

300.0

Exp. m

4.52

4.57

4.00

20.79

4.00

4.40

4.64

4.99

83.77

48.45

323.0

Mean

4.36

4.41

4.06

20.05

4.18

4.14

4.46

5.04

85.32

45.92

311.5

CV

1.81

4.14

1.89

1.02

2.10

4.57

2.37

2.59

13.42

5.93

3.35

−3.65

−3.39

1.58

−3.57

4.54

−6.02

−3.81

0.97

1.85

−5.22

−3.56

Rel. err

n.d.

17OHP 17PE

11.77

23.0 4.51

n.d.: not detected, spiked: spiked concentration in ng/ml, exp. m: expected mean in ng/ml, mean: measured mean in ng/ml, CV in %, rel. err: relative error in %, n = 6

Table 2 Intra- and inter-assay precision of the GC-MS method for profiling plasma steroid T

4A

AD

DHEA

DHT

17OHP

17PE S

B

Prog

F

Intra-assay precision  Mean 0.56 0.28  CV 2.60 6.82

0.45 1.67

9.56 1.30

0.55 4.86

0.47 2.26

0.87 1.73

0.63 5.47

8.33 1.50

12.17 0.82

125.7 1.72

Inter-assay precision  Mean 0.57 0.29  CV 2.20 9.05

0.45 2.15

9.56 1.86

0.56 5.49

0.48 3.18

0.88 1.71

0.62 5.27

8.43 3.13

12.10 0.52

122.2 4.36

Mean: mean in ng/ml, CV in %, n = 6

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Alberto Sánchez-Guijo et al.

Table 3 Sensitivities of HFB derivatives of plasma steroids T

4A

AD

DHEA

DHT

17OHP

17PE

S

B

Prog

F

Amount

1 pg

1 pg

1 pg

5 pg

1 pg

1 pg

1 pg

1 pg

10 pg

10 pg

400 pg

S/N

10

7

10

20

4

16

16

5

42

32

22

Amount: absolute amount of analyte on column, S/N: signal-to-noise ratio

Coefficients of variation (CV) for intra-assay precision were between 0.82 % (Prog) and 6.82 % (4A) and for inter-assay precision between 0.52 % (Prog) and 9.05 % (4A). Table 3 provides the sensitivities of the analytes in this assay. Figure  9 shows the steroid profile of a plasma sample. Corresponding ion traces of analyte and deuterated internal standard are superimposed. 5.3  Significance and Perspective

The judicious choice of an appropriate method of derivatization is of primary concern not only for GC analysis but also for sensitivity and specificity of mass fragmentation. HFB esters are stable and yield high mass increments, reducing effectively the interfering background noise. Hence, the use of HFB-derivatives allowed for excellent sensitivity. In contrast to TMS derivatives, there is less contribution from endogenous isotopes (e.g., from silicone). In Fig. 9, the peaks are essentially clear, thus indicating excellent specificity. In the spike experiment, good agreement between the values found and the amounts of analyte added revealed excellent accuracy. Likewise, the method shows good reproducibility. Steroid analysis by GC-MS can be used as a complementary analytical technique with the highest specificity whenever problems from matrix effects or cross-reactivity are likely to arise or suspicious results need to be rechecked. Since expensive analytical instrumentation is needed and extensive specialist knowledge in steroid biochemistry and steroid analytics are required, GC-MS steroid analysis are at best carried out in a small number of highly specialized supraregional laboratories (referral centers, reference centers).

Fig. 9 Chromatogram of the steroid profile of a plasma sample. m/z 680.4/683.4 for T/d3-T (retention time tR 11.34 min/11.32 min; concentration c 0.64 ng/ml), m/z 482.3/484.3 for 4A/d2-4A (tR 11.42 min/11.40 min; c 0.38 ng/ml), m/z 470.3/473.3 for AD/d3-AD (tR 10.37 min/10.34 min; c 0.14 ng/ml), m/z 270.2/272.2 for DHEA/ d2-DHEA (tR 11.47 min/11.45 min; c 1.05 ng/ml), m/z 414.3/417.3 for DHT/d3-DHT (not detected/12.09 min), m/z 465.4/469.4 for 17OHP/d4-17OHP (tR 12.60 min/12.64 min; c 0.44 ng/ml), m/z 467.4/471.4 for 17PE/ d4-17PE (tR 12.62 min/12.57 min; c 0.62 ng/ml), m/z 465.2/467.2 for S/d2-S (tR 14.60 min/14.58 min; c 0.88 ng/ml), m/z 489.3/491.3 for F/d2-F (tR 14.70 min/14.69 min; c 247.4 ng/ml), m/z 510.3/518.3 for Prog/ d9-Prog (tR 13.77 min/13.68 min; c 8.60 ng/ml), and 720.4/726.4 for B/d8-B (tR 16.05 min/15.99 min; c 3.92 ng/ml)

Introduction to GCMS

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Acknowledgments The excellent technical assistance of Mrs. Birgit Wardega is gratefully appreciated. Stefan A. Wudy thankfully acknowledges funding from the “Deutsche Forschungsgemeinschaft (DFG)” during the last 20 years, having enabled constant progression in analytical method development. References 1. Makin HLJ, Gower DB (eds) (2010) Steroid analysis, 2nd edn. Springer, London 2. Grob RL, Barry EF (eds) (2004) Modern practice of gas chromatography, 4th edn. Wiley, Hoboken, NJ 3. Shackleton CHL (1985) Mass spectrometry: application to steroid and peptide research. Endocr Rev 6:441–486 4. Wudy SA, Hartmann MF (2004) Gas ­chromatography-mass spectrometry profiling of steroids in times of molecular biology. Horm Metab Res 36:415–422 5. Wudy SA (1990) Synthetic procedures for the preparation of deuterium-labeled analogs

of naturally occurring steroids. Steroids 55:463–471 6. Wudy SA, Hartmann M, Homoki J (2002) Determination of 11-deoxycortisol (Reichstein’s compound S) in human plasma by clinical isotope dilution mass spectrometry using benchtop gas chromatography-mass selective detection. Steroids 67:851–857 7. Wudy SA, Wachter UA, Homoki J, Teller WM, Shackleton CH (1992) Androgen metabolism assessment by routine gas chromatography/ mass spectrometry profiling of plasma steroids. Part 1, unconjugated steroids. Steroids 57: 319–324

Chapter 4 Tandem Mass Spectrometry in Hormone Measurement Helen P. Field Abstract Mass spectrometry methods have the potential to measure different hormones during the same analysis and have improved specificity and a wide analytical range compared with many immunoassay methods. Increasingly in clinical laboratories liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays are replacing immunoassays for the routine measurement of testosterone, 17-hydroxyprogesterone, and other steroid hormones. Reference LC-MS/MS methods for steroid, thyroid, and peptide hormones are being used for assessment of the performance and calibration of commercial immunoassays. In this chapter, the general principles of tandem mass spectrometry and examples of hormone assays are described. Key words LC-MS/MS, Adrenal steroids, Gonadal steroids, Thyroid hormones, Insulin

1

Introduction Currently many of the measurements of steroid and peptide hormones for routine clinical practice are performed using automated immunoassays. For many decades alternative methods including extraction (indirect) isotopic immunoassays or gas chromatographymass spectrometry (GC-MS) assays for steroid hormones have also been used in specialized referral laboratories. By utilizing two stages of mass analysis, tandem mass spectrometry is a particularly selective technique for the measurement of small molecules and metabolites in biological fluids and tissues. Liquid chromatographytandem mass spectrometry (LC-MS/MS) was first used successfully in neonatal screening programs [1, 2] and over the last 10 years LC-MS/MS technology has been implemented in both general and specialized hospital laboratories for measurement of immunosuppressant drugs, steroid hormones, and drugs of abuse in clinical samples [3]. Most of the hormone assays using tandem mass spectrometry are developed in-house by hospital laboratories or by collaboration with the instrument manufacturers. Guidelines and reviews are available that describe the requirements for the development and

Michael J. Wheeler (ed.), Hormone Assays in Biological Fluids: Second Edition, Methods in Molecular Biology, vol. 1065, DOI 10.1007/978-1-62703-616-0_4, © Springer Science+Business Media New York 2013

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Helen P. Field

validation of tandem mass spectrometry assays [4–9]. Demonstration of assay specificity, sensitivity, accuracy, precision, linearity, carryover, and stability is needed. Preferably the methods should be validated with a reference measurement procedure or a reference material though this is rarely described. Information about the types of sample and collection tubes tested and grades of reagents may be limited in published methods. At the moment inhouse methods are not subject to the regulation required for commercial in vitro diagnostic methods in Europe but this situation may change.

2

The LC-MS/MS System The most widely used sample inlet for tandem mass spectrometry methods is a liquid chromatography system. GC-MS/MS analyzers are available but are rarely used for routine hormone assays [10–12]. Different models and types of LC-MS/MS analyzer are available. Factors to be considered include the range of analyses to be performed, required mass accuracy, mass resolution, or sensitivity of response. At present the triple-quadrupole LC-MS/MS is used for the majority of hormone assays; Fig. 1 shows a block diagram of its components. In summary: ●

Sample flows in the volatile mobile phase from the liquid chromatography system to the ionization source (see Subheading 2.1).

●

Gas-phase ions of the analyte are formed in the ionization source (see Subheading 2.2).

●

Ions travel to the first mass analyzer where they are separated in electric fields according to their mass-to-charge (m/z) ratio (see Subheading 2.3).

●

Ions passing from the first mass analyzer are fragmented in the collision cell.

Fig. 1 Components of a typical LC-MS/MS system

Tandem Mass Spectrometry

2.1 Liquid Chromatography

47

●

Fragment (product) ions pass from the collision cell to the second mass analyzer where they are separated on the basis of their m/z ratio.

●

Ions passing from the second mass analyzer are recorded and quantified by the detector.

●

The detector sends data to the computer software for processing of the m/z ratios and their relative abundance.

●

Computer software controls all the components and acquisition, processing, and storage of data (see Subheading 2.4).

Separation of hormones by liquid chromatography is achieved through partition between the mobile phase and the stationary phase in the LC column. The outlet from the LC column is coupled directly to the mass spectrometer and during the chromatography the separated components of the sample in mobile phase travel sequentially to the MS/MS for analysis. The increased specificity conferred by MS/MS analysis may enable the use of simpler chromatography methods. However selective sample preparation and/or prolonged chromatographic separation may be required to minimize matrix effects (see Subheading 2.7). Also the presence of isomeric or isobaric compounds may influence the choice of chromatography conditions— for example during detection of adrenal and gonadal steroids such as 17-hydroxyprogesterone (17OHP) and 11-desoxycorticosterone or testosterone and epitestosterone. Both conventional particle size (2.5–5 μm) and small-particle (sub-2 μm) analytical LC columns are suitable for hormone methods. Small-particle columns are used in ultrahigh-pressure liquid chromatography (UHPLC/UPLC) systems which require specialized LC pumps and fittings for the high back pressures generated (~15,000 psi, 1,000 bar). Resolution is improved and the narrow peaks have improved the signal-to-noise ratio which is advantageous for MS/MS methods. Faster chromatography is possible as higher flow rates may be used without loss of resolution. Core-shell particle columns may provide a lower pressure alternative to the sub-2 μm particle columns. Most methods use reverse-phase (RP) chromatography, usually with C18 columns, but shorter chain lengths or phenyl-hexyl phases are useful alternatives. Applications requiring high flow rates may use monolithic columns. An in-line frit is helpful to prevent blockages from particulate matter and a guard column is helpful to protect the analytical column from nonpolar inert compounds in the sample extract. Heating the column in a column oven improves the precision of retention time. Polar mobile phases for LC-MS/MS RP chromatography contain either methanol or acetonitrile mixed with water. MS-grade solvents are widely available but LC grade may be acceptable for some applications. Ammonium acetate is a suitable volatile buffer

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Helen P. Field

and formic acid may be added to enhance protonation. Unsuitable additives for mass spectrometry methods include phosphate salts and inorganic acids. Typically gradient elution is used where the strength of the mobile phase increases with time during the separation. Gradient elution optimizes separation of multiple analytes and improves peak symmetry. The gradients may be stepped-, linear-, or curve-controlled by the software used. After sample preparation, typically up to 50 μL of extract is injected into the sample loop of the auto sampler. Inserting a switching valve between the LC and the mass spectrometer and diverting the LC flow to waste during initial and wash stages of the chromatography minimize contamination of the ion source. 2.2

Ion Source

For detection by mass spectrometry gas-phase ions of the analyte must first be formed in the ionization source [13, 14]. Three modes of ionization may be used for hormone analysis—electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photoionization (APPI). All are “soft” ionization techniques and generally result in little fragmentation of the hormone molecule. However conjugates of hormones may be fragmented in the ion source—for example free hormone is ionized from sulfated steroid hormone [15]. Choice of ionization mode and optimization of the parameters used in the ion source for temperature and gas flows are important parts of method development [16]. APCI and APPI are useful for the ionization of less polar or nonpolar small molecules. Currently, steroid hormone methods are divided mainly between the ESI and APCI modes of ionization and in most analyzers the ion sources are easily interchangeable. For ESI the sample in volatile mobile phase is passed through a narrow metal capillary at flows up to ~1 mL/min (see Fig. 2). A high voltage (~4 kV) is applied to the capillary. The capillary is held within a larger bore tube allowing a coaxial stream of gas to be applied and as the sample flow exits from the capillary it is nebulized

Fig. 2 Electrospray ionization. Courtesy of AB Sciex Pte. Ltd., redrawn by G Kemp

Tandem Mass Spectrometry

49

Fig. 3 Atmospheric pressure chemical ionization. Courtesy of AB Sciex Pte. Ltd., redrawn by G Kemp

to produce a fine aerosol of charged droplets. With the flow of drying gas and heating, the droplets evaporate (desolvation). As the droplets decrease in size the sample ions in the droplet are forced together until repulsion causes the solvent-free ions to be ejected from the surface. A portion of the gas-phase ions are then extracted through a small orifice into the ion transfer region of the mass spectrometer. The remainder of the vapor is removed by the vacuum pumps. Ionization may be performed in positive or negative mode to add or remove a proton from the molecule and produce a positively or a negatively charged ion. ESI is suitable for both low and higher molecular weight compounds, such as peptides and proteins. Lower mass molecules such as steroid hormones will be singly charged; however higher mass molecules may be multiply charged. For APCI, the ionization process occurs in stages (see Fig. 3). Initially the sample flow is nebulized and desolvated and molecules from the sample flow are transferred into the gas phase. The corona discharge around the corona needle charges molecules of solvent which then act as intermediaries to transfer the charge to the molecules of analyte. The mass limit is ~1,000 Da for APCI; this process is also less suitable for thermally labile compounds. APPI may be direct or solvent (dopant) assisted and typically forms a protonated ion [17]. 2.3 Ion Separation and Detection

Ions are transferred from the atmospheric pressure region of the source into an intermediate vacuum region and are focussed using a radio frequency (RF) lens. The ions then travel to the first mass analyzer where they are separated on the basis of their m/z ratio [18, 19]. If the ions are singly charged the m/z ratio equals [M+H]+ for a positive ion and [M−H]− for a negative ion. The m/z ratio of multiply charged ions would equal [M+nH]n+/n. The mass analyzers and detector are maintained under high vacuum so that ions travel through the ion path to the detector without hindrance from molecules of air.

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Helen P. Field

Three types of mass analyzers may be used for clinical diagnostics—the time-of-flight (TOF), ion trap, and quadrupole (Q) mass analyzers. Each has different characteristics for mass accuracy, mass resolution, and m/z range. For tandem mass spectrometry two ion separation steps are performed in series; this may be in space using triple-quadrupole analyzers (QqQ) or in time using the ion trap analyzer. MS/MS analysis enhances specificity as it provides increased structural information about the analyte molecule. The triple-quadrupole MS/ MS offers low-to-medium mass resolution, moderate mass accuracy, moderate sensitivity for full-spectrum analysis, and high sensitivity for selected/multiple reaction monitoring mode. The quadrupole mass analyzer consists of four parallel rods connected as two pairs with opposite potentials. DC and RF voltages are applied such that only ions of one m/z ratio will oscillate in a stable trajectory through the rods to reach the collision cell or detector. Ions of other m/z ratios will either be removed before the quadrupole region or move into the rods but with an unstable trajectory so that the ions strike the rods and are removed. A mass spectrum of ions and their relative abundance is produced by systematically varying the voltages applied. The second “quadrupole” (Q2 or q) is modified to form part of the collision cell. In the collision cell the ions passing from the first mass analyzer are fragmented under controlled conditions by colliding with a neutral target gas—termed collision-induced dissociation (CID). The degree of fragmentation is dependent on the speed of the ions as they enter the collision cell, the number of collisions the ions undergo with the target gas, and the structure of the ions. The fragmented ions then travel into the second quadrupole mass analyzer where they are separated according to their m/z ratios, and finally the ions are counted by the detector. Table 1 summarizes the versatility of the triple-quadrupole instruments. Setting the first quadrupole (Q1) or the third quadrupole (Q3) in different modes will allow detection of families of compounds or a single compound as required. SRM or multiple reaction monitoring (MRM) mode is used for quantitative hormone methods. It is the most sensitive mode because the mass analyzers are set for restricted ions during the scan cycle time. The dwell time for each transition of precursor ion to product ion is usually set to achieve ~15 data points across a chromatographic peak. It is advisable to avoid the less specific MRM transitions, e.g., the water loss transition. In MRM methods, three characteristics of the analyte are used for identification—these are the retention time on the LC column, the mass-to-charge ratio of the precursor ion, and the mass-tocharge ratio of one (or more) of the fragment ions arising from the precursor ion (see Fig. 4). Time windows containing panels of MRM transitions may be set at discrete retention times across the chromatography so that different groups of compounds are detected.

Tandem Mass Spectrometry

51

Table 1 Summary of MS/MS modes for triple-quadrupole analyzer Type of MS/MS experiment

Q1 setting

Full scan

Scanning N

Scanning

Method development

Product ion scan

Fixed

Scanning

Structural information, method development

Neutral loss scan

Scanning Y

Offset Structurally related compounds that lose a scanning common neutral group, e.g., amino acids

Precursor ion scan

Scanning Y

Fixed

Method development or for structurally related compounds that give a common fragment ion, e.g., acyl carnitines

Fixed

Targeted quantitative analyses, e.g., steroid hormones. High specificity and sensitivity

Selected/multiple Fixed reaction monitoring (SRM/MRM)

Collision cell Q2, q Q3 setting Type of application

Y

Y

Fig. 4 Multiple reaction monitoring. Courtesy of AB Sciex Pte. Ltd., redrawn by G Kemp

2.4

Data Handling

Bidirectional interfacing of the mass spectrometer computer to the laboratory computer is desirable for laboratories analyzing large number of samples. This process is currently underdeveloped in comparison with the user-friendly systems available for core biochemistry analyzers. Optional software restricted to routine analytical processes may simplify operation for less experienced users [20]. Software which flags data out of specified limits for retention time, ion ratios, and other parameters is available [21].

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Helen P. Field

Maintenance

2.6 Preparation of Samples for Hormone Assays

It is important to regularly clean the ion source and ion transfer region. Use of high-quality solvents and consumables is essential to maintain the performance of the analyzer. Troubleshooting to detect the cause of changes in performance is time consuming and expertise needs to be developed. Clinical laboratories require timely technical support from the manufacturers and this is one area that is gradually being improved. However if rapid turnaround times are necessary then it is advisable to have access to a backup analyzer. Hormones are most commonly measured in plasma, serum, urine, or salivary samples. For these complex matrices, sample preparation is necessary to remove salts, proteins, and other interfering substances. Protein precipitation, solvent extraction, and solidphase extraction are suitable techniques for preparing biological fluids before measurement of steroid hormones. The procedures are adapted for the concentration range and matrix of the analyte(s) and the response of the MS/MS detector. Protein precipitation is a rapid and easy procedure for serum samples. Typically the serum sample is diluted with two parts of precipitation reagent, followed by vigorous mixing before highspeed centrifugation. A portion of the supernatant is then sampled in the LC. Precipitation reagents include methanol, acetonitrile, or salts such as zinc sulfate. The main drawback of this procedure is that salts and other endogenous components of the serum matrix, e.g., phospholipid, remain in solution and these may affect the ionization of the analyte to a variable degree from sample to sample (see Subheading 2.7). Protein precipitation may be used before another procedure for an analyte that is strongly protein bound. Commercial 96-well protein precipitation plates are suitable for manual or automated procedures. Organic solvent extraction or solid-phase extraction procedures enable removal of more of the sample matrix and concentration of the analyte in the final extract. Solvent extraction produces a “cleaner” extract than protein precipitation but is more labor intensive and time consuming. A high ratio of solvent to sample is required to avoid emulsions. The sample is usually vortex-mixed with the solvent for several minutes. Separation of the solvent and aqueous layers is achieved by centrifugation or by freezing the aqueous phase. After decanting, the organic solvent is removed using vacuum or nitrogen evaporators and then the dried residue containing the analyte is reconstituted in mobile phase. Methyl tertbutyl ether (MTBE) is widely used for steroid hormone methods and extracts less phospholipid from serum than other organic solvents [37]. Microtiter 96-well filter plates which remove phospholipid during solvent extractions are also available [21]. Solid-phase extraction procedures may be performed manually or as automated off-line or online techniques. Costs for the

Tandem Mass Spectrometry

53

cartridges or columns may be sizable compared with the reagent costs of the other procedures. Online solid-phase extraction (also termed two-dimensional chromatography) uses a pre-column to retain the analyte(s) from the sample flow and other components of the matrix pass through the pre-column to waste. After valve switching, analytes are back-flushed from the pre-column to the analytical LC column and then eluted to the mass spectrometer [22]. Systems developed by the laboratory usually require an initial sample preparation step, an additional LC pump, and switching valve, and use commercial pre-columns such as the Oasis or Chromolith columns. Commercial online systems include the TurboFlow TLX which uses a single turbulent flow chromatography column to prepare the samples [23] and the Symbiosis Prospekt-2 which uses individual solid-phase extraction cartridges for each sample [24]. Alternative sample preparation procedures may affect assay imprecision [25]. 2.7

Matrix Effects

Matrix effects occur when other substances are present in the sample flow which compete with the analyte for ionization and suppress the formation of analyte ions and therefore decrease the signal obtained (ion suppression). Ion enhancement causing an increase in signal may also be found. The accuracy, precision, and lower limit of quantitation of mass spectrometry assays may be compromised by matrix effects [26–31]. ESI is most prone to ion suppression effects. To minimize ion suppression it is important to separate the compound of interest from sample matrix which is not retained by the LC column or from other interfering endogenous or exogenous compounds. A common source of matrix effects in serum is phospholipids [37]. Impurities in LC solvents, compounds in plastics, and blood sampling tubes may also cause ion suppression or interferences [26, 33, 34]. The matrix effects may be assessed qualitatively or quantitatively [26, 29]. For qualitative assessment, a dilute solution of analyte is infused post column into the flow of either “analyte-free” sample extracts or solvent. Baseline fluctuations are then compared. Figure 5 shows the comparison of two chromatograms for a transition of 17OHP for extracts after protein precipitation of an analyte-free serum extract and a solvent blank. A typical area of ion suppression occurs at around 0.75–1 min and the ion signal from the sample extract falls away—this is typical of ion suppression due to the sample matrix which was not retained by the LC column. Ion suppression or ion enhancement may also occur at other times throughout the chromatographic analysis. In this method the retention time for 17OHP is 5.3 min; therefore it is separated from any areas of ion suppression or interference. A means of quantitatively assessing matrix effects was described by Matuszewski [29]. The response of an analyte in solvent is compared with the response of the analyte spiked into different

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Helen P. Field

Fig. 5 Ion suppression profile for 17-hydroxyprogesterone in serum prepared using protein precipitation

analyte-free sample matrices before and after the sample preparation process. This allows an indication of the absolute and the relative matrix effects across different samples. Recent reports review analytical strategies to reduce matrix effects [30, 31, 37]. 2.8

Derivatization

A chemical derivatization procedure modifies the hormone molecule. It is mainly used to improve the detection limit of the hormone assays or for compounds that do not ionize well or exhibit characteristic fragmentation [16, 38–41]. Excess derivatization reagent is usually removed before chromatography so that the processing time and complexity of the assay will be increased.

2.9

Quantitation

Analyses are usually run singly in batch mode. Calibration is required for each batch because of day-to-day variations in ionization, or in the solvents used for the mobile phase. Internal standardization with a stable isotope-labelled compound is most commonly used for quantitative analysis due to variable withinsample recovery. For the calibration line the ratio of analyte peak area (height) to internal standard peak area (height) is plotted against the concentration of the analyte. The isotope-labelled internal standard is closely related to the analyte and if it co-elutes with the analyte allows correction of variation in ion suppression or enhancement from sample to sample [42, 43]. For routine clinical methods the internal standard is used at a single concentration and is added at the start of the sample preparation procedure allowing compensation for losses at any stages in the procedure. Some assays

Tandem Mass Spectrometry

55

include a second labelled internal standard added at other stages of the procedure to monitor breakdown or conversion of the analytes [44, 45]. For reference methods the quantity of internal standard is adjusted to the concentration of analyte in each sample to obtain a 1:1 ratio. Methods mainly use a deuterated (2H) or 13C-labelled internal standard for each analyte if available. Two to eight atoms of the analyte molecule may be labelled. The endogenous 13C2 isotope of a hormone may adversely affect quantitation by increasing the signal of a doubly labelled internal standard [32]. Exchange of deuterium with hydrogen may occur under some conditions and the purity of the deuterated reagent should be checked over time. A deuterated internal standard may partially resolve from the analyte on a reversed-phase column due to a slight alteration in lipophilicity (isotope effect). Reports describing drug measurements have described circumstances in which deuterated labelled internal standards did not correctly adjust the analyte response. In the first report, the slight difference in retention time and a nearby matrix effect in a particular batch of plasma affected the signals of the internal standard and the analyte differently [46]. Modifications to resolve this included using a smaller volume of sample and dilution of the final extract. In the second report, residues of solvent remaining in the sample extract suppressed the respective signals to different extents [47]. Broad or split chromatographic peaks or an increase in background signal may indicate the presence of interferences. The stability of retention times, ion signals, and response ratios should be monitored. To further improve analytical specificity, ion ratios of the analyte or internal standard may be calculated [48]. The ratios of the responses of the main and secondary MRM transition found in the calibrators are used to define the acceptable limits for the unknown samples—if the ratio in a patient sample is out of the acceptable range it is likely that some interference is present. Ion ratios in ~2 % of testosterone samples from female patients were outside the limits set (±29 %) [28]. The limits to use for LC-MS/MS methods are still under discussion: for example the ion ratios used for detecting hormone residues in animal tissues range from ±20 to 50 % depending on their relative intensity to the base peak or ±15 % for drug analysis [49–51]. Another strategy to improve specificity is to use different collision energies for an MRM transition to identify interference from co-eluting isomers; for example the positional isomers prednisolone and cortisone may be distinguished using this approach [28]. 2.10 Automation and Further Developments

At present the workflow of most mass spectrometry procedures is largely manual and time consuming compared with the workflow of automated immunoassay analyses. Consequently there is interest in

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increasing the use of robotics, integration of sample preparation systems to the mass spectrometer, and development of a fully automated system [22, 52, 53]. Another challenge is to increase the throughput of mass spectrometry assays and this may be achieved in several ways. The first is by multiplexing several LCs which are synchronized to a single MS/MS detector. Multiple analyses are run simultaneously and the MS/MS detector collects data from each LC within staggered time windows [23]. Alternatively higher throughput may be achieved by combining analysis of five samples at a time through one LC-MS/MS. Samples are prepared using different mass tags, combined and analyzed together, and the mass differences allow calculation of the concentration of analyte and internal standard in each respective sample [54]. LC-chip MS microfluidics is a recent development for analysis of small molecules and peptides [55]. Continuing developments of the technology include enhancing the efficiency of ionization, increasing the efficiency of ion transfer, and increasing the sensitivity of the detector. Hybrid tandem mass spectrometers, such as the Q-TOF or Q-linear ion trap (capable of MS/MS/MS analysis), may enhance selectivity and their use for clinical assays is likely to increase. The combination of ion mobility measurements and tandem mass spectrometry may have advantages in reducing background noise or separating isomers and isobars [56].

3

Examples of the LC-MS/MS Analysis of Hormones

3.1 Steroid Hormones

Isotope dilution GC-MS is regarded as the reference method for measurement of steroid hormones and has been used extensively since the 1970s [57]. It remains a useful research and discovery tool as every steroid present will be detected [58]. In a series of reports Tai and colleagues have described LC-MS/MS candidate reference methods for cortisol, estradiol, progesterone, and testosterone [59–62]. LC-MS/MS assays developed for routine clinical practice may be targeted for a single steroid or a panel of steroids [63–67] and utilize the range of procedures for sample preparation or chromatography described above. Methods for certain steroid hormones are described in later chapters. It is anticipated that LC-MS/MS assays will be used both as the reference technique for the measurement of steroid hormones and as the “gold standard” assay for routine clinical practice—for example for measuring serum testosterone in women, children, and hypogonadal men. However, comparison of routine clinical LC-MS/MS methods with reference assays has revealed variable accuracy and imprecision leading to awareness that although the performance of LC-MS/MS assays may be superior to particular immunoassays further improvements are required [68–71].

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In part the variability may be due to the use of different calibration materials as the standardization of steroid hormone assays is challenging [25, 72]. If the calibration of mass spectrometry methods is accurate and traceable to certified reference material, then the reference data derived from suitable populations of patients using mass spectrometry assays should be transferable [16, 73–76]. This would allow harmonization of the interpretive data provided by clinical laboratories which would be advantageous to both patients and their clinicians. Immunoassay (IA), GC-MS, and LC-MS/MS techniques each have benefits and drawbacks and are likely to coexist as complementary techniques for hormone assays for some time yet. A brief summary of the advantages and challenges of LC-MS/MS methods in comparison with immunoassay or GC-MS methods is given in Tables 2 and 3. 3.1.1 Testosterone

Automated immunoassay platforms provide convenient and rapid direct serum testosterone assays. However, clinical laboratories providing a specialist endocrinology service currently use mass spectrometry methods for measurement of serum testosterone. The impetus for adoption of the technology was dissatisfaction with the quality of the direct immunoassays at low concentrations of testosterone. The poor accuracy and reproducibility of immunoassays compared with GC-MS or LC-MS/MS methods were highlighted in two reports [68, 69]. Publication of the reports prompted critical editorials, followed by guidelines prepared by expert panels [77–81] and a project to standardize testosterone measurements [72]. As part of the standardization initiative, reference methods for serum testosterone have been developed and reference materials with assigned values will be made available to other laboratories. A number of routine methods suitable for measuring total testosterone in clinical samples have been reported [16, 23, 83–90]. Most of the methods measure the protonated ion of testosterone using ESI. One method used 50 μL serum and a protein precipitation procedure to measure testosterone between 0.3 and 100 nmol/L [82]. Solvent extraction procedures utilizing 200– 500 μL of serum achieved detection ranging from 0.06 to 0.25 nmol/L [83, 84, 86, 87]. Singh described a method with a high throughput of 30 samples/hour [23]. Samples were prepared by protein precipitation and then analyzed using two-dimensional chromatography with four LCs multiplexed to the MS/MS. Another multiplexing procedure was used to measure total testosterone and then free testosterone concentrations were calculated using tracer equilibrium dialysis [89]. A sensitive method which achieved detection to 0.035 nmol/L involved preparing oxime derivatives of testosterone, and used a rapid 3-min chromatographic separation on a C18 column [16].

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Table 2 Advantages and challenges of LC-MS/MS vs. immunoassay (IA) methodology Advantages of LC-MS/MS over IA

Comments

High selectivity

Immunoassay methods may cause misleading results due to matrix effects, cross-reactions to related compounds, hook effects, and autoantibody and heterophilic antibody interferences

Sensitivity equivalent or better

But remains challenge for low-concentration hormones (low pmol/L range)

Wide analytical range

Particularly compared with automated IAs, less need for dilution and reanalysis of sample

Improved imprecision

Performance of some direct immunoassays is poor at low concentrations of hormones

Possible to measure multiple hormones in the same analysis

Advantageous for pediatric samples

Versatile analyzer

May measure a range of small molecules such as urine drugs of abuse, urine metanephrines, whole-blood immunosuppressants and serum steroids on the same analyzer, potential for rationalization of tests to single analytical platform

Low reagent costs for in-house methods

Compared with low-throughput specialist commercial immunoassays

Assay agreement and reference range comparability

IA ranges assay-specific, new ranges using suitable populations required for MS methods

Matrix independent

Simpler modifications to methods for serum, saliva, urine, CSF matrices

Internal standardization

Compensation for procedural losses, not possible for most extraction IA

Stability of calibration as using pure compound

Reformulation of IAs is drawback for longitudinal studies and research

More rapid development of new assays

In vitro diagnostics regulations not currently applied

Challenges of LC-MS/MS vs. IA

Comments

Capital cost of the analyzer is considerable

~£200K (~$315K, € 250K) for a triple-quadrupole analyzer

Cost of support contracts is significant

~10 % of the initial costs per year

Few LC-MS/MS methods are available as Costs may be higher than for laboratory in-house methods commercial kits Require specialist skilled staff to develop and maintain methods

Challenging for smaller laboratories

Costs of sample preparation time are considerable

Though may be similar to time required for nonautomated immunoassays (continued)

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Table 2 (continued) Challenges of LC-MS/MS vs. IA

Comments

Front-end automation less developed

Manual workflow may be more susceptible to errors

Technical support not available 24/7

Increases downtime

Complex operation and maintenance, As most are in-house methods the manufacturers cannot skill is needed for troubleshooting assay offer full support performance problems Control of laboratory temperature, ventilation, and environment is important to prevent drift and other problems during operation

Specific requirements for vacuum pumps and gas supply

Throughput lower and assay turnaround times usually longer

Though may be comparable or better than nonautomated immunoassays

Costs of disposal of organic solvents and other chemicals

Disposal of radioisotopes is required for radioimmunoassays

Usually batch analysis

Need to calibrate each batch

Data analysis and interface to laboratory computer underdeveloped

More time consuming to report data

Volume of sample required

Newer models of MS have improved response

Table 3 Advantages and challenges of LC-MS/MS vs. GC-MS methodology Advantages of LC-MS/MS over GC-MS

Comments

Selectivity is increased by using two stages of mass analysis

However specificity remains challenge for MS/MS methods using complex matrices

Potential to measure wider mass range of Mass range GC-MS ~1,000 compounds—e.g., small molecules to peptides The new generation of analyzers are more integrated and robust and the software is easier to use by all grades of staff

Easier operation for routine laboratories

Suitable technique for analysis of polar molecules, thermally labile compounds, conjugated compounds without hydrolysis

Possible to measure free and conjugated steroid hormones together

No requirement to make molecules volatile or to use the chemical derivatization reagents which are expensive and toxic

LC sample preparation is typically simpler, though derivatization may be used for LC-MS/MS methods to increase the signal

Run times are faster

MS/MS is suitable for screening or other clinical laboratories with high workloads

Possible to multiplex several LC to one MS/MS detector to increase throughput (continued)

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Table 3 (continued) Advantages of LC-MS/MS over GC-MS

Comments

Requires smaller volume of sample

Clinical need for pediatric assays, GC-MS assays usually require 0.5–1 mL serum

CE-marked kits available

Useful for smaller laboratories, may improve system robustness

Challenges of LC-MS/MS vs. GC-MS

Comments

Targeted analysis

Less useful for metabolomics/research

Library searching unknowns

GC-MS library searching more advanced and libraries less transferable between types of LC-MS/MS analyzer

Ion suppression/enhancement

Affects assay variability and sensitivity

Separation of isobaric/isomeric compounds

Need to alter chromatography

Peaks wider

Ultrahigh-pressure (UHPLC) chromatography with small-particle columns gives sharper peaks and improves signal:noise, also increases throughput

Imprecision

Probably multifactorial—likely ionization process one key factor

Increased cost analyzer

GC-MS ~25 % cost of LC-MS/MS

Ion ratios (±30 %) were also compared to check for interferences. The authors derived reference intervals for girls and boys at Tanner stages 1–5. A later modification included analysis of androstenedione and DHEA [74]. Other derivatization reagents suitable for testosterone are hydroxylamine [85] and methoxylamine hydrochloride [88]; the latter assay showed close agreement with a reference GC-MS assay. A procedure for measuring free testosterone prepared by ultrafiltration has also been reported [91]. In women the testosterone reference range is decreased by ~1 nmol/L using mass spectrometry methods [74–76, 84]. As part of the standardization project reference ranges for testosterone in men have been derived from samples collected for the Framingham Heart Study [92]. A method comparison of four routine LC-MS/MS procedures with a reference GC-MS method [70] revealed that the assays demonstrated good specificity, reproducibility, and fairly good accuracy. The imprecision and accuracy were greatly improved compared to data reported for immunoassays [68, 69, 85]. However the performance of LC-MS/MS assays could be improved further and there is a need to validate in-house assays to reference material or procedures [25]. Comparison of LC-MS/MS and GC-MS/MS methods with a reference LC-MS/MS method also confirmed these findings [71].

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Serum testosterone has been measured jointly with other steroid hormones—for example androstenedione [74, 84, 93], dihydrotestosterone [36, 93–95], 17OHP [15, 96–99], DHEA, and DHEAS [15, 74, 97]. There has been a resurgence of interest in measuring steroid hormones in saliva using mass spectrometry methods [100–102]. Salivary testing has advantages in being relatively noninvasive and less stressful for patients and it is easier to collect samples throughout the day or as necessary if a cyclical disorder is suspected. Detecting contamination of salivary samples with gingival blood is important as the respective concentrations of hormones are dissimilar. Unconjugated steroid hormone concentrations in saliva reflect the unbound (free) hormone concentrations in blood and therefore require more sensitive mass spectrometry methods. Testosterone and cortisol have been measured in saliva samples from healthy adult men with corresponding lower limits of quantitation of 17 and 34 pmol/L [103]. Early morning salivary testosterone concentrations ranged from 170 to 450 pmol/L and then decreased over the day. In a larger group of healthy male volunteers the range of salivary testosterone concentrations was 73–343 pmol/L [104]. A recent pilot study combined the assay of salivary melatonin, cortisol, and testosterone [105]. Measurement of conjugated anabolic steroids in urine enables assessment of exogenous use by athletes [106]. Hybrid tandem mass spectrometers, the Q-linear ion trap, or Q-TOF may be used for this application [107–109]. 3.1.2 Dihydrotestosterone

Measuring testosterone and dihydrotesterone (DHT) simultaneously in serum is useful for the diagnosis of 5-alpha reductase deficiency in children and also for assessing the testosterone:DHT ratio in men with prostatic hypertrophy and prostate cancer. DHT does not ionize as well as testosterone and it has different fragmentation patterns to testosterone and other related steroids [110–112]. The concentrations of DHT are ~10 % of circulating testosterone concentrations, so until recently it was not possible to develop mass spectrometry assays that used low volumes of serum [94]. Kalhorn derivatized DHT and testosterone with hydroxylamine and measured adducts formed with acetonitrile during ESI [111]. A method for underivatized DHT and testosterone [36, 93] was used to measure values across the menstrual cycle and in postmenopausal women [113]. A semi-automated UPLC-MS/ MS method has also been developed for studies on testosterone replacement therapy in hypogonadism [95]. The limit of quantitation for the androgens was 0.015 nmol/L after derivatization with picolinic acid [114]. Urinary glucuronides of DHT and testosterone may be measured utilizing an online column switching method [115].

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3.1.3 17-Hydroxyprogesterone

Detection of 21-hydroxylase deficiency, the most common form of congenital adrenal hyperplasia (CAH), is by measurement of serum 17OHP. Immunoassays are commonly used but show interference from related adrenal steroids and conjugates and method-related variance; overall in screening programs the false-positive rate is unacceptably high [116–118]. Applying second-tier LC-MS/MS assays and the calculation of ratios of 17OHP to other steroid hormones have significantly reduced the false-positive rate [38, 119–125]. Laboratories are also increasingly using mass spectrometry assays for detection and monitoring of 21-hydroxylase deficiency in serum samples from infants, children, and adults [39, 126–128]. Recent reports, using LC-MS/MS analyzers with increased sensitivity, describe methods for targeted panels of 3–12 steroid hormones; run times for the panels ranged from 5 min (using UHPLC) to 21 min [15, 40, 73, 96–99, 129–132]. Two methods included a derivatization step to improve the limit of quantitation [39, 40]. Quadrupole ion trap analyzers were employed for some of the methods [15, 131]. Rarer forms of CAH may also be detected using these panels of steroid hormones [40, 97, 98, 124, 129–132].

3.1.4 Cortisol

Assays for total or free serum cortisol, urinary free cortisol, and salivary cortisol measurements have been published. These include a candidate reference method for serum cortisol [59] and methods suitable for routine analysis; several combined the estimation of cortisone [133–136]. Free serum cortisol measurements may be useful in patients with low concentrations of cortisol-binding globulin [137–140]. Significantly lower results for urinary free cortisol were obtained using mass spectrometry assays compared with immunoassays [141–146]. Isobaric interferences from a structurally related compound, a metabolite of prednisolone, and an unrelated drug fenofibrate have been observed [142, 143]. Measuring cortisol in saliva samples collected at midnight has also been evaluated as a screening test for Cushing’s syndrome [147–155]. As different diagnostic cutoffs were used, a range of sensitivity and specificity values were reported. Other researchers have described methods for simultaneously measuring endogenous and synthetic corticosteroids in serum, urine, or saliva samples. The assays were used for clinical management, or for detection of banned substances in athletes or agricultural livestock [156–161]. Combined measurement of serum corticosteroids and the immunosuppressant mycophenolic acid was used for monitoring of patients after renal transplant [159].

3.1.5 Estrogens

A sensitive and specific estradiol assay is needed for the investigation of disorders of puberty, measurement in men and postmenopausal women, or monitoring suppression of estrogens during treatment of cancer. The accuracy of direct automated estradiol

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63

immunoassays is not adequate at the expected concentrations, typically serum estradiol 21 years 0.8–2.7 ng/dL (10.3–34.8 pmol/L) First trimester of pregnancy 0.7–2.0 ng/dL (9.0–25.7 pmol/L) Second trimester of pregnancy 0.5–1.6 ng/dL (6.4–20.6 pmol/L) Third trimester of pregnancy 0.5–1.6 ng/dL (6.4–20.6 pmol/L)

23. Based on data on 200 euthyroid individuals, the manufacturer reports a reference interval of 62–154 nmol/L. 24. The manufacturer reports the assay to have a minimum detectable concentration of 5 nmol/L, based on 2 SDs above the mean value obtained from repeat measures of the zero calibrant. 25. The manufacturer states that the pipetting of calibrants, controls and serum specimens should take no longer than 10 min. This may be impractical, in which case, it is recommended that the microtiter plate is divided into two sections, and the calibrants and the controls are replicated in each half. 26. Calibrant values are quoted in pg/mL. For conversion to SI units, 1  pg/mL × 1.536 = 1  pmol/L. 27. Based on data on 110 euthyroid individuals, the manufacturer reports a reference interval of 1.4–4.2 pg/mL (2.15– 6.45 pmol/L) for adults. For pregnant individuals, an interval of 1.8–4.2 pg/mL (2.76–6.45 pmol/L) has been quoted based on data from 75 specimens. 28. Based on 2 SDs above the mean value obtained from repeated measures of the zero calibrant, the detection limit of the assay is reported as 0.05 pg/mL (0.077 pmol/L). The manufacturer reports intra-assay CVs of 4.9, 3.6 and 3.1 % at FT3 concentrations of 1.85, 4.49 and 8.00 pg/mL (equivalent to 2.84, 6.90 and 12.29 pmol/L), respectively, based on 24 repeated measures. Inter-assay CVs are 13.1, 7.9 and 10.2 % at FT3 concentrations of 2.16, 5.09 and 9.13 pg/mL (equivalent to 3.32, 7.82 and 14.02 pmol/L), respectively, based on 12 repeated measures. 29. The assay is calibrated against the International Standard for Thyroxine Binding Globulin 88/638, established by the Expert Committee on Biological Standardization of the World Health Organization.

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30. From 20 repeated measures, the manufacturer reports intra-­ assay CVs of 3.6, 9.3 and 8.2 % at TBG concentrations of 4.3, 11.8 and 19.6 μg/mL, respectively. Inter-assay CVs based on ten separate assays are reported as 6.7, 9.0 and 4.8 % at TBG concentrations of 4.6, 12.1 and 21.1 μg/mL, respectively. The sensitivity of the assay is reported as 1.0 μg/ mL, and the assay is linear up to a TBG concentration of 340 μg/mL. 31. The quoted TBG reference intervals for this kit are 12–26 μg/mL for males, and 11–27 μg/mL for females. 32. Once opened, unused strips of wells, calibrant and control materials, antibody conjugate, and enzyme substrate may be stored at 2–8 °С until expiry date if they are resealed well. 33. Turbid specimens should be centrifuged before the assay. Grossly lipemic, grossly hemolyzed, or grossly turbid specimens should not be analyzed. 34. For the results obtained from the calibration curve, the 101-­ fold initial dilution has already been taken into consideration. 35. For this, the absorbance reading obtained on the positive control is used to determine a threshold absorbance. ­ Absorbance values above this threshold are considered to be positive. This threshold absorbance is calculated by multiplying the absorbance reading of the positive control by a factor. For each assay kit, this factor is quoted in the kit’s lot-specific QC Certificate. 36. The assay has been calibrated against the NIBSC standard 66/387. The detection limit of the assay, from 3 SDs above the mean values from 24 repeated measures of the sample buffer, is reported as 0.3– 0.4  IU/mL = equivocal; and >0.4  IU/mL = positive. 42. The assay has been calibrated against the WHO standard 90/672. The detection limit of the assay, based on 3 SDs above the mean values from repeated measures of a sample with no anti-TSH antibody, is given as 0.1 IU/L. However, the functional sensitivity of the assay is reported as 0.3 IU/L, and this should be used for the evaluation of results. The intra-­ assay CV is 16 % at an anti-TSH antibody concentration of 0.26 IU/L, and is ≤6 % at concentrations of 0.8–21.0 IU/L. Inter-assay CVs of 14, 12, 8 and 6 % have been reported for anti-TSH antibody concentrations of 0.3, 0.7, 1.8 and 6.0 IU/L, respectively. References 1. Association of Clinical Biochemistry, British Thyroid Association, British Thyroid Foundation (2006) UK guidelines for the use of thyroid function tests. http://acb.org.uk/ docs/tftguidelinefinal.pdf (Accessed July 2013) 2. Stockigt J (2003) Assessment of thyroid function: towards an integrated laboratory—clinical approach. Clin Biochem Rev 24:109–122 3. Beckett G, Toft T, O’Kane M (2007) Thyroid function tests in the UK. www.acb.org.uk/ docs/TFTprofile.pdf (Accessed July 2013) 4. Snyder PJ, Utiger RD (1972) Inhibition of thyrotropin response to thyrotropin-releasing hormone by small quantities of thyroid hormones. J Clin Invest 51:2077–2084 5. Vagenakis AG, Rapoport B, Azizi F et al (1974) Hyperresponse to thyrotropin-­ releasing hormone accompanying small decreases in serum thyroid hormone concentrations. J Clin Invest 54:913–918 6. Spencer CA, Takeuchi M, Kazarosyan ME (1995) Interlaboratory differences in f­ unctional sensitivity of immunometric assays of thyrotropin and impact on relaibility of measurement of subnormal concnetrations of TSH. Clin Chem 4:367–374 7. Spencer CA, Takeuchi M, Kazarosyan M (1996) Current status and performance goals for serum TSH assays. Clin Chem 42:140–145

8. Hay ID, Bayer MF, Kaplan MM et al (1991) American Thyroid Association assessment of current free thyroid hormone and thyrotropin measurementsand guidelines for future clinical assays. Clin Chem 37:2002–2008 9. Mendel CM (1989) The free hormone hypothesis: a physiolocally based mathematical model. Endocr Rev 10:232–274 10. Mendel CM (1992) The free hormone hypothesis. Distinction from the free hormone transport hypothesis. J Androl 13:107–116 11. Ekins R (1990) Measurements of free hormones in blood. Endocr Rev 11:5–46 12. Ekins R (1992) The free hormone hypothesis and measurement of free hormones. Clin Chem 38:1289–1293 13. Yue B, Rockwood AL, Sandrock T et al (2008) Free thyroid hormones in serum by direct equilibrium dialysis and online solid-pahse extraction-liquid chromatography/tandem mass spectrometry. Clin Chem 54:642–651 14. Csako G, Zweig MH, Glickman J et al (1989) Direct and indirect techniques for free thyroxin compared in patients with nonthyroidal illness. I. Effect of free fatty acids. Clin Chem 35:102–109 15. Nelson JC, Weiss RM (1985) The effects of serum dilution on free thyroxine (T4) concentration in the low T4 syndrome of nonthyroidal illness. J Clin Endocrinol Metab 61:239–246

Thyroid Hormone Assays 16. Holm SS, Andreasen L, Hansen SH et al (2002) Influence of adsorption and deproteination on potential free thyroxine reference methods. Clin Chem 48:108–114 17. Tikanoja SH (1990) Ultrafiltration devices tested for use in a free thyroxine assay validated by comparison with equilibrium dialysis. Scand J Clin Lab Invest 50:663–669 18. Romelli PB, Penrisi F, Vancheri L (1979) Measurement of free thyroid hormones in serum by column adsorption chromatography and radioimmunoassay. J Endocrinol Invest 2:25–40 19. Jonklaas J, Kahric-Janicic N, Soldin OP et al (2009) Correlations of free thyroid hormones measured by tandem mass spectrometry and immunoassay with thyroid-stimulating hormone across 4 patient populations. Clin Chem 55:1380–1388 20. Gu J, Soldin OP, Soldin SJ (2007) Simultaneous quantification of free triiodothyronine and free thyroxine by isotope dilution tandem mass spectrometry. Clin Biochem 40:1386–1391 21. Christofides ND, Midgley JE (2009) Inaccuracies in free thyroid hormone measurement by ultrafiltration and tandem mass spectrometry. Clin Chem 55:2228–2229 22. Ross HA, Bernardm TJ (1992) Is free thyroxine accurately measurabe at room temperature? Clin Chem 38:880–887 23. van der Sluijs Veer G, Vermes I, Bonte HA et al (1992) Temperature effects on free thyroxine measurements: analytical and clinical consequences. Clin Chem 38:1327–1331 24. Csako G, Zwieg MH, Glickman J et al (1989) Direct and indirect techniques for free thyroxin compared in patients with nonthyroidal illness. II. Effect of prealbumin, albumin and thyroxinbinding globulin. Clin Chem 35:1655–1662 25. Stockigt JR, Lim CF (2009) Medications that distort in vitro tests of thyroid function, with particular reference to estimates of serum free thyroxine. Best Pract Res Clin Endocrinol Metab 23:753–767 26. Midgley JEM (2001) Direct and indirect free thyroxine assay methods: theory and practice. Clin Chem 47:1353–1363 27. Burr WA, Evans SE, Lee J et al (1979) The ratio of thyroxine to thyroxine-binding globulin measurement in the evaluation of thyroid function. Clin Endocrinol 11:333–342 28. Attwood EC, Atkin GE (1982) The T4: TBG ratio: a re-evaluation with particular reference to low and high serum TBG levels. Ann Clin Biochem 19:101–103

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29. Szpunar WE, Stoffer SS, DiGiulio W (1987) Clinical evaluation of a thyroxine binding globulin assay in calculationg a free thyroxine index in normal, thyroid disease and sick euthyroid patients. J Nucl Med 28:1341–1343 30. Nelson JC, Tomei RT (1989) Dependence of the thyroxin/thyroxin-binding globulin (TBG) ratio and the free thyroxin index on TBG concentrations. Clin Chem 35:541–544 31. Wilcox RB, Nelson JC, Tomei RT (1994) Heterogeneity in affinities of serum proteins for thyroxine among patients with non-­ thyroidal illness as indicated by the serum free thyroxine response to serum dilution. Eur J Endocrinol 131:9–13 32. Evans SE, Burr WA, Hogan TC (1977) A reassessment of 8-anilino-1-naphthalene sulphonic acid as a thyroxine binding inhibitor in the radioimmunoassay of thyroxine. Ann Clin Biochem 14:330–334 33. Mariotti S, Caturegli P, Piccolo P et al (1990) Antithyroid peroxidase autoantibodies in thyroid diseases. J Clin Endocrinol Metab ­ 71:661–669 34. Feldt-Rasmussen U, Hoier-Madsen M, Bech K et al (1991) Anti-thyroid peroxidase antibodies in thyroid disorders and non-thyroid autoimmune diseases. Autoimmunity 9:245–253 35. Gilmour J, Brownlee Y, Foster P et al (1991) The quantitative measurement of autoantibodies to thyroglobulin and thyroid peroxidase by automated microparticle based immunoassays in Hashimoto’s disease, Graves’ disease and a follow-up study on postpartum thyroid disease. Clin Lab 46:57–61 36. Finke R, Bogner U, Kotulla P et al (1994) Anti-TPO antibody determinations using different methods. Exp Clin Endocrinol 102:145–150 37. Feldt-Rasmussen U (1996) Analytical and clinical performance goals for testing autoantibodies to thyroperoxidase, thyroglobulin and thyrotropin receptor. Clin Chem 42:160–163 38. Smith BR, Sanders J, Furmaniak J (2007) TSH receptor antibodies. Thyroid 17:923–938 39. Ando T, Latif R, Davies TF (2005) Thyrotropin receptor antibodies: new insights into their actions and clinical relevance. Best Pract Res Clin Endocrinol Metab 19:33–52 40. Mizutori Y, Chen CR, Latrofa F et al (2009) Evidence that shed thyrotropin receptor A subunits drive affinity maturation of autoantibodies causing Graves’ disease. J Clin Endocrinol Metab 94:927–935

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41. Ajjan RA, Weetman AP (2008) Techniques to quantify TSH receptor antibodies. Nat Clin Pract Endocrinol Metab 4:461–468 42. Sinclair D (2008) Analytical aspects of thyroid antibodies estimation. Autoimmunity 41:46–54 43. Smith BR, Bolton J, Young S et al (2004) A new assay for thyrotropin receptor antibodies. Thyroid 14:830–835 44. Kamijo K, Ishilawa K, Tanaka M (2005) Clinical evaluation of 3rd generation assay for thyrotropin receptor antibodies: the

­ 22-biotin-­based ELISA initiated by Smith. M Endocr J 52:525–529 45. Zöphel K, Roggenbuck D, von Landenberg P et al (2010) TSH receptor antibody (TRAb) assays based on the human monoclonal autoantibody M22 are more sensitive than bovine TSH based assays. Horm Metab Res 42: 65–69 46. Theodoraki A, Jones G, Parker J et al (2011) Performance of a third generation TSH-­receptor antibody (TRAb) in a UK clinic. Clin Endocrinol 75:127–133

Chapter 6 The Measurement of LH, FSH, and Prolactin Michael J. Wheeler Abstract In the clinical laboratory, the reproductive hormones are probably the second most commonly measured hormones after the thyroid hormones. More than 300 laboratories participate in the UK National External Quality Control Scheme. In addition, investigations into reproduction and fertility in humans and animals remain a major area of research. Illustrative methods are described for the three reproductive hormones (luteinizing hormone, follicle-stimulating hormone, prolactin). Radioimmunoassay, immunoradiometric, and enzyme assays are described to give a wide choice of assay formats. There are many commercial assays available and illustrative ones are described. Possible interferences are discussed and procedures for investigating their presence and removal are given. Key words LH, FSH, Prolactin, Gonadotropins, Immunoassay, Macroprolactin, Heterophilic antibodies

1

Introduction The reproductive peptide hormones, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prolactin are secreted from the anterior pituitary. Secretion of LH and FSH are stimulated by gonadotropin-releasing hormone from the hypothalamus and inhibited by negative feedback control by the reproductive steroids and inhibins from the gonads [1]. Prolactin is under the inhibitory control of dopamine. Increased prolactin secretion results from anti-dopaminergic drugs, stress, pituitary adenomas, pregnancy, and suckling. LH and FSH act on the gonads of both sexes stimulating steroidogenesis and gamete maturation. Although prolactin is involved in milk production in the breast, there is evidence that it also influences steroidogenesis in the gonad as well as the adrenal gland [2–4]. In vitro experiments suggest that there are optimum levels of prolactin for normal steroidogenesis and both low and high levels result in reduced steroidogenesis. Patients with a prolactin-secreting tumor of the pituitary often have reduced gonadal activity leading to low testosterone levels in men and

Michael J. Wheeler (ed.), Hormone Assays in Biological Fluids: Second Edition, Methods in Molecular Biology, vol. 1065, DOI 10.1007/978-1-62703-616-0_6, © Springer Science+Business Media New York 2013

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oligomenorrhea or amenorrhea in women. Because of their involvement in reproductive function these three peptides are commonly measured in the investigation of infertility and gonadal dysfunction in humans and other animals [5–8]. The availability of MAbs and a solid-phase sandwich technique led to the development of noncompetitive or reagent excess assays. In the sandwich the two antibodies are raised against two different epitopes of the antigen increasing the specificity of the assay. One antibody, the capture antibody, is bound to a solid phase and the other antibody is iodinated or conjugated to an enzyme. In the latter case, after the final incubation, substrate is added to give a color, chemiluminescence, or fluorescence. Other labels are also used, for example the rare earth chelates europium and terbium. As these assays do not rely on competition for epitopes between the two antibodies excess reagent may be added to speed up reactions. These assays have been referred to as reagent excess assays. Today commercial assays use either two MAbs or a polyclonal and an MAb. Both MAbs and polyclonal antibodies can be purchased from several suppliers and it is relatively simple to prepare tracer. Details for the latter have been given in Chapter 2. Automated immunometric assays using reagent excess, controlled incubation at 37 °C, and precision timing and engineering under sophisticated software have allowed manufacturers to reduce the assay time dramatically. Some automated assays take as little as 9 min to complete. The automated assays for LH, FSH, and prolactin are nonradioactive, mostly chemiluminescent, immunometric assays and virtually every clinical laboratory measures them on a daily basis. Therefore, it is not difficult to find a laboratory willing to measure small to medium number of samples saving the researcher the need to develop and/or set up assays. It is only worthwhile establishing an assay when large numbers of samples are to be analyzed over a long period of time. Simple manual assays are available from commercial companies. Human assays cannot be used to measure these hormones in lower vertebrates, such as rats and mice, because there are significant differences in hormone structure. Rat assays are available commercially and one is described in Subheading 3.2 but for other animals commercial assays may not be available. In these cases assays will need to be developed or other assays modified [6–8]. Although there have been significant changes in immunoassay technology, there has been remarkably little change in the values produced by these assays. LH is perhaps the exception because immunometric assays have been associated with a decrease in the reported LH concentration in blood of about 30 %. This needs to be kept in mind if moving from a competitive immunoassay to an immunometric assay. Despite the sophistication of modern assays, they are all prone to interference [9, 10], which can give rise to erroneous and

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Table 1 Examination of interference in LH and FSH assays LH IU/L FSH IU/L

Post-blocking

Mean on dilution

50/F

11.0 99.0

21.5 105

21.5 106

42/F

84.0 4.4

7.6 7.9

90.6 4.6

Case

misleading results [11]. Where assays are used for clinical studies such results could lead to misdiagnosis and wrong treatment. Interferences that can influence the results from immunoassays have recently been reviewed [12]. Heterophilic antibodies are antibodies in the patient that react to antibody species and other components in the assay. Anti-animal antibodies (AMA) are often referred to as heterophiles but it has been suggested that these should be recognized as a separate group [13]. All assays that use MAbs are subject to interference by heterophilic and AMA antibodies. These latter antibodies may bind to either or both of the MAbs interfering with the binding of the monoclonal antibody to the hormone. Ismail et al. [14] have reported such interference in the LH, FSH, and TSH assays used in their laboratory. They found that interference could occur in any of the three assays but not necessarily all three at once. Three approaches have been suggested to investigate this type of interference [15]. Suspicious samples may be sent away to another laboratory for analysis in a different assay system. However, Ismail et al. [14] found one case where two assays for TSH gave suspicious results and this could also occur in assays for other hormones. Secondly a sample with a suspicious result could be serially diluted in assay diluent, hormone-free serum, or zero calibrator. When there are interfering proteins or IgGs present there is frequently, but not always, a lack of parallelism. At least two dilutions should be carried out and preferably three. Finally interference may be removed by the use of heterophilic antibody blocking tubes. Table 1 shows the results for two samples where the initial results for LH and FSH were doubted. Serial dilution of samples and the use of blocking tubes to remove heterophilic antibodies was carried out on both samples. In both samples the LH result was found to be erroneous but this was shown in sample two only using the blocking tubes demonstrating that more than one approach should be used. A method is described below using blocking tubes. In prolactin assays, a more common problem is the presence of macroprolactin. Three main forms of prolactin circulate in the body: monomeric (MW 23 kD), big (MW 50–60 kD), and big, big

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Prolactin conc. mU/L)

50

Macroprolactin

40

Albumin

30 Prolactin 20

Albumin

10 Monomeric prolactin 0

30

35

40

45

50

55

Eluent volume (mL)

Fig. 1 Separation of macroprolactin and monomeric prolactin in a patient sample on Sephacryl gel

prolactin (MW 150–170 kD) [13]. The latter form has been shown to be bound to an IgG antibody and is termed macroprolactin. Although it shows biological activity in vivo, it is thought to be too big to enter cells or bind to receptors, and therefore in vitro it is not biologically active [16]. However, some recent reports have questioned this [17], although it is unclear whether the prolactin level still remains above the reference range after treatment with polyethylene glycol (PEG). It is now apparent that all assays are affected by the presence of macroprolactin, although some assays detect it more readily than others. This is true for both manual and automated assays. In clinical laboratories the concentration at which the presence of macroprolactin is investigated varies between laboratories but a large proportion use 700 mIU/L as the cutoff level. The presence of macroprolactin has been investigated in two ways. One is by precipitation of the macroprolactin with PEG 6000 [18], and the other by the more laborious gel filtration on a column of Sephadex [19]. Methods using these approaches are described here and an example of the prolactin profile following an analysis of a sample on a Sephacryl column is shown in Fig. 1.

2

Materials

2.1 Simple Radioimmunoassays for LH, FSH, and Prolactin

1. Standards: May be obtained from the National Institute of Biological Control, South Mimms, UK. 2. Polyclonal antibody.

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3. Peptides for iodination: National Institute of Biological Control, South Mimms, UK. 4. Donkey or goat anti-rabbit immunoglobulin (IgG). 5. Nonimmune rabbit serum. 2.2 Shortened Radioimmunoassays for LH, FSH, and Prolactin

1. Standard: As above. 2. Polyclonal antibodies. 3. Peptide for iodination: As above. 4. PEG 6000. 5. Donkey or goat anti-rabbit serum. 6. Nonimmune rabbit serum.

2.3 Human FSH IRMA (IBL International Gmbh, Hamburg, Germany).

1. Antibody-coated tubes: Polystyrene tubes coated with monoclonal antibody to FSH. 2. Iodinated monoclonal antibody: Liquid anti-FSH antibody in borate phosphate buffer with bovine serum albumin, sodium azide, and an inert red dye. 3. Calibrators: Zero plus six concentrations in bovine serum albumin. 4. Wash solution: Tris–HCl. 5. Quality control samples: Two levels in human serum.

2.4 Human LH ELISA (IBL ImmunoBiological Laboratories, Hamburg, Germany)

1. Microtiter plate: 12 × 8-well break-apart strips coated with monoclonal antibody to LH. 2. Calibrators: Six including a zero calibrator. 3. Anti-LH antiserum: 11 mL conjugated to horseradish peroxidase. 4. Substrate solution: 14 mL tetramethylbenzidine solution. 5. Stop solution: 14 mL 0.5 M sulfuric acid.

2.5 Rat EIA for Prolactin (Cayman Chemical Company, MI, USA)

1. Microtiter plate: 96-well plate coated with mouse anti-rabbit IgG. 2. Rat prolactin tracer: Lyophilized. 3. Rat prolactin standard: Lyophilized. 4. Rat prolactin antiserum: Lyophilized. 5. Assay buffer: Liquid. 6. Wash buffer: Liquid concentrate. 7. Ellman’s reagent: One vial lyophilized. 8. Tween-20 detergent. 9. Quality control (QC) samples: Two vials lyophilized.

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2.6 Use of Blocking Tubes to Remove Heterophilic Antibody Interference

1. Heterophilc blocking tubes.

2.7 PEG Precipitation Method for Investigating the Presence of Macroprolactin in Samples

1. PEG 6000 (Sigma-Aldrich Company Ltd).

2.8 Column Chromatography Method for Investigating the Presence of Macroprolactin in Samples

1. Sephacryl S-300 (Pharmacia Ltd, Milton Keynes, UK).

3

Methods

3.1 Simple 4-day Radioimmunoassays for LH, FSH, or Prolactin (See Notes 1 and 2)

1. Label up tubes in duplicate for total tubes, nonspecific binding (NSB) tubes, standards, and samples. 2. Add 100 μL standard and sample to appropriate tubes. 3. Add 100 μL antibody dilution to all tubes except total and NSB tubes. 4. Mix well on rotamixer or multitube mixer. 5. Incubate overnight at 4 °C. 6. Add 100 μL radioactive hormone solution to all tubes. 7. Mix well. 8. Incubate overnight at 4 °C. 9. Add 500 μL second antibody solution to all tubes except total tubes (see Note 3). 10. Mix well. 11. Incubate overnight at 4 °C. 12. Centrifuge all tubes except total tubes at 1,500 × g. 13. Aspirate or decant all tubes except total tubes. 14. Count the radioactivity in the precipitate in all tubes and also the total tubes. 15. Construct standard curve by plotting percent binding of label in the standards against standard concentration. 16. Read samples from standard curve (see Notes 4 and 5).

LH, FSH, PRL

3.2 Shortened Radioimmunoassays for LH, FSH, or Prolactin

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1. Label up tubes in duplicate for total tubes, NSB tubes, standards, and samples. 2. Add 100 μL sample or standard to appropriately labeled tube. 3. Add 200 L assay buffer to all tubes except total tube. 4. Add 100 L antibody solution to all tubes except total tube. 5. Mix tubes well on rotamixer or multitube mixer. 6. Incubate overnight at 4 °C. 7. Add 100 μL iodinated hormone to all tubes. 8. Mix well. 9. Incubate at room temperature for 6 h. 10. Add 500 μL separating solution (PEG second antibody solution). 11. Mix well. 12. Incubate at room temperature for 15 min. 13. Centrifuge at 1,500 × g for 20 min. 14. Decant supernatant from all tubes except total tubes and count for 60 s. 15. Construct standard curve by plotting percent binding of label in the standards against standard concentration. 16. Read samples from standard curve (see Note 4).

3.3 Human FSH IRMA

The method is a solid-phase immunoradiometric assay. Monoclonal anti-FSH antibody is coated on the inside of a polystyrene assay tube. Sample is added and the endogenous FSH is bound to the immobilized monoclonal antibody 1. Iodine-125-labeled antiFSH monoclonal antibody 2 is added and a sandwich is formed with the endogenous FSH at the center. Excess reagents are washed away and the radioactivity bound is counted. The amount of radioactivity is proportional to the amount of hormone present in the sample. 1. Label tubes in duplicate for total, calibrators, QCs, and patient samples. 2. Pipet 100 μL of calibrator, QC, and patient sample into appropriate tubes (see Notes 6 and 7). 3. Add 50 μL iodinated FSH to all tubes. 4. Shake for 60 min on a rack shaker. 5. Decant or aspirate off liquid. 6. Add 2 mL wash buffer to all tubes except total tubes. 7. Repeat steps 5 and 6. 8. Decant or aspirate the liquid as completely as possible. 9. Count the radioactivity in the tubes for 1 min.

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10. Draw a calibration curve on semilogarithmic paper after plotting counts per minute against concentration of the calibrators. 11. Read off concentrations of samples and QCs from the calibration curve (see Note 8). 3.4

Human LH ELISA

The assay is microtiter based with a MAb directed against the β-subunit of LH coated onto the inside of the wells. Patient sample is incubated in the wells and the LH becomes bound to the bound antibody. A second antibody directed against the α-subunit of LH and conjugated to horseradish peroxidase is then added. A sandwich is formed with the LH at the center. The amount of conjugate bound is determined by adding a substrate for the enzyme. The color that develops is proportional to the concentration of LH. 1. The desired number of microtiter wells is fixed into the holder supplied (see Note 9). 2. 25 μL standard, control, and patient sample are added to the appropriate wells (see Note 10). 3. 100 μL enzyme conjugate is added to all the wells. 4. Mix the plate well for 10 s. 5. Seal with adhesive foil and incubate for 30 min at room temperature. 6. Shake out contents and wash five times with distilled water. 7. 100 μL substrate solution are added to all wells. 8. Incubate for 10 min at room temperature. 9. Add 50 μL stop solution to all wells and mix. 10. Read at 450 ± 10 nm with a microtiter plate reader within 10 min of adding stop solution (see Note 11).

3.5 Rat EIA for Prolactin

Rat prolactin has been labeled with acetylcholinesterase (AChE). This tracer competes with prolactin in the sample for binding sites of a specific rabbit anti-rat prolactin polyclonal antibody. The complex formed binds to mouse monoclonal anti-rabbit antibody bound to the wells of a microtiter plate. After washing, Ellman’s Reagent is added to the wells followed by AChE that reacts with the Ellman’s Reagent to form a yellow color. The intensity of the color is proportional to the amount of prolactin present in the sample. Both serum and plasma samples may be used. 1. Add 100 μL assay buffer to the blank wells (see Note 12). 2. Add 50 μL assay buffer to the maximum (zero) binding wells. 3. Add 50 μL of each standard in duplicate to the standard wells. 4. Add 50 μL QC sample to the two QC wells. 5. Add 50 μL of each sample in duplicate. 6. Add 50 μL AChE tracer to each well except blank wells.

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7. Add 50 μL rat prolactin antiserum to all wells except the blank wells. 8. Cover the plate and incubate overnight at room temperature. 9. Tip out the well contents and shake. 10. Wash the wells five times with 300 μL reconstituted wash buffer. 11. Add 200 μL Ellman’s Reagent to each tube. 12. Incubate in the dark at room temperature for about 90 min (see Note 13). 13. Read the developed color in a plate reader at a wavelength between 405 and 414 nm. 14. Calculate the mean absorbance for all samples, calibrators, and QCs and subtract the blank absorbance. 15. Construct a standard curve on semilogarithmic paper from the absorbance of the calibrators. 16. Read the concentrations of the samples from the calibration curve (see Note 14). 3.6 Use of Blocking Tubes to Remove Heterophilic Antibody Interference

1. Holding the tube upright, gently tap the bottom of the tube on a hard surface to bring the blocking reagents to the bottom of the tube. 2. Uncap the blocking tube and add 500 μL serum to the bottom of the tube. 3. Cap the blocking tube and invert five times to mix the sample with the blocking reagent. 4. Incubate at room temperature for 60 min. 5. Assay the treated sample along with an untreated aliquot of the same sample and compare the results. 6. Where the results are significantly different, interference may be considered present.

3.7 PEG Precipitation Method for Investigating the Presence of Macroprolactin in Samples

1. Prepare a solution of 25 % PEG 6000. 2. Add 200 mL PEG solution to 200 mL patient serum (see Notes 15 and 16). 3. Mix well and centrifuge at 1,500 × g for 30 min. 4. Measure the prolactin concentration in the treated sample. 5. Correct for dilution (see Note 17) and calculate the percentage of prolactin (see Note 18). As suggested previously, some assays detect macroprolactin more commonly, e.g., Wallac DELFIA, but this does not mean that such an assay will always give a higher result than an assay that detects macroprolactin less frequently, e.g., Siemens Advia Centaur. Figure 1 shows a chromatographic profile of a sample that gave a

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result of 1,800 mIU/L (reference range for normals