Liquid chromatography: applications 9780124158061, 0124158064, 9780124158665, 0124158668, 9781283948272, 1283948273

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LIQUID CHROMATOGRAPHY: APPLICATIONS

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LIQUID CHROMATOGRAPHY: APPLICATIONS

SALVATORE FANALI PAUL R. HADDAD COLIN F. POOLE PETER SCHOENMAKERS DAVID LLOYD

AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO

Elsevier 225, Wyman Street, Waltham, MA 02451, USA The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands Copyright Ó 2013 Elsevier Inc. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: [email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at http://elsevier.com/locate/ permissions, and selecting Obtaining permission to use Elsevier material Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made Library of Congress Cataloging-in-Publication Data Application Submitted British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-415806-1 For information on all Elsevier publications visit our web site at store.elsevier.com Printed and bound in USA 13 14 15

10 9 8 7 6 5 4 3 2 1

Contents Contributors xi

1. Affinity Chromatography 1 D.S. HAGE, J.A. ANGUIZOLA, R. LI, R. MATSUDA, E. PAPASTAVROS, E. PFAUNMILLER, M. SOBANSKY AND X. ZHENG

1.1. Introduction 1 1.2. Basic Components of Affinity Chromatography 3 1.3. Bioaffinity Chromatography 5 1.4. Immunoaffinity Chromatography 6 1.5. Dye-Ligand and Biomimetic Affinity Chromatography 9 1.6. Immobilized Metal-Ion Affinity Chromatography 11 1.7. Analytical Affinity Chromatography 11 1.8. Miscellaneous Methods and Newer Developments 14 Acknowledgment 17 References 17

2.

Derivatization in Liquid Chromatography 25 C.F. POOLE

2.1. Introduction 25 2.2. Reagent Selection 27 2.3. Postcolumn Reaction Detectors 2.4. Conclusions 52 References 52

49

3. Validation of Liquid Chromatographic Methods 57 K.L. BARNETT, B. HARRINGTON AND T.W. GRAUL

3.1. Traditional Method Validation 58 3.2. Quality by Design and Analytical Methods 3.3. Conclusion 71 References 72

v

64

vi

CONTENTS

4. Liquid Chromatographic Separation of Enantiomers 75 B. CHANKVETADZE

4.1. Introduction 75 4.2. A Short History of Chiral Separations By Liquid Chromatography 77 4.3. Materials for the Liquid Chromatographic Separation of Enantiomers 78 4.4. Modes of Liquid Chromatographic Separation of Enantiomers 82 4.5. Separation of Enantiomers By Supercritical Fluid Chromatography 86 4.6. Summary and Future Trends 87 References 88

5. Liquid Interaction Chromatography of Polymers 93 W. RADKE AND J. FALKENHAGEN

5.1. Introduction 94 5.2. Theoretical Aspects of Isocratic Liquid Chromatography of Polymers 5.3. Applications of Liquid Chromatography of Polymers 98 5.4. Hyphenated Techniques 107 5.5. Summary 121 References 122

94

6. Amino Acid and Bioamine Separations 131 Y. MIYOSHI, T. OYAMA, R. KOGA AND K. HAMASE

6.1. Introduction 132 6.2. Direct Separation of Amino Acids and Amines 132 6.3. Indirect Separation of Amino Acids and Amines 135 6.4. Enantioselective Liquid Chromatographic Analysis of Amino Acids 141 6.5. Conclusions 144 References 145

7. Protein and Peptide Separations 149  J. GIACOMETTI AND D. JOSIC

7.1. Introduction 149 7.2. Methods of Protein Liquid Chromatography 7.3. Conclusions 179 Acknowledgments 180 References 180

151

8. Glycans and Monosaccharides 185 L. ROYLE

8.1. Introduction 185 8.2. Types of Glycans 186

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CONTENTS

8.3. Analysis and Characterization of Glycans 188 8.4. Monosaccharide Composition Analysis 196 8.5. Conclusions 201 References 201

9. Separation of Lipids 203 P. DONATO, P. DUGO AND L. MONDELLO

9.1. Introduction and Contents 204 9.2. Definitions and Classification 205 9.3. Structures and Occurrence 206 9.4. Sample Extraction and Handling 218 9.5. Lipid Analysis by Liquid Chromatography 224 9.6. Conclusions and Future Perspectives 243 References 244

10.

Forensic Toxicology

249

C. KOSTAKIS, P. HARPAS AND P. STOCKHAM

10.1. General Drug Screening 251 10.2. Liquid Chromatography-Mass Spectrometry in Forensic Toxicology 255 10.3. Testing for Driving Under the Influence of Drugs Using Oral Fluid 268 10.4. Toxicological Analysis of Hair in the Investigation of Drug Facilitated Crimes 274 10.5. Targeted Poisons 277 10.6. Conclusions 282 References 283

11. Compositional Analysis of Foods 295 ´N ˜ EZ AND A. CIFUENTES M. HERRERO, M. CASTRO-PUYANA E. IBA

11.1. Introduction 296 11.2. Carbohydrates 298 11.3. Vitamins 301 11.4. Amino Acids, Peptides, and Proteins 306 11.5. Lipids 307 11.6. Minor Components of Food 309 11.7. Food Additives 311 11.8. Conclusions and Future Trends 314 Acknowledgment 315 References 315

12.

Multiresidue Methods for Pesticides and Related Contaminants in Food 319 ´ NDEZ AND M. IBA ´N ˜ EZ F. HERNA

12.1. Introduction 319 12.2. Sample Treatment 322

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CONTENTS

12.3. Matrix Effects In LCeMS Analysis 324 12.4. Method Validation 325 12.5. Analysis of Samples 328 12.6. Individual Methods for Specific Compounds 330 12.7. LCeTOF MS in the Field of Pesticide Residue Analysis 331 References 333

13.

Environmental Analysis: Persistent Organic Pollutants 337 L.C. SANDER, M.M. SCHANTZ AND S.A. WISE

13.1. Polycyclic Aromatic Hydrocarbons 341 13.2. Chlorinated Aromatic Compounds 346 13.3. Pesticides 347 13.4. Brominated Flame Retardants 352 13.5. Perfluoroalkyl Compounds 360 13.6. Reference Materials 372 13.7. Concluding Remarks 373 13.8. Disclaimer 375 References 375

14.

Environmental Analysis: Emerging Pollutants 389

´ M. PETROVIC, M. FARRE´, E. ELJARRAT, M.S. DI´AZ-CRUZ AND D. BARCELO

14.1. Introduction 390 14.2. General Trends 391 14.3. Target Analysis of Specific Contaminant Groups Using LCeMS 393 14.4. Conclusions 403 References 404

15.

Analysis of Natural Toxins

411

P. OTERO, P. RODRI´GUEZ, A.M. BOTANA, A. ALFONSO AND L.M. BOTANA

15.1. Introduction 411 15.2. Tetrodotoxin 415 15.3. Lipophilic Marine Toxins 418 15.4. Saxitoxin and Analogs 425 References 426

16.

Liquid Chromatography in the Pharmaceutical Industry 431 R. SZUCS, C. BRUNELLI, F. LESTREMAU AND M. HANNA-BROWN

16.1. The Role of Separation Science in Pharmaceutical Drug Development 16.2. Increasing Chromatographic Resolution 433 16.3. Chromatographic Method Development: RPLC 444 Acknowledgments 452 References 453

432

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CONTENTS

17. Determination of Veterinary Drug Residues in Foods by Liquid ChromatographyeMass Spectrometry: Basic and Cutting-Edge Applications 455 M.D. MARAZUELA AND S. BOGIALLI

17.1. Introduction 456 17.2. Options in Veterinary Residue Analysis using LCeMS 17.3. Conclusions 471 References 472

459

18. Analysis of Vitamins by Liquid Chromatography 477 A. GENTILI AND F. CARETTI

18.1. Introduction 478 18.2. Liquid Chromatographic Determination of Water-Soluble Vitamins 479 18.3. Liquid Chromatographic Determination of Fat-Soluble Vitamins 489 18.4. Multivitamin Methods 497 References 510

19. Applications of Liquid Chromatography in the Quality Control of Traditional Chinese Medicines, an Overview 519 Y. SHEN, T.A. VAN BEEK, H. ZUILHOF AND B. CHEN

19.1. Introduction 520 19.2. Liquid Chromatographic Analysis of Traditional Chinese Medicines 520 19.3. Conclusions 535 Acknowledgments 536 References 537

20.

Analysis of Neurotransmitters and Their Metabolites by Liquid Chromatography 541 K.E. BOSSE, J.A. BIRBECK, B.D. NEWMAN AND T.A. MATHEWS

20.1. Introduction 542 20.2. Biogenic Amines 556 20.3. Acetylcholine 568 20.4. Amino Acids 573 20.5. Purines: Adenosine Triphosphate and Adenosine 579 20.6. Endocannabinoids 582 20.7. Neuropeptides 585 20.8. Multianalyte Monitoring of Neurotransmitters from Diverse Classes 590 20.9. Clinical Applications of Microdialysis Sampling and Liquid ChromatographicBased Analysis 592 20.10. Conclusions 596 References 596

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CONTENTS

21. Clinical Chemistry, Including Therapeutic Drug Monitoring and Biomarkers for Diagnosis 611 G. BAIRD

21.1. Introduction 612 21.2. Preanalytical Specimen Handling in Clinical Liquid Chromatography 613 21.3. Separation Technologies in Clinical Liquid Chromatography 613 21.4. Detection Technologies in Clinical Liquid Chromatography 617 21.5. Applications 620 21.6. Conclusion 628 References 628

22.

Speciation and Element-Specific Detection

633

B. MICHALKE AND V. NISCHWITZ

22.1. Introduction 634 22.2. Sampling 635 22.3. Sample Storage and Processing 636 22.4. Speciation Approaches: Direct Methods or Hyphenated Techniques 637 22.5. Interfacing: Nebulizers, Use of Internal Standard and Postcolumn Dilution 641 22.6. Element-Selective Detection 643 22.7. Quantification and Quality Control 646 References 648

Index 651

Contributors A. Alfonso Departamento de Farmacologı´a, Facultad de Veterinaria J.A. Anguizola Chemistry Department, University of Nebraska, Lincoln D. Barcelo´ King Saud University, Riyadh, Saudi Arabia G. Baird Department of Laboratory Medicine, University of Washington, Seattle, Washington, USA K.L. Barnett Analytical Research and Development, Pfizer Inc., Groton, CT J.A. Birbeck Department of Chemistry, Wayne State University, Detroit, Michigan S. Bogialli Department of Chemistry, University of Padua, Padova, Italy K.E. Bosse Department of Chemistry, Wayne State University, Detroit, Michigan A.M. Botana Departamento de Quı´mica Analı´tica, Facultad de Ciencias; Campus de Lugo, USC, Lugo, Spain L.M. Botana Departamento de Farmacologı´a, Facultad de Veterinaria C. Brunelli Analytical R&D, Pfizer Global R&D, Sandwich, Kent, UK F. Caretti Department of Chemistry, Faculty of Mathematical, Physical and Natural Sciences, University of Rome La Sapienza, Italy M. Castro-Puyana Laboratory of Foodomics, Institute of Food Science Research (CIAL-CSIC), Madrid, Spain B. Chankvetadze Institute of Physical and Analytical Chemistry, School of Exact and Natural Sciences, Tbilisi State University, Georgia B. Chen Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research, Ministry of Education, Hunan Normal University, Changsha China A. Cifuentes Laboratory of Foodomics, Institute of Food Science Research (CIAL-CSIC), Madrid, Spain M.S. Dı´az-Cruz Department of Environmental Chemistry, Institute of Environmental Assessment and Water Research (IDAEA-CSIC), Barcelona, Spain P. Donato University Campus Bio-Medico, Rome, Italy; University of Messina, Messina, Italy P. Dugo University of Messina, Messina, Italy; University Campus Bio-Medico, Rome, Italy E. Eljarrat Department of Environmental Chemistry, Institute of Environmental Assessment and Water Research (IDAEA-CSIC), Barcelona, Spain J. Falkenhagen BAM Bundesanstalt fu¨r Materialforschung und epru¨fung, Berlin, Germany

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xii

CONTRIBUTORS

M. Farre´ Department of Environmental Chemistry, Institute of Environmental Assessment and Water Research (IDAEA-CSIC), Barcelona, Spain A. Gentili Department of Chemistry, Faculty of Mathematical, Physical and Natural Sciences, University of Rome La Sapienza, Italy J. Giacometti Department of Biotechnology, University of Rijeka, Croatia T.W. Graul Analytical Research and Development, Pfizer Inc., Groton, CT D.S. Hage Chemistry Department, University of Nebraska, Lincoln K. Hamase Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan M. Hanna-Brown Analytical R&D, Pfizer Global R&D, Sandwich, Kent, UK; Warwick Centre for Analytical Science, University of Warwick, UK P. Harpas Forensic Science South Australia, Adelaide, Australia B. Harrington Nonclincial Statistics, Pfizer Inc., Groton, CT F. Herna´ndez Research Institute for Pesticides and Water, University Jaume I, Castello´n, Spain M. Herrero Laboratory of Foodomics, Institute of Food Science Research (CIAL-CSIC), Madrid, Spain E. Iba´n˜ez Laboratory of Foodomics, Institute of Food Science Research (CIAL-CSIC), Madrid, Spain M. Iba´n˜ez Research Institute for Pesticides and Water, University Jaume I, Castello´n, Spain D. Josic Warren Alpert Medical School, Brown University, Providence, Rhode Island; Department of Biotechnology, University of Rijeka, Croatia R. Koga Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan C. Kostakis Forensic Science South Australia, Adelaide, Australia; Flinders University, Bedford Park, Australia F. Lestremau Analytical R&D, Pfizer Global R&D, Sandwich, Kent, UK R. Li Chemistry Department, University of Nebraska, Lincoln M.D. Marazuela Department of Analytical Chemistry, Faculty of Chemistry, Universidad Complutense de Madrid, Spain T.A. Mathews Department of Chemistry, Wayne State University, Detroit, Michigan R. Matsuda Chemistry Department, University of Nebraska, Lincoln B. Michalke Helmholtz Zentrum Muenchen, German Research Center for Environmental Health, Institute of Ecological Chemistry, Neuherberg, Germany Y. Miyoshi Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan L. Mondello University of Messina, Messina, Italy; University Campus Bio-Medico, Rome, Italy

CONTRIBUTORS

xiii

B.D. Newman Department of Chemistry, Wayne State University, Detroit, Michigan V. Nischwitz LGC Limited, Teddington, Middlesex, UK P. Otero Departamento de Farmacologı´a, Facultad de Veterinaria T. Oyama Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan E. Papastavros Chemistry Department, University of Nebraska, Lincoln M. Petrovic Catalan Institute of Water Research (ICRA), Girona, Spain; Department of Environmental Chemistry, Institute of Environmental Assessment and Water Research (IDAEA-CSIC), Barcelona, Spain E. Pfaunmiller Chemistry Department, University of Nebraska, Lincoln C.F. Poole Department of Chemistry, Wayne State University, Detroit, Michigan W. Radke Fraunhofer-Institut fu¨r Betriebsfestigkeit und Systemzuverla¨ssigkeit LBF, Bereich Kunststoffe, Darmstadt, Germany P. Rodrı´guez Departamento de Farmacologı´a, Facultad de Veterinaria L. Royle Ludger Ltd., Oxford, UK L.C. Sander Chemical Sciences Division, National Institute of Standards and Technology (NIST), Gaithersburg, MD M.M. Schantz Chemical Sciences Division, National Institute of Standards and Technology (NIST), Gaithersburg, MD Y. Shen Laboratory of Organic Chemistry, Wageningen University, the Netherlands; Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research, Ministry of Education, Hunan Normal University, Changsha China M. Sobansky Chemistry Department, University of Nebraska, Lincoln P. Stockham Forensic Science South Australia, Adelaide, Australia; Flinders University, Bedford Park, Australia R. Szucs Analytical R&D, Pfizer Global R&D, Sandwich, Kent, UK; Pfizer Analytical Research Centre, Ghent University, Ghent, Belgium; Irish Separation Sciences Cluster, Dublin City University, Dublin, Ireland T.A. van Beek Laboratory of Organic Chemistry, Wageningen University, the Netherlands S.A. Wise Chemical Sciences Division, National Institute of Standards and Technology (NIST), Gaithersburg, MD X. Zheng Chemistry Department, University of Nebraska, Lincoln H. Zuilhof Laboratory of Organic Chemistry, Wageningen University, the Netherlands

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C H A P T E R

1 Affinity Chromatography D.S. Hage, J.A. Anguizola, R. Li, R. Matsuda, E. Papastavros, E. Pfaunmiller, M. Sobansky, X. Zheng Chemistry Department, University of Nebraska, Lincoln O U T L I N E 1.1. Introduction

1

1.2. Basic Components of Affinity Chromatography

3

1.3. Bioaffinity Chromatography

5

1.4. Immunoaffinity Chromatography

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1.5. Dye-Ligand and Biomimetic Affinity Chromatography

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1.6. Immobilized Metal-Ion Affinity Chromatography

11

1.7. Analytical Affinity Chromatography

11

1.8. Miscellaneous Methods and Newer Developments

14

Acknowledgement

17

References

17

1.1. INTRODUCTION Affinity chromatography is a type of liquid chromatography in which a biologically related agent is used in a column as a stationary phase to purify or analyze the components of a sample [1e4]. The ability of this method to selectively bind and purify its target compounds is based on Liquid Chromatography: Applications http://dx.doi.org/10.1016/B978-0-12-415806-1.00001-2

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Copyright Ó 2013 Elsevier Inc. All rights reserved.

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1. AFFINITY CHROMATOGRAPHY

the specific and reversible interactions present in many biological systems, such as the binding of a hormone to a receptor or an antibody to its antigen. To develop a method based on affinity chromatography, one of the pair of interacting species is first immobilized to a solid support, such as agarose beads or silica particles. The immobilized agent, called the affinity ligand, acts as the stationary phase for the affinity column [1,3]. The other interacting compound is then injected onto the affinity column or applied in the presence of an application buffer, which allows the desired target to bind to the immobilized ligand (see Figure 1.1) [5]. After nonretained sample components have been washed from the column, the retained target analyte is typically released in the presence of an elution buffer [2,3,5]. If the retained compound has only weak or moderate binding to the immobilized ligand, it is also possible to use the application buffer to elute this target under isocratic conditions; this approach is known as

FIGURE 1.1 Typical scheme for the application of a sample to an affinity column, elution of the retained targets, and regeneration of the affinity column [1].

1.2. BASIC COMPONENTS OF AFFINITY CHROMATOGRAPHY

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weak-affinity chromatography (WAC) [5e11]. As the target elutes, it is collected for further use or analyzed by an on-line or off-line detector. The column is regenerated by reapplying the application buffer before the next sample injection.

1.2. BASIC COMPONENTS OF AFFINITY CHROMATOGRAPHY The success of any affinity separation depends largely on the selection of the ligand immobilized in the column. A summary of common ligands used in affinity chromatography is given in Table 1.1. Many of the ligands used in affinity chromatography are obtained from a biological source, such as antibodies, serum proteins, and lectins. Other binding agents that are useful in affinity chromatography are boronic acid, metal chelates, and triazine dyes, which are synthetic agents or inorganic molecules that can be employed as ligands [1e4]. The affinity ligands can be divided into two main categories: highspecificity ligands and general ligands [3]. High-specificity ligands are binding agents that retain only one or a few closely related targets; these ligands are used when the goal is to isolate or separate a specific solute. Examples of high-specificity ligands include antibodies for the binding of TABLE 1.1

Common Ligands Used in Affinity Chromatography

Type of ligand

Retained targets

BIOLOGICAL LIGANDS Antibodies

Antigens (drugs, hormones, peptides, proteins, viruses, and cell components)

Inhibitors, substrates

Enzymes

Cofactors and coenzymes Lectins

Sugars, glycoproteins, and glycolipids

Nucleic acids

Complementary nucleic acids and DNA/RNA-binding proteins

Protein A/protein G

Antibodies

NONBIOLOGICAL LIGANDS Boronates

Sugars, glycoproteins, and diol-containing compounds

Triazine dyes

Nucleotide-binding proteins and enzymes

Metal ion chelates

Metal-ion-binding amino acids, peptides, and proteins

Source: This table is based on information provided in Refs. [1e4].

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1. AFFINITY CHROMATOGRAPHY

antigens, substrates or inhibitors for separating or binding enzymes, and single-strand nucleic acids for the retention of complementary sequences of DNA or RNA [1]. General ligands are binding agents that retain a class of related molecules or structurally similar targets. Examples of general ligands are lectins and boronates for binding carbohydrate-containing agents, some types of dyes for the retention of enzymes and proteins, and protein A or protein G for the binding of immunoglobulins [3]. When designing a system for use with affinity chromatography, an important aspect to consider is the selection of the support material utilized for ligand attachment. This support material should have low nonspecific binding for sample components but be easy to modify for chemical activation and ligand attachment. In addition, the support should be able to withstand the pressures and flow rates used in the separation [1]. Agarose is often used as a support in traditional affinity columns [1]. This material consists of polymeric chains comprised of Dgalactose and 3,6-anhydro-L-galactose [12]. Another common type of polysaccharide support is cellulose. Work has also been conducted in the use of affinity ligands with supports for high-performance liquid chromatography (HPLC), resulting in a method known as high-performance affinity chromatography (HPAC) or high-performance liquid affinity chromatography (HPLAC) [1]. Supports utilized in this latter method include silica particles, modified polystyrene supports, silica monoliths, and organic-based monoliths [12e15]. Several techniques are available for attaching a ligand to a chromatographic support. These techniques include both covalent and nonspecific immobilization methods [16]. Nonspecific immobilization techniques involve the physical adsorption of a ligand to a support [17,18]. Biospecific adsorption is a form of noncovalent immobilization that uses the binding between two ligands, one of which has been previously bound to the support and the second of which is adsorbed to the first ligand and used to bind the final desired target. An example of this approach is in the use of avidin or streptavidin for the adsorption of biotinylated proteins [16,19]. Another example is the use of immobilized protein A or protein G to adsorb antibodies for use in the creation of immunoaffinity supports [16,20,21]. Covalent immobilization involves the chemical attachment of a ligand to a chromatographic support. In this method, the support must first be activated for ligand attachment. Several functional groups can be used for covalent immobilization. These methods may involve amine groups, carboxyl groups, sulfhydryl groups, hydroxyl groups, or aldehyde groups [16]. The application buffer used with an affinity column should have an appropriate pH and ionic strength to promote binding between the immobilized ligand and target [5]. The elution buffer is a mobile phase that disrupts the binding of the target with the ligand. This elution may be

1.3. BIOAFFINITY CHROMATOGRAPHY

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accomplished by changing the pH, ionic strength, or amount of organic solvent in the mobile phase. Such an approach, referred to as nonspecific elution, is often used in analytical methods for the quick removal of a target from an affinity column. Alternatively, a competing agent may be placed into the mobile phase to displace the target from the column by binding with either the target or ligand, thus preventing their further interaction. This method is called biospecific elution. Although biospecific elution is often slower than nonspecific elution, it is useful when gentle removal and affinity purification of an active target are desired [5].

1.3. BIOAFFINITY CHROMATOGRAPHY Bioaffinity chromatography is a type of affinity chromatography that employs a biological ligand as the stationary phase [22]. This technique was first used in 1910 by Starkenstein to purify a-amylase using insoluble starch. Bioaffinity chromatography is now commonly used as a purification technique for numerous compounds [23]. Because many biological agents are found in nature, bioaffinity chromatography is one of the most diverse forms of affinity chromatography. Several examples of biological agents that can be used as ligands in bioaffinity applications are listed in Table 1.1. Many of these agents are commercially available in an immobilized form for use in affinity columns [3,22,24]. Enzyme purification was the first application of bioaffinity chromatography [23] and remains an important use of this technique. In this type of separation, ligands, such as enzyme inhibitors, coenzymes, or cofactors, are used to purify and separate enzymes [25]. For instance, in 1968 and the first report of “modern” affinity chromatography, Cuatrecasas, Wilchek, and Anfinsen employed specific enzyme inhibitors to selectively isolate enzymes [1,4]. A more recent example was the use of a support containing flavin mononucleotides for the purification of flavin adenine dinucleotide synthetase [26]. Other examples have included the use of mono-, di-, and triphosphate nucleotides for the purification of kinases and the use of nicotinamide adenine dinucleotide for the isolation of dehydrogenases [26,27]. Lectins are another group of ligands that are frequently found in bioaffinity chromatography. Lectins are nonimmune system proteins able to bind to specific carbohydrate groups [22]. The most common lectins used in bioaffinity chromatography are concanavalin A (Con A), wheat-germ agglutinin (WGA), and jacalin [25]. Con A has the ability to bind to targets that contain a-D-mannose or a-D-glucose residues [25]. WGA specifically binds to D-N-acetylglucosamine residues, and jacalin binds to galactose or mannose residues [25,28]. The most popular application for lectins has been in glycomics, where these ligands are

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1. AFFINITY CHROMATOGRAPHY

used to isolate and separate polysaccharides, glycoproteins, glycopeptides, and glycolipids [24,25]. For instance, supports containing multiple lectins (e.g., Con A, WGA, and jacalin) have been used for the separation of glycoproteins in plasma samples to help map glycosylation patterns for disease detection [29,30]. Immunoglobulin-binding proteins are another class of ligands that are employed in bioaffinity chromatography. Protein A and protein G are two examples of such binding agents. Protein A is produced by Staphylococcus aureus and protein G is produced by group G streptococci [22,31e33]. Both proteins bind to the Fc region of immunoglobulins [22,25], which makes these ligands useful for antibody purification and the biospecific adsorption of antibodies to affinity supports [25]. Protein A and protein G have some differences in the species and classes of antibodies to which they will bind [22,31e33]. For example, protein A can bind to human antibodies that belong to the classes of immunoglobulin G (IgG), IgM, and IgA; however, protein G can bind to only IgG-class antibodies from humans [22,31e33]. Both these proteins bind strongly to immunoglobulins at a neutral pH but dissociate from their targets at a mildly acidic pH [25]. Hybrid forms of proteins A and G, such as protein A/G, are also available, which can increase the types of immunoglobulin to which this type of ligand is able to bind [22,34]. Nucleic acids and polynucleotides are additional binding agents that can be used in bioaffinity chromatography. This combination produces a method referred to as DNA affinity chromatography [35e37]. Methods in DNA affinity chromatography can be classified into two categories: nonspecific techniques and sequence-specific techniques. Ligands used in nonspecific DNA chromatography are generally composed of fragmented nuclear DNA (e.g., salmon sperm DNA or calf thymus DNA) [35]. In this technique, DNA binding proteins can be bound to the immobilized ligand for their separation from other sample components [35]. Sequencespecific DNA chromatography is used for the purification and isolation of targets that bind to specific sequences of the polynucleotide utilized as the ligand [35e37].

1.4. IMMUNOAFFINITY CHROMATOGRAPHY Immunoaffinity chromatography (IAC) is a special type of bioaffinity chromatography that uses antibodies or antibody-related molecules as the stationary phase. IAC columns typically have strong and selective binding between antibodies and their target antigens. This feature, as well as the ability to produce antibodies against a wide range of targets, has made IAC popular for use in both the purification and analysis of chemicals in a variety of complex samples [38e40].

1.4. IMMUNOAFFINITY CHROMATOGRAPHY

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The most common technique used in IAC is the oneoff elution mode, as illustrated earlier in Figure 1.1 [39,40]. In this format, the sample of interest is injected onto the IAC column under conditions in which the target has a strong affinity to antibodies immobilized within the column. Only the target and closely related compounds are retained on the IAC column, while other sample components are not retained. After the nonretained sample components have been washed away, an elution buffer is applied to the column to dissociate the bound target from the column. In this format, it is often easy to obtain baseline resolution between the nonretained and retained peaks by changing the time at which the elution buffer is applied. Proteins, glycoproteins, carbohydrates, lipids, bacteria, viral particles, drugs, and environmental agents have all been isolated by IAC through the use of this format [38,40e45]. The main advantage of this approach is its speed and simplicity, especially when it is carried out as part of an HPLC system. The use of antibodies or related binding agents with HPLC supports is referred to as high-performance immunoaffinity chromatography (HPIAC) [46,47]. Chromatographic (or flow-injection) immunoassays represent another important mode of IAC. This technique utilizes immobilized antibodies, or antigens, in a column to perform various competitive or noncompetitive immunoassays. Detection in these methods involves the use of a labeled antibody or labeled analyte analog (known as the label) to indirectly measure the amount of a target in a sample, as demonstrated in Figure 1.2. The detection format may be based on absorbance,

FIGURE 1.2 Chromatographic immunoassay based on a sandwich, or two-site immunometric, format.

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1. AFFINITY CHROMATOGRAPHY

fluorescence, chemiluminescence, electrochemical activity, radioactivity, or thermal measurements. The label may consist of an enzyme, fluorescent tag, chemiluminescent agent, liposome, or radioisotope, among other possibilities [46,47]. Competitive binding immunoassays involve competition between a target in a sample and a labeled analyte analog (or label) for a limited amount of antibodies that are capable of binding to both the target and labeled analog. After they have been allowed to compete for binding sites on the antibodies, the bound and free portions of the target and labeled analog are separated. The amount of labeled analog in the bound or free faction is then measured. The absence of any target results in the maximum amount of labeled analog in the bound fraction. An increase in the amount of target results in a decrease in the level of this bound labeled analog, which provides a signal that is indirectly related to the amount of target in the original sample. Several formats are available for a competitive binding immunoassay in IAC or HPIAC. The simultaneous injection format involves injection of the target and a labeled analog at the same time onto an immobilized antibody column [47,48]. In the sequential injection format, the sample is injected first, followed later by injection of the labeled analog [47,49]. A displacement immunoassay involves the adsorption of a large amount of labeled analog to an immobilized antibody column, followed by the displacement of some of this labeled analog upon sample injection [47]. Simultaneous injection immunoassays have been utilized in the analysis of human serum albumin (HSA), IgG, theophylline, caffeine, and atrazine. Sequential injection immunoassays have also been used to measure HSA, IgG, and atrazine. Displacement immunoassays have been employed for the analysis of cocaine, benzoylecgonine, di- and trinitrotoluene, and cortisol [47]. Noncompetitive immunoassays (or immunometric assays) can be performed using IAC or HPIAC. There are two main types of noncompetitive immunoassays: the one-site immunometric assay [10] and the sandwich immunoassay, or two-site immunometric assay [47]. A one-site immunometric assay is carried out by incubating a sample with a known excess of labeled antibodies that are specific for the target. This mixture is then applied to a column containing an immobilized analog of the target. The column is used to capture the nonbound portion of the labeled antibodies while allowing antibodies that are bound to the target to pass through unretained. Measurement of the target is performed indirectly by monitoring either the change in response for the unretained labeled antibodies or by monitoring the amount of retained antibodies captured by the column and released during the elution step. This type of analysis has been utilized in the measurement of digoxin, thyroxine, and 17b-estradiol [47].

1.5. DYE-LIGAND AND BIOMIMETIC AFFINITY CHROMATOGRAPHY

9

Sandwich immunoassays use two antibodies that can simultaneously bind to the same target. One of these antibodies is immobilized to the support in an IAC column and is used to extract the target from a sample. The second antibody is labeled for detection and is either mixed with the sample prior to application or applied to the column after the target is bound to the IAC column. The binding of the target by the two antibodies creates a “sandwich” complex in the column, as illustrated in Figure 1.2. After this complex has been created, an elution buffer is passed through the column to dissociate the target and labeled antibodies. Measurement of the labeled antibodies that elute with the target from the column give a response that is directly proportional to the amount of target. Sandwich immunoassays have been reported for the determination of HSA, IgG, parathyroid hormone, and human chorionic gonadotropin [47]. Another use of IAC columns is to monitor a target that is eluting from other types of columns. This approach is referred to as postcolumn immunodetection [39,47]. The technique typically involves the use of a postcolumn reactor and an immobilized antibody or antigen column attached to the outlet of an analytical HPLC column. As the target elutes from the analytical column, it is mixed with an excess of labeled antibodies that can bind to the target. The remaining, unbound labeled antibodies are removed from this mixture by using a column that contains an immobilized analog of the target. The unretained labeled antibodies that are already bound to the target pass through the analog column and give a signal proportional to the target’s concentration. This type of detection has been used for various applications, including the measurement of growth-hormone-releasing factor, digoxin, digoxigenin, and human granulocyte colony-stimulating factor [40].

1.5. DYE-LIGAND AND BIOMIMETIC AFFINITY CHROMATOGRAPHY Synthetic dyes and chlorotriazine-linked biomimetic ligands are another group of ligands that can be used in affinity chromatography. These ligands have been used in numerous applications to purify enzymes and proteins. Dyes and biomimetic ligands are easy to immobilize, inexpensive, stable, and provide stationary phases with high binding capacities. These features make such ligands of interest in largescale or high-throughput separation techniques for the development of protein-based drugs or the examination of protein libraries [50e54]. Dyeligand affinity chromatography often uses triazine dyes to purify albumin and other blood proteins, as well as enzymes and pharmaceutical proteins [50,52,54]. Figure 1.3 shows some typical dye ligands used in dye-affinity chromatography, including Cibracron Blue 3GA and Procion Blue. These

10

1. AFFINITY CHROMATOGRAPHY

FIGURE 1.3

Structures of Cibacron Blue 3GA and Procion Blue, two common binding agents used in dye-ligand affinity chromatography [52].

dyes have two units joined through an amino-bridge. The first unit usually contains an anthraquinone group but can also contain an azo or phthalocyanine group. The second unit usually contains a triazine and forms a scaffold for the binding domain and groups that can be used to attach the ligand to a support [50,53,55]. These dye-ligands often have negatively charged sulfonic groups, which gives them some cationexchange properties [52,56]. Retained proteins may be dissociated from these columns by using nonspecific elution; however, biospecific elution through the addition of a competing agent to the mobile phase is usually the preferred approach [52,57]. To increase the speed and efficiency of dye-ligand purifications, a method known as polymer-shielded dye-affinity chromatography is sometimes employed [58,59]. In this technique, the stationary phase is treated with a water-soluble polymer to prevent nonspecific interactions between proteins and the dye. This method has been effectively used in the purification of enzymes [59]. Other applications of dye ligands include their use in removing toxic macromolecules from biological fluids, such as prion proteins, human immunodeficiency virus-1, and hepatitis B particles [60e62]. There is also growing interest in using computational and combinatorial chemistry, in a method known as biomimetic affinity chromatography, to develop improved dye-based ligands and related binding agents for use in the purification of pharmaceutical proteins [50].

1.7. ANALYTICAL AFFINITY CHROMATOGRAPHY

11

1.6. IMMOBILIZED METAL-ION AFFINITY CHROMATOGRAPHY Immobilized metal-affinity chromatography (IMAC) is also known as metal-chelate affinity chromatography (MCAC). This method was first proposed by Porath et al. in 1975 [63] and is based on the specific interactions between immobilized metal ions and amino acid residues, such as histidine, tryptophan, and cysteine in proteins or peptides [63]. IMAC has become an important tool for the detection and purification of metalloproteins, histidine-tagged proteins, and phosphorylated proteins. Areas in which this method is now used include proteomics [64e66], work with recombinant proteins [67e69], and disease diagnosis [70,71]. The stationary phase in IMAC consists of an immobilized chelating ligand that is complexed with a metal ion. The chelating ligand is attached covalently to a support and used to entrap metal ions via coordinate binding. Agarose was the first support utilized in IMAC, but supports based on other materials, such as silica, are also now common [72,73]. Recent reports have further described the development of supports for IMAC based on cryogels, silica monoliths, and polymethacrylate monoliths [74e76]. Examples of chelating ligands that can be used in IMAC are iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), carboxymethylated-aspartic acid (CM-Asp), 8-hydroxyquinoline, ortho-phosphoserine, and N,N,N’tris(carboxymethyl)ethylenediamine [72,73]. IDA is the most common chelating agent in IMAC [72], followed by NTA [77] and CM-Asp [78] (see examples in Figure 1.4). According to the principles of Lewis acids and bases, the metal ions that are used with these chelating groups can be divided into three categories. Hard metal ions prefer to bind to targets that contain phosphorus, aliphatic nitrogen, and oxygen. Soft metal ions prefer targets that contain sulfur. “Borderline” metal ions tend to bind targets that contain aromatic nitrogens, oxygens, and sulfur groups [72,79]. Columns containing IDA or NTA complexed with Ni(II), Cu(II), Zn(II), Fe(III), or Ga(III) are typically used for the isolation and purification of histidine-tagged proteins and phosphorylated proteins [65,70,71,80,81]. Zr(IV)- and Ti(IV)-IMAC columns are useful for phosphopeptide isolation and phosphoproteome analysis [75,82].

1.7. ANALYTICAL AFFINITY CHROMATOGRAPHY The terms analytical affinity chromatography and quantitative affinity chromatography refer to the use of an affinity column to provide information on the kinetics, thermodynamics, or mechanism of a biological

12

1. AFFINITY CHROMATOGRAPHY

FIGURE 1.4 IMAC supports based on iminodiacetic acid (IDA) or nitrilotriacetic acid (NTA) as chelating agents for metal ions [72,77].

interaction. One approach for this type of study is zonal elution. In this technique, a small plug of a target is injected onto an affinity column that contains an immobilized ligand that binds to this target. The retention time and elution profile of the target are then used to obtain data on the interaction between the analyte and the ligand [83]. This technique was first employed by Andrews, Kitchen, and Winzor in 1973 and Dunn and Chaikin in 1974 for studying lactate synthetase and staphyloccal nuclease [84,85]. Since then, zonal elution has been utilized to examine enzymeinhibitor binding, proteineprotein interactions, and drugeprotein binding [83,86e90]. Applications of zonal elution include its use in comparing the relative affinities of a ligand for injected solutes, determining the strength of these interactions, studying the effects of changing reaction conditions (e.g., pH, solvent, temperature) on soluteeligand binding, and determining the number and location of binding sites for a solute on a ligand [83]. Displacement and competitive binding studies are the most frequent uses of zonal elution in analytical affinity chromatography. An example of such a study is shown in Figure 1.5(a) [91]. In this type of application, a fixed concentration of a competing agent is placed into the mobile phase. The retention of an injected target depends on the concentration of the competing agent, the ability of the target and competing agent to bind the ligand, and the relative amount of active ligand present in the column [90]. It is possible, with this type of study, to measure the equilibrium

1.7. ANALYTICAL AFFINITY CHROMATOGRAPHY

13

FIGURE 1.5 Typical chromatograms obtained by analytical affinity chromatography on an HSA column during (a) zonal elution competitive binding experiments, in which Swarfarin was the injected analyte and racemic verapamil was a mobile phase additive, and (b) frontal analysis experiments, in which S-verapamil was applied as the target. The concentrations shown represent the amount of (a) racemic verapamil or (b) S-verapamil applied to the column. Source: Reproduced with permission from Ref. [91].

constants for the ligand with the target and competing agent and to determine the type of competition occurring between these two solutes for the ligand. Frontal analysis, also known as frontal affinity chromatography (FAC), is another method employed in analytical affinity chromatography. In this technique, a solution containing a known concentration of a target is continuously applied to an affinity column. As the immobilized ligand becomes saturated with the applied target, a breakthrough curve is obtained. This type of curve is shown in Figure 1.5(b). If this experiment is done under conditions that allow a local equilibrium to be established, the moles of target required to reach the mean position of the breakthrough

14

1. AFFINITY CHROMATOGRAPHY

curve can be related to the target’s concentration, the number of binding sites in the column, and the equilibrium constants for the soluteeligand interaction [83]. This method was first used for affinity chromatography in 1975 by Kasai and Ishii [92]. A major advantage of frontal analysis over zonal elution is that the former method can simultaneously provide information on both the equilibrium constants for a soluteeligand interaction and the binding capacity of the column. However, frontal analysis also usually needs a larger amount of target than zonal elution for a binding study [89]. Several other methods have been developed or modified for use in affinity chromatography to obtain information on soluteeligand interactions. Approaches that have been developed for kinetic studies include band-broadening measurements, the split-peak method, peak-fitting methods, peak-decay analysis, and free-fraction analysis [87,88,89,93,106]. These methods have been used in analyzing the rates of drug interactions with serum proteins [107e111], the binding of lectins with sugars [87], the interaction rates between antibodies and antigens or antibody binding proteins [94e96], and the interactions of various solutes with immobilized receptor membrane affinity columns [97e101]. Affinity chromatography has further been used for kinetic and thermodynamic studies in the area of chiral separations based on enzymes or serum proteins as stationary phases [102,103]. For example, band-broadening measurements have been used to study the kinetics of protein-based chiral stationary phases, [94,104], and free-fraction measurements have been employed to analyze the binding of chiral compounds with proteins in solution [97].

1.8. MISCELLANEOUS METHODS AND NEWER DEVELOPMENTS In addition to the methods already described, many other techniques use affinity chromatography for new and improved separation or analysis methods. One example is the combined use of affinity ligands and columns with mass spectrometry in either off-line or on-line methods for work with complex samples. For instance, immunoaffinity columns have been used for sample pretreatment with gas chromatographyemass spectrometry (GCeMS) and liquid chromatographyemass spectrometry (LCeMS). In the case of GCeMS, the analytes must be derivatized after extraction to improve their stability or volatility prior to their analysis by gas chromatography. Affinity chromatography can be coupled on-line with electrospray ionization (ESI) as long as the elution buffer contains only volatile salts, such as ammonium acetate or ammonium formate. For work with matrix-assisted laser desorptioneionization time-of-flight mass spectrometry (MALDIeTOF MS), off-line analysis can be conducted

1.8. MISCELLANEOUS METHODS AND NEWER DEVELOPMENTS

15

by using an affinity column to separate sample components, which are then collected as fractions and combined with the MALDI matrix. On-line procedures are possible if an affinity ligand is immobilized onto a MALDI target and used to bind specific compounds from a sample. Fast atombombardment mass spectrometry (FAB MS) can also be used with off-line or on-line affinity extraction [111]. Several recent examples combine mass spectrometry with affinity ligands. For instance, antibody-conjugated nanoparticles have been utilized for the affinity extraction of plasma antigens prior to analysis by MALDIeTOF MS [112]. Magnetic nanoparticles have also been used as laser desorptioneionization components and as solid-phase extraction probes in MALDIeTOF MS. A nanoprobe-based assay has been employed for the robotic screening of small molecules in serum, based on the weak interactions between mannose and Con A [113]. These types of techniques tend to be relatively simple, cost effective, and rapid and are of interest for targeted proteome profiling [114]. In addition, surface-noncovalent-affinity mass spectrometry (SNAeMS) has been used to isolate, enrich, and sequence glycosaminoglycans that bind to specific proteins [115]. In recent years, affinity columns and ligands have also been incorporated into miniaturized devices. Examples include devices such as nucleic-acid microarrays, protein arrays, antibody arrays, and the use of affinity ligands in microfluidic devices for capillary electrophoresis or liquid chromatography. Nucleic-acid-based microarrays have been useful in genomic studies, and protein arrays (e.g., antibody arrays) have been employed for proteomics. These systems have been used with various detection methods, including absorbance, fluorescence, chemiluminescence, electrochemical detection, and mass spectrometry [116]. Affinity chromatography has been utilized in miniaturized lab-on-a-valve (LOV) devices [117]. For example, the separation, binding, and elution of immunoglobulins on beads coated with protein G were studied using dual-phase detection. This involved optical fibers placed at the outlet of the affinity column to monitor eluted compounds and at the inlet to monitor analyte binding [118]. In another example, biotinylated, singlestrand DNA was captured on a solid support containing streptavidincoated agarose beads, giving a limit of detection of 111 pg single-strand DNA when coupled with on-column fluorimetric detection [119]. Another area of growing interest has been in the creation of synthetic ligands for affinity methods. As an example, molecularly imprinted polymers (MIPs) have been examined as an alternative to antibodies for use in affinity separations, binding assays, and biosensors. Binding and recognition sites are formed during the preparation of these polymers by a process like the one illustrated in Figure 1.6. During this synthesis, functional monomers form a complex with a template, which is usually

16

1. AFFINITY CHROMATOGRAPHY

FIGURE 1.6 General process for the preparation of a molecularly imprinted polymer [121].

chosen to be the target analyte. Cross-linking of the monomers creates a polymer about this template. The template is then extracted, leaving behind a binding site that recognizes and retains the target when it is applied to the support. Although MIPs have been used in some applications for affinity chromatography [120], most of their use up to the present has involved solid-phase extraction methods [121]. MIPs have recently been employed in LOV microdevices [117], such as for the solidphase extraction of riboflavin from food [122] and chlorotriazine herbicides plus their metabolites from environmental samples [123]. Another type of relatively new affinity ligand is an aptamer. Aptamers are nucleic acid ligands that have been designed to be selective for a specific target. Aptamers are generated by separating oligonucleotides that bind to the desired target from a large, random pool of single-strand DNA or RNA. These oligonucleotides are then enriched by a process called SELEX (i.e., the systematic evolution of ligands by exponential

REFERENCES

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enrichment) [35,50]. The SELEX process begins with a protein or other target immobilized in a column. A random library of oligonucleotides is passed through the column, and the ones that are bound are later eluted and amplified. Additional rounds of selection and enrichment are carried out with fresh columns until an aptamer with the desired binding strength is obtained. Aptamers have been used in affinity chromatography to purify a recombinant human L-selectin-immunoglobulin fusion protein from Chinese hamster ovary cells [124] and are promising for use in pharmacology [125], chemical analysis [126,127], and cell biology [50]. In addition, aptamers have been used in microfluidic chips for affinity extraction, separation, and detection. Microfluidic chips have been further used to study aptamer-target interaction and for aptamer selection [128]. Aptamers have been immobilized within monoliths [129,130] and used in biosensors with modified nanoparticles [131].

Acknowledgment This word was supported, in part, by the National Institutes of Health under grants R01 DK069629 and R01 GM044931 and by the NSF/EPSCoR program under grant EPS-1004094.

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[75] Hou C-Y, Ma J-F, Tao D-Y, Shan Y-C, Liang Z, Zhang L-H, et al. Organiceinorganic hybrid silica monolith based immobilized titanium ion affinity chromatography column for analysis of mitochondrial phosphoproteome. J Proteome Res 2010;9:4093e101. [76] Peterka M, Jarc M, Banjac M, Frankovic V, Bencina K, Merhar M, et al. Characterisation of metal-chelate methacrylate monoliths. J Chromatogr A 2006;1109:80e5. [77] Hochuli E, Bannwarth W, Dobeli H, Gentz R, Stuber D. Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent. Biotechnol 1988;6:1321e5. [78] Arnold FH. Metal-affinity separationsea new dimension in protein processing. Biotechnol 1991;9:151e6. [79] Ueda EKM, Gout PW, Morganti L. Current and prospective applications of metal ionprotein binding. J Chromatogr A 2003;988:1e23. [80] Vorackova I, Suchanova S, Ulbrich P, Diehl WF, Ruml T. Purification of proteins containing zinc finger domains using immobilized metal ion affinity chromatography. Protein Expres. Purif 2011;79:88e95. [81] Ye J-Y, Zhang X-M, Young C, Zhao X-L, Hao Q, Cheng L, et al. Optimized IMAC protocol for phosphopeptide recovery from complex biological samples. J Proteome Res 2010;9:3561e73. [82] Zou H-F, Ye M-L, Jiang X-N, Han G-G, Wang F- J. Development of technology and methods for large-scale phosphoproteome analysis. Pacifichem 2010. Honolulu, HI: International Chemical Congress of Pacific Basin Societies; December 15e20, 2010. ANYL-482. [83] Hage DS, Chen J. Quantitative affinity chromatography: practical aspects. In: Hage DS, editor. Handbook of affinity chromatography. 2nd ed. Boca Raton, FL: Taylor & Francis, CRC Press; 2006. p. 596e628. [84] Andrews P, Kitchen BJ, Winzor D. Use of affinity chromatography for the quantitative study of acceptor-ligand interactions: the lactose synthetase system. Biochem J 1973;135:897e900. [85] Dunn BM, Chaiken Im. Quantitative affinity chromatography. Determination of binding constants by elution with competitive inhibitors. Proc Natl Acad Sci USA 1974;71:2382e5. [86] Winsor DJ. Quantitative affinity chromatography: recent theoretical developments. In: Hage DS, editor. Handbook of affinity chromatography. 2nd ed. Boca Raton, FL: Taylor & Francis, CRC Press; 2006. p. 630e62. [87] Chaiken IM, editor. Analytical affinity chromatography. Boca Raton, FL: CRC Press; 1987. [88] Bertucci C, Pistolozzi M, Felix G, Danielson UH. HSA binding of HIV protease inhibitors: a high-performance affinity chromatography study. J Sep Sci 2009;32:1625e31. [89] Schiel JE, Joseph KS, Hage DS. Biointeraction affinity chromatography: general principles and recent development. Adv Chromatogr 2010;48:145e93. [90] Hage DS, Jackson A, Sobansky MR, Schiel JE, Yoo MJ, Joseph KS. Characterization of drug-protein interactions in blood using high-performance affinity chromatography. J Sep Sci 2009;32:835e53. [91] Mallik R, Yoo MJ, Chen S, Hage DS. Studies of verapamil binding to human serum albumin by high-performance affinity chromatography. J Chromatogr B 2008;876:69e75. [92] Kasai K, Ishii S. Quantitative analysis of affinity chromatography of trypsin. A new technique for investigation of protein-ligand interaction. J Biochem 1975;77:261e4. [93] Hage DS, Walters RR, Hethcote HW. Split-peak affinity chromatographic studies of the immobilization-dependent adsorption kinetics of protein A. Anal Chem 1986;58:274e9.

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[94] Yang J, Hage DS. Effect of mobile phase composition on the binding kinetics of chiral solutes on a protein-based HPLC column: interactions of D- and L-tryptophan with immobilized human serum albumin. J Chromatogr A 1997;766:15e25. [95] Denizot FC, Delaage MS. Statistical theory of chromatography: new outlooks for affinity chromatography. Proc Natl Acad Sci USA 1975;72:4840e3. [96] Talbert AM, Tranter GE, Holmes E, Francis PI. Determination of drug-plasma protein binding kinetics and equilibria by chromatographic profiling: exemplification of the method using L-tryptophan and albumin. Anal Chem 2002;74:446e52. [97] Clarke W, Schiel JE, Moser A, Hage DS. Analysis of free hormone fractions by an ultrafast immunoextraction/displacement immunoassay: studies using free thyroxine as a model system. Anal Chem 2005;77:1856e66. [98] Ohnmacht CM, Schiel JE, Hage DS. Analysis of free drug fractions using near infrared fluorescent labels and an ultrafast immunoextraction/displacement assay. Anal Chem 2006;78:7547e56. [99] Mallik R, Yoo MJ, Brisoe CJ, Hage DS. Analysis of drug-protein binding by ultrafast affinity chromatography using immobilized human serum albumin. J Chromatogr A 2010;1217:2796e803. [100] Moaddel R, Wainer IW. Conformational mobility of immobilized proteins. J Pharm Biomed Anal 2007;43:399e406. [101] Jozwiak K, Haginaka J, Moaddel R, Wainer IW. Displacement and nonlinear chromatographic techniques in the investigation of noncompetitive inhibitors with an immobilized a3b4 nicotinic acetylcholine receptor liquid chromatographic stationary phase. Anal Chem 2002;74:4618e24. [102] Patel S, Wainer IW, Lough WJ. Affinity-based chiral stationary phases. In: Hage DS, editor. Handbook of affinity chromatography. 2nd ed. Boca Raton, FL: Taylor & Francis, CRC Press; 2006. p. 571e92. [103] Patel S, Wainer IW, Lough WJ. Chromatographic studies of molecular recognition and solute binding to enzymes and plasma proteins. In: Hage DS, editor. Handbook of affinity chromatography. 2nd ed. Boca Raton, FL: Taylor & Francis, CRC Press; 2006. p. 663e83. [104] Loun B, Hage DS. Chiral separation mechanisms in protein-based HPLC columns, II. Kinetic studies of R- and S-warfarin binding to immobilized human serum albumin. Anal Chem 1996;68:1218e25. [105] Schiel JE, Hage DS. Kinetic studies of biological interactions by affinity chromatography. J Sep Sci 2009;32:1507e22. [106] Schiel JE, Ohnmacht CM, Hage DS. Measurement of drug-protein dissociation rates by high-performance affinity chromatography and peak profiling. Anal Chem 2009;81:4320e33. [107] Tong Z, Schiel JE, Papastavros E, Ohnmacht CM, Smith QR, Hage DS. Kinetic studies of drug-protein interactions by using peak profiling and high-performance affinity chromatography: examination of multi-site interactions of drugs with human serum albumin columns. J Chromatogr A 2011;1218:2065e71. [108] Moore RM, Walters RR. Peak-decay method for the measurement of dissociation rate constants by high-performance affinity chromatography. J Chromatogr A 1987;384:91e103. [109] Chen J, Schiel JE, Hage DS. Noncompetitive peak decay analysis of drug-protein dissociation by high-performance affinity chromatography. J Sep Sci 2009;32:1632e41. [110] Yoo MJ, Hage DS. Use of peak decay analysis and affinity microcolumns containing silica monoliths for rapid determination of drug-protein dissociation rates. J Chromatogr A 2011;1218:2072e8. [111] Briscoe CJ, Clarke W, Hage DS. Affinity mass spectrometry. In: Hage DS, editor. Handbook of affinity chromatography. 2nd ed. Boca Raton, FL: Taylor & Francis; 2006. p. 737e61.

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[112] Lin P-C, Chou P-H, Chen S-H, Liao H-K, Wang K-Y, Chen Y-J, et al. Ethylene glycolprotected magnetic nanoparticles for a multiplexed immunoassay in human plasma. Small 2006;2:485e9. [113] Lin P-C, Tseng M-C, Su A-K, Chen Y-J, Lin C- C. Functionalized magnetic nanoparticles for small-molecule isolation, identification, and quantification. Anal Chem 2007;79:3401e8. [114] Wei C, Yamato M, Wei W, Zhao X, Tsumoto K, Yoshimura T, et al. Genetic nanomedicine and tissue engineering. Med Clin N Am 2007;91:889e98. [115] Prabhakar V, Capila I, Sasisekharan R. Glycosaminoglycan characterization methodologies: probing biomolecular interactions. Meth Mol Biol 2009;534:331e40. [116] Phillips TM. Microanalytical methods based on affinity chromatography. In: Hage DS, editor. Handbook of affinity chromatography. 2nd ed. Boca Raton, FL: Taylor & Francis; 2006. p. 763e87. [117] Miro M, Oliveira HM, Sgundo MA. Analytical potential of mesofluidic lab-on-a-valve as a front end to column-separation systems. Trends Anal Chem 2011;30:153e64. [118] Gutzman Y, Carroll AD, Ruzicka J. Bead injection for biomolecular assays: affinity chromatography enhanced by bead injection spectroscopy. Analyst 2006;131:809e15. [119] Decuir M, Laehdesmaeki I, Carroll AD, Ruzicka J. Automated capture and on-column detection of biotinylated DNA on a disposable solid support. Analyst 2007;132:818e22. [120] Haginaka J. Monodispersed, molecularly imprinted polymers as affinity-based chromatography media. J Chromatogr B 2008:3e13. [121] Haupt K. Molecularly imprinted polymers: artificial receptors for affinity separations. In: Hage DS, editor. Handbook of affinity chromatography. 2nd ed. Boca Raton, FL: Taylor & Francis; 2006. p. 837e56. [122] Oliveira HM, Segundo MA, Lima JLFC, Miro M, Cerda V. Exploiting automatic online renewable molecularly imprinted solid-phase extraction in lab-on-valve format as front end to liquid chromatography: application to the determination of riboflavin in foodstuffs. Anal Bioana. Chem 2010;3(97):77e86. [123] Boonjob W, Yu Y, Miro M, Segundo MA, Wang J, Cerda V. Online hyphenation of multimodal microsolid phase extraction involving renewable molecularly imprinted and reversed-phase sorbents to liquid chromatography for automatic multiresidue assays. Anal Chem 2010;82:3052e60. [124] Romig TS, Bell C, Drolet DW. Aptamer affinity chromatography: combinatorial chemistry applied to protein purification. J Chromatogr B 1999;731:275e84. [125] Bock LC, Griffin LC, Latham JA, Vermaas EH, Toole JJ. Selection of single-stranded DNA molecules that bind and inhibit human thrombin. Nature 1992;355:564e6. [126] German I, Buchanan DD, Kennedy RT. Aptamers as ligands in affinity probe capillary electrophoresis. Anal Chem 1998;70:4540e5. [127] Potyrailo RA, Conrad RC, llington AD, Hieftje GM. Adapting selected nucleic acid ligands (aptamers) to biosensors. Anal Chem 1998;70:3419e25. [128] Xu Y, Yang X, Wang E. Review: aptamers in microfluidic chips. Anal Chim Acta 2010;683:12e20. [129] Zhao Q, Li X-F, Shao Y, Le XC. Aptamer-based affinity chromatographic assays for thrombin. Anal Chem 2008;80:7586e93. [130] Zhao Q, Li X-F, Le XC. Aptamer-modified monolithic capillary chromatography for protein separation and detection. Anal Chem 2008;80:3915e20. [131] Mairal T, Oezalp VC, Sanchez PL, Mir M, Katakis I, O’Sullivan CK. Aptamers: molecular tools for analytical applications. Anal Bioanal Chem 2008;390:989e1007.

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C H A P T E R

2 Derivatization in Liquid Chromatography C.F. Poole Department of Chemistry, Wayne State University, Detroit, Michigan O U T L I N E 2.1. Introduction

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2.2. Reagent Selection 2.2.1. Reagents for UVeVisible Detection 2.2.2. Reagents for Fluorescence and Chemiluminescence Detection 2.2.3. Reagents for Electrochemical Detection 2.2.4. Reagents for Mass-Spectrometric Detection 2.2.5. Reagents for the Formation of Diastereomers 2.2.6. Multifunctional Reagents for the Formation of Cyclic Derivatives 2.2.7. Solid-Phase Analytical Derivatization

27 27 31 38 40 43

2.3. Postcolumn Reaction Detectors 2.3.1. Photoreactors

49 51

2.4. Conclusions

52

References

52

46 48

2.1. INTRODUCTION Derivatization represents an added step in the analysis of a sample and is justified only when it facilitates the isolation, separation, or detection of Liquid Chromatography: Applications http://dx.doi.org/10.1016/B978-0-12-415806-1.00002-4

25

Copyright Ó 2013 Elsevier Inc. All rights reserved.

26

2. DERIVATIZATION IN LIQUID CHROMATOGRAPHY

a compound or leads to more-robust results by enhancing the stability of the compound, reduces matrix interferences, improves the reproducibility of the method, or simplifies the operational steps in the method. Many compounds lack a suitable chromophore (UV-Visible detection), fluorophore (fluorescence and chemiluminescence detection), electrophore (electrochemical detection) or possess low ionization efficiently (mass spectrometric detection) for detection at anticipated sample concentrations. For compounds with reactive functional groups, simple chemical reactions allow the required detection characteristics to be acquired by the compound by modifying its chemical structure to that required for facile detection or serves to minimize matrix interference by moving the compound to a position in the chromatogram where interference in the detector response is minimal. Replacing a reactive functional group (or groups) with a substituent of different polarity, and perhaps, a relatively complex structure, alters the separation characteristics of the compound, and if favorable, might lead to an improvement in the separation, but just as easily, might make the compounds more difficult to separate or extend the separation time. Individual reagents behave differently in this respect, and a suitable reagent to enhance sample detection must also not lead to an inferior or unacceptable separation. Since derivatizing reactions can be performed either precolumn, and affect the separation of the compounds, or postcolumn, and affect only the detection step, there is some flexibility in the selected approach. Occasionally, the derivatization reaction is performed close to the start of an isolation procedure to increase the recovery of the analytes, to ensure their stability to the conditions employed in the isolation procedure, or to increase the selectivity of the method for the target compounds. For precolumn derivatization, the selected reaction must be quantitative, or nearly so, and free from by-products. Reaction conditions can usually be optimized free of time constraints. When possible, samples are processed in batches with a high level of automation and control of the reaction conditions but can also be performed manually or for individual samples. In general, a simple method must be available to separate excess reagent and other products from the derivatives, if these interfere in the separation or detection of the derivatives. This is quite likely if the reagent and derivative share a common core structure responsible for the detector response. Postcolumn reactions occur in a continuous-flow reactor and need not be quantitative, so long as they are reproducible. Reaction times are usually constrained by the design of the reactor and should be sufficiently fast that the column resolution is not destroyed by diffusion in the reactor device. Although artifact formation is rarely a problem, both the reagent and by-products (if any) must either not respond to the detector under the same conditions used to detect the analytes or must be easily separated

2.2. REAGENT SELECTION

27

from the analytes after derivatization and before detection. The latter operation can be performed by extraction in a solvent-segmented stream but adds to the complexity of the reactor design. Although postcolumn derivatization allows independent optimization of the conditions for separation and detection, it is not uncommon for the optimum solvent composition for the separation to be incompatible with the requirements of the derivatization reaction. This may limit some applications of postcolumn detection.

2.2. REAGENT SELECTION A derivatizing reagent can be considered to comprise two parts: a reactive functional group that controls the rate, extent, and selectivity of the chemical reaction (derivative formation) and a structural unit that provides a favorable detector response to the derivative and, in precolumn reactions, modifies the isolation and separation properties of the analytes. Some typical reagent functional groups and the complementary analyte functional groups they react with are summarized in Table 2.1 [1e4]. In selecting a reagent based on one of these groups, important considerations are the reaction time, the completeness of the reaction, the conditions required for the reaction and whether these affect the stability of the analytes, the stability of the derivatives (storage properties), and the ease with which excess reagent can be separated from the derivatives. Depending on the sample type, whether the reaction requires completely anhydrous conditions or occurs in aqueous solution can be an important factor. The desire to use fast and quantitative reactions places a restriction on the number of reagents that are commonly used and the types of analyte functional groups that can be easily derivatized.

2.2.1. Reagents for UVeVisible Detection The majority of reagents used to introduce a chromophore contain an aromatic ring with different substituents to optimize the absorption wavelength or the molar absorption coefficient. Ideally, the chromophoric group should have a large molar absorption coefficient (>10,000) at some convenient wavelength for detection limits at the low nanogram level. Some representative chromophores are summarized in Table 2.2. There is a preference for reagents that absorb at relatively long wavelengths, since this minimizes interferences from the sample matrix and solvents used in the separation. Since most solvents absorb strongly at wavelengths 240 nm are preferred. Many reagents forming colored derivatives possess a higher

28

2. DERIVATIZATION IN LIQUID CHROMATOGRAPHY

TABLE 2.1 Classification of Reactions Based on the Reactive Functional Group of the Reagent Reagent reactive functional group

Analyte functional group

Reaction product

R-OH

R-CH2OR

R-COOH

R-CH2COOR

R-NHR

R-CH2NR2

R-OH

R-COCH2OR

R-COOH

R-COCH2OCOR

R-NHR

R-COCH2NR2

R-SH

R-COCH2SR

R-OH

A-OR

R-NH-R

A-NR2

R-SH

A-S-R

R-OH

R-NHCOOR

R-NCO

R-NHCOOR

R-OH

R-COOR

R-NHCO-R

R-NR(COR)2

R-NH-R

R-CONR2

R-SH

R-COSR

R-OH

R-COOR

R-NH2

R-CHN-R

R-NHNH2

R-NHNCHR

R-SO2NHNH2

R-SO2NHNCHR

R-CHO

R-CH2NHR (after reduction)

R-COOH

R-NHCOR

R-CO-R

R-NCR2

ACTIVATED HALIDE R-CH2Br

R-COCH2Br

A-Cl

ACYL AZIDE R-CON3

ACYL HALIDE R-COCl

ACYL NITRILE R-COCN ALDEHYDE R-CHO

AMINE R-NH2

29

2.2. REAGENT SELECTION

TABLE 2.1 Classification of Reactions Based on the Reactive Functional Group of the Reagentdcont’d Reagent reactive functional group

Analyte functional group

Reaction product

R-OH

R-COOR

R-NH2

R-CONHR

R-NH-R

R-CONR2

R-OH

R-COOR

R-NH2

R-CONHR

R-OH

R-OCOOR

R-NH2

R-OCONHR

R-NHR

R-OCONR2

R-NHCOR

R-OCONRCOR

R-COOH

R-CHOCOR

R-CHO

R-CONHNCHR

R-COOH

R-CONHNHCOR

R-COO

R-COOR

R-CHO

RONCHR

R-CO-R

RONCR2

R-OH

R-NHCOOR

R-NHR

R-NHCONR2

R-NHR

RNHCSNR2

R-SH

R-NHCSSR

R-COOH

R-COOR

ANHYDRIDE R-COOCO-R

CARBOXYLIC ACID R-COOH

CHLOROFORMATE R-OCOCl

DIAZOMETHANE R-CHN2 HYDRAZIDE R-CONHNH2

HYDROXYL R-OH HYDROXYLAMINE RONH2

ISOCYANATE R-NCO

ISOTHIOCYANATE R-NCS

ISOUREA R2HCNCRNHCHR2

(Continued)

30

2. DERIVATIZATION IN LIQUID CHROMATOGRAPHY

TABLE 2.1 Classification of Reactions Based on the Reactive Functional Group of the Reagentdcont’d Reagent reactive functional group

Analyte functional group

Reaction product

R-NH2

R-CR2

R-NHNH2

R-NHNCR2

R-SO2NHNH2

R-SO2NHNCR2

R-OH

R-SO2OR

R-NH2

R-SO2NHR

R-NHR

R-SO2NR2

R-NHCOR

R-SO2NRCOR

R-CHO

R-SO2NHNCHR

R-COOH

R-SO2NHNHCOR

R-CO-R

R-SO2NHNCR2

KETONE R-CO-R

SULFONYL CHLORIDE R-SO2Cl

SULFONYL HYDRAZINE R-SO2NHNH2

TRIFLUOROMETHYLSULFONATE R-SO3CF3

R-COOH

R-COOR

Note: R is any part of the reagent or analyte not directly involved in the reaction and A is an aromatic ring containing the reactive functional group for the reagent.

TABLE 2.2 Some Common Chromophores and Their Properties Chromophore

Wavelength for maximum absorption (nm)

Molar absorption coefficient at 254 nm

4-Nitrophenyl

265

5,200 >10,000

3,5-Dinitrobenzyl 4-Chlorobenzoate

236

6,300

4-Nitrobenzoate

254

>10,000 >10,000

2,4-Dinirophenyl Toluoyl

236

5,400

Anisyl

262

16,000

Phenacyl

250

10,000

4-Bromophenacyl

260

18,000

2-Naphthacyl

248

12,000

2.2. REAGENT SELECTION

31

molar absorption in the UV region, and, in general visible detection is less common. However, visible detection can provide higher selectivity for analytes with a high matrix burden, since most organic compounds are transparent in the visible region. Some common reagents for derivatizing specific analyte functional groups are summarized in Table 2.3 [1e12]. Alcohols are usually converted to esters by reaction with an acid chloride in the presence of a base catalyst. For compounds with labile functional groups, reaction with a phenyl isocyante to form phenylurethanes can be performed under mild conditions. Alcohols in aqueous solution can be derivatized with 3,5dinitrobenzoyl chloride. Amines are usually derivatized with an acid chloride to form phenyl substituted amides or sulfonamides. Sanger’s reagent, 2,4-dinitro-1-fluorobenzene, originally introduced for the identification of N-terminal amino acid residues in proteins, has been widely used for the derivatization of amines. Carbamate and phenylacetate reagents can be used to derivatize amines under mild conditions without a catalyst. Phenyl isothiocyanate is commonly used for the precolumn derivatization of amino acids (phenylthiocarbamyl derivatives) for separation by reversed-phase liquid chromatography [13]. Alkylation of carboxylic acids with haloalkyl reagents in the presence of a catalyst in aqueous or anhydrous conditions is widely used [6]. O-(4-nitrobenzyl)N,N’-diisopropylurea facilitates the derivatization of carboxylic acids under mild conditions without addition of a catalyst. Aldehydes and ketones can be selectively derivatized in the presence of other functional groups using reagents with a hydroxylamine or hydrazine reactive group [9]. Aldehyde groups in monosaccharides and more complex glycogens are detected after derivatization with a number of reagents containing a reactive amine group [10].

2.2.2. Reagents for Fluorescence and Chemiluminescence Detection Fluorescence detection is used for those methods that require either low detection limits or higher selectivity. Few organic compounds are naturally fluorescent and nonspecifc interferences in the determination of fluorescent compounds are reduced compared with UVeVisible absorption methods. In addition, the fluorescence process requires excitation at one wavelength and emission at a different (longer) wavelength, which further enhances both selectivity and detectability. The choice of excitation and emission wavelengths provides a possible mechanism to distinguish between fluorescent compounds in a mixture improving selectivity. The longer wavelength of the emission signal minimizes the contribution of scattered light from the excitation wavelength, resulting in low background noise and the possibility of

32

2. DERIVATIZATION IN LIQUID CHROMATOGRAPHY

TABLE 2.3 Reagents for the Introduction of a Chromophore into Compounds with Complementary Reactive Functional Groups Analyte functional group

Derivatizing reagent

Reference

Hydroxyl

3,5-Dinitrobenzyl chloride

[1,2]

Benzoyl chloride

[1,2]

4-Nitrobenzoyl chloride

[1,2]

4-Nitrophenyl chloroformate

[3]

Phenyl isocyanate

[1,2]

4-Dimethylaminophenyl isocyanate

[3]

3,5-Dinitrobenzyl chloride

[1,2]

4-Toluenesulfonyl chloride

[3]

Diazo-4-aminobenzonitrile

[3]

4-Bromophenacyl bromide

[6]

O-(4-nitrobenzyl)-N,N’diisopropylisourea

[1,2]

N-Chloromethyl-4-nitrophthalimide

[3]

Methylphthalimide

[3]

Naphthyldiazomethane

[6]

4-Nitrobenzyl bromide

[6]

4-Methoxyaniline

[6]

1-Naphthylamine

[6]

2,4-Dinitrophenylhydrazine

[9]

4-Nitrobenzylhydroxylamine

[1,2]

Isocyanates

N-4-Nitrobenzyl-N-n-propylamine

[1,3]

Thiols

4-Dimethylaminoazobenzene-4’sulfonyl chloride

[9,11,12]

1-Benzyl-2-chloropyridinium bromide

[10]

N-Ethylmaleimide

[10]

2-Chloro-1-methylquinolinium tetrafluoroborate

[10]

3,5-Dinitrobenzoyl chloride

[1,2]

4-Methoxybenzoyl chloride

[1,2]

n-Succinimidyl-4-nitrophenylacetate

[3]

4-Nitrobenzoyl chloride

[1,2]

Phenol

Carboxylic acid

Ketones and aldehydes

Amines

33

2.2. REAGENT SELECTION

TABLE 2.3 Reagents for the Introduction of a Chromophore into Compounds with Complementary Reactive Functional Groupsdcont’d Analyte functional group

Derivatizing reagent

Reference

4-Toluenesulfonyl chloride

[1,2]

2-Naphthacyl bromide

[1,2]

2,4-Dinitro-1-fluorobenzene

[1e3]

2-Naphthalenesulfonyl chloride

[1e3]

phenyl isocyanate

[8]

4-(Dimethylamino)benzaldehyde

[8]

using higher amplification to maximize the signal. This, combined with the higher excitation energy of sources used for fluorescence, is largely responsible for the two or more orders of magnitude increase in signal that allows the detection of fluorescent compounds at low pictogram amounts. The fluorescence signal, however, is subject to a number of matrix interferences that can affect both the signal intensity and the emission wavelength [14]. Impurities in the mobile phase, particularly oxygen, may quench the signal from low concentrations of fluorescent compounds, even after solvent degassing. Provided that the mobile phase contains a minimum of 1% (v/v) methanol, oxygen can be efficiently removed by a short precolumn containing an immobilized catalyst located between the pump and the injector [15]. Both the emission wavelength and fluorescence intensity of ionizable fluorophores are critically dependent on pH and solvent hydrogen-bonding interactions. Intensity changes on an order of magnitude and large shifts in the emission wavelength are possible for neutral fluorophores that undergo strong solvent interactions. Many fluorophores exhibit significant temperature dependence with an increase in temperature of 1 C causing a 1e2% decrease in fluorescence intensity and short-term temperature fluctuations increasing the background detector noise. Coeluting components of the sample matrix can also modify the observed response compared with standards prepared in neat solvent. Reagents containing fluorophores can have complex structures and some representative reagents are shown in Figure 2.1 [1e6,16e20]. A number of reagents have a common fluorophore and different reactive functional groups. This is because the number of stable and intense fluorophores is not that large, while the number of potential applications can be significantly increased by variation in the reactive group. When selecting a reagent, those forming derivatives with a relatively high

34

2. DERIVATIZATION IN LIQUID CHROMATOGRAPHY

FIGURE 2.1

Reagents for introducing a fluorophore into compounds with a complementary reactive functional group. Reagents: 1 ¼ dansyl chloride; 2 ¼ dabsyl chloride; 3 ¼ dansyl hydrazine; 4 ¼ fluorescamine; 5 ¼ 2,4-dihydro-6,7-dimethoxy-4-methyl-3-oxoquinoxaline-2-carbonyl azide; 6 ¼ o-phthalaldehyde; 7 ¼ fluorenylmethyloxycarbonyl chloride; 8 ¼ 4-bromomethyl-7-methoxycoumarin; and 9 ¼ 4-chloro-7-nitrobenzofurazan.

quantum efficiency and long-wavelength fluorescence are the most useful. Fluorophores with a large Stoke’s shift (difference between the excitation and emission maxima) are usually favored and provide higher selectivity when analyzing complex mixtures. Some common reagents for derivatizing compounds with reactive functional groups are summarized in Table 2.4 [1e6,16e27]. Reagents containing a methoxycoumarin, dimethylaminonaphthalene, or benzoxadiazole fluorophore usually provide lower detection limits than those containing polycyclic aromatic hydrocarbon or acridine fluorophores. Dansyl chloride (and its analogs) is a popular multipurpose reagent [1e3]. Dansyl chloride forms derivatives with primary and secondary amines readily, less rapidly with phenols and imidazoles, and very slowly with alcohols. The reaction media is usually an aqueous organic solution (e.g., 1:1 acetoneewater) buffered to pH 9.5e10. Several analogs of dansyl chloride are also in use. 5-Dibutylaminonaphthalenesulfonyl chloride forms derivatives with better storage capability and facilitates the extraction of hydrophilic compounds. Dansyl hydrazine is a selective reagent for the detection of aldehydes and ketones (it will also form

35

2.2. REAGENT SELECTION

TABLE 2.4 Reagents for the Introduction of a Fluorophore into Compounds with Complementary Reactive Functional Groups Analyte functional group

Derivatizing reagent

Reference

Hydroxyl

4-Dimethylamino-1-naphthoyl nitrile

[1e3]

9-Anthronyl nitrile

[1e3,22]

7-[(Chlorocarbonyl)methoxy]-4methylcoumarin

[1,5]

3,4-Dihydro-6,7-dimethoxy-4-methyl3-oxo-quinoaline-2-carbonyl azide

[3e5]

1-Naphthyl isocyanate

[23]

4-(7-diethylaminocoumarin-3-yl)benzoyl cyanide

[24]

5-Dimethylaminonaphthalene-1-sulfonyl chloride

[1,2]

4-Bromomethyl-7-methoxycoumarin

[1,2]

4-Bromomethyl-7-methoxycoumarin

[1e3,6]

9-(Chloromethyl)anthracene

[6]

9-Anthradiazomethane

[6]

9-(Hydroxymethyl)anthracene

[3,6]

2-Bromoacetyl-6-methoxynaphthalene

[25]

5-Dimethylaminonaphthalene1-sulfonylhydrazine

[1e3]

2-Aminooxy-N-[3-(5dimethylaminonaphthalene)-1sulfonylaminoprpyl]acetamide

[26]

4-Hydrazino-7-nitrobenzofurazan

[19]

N-(9-Acridinyl)maleimide

[12]

N-[4-(2-benzoxazoyl)phenyl]maleimide

[12]

4-(Aminosulfonyl)-7-fluorobenzo-2-oxa1,3-diazole

[12]

5-Dimethylaminonaphthalene-1-sulfonyl chloride

[1e3]

1-Pyrenesulfonyl chloride

[27]

Fluorescamine

[1e3]

o-Phthalaldehyde

[17]

Naphthalene-2,3-dicarboxaldehyde

[20]

Phenol

Carboxylic acids

Ketones and aldehydes

Thiols

Amines

(Continued)

36

2. DERIVATIZATION IN LIQUID CHROMATOGRAPHY

TABLE 2.4 Reagents for the Introduction of a Fluorophore into Compounds with Complementary Reactive Functional Groupsdcont’d Analyte functional group

Derivatizing reagent

Reference

4-Fluoro-7-nitrobenzofurazan

[19]

Fluorenylmethyloxycarbonyl chloride

[17]

3-Chlorocarbonyl-6,7-dimethoxy-1-methyl2(1H)- quinoxalinone

[27]

derivatives with carboxylic acids) and dansyl aziridine is a selective reagent for the detection of thiols. Several reagent-containing maleimide or iodoacetamide groups that form adducts with thiols are also widely used [12]. Numerous reagents have been described for the detection of amines, and in particular, for the on-line detection of amino acids separated by liquid chromatography [17,18,27]. Fluorescamine reacts with water, alcohols, and amines but forms only stable fluorescent products with primary amines. Quantitative reaction in an aqueouseorganic solvent mixture buffered at pH 8e9.5 occurs in less than a minute, facilitating postcolumn detection strategies; hydrolysis of the reagent occurring concurrently. o-Phthalaldehyde reacts rapidly, 1e2 min, with amines in an aqueous solution buffered to pH 10 in the presence of a thiol (2-mercaptoethanol, ethanthiol, etc.) to form a highly fluorescent 1-(alkylthio)-2-alkylisoindole, Figure 2.2. This reaction can be used for both precolumn and postcolumn derivatization of amino acids. Isoindole derivatives are unstable and cannot be stored [20]. In some sources it is claimed that the isoindole derivatives formed by naphthalene-2,3-dicarboxaldehde in the presence of the cyanide ion are more stable and a better choice as a precolumn derivatizing reagent for amines [20]. When stability of the derivatives is important, amide derivatives containing an acridine or carbazole fluorophore provide a further alternative [21]. These are conveniently prepared using reagents with a reactive acetyl chloride group. Fluorenylmethyloxycarbonyl chloride reacts with amines in basic solution in less than one

FIGURE 2.2 Reaction of o-phthalaldehyde in the presence of 2-mercaptoethanol to form a highly fluorescent 1-(alkylthio)-2-alkylisoindole.

2.2. REAGENT SELECTION

37

minute. The derivatives can be stored but should be isolated from excess reagent, since the derivatives and reagent have similar fluorescent properties. Alternatively, the reaction can be quenched by adding an excess of an amine, easily separated from the derivatives of interest. 4-Chloro7-nitrobenzofurazan forms strongly fluorescent derivates with amines but only weakly or nonfluorescent products with anilines, phenols, and thiols, often in poor yield [19]. Although the reagent is nonfluorescent, it interferes in the fluorescence of the derivatives and excess reagent should be removed prior to separation. The 4-fluoro-7-nitrobenzofurazan analog is more reactive and has replaced 4-chloro-7-nitrobenzofurazan in many of its conventional applications. Only reagents with a hydroxyl group can be considered chemically selective for the derivatization of carboxylic acids. These reactions usually require an activating agent as a catalyst. Reagents containing the diazoalkane group react rapidly and smoothly with carboxylic acids in aqueous or anhydrous solvents to form esters [6]. Diazoalkane reagents do not store well and may require purification before use [16]. Other common reagents for derivatizing carboxylic acids are alkylating reagents which simultaneously react to various extents with phenols, thiols, and amides. 4-Bromomethyl-7-methoxycoumarin in anhydrous acetone smoothly alkylates carboxylic acids in the presence of potassium carbonate solvolysed by a crown ether catalyst [6,16]. Coumaric acid salts formed by the base catalysed solvolysis of the lactone ring of the reagent are potential interfering fluorescent by-products. Reagents for the derivatization of alcohols vary significantly in their reaction rates. Reagents with acid chloride or nitrile groups require anhydrous conditions. 3,4-Dihydro-6,7-dimethoxy-4-methyl-3-oxo-quinoxaline-2-carbonyl azide is the only choice for tertiary alcohols [3e5]. Many of the reagents used to derivatize alcohols also react with other functional groups, and this can be a problem when specificity is desired or chromatograms are complicated by a large number of unrelated compounds [5]. Detection limits for alcohol derivatives are also frequently no more than modest, leaving some scope for the development of new reagents. The limiting factor for the measurement of fluorescence is background noise resulting from stray light and instability of the source. A detection method that does not require optical excitation, such as chemiluminescence, should be capable of lower detection limits, perhaps by one to three orders of magnitude, as well as affording a wider linear response range [5,28e30]. Chemiluminescence is the emission of light observed when a chemical reaction yields an electronically excited intermediate or product, which either luminesces (direct chemiluminescence) or donates its energy to a compound containing a fluorophore responsible for the emission (indirect or sensitized chemiluminescence). Except for the

38

2. DERIVATIZATION IN LIQUID CHROMATOGRAPHY

excitation process, the production of luminescence is the same as for fluorescence. The compounds of interest are either derivatized with a reagent containing a chemiluminergenic group (direct method) or reacted with a conventional fluorogenic reagent (indirect method), which after separation, generates chemiluminescence in a postcolumn reaction. The first approach could be considered general, while for the second approach, only specific fluorophores generate meaningful chemiluminescence. The most popular of the chemiluminergenic reagents are based on luminol (5-amino-2,3-dihydro-1,4-phthlazine-1,4-dione) or isoluminol (6-amino analog), which react with an oxidizing agent, such as hydrogen peroxide and potassium hexacyanoferrate (III), in an alkaline medium, in a postolumn reactor, to produce chemiluminescence from the resultant excited aminophthalate dianion. For the indirect approach, the peroxyoxalte chemiluminescence reaction is the most widely used. This reaction involves the oxidation of an aryl oxalate ester with hydrogen peroxide, leading to the formation of an energy-rich intermediate(s) capable of exciting a large number of fluorophores. The intermediate forms a charge transfer complex with the fluorophore, donating one electron to the intermediate, which is transferred back to the fluorophore, raising it to an excited state, which relaxes by emission (and other processes). The process is efficient for fluorophores with high quantum efficiency, a low oxidation potential, and low singlet energy. As a consequence, it is not effective for all fluorophores. Electrochemically produced chemiluminescence employing the oxidation of the tris(2,2’-bipyridyl)ruthenium (II) complex as the reagent species provides an alternative excitation mechanism but has found few applications in liquid chromatography. Chemiluminescence applications are not as common as those based on conventional fluorescence. The much lower detection limits are not always needed, and the greater complexity of the postcolumn reaction conditions detracts from its use for routine methods. The chemiluminescence emission is strongly dependent on the reaction conditions (reagent concentration, pH, solvent composition, temperature, time, etc.), and fluctuations in these conditions have a major effect on precision.

2.2.3. Reagents for Electrochemical Detection Electrochemical detection complements fluorescence detection in providing an alternative approach to obtain low detection limits (nanomolar) and improved selectivity compared with UVeVisible detection. The common reagents used to install an electrophore into compounds with a reactive functional group are the same as those used to introduce a chromophore or fluorophore, Table 2.5 [31e33]. The isoindole derivatives of amines and amino acids formed by reaction with o-phthalaldehyde (postcolumn) or naphthalene-2,3-dicarboxaldehyde (precolumn)

TABLE 2.5

Reagents for the Introduction of an Electrophore into Compounds with Complementary Reactive Functional Groups

Analyte functional group

Derivatizing reagent

Hydroxyl

4-Nitrophenacyl chloride 3-Ferrocene acyl azide

Carboxylic acid

Amines

[1e3,31] 400

[33]

Ferrocenecarbonyl chloride

[33]

1-(2,5-dihydroxyphenyl)bromoethanone

[1e3]

2-Methyl-1,4-naphthaquinone

[32]

3-Bromo-1,1-dimethylferrocene

[33]

4-Aminophenol

700

[1e3,31]

4-Nitrophenylhydrazine

800e1100

[1e3,33]

2,4-Dinitrophenylhydrazine

700

[1e3,33]

o-Phthalaldehyde

500e850

[33]

Naphthalene-2,3-dicarboxaldehyde

700

[20]

Ferrocenecarboxylic acid

400e500

[33]

Ferrocenesulfonyl chloride

[33]

Ferroceneisothiocyanate

[33]

Trinitrobenzenesulfonic acid

Thiols

Reference

e600 to e850

[31] [31]

N-(4-dimethylaminophenyl)maleimide

[12]

Maleimide

[31] 900e1000

[33]

39

4-Chloro-7-nitrobenzofurazan

N-(Ferrocenyl)maleimide

2.2. REAGENT SELECTION

Ketones and aldehydes

Potential vs Ag/AgCl (mV)

40

2. DERIVATIZATION IN LIQUID CHROMATOGRAPHY

can be determined at low levels with an aperometric detector as an alternative to fluorescence detection [31]. A useful feature of this reaction is that the reagents are not electrochemically active at the potential used to detect the derivatives. A number of reagents containing ferrocene with different reactive groups facilitate reactions with a wide range of compounds containing complementary reactive functional groups forming derivatives with a relatively low oxidation potential (200e600 mV) [33]. This allows selective detection of the ferrocene derivatives, even in the presence of other aromatic compounds. In general, reagents for oxidative electrochemical detection are preferred, due to operational problems in the reduction mode resulting from the difficulty of excluding oxygen from the solvents used to prepare samples and mobile phases. Electrochemical detection is best performed in a buffered aqueous solution, and the presence of organic solvent and variations in pH and ionic strength can degrade the detector response. To minimize changes in the detector response accompanying changes in the mobile phase composition, separations are usually limited to the isocratic mode. Excess reagent and reaction by-products are a possible source of interference that may require isolation of the derivatives prior to detection. From the perspective of typical applications, electrochemical detection of compounds in their native form (when they contain an electrophore) is more common than the detection of compounds containing an electrophore introduced by derivatization. Also, the number of applications in which the electrochemical detector was used for the detection of electrophoric derivatives is only a small fraction of those employing fluorescence detection and fluorophore-containing derivatives.

2.2.4. Reagents for Mass-Spectrometric Detection Many laboratories currently use mass spectrometry for detection based on one or more characteristic mass ions or for identification by fragmentation of one or more characteristic mass ions [14]. When used in combination with liquid chromatography the most important ionization methods are electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI). Both are soft ionization methods that produce molecular ions, protonated or deprotonated molecular ions, or catonized molecular ion adducts with very little excess energy and rather limited fragmentation. ESI and APCI differ in several ways, but of note for developing a role for derivatization for mass-spectrometric detection is that ionization in ESI takes place in solution, with ions being expelled into the gas phase, while APCI ionization employs reactions with a plasma of thermalized electrons and solvent-derived ions occurring in the gas phase. In terms of application, the two techniques are complementary rather than competitors. ESI is capable of generating multiply charged

2.2. REAGENT SELECTION

41

ions that allow the identification of large molecules on mass spectrometers of limited scan range. This is of value for the analysis of biopolymers and similar compounds but is not important (or useful) for the analysis of compounds of low mass. In the majority of cases, ESI is selected for the analysis of compounds that exist in an ionic form in solution (this includes compounds that can be easily ionized by buffering the mobile phase or readily form ion adducts with additives added to the mobile phase). APCI is selected for compounds of low to medium polarity and, in particular, for compounds containing elements or groups with a high proton or electron affinity. Derivatization to enhance ionization efficiency is far less common in the case of APCI for small molecules than with ESI. In both cases, derivatization is employed primarily to enhance the ionization efficiency of compounds that are otherwise difficult to detect, to minimize interference in the ionization process by matrix components (largely ion suppression problems), and to produce stable product ions in tandem mass spectrometry for compound class identification or low-level detection using selected reaction monitoring. A selection of derivatizing reagents for mass-spectrometric detection is summarized in Table 2.6 [34e44]. Some compounds of modest polarity containing hydroxyl, aldehyde, and ketone functional groups are usually poorly ionized by ESI, and derivatives with a charged or chargeable group can dramatically improve detection limits. Low-mass carboxylic acids can be determined as negative ions, but sensitivity is usually low due to high background noise. Lower detection limits are possible after derivatization by moving the characteristic mass ions to regions were background contributions are reduced. Compounds having an amino group are easily protonated under acidic conditions and suitable for ESI mass spectrometry. Derivatization can improve their separation and lower detection limits by minimizing matrix interferences. To enhance the ionization efficiency in ESI, the derivative should contain either an ionic group or functional group that is easily charged under ESI conditions. In addition, either the reagent or analyte should contain a low polarity region, since during the ESI process, compounds of low polarity accumulate at the surface of the drop and transfer to the gas phase more readily than those in the drop interior. For low mass compounds, derivatives of higher molecular mass are preferred, as they produce characteristic ions in the higher mass range, where noise caused by matrix ions is lower and a cleaner signal is obtained. For high-mass compounds, low-mass derivatives are generally preferred. The size and hydrophobicity of the reagent also influences the chromatography. For almost all applications described so far, reversed-phase liquid chromatography was utilized for the separation. The derivative employed should be tailored to provide suitable separation and mass-spectrometry properties, and for compounds with the same reactive functional groups but

42

2. DERIVATIZATION IN LIQUID CHROMATOGRAPHY

TABLE 2.6 Reagents for the Introduction of Ionic or Ionizable Groups into Compounds with Complementary Reactive Functional Groups for Detection by Mass Spectrometry Analyte functional group Derivatizing reagent

Reference

Hydroxyl or phenol

4-(4-Methyl-1-piperazyl)-3-nitrobenzoyl azide

[42]

Dansyl chloride

[37,39]

Ferrocenecarbonyl chloride

[33]

Tris(2,4,6-trimethoxyphenyl)phosphonium propylamine bromide

[40]

Aminoarenesulfonic acid

[34]

3-Hydroxy-1-methylpiperidine

[37]

2-Hydrazinopyridine

[44]

2,4-dinitrophenylhydrazine

[39]

2-Hydrazino-1-methylpyridine

[41]

Girard P and T reagents

[37]

5-N-(succinimidoxy-5-oxopentyl) triphenylphosphonium bromide

[38]

p-N,N,N-trimethylammonioanilyl N’hydroxysuccinimidylcarbamate iodide

[43]

9-Fluorenylmethoxycarbonyl chloride

[34,37]

Dansyl chloride

[34,37]

2-Fluoro-1-methylpyridinium 4-toluenesulfonate

[34]

Carboxylic acid

Ketones and aldehydes

Amines

Thiols

different size, different derivatizing reagents may be required. The ESI process is generally more efficient for mobile-phase compositions containing a high volume fraction of organic solvent and the optimum separation conditions by liquid chromatographyemass spectrometry (LCeMS) may be different to those selected for optical detection. Highmass derivatizing reagents may react either slowly or incompletely with some compounds, due to steric hindrance. This can be a limitation for some applications or require more complex protocols using internal standards to improve precision. Tandem mass spectrometry is being increasingly used for trace analysis and identification. Derivatives can assist in this process by fragmenting into a small number of stable and intense product ions [37e39,44]. If the product ion is characteristic of the derivative, then the parent ions for all compounds that form a similar derivative can be identified. Alternatively, selected reaction monitoring of characteristic

2.2. REAGENT SELECTION

43

product ions is used to facilitate the detection of target compounds at low levels in complex mixtures only partially separated by liquid chromatography. For derivatives with an ionic group, the fixed charge may be responsible for directing fragmentation, producing intense characteristic ions suitable for low-level detection.

2.2.5. Reagents for the Formation of Diastereomers Enantiomers are stereoisomers that behave identically in an achiral environment but may exhibit different properties in a chiral environment. These differences in a chiral environment explain the general interest in enantiomers in biology and the environment. They also provide the mechanism that allows their separation by liquid chromatography. The interaction or reaction between single enantiomers to form diasteromers is the basis of their separation by liquid chromatography. In practice, two complementary strategies based on this mechanism are used. The most popular strategy, the direct method, involves the formation of transient diastereomer association complexes between a mixture of enantiomers and a chiral stationary phase or chiral additive in the mobile phase (see Chapter 4 for details). The alternative approach, the indirect method, involves formation of diasteromers by a chemical reaction employing a single enantiomer reagent. The last two decades have witnessed enormous advances in the direct approach, which tends to dominate enantiomer separations today. However, the formation of covalent diastereomer derivatives remains a viable option for many compounds, and sometimes it is the only option available. The formation of diasteriomers by itself does not guarantee that a separation can be obtained; it merely establishes that a separation may be possible using a conventional (achiral) separation system. A useful separation depends on the differences in physical properties of the diastereomers, the extent to which these differences affect the relative distribution of the diasteromers in a biphasic separation system, and the chromatographic efficiency of the separation system. Several factors need to be considered in the selection of a suitable single enantiomer reagent for a particular application [2,45,46]. The analyte must contain at least one functional group capable of reacting with the derivatizing reagent. Several hundred single enantiomer reagents have been described for reaction with amine, alcohol, phenol, and carboxylic acid functional groups, mainly, as well as a smaller number for reaction with aldehydes, ketones, thiols, epoxides, and the like, Table 2.7 [2,45e50]. Some representative structures are shown in Figure 2.3. The most widely used reactions employ relatively mild conditions and short reaction times, to minimize racemization. The diastereomer transition states should have similar conversion rates for the enantiomers; otherwise, differences in

44

2. DERIVATIZATION IN LIQUID CHROMATOGRAPHY

TABLE 2.7 Single Enantiomer Reagents for the Formation of Diastereomers with Enantiomers Containing a Complementary Reactive Functional Group Analyte functional group

Derivatizing reagent

Reference

Hydroxyl

a-Methoxy-a-(trifluoromethyl)phenylacetic acid

[2,46]

2-t-Butyl-2-methyl-1,3-benzodioxazolo-4carboxylic acid

[48]

2-Phenylproionyl chloride

[2,46]

1-Phenylethyl isocyanate

[46,47]

1-(1-Naphthylethyl) isocyanate

[48]

Menthyl chloroformate

[47]

1-(9-Fluorenyl)ethyl chloroformate

[48]

2-Butanol

[2,46]

Menthyl alcohol

[2,46]

1-Phenylethanol

[46,47]

a-Methyl-4-nitrobenzylamine

[45]

1-(4-Dansylaminophenyl)ethylamine

[2]

2-[4-(1-Aminoethyl)phenyl]-6-methoxybenzoate

[45]

1-(4-Nitrophenylsulfonyl)propionyl chloride

[50]

a-Methoxy-a-(trifluoromethyl)phenylacetyl chloride

[45]

(2,3,4,6-Tetra-O-acetyl)-b-glucopyranosyl isothiocyanate

[2,45,46]

1,3-Diacetoxy-1-(4-nitrophenyl)-2-propyl isothiocyanate

[47]

1-Phenylethyl isocyanate

[46,47]

Carboxylic acid

Amines

4-(3-Isothiocyanatopyrrolidin-1-yl)-7-(N,Ndimethylaminosulfonyl)2,1,3-benzoxadiazole

[47]

1-Fluoro-2,4-dinitrophenyl-5-alaninamide

[49]

1-(Fluorenyl)methyl chloroformate

[45]

Menthyl chloroformate

[45]

2.2. REAGENT SELECTION

45

FIGURE 2.3 Reagents for the separation of enantiomers by forming diasereomeric derivatives with compounds containing complementary reactive functional groups. Reagents: (I) 1-(1-naphthylethyl) isocyanate; (II) 1-(4-dimethylamino-1-naphthyl)ethylamine; (III) 1-(4-dimethylamino-1-naphthyl)acetic acid; (IV) a-methoxy-a-methyl-1-naphthalenecarboxylic acid; (V) a-methoxy-a-trifluoromethylphenacetyl chloride; (VI) carboxylic acid analog of (V); (VII) a-methoxy-4-nitrobenzylamine; (VIII) acid chloride analog of (VII); (IX) camphor-10-sulfonyl chloride; and (X) neomenthyl isothiocyanate.

reaction rates may result in kinetic resolution. The diastereomers should also have similar stability in the reaction media. Diasteromers can have different spectrophotometric properties, and calibration of the individual diastereomers may be required for quantitative analysis. For amine-activated carboxylic acids (e.g., acid chlorides), chlorofrormates (forming carbonate derivatives), isocyantes (forming urea derivatives), and isothiocyantes (forming thiourea derivatives) are popular general choices. The isocyanate group is selective toward primary amines and secondary amines under mild conditions, no other free functional groups need be protected, and the thiourea derivatives produced allow sensitive UV detection [47]. Marfey’s reagent (1-fluoro-2,4-dinitrophenyl-5-L-alanineamide) is popular for the separation of peptides and related compounds, forming aniline derivatives with good UV detection properties [50]. A number of reagents are used for the introduction of fluorophores into enantiomers combining separation

46

2. DERIVATIZATION IN LIQUID CHROMATOGRAPHY

with low-level detection [48e50]. The reaction of amino acids with o-phthalaldehyde and a chiral thiol (e.g., N-butyrylcysteine) results in the formation of highly fluorescent diasteromeric isoindole derivatives [45]. For alcohols, activated carboxylic acids or acyl nitrile reagents (forming ester derivatives), chloroformates (forming carbonate derivatives) and isocyanates (forming carbamate derivatives) are widely used. Reagents containing an acid chloride, anhydride, or acyl cyanide groups are suitable for the derivatization of hydroxyl groups under anhydrous conditions with a catalytic amount of organic base. Because of the lower reactivity of hydroxyl groups with isocyanates and chloroformates, a catalyst (organic base) is usually required. The most frequently used approaches for derivatizing carboxylic acids are esterification with a number of single enantiomer alcohols or formation of amides with single enantiomer amines. Esterification reactions generally require harsh conditions, and this needs to be considered if either the analyte or diasteromeric derivative is conformationally labile or unstable. The selection of the reactive functional group of the reagent is quite straightforward but the choice of the correct chiral substituent to facilitate the separation of the diesteromers is often arrived at empirically. Separation is usually favored if the diasteromers have conformationally rigid groups attached to the asymmetric centers and a large size difference between the groups. The distance between the asymmetric centers should be short and ideally less than three bonds. Both reversed-phase and liquidesolid chromatography are used for the separation of diastereomers, with liquidesolid chromatography favored for moderately polar diastereomers, lacking strong hydrogen-bonding functional groups. This takes advantage of the greater stereoselectivity of the adsorption mechanism on surfaces with immobilized active sites. From a practical point of view, single enantiomer reagents of high purity (to detect the presence of 0.5% of a minor enantiomer in a mixture of enantiomers requires a reagent with at least 99.9% enantiomer purity) and with a reasonable shelf life is required. It is advantageous if the derivatizing reagent is available in both the R and S form, to allow elution order reversal, to facilitate the quantification of the minor enantiomer by eluting it before the major enantiomer.

2.2.6. Multifunctional Reagents for the Formation of Cyclic Derivatives Liquid chromatography is well suited to the separation of cations directly or after formation of stable metal chelates with reasonable solubility in organic solvents [51]. The most widely used chelating reagents are the dithiocarbamates, dithionates, ketoamines, salicylaldimines, dialkyl thiophosphonates, 8-hydroxyquinolines, and tetradentate Schiff bases. The stability of the metal chelates cover a wide

2.2. REAGENT SELECTION

47

range, and some are kinetically labile under typical separation condition used for liquid chromatography. Others may undergo ligand substitution reactions with metal components of the separation system. These considerations tend to define the chelating reagent chosen for the separation of a particular series of cations. Most metal chelates also exhibit strong UVeVisible absorption facilitating their detection. The separation conditions for many metals by ion-exchange chromatography are well known. In this case, chelation reactions are often employed in a postcolumn reactor for multielement detection with detection limits at the nanogram level [51,52]. The reagent is usually a chelating dye, producing strongly absorbing metal complexes that absorb at a different wavelength to the reagent. For a series of metals, it is advantageous if the wavelength maxima for the metal complexes are reasonably close together, so that a single compromise detection wavelength can be used. Common reagents for multielement detection are 4-(2-pyridyl)-azoresorcinol, arsenazo dyes, xylenol organge, dithizone, and dithiocarbamates. Most of the metals that can be separated by ion chromatography can be determined with one or more of these reagents. Bifunctional organic compounds are characterized by the presence of at least two reactive functional groups on a molecular framework that places these groups into close proximity to one another. As such they do not constitute a defined chemical class but examples are found within almost all of the common classes of functionalized organic compounds, some of which are of biomedical or environmental interest. Typically, these are compounds containing reactive functional groups on 1,2-, 1,3- or 1,4-carbon atoms in an alkyl chain or 1,2-disubstituted aromatic rings. Specific reagents have been developed to react with these functional groups, forming cyclic derivatives [2,3,53e55]. Reagents used to form cyclic derivatives can be organized into two classes: reagents that are highly selective for specific functional groups and general reagents that react with a wider range of compounds. The specific reagents, although very useful, is not discussed further in this section, because each reaction is its own story and there is little to generalize. The reaction of o-phthalaldehyde in the presence of a thiol with primary amines to form a cyclic isoindole derivative (see Section 2.2.2) is an example of this reagent type. The formation of acetals and ketals by the reaction of aldehydes and ketones with diols, quinoxalinols from the reaction of diamines with a-keto acids, and thiohydantoins formed by amino acids and phenyl isocyanate are further examples. The most versatile of the general reagents are the boronic acids, which react with hydroxyl, amine, thiol, and carboxylic acid functional groups, as shown in Figure 2.4. Their dominant position as a derivatizing reagent for bifunctional compounds is a consequence of their versatility, ease of

48

2. DERIVATIZATION IN LIQUID CHROMATOGRAPHY

RCH-XH (CH2)n RCH-XH

RCH-X +

(HO)2BR1

(CH2)n

BR1

RCH-X

FIGURE 2.4 Formation of cyclic boronates by reaction of a boronic acid with a bifunctional compound (X [ O, N, S, or CO2 and n [ 0, 1, or 2). Varying the structure of R1 allows different detection options for the derivatives.

reaction under mild conditions, and the possibility of varying the substituent group on the boronic acid to facilitate a wide range of detection options. Some derivatives are labile in aqueous solution, which can be a problem for their separation by liquid chromatography [56].

2.2.7. Solid-Phase Analytical Derivatization Derivatization reactions can be used at an early stage in sample preparation to improve the efficiency or selectivity of the isolation step when using solid-phase extraction, solid-phase microextraction, liquide liquid extraction, or liquidephase microextration [57,58]. When a solid phase is used for extraction the derivatization reaction can be performed after elution of the analytes from the extraction column and occurs in solution (conventional reaction) or in the presence of the sorbent prior to elution of the derivatives (heterogeneous reaction). The latter approach called solidephase analytical derivatization, combines the extraction and derivatization steps into a single procedure, in one of several ways [59,60]. The sorbent could be coated with the derivatizing reagent and the reagent-coated sorbent used to enhance the extraction process as well as simultaneously (or subsequently if a change of conditions is required) perform the derivatization reaction [61]. A typical example is the determination of volatile aldehydes and ketones by gas-phase trapping of the analytes on a sorbent coated with 2,4-dinitrophenylhydazine and subsequent elution of the hydrazone derivatives [62]. Alternatively, the analytes could be extracted by the sorbent and the sorbent immobilized analytes reacted with the derivatizing reagent in a two-step process. This is the most common arrangement for solid-phase extraction and solidphase microextraction procedures [63,64]. To be effective, it is generally required that, after the reaction, the derivatives can be isolated by a sequence of solvent rinses with minimal contamination by reagents and by-products of the reaction. Full automation of the extraction, derivatization, and separation by liquid chromatography has been demonstrated using a short precolumn connected to the separation column by a multifunctional valve, together with associated pumps and other equipment to introduce the reagent to the precolumn, transfer the derivatives to the

2.3. POSTCOLUMN REACTION DETECTORS

49

analytical column, and clean and prepare the precolumn for the next extraction and reaction [64]. Reactions employing physically adsorbed reagents are most often used. In earlier times, solid-phase reagents utilizing ionic or covalently attached reagents to a polymeric support were used for precolumn and postcolumn reactions, but these are little used today, although solid-phase reagents continue to be used in synthetic organic chemistry [65]. In spite of its attractive features solid-phase analytical derivatization is not widely used at present. It is most useful for trace analysis but requires careful optimization. Reaction yields are rarely quantitative (although reproducible) and background contamination from by-products derived from the extraction sorbent can be a problem. Most applications so far have been set up with gas chromatography as the determinant step rather than liquid chromatography.

2.3. POSTCOLUMN REACTION DETECTORS Postcolumn reaction systems are convenient for on-line derivatization of target compounds to facilitate their detection, often at low concentrations [14]. Reactions must be reasonably fast ( Fcr, the initially precipitated polymer dissolves directly into a nonadsorbed state and migrates with the surrounding solvent front, resulting in Fel z Fsol. At first glimpse, it appears only as a mechanistic detail whether elution occurs at Fel z Fsol or at Fel z Fcr, since both Fcr and Fsolv depend on the chemical structure of the polymer. However, as explained already, adorption-controlled gradient elution, that is, Felz Fcr, results in molarmass-independent elution, while solubility control depends more strongly on molar mass. Therefore, a separation based on solubility is more strongly affected by molar-mass effects, rendering interpretation of the chromatograms more difficult, due to coelution of macromolecules differing in molar mass and, say, chemical composition. Additionally, the question of precipitationedissolution or adsorptionedesorption control in gradient elution is directly related to the question of, whether, for the mobile and stationary phase under investigation, critical conditions can be achieved at all. If gradient elution is determined by precipitationedissolution, that is, Fcr< Fsol ¼ Fel, the polymer experiences no or a too low adsorption to be sufficiently retained in order to compensate for

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size exclusion effects. Consequently, isocratic elution always results in an SEC-like elution order and no critical conditions can be adjusted in such a system. In the recent years, chromatographic separations using interaction chromatography were also performed at temperatures above 140 C, allowing separations of polyolefin homo- and copolymers [31e34]. In an overwhelming number of gradient applications, the solvent strength is varied by changing the composition of the mobile phase. Temperature-gradient interaction chromatography (TGIC), as introduced by Chang and Lee, uses temperature variations during the chromatographic experiment to adjust the interaction of the polymer with the stationary phase [35e38]. TGIC allows controlling much smaller changes in eluent strength than can be done by solvent variation. This allows more delicate separations to be achieved. On the other hand, TGIC does not allow for large variations in interaction strength, due to the limitations of temperature variation. Therefore, separations are limited to polymers of very similar interaction strength. There is a fundamental difference between solvent and temperature gradients. In the latter case, the change in eluent strength is applied to all macromolecules within the column at the same time and is decoupled from the flow rate. In contrast, by changing the eluent composition, molecules closer to the column inlet experience the eluent change earlier than those that have already started moving in the column. As a consequence, the elution order in TGIC depends on the time rate of change of temperature relative to the applied flow rate. For a fast temperature increase and a low flow rate, a SEC-like elution order is observed, while high flow rates and shallow temperature gradients result in an increase of elution time with molar mass. A proper adjustment of the flow rate at a given temperature gradient can be used to obtain a nearly molar-mass-independent elution order [39]. TGIC has been applied to the separation of polystyrenes of different topologies [20e22,40,41], functionalized polystyrenes [42], a blend of polystyrene and polyisoprene [42], miktoarm star polymers [43], and separations according to tacticity [44].

5.3.4. Barrier Methods The strong dependency of elution volume on eluent composition, results in unstable chromatographic conditions, especially for high molar-mass samples. Even small changes in eluent composition, which might occur from selective evaporation of one solvent component or moisture uptake from air, might be sufficient to change the elution of high molar-mass polymers from SEC to adsorption mode and vice versa. As a result, the majority of LCCC separations are performed for

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molar masses below approximately 200 kg/mol, although critical conditions have been realized also at higher molar masses as well. These problems incited Berek to develop another way to establish a molar-mass-independent elution, using the so-called limiting conditions [45e47]. Liquid chromatography at limiting conditions (LCeLC) refers to isocratic elution where the polymer is dissolved in a solvent that differs from the actual eluent. There is a close relation between LCeLC and the breakthrough effects sometimes observed in GELC, when the sample solvent differs from the eluent composition at the beginning of the gradient [48]. The most promising LCeLC experiments were performed at limiting conditions of desorption (LCeLCD). Chromatography under LCeLCD uses a strong eluent, resulting in steric exclusion of the polymer from the pores of the stationary phase; that is, the polymer molecules migrate at a higher velocity than the surrounding solvent molecules. If a weak eluent has been injected before the sample, the polymer molecules might catch up with this eluent. Those polymer molecules for which this injected eluent is a desorli penetrate the solvent front, while polymer molecules adsorbed from the injected eluent accumulate at the back of the injected eluent. Thus, the injected eluent presents a barrier for those molecules, which elute directly after it, irrespective of molar mass. The method has been used for the separation of homopolymer blends [49], separation by stereoregularity [50], and to quantify homopolymers in blockcopolymer samples [51]. It has been shown that LCeLCD allows the detection of very low amounts of polymeric impurities [49].

5.3.5. Size-Exclusion Chromatography Gradients Application of chromatography at limiting conditions requires selection of a suitable barrier solvent and width, allowing penetration of one type of macromolecules, while the barrier should be sufficiently weak to hold back the other polymer type. This limits the application of LCeLC to samples whose components exhibit clear differences in adsorption strength, such as blends. To separate more components or components whose adsorptivity differs only slightly, it would therefore be required to adjust a variety of barriers of different or even continuously decreasing eluent strength, such that the polymer molecules automatically adjust at a proper barrier composition. This approach, named SEC gradients, was recently applied to the characterization of polymer blends [52,53]. Actually, SEC gradients are a reinvention of the zone precipitation proposed by Porath in the early 1960s [54]. The name SEC gradients results from the fact that the separation is performed using a gradient, but elution occurs in the elution volume range typical for SEC separations.

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In SEC gradients, the sample is dissolved in a good solvent and is injected at the end of the gradient under strong eluent conditions, in contrast to conventional GELC. Since the polymer is surrounded by a strong eluent, it experiences SEC conditions and migrates with a higher velocity than the surrounding solvent. Thus, it experiences conditions of continuously decreasing eluent strength. This overtaking of the eluent continues until the molecules reach an eluent composition defining a threshold at which the molecule is either adsorbed to (Fcr) or precipitated (Fsolv) onto the stationary phase. At this point, the polymer continues migrating at the eluent velocity, exiting the column at the threshold composition. Since both thresholds (Fcr , Fsolv) depend on the chemical composition of the polymer molecules, a separation by chemical composition occurs. As an example, Figure 5.5 shows the SEC-gradient separation of poly(n-butyl acrylate-co-acrylic acid)s of different acrylic acid content but similar molar masses. Compared to the elution volume at SEC conditions in pure dimethyl acetamide (DMAc, V z 6 ml), the samples are retarded. The applied SEC gradient runs from low polarity to higher polarity. The polymer molecules experience the gradient in the opposite order, as they migrate into the gradient from its end. Consequently, the more polar polymer molecules containing a larger amount of acrylic acid reach their

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FIGURE 5.5 Normalized SEC-gradient chromatograms of poly(n-butyl acrylate-coacrylic acid) containing 20 (solid line), 40 (broken line), and 60% (dotted line) acrylic acid. ˚ , 300  8 mm L  i.d., Lines, Stationary phase: PSS-Proteema (3 mm particle size, 100 A gradient: 96/4 CHCl3/DMAc to 0/100 in 6 min, injection 6 min after gradient start. Concentration: 1 g/l in pure DMAc. Source: Data extracted Ref. [55].

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adsorption threshold earlier than those of lower acrylic acid content. That is why they elute later than the less-polar molecules. It should be noted that the dissolution of the samples of higher acrylicacid content requires the addition of DMAc to chloroform. When running a conventional gradient from chloroform to DMAc, breakthrough peaks are observed, since the polar DMAc prevents adsorption of the polymer molecules onto the stationary phase.

5.4. HYPHENATED TECHNIQUES The preceding chromatographic techniques allow polymer separations according to various structural parameters. However, since complex polymer samples are heterogeneous with respect to different structural features, coelution of molecules of different structures might still occur, even if, in an ideal case, a separation by one structural parameter was realized. For example, a separation according to end-group functionality might be realized by LCCC at the cost, however, of coelution of chains differing in molar mass. Another problem is that chromatography with conventional concentration detectors like the refractive index (RI), UV, or evaporative light scattering (ELS), in the first place, provides a separation resulting in various peaks, the underlying structures of which are not known. That is why hyphenated techniques are increasingly used in polymer characterization. Hyphenation might involve the combination of a chromatographic separation with one or more structure-sensitive detection techniques or the combination of various separation techniques [56,57], rendering separations according to more than only one structural feature. The most frequently applied combinations of hyphenation are schematically depicted in Figure 5.6 [56,57].

5.4.1. Two-Dimensional Liquid Chromatography Conventionally, in the first dimension of two-dimensional LC, the molecules are separated according to other features than molar mass by applying methods of interaction chromatography (IC). Such features can be a functionality-type distribution (FTD), a copolymer-composition distribution (CCD), topology distribution (TD, e.g., linear, cyclic, or branched structures), or even a separation by microstructure. Often, liquid chromatography at critical conditions is applied, but also liquidadsorption chromatography (LAC), gradient-elution liquid chromatography, or temperatureegradient interaction chromatography (TGIC) can act as the first dimension. Using a transfer valve, the separated fractions are transferred on-line from the first into the second chromatographic

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FIGURE 5.6 Coupling of separation methods with spectroscopic and spectrometric techniques. Source: Adapted from J. Falkenhagen, S. Weidner, Hyphenated Techniques. In: C. Barner-Kowollik, T. Gruendling, J. Falkenhagen, S. Weidner (Eds.), Mass Spectrometry in Polymer Chemistry. Wiley-VCH Verlag GmbH Co. KGaA, Weinheim, Germany, 2012, p. 500. Ó (2112), used with permission from Wiley-VCH (Ref. [58]).

dimension, in which the polymers are usually separated according to their molar mass by SEC. To obtain a comprehensive characterization of the sample, the flow rates in both dimensions have to be carefully adjusted, depending on the time required for the second dimension analysis and the transfer volumes. Since the rate-determining step is the duration of the separation in the second dimension (typically SEC), highspeed SEC is increasingly applied for this purpose. Therefore, columns differing in column dimension from the conventional ones are used. Using such columns, two-dimensional analysis could be accelerated considerably [59]. The two-dimensional setup for an ICeSEC combination is shown in Figure 5.7. Comprehensive two-dimensional liquid chromatographyesizeexclusion chromatography (LC  SEC) was refined considerably over the last 20 years. It allows characterizing by-products and impurities,

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FIGURE 5.7 Setup for two-dimensional LC. Source: Adapted from J. Falkenhagen, S. Weidner, Hyphenated Techniques. In: C. Barner-Kowollik, T. Gruendling, J. Falkenhagen, S. Weidner (Eds.), Mass Spectrometry in Polymer Chemistry. Wiley-VCH Verlag GmbH Co. KGaA, Weinheim, Germany, 2012, p. 500. Ó (2112), used with permission from Wiley-VCH (Ref. [58]).

such as homopolymers from copolymers, cyclic from linear structures, or copolymer compositions depending on polymer chain length [18,60e72]. As an example, the two-dimensional separation of a mixture of end-functionalized poly(methyl methacrylate) polymers with UV and ELS detection is demonstrated in Figure 5.8. Comparing the elution times of peaks 1 and 2, a slight decrease is observed with increasing molar mass, indicating that critical conditions at which the retention is independent of molar mass are not perfectly met. That is, the separation in the first dimension was carried out not at but close to the critical conditions of PMMA. In the second dimension (SEC), a separation by molar mass was realized. Since, at critical conditions, the influence of molar mass on the elution vanished, the retention should be determined by the functionality of the chains. Therefore, a clear separation of the various functionalized PMMAs is obtained. The information on the molar-mass distributions of the fractions corresponding to a given functionality is obtained by the subsequent second-dimension SEC analysis. However, the example also shows that, for practical

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FIGURE 5.8 LCCC 3 SEC chromatograms of a mixture of PMMA standards (peak 1: PMMA 620, peak 2: PMMA 5270; nonfunctional), monofunctional RAFT polymers (peak 3: PMMA-OH 3310, peak 4: PMMA-OH 13,950) and difunctional PMMA (peak 5: MD-1000 (commercial telechelic PMMA with two OH groups was obtained from Tego Chemie (Essen, Germany)) at near-critical conditions. (a) UV 220 nm, (b) ELSD. LC ˚ bare silica; 56% ACN in DCM, 4 ml/min. columns: two 150 mm 3 1.0 mm i.d., 3 mm, 100 A ˚ plus 6 mm oligopore, two times 50 mm 3 4.6 mm i.d.; fresh SEC columns: 5 mm 100 A nonstabilized THF, 0.9 ml/min. Source: Reprinted from X. L. Jiang, A. van der Horst, V. Lima, P. J. Schoenmakers, Comprehensive two-dimensional liquid chromatography for the characterization of functional acrylate polymers, Journal of Chromatography A, 1076 (2005):51e61, Ó (2005), used with permission from Elsevier (Ref. [73]).

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applications of LCCC  SEC, it is often not strictly necessary to work at exactly the critical eluent composition to determine the dependency of molar mass on functionality type [73]. The combination of different IC separation mechanisms in an orthogonal way is quite useful for characterization of certain materials, although not frequently applied for polymers. Raust et al. [61] investigated the endgroup functionality of fatty alcohol ethoxylates by gradient LC on a nonpolar stationary phase as a first dimension and its correlation with ethoxylate chain length by isocratic LAC on a polar column in the second dimension. Im et al. presented a two-dimensional LC separation of branched polystyrenes heterogeneous with regard to molar mass and the number of branches [74]. TGIC was employed in the first dimension to separate the branched polymer according to the molecular weight. In the second dimension, LCCC at the critical conditions of linear polystyrene (PS) was applied to separate the branched PS in terms of the number of branches. It was demonstrated that in this case RP-TGIC  LCCC accomplishes a better resolution in molar mass than the common LCCC  SEC configuration, due to the higher selectivity toward the molar mass of TGIC as compared to SEC [74]. Ahn et al. used on-line two-dimensional liquid chromatography with triple detection for the characterization of star-shaped polystyrenes [75]. They proposed a TGIC separation according to molar mass in the first dimension followed by the subsequent SECetriple-detection characterization of the chain-size distribution of the eluting fractions, which have been assumed to be homogeneous with respect to molar mass. A rather recent development is two-dimensional, high-temperature liquid chromatography (HTLC) [76e79], which is essential for characterizing polymers having low solubilities at room or slightly elevated temperature, such as polyolefines. Roy et al. reported on comprehensive two-dimensional, high-temperature liquid chromatography of random ethylene/octane copolymers [80]. Separation based on octene content was achieved in the first dimension by high-temperature gradient liquid chromatography, while molar-mass determination was realized in the second dimension by high-temperature SEC. The essential advantages of the gradient HTLC approach as compared to separations based on crystallinity (CRYSTAF, TREF, or CEF) are the higher sample throughput and the applicability to both semicrystalline and amorphous polymers [80].

5.4.2. Liquid ChromatographyeFourier-Transform Infrared Spectroscopy Hyphenation of Fourier-transform infrared spectroscopy (FTIR) with different modes of liquid chromatography is a useful approach to

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identifying variations in chemical structure with elution volume. These variations might results from changes in functionality or copolymer composition, microstructure, tacticity, or branching. Another application of LCeFTIR is the identification of additives in polymeric materials [81]. Two main approaches are employed in LCeFTIR coupling, either a flow cell or solvent elimination by special deposition interfaces. Both methods have their advantages and drawbacks concerning handling, sensitivity, repeatability, and qualitative and quantitative interpretation. The major drawback of the flow-cell setup is the strong absorption bands of most LC solvents, causing problems especially when coupled to GELC. Thus, only polymers having adsorption bands within the free regions of the solvent can be identified. That is why existing on-flow systems are mostly restricted to high-temperature SEC in trichlorobenzene [82] or other suitable solvents as eluents [83]. However, the constant optical pathway of the flow cell approach allows for absolute quantification of analyte concentrations. The drawback of solvent adsorption bands has been overcome by the use of interfaces that eliminate the solvent after the chromatographic separation, resulting in deposition of the nonvolatile components (e.g., polymers) on a moving target (e.g., germanium disc) at different positions. For solvent elimination interfaces, different principles are described: (1) a pneumatic nozzle with a heated gas evaporation system, (2) an ultrasonicevacuum nebulizer [84,85] (3) electrospray transfer devices combined with a heated gas evaporation system [86,87]. Having deposited the nonvolatile sample, the solvent-free IR spectra can be recorded at each position of the track, The removal of the solvent allows one to apply this technique in gradient elution. The decoupling of the deposition from the data acquisition step allows for longer FTIR scanning times, resulting in better signal-to-noise ratios for the acquired spectra. However, due to the changing film thicknesses and film width, absolute quantification of analyte concentrations is rather difficult to perform. But, determination of composition and its variations with elution volume can be obtained if IR bands for the different structural units can be identified, which differ in their relative intensities. In addition, the deposited track needs to be homogenous perpendicular to the spraying direction. This sometimes represents a problem when applying mixed solvent, due to the influence of mixed solvents on the film-formation process. Trace homogeneity depends on the LC flow rate and nebulizing system [88]. Coulier, Kaal, and Hankemeier investigated the degradation of poly(bisphenol A)carbonate using both flow cell and solvent elimination. For the extraction of relevant information from the large data sets of the flow-cell coupling, multivariate data analysis (MVDA) is essential. This approach revealed chemical differences due to degradation that could not be detected by other means [89].

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Piel et al. [90] used the flow-cell principle for characterization of shortchain branching of high-density polyethylene copolymers as a function of SEC elution time. High resolution in SEC was obtained by a very low sample concentration. The FTIR signal-to-noise ratio could be increased by applying a bandpass filter, which enables new possibilities in investigating high molecular-weight samples. Samples even of very broad MWD and low degrees of branching were analyzed in detail. Very recently, a new SECeFTIR coupling method was utilized for the characterization of polyurethane-based multiblock copolymers with respect to structure and size. The use of a mathematical solvent suppression technique, together with a newly developed flow cell, overcame the problems arising from conventional flow cells and SEC solvents. The approach allows determination of the exact composition of each polymer structure, showing the potential of the newly introduced on-line SECeFTIR technique for elucidating polymer structures [91,92].

5.4.3. Liquid ChromatographyeNuclear Magnetic Resonance Spectroscopy If structure elucidation is required, most commonly a NMR spectrum of the bulk sample provides the first structural information. NMR is applied for determination of the chemical constitution of the monomer units, tacticity, end groups, degree of branching, microstructure, and sequence length distribution. One of the main advantages of NMR is the ability to provide exact quantitative results, without detector calibration. Therefore, the coupling of LC to NMR should show a high potential for polymer characterization. However, rather high sample concentrations are required, due to the relatively low sensitivity of NMR as compared to MS or FTIR. So, laborious off-line fractionations are often required, especially if carbon 13C NMR is to be applied. That is why 1H NMR is more commonly applied for both off-line and on-line coupling to LC. NMR usually uses deuterated solvents, which would make LCeNMR a highly expensive technique. Therefore, on-line LCeNMR is performed mostly by applying nondeuterated solvents using special pulse sequences, which allow suppression of the solvent resonances. However, the resonances of the investigated molecules may be close to the solvent signals and might be disturbed or suppressed as well. Furthermore in gradient applications, changes of the solvent composition exceeding 2e3%/min might cause problems, due to solvent mixing in the flow cell, resulting in magnetic field inhomogeneities. LCeNMR coupling can be realized by four operation modes: on flow, stop flow, time sliced, and loop collection. Since, in the on-flow mode, only a relatively low number of NMR pulses can be acquired for a peak or

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fraction, only spectra of major components can obtained with high resolution. However, the solvent flow of the chromatographic run can be interrupted at defined times (stop flow) or fractions can first be stored in loops and analyzed after the completion of the chromatographic experiment, allowing use of a larger number of pulses to increase the quality of the acquired spectra. In addition, these techniques allow analyzing different slices from the same peak, to identify structural variations across the peak, such as changes in chemical composition. The advantages and limitations of LCeNMR coupling are summarized by Elipe in Reference [93]. The tacticity distribution of stereo-regular poly(ethyl methacrylate)s was investigated by Kitayama et al. using on-flow LCeNMR [94]. Blechta et al. compared LCeNMR and SECeNMR using 29SIeNMR to investigate hydride terminated polydimethylsiloxane in a stopped-flow arrangement [95]. On-line hyphenation of critical chromatography and 1HeNMR was applied by Sinha, Hiller, and Pasch [96] for the analysis of blends of polystyrene (PS) and polyisoprene (PI). At the critical conditions of either PS or PI, a perfect separation of the blend components was achieved and the molar masses could be determined. Figure 5.9 shows

FIGURE 5.9 Two-dimensional plot of the on-flow LCCCeNMR separation of blends of PS and PI, separation at critical conditions of PS, samples 1e5 are indicated by numbers. Source: Reprinted from P. Sinha, W. Hiller, H. Pasch, Characterisation of blends of polyisoprene and polystyrene by on-line hyphenation of HPLC and 1-H-NMR: LC-CC-NMR at critical conditions of both homopolymers. Journal of Separation Science, 33 (2010) 3494e3500. Ó (2010), used with permission from Wiley-VCH (Ref. [96]).

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the two-dimensional plot at LCCC conditions for PS (x axis: chemical shift in NMR; y axis: retention time in LCCC). Peaks 1e5 of the 1,4- and 3,4-resonances of PI correspond to different molar masses of polyisoprene. In addition, the NMR analysis allowed the detailed characterization of the chemical composition and the PI microstructure [96]. Hiller et al. demonstrated the separation and microstructural characterization of polyisoprene blends consisting predominantly of 3,4- and 1,4-isoprene units at critical conditions of 1,4-PI by applying LCCCe1HeNMR on flow coupling. Application of LCeNMR allowed the identification and quantification of all three isomeric species, such as 1,2-, 1,4-, and 3,4-isoprene within the different chromatographic fractions [97]. Recently, PI-b-PMMA copolymers were investigated by on-flow SECeNMR. It was demonstrated that the NMR detector is able to differentiate the syndiotactic and atactic triads of the PMMA block as well as the 1,2-, 1,4-, and 3,4-PI units of the PI block. The major advantage of SECeNMR is the ability to detect both comonomers simultaneously allowing a comprehensive molar-mass analysis of the copolymers, including average chemical composition distributions. It was also possible to apply a simulation analysis on a sample with a bimodal shape caused by the remaining PI homopolymer. This mathematical method allowed quantifying the amount of homopolymer in the sample as well as determining the correct molar masses for the co- and homopolymer [98]. However, LCeNMR remains a cost-intensive coupling technique. Therefore, it is more suitable for research purposes than for typical routine analysis in industrial applications.

5.4.4. Liquid ChromatographyeMass Spectrometry Mass spectrometry is a fast and powerful analytical technique to characterize polymers. The principle is based on the ionization of molecules followed by the separation of the ionized species according to their mass-to-charge ratio (m/z). Soft ionization techniques prevent undesirable fragmentation of the polymer molecules. The different ionization methods include matrix-assisted laser desorptioneionization (MALDI), electrospray ionization (ESI or IonSpray), or atmospheric pressure chemical ionization (APCI). Should fragmentation occur, it can be suppressed to a large extent, say, by either applying lower laser power or using an excess of matrix in the MALDI technique or by lowering the voltage in the ESI process. High sensitivity, high resolution, and the analysis of relatively broad mass ranges are accomplished by timeof-flight (TOF) instrumentation, where ions are accelerated by an electric field of known strength. The TOF mass analyzer, which is frequently used

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in conjunction with MALDI or ESI instrumentation, can be a linear flight tube or a reflectron. The ion detector typically consists of a microchannelplate (MCP) detector or a fast secondary-emission multiplier (SEM). Using mass spectrometry information, the exact molar mass and molarmass distribution of the intact macromolecules can be obtained. The high mass resolution of modern instruments allows information on end groups and polymer structure to be obtained. The most commonly used ionization techniques for characterization of polymers are MALDI and ESI, while APCI applications for polymers are rather limited [99,100]. Both, MALDI and ESI exhibit their advantages and drawbacks and are perfectly suited to complement each other, especially in combination with an upstream liquid-chromatographic separation. ESIeMS can be relatively easily coupled on-line to LC but is limited to relatively low molar masses for singly charged species (m/z  4000). The formation of multiple charged ions allows analyzing macromolecules of higher molar masses. However multiple charged ions obtained during the ionization process, result in very complex mass spectra for polymers. These spectra are difficult to interpret, especially for copolymers. The outstanding benefit of LCeESIeQeTOFeMS compared to MALDIe TOFeMS is the significantly higher mass accuracy. On-line coupling of LCeESIeMS has special demands on the chromatographic conditions. Solvents should be protic or polar; otherwise, acids or salts have to be added to facilitate ionization. However, only volatile buffers can be used. Low solvent flow rates, which are usually realized by splitting the eluent stream after the columns, are mandatory. Also, the limitation of ESIeMS to polymers with relatively high polarity has to be mentioned. A variety of LCeESIeMS applications are described in several reviews [101e107]. In contrast to ESI, the MALDI process provides singly charged ions. MALDIeMS allows analyzing polymers of slightly lower polarity than required for ESIeMS. Synthetic polymers of up to 70e100 kDa can be routinely analyzed. However, the dispersity (Mw/Mn) of the investigated sample should not exceed approximately 1.2. Higher dispersities albeit the so-called mass discrimination (low-mass bias), a typical problem of MS techniques, which complicates direct application of MALDIeMS for most industrial polymers. The MS response is influenced by not only broad molar-mass distributions but also the chemical structure of the repeating units and the type of end group. Hence, a MS spectrum of a complex polymer sample does not always provide a true picture of the polymer distribution. Obtaining quantitative results is very difficult, and the repeatability is poor, as it is additionally influenced by sample spot inhomogeneities caused by the sample preparation. The most common procedure to prepare a sample for MALDIeMS, called dried-droplet technique, implies the deposition of dissolved matrix and sample on the target,

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forming layers (sandwich technique) or together in one droplet. However, certain solventematrixesample combinations lead to segregation or even crystallization within the spot, due to solubility differences and evaporation effects. Segregation might also occur between higher and lower molar-mass fractions. These effects can be visualized by imaging mass spectrometry [108]. To get more accurate results requires obtaining a homogeneous thin-film layer on the MALDI target. Solvent-free samplepreparation techniques produce the best results for this purpose and may suppress the undesired effects [101,102,109e111]. Therefore, compared to LCeESIeMS, the eluent and buffers play a minor role in LCeMALDIeMS. The coupling of MALDIeMS with LC usually is carried out in an offline mode. The LC fractions are collected and analyzed after suitable sample preparation [109,112,113]. A first systematic investigation on the application of SECeMALDI analysis of polystyrene, polycarbonate, and aromatic polyester resins was accomplished by Nielen and Malucha [114]. Poly(dimethylsiloxanes) were characterized by Montaudo and coworkers [115]. They also applied off-line SECeMALDI coupling for the analysis of different copolymers and compared the results with SECeNMR [116e119]. Off-line coupling of interaction chromatography and MALDI was first performed in the 1990s by investigating polyethers [120e123] and polyesters. A summary of applications is given in reference [124]. Off-line coupling of LC and MALDI is a powerful tool for identifying the structure corresponding to eluting peaks. However, sample preparation is tedious, especially when a large number of fractions are to be analyzed. Therefore, several commercial or homemade interfaces were described for semi-on-line coupling. These interfaces in addition allow reducing the main drawback of MALDIdthe sample matrix spot inhomogeneitydby solvent-free dry-spray approaches, which are described by Hanton et al. in ref. [125]. A summary of coupling techniques is given in Figure 5.10. Applying semi-on-line coupling techniques, particularly spraying, provides reproducible results. The coupling to an electrospray deposition interface is shown in Figure 5.11. The matrix can be added to the eluent stream by an injection pump with flow rates of 5e10 ml/min-1 or it can be predeposited on the target in a preceding step. The knowledge of the true molar masses of the eluting peaks significantly eases structural identification. It is even possible to calibrate the SEC system using MALDIe or ESIeTOFeMS [103] by semi-on-line coupling. However, care must be taken when calculating molar-mass averages from MS data alone, due to the intensity bias by ionizability and molar mass. Micro-SEC calibration and copolyester characterization by SECeMALDI coupling via a spotting interface was performed by Nielen

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FIGURE 5.10 Scheme of basic LCeMS coupling principles. Source: Reprinted from J. Falkenhagen, S. Weidner, Hyphenated Techniques. In: C. Barner-Kowollik, T. Gruendling, J. Falkenhagen, S. Weidner (Eds.), Mass Spectrometry in Polymer Chemistry. Wiley-VCH Verlag GmbH Co. KGaA, Weinheim, Germany, 2012, p. 500. Ó (2112), used with permission from WileyVCH. (Ref [58]).

[127]. Fatty alcohol ethoxylates were spotted and characterized by Spriestersbach, Rode, and Pasch [128]. Other applications of semi-on-line LCeMALDI coupling via a spraying interface for synthetic polymers involve copolyesters [129], silsesquioxanes [130], and hydroxylterminated polydimethylsiloxane [131,132]. Reference [103] summarizes different applications, comparing their advantages and drawbacks. Coupling of interaction chromatography to MALDIeESIeMS can be used to create two-dimensional plots comparable to two-dimensional LC (IC  SEC) [129]. Figure 5.12 shows that similar plots were obtained for copolyesters of adipic acid, neopentylglycol, and hexanediol when LCCC is combined with SEC (left) or MALDIeMS (right). While the separation

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FIGURE 5.11

Electrospray deposition interface. Source: Reprinted with permission from S. M. Weidner, J. Falkenhagen, LC-MALDI-TOF Imaging MS: A New Approach in Combining Chromatography and Mass Spectrometry of Copolymers, Analytical Chemistry, 83 (2011) 9153e9158. Ó (2011) American Chemical Society (Ref. [126]).

FIGURE 5.12 Left: Two-dimensional plot combining the structural information of separation by LCCC mode and molar-mass information of 100 SEC chromatograms. Right: Two-dimensional plot combining the structural information of separation at LCCC mode and molar¼mass information of 12 MALDIeTOF mass spectra recorded after spraying the LCCC run continuously onto the MALDI target. Source: Reprinted with permission from S. Weidner, J. Falkenhagen, R. P. Krueger, U. Just, Principle of two-Dimensional Characterization of Copolymers. Analytical Chemistry, 79 (2007) 4814e4819. Ó (2007) American Chemical Society (Ref. [129]).

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in LCCC separates by the different end groups, the second-dimension separation is based on size (SEC) or mass (MALDI), which are correlated with each other. Two important advantages can be asserted from Figure 5.12: the timeconsuming SEC separation can be replaced by MS, and no specific standards are required to obtain true molar masses when using MS as a second dimension. Due to the large amount of data acquired using LCeMS coupling as a two-dimensional technique, optimized software tools are required for data mining. Two approaches on how information on correlations between elution volume and molar mass can be expeditiously retrieved from chromatographic runs with MALDI coupling are demonstrated in Refs. [87,129,133]. Recently MALDIeTOF imaging mass spectrometry was presented as a new detector for polymer chromatography. The individual retention behavior of single structural units of polyethylene oxide (PEO) e polypropylene oxide (PPO) copolymers and changes of the copolymer composition could be monitored. Composition-specific calibration curves were obtained by displaying the copolymer ion intensity data as a function of elution volume. This approach also allows for adaption of separation conditions. In combination with electrospray deposition, homogeneous sampleematrix traces of surprisingly high resolution were obtained [126]. Figure 5.13 shows a series of ion intensity plots for copolymers containing constant PPO and varying PEO numbers. Calibration curves show size exclusion behavior for PEOs and adsorption for PPOs. Often mass data alone do not permit exact structural assignment, as the same mass can result from different microstructures of copolymers

FIGURE 5.13

(a) Set of ion intensity plots of PPOePEO copolymers (having constant PPO and varying PEO numbers) and (b) transformed data used for specific calibration. Source: Reprinted with permission from S. M. Weidner, J. Falkenhagen, LC-MALDI-TOF Imaging MS: A New Approach in Combining Chromatography and Mass Spectrometry of Copolymers. Analytical Chemistry, 83 (2011) 9153e9158. Ó (2011) American Chemical Society (Ref. [126]).

5.5. SUMMARY

121

having the same composition. Tandem mass spectrometers can provide additional analytical information. The first TOF mass spectrometer isolates precursor ions of a selected m/z ratio, which are subsequently fragmented at higher collision energy. The second TOFeMS analyzes the fragment ions formed. The primary structure of the fragments and typical fragmentation patterns allow reconstruction and differentiation of the macromolecules, such as the distinction between block and random copolymers. A prior LC separation offers additional advantages for MS/MS detection especially for copolymer analysis. The LC separation effectively removes other structures having similar masses to the structure to be characterized. Thus, the resulting fragment pattern is not blurred by the fragments of uninteresting compounds. LCeMS/MS applications for polymers are published in numerous reviews [58,101,102,110,112,134]. The coupling of MS to LC, regardless of the separation principle, results in a reduction of spectra complexit,y due to a reduction of heterogeneity. Prior SEC separation reduces the molar mass dispersity of the sample. LCCC as the first dimension reduces heterogeneity due to different end groups and the chemical heterogeneity within the MS fraction. This enables partial quantification by MS, since problems resulting from different ionizabilities are minimized. LACeGELC reduces the complexity of copolymers, and composition information is more easily accessible from the mass spectra. In addition, problems associated with ion suppression caused by concentration differences are reduced. Thus, trace components can be analyzed too. LC and MS complement one another in a mutual manner. LC not only clarifies mass spectra, but vice versa, mass spectrometry allows simplifying LC optimization, for example, adjusting critical conditions of adsorption for polymers [107]. Furthermore, a series of MS spectra, such as that recorded by imaging techniques, can provide information on the underlying separation mechanisms [126]. A comprehensive summary of ionization techniques, data processing, sample preparation, imaging techniques, copolymer characterization, and elucidation of reaction mechanisms is demonstrated in several references [58,101,102].

5.5. SUMMARY The increasing structural complexity of polymeric materials requires new and modern analytical tools. Methods of interaction chromatography have become increasingly popular over the last decade, allowing separations by other structural features than molar mass. Hyphenation of different chromatographic modes or chromatography with spectroscopic

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or spectrometric techniques enables obtaining detailed information on the eluting fractions. Therefore, characterization of the complex distributions of modern polymeric materials has become possible.

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6 Amino Acid and Bioamine Separations Y. Miyoshi, T. Oyama, R. Koga, K. Hamase Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan O U T L I N E 6.1. Introduction

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6.2. Direct Separation of Amino Acids and Amines 6.2.1. Postcolumn Colorimetric Derivatization with Ninhydrin 6.2.2. Postcolumn Fluorescence Derivatization with o-Phthalaldehyde 6.2.3. Postcolumn Fluorescence Derivatization with Fluorescamine 6.2.4. ESIeMS/MS Determination of Underivatized Amino Acids

132 132 133 134 134

6.3. Indirect Separation of Amino Acids and Amines 6.3.1. Derivatization with UVeVis Reagents 6.3.2. Derivatization with Fluorescent Reagents 6.3.3. Derivatization for Mass Spectrometric Detection

135 135 137 140

6.4. Enantioselective Liquid Chromatographic Analysis of Amino Acids 6.4.1. 1-Fluoro-2,4-Dinitrophenyl-5-L-Alanine Amide (Marfey’s Reagent) 6.4.2. o-Phthalaldehyde plus Chiral Thiols 6.4.3. (þ)-1-(9-Fluorenyl)Ethyl Chloroformate 6.4.4. Cyclodextrin-Bonded Chiral Stationary Phase 6.4.5. Cinchona-Alkaloid-Bonded Chiral Stationary Phase 6.4.6. Two-Dimensional Liquid Chromatographic Analysis of Amino Acid Enantiomers

142 142 143 143 143 144 144

6.5. Conclusions

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References

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Liquid Chromatography: Applications http://dx.doi.org/10.1016/B978-0-12-415806-1.00006-1

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Copyright Ó 2013 Elsevier Inc. All rights reserved.

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6.1. INTRODUCTION Amino acids and bioamines play crucial roles in living systems, and liquid chromatographic separationedetermination techniques for amino acids and bioamines are essential tools for a wide range of research areas, including life science, food chemistry, environmental analysis, and pharmaceutical science. Liquid chromatographic methods for the determination of amino acids and amino compounds are mainly classified into two approaches. The direct separation of amino acids and amines followed by postcolumn derivatization with colorogenic or fluorogenic reagents or direct separation followed by mass spectrometric detection. The second approach is the indirect separation of amino acids and amines following precolumn derivatization with reagents suitable for the detection of these compounds with various detectors. In this chapter, we provide an overview of the liquid chromatographic analysis of amino acids and biological amines, including enantioselective analysis of amino acids.

6.2. DIRECT SEPARATION OF AMINO ACIDS AND AMINES Most amino acids and amino compounds do not have strong UVeVis absorbing groups nor fluorescent moieties, therefore, derivatization processes are useful for the sensitive analysis of these compounds. The direct separation of these compounds by liquid chromatography using a postcolumn derivatization system is a simple approach, because tedious precolumn derivatization steps are not needed and most of the postcolumn derivatization methods are easily automated. For the liquid chromatographic separation of amino acids and amines, cation-exchange columns are usually employed, and ninhydrin, o-phthalaldehyde, and fluorescamine are widely used as postcolumn derivatization reagents. Sensitive LCeMS/MS methods for the detection of native amino acids have also been reported.

6.2.1. Postcolumn Colorimetric Derivatization with Ninhydrin Ninhydrin (2,2-dihydroxyindane-1,3-dione) reacts with primary and secondary amino compounds to form characteristic colored compounds. Since its discovery by Ruhemann in 1910 [1], this colorimetric reaction has been widely used for the detection of amino acids, peptides, proteins, and amines. By the reaction with primary amino acids, a typical

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133

blue-violet-colored compound (Ruhemann purple, Figure 6.1) is formed [2,3], and a yellow-colored compound by the reaction with proline and hydroxyproline. Normally, the amino acids are separated by the cationexchange column, and the column effluent is mixed with ninhydrin reagent. The mixture is then pumped through a reaction coil set in a boiling-water bath, and the blue-violet color is monitored at 570 nm. For the determination of proline and hydroxyproline, the yellow color is monitored at 440 nm. Fully automated LC systems originally established by Spackman, Stela, and Moore in 1958 [4] have been used with various improvements in the natural sciences for the determination of amino acids. Because the ninhydrin postcolumn-labeling LC method is reproducible and accurate, it is still one of the most useful techniques for the determination of amino acids.

6.2.2. Postcolumn Fluorescence Derivatization with o-Phthalaldehyde A highly sensitive fluorescence reaction of amino acids was identified by Roth in 1971 [5]. After trials using o-diacetylbenzene with reducing agents, o-phthalaldehyde (OPA) was found to be a suitable fluorogenic reagent for the sensitive determination of primary amines and amino acids. OPA reacts with most of the amino acids (except cysteine, proline, and hydroxyproline) under alkaline conditions in the presence of reducing reagents to generate a bright blue fluorescence. As reducing reagents, alkylthiols, such as 2-mercaptoethanol, are widely used. The structure of the fluorescent adduct formed by the reaction of OPA and thiols with amines was determined by Simons and Johnson in 1976 [6] as 1-alkylthio-2-alkyl-substituted isoindoles (Figure 6.1). Because OPA,

+

Ninhydrin

+

+

OPA

+

Fluorescamine

FIGURE 6.1

Reaction of postcolumn derivatization reagents with amino acids.

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6. AMINO ACID AND BIOAMINE SEPARATIONS

and the reducing reagents, alkylthiols, do not fluoresce, the OPA reaction could be used as a postcolumn fluorescence derivatization procedure for amino compounds [7]. In the OPA postcolumn derivatization procedure, amino acids are separated by a cation-exchange column similar to the ninhydrin method, and the column effluent is then mixed with the OPA reagent. Because the reaction of OPA with primary amino compounds is sufficiently rapid (within a few minutes) even at room temperature, the heating system essential for the ninhydrin reaction is unnecessary. After the reaction, the blue fluorescent derivatives are determined at 440e460 nm with excitation at 340 nm. The OPA method is 5e10 times more sensitive than the ninhydrin and fluorescamine methods.

6.2.3. Postcolumn Fluorescence Derivatization with Fluorescamine Fluorescamine was developed by Weigele et al. in 1972 [8], based on the fact that strongly fluorescent pyrrolinones were formed by the reaction of ninhydrin, phenylacetaldehyde, and primary amines. The reagent, 4-phenylspiro[furan-2(3H),1’-phthalan]-3,3’-dione (fluorescamine), is nonfluorescent, and it reacts with primary amines, amino acids, and peptides under aqueous conditions in a few minutes at room temperature to form intensely fluorescent substances (Figure 6.1). On the other hand, nonfluorescent derivatives are formed by the reaction of fluorescamine and secondary amino compounds. Therefore, fluorescamine can be used for the selective determination of primary amino compounds, and the fluorophore produced by the reaction is the expected pyrrolinone. Because the reaction is sufficiently rapid and the hydrolysis products are nonfluorescent, the fluorescamine reaction is applicable for the postcolumn fluorescence derivatization of primary amino compounds [9]. The amino acids are separated by a cationexchange column similar to the ninhydrin method, and the column effluent is mixed with an alkaline-buffered solution and fluorescamine reagent. The fluorescent derivatives are detected at 480 nm with excitation at 390 nm.

6.2.4. ESIeMS/MS Determination of Underivatized Amino Acids LCeMS/MS methods are powerful tools for the determination of various compounds in complex biological matrices. For the determination of amino acids by MS/MS detection, a volatile mobile phase is required, and therefore, an ion-pairing reversed-phase LC system with a volatile

6.3. INDIRECT SEPARATION OF AMINO ACIDS AND AMINES

135

acid modifier is generally used. As an example, 76 amino acids of biological interest, including all 20 proteinogenic amino acids, can be analyzed within 15 min using LCeMS/MS [10]. The separation was performed on a short C18 column packed with 3-mm particles, and tridecafluoroheptanoic acid (TDFHA) was used as the ion-pairing agent. After the separation, the underivatized amino acids were monitored by a positive-mode ESIeMS/MS detection system, and the lower limit of quantification was from 250 fmol to 50 pmol. Considering that the lower limit of quantitation using fluorescence derivatization is around 10 pmol [7e9], the LCeMS/MS system enables the rapid and simultaneous determination of a large number of amino acids with acceptable sensitivity.

6.3. INDIRECT SEPARATION OF AMINO ACIDS AND AMINES For the liquid chromatographic separation of amino acids and amino compounds, indirect separation techniques after precolumn labeling of the amino group are also widely used. By the precolumn derivatization approach, amino compounds are converted into structures that are suitable for separation and suitable for detection by various sensitive detectors. For the separation of the labeled amino compounds, a wide variety of separation columns, including reversed-phase, can be used. Concerning detection, UVeVis absorbance, fluorescence, and also MS (MS/MS) detectors are widely used, depending on the properties of the derivatization reagents.

6.3.1. Derivatization with UVeVis Reagents Amino acids and many small biological amines usually do not have strong absorbing or fluorescent moieties, thus derivatization approaches are often used for their determination. Because UVeVis absorption is one of the most widely used techniques in liquid chromatography, various UVeVis pre-column derivatization reagents for amino compounds have been reported. Below, we have summarized examples of some widely used reagents. Phenyl Isocyanate and Phenyl Isothiocyanate Isocyanates and isothiocyanates react with primary and secondary amino compounds, and a variety of derivatization reagents have been reported. Both aliphatic and aromatic amines react with isocyanates to form N,N’-disubstituted ureas (Figure 6.2). As an example, aliphatic amines derivatized with phenyl isothiocyanate (PITC) were nicely

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+

+

PIC NBD-F

+

DABS-Cl

+

+

AQC

PITC

DNS-Cl

Fmoc-Cl

PSE

APDS

SFP

FIGURE 6.2 Reaction of precolumn derivatization reagents with amino acids.

separated by gradient elution with a water-acetonitrile mobile phase on an ODS column [11]. The derivatives were monitored by UV absorbance at 240 nm. PITC is well-known because of the pioneering wok of Edman, who established its use for peptide sequencing [12e14]. PITC is also widely used for the determination of amino acids [15]. PITC reacts with primary and secondary amino compounds, including amino acids, under alkaline conditions to form phenylthiocarbamyl (PTC) derivatives. All the PTC derivatives of 20 proteinogenic amino acids and 22 other physiological amino acids can be separated within 60 min by reversed-phase liquid chromatography and detected at 254 nm [16]. 4-N,N-Dimethylaminoazobenzene-4’-Sulfonyl Chloride In 1975, Lin and Chang designed a sensitive chromophoric labeling reagent, 4-N,N-dimethylaminoazobenzene-4’-sulfonyl chloride (DABSCl), for the determination of amino acids [17]. The reagent, DABS-Cl, reacts with primary and secondary amino groups (Figure 6.2), and the dabsylated amino compounds are detected by their absorbance at around 450 nm. All proteinogenic amino acids including proline are easily derivatized within 10 min at 70 C. The resultant dabsyl derivatives of 20 proteinogenic amino acids were separated by various reversed-phase columns within about 30 min and detected simply by their absorbance in the visible region [18,19]. Because the dabsyl amino acid derivatives are

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137

stable and have sufficient sensitivity, DABS-Cl is one of the more promising reagents for the quantitative and qualitative analysis of amino compounds.

6.3.2. Derivatization with Fluorescent Reagents Precolumn fluorescence derivatization is one of the most useful procedures for the sensitive determination of amino acids and bioamines. A large number of fluorescence labeling reagents have been reported with various combinations of fluorescent moieties and groups that are reactive with amines. The more useful ones are the fluorogenic reagents in which the reagent itself is nonfluorescent and the derivatives highly fluorescent. Because fluorescence detection is sensitive and selective by setting both the excitation and emission wavelengths, it has been widely used for the determination of trace levels of compounds in complicated biological matrices. o-Phthalaldehyde The OPA reagent was first reported in 1971 by Roth as a postcolumn fluorogenic reagent for amines [5] and has been widely used for the sensitive determination of primary amino compounds. However, the fluorescent derivatives are not sufficiently stable, and it is sometimes difficult to obtain reproducible results using the postcolumn derivatization system. A precolumn derivatization technique has also been developed using OPA in the presence of alkylthiol compounds such as 2-mercaptoethanol. OPA rapidly reacts with primary amino compounds within 2 min at room temperature, and the derivatives can be separated by reversed-phase liquid chromatography [20]. Fluorescence detection of the derivatives is performed at 440 nm (emission wavelength) with excitation at 330 nm. Because OPA does not react with secondary amino compounds, proline and hydroxyproline can not be determined by this method. Replacement of 2-mercaptoethanol with other thiols, such as 2-ethanethiol [21] and 3-mercaptopropionic acid [22], produced more stable fluorescent derivatives. 1-Dimethylaminonaphthalene-5-Sulfonyl Chloride During the course of protein chemistry research looking for fluorescent compounds sensitive to the surrounding hydrophobicity, Weber designed 1-dimethylaminonaphthalene-5-sulfonyl chloride (DNS-Cl) in 1952 [23]. DNS-Cl reacts with primary and secondary amino compounds (Figure 6.2) to form a fluorescent derivative with a yellowgreen fluorescence [24]. DNSeamino acids can be separated by both normal-phase and reversed-phase liquid chromatography [25] and

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detected by their fluorescence emission at 500e530 nm with excitation at around 350 nm. The fluorescence intensities of the DNS derivatives are stronger in organic solvents than those in the aqueous solutions frequently used for reversed-phase liquid chromatography. Nevertheless, DNS-Cl is still one of the most widely used reagents, and many applications have been reported for the determination of various amino acids and bioamines. 9-Fluorenylmethyl Chloroformate In 1972, Carpino and Han designed a novel reagent, 9-fluorenylmethyl chloroformate (Fmoc-Cl; Figure 6.2), for the protection of the amino group during peptide synthesis [26]. Moye and Boning established, in 1979 [27], an LC method using Fmoc-Cl for the determination of primary and secondary amines, and since then, this reagent has been widely used as a fluorescent labeling reagent for amino compounds [28]. Fmoc-Cl rapidly reacts with amino compounds (within 1 min) in alkaline conditions, and the derivatives can be determined by the fluorescence emission at 310 nm with excitation at 260 nm. Because Fmoc-Cl is highly fluorescent, the excess reagent and its fluorescent hydrolysis side products should be removed prior to separation and detection [28]. The resultant amino acid derivatives can be separated by reversed-phase liquid chromatography. 6-Aminoquinolyl-N-Hydroxysuccinimidyl Carbamate A highly reactive and sensitive derivatization reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) was reported by Cohen and Michaud in 1993 [29]. By reaction with this reagent, amino compounds are converted into urea derivatives (Figure 6.2), that can be separated by reversed-phase liquid chromatography. Fluorescence detection of AQCeamino acids is carried out at 395 nm with excitation at 250 nm (Figure 6.3). Because the emission wavelength of the AQCeamino acid is different from that of 6-aminoquinolone, a major by-product of the derivatization reaction, selective determination of amino compounds can be performed without removing excess reagent. Furthermore, the derivatization reagent AQC is immediately hydrolyzed to 6-aminoquinolone, N-hydroxysuccinimide and carbon dioxide. Therefore, the reaction mixture could be directly injected into the LC system without quenching the reaction. 4-Fluoro-7-Nitro-2,1,3-Benzoxadiazole During research on antileukemic compounds having the 4-nitrobenzo2-oxa-1,3-diazole (NBD) moiety, Ghosh and Whitehouse noticed that some 7-amino derivatives of the NBD analogs were highly fluorescent. In 1968, they reported a novel fluorogenic reagent, 7-chloro-4-nitrobenzo-2-oxa-1,

139

6.3. INDIRECT SEPARATION OF AMINO ACIDS AND AMINES

Phe Leu

Fluorescence intensity

Ile Val

Met Tyr Lys Ser His Glu NH3 Gly Asp

Arg Ala Thr

Pro

AMQ

10

Cys

15

20

25

30

(min)

FIGURE 6.3

Reversed-phase LC separation of AQC-labeled amino acids. Separation was generated on a Waters Nova-Pak C18 column. Source: Reproduced from reference [29] with permission.

3-diazole (equal to 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl)), for the determination of amino compounds [30]. As a highly reactive analog of NBD-Cl, Imai and Watanabe reported 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) in 1981 [31], which has fluorine at the p-position to the nitro group. The fluorogenic reagent NBD-F rapidly reacts with both primary and secondary amino compounds within 5 min under mild conditions (pH 7e8, 50e60 C) to produce highly fluorescent derivatives (Figure 6.2). The NBD derivatives of all proteinogenic amino acids can be separated by reversed-phase liquid chromatography, and highly sensitive detection can be carried out (except for tryptophan) at 530 nm with excitation at 470 nm [32,33]. 4-(1-Pyrene)Butyric Acid N-Hydroxysuccinimide Ester (Intramolecular Excimer-Forming Fluorescence Derivatization of Polyamines) Dipyrene derivatized molecules are known to form intramolecular excimers. The excimer exhibits a fluorescence emission at a wavelength longer than that of the monomer, and selective analysis of polypyrenelabeled compounds can be carried out using the excimer fluorescence.

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In 2000, Nohta et al. described a method for the determination of biologically active polyamines by intramolecular excimer-forming derivatization with 4-(1-Pyrene)butyric acid N-hydroxysuccinimide ester (PSE) [34]. By this method, dipyrene-labeled putrescine, cadaverine, spermidine, and spermine could be separated by reversed-phase liquid chromatography and specifically detected by the excimer fluorescence at 475 nm with excitation at 345 nm. The excimer fluorescence-emission wavelength is far different from that of the monomer fluorescence-emission wavelength (375 nm) derived from the excess PSE reagent, the hydrolysate product (4-(1pyrene)butyric acid), and other monopyrene-labeled derivatives. In real biological samples, various monopyrene-labeled derivatives are formed by reaction with PSE and severely interfere with the determination of polyamines.

6.3.3. Derivatization for Mass Spectrometric Detection Various derivatization techniques are also useful for the MS (MS/MS) detection of amino compounds. Although native amines or amino acids could be detected by MS detection, precolumn derivatization procedures are also frequently used to obtain better separation by the widely used reversed-phase columns. By using precolumn derivatization with reagents suitable for MS or MS/MS, a highly sensitive and selective determination of amino compounds can be performed. 6-Aminoquinolyl-N-Hydroxysuccinimidyl Carbamate AQC is one of the best precolumn fluorescence derivatization reagents for amino compounds [29]. Currently, MS/MS detection is frequently used for the selective determination of biological substances in complex mixtures. The reagent AQC reacts with primary and secondary amines to form aminoquinoline-labeled compounds via a carbamide linkage. These derivatives are separated by reversed-phase liquid chromatography and can be monitored by electrospray ionizationemass spectrometry. The loss of the aminoquinoline tag occurs readily and can be monitored by MS/MS detection, thus, metabolite analysis of amino compounds can be carried out [35]. 3-Aminopyridyl-N-Hydroxysuccinimidyl Carbamate In 2009, Shimbo et al. designed a new precolumn derivatization reagent, 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS; Figure 6.2), for the LCeMS/MS determination of amino compounds [36]. Amino compounds easily react with APDS under mild alkaline conditions at 55 C within 10 min and can be directly injected into the reversedphase LC system. The derivatized molecules are monitored by the common fragment ion (m/z 121) derived from the aminopyridyl moiety

6.4. ENANTIOSELECTIVE LIQUID CHROMATOGRAPHIC

141

FIGURE 6.4 Overlaid mass chromatograms (a) of APDS-tagged 105 amino compounds. An inertsil C8-3 column was used for the separation. (b) Typical chromatograms of APDS-tagged amino acids with the same mass. Source: Reproduced from reference [36] with permission.

of the reagent. By using the rapid gradient procedure, more than 100 analytes could be selectively determined with high sensitivity within 10 min (Figure 6.4).

6.4. ENANTIOSELECTIVE LIQUID CHROMATOGRAPHIC ANALYSIS OF AMINO ACIDS Most amino acids have a chiral center at the alpha position, resulting in and D-enantiomers. High-performance liquid chromatography is the most widely used technique for the separation of amino acid enantiomers. For that purpose, various chiral derivatizing reagents and chiral stationary phases are frequently used. The following topics are typical examples of the LC separation and determination of amino acid enantiomers. L-

6.4.1. 1-Fluoro-2,4-Dinitrophenyl-5-L-Alanine Amide (Marfey’s Reagent) In 1984, a new reagent, 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA), was reported by Marfey for the enantiomeric separation of amino acids [37]. FDAA contains the enantiomerically pure L-alanine moiety in the reagent and reacts with amino acids to form diastereomers (Figure 6.5). As of now, many Marfey reagent analogs have been reported

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*

*

+

FDAA

*

+

*

OPA

* +

*

FLEC FIGURE 6.5 Reaction of chiral derivatization reagents with amino acid enantiomers.

[38,39]. By using a reversed-phase LC system, the enantiomers of FDAAederivatized amino acids could be separated and detected by their absorbance at 340 nm.

6.4.2. o-Phthalaldehyde plus Chiral Thiols OPA was originally established for the postcolumn derivatization of amino compounds [5], then a precolumn derivatization procedure was also reported using 2-mercaptoethanol as a thiol compound [20]. Because the fluorescent derivative is an adduct of OPA, amino acid, and thiol, the use of an optically active thiol compound results in the formation of a diastereomer (Figure 6.5). In 1984, Aswad reported a method using N-acetyl-Lcysteine [40], wherein aspartic acid enantiomers could be sufficiently separated. By a similar method, the enantiomers of 21 amino acids including 17 proteinogenic amino acids can be separated by reversedphase liquid chromatography in about 60 min [41,42], and the derivatives were detected by their fluorescence at 443 nm with excitation at 344 nm. In 1994, Bru¨ckner et al. reported a set of chiral thiol compounds, N-isobutyryl-L-cysteine (IBLC) and N-isobutyryl-D-cysteine (IBDC), that could be utilized for the OPA procedure [43]. These thiol compounds enable complete separation of proteinogenic amino acid enantiomers in a single reversed-phase LC run within 70 min, and the elution order of the enantiomers could be reversed by the replacement of IBLC with IBDC.

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143

6.4.3. (D)-1-(9-Fluorenyl)Ethyl Chloroformate In 1987, Einarsson et al. designed a new chiral derivatization reagent [44], (þ)-1-(9-fluorenyl)ethyl chloroformate (FLEC). This reagent is an analog of Fmoc-Cl [26], which is widely used as a fluorescence-labeling reagent for amino compounds. FLEC contains an ethyl group instead of the methyl group of Fmoc-Cl and, thus, has a chiral carbon in its structure. FLEC reacts with amino acids (Figure 6.5) within 4 min at room temperature, and the derivatives can be detected by their fluorescence at 310 nm with excitation at 260 nm. By using a reversed-phase column, the enantiomers of 17 primary amino acids could be separated within about 70 min in a single run [44].

6.4.4. Cyclodextrin-Bonded Chiral Stationary Phase Cyclodextrins (CDs) are useful selectors for the separation of enantiomers, and various chiral stationary phases containing a CD moiety have been reported. Armstrong et al. reported the direct enantiomer separation of several amino acids using an a-CD bonded stationary phase [45]. The linkage of the a-CD bonded chiral stationary phase is hydrolytically stable, and by using the enantioselective column, 22 amino acid analogs including tryptophan, phenylalanine, and tyrosine could be separated.

6.4.5. Cinchona-Alkaloid-Bonded Chiral Stationary Phase Cinchona alkaloids are low molecular-weight chiral selectors, and several enantioselective columns having quinine and quinidine analogs have been reported. In 2011, Reischl et al. reported an enantioselective and chemoselective method for the determination of all proteinogenic amino acids [46]. The amino acids were derivatized with succinimidyl ferrocenyl propionate (SFP; Figure 6.2) and separated by a quinine-based weak chiral anion-exchange column, QD-AX. With the mobile phase containing ammonium formate, the enantiomers of all 20 proteinogenic amino acids except the achiral glycine were separated within 10 min. Additionally, by using MS/MS detection, the chemoselective analysis of all amino acids could be performed in a single LC run.

6.4.6. Two-Dimensional Liquid Chromatographic Analysis of Amino Acid Enantiomers For the determination of trace amounts of D-amino acids in complicated biological matrices, two-dimensional LC with two separation modes is a straightforward approach. As an example, a small amount of D-alanine

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Auto sampler

Microboremonolithic-ODS

D

Waste

First dimension

Fluorescence intensity

P P

0

Ser Gln Arg Asp Gly alloThr

20

2D Fluorescence intensity

Asn

His

0

L

His

15

30

Asp

L

20

60 (min)

0

L

Gln

D 10

0

10

10 0

Arg D

10 L

Glu

0

10

0

10

L 20

10

L

Thr

D

D 40 0

L

D

0

allo-Thr

Gly

D 0

Glu Thr

Ser

L

Waste

Second dimension

40 Asn D

D

D

Multiloop valve

P P

1D

Narrow boreenantioselective column

HPV

D 20

0

10

FIGURE 6.6 Flow diagram of an enantioselective two-dimensional LC system and the chromatograms obtained for rat urine. Enantiomers of NBDeamino acids were separated on a Sumichiral OA-2500S column Source: Reproduced from reference [50] with permission.

(as a sensitive fluorescence derivative with NBD-F) could be determined by the combination of a reversed-phase column and a Pirkle-type enantioselective column with naphthylglycine as the chiral selector [47]. By using this system, the fraction of NBDealanine (as the mixture of D plus L forms) was on-line collected to the loop device and automatically transferred to the enantioselective column to separate the D- and L-enantiomers. Using a microbore ODS column and a narrow bore enantioselective column, a more sensitive two-dimensional LC analysis of the NBDeamino acid enantiomers could be carried out [48]. These two-dimensional LC systems could be expanded to the simultaneous determination of multiple D-amino acids by adopting a multiloop device (Figure 6.6), and the sensitive and selective analysis of various amino acid enantiomers in real biological samples was accomplished in a single LC run [49,50].

6.5. CONCLUSIONS Liquid chromatographic separation techniques are useful for the simultaneous determination of various amino acids and bioamines.

REFERENCES

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Concerning real-world samples, uncountable numbers of interfering substances, including peptides and other amino compounds, are present in the complicated biological matrices, such as tissues and physiological fluids. Therefore, selective liquid-phase separation techniques in combination with sensitive detection systems are indispensable approaches. The development of the novel reagents as well as novel separation techniques have definitely contributed (and will also continue to contribute) to expanding the frontiers of amino acid and bioamine research.

References [1] Ruhemann S. Cyclic di- and tri-ketones. J Chem Soc 1910;97:1438e49. [2] Ruhemann S. Triketohydrindene hydrate, Part V. The analogues of uramil and purpuric acid. J Chem Soc 1910;97:1486e92. [3] West R. Siegfried Ruhemann and the discovery of ninhydrin. J ChemEduc 1965;42:386e7. [4] Spackman DH, Stein WH, Moore S. Automatic recording apparatus for use in the chromatography of amino acids. Anal Chem 1958;30:1190e206. [5] Roth M. Fluorescence reaction for amino acids. Anal Chem 1971;43:880e2. [6] Simons Jr SS, Johnson DF. The structure of the fluorescent adduct formed in the reaction of o-phthalaldehyde and thiols with amines. J Am Chem Soc 1976;98: 7098e9. [7] Benson JR, Hare PE. o-Phthalaldehyde: Fluorogenic detection of primary amines in the picomole range. Comparison with fluorescamine and ninhydrin. Proc Nat Acad Sci USA 1975;72:619e22. [8] Weigele M, Debernardo SL, Tengi JP, Leimgruber W. A novel reagent for the fluorometric assay of primary amines. J Am Chem Soc 1972;94:5927e8. [9] Udenfriend S, Stein S, Bo¨hlen P, Dairman W, Leimgruber W, Weigele M. Fluorescamine: a reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range. Science 1972;178:871e2. [10] Piraud M, Vianey-Saban C, Petritis K, Elfakir C, Steghens JP, Bouchu D. Ion-pairing reversed-phase liquid chromatography/electrospray ionization mass spectrometric analysis of 76 underivatized amino acids of biological interest: a new tool for the diagnosis of inherited disorders of amino acid metabolism. Rapid Comm Mass Spec 2005;19:1587e602. [11] Bjo¨rkqvist B. Separation and determination of aliphatic and aromatic amines by highperformance liquid chromatography with ultraviolet detection. J Chromatogr 1981;204:109e14. [12] Edman P. A method for the determination of the amino acid sequence in peptides. Arch Biochem 1949;22:475e6. [13] Edman P. Method for determination of the amino acid sequence in peptides. Acta Chem Scand 1950;4:283e93. [14] Edman P, Begg G. A protein sequenator. Euro J Biochem 1967;1:80e91. [15] Heinrikson RL, Meredith SC. Amino acid analysis by reverse-phase high-performance liquid chromatography: precolumn derivatization with phenylisothiocyanate. Anal Biochem 1984;136:65e74. [16] Cohen SA, Bidlingmeyer BA, Tarvin TL. PITC derivatives in amino acid analysis. Nature 1986;320:769e70. [17] Lin JK, Chang JY. Chromophoric labeling of amino acids with 4-dimethylaminoazobenzene-4’-sulfonyl chloride. Anal Chem 1975;47:1634e8.

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[18] Knecht R, Chang JY. Liquid chromatographic determination of amino acids after gasphase hydrolysis and derivatization with (dimethylamino)azobenzenesulfonyl chloride. Anal Chem 1986;58:2375e9. [19] Jansen EHJM, Berg RHVD, Both-Miedema R, Doorn LB. Advantages and limitations of pre-column derivatization of amino acids with dabsyl chloride. J Chromatogr 1991;553:123e33. [20] Lindroth P, Mopper K. High performance liquid chromatographic determination of subpicomole amounts of amino acids by precolumn fluorescence derivatization with o-phthaldialdehyde. Anal Chem 1979;51:1667e74. [21] Fleury MO, Ashley DV. High-performance liquid chromatographic analysis of amino acids in physiological fluids: on-line precolumn derivatization with o-phthaldialdehyde. Anal Biochem 1983;133:330e5. [22] Godel H, Graser T, Fo¨ldi P, Pfaender P, Fu¨rst P. Measurement of free amino acids in human biological fluids by high-performance liquid chromatography. J Chromatogr 1984;297:49e61. [23] Weber G. Polarization of the fluorescence of macromolecules 2. Fluorescent conjugates of ovalbumin and bovine serum albumin. Biochem J 1952;51:155e67. [24] Hartley BS, Massey V. The active centre of chymotrypsin 1. Labelling with a fluorescent dye. Biochim Biophys Acta 1956;21:58e70. [25] Bayer E, Grom E, Kaltenegger B, Uhmann R. Separation of amino acids by high performance liquid chromatography. Anal Chem 1976;48:1106e9. [26] Carpino LA, Han GY. The 9-fluorenylmethoxycarbonyl amino-protecting group. J Organ Chemy 1972;37:3404e9. [27] Moye HA, Boning Jr AJ. A versatile fluorogenic labelling reagent for primary and secondary amines: 9-Fluorenylmethyl chloroformate. Anal Lett 1979;12:25e35. [28] Einarsson S, Josefsson B, Lagerkvist S. Determination of amino acids with 9-fluorenylmethyl chloroformate and reversed-phase high-performance liquid chromatography. J Chromatogr 1983;282:609e18. [29] Cohen SA, Michaud DP. Synthesis of a fluorescent derivatizing reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and its application for the analysis of hydrolysate amino acids via high-performance liquid chromatography. Anal Biochem 1993;211:279e87. [30] Ghosh PB, Whitehouse MW. 7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole: a new fluorigenic reagent for amino acids and other amines. Biochem J 1968;108:155e6. [31] Imai K, Watanabe Y. Fluorimetric determination of secondary amino acids by 7-fluoro4-nitrobenzo-2-oxa-1,3-diazole. Anal Chim Acta 1981;130:377e83. [32] Watanabe Y, Imai K. High-performance liquid chromatography and sensitive detection of amino acids derivatized with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole. Anal Biochem 1981;116:471e2. [33] Hamase K, Homma H, Takigawa Y, Fukushima T, Santa T, Imai K. Regional distribution and postnatal changes of D-amino acids in rat brain. Biochimit Biophys Acta 1997;1334:214e22. [34] Nohta H, Satozono H, Koiso K, Yoshida Y, Ishida J, Yamaguchi M. Highly selective fluorometric determination of polyamines based intramolecular excimer-forming derivatization with a pyrene-labeling reagent. Anal Chem 2000;72:4199e204. [35] Boughton BA, Callahan DL, Silva C, Bowne J, Nahid A, Rupasinghe T, et al. Comprehensive profiling and quantitation of amine group containing metabolites. Anal Chem 2011;83:7523e30. [36] Shimbo K, Oonuki T, Yahashi A, Hirayama K, Miyano H. Precolumn derivatization reagents for high-speed analysis of amines and amino acids in biological fluid using liquid chromatography/ electrospray ionization tandem mass spectrometry. Rapid Comm Mass Spec 2009;23:1483e92.

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[37] Marfey P. Determination of D-amino acids, II. Use of a bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene. Carlsberg Res Comm 1984;49:591e6. [38] Bru¨ckner H, Gah C. High-performance liquid chromatographic separation of DL-amino acids derivatized with chiral variants of Sanger’s reagent. J Chromatogr 1991;555:81e95. [39] Bhushan R, Bru¨ckner H. Use of Marfey’s reagent and analogs for chiral amino acid analysis: assessment and applications to natural products and biological systems. J Chromatogr B 2011;879:3148e61. [40] Aswad DW. Determination of D- and L-aspartate in amino acid mixtures by highperformance liquid chromatography after derivatization with a chiral adduct of o-phthaldialdehyde. Anal Biochem 1984;137:405e9. [41] Buck RH, Krummen K. Resolution of amino acid enantiomers by high-performance liquid chromatography using automated pre-column derivatisation with a chiral reagent. J Chromatogr 1984;315:279e85. [42] Nimura N, Kinoshita T. o-Phthalaldehyde-N-acetyl-L-cysteine as a chiral derivatization reagent for liquid chromatographic optical resolution of amino acid enantiomers and its application to conventional amino acid analysis. J Chromatogr 1986;352:169e77. [43] Bru¨ckner H, Haasmann S, Langer M, Westhauser T, Wittner R, Godel H. Liquid chromatographic determination of D- and L-amino acids by derivatization with o-phthaldialdehyde and chiral thiols: applications with reference to bioscience. J Chromatogr A 1994;666:259e73. [44] Einarsson S, Josefsson B, Mo¨ller P, Sanchez D. Separation of amino acid enantiomers and chiral amines using precolumn derivatization with (þ)-1-(9-fluorenyl)ethyl chloroformate and reversed-phase liquid chromatography. Analy Chem 1987;59:1191e5. [45] Armstrong DW, Yang X, Han SM, Menges RA. Direct liquid chromatographic separation of racemates with an a-cyclodextrin bonded phase. Anal Chem 1987;59:2594e6. [46] Reischl RJ, Hartmanova L, Carrozzo M, Huszar M, Fru¨hauf P, Lindner W. Chemoselective and enantioselective analysis of proteinogenic amino acids utilizing N-derivatization and 1-D enantioselective anion-exchange chromatography in combination with tandem mass spectrometric detection. J Chromatogr A 2011;1218:8379e87. [47] Morikawa A, Hamase K, Zaitsu K. Determination of D-alanine in the rat central nervous system and periphery using column-switching high-performance liquid chromatography. Anal Biochem 2003;312:66e72. [48] Miyoshi Y, Hamase K, Tojo Y, Mita M, Konno R, Zaitsu K. Determination of D-serine and D-alanine in the tissues and physiological fluids of mice with various D-aminoacid oxidase activities using two-dimensional high-performance liquid chromatography with fluorescence detection. J Chromatogr B 2009;877:2506e12. [49] Hamase K, Morikawa A, Ohgusu T, Lindner aitsu WK, aitsu K. Comprehensive analysis of branched aliphatic D-amino acids in mammals using integrated multi-loop two-dimensional column-switching high-performance liquid chromatographic system combining reversed-phase and enantioselective columns. J Chromatogr A 2007;1143:105e11. [50] Hamase K, Miyoshi Y, Ueno K, Han H, Hirano J, Morikawa A, et al. Simultaneous determination of hydrophilic amino acid enantiomers in mammalian tissues and physiological fluids applying a fully automated micro-two-dimensional highperformance liquid chromatographic concept. J Chromatogr A 2010;1217:1056e62.

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C H A P T E R

7 Protein and Peptide Separations J. Giacometti *, D. Josic *,y *

y

Department of Biotechnology, University of Rijeka, Croatia Warren Alpert Medical School, Brown University, Providence, Rhode Island, O U T L I N E

7.1. Introduction

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7.2. Methods of Protein Liquid Chromatography 7.2.1. Size-Exclusion Chromatography 7.2.2. Ion-Exchange Chromatography 7.2.3. Methods Based on the Hydrophobic Interaction 7.2.4. Affinity Chromatography 7.2.5. Chromatography on Hydroxyapatite 7.2.6. Chromatography on Monolithic Supports 7.2.7. Displacement Chromatography

151 151 153 158 166 173 175 177

7.3. Conclusions

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Acknowledgments

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References

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7.1. INTRODUCTION The development of techniques and methods for protein purification is very important for bioscience and biotechnology. Various chromatographic techniques with different selectivities are powerful methods for the purification of biomolecules, especially the separation of proteins. Liquid Chromatography: Applications http://dx.doi.org/10.1016/B978-0-12-415806-1.00007-3

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Copyright Ó 2013 Elsevier Inc. All rights reserved.

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New developments in high-resolution liquid chromatography made this method compatible with mass spectrometry and an indispensable tool in proteomic research. In general, a chromatographic separation depends on the differential partition of proteins between the stationary phase (the chromatographic medium or the adsorbent) and the mobile phase (the buffer or organic solvent). Several liquid chromatographic methods are used for the separation of proteins. They differ mainly in the type of stationary phase employed (Table 7.1). A wide variety of materials have been used as chromatographic matrices for protein separations, classified as inorganic supports, synthetic organic polymers, or polysaccharides. Traditional standard chromatography media as well as modern high-performance media are included among these groups, as shown in Figure 7.1 [1]. All these bulk materials are designed to fulfill a general matrix requirement for protein chromatographydto minimize the interaction between the sample and the surface of the support. A combination of optimal chemical and physical properties is achieved by the use of hydroxyl or amino groups, and such standard chromatographic media, based on neutral polysaccharides and modified polyamides, are widely used for protein separations [2].

TABLE 7.1 Methods of Protein Liquid Chromatography Type of separation

Type of chromatography

Size and shape

Gel filtration (GF)/size-exclusion chromatography (SEC)

Net charge

Ion-exchange chromatography (IEX)

Hydrophobicity

Hydrophobic-interaction chromatography (HIC) Reversed-phase chromatography (RPC)

Biological function

Affinity chromatography (AC)

Interaction with ligands Metal binding

Pseudo-affinity chromatography Immobilized metal ion affinity chromatography (IMAC)

Other

Chromatography on hydroxyapatite (HT/HTP)

FIGURE 7.1 Chromatographic materials for protein separation.

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Sample preparation and extraction procedures are an important prepurification step. Different methods are used in the purification of a target protein, such us precipitation, preparative centrifugation, twophase systems, electrophoresis, or chromatography. In chromatography, proteins are separated on the basis of their different affinity to a stationary or a mobile phase. Purification of proteins is often associated with a number of steps in order to obtain higher purity, defined as a purification strategy. However, all of these steps are associated with sample loss. On the other hand, a single chromatographic step is most often insufficient, and it is necessary to apply several steps to achieve the desired purity of the target molecule. To meet these criteria, further development of higher selectivity and effectiveness of chromatographic supports is still required. To determine the conditions for best performance, it is also necessary to optimize the chromatographic steps during protein purification [3]. In addition, sometimes, it is necessary to remove solvent from the eluate to achieve higher concentrations of stable proteins in a final form, ready for the intended application. That requires an additional step in the product preparation (e.g., freeze drying, ultrafiltration, or dialysis). Recombinant DNA techniques are widely used in the production of therapeutic proteins, and the problems associated with solubility, structural integrity and biological activity, and contamination with host proteins have to be reduced to a minimum. Consequently, the development of fast, reliable analytical assays is essential for monitoring the progress of a purification process and assessing its effectiveness (yield, biological activity, recovery) [4]. Until the end of the last century, bulk chromatographic materials containing porous or nonporous particles were used. Monolithic materials made of synthetic or natural polymers and silica-based monoliths with similar surface chemistry have also been used as additional chromatographic supports in the last 15e20 years. Due to the rapid interaction between the sample components and the surface, monolithic materials enable very fast chromatographic separation of large molecules such as proteins and nucleic acids and nanoparticles such as viruses and protein aggregates [5,6].

7.2. METHODS OF PROTEIN LIQUID CHROMATOGRAPHY 7.2.1. Size-Exclusion Chromatography The matrices in size-exclusion chromatography (SEC) consist of porous particles, and the separation of proteins is achieved according to the size

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FIGURE 7.2 Principles of size-exclusion chromatography (SEC). In this chromatographic method, the analyte does not interact with the surface of the stationary phase. Separation is achieved by the differential penetration and exclusion of the sample components in and out of the pores of the packing material. Particles of different sizes elute at different rates. Small molecules, which can penetrate into the pores of the stationary phase, elute later. On the other hand, a very large molecule, which cannot penetrate into the pore system, elutes earlier, in the dead volume of the column. The molecules of intermediate size, which can partially penetrate the pores of the stationary phase elute in the intermediary time, between very large and very small molecules.

and shape of the protein (see Figure 7.2) that passes through a packed column. This technique is sometimes referred as gel-filtration, molecular sieve, or gel-permeation chromatography. SEC can be used in LC for the analysis of sample components if there are sufficient molecular-weight differences among solutes [7]. The selectivity of porous bulk materials in SEC depends solely on their porosity. Consequently, an appropriate matrix for SEC is a support with reduced adsorptive properties such as natural (mostly agarose or dextran) and synthetic polymers (mostly polyacrylamide). The pore size of chromatographic materials (e.g., Sephadex) depends on the degree of crosslinking in the particular polymer. Predominantly, hydroxyl groups coverage is favorable for use in the fractionation of hydrophilic proteins. After addition of water, some polymers such as agarose form gels spontaneously. To prevent of denaturation of proteins, macroporous silica, used as a support for SEC, must be coated with a protecting hydrophilic layer [7].

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To achieve an optimal and reproducible separation, proper selection of a gel with a suitable separation range is necessary. In general, resins are classified based on the different hypothetical sizes of globular proteins. The residence of proteins and their separation in SEC depends on their size and shape. In general, the gels with smaller pores are used for fast desalting procedures (e.g., peptide purification), and the gels with larger ones are used for separation of small- and middle-sized proteins. Stationary phases containing particles with very large pore sizes are used for purification of biological complexes [8]. Uniformity of pore distributions within the gel is also crucial for the optimal separation of proteins [7]. Contrary to other chromatographic media, the selectivity of SEC matrix is not dependent on adjusting the composition of the mobile phase. To preserve the structure and biological activity of proteins, the sample can sometimes require a buffer solution of defined pH and ionic composition. However, high salt concentration in the mobile phase should be avoided to prevent the undesirable interaction of proteins with the support [7]. Compared to other chromatographic methods, both capacity and resolution in SEC are relatively low. An additional problem is the degree of sample dilution. SEC is a useful and simple method for the separation of multimers. An advantage of this approach is the high biological activity of proteins after SEC due to the absence of surface interactions of the analyte with the chromatographic support. Sometimes, this method is also used as the final polishing step in cases of low sample volume. Finally, SEC has a wide range of applicability both in preparative (buffer exchange and protein fractionation) and analytical protein purification [9]. Finally, size-exclusion chromatography coupled with more than one detector, such as laser light-scattering (LS) and refractive-index (RI) detection, provides an excellent approach for determination of the molecular weight (MW) of proteins [10].

7.2.2. Ion-Exchange Chromatography Ion-exchange chromatography (IEX) has been used for more than 50 years for the separation and purification of proteins. In comparison with other chromatographic methods, 40% of all protein separations are related to IEX, 18% to SEC, and 29% to affinity chromatography (including immobilized-metal-affinity and dye-affinity chromatography) [1]. IEX is a method for purification of proteins based on ionic interactions between proteins and surface charges opposite to those of the charged groups on an ionic resin (see Figure 7.3). In cation-exchange chromatography, positively charged molecules are attracted to a negatively charged

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FIGURE 7.3

Principles of ion exchange chromatography (IEX). Biomolecules exhibit different degrees of interaction with charged chromatography media according to differences in their overall charge, charge density, and surface charge distribution. The separation process is based on the formation of ionic bonds between the charged groups on biomolþ 3e 2e þ e ecules (mostly, eNHþ 3 , ¼NH2 , hNH , eCOO , PO4 , SO3 ), and an ion-exchange support with the opposite charge. Nonbound biomolecules (i.e., neutral molecules with no electrical charge or molecules with the same charge as the ion-exchange support) are removed by washing, and bound biomolecules are recovered by elution with a buffer of either higher ionic strength or altered pH.

solid support. Conversely, in anion-exchange chromatography, negatively charged molecules are attracted to a positively charged solid support. The separation occurs due to competition between proteins with different charges on an ion-exchange resin. Proteins are complex ampholytes whose charges depend on the proportions of the amino acid residues in their structure as well as the acidity of the aqueous separation media. The isoelectric point (pI) of a protein depends on the structural proportion of ionizable amino acids. Positive charges are typically found when the pH of the protein solution is below 8, due to the N-terminal amine and basic residues such as arginines, lysines, and histidines. Similarly, negative protein charges typically exist above pH 6 and are due to the C-terminal carboxyl group and acidic (e.g., aspartate and glutamate) residues. The charged groups are almost always on the surface of proteins, except in

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case of metalloproteins, where the metal ion (usually inside the molecule) is usually coordinated by ligands (amino acid residues of the protein) [11]. A stoichiometric model describes the relationship between the charged groups in a protein and the stationary phase. The number of charged groups of the protein binds to the same number of oppositely charged groups of an ionic exchanger, and counterions are released both from the protein and the ion exchanger (see Figure 7.3). Protein retention on an ionic surface depends on the protein charge, surface charge, and the charge characteristics of the surrounding medium. To describe this phenomenon, Kopaciewicz et al. [12,13] developed a nonmechanistic model that shows a positive correlation between protein retention and the number of charges associated with the adsorption-desorption process. Generally, anion-exchange chromatography (AEX) is used at pH values above the isoelectric point of the protein of interest, while cationexchange chromatography (CEX) is performed below the isoelectric point. At low pH or even at very high ionic strength, proteins may adsorb very strongly to an ion exchanger. This probably occurs due to an increase in the number of hydrogen bonds [11]. The interaction between a protein and an ion exchanger depends not only on the net charge and the ionic strength but also on the surface charge distribution and conformation of the protein. Some structural changes can affect the separation by IEX. Urea is widely employed to facilitate protein separations in ion exchange chromatography at various scales. Hou et al. [14] indicated that the retention times correlate well with structural changes and they are more sensitive to the change of the tertiary structure. The properties of the ion exchanger also influence the protein separation. Depending on the functional group, ion exchangers are classified as weak or strong. Use of strong ion exchangers, such as sulfonate (CEX) and quaternary ammonium (AEX), with pKa values outside the pH range for work with proteins (i.e., pH 4e10) result in the charge of the ion exchanger remaining the same despite changes in mobile phase pH. These ion exchangers are applicable in case of weakly ionizable proteins. The benefits of the use of weak ion exchangers are related to a reduced tendency for sample denaturation, less ability to bind impurities, as well as enhanced resolution. In general, two methods are applicable in the elution strategy in IEX: changing the pH of the eluting buffer, and increasing the ionic strength by addition of salts, mostly NaCl. The most common active groups related to the ion exchange chromatographic matrices are carboxyl (eCOOH), sulfonyl (eSO3H), secondary or tertiary amines (eNH2, eNRH), and a quaternary amino

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group (-NþR2H). The ion exchangers are classified as weak-cation, strongcation, weak-anion, and strong-anion exchangers, respectively. Lendero et al. [15,16] studied a universal nondestructive, noncontaminating method for the characterization of ion-exchange chromatographic columns. This method is based on making a step change in ionic strength of buffer solutions with the same pH in the ion exchange columns and can be used for identification and determination of the type of ion exchange groups on all sorts of ion exchangers (see Figure 7.4). This investigation resulted in the observations that, after the step change from TriseHCl buffer to TriseHCl buffer with sodium chloride: (1) the effluent pH for strong-anion-exchange columns remains nearly the same or rises by less than 0.5 pH units in the shape of sharp and relatively short peak;

FIGURE 7.4 A scheme to distinguish among different active groups on the most common ion exchangers. To distinguish among different ion exchange groups on the convection interaction media supports (CIM), TriseHCl buffer pH 7.4 was pumped through the column. The change in pH values of the solution (TriseHCl buffer with NaCl) and at the column outlet were measured. The pH profiles versus elution volume normalized to the column volume were compared in three parts of the profile: (a) at low ionic-strength buffer (the effluent pH for the weak-anion exchanger DEAE was lower than for the other ion exchangers), (b) at high ionic-strength buffer (the pH of the cation exchangers reduced, whereas the pH of the strong anion exchanger remained the same), and (c) the two cation exchangers were classified by the duration of the pH drop (for the weak cation exchanger, the pH drop lasted longer than for the strong cation exchanger). Source: Printed from the Ref. [15] with permission.

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(2) for weak-anion-exchange columns, it raises more than 1 pH unit and this lasts several column volumes (CV); (3) for cation-exchange columns, it drops by more than 1 pH unit. In practice, the strategy based on the ionic strength by the addition of NaCl is the method of choice. As a rule, more weakly charged proteins are eluted at lower salt concentrations, while the more strongly charged proteins are eluted at higher salt concentrations. Stepwise elution is often used for recovery of a concentrated protein especially in preparative chromatography. In this case, the optimization of gradient conditions is needed [17e19]. Finally, IEX is one of the most used separation techniques in protein purification, for an advantage of this technique is that the elution normally takes place under mild conditions, and the protein can maintain its native conformation during the chromatographic process. Limited selectivity is the major disadvantage of this method [20]. To establish the structural and functional relationships in the characterization of all human proteins, of greatest importance are proteins of the blood. Blood plasma contains an unusually small group of highabundance proteins, namely, serum albumin, immunoglobulins, transferrin, and a-2-macroglobulin. The amount of these proteins is about 85% of the total serum protein, and they often interfere with the identification of proteins of lower abundance. In proteomics, characterization of the blood proteome requires extensive fractionation prior to mass spectrometry analyses, and the removal of high-abundance proteins and subsequent enrichment of low-abundance proteins is a crucial step. For this purpose, IEX can be combined with other chromatographic methods, mostly with SEC and chromatography on hydrophobic resins [21,22]. As shown in Figure 7.5, CEX and AEX, used after SEC and before hydrophobic interaction chromatography (HIC), were applied in the separation of human plasma proteins. After the initial fractionation steps using ammonium sulfate precipitation and SEC, in the next steps, cationand anion-exchange chromatography resulted in the highest number of identified proteins in the human plasma proteome [23]. In proteomics, multidimensional chromatographic separation and analysis of the proteins are performed at the peptide level, after proteolytic digestion of the entire proteins extracted from tissue samples (the bottom-up approach) [24]. Strong cation-exchange chromatography (SCEX) is one of the frequently used liquid-chromatography strategies, where it has been shown that peptides are eluted according to their charge in a defined process. Compared to the classical strong cation exchange followed by ion-pair reversed-phase (SCX  IPeRPLC) approach, the reversed-phase ion-pair reversed-phase (RP  IPeRPLC) showed a more homogenous distribution of eluted peptides at high pH [25,26].

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FIGURE 7.5

(a) Cation-exchange chromatography trace (UV 280 nm, black line) of the interim Analyte Library fraction of 65%-G3 (65% ammonium sulfate saturation, third pooled gel-filtration fraction). Conditions: HiTrap SP HP column with 0 to 0.5 M KCl (in 50 mM phosphate buffer, pH 5.5) gradient elution, flow rate: 1 ml/min, 1-ml fractions. (b) Anion-exchange chromatography trace (UV 280 nm, dark line) of the cation exchange chromatography flow-through of fraction 75%-G4 (75% ammonium sulfate saturation, fourth pooled gel filtration fraction). Conditions: HiTrap Q HP column, 0e0.5 M KCl (in 20 mM TriseHCl, pH 8.5) gradient elution; flow rate, 1 ml/min, 1-ml fractions. Source: Reprinted from Ref. [23] with permission.

7.2.3. Methods Based on the Hydrophobic Interaction As shown in Figure 7.5, proteins can be separated according to differences in their surface hydrophobicity using two methods: hydrophobic-interaction chromatography and reversed-phase chromatography. Hydrophobic Interaction Chromatography Hydrophobic-interaction chromatography (HIC) is a powerful method for protein purification based on the reversible interaction between the protein surface and a hydrophobic chromatographic sorbent [27]. This technique is used in protein purification as a complement to other separation techniques and as a next step after ammonium sulfate precipitation of the sample or after IEX separation. Van der Waals forces are the major contributing factor to the hydrophobic interactions [28]. Using HIC, the structural damage to protein

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molecules is minimized and its biological activity is maintained, due to a weaker interaction than affinity, ion-exchange, or reversed-phase chromatography (RPC) [29,30]. Hydrophobicity is repulsion between a nonpolar compound and a polar environment, such as water. The distribution and hydrophobicity of hydrophobic amino acids are characteristic of each protein, so a specific separation is possible with use of hydrophobic supports. Different models are used for the description of protein hydrophobicity and hydrophobic interactions, and different theories are proposed for the retention mechanism of proteins in HIC [31]. In addition to the important role of water in the strengtheningeweakening of the hydrophobicity, these interactions can be induced by changing the ionic strength, presence of organic solvents, temperature, and the pH value of the chromatographic media. Based on these properties, the HIC separation occurs by the modulation of the frequency and distribution of surface-exposed hydrophobic aminoacid residues, the hydrophobicity of the medium, the nature and composition of the sample, as well as type and concentration of salt in the mobile phase (see Figure 7.6). HIC is most strongly affected by the specific effects of the salts. To enhance protein binding to the resin at the weakly hydrophobic ligand (such as phenyl group), a salting-out salt is required. Ammonium sulphate ((NH4)2SO4) and sodium sulphate (Na2SO4) are the most-utilized

FIGURE 7.6 Typical ordering of cations and anions in a Hofmeister series. Source: Reprinted from Ref. [36] with permission.

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salts in HIC. Since binding of proteins to the HIC resin depends on the hydrophobicity of the proteins and salt concentration, both binding and elution of proteins are modulated by the (NH4)2SO4 concentration. Those salts that interact unfavorably with the proteins or increase the surface tension enhance binding, while those salts that interact favorably with the proteins, enhance elution. The unfavorable interaction increases with salt concentration, meaning that binding to HIC resins occurs at higher salt concentration and lower salt concentration weakens binding, leading to elution of proteins bound at high salt concentration [32]. Amino acids can be hidden into the hydrophobic core of proteins or may be on the surface. Both anions and cations can be sorted, according to the Hofmeister (lyotropic) series in a list of salts that influence the water tension and consequently the interaction between proteins and the HIC adsorbent [33]. Salt effects in protein precipitation and HIC were investigated for a broad combination of proteins, salts, and HIC resins in the study of Nfor et al. [34]. The interaction between hydrophobic proteins and a HIC medium is significantly influenced by the presence of certain salts in the running buffer. The most frequently used salt for the preparation of the mobile phase is (NH4)2SO4. [35]. The high salt concentration enhances the interaction between the hydrophobic components of the sample and the chromatography medium, and the lower salt concentration weakens the interaction. These interactions are a result of the presence of side chains of hydrophobic amino acids. They do not form hydrogen bonds with water and are not surrounded by water molecules. As a result of this phenomenon, the hydrophobic interaction depends on the behavior of the water molecules rather than on direct attraction between the hydrophobic molecules with the support. To and Lenhoff [37] investigated the contribution of protein solubility to retention in HIC using isocratic elution with four commercially available agarose media and four model proteins (ribonuclease A, lysozyme, myoglobin, and ovalbumin). Various retention trends (type of adsorbents, type and concentration of salt, pH) were tested as a function of the properties of each protein and the importance of proteinesurface interaction or conformational change for protein binding was found. Selectivity of HIC depends on the nature of the ligands and the matrix (the degree of ligand substitution on the matrix), the nature of the target protein, as well as the type and concentration of salt used in the mobile phase [38]. Proteins with the highest degree of hydrophobicity are most strongly retained and are eluted at the end of the chromatogram. HIC media are composed of resins that contain alkyl or aryl groups coupled to an inert, porous matrix made of spherical particles with a high internal surface area. Agarose is the main matrix suitable for preparing

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adsorbents for HIC [39]. Silica or organic polymer resins (e.g., phenyl and butyl) are also used extensively [40]. The pH of the buffers used in HIC separations is important because of the adsorption of proteins to the chromatographic support. Increase in the pH value (up to 9e10) of the mobile phase also decreases the hydrophobic interactions between proteins and the hydrophobic ligands, due to the change in charge of the protein. At high pH, silica-based supports are unstable and inadequate for protein purification. Generally, lowering the temperature promotes protein elution. Therefore, labile proteins should be separated at low temperatures [40]. Separations by HIC are often designed using nearly opposite conditions to those used in IEX. The sample is loaded in a buffer containing a high concentration of salt, which makes this method very useful as a subsequent step after proteins are eluted from ion-exchange columns by use of buffers with high salt concentration. The proteins are eluted from the HIC resin as the concentration of the salt in the buffer is decreased. Frequently, they are ready for the next purification step by IEX without further buffer exchange (Figure 7.7). Reversed-Phase Chromatography Due to the excellent resolving power, convenience, versatility, stability, and reproducibility, reversed-phase liquid chromatography (RPLC) is one of most important techniques for protein separations and the method of choice for peptide separations. RPLC has been applied on the nano, micro, and analytical scales and can be scaled up for preparative purification at the industrial scale [41,42]. In contrast to reversed phase chromatography, mechanisms of the ion-exchange and hydrophobic-interaction chromatography for peptide and protein separation is based on differences in surface hydrophobicity or surface charge [41]. Because of its compatibility with mass spectrometry (MS), RPLC is an indispensable tool in proteomic research. The increase in resolution offered by LC separation greatly enhances MS detection of sample components, and high-resolution separation reduces ion suppression in MS [42]. Reversed-phase chromatography is a separation method based on the hydrophobicity of the protein. In RPC, the hydrophobic stationary phase is based on silica gel or a synthetic polymer. In recent years, instead of bulk materials for column packing, polymer- or silica gel-based monolithic stationary phases have also been used [43]. In the presence of aqueous buffers, the solute mixture is applied at the start to the sorbent, then the solutes are eluted by the addition of organic solvent to the mobile phase. The stationary phase bears the hydrophobic ligands, mainly C4e, C8e, or C18ealkyl chains. The mobile phase contains water and a water-miscible organic solvent, such as methanol, acetonitrile, or isopropanol. Elution is continued by

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FIGURE 7.7 Methods based on the interaction between surface and proteins. Both methods involve the separation of molecules on the basis of hydrophobicity. (a) Hydrophobic interaction chromatography (HIC) is based on the adsorption of biomolecules to a weakly hydrophobic surface at high salt concentrations. Elution is carried out with a descending salt gradient. (b) The protein separation in the reversed-phase chromatography (RPLC) depends on the hydrophobic binding of the solute molecule from the mobile phase to the immobilized hydrophobic ligands attached to the stationary phase (i.e., the sorbent).The solutes are eluted in order of increasing molecular hydrophobicity. Elution can carried out by either isocratic conditions (at the constant concentration of organic solvent) or gradient elution (the amount of organic solvent is increased over a period of time).

isocratic conditions at the constant organic solvent concentration or by gradient elution by increasing of the amount of organic solvent over a period of time. Acid (usually, formic, acetic, or trifluoroacetic acid) is added to the mobile phase to render the proteins and peptides positively charged and to reduce undesirable interactions with the stationary phase. Analytical applications range from the assessment of purity of peptides following solid phase peptide synthesis to the analysis of tryptic maps of proteins, while preparative RPLC is used for the micropurification of protein fragments for sequencing to large-scale purification of synthetic peptides and recombinant proteins [44e46]. The numbers of applications of RPLC in peptide and protein purification continue to expand at an extremely rapid rate. Mant and Hodges [47] developed more quantitative selectivity parameters for peptide separations using RPLC for control de-novo design of synthetic peptide standards with the same (amino acid) composition and minimal sequence variation (SCMSV). They used a range of stationary phases (C8 and C18 alkyl, polar embedded, polar endcapped, ether-linked phenyl, and phenylhexyl) such as those already available for small molecules, further enhancing the universal value of

7.2. METHODS OF PROTEIN LIQUID CHROMATOGRAPHY

163

utilizing peptide standards to compare column performance in the separation of peptide mixtures. A reversed-phase column was used to purify the ErITC-labeled tryptic fragments, unlabeled peptides, and peptides labeled with erythrosine (see Figure 7.8).

FIGURE 7.8 Separation of tryptic peptides by RPLC on a Merck 250-3 Lichrospher WP300 RP-18 (5-mm) column. Absorbance of the eluate was recorded at 210 nm to detect all peptides and at 530 nm to detect erythrosin-labeled peptides. The upper chromatogram shows the peptide separation with a gradient of 2e90% acetonitrile in 25 mM KH2PO4 at pH 7.0. The lower chromatogram shows the rechromatography of the fraction with a retention time of 70e73 min (the bar in the upper graph) in the first step under isocratic conditions in a mixture of equal volumes of acetonitrile and 25 mM KH2PO4 pH 7.0. The peak at 13.0e13.3 min (the bar in the lower graph) was used for amino-acid sequence analysis. Source: Reprinted from Linnertz et al. [48], with permission.

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7. PROTEIN AND PEPTIDE SEPARATIONS

Reversed-phase chromatographic separation involves loading of a protein mixture onto a hydrophobic stationary phase in water or a wateresolvent mixture, usually containing very dilute acid (usually 0.1% formic or trifluroacetic acid; TFA). The elution of bound components occurs by increasing the organic solvent (usually, acetonitrile or methanol) concentration (see Figure 8.8). According to the theory, HIC and RPC are related techniques, since both are based on interactions between hydrophobic regions (amino acids) in protein molecules and hydrophobic ligands of a chromatographic support. However, experimentally, these techniques are different in the following ways: 1. The surface of the RPC supports is usually more hydrophobic than that of the HIC medium. 2. In HIC, concentrated salt solutions are used for sample application. In RPC, proteins and peptides are dissolved in water or a watereorganic solvent mixture, usually containing a dilute acid (see previously). 3. Because of the stronger hydrophobic interaction of proteins with the stationary phase, organic solvents are used for RPC elution. Elution in HIC occurs by decreasing the salt concentration in the mobile phase. 4. The use of organic solvents in RPC frequently leads to denaturation and loss of biological activity of biopolymers, especially of highmolecular weight proteins [49]. 5. Temperature, especially elevated temperature, is an important variable in controlling selectivity in RPC separations of proteins. In routine use, most HIC separations are performed at room temperature. RPC provides excellent resolution of complex protein mixtures at analytical and micro- and nano-analytical scale (see Figure 7.9). This chromatographic method also has great potential for highresolution purification as well as for low-resolution desalting steps, especially for sample preparation for proteomics applications [51]. Combined with other chromatographic methods, especially ion-exchange chromatography, and as a component (dimension) in so-called multidimensional liquid chromatography (MDLC), RPC is a key method for separation, identification, and characterization of proteins in very complex mixtures [52]. The performance of a typical RPC separation of proteins is summarized in Figure 7.9. In analytical-scale RPC separations, a continuous linear gradient is often used, as shown in Figure 7.9(a), while preparativescale separations typically employ an optimized elution scheme combining isocratic elution and gradients with different slopes, see Figure 7.9(b). A successful RPeHPLC separation is influenced by many parameters. Consequently, to meet the requirements for an application, the RPC separation has to be optimized.

7.2. METHODS OF PROTEIN LIQUID CHROMATOGRAPHY

165

FIGURE 7.9 Analytical RPLC of a crude mixture of proteins from rabbit skeletal muscle: tropomyosin (Tm) and components of troponin (Tn) complex, comprising troponin T (TnT), troponin I (TnI), and troponin C (TnC). (a) Analytical RPLC. Column, ˚ pore size; Agilent TechnolZorbax SB300 C8 (150  4.6 mm i.d., 5-mm particle size, 300 A ogies). Conditions: linear AeB gradient (1% B/min starting from 25% acetonitrile) at a flow rate of 1 ml/min. Eluent A, water containing 0.05% TFA; Eluent B, acetonitrile containing 0.05% TFA. (b) Preparative RPLC (optimized after scaling-up experiments). Column, ˚ pore size, from Waters, in column of mBondapack C8 packing, 5-mm particle size, 300 A dimensions 280  50 mm i.d. Eluent A, water containing 0.05% TFA; Eluent B, acetonitrile containing 0.05% TFA. Sample load, 5700 mg in 440 ml water containing 0.05% TFA (w13 mg protein/ml); following sample loading at 22 ml/min, a 10-min isocratic hold (at constant solvent concentration) with water containing 0.05% TFA, followed by linear AeB gradient (1.7% acetonitrile/min) up to 25% acetonitrile, then 0.1% acetonitrile/min up to 35% acetonitrile and, finally, 0.5% acetonitrile/min up to 55% acetonitrile. The positions of the individual components identified following fraction analysis are denoted in histograms. Source: Reprinted from Mant and Hodges [50], with permission.

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Choosing the optimal support and column dimensions is crucial for a successful chromatographic separation. The selection of an RPC support must be made empirically. The most critical factor in choosing the appropriate stationary phase is sample hydrophobicity. For separation of highly hydrophobic components, a less-hydrophobic stationary phase should be used to facilitate the elution. Proteins that bind strongly to a more hydrophobic support, bind more weakly to a less hydrophobic medium, and also are eluted at lower concentrations of organic solvent. The most important characteristics of the chromatographic support are surface chemistry, alkyl chain density, particle size, pore size, and mechanical stability. To balance high efficiency and short separation time, spherical nonporous or porous particles with diameters between 1.5 and 5 mm packed in short columns (3 to 5 cm long) are often used. The efficiency, but also the back pressure, of the column rises with smaller particle diameters. For large-scale preparative separations, porous particles with larger diameters are used. Use of chromatographic supports with a larger particle size allows the use of higher flow rates at lower back pressure, especially at the early stages of protein or peptide purification.

7.2.4. Affinity Chromatography Affinity chromatography is a specific purification technology based on biological function or individual chemical structure. The application of this technique is in separation of active biomolecules from denaturated or functionally different forms in the isolation of pure proteins present at low concentration and also for removing specific contaminants [53]. Proteins are separated on the basis of reversible specific interactions between proteins and a ligand coupled to a chromatography matrix (see Figure 7.10). The interaction between target molecules and binding sites with complementary surfaces to their ligands can involve different intermolecular chemical bonds, such as a combination of electrostatic or hydrophobic interactions, van der Waals forces, and hydrogen bonds. However, a specific ligand is covalently attached to an inert chromatographic matrix. This allows a high selectivity and high resolution as well as high capacity for the proteins of interest. Recovery of active material is generally very high, which enables the purification of a protein on the order of several thousand-fold. The advantage of this technique compared with others is the shortening of the purification process. On the other hand, the elution of the target molecule that binds to the affinity ligand can sometime be difficult. It can be achieved by addition of a molecule that binds competitively to the ligand (e.g., a sugar in the case

7.2. METHODS OF PROTEIN LIQUID CHROMATOGRAPHY

FIGURE 7.10

167

Typical affinity purification.

of lectin-affinity chromatography) or by a drastic change in the pH value, ionic strength, or polarity of the eluent [54]. Hydrophilic and neutral matrices are favored because they prevent nonspecific interactions between proteins and the gel matrix. The matrix should be macroporous, which means that the protein should be accessible for binding with the immobilized ligand. Agarose is the most-used material for affinity matrices. Specific ligands are covalently bound to the chromatographic matrix. These ligands are selective and bind proteins such as antibodies, protein receptors, and some enzyme inhibitors. Staphylococcal protein A is one of the first discovered IgG binding molecules and has been extensively used as a biospecific ligand. Today, protein A chromatography remains the most common technique for antibody purification on a large scale, and its further application will have great importance for both small- and large-scale purification of native polyclonal and monoclonal and recombinant antibodies [54]. Various methods can be used for activation of an affinity adsorbent. They differ on the binding chemistry of the ligand and adsorbents and on the need for a spacer arm. The activation occurs by the introduction of an electrophilic group into the matrix. In the next step, the ligand reacts with

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7. PROTEIN AND PEPTIDE SEPARATIONS

nucleophilic groups, such as amino, thiol, and hydroxyl residues (see Table 7.2). Ligands that contain a coupled amino group, attached by the ester bond, give a very stable amide linkage. The amide bond is stable up to pH 13. That makes the N-hydroxysuccinimide-activated Sepharose (NHS) supports, such as Sepharose, suitable for applications at high pH. Cyanogen bromide reacts with hydroxyl groups on Sepharose to form reactive cyanate ester groups. Under mild conditions, proteins, peptides, amino acids, and nucleic acids can be coupled to a CNBr-activated matrix via primary amino groups or similar nucleophilic groups. In weakly alkaline conditions (pH 9e10), CNBr-activated supports with primary amines give an isourea derivative. The isourea linkage of the ligand causes several problems during the purification procedure, including nonspecific binding, due to the charge and the leakage of ligand from the instability of the isourea bond [55]. In the presence of a coupling reagent, such as carbodiimide, ligands are coupled in a simple one-step procedure. Epoxy-activated matrices, such as Sepharose 6B, are used for coupling ligands that contain hydroxyl, amino, or thiol groups. Because of the long hydrophilic spacer arm, it is useful for coupling small ligands, such as choline, ethanolamine, and sugars. Three types of interactions for stability of the bound protein help in choosing the suitable eluant: pH change, adjustments of the ionic strength of the buffer, and addition of a competitor (with a higher concentration and higher affinity to the active groups on the surface of the support). Affinity chromatography has a wide range of applications for protein purification, such as immunoaffinity, purification of immunoglobulins, purification of glycoproteins, DNA-binding proteins, receptor proteins, enzymes, cell isolation, and nucleotide isolation [53]. Pseudo-Affinity Chromatography Since no biospecific interactions are characteristic of affinity separations, immobilized metal-affinity chromatography) and chromatography on synthetic dye ligands are usually called pseudo-affinity techniques. Immobilized Metal-Affinity Chromatography Immobilized metal-affinity chromatography is a separation technique that has proven to be an efficient and versatile technology for the isolation and purification of industrial enzymes as well as proteins that are of commercial importance or used in research fields, such as genetics, molecular biology, and biochemistry. IMAC is based on the formation of immobilized metal complexes, formed by the reaction between metal ions with chelating ligands that are attached to a chromatographic support (e.g., Sepharose). The separation

169

7.2. METHODS OF PROTEIN LIQUID CHROMATOGRAPHY

TABLE 7.2 Overview of Some Commonly Used Immobilization Procedures Coupling through the primary amine of a ligand O O Sepharose

O

CH2

CH

CH2

NH (CH 2) 5

C

O

N

OH O

O O Sepharose

O

CH2

CH

CH2

NH (CH 2) 5

C

O

+

N

R-NH2

OH O O O Sepharose

O

CH2

CH

CH2

NH (CH 2) 5

C

NH

R

+

HO

N

OH O

Activation by N-hydroxysuccinimide-activated Sepharose (NHS)

CNBr-activated Sepharose

NH OH

OH

CNBr

O

Sepharose

Sepharose

RNH2

C

CH3 OH

isourea

Activation by cyanogen bromide and coupling to the activated matrix.

(Continued)

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7. PROTEIN AND PEPTIDE SEPARATIONS

TABLE 7.2 Overview of Some Commonly Used Immobilization Proceduresdcont’d Coupling small ligands through amino or carboxyl groups via a spacer arm O Sepharose

O

CH2

CH CH2

NH (CH 2) 5

C

OH

Sepharose

O

CH2

OH

ECH Sepharose

CH2

NH

(CH 2) 5

NH2

EAH Sepharose

NH R1

O R - COOH + R1 N

C

N

R C

R2

O C N

+ R3 NH2 N

O

O

NH R1

R C O C

R2 R and R3 is matrix or ligand

Active ester

Carbodiimide

O

CH OH

R C

NH R3 + R1 NH C NH

R2

Urea derivate (side-product)

Peptide bond

Active ester

R2

Coupling through hydroxy, amino, or thiol groups via a 12-carbon spacer arm Sepharose

O CH2

HC OH

CH2

O

(CH 2) 4

O

CH2 HC

CH2 O

7.2. METHODS OF PROTEIN LIQUID CHROMATOGRAPHY

171

TABLE 7.2 Overview of Some Commonly Used Immobilization Proceduresdcont’d

Source: Reproduced with permission from ref [56].

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7. PROTEIN AND PEPTIDE SEPARATIONS

FIGURE 7.11

Protein adsorption and desorption from the matrix. The adsorption of proteins in IMAC is based on the coordination between an immobilized metal ion and electron donor groups from the protein surface. Most commonly used are the transitionmetal ions (Cu2þ, Ni2þ, Zn2þ, Co2þ, Fe3þ) as electron-pair acceptors (or electrophiles). Electron-donor atoms (or nucleophiles such as N, S, O) present in the chelating compounds attached to the chromatographic support are capable of forming metal chelates. Elution (desorption) of the target protein is achieved by protonation (using elution buffers with lower pH or lowering pH gradients) and ligand exchange of the metal ion by a stronger chelator (such as EDTA).

of proteins is possible through interaction of specific metal binding sites in the molecule (amino-acid side chains on the protein surface or specifically developed tags, mainly histidine residues) [57]. The selectivity of IMAC depends on the choice of metal ion, chelating ligand, absorption and elution conditions, and the modification of the target protein. A schematic diagram showing protein adsorption and desorption from the matrix is shown in Figure 7.11. Histidine is the amino acid that shows the strongest interaction with metals. Electron donor groups on the imidazole ring in histidine form coordination bonds with the immobilized transition metal. Since many proteins contain amino acids, such as Cys, Trp, Phe, and Tyr, located at the surface, that are suitable for interaction with metal ions, it is expected that almost all proteins are capable of binding to metal chelators. The strength of interaction depends on the number coordinative bindings between the metal ion and protein. In general, the supports in IMAC are the same as those applied in affinity chromatography. Beaded agarose is the predominantly used support. Ligands like iminodiacetic acid (IDA) and nitrilotriacetic acid (NTA) complexed with different metal ions such as Cu(II) or Ni(II) have been used widely, and gels incorporating these ligands are available from commercial suppliers. In general, the supports in IMAC are the same as in affinity chromatography [58]. Divalent ions, such as the transition metals Fe2þ, Co2þ, Ni2þ, Cu2þ and Zn2þ, are most commonly used. The affinities of many retained proteins and their respective retention times, such as in the IDA chelator, are in the following order: Cu2þ > Ni2þ > Zn2þ > Co2þ. The loss of metal ions at lower pH values leads to reduced adsorption capacity of the sorbent and can also cause damage to the target proteins by metal-catalyzed reactions.

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173

Buffers of relatively high ionic strength (containing 0.1 to 1.0 M NaCl) are used to reduce nonspecific ionic adsorption. A preelution step with a low concentration of imidazole or EDTA can be used for the elution of various contaminating proteins that contain histidine residues [59]. The benefits of IMAC separation technologies are related to ligand stability, high protein loading, mild elution conditions, simple column regeneration, and low cost. These are reasons for the wide application of this technology in protein separation. An advantage of this technique is that it is also applicable under denaturing conditions. Such conditions are often necessary when recombinant proteins are highly expressed in the cell culture system in the form of inclusion bodies (e.g., in E. coli). However, IMAC is not the preferred technique for the production of therapeutic proteins in substantial quantities, mostly due to problems with reproducibility and loss of metal ions during protein elution [59].

7.2.5. Chromatography on Hydroxyapatite Hydroxyapatite (HT), (Ca5(PO4)3OH)2, is a form of calcium phosphate used in the chromatographic separation of biomolecules. Five calcium doublets (Ca sites) and pairs of eOH containing phosphate triplets (P sites) are arranged in a repeating geometric pattern. Hydroxyapatite has unique separation properties, such as high selectivity and resolution, and can be used in separations of proteins by electrophoretic and chromatographic techniques. Applications of hydroxyapatite chromatography include the purification of different subclasses of monoclonal and polyclonal antibodies, antibody subtypes with different chain compositions, antibody fragments, isozymes, supercoiled DNA from linear duplexes, and differentiating single-strand from double-strand DNA [60]. Ceramic hydroxyapatite (CHT) is a spherical, macroporous form of hydroxyapatite. Compared to most other chromatography adsorbents, CHT is unusual in that it is both the ligand and the support matrix. Two types of CHT ceramic hydroxyapatite, Type I and Type II, have elution characteristics similar to crystalline hydroxyapatite but also have some important differences. CHT Type I has a higher protein-binding capacity and better capacity for acidic proteins, while CHT Type II has a lower overall protein-binding capacity but has better resolution for nucleic acids and certain proteins. The Type II material also has a very low affinity for albumin and is especially suitable for the purification of many species and classes of antibodies. Hydroxyapatite contains two types of binding sites, positively charged calcium (Ca2þ) and negatively charged phosphate (PO34 ) groups. These

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7. PROTEIN AND PEPTIDE SEPARATIONS

FIGURE 7.12 CHT binding mechanisms.

sites are distributed into the crystal structure of the matrix. Cation exchange occurs by positively charged proteins with the negatively charged phosphates, and analogously, they are repulsed by the calcium sites (see Figure 7.12). The ion-exchange interactions are weakened by the addition of neutral salts, such as NaCl or phosphate buffer (from mobile phase), or an increase in pH.

7.2. METHODS OF PROTEIN LIQUID CHROMATOGRAPHY

175

7.2.6. Chromatography on Monolithic Supports In proteomic applications and for in-process analysis in biotechnology, analytical columns with high efficiency and short analysis times are required. Hence, high-speed packing materials are in demand. If porous materials are used, separation speed is limited by mass transfer between the mobile phase and stationary liquid in the pores of the chromatographic support. For nonporous materials, the binding of sample components and subsequent elution occurs on the surface of the support so separation speed is minimally limited by mass transfer. To increase the performance of nonporous chromatographic material, particles with very small diameter (less than 3 mm, e.g., Tosoh Bioscience, Tokyo, Japan, offers polymer-based nonporous columns and bulk materials) are used, and high column back pressure is often the limiting factor. Frequently, monoliths are an alternative to columns packed with bulk stationary phases. The structure of monolithic materials (see Figure 7.13) allows very fast mass transfer during chromatographic separation that is hardly limited by the flow rate. Because of the low black pressure and fast mass transfer, monolithic columns can be run at very high speed (see Figure 7.14). As a result,

FIGURE 7.13 Comparison of particle-based chromatography and chromatography on compact, porous units. Source: Reprinted from Ref. [61] with permission.

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7. PROTEIN AND PEPTIDE SEPARATIONS

FIGURE 7.14 Comparison of a conventional porous particle medium (Mono Q) with a CIM disk with regard to their separation performance under fast gradient elution conditions: (a) a Mono Q anion exchange column 50 3 5 mm i.d.; (b) a QA anion exchange CIM disk 3 3 10 mm i.d. Conditions: buffer A, 10 mM Tris, pH 7.4; buffer B, buffer A D 0.5 M NaCl. Gradient time: (a) 0e100% buffer B in 4 min; (B) 0e100% buffer B in 6 sec. Flow rate: (a) 1 ml/min; (b) 10 ml/min. Back pressure: (a) 0.9 MPa; (B) 0.8 MPa. Calibration solution: myoglobin (peak 1), conalbumin (peak 2), and soybean trypsin inhibitor (peak 3) in buffer A. Source: Reprinted from Ref. [64] with permission.

separation time is up to one order of magnitude faster than that of columns packed with bulk particles [51,62e63]. Similar to columns packed with spherical particles, monolithic columns are silica or polymer based. In the reversed-phase mode, monolithic columns are mostly used for protein and peptide separation in proteomics applications [51]. In the ionexchange and hydrophobic-interaction modes, monolithic columns are used for analytical and preparative separation of large biopolymers, such as proteins and nucleic acids, as well as nanoparticles, such as plasmids and viruses [65]. Hennessy et al. [66] describe a general procedure for the generation of peptide maps of proteins with monolithic silica-based columns. The use of reversed-phase monolithic sorbents has been demonstrated to significantly reduce separation times for peptide map analysis, thus enabling increased sample throughput (see Figure 7.15). The obtained difference between the Chromolith Performance RP18e column and the SpeedROD 50 mm or SpeedROD 100 mm columns

7.2. METHODS OF PROTEIN LIQUID CHROMATOGRAPHY

177

FIGURE 7.15

RPLC separation of tryptic peptide fragments of equine cytochrome c (peaks 1e10) on Chromolith SpeedROD RP-18e (a) 100 mm and (b) 50 mm. Source: Reprinted from Ref. [66] with permission.

is the pore size. The wide-pore monolith influences the flow rate. Thus, compared to the SpeedROD 50 mm, where total separation time for the 10 tryptic peptides was about 10 min, the longer SpeedROD 100-mm column could be exploited to generate very fast separations; for example, total separation time for the 10 tryptic peptides was less than 4 min, see Figure 7.15(a), at the same conditions (flow rate of 8 ml/min).

7.2.7. Displacement Chromatography Displacement chromatography was first discovered by Tiselius, who also classified the three modes of chromatography as frontal, elution, and displacement [67]. More than 30 years ago, this technique was employed by Horvath, who first used the modern high-performance columns and equipment [68]. The principle of displacement chromatography for separation is based on the Langmuir isotherm. Only a finite number of sites are on the chromatographic support (stationary phase) for the binding of sample components, and if a site is occupied by one molecule, it is not available to the other sample components. Because the number of binding sites is limited, they are saturated when the concentration of the molecules in the sample is large in comparison to the dissociation constant for the sites.

178

7. PROTEIN AND PEPTIDE SEPARATIONS

The displacer is a molecule with a high affinity to the given chromatographic support, and it will compete more effectively for binding sites. It causes the displacement of the sample molecules and their enrichment in the lower-affinity solute. In elution chromatography, the retention of the substances that have to be separated is controlled by adjusting the composition of the mobile phase. The resolution in elution chromatography is generally better if the substance has a higher affinity to the mobile phase and the peaks are strongly retained. On the other hand, the conditions that give good resolution of the early peaks lead to strong retention of the late ones, to long separation times, and to the broadening of later peaks unless gradient elution is employed. The use of a gradient adds to the complexity of the chromatographic process, particularly on a large scale. Solutes separated in the displacement mode form sharp-edged zones, and peak spreading is less emphasized. The separated zones in this kind of chromatography are self-sharpening. This means that, if a component gets ahead of its band, it enters a zone in which it is more strongly retained and runs more slowly until the original zone catches up. Furthermore, since the loadings in displacement chromatography are deliberately high, more material can be separated on a smaller column, with the purified components recovered at significantly higher concentrations. The displacer is selected to have higher a affinity for the stationary phase than the sample components. The retention conditions in displacement chromatography depend on the displacer concentration. Consequently, the conditions that allow high retention can be employed without a gradient operation, and the displacement pushes all components of the sample out of the column in the designed run time [69]. One of the biggest advantages of displacement chromatography, is that this chromatography mode is well suited for the purification of components from dilute solutions. The disadvantages are the formation of overlapping zones between the separated components, and additionally, the removal of the displacer and column regeneration frequently limits the throughput [70]. Displacement chromatography is a very efficient technique for the purification of proteins from complex mixtures at high column loadings (see Figure 7.16). An important advance in the development of displacement chromatography was the discovery of low molecular-mass displacers such as 4-toluenesulfonic acid salts, streptomycin sulfate A (and similar substances), chloroquine diphosphate, and other small molecules instead of the large polyelectrolyte polymers [69,71,72]. These displacers can be readily separated from the protein after the separation, such as by size-exclusion chromatography or simple ultrafiltration. Their adsorption is also salt dependent, and it significantly facilitates the column regeneration. Displacement chromatography is well suited for the

7.3. CONCLUSIONS

179

FIGURE 7.16 Selective displacement of the antigen vaccine protein (AVP). Displacer: 35 mM p-toluenesulfonic acid (PTS). Mobile phase: 20 mM, pH 7.0 Tris buffer. Column: 10  290 mm DEAE Fast Flow Sepharose. Loading: ~6.5 ml of AVP feedstock per ml of column volume. Flow rate: 1 ml/min. Fraction size: 1 ml. AVP fractions are numbered from 1 to 16. Source: Reprinted from Ref. [80] with permission.

preparative separation of large quantities of proteins from complex mixtures by use of relatively small columns. This mode of chromatography can be performed with a variety of chromatographic resins, such as ion exchange, hydrophobic interaction, reversed phase, hydroxyapatite and pseudo affinity mode phases [71e75], and by use of particlebased as well as monolithic supports [76], for the separation of variety of proteins [77e79].

7.3. CONCLUSIONS • Chromatography is an indispensible method for protein separation on the analytical and preparative scales. • Ion-exchange, hydrophobic-interaction chromatography; chromatography on hydroxyapatite; and different types of affinity and pseudo-affinity chromatography are the most frequently used methods for isolation of nondenatured, physiologically active proteins. • Use of the organic solvents necessary in reversed-phase chromatography frequently causes protein denaturation and loss of physiological activity. • Monoliths are newly introduced chromatographic supports that enable very fast and effective separation of macromolecules, especially large proteins and protein complexes.

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7. PROTEIN AND PEPTIDE SEPARATIONS

• Because of the high grade of the concentrating effects, displacement chromatography is well suited for the purification of proteins from dilute solutions.

Acknowledgments This work was supported by the Croatian Ministry of Science, Projects No. 335-0000000-0221 and the National Science Foundation of Republic of Croatia. Neither of the authors has conflicts of interest with respect to this work.

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C H A P T E R

8 Glycans and Monosaccharides L. Royle Ludger Ltd., Oxford, UK O U T L I N E 8.1. Introduction

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8.3. Analysis and Characterization of Glycans 8.3.1. Glycan Release 8.3.2. Fluorescent Labelling of Glycans 8.3.3. Hydrophilic Interaction Liquid Chromatography 8.3.4. Weak Anion-Exchange Liquid Chromatography 8.3.5. Exoglycosidase Sequencing 8.3.6. Reversed-Phase Liquid Chromatography 8.3.7. Porous Graphitic Carbon

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8.4. Monosaccharide Composition Analysis 8.4.1. Hydrolysis of Monosaccharides 8.4.2. Labeling and Analysis of Monosaccharides

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8.5. Conclusions

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8.1. INTRODUCTION Glycosylation is the most-common form of posttranslational modification found on proteins and is critical for a wide range of functions, including cellecell interaction, immune defense, inflammation, and disease progression. Sugars are ideal for coding these complex Liquid Chromatography: Applications http://dx.doi.org/10.1016/B978-0-12-415806-1.00008-5

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Copyright Ó 2013 Elsevier Inc. All rights reserved.

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recognition patterns, because just a few simple building blocks can be assembled together in a large variety of different ways. The most common monosaccharides found in glycans are N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), galactose (Gal), glucose (Glc), mannose (Man), fucose (Fuc), and sialic acids. These monosaccharides can be linked together via the hydroxyl groups at different positions on the sugar ring (e.g., at the 2, 3, 4, or 6 position on a hexose), these anomeric linkages can also be in either the alpha or beta conformation. Add to this the ability of sugars to form branched rather than just linear structures, and the number of possible structures increases enormously. Full detailed characterization of these oligosaccharides is significanlty more of a challenge than sequencing linear chains of amino acids from proteins or nucleotides from DNA. Identifying, for example, whether a galactose is linked b1-3, b1-4, or a1-3 can be crucial to the function of a glycan: These small differences are akin to the differences between the written words big, dig, and pig, where a small change in the conformation of the first letter of the word completely changes its meaning. In addition, unlike proteins, which are synthesized directly from a specific gene sequence, glycosylation is controlled by a number of factors, such as the presence of specific glycosyltransferases or glycosidases and the availability of the appropriate substrates. Even small changes in physiological state or cell culture conditions can result in alterations in the glycans produced. This means that, although a glycoprotein may have a single amino acid sequence, it has a heterogeneous collection of differently glycosylated variants, termed glycoforms. This is complicated even further when there are a number of different glycosylation sites within the protein, which may or may not be glycosylated.

8.2. TYPES OF GLYCANS The glycans attached to proteins fall into two categories [1,2], but the techniques used for their analysis are the same: 1. N-glycans are linked to proteins via an amide bond to the asparagine (Asn) residue in an AsnXSer/Thr motif (where X is any amino acid except Pro). All N-linked glycans have the common core pentasaccharide Mana1,3(Man a1-6)Manb1,4GlcNAcb1,4GlcNAc (abbreviated to Man3GlcNAc2) and are either of the complex, oligomannose, or hybrid type (Figure 8.1). Complex glycans have GlcNAc residues linked to the mannose arms of the Man3GlcNAc2, forming bi-, tri-, or tetra-antennary complex glycans, which can be further elongated by other monosaccharides. Oligomannose N-glycans typically have between five and nine mannose residues attached to the

8.2. TYPES OF GLYCANS

FIGURE 8.1

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Glycan structures drawn using the symbolic nomenclature from University of Oxford (UOXF), Consortium of Functioal Glycomics (CFG), or text only. The UOXF nomenclature uses open symbols for hexoses, closed symbols for N-acetylhexosamines, dots inside the symbol for deoxy sugars, and stars for sialic acids; the line angles represent linkage positions; and anomericity is indicated by a full line to represent a beta linkage and a dashed line to represent an alpha linkage. The CFG nomenclature uses a range of colored symbols for the monosaccharides (yellow for Gal and GalNAc; blue for Glc and GlcNc) with linkage positions and anomericity written between them. The software tool GlycanBuilder (available at www.glycoworkbench.org/) is designed for rapid drawing of glycan structures in these alternative symbolic notations and for simple switching between the different outputs as required [3].

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GlcNAc2 core. Hybrid glycans are those in which only the Mana1,3arm of the core has GlcNAc antennae attached. 2. O-glycans are attached to proteins via the hydroxyl group of serine or threonine. The most common O-glycans have GalNAc as the core sugar. Eight core structures have been identified as extensions of this core GalNAc, these can be elongated and branched to form complex mucin-type glycans (Figure 8.1). Mannose, fucose, xylose, or GlcNAc can also be found directly attached to serine or threonine on some proteins. Glycans are also found attached to lipids, where the lipid portion of the molecule is embedded in the membrane bilayer. There are two major types: 1. Glycophospholipids, built on a phosphatidylglycerol core, which anchor proteins to the cell surface. 2. Glycosphingolipids (GSLs), where the sugar head group is linked to ceramide. The GSL head group can be elaborately glycosylated (Figure 8.1) in a similar manner to N- and O-glycans and are involved in cellecell interactions.

8.3. ANALYSIS AND CHARACTERIZATION OF GLYCANS Characterization of glycosylation is required to understand structuree activity relationships. For example, removal of the core fucose from the N-glycan on the heavy chain of IgG1 can significantly enhance the FcgRIIIa binding affinity, resulting in increased antibody-dependent cellular cytotoxicity activity [4]. Many biopharmacuticals are produced in nonhuman cell lines, which have the potential to add nonhuman-type glycosylation. This may give rise to altered immunogenicity that can lead to clinical consequences, such as reduced efficacy, altered pharmacokinetics, general immune and hypersensitivity reactions, and neutralization of the natural counterpart. Identification of these critical quality attributes (CQAs) is crucial, and optimization and monitoring of these CQAs throughout the drug cycle ensures increased product efficacy and safety as well as reducing the risk of batch rejects. Due to the complexity and heterogeneity of glycans, they are usually removed from the protein and analyzed separately. For example, human IgG has one conserved N-glycosylation site on each heavy chain at Asn 297, where there are 36 possible glycan structures: The largest glycan structure is a di-sialylated, di-galactosylated, bisected, core-fucosylated biantennary glycan, and the rest of the population consists of smaller, lessprocessed glycans lacking some of these monosaccharides [4].

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The quantities of glycans available for analysis are usually quite low, so highly sensitive detection systems are required. Sugars do not absorb UV light very strongly, so analysis of underivatizd glycans is performed by high-performance anion-exchange chromatography with pulsedamperometric detection (HPAEC PAD) [5];, however, this method requires picomole quantities of glycans and detects two peaks for each glycan (the alpha and beta epimers). Mass spectrometry can be used to detect glycans (labeled or unlabeled) either by themselves, or in combination with LC separation [6]; however, it should be noted that m/z detection is not necessarily linear across different compositions of glycans, so it is not best suited for quantitative work. The prevalent method of detection is by fluorescence, where labeling the reducing terminus of the glycan with a fluorescent tag allows for detection at femtomole levels and provides for quantitative measurement, as there is one tag per glycan, irrespective of the number of constituent monosaccharides. The most commonly used fluorescent label is 2-aminobenzamide (2AB), although 2-aminobenzoic acid (2-AA) and 2-aminopyridine (PA) are also used. Several HPLC stationary phases are used for the separation of glycans, including amide-bonded hydrophilic interaction (HILIC), reversed phase (RP), weak anion exchange (WAX), and porous graphitized carbon (PGC). Due to the heterogeneous nature and complexity of many glycan populations, analysis may require the use of several orthogonal methods [7].

8.3.1. Glycan Release Glycans can be removed by either chemical or enzymatic methods. The advantages of enzymatic release are that (a) no chemical alterations are made to the sugars and (b) the glycans retain a free reducing terminal that can easily be labeled with a fluorophore for highly sensitive detection. The most widely used method for releasing N-glycans is by the endoglycosidase peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase (EC 3.5.1.52), which is commonly known as PNGase F [8]. The three-dimensional structure of a glycoprotein can inhibit the action of PNGase F, so the glycoprotein should be reduced and denatured by sodium dodecyl sulfate (SDS) and mercaptoethanol before digestion. The PNGase F is able to directly release N-glycans from glycoproteins that are in solution with nonionic surfactants, such as NP-40 and Triton-X100; immobilized on a membrane; or immobilized in an SDS-PAGE gel. However, PNGase F is unable to remove N-glycans with an a1-3-linked core fucose found on many plant-derived glycans; these require the glycoprotein to be digested into peptides before PNGase A is able to remove the glycans. N-glycans can also be released chemically by hydrazine (85 C to 100 C, 5e16 hr), but during this process, any N-acyl

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groups are removed (i.e., N-acetyl and N-glycolyl) from GalNAc, GlcNAc, and sialic acids. These sugars are then re-N-acetylated, but any N-glycolyl sialic acids are converted to N-acetyl sialic acid [9]. Unfortunately, as yet, there is no generic O-glycanase for removing intact O-glycans. The only O-glycanases available are limited to the release of the neutral core 1 disaccharide Galb1-3GalNAc. Therefore, chemical methods have to be used for the full release of O-glycans. Release by hydrazinolysis, using milder conditions (60 C, 6 hr) than for N-glycans, generates O-glycans with free reducing termini, which can be fluorescently labeled. Other chemical release methods based on reductive b elimination produce glycans where the terminal sugar has been converted to an alditol, so they cannot be fluorescently labeled [10]. The enzyme ceramide-glycanase can be used to cleave the oligosaccharide head group from glycosphingolipids to produce glycans with free reducing termini [11].

8.3.2. Fluorescent Labelling of Glycans A number of fluorescent aromatic amines [12] are available for labeling the reducing terminus of glycans by means of reductive animation via formation of a Schiff’s base. The Schiff-base formation is facilitated by acid (usually, a DMSOeacetic acid mixture), then is reduced to a stable derivative, typically using cyanoborohydride as the reductant [13]. Simple to use kits are available for derivatization with the most-common labels: 2-aminobenzamide (2AB), 2-aminobenzoic acid (2-AA), and 2-aminopyridine (PA). This high-efficiency, nonselective labeling of the reducing terminus of the glycan allows for relative molar quantitation of individual glycans at femtomole levels.

8.3.3. Hydrophilic Interaction Liquid Chromatography Hydrophilic interaction liquid chromatography (HILIC) using amide bonded phase columns, such as TSKgel amide-80, is the most common method used to separate and analyze glycans. The fluorescently labeled glycans are applied to the column in an acetonitrile-50 mM ammonium formate pH 4.4 mixture (e.g., 65% acetonitrile for N-glycans or 80% acetonitrile for O-glycans) and are eluted by increasing amounts of aqueous solvent. The larger the glycan, the more hydroxyl groups it possesses and the more interaction it has with the stationary phase, thus larger glycans generally elute later, with some additional separation of stereo isomers. A glucose homopolymer (GHP) ladder of partially hydrolyzed dextran is run as an external standard (Figure 8.2), so that the elution positions of glycan peaks can be expressed as glucose units (GU)

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FIGURE 8.2 Separation of 2AB-labeled N-glycans by standard liquid chromatography. Column: LudgerSep-N2, 3 mm particle size, 4.6  150 mm. Instrument: Waters Alliance 2795 with 2475 fluorescence detector. Solvent A: 50 mM ammonium formate, pH 4.4. Solvent B: Acetonitrile. Flow rate: 1 ml/min. Gradient: 0 to 22 min 35 to 46% A, 22 to 22.5 min 46 to 100% A, 22.5 to 24.5 min 100% A, 24.5 to 26 min 100 to 35% A, 26 to 30 min 35% A. Temp: 35  C. Detection: lex = 330 nm, lem 420. Samples: (top trace) 2AB-labeled glucose homopolymer (GHP) ladder, which is used as an external standard; (middle trace) 2AB-labeled N-glycans released from human IgG; (bottom trace) 2AB-labeled N-glycans released from bovine fetuin. Retention times of glycans shown as peak labels in the chromatograms and are expressed in terms of glucose units (GU) by reference to the GHP ladder.

by comparison with the elution times of the GHP. This can be done automatically within chromatography software (e.g., Waters Empower GPC option) or in a spreadsheet using a polynomial distribution or cubic spline fit. These GU values are reproducible and predictive, because each monosaccharide in a given linkage contributes a set increment to the GU value. This allows for preliminary assignment of structures by comparison of GU values for unknown glycans with glycan standard GU values within databases: for N-glycans, GlycoBase (http://glycobase.nibrt.ie/); for O-glycans, UnicarbDB (www.unicarb-db.com/).

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Fingerprinting of the whole undigested glycan pool using HILIC profiles can often be sufficient for comparing the glycosylation between batches of glycoproteins where the range of possible glycans is well established. However, because a number of glycan structures can coelute, it is essential to confirm preliminary assignments using complementary methods, such as exoglycosidase sequencing or mass spectrometry [6]. Biopharmaceuticals tend to fall into two groups, those with IgG type N-glycosylation (biantennary with 0 to 2 galactoses and little sialylation), and those with more complex, larger sialylated glycans. Figure 8.2 shows an example of HILIC profiles from human IgG glycans and bovine fetuin, which has larger, complex sialylated glycans. A biantennary glycan with one galactose can have the galactose on either of the two antennae (i.e., linked to the a1-3 mannose arm or to the a1-6 mannose arm). These isomers are just separated by liquid chromatography on a 3-mm particle-packed column (Figure 8.2, IgG, GU 6.6 and 6.7) but can be baseline separated using a 1.7-mm particle-packed column (Figure 8.3, IgG, GU 6.8 and 6.9). A similar increase in resolution is

FIGURE 8.3 Separation of 2AB-labeled N-glycans by UPLC. Column: ACQUITY UPLC BEH Glycan, 1.7-mm particle size, 2.1  100 mm. Instrument: Waters ACQUITY UPLC H-Class with UPLC-optimized fluorescence (FLR) detector. Solvent A: 50 mM ammonium formate, pH 4.4. Solvent B: acetonitrile. Flow rate: 0.4 ml/min. Gradient: 0 to 45 min 28 to 43% A, 45 to 48 min 43 to 100% A, 48 to 49 min 100% A, 49 to 50 min 100 to 28% A, 50 to 60 min ( or to 80 min for fetuin run) 28% A. Temp: 35 C. Detection: lex ¼ 330 nm, lem 420. Samples: (top trace) 2AB-labeled N-glycans released from human IgG; (bottom trace) 2ABlabeled N-glycans released from bovine fetuin. Retention times of glycans are expressed in terms of glucose units (GU) by reference to a GHP ladder.

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observed for the large sialylated tetra antennary glycans in fetuin, even though longer run times are required to elute the charged glycans from the 1.7-mm particle-packed BEH amide column. The 3-mm particle-packed amide column and the 1.7-mm particle-packed BEH amide column have slightly different stationary phases, so some change in selectivity would be expected with consequent alterations in GU values. However, the GU values of the neutral glycans from IgG are very similar between the two columns, although the GU values of the acidic glycans were much higher on the BEH column. Thus, although the increased resolution of the 1.7-mm particle-packed BEH column is welcomed, it is important to also run characterized glycans to build up information on their GU values, as they will be different from those in a standard LC database.

8.3.4. Weak Anion-Exchange Liquid Chromatography Weak anion-exchange liquid chromatography (WAX) using diethylaminoethyl cellulose (DEAE) positively charged resins can be used to separate glycans on the basis of charge. Sialic acids are the most-common negatively charged monosaccharides and are very important to the function of glycans. For instance, sialylation can increase the anti-inflammatory activity of IgG and affect the serum half-life of glycoproteins by preventing clearance via the asialoglycoprotein receptors in the liver. Highly sialylated N-glycans increase the potency and enhance the half-life of erythropoietin (EPO), which stimulates red-blood-cell production, so detailed characterization of these glycans is essential for understanding which CQAs should be monitored during production of such drugs [14]. Full characterization of the different glycans in these heterogeneous mixtures of highly sialylated complex glycans requires a combination of separation methods. Figure 8.4 illustrates how this can be achieved, using fetuin N-glycans as an example. The N-glycans are separated into differently charged fractions by WAX, then analyzed by HILIC to obtain GU values. These can be further analyzed using exoglycosidase sequencing. In addition to negatively charged sialic acids, glycans occasionally contain sulfated or phosphated sugars, which also carry a negative charge. If the sample is desialylated by sialidase then analyzed by WAX, all the negatively charged sialylated peaks will no longer be present, so if any peaks remain (compared to a negative control), these are likely to be from sulfated or phosphated glycans.

8.3.5. Exoglycosidase Sequencing Although the GU value can give a preliminary idea of which glycans are present, several structures are possible with the same or similar GU

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FIGURE 8.4 Separation of fetuin N-glycans by WAX, followed by HILIC. WAX column: LudgerSep-C3, 10 mm, 7.5  75 mm. Solvent A: 20% acetonitile in water. Solvent B: 500 mM ammonium formate, pH 4.4, 20 % acetonitrile. Flow rate: 0.4 ml/min. Gradient: 0 to 5 min 100% A, 5 to 21 min 100 to 96% A, 21 to 61 min 96 to 75% A, 61 to 72 min 75 to 60% A, 72 to 75 min 60% A; 15 to 76 min 60 to 100% A at 0.8 ml/min; 76 to 90 min 100% A 0.8 ml/min. Temp: 35 C. Detection: lex ¼ 330 nm, lem 420. HILIC conditions as in Figure 8.2. 2AB-labeled N-glycans were separated by charge on WAX (left-hand traces), the differently charged fractions were then run on HILIC (right-hand traces). N ¼ neutral, 1S to 4 S ¼ 1 to 4 sialic acids.

values. To characterize the glycans further, exoglycosidase sequencing can be performed. A range of exoglycosidases are available for the removal of monosaccharides in specific linkages. As each monosaccharide in a specific linkage adds a given amount to the GU value of a given glycan, changes in GU values following digestions can be used to allocate which structures are present. For example, from the WAX separation of fetuin N-glycans (Figure 8.4), we know that the majority of glycans carry three sialic acids. However, there are at least four peaks in the HILIC profile of this fraction, and each of the three sialic acids can be a2-3 or a2-6 linked. Digestions of the tri-sialylated fraction are shown in Figure 8.5. Digestion with a2-3 sialidase changes the profile, indicating that some of the sialic acids are a2-3 linked. Further digestion with sialidase, which also removes a2-6-linked sialic acids, collapses the glycans into one peak, indicating that a mixture of both a2-3 and a2-6 sialic acids are present on the glycans from the tri-sialylated fraction. The structures digest to a neutral peak at GU 8.3 after removal of sialic acids. This peak at GU 8.3

8.3. ANALYSIS AND CHARACTERIZATION OF GLYCANS

195

FIGURE 8.5 Exoglycosidase sequencing of the tri-sialylated fraction from fetuin N-glycans. HILIC traces from the tri-sialylated fraction collected following WAX separation of fetuin 2AB-labeled N-glycans (see Figure 8.4) were digested with (a) sialidase specific for a2-3 sialic acid, (b) sialidase that removes all sialic acids, and (c) sialidase plus b1-4 galactosidase. Separation conditions as in Figure 8.2. Numbers refer to GU values of peaks. Arrows indicate which peaks digest.

is a mixture of two structures, both of which are tri-antennary structures with three galactoses. These structures are distinguished from each other by digestion with a b1-4 galactosidase, which removes three Gal (3  ~0.8 GU), moving the peak to GU 5.8 (which has a GU value consistent with the tri-antennary core structure GalNAc3Man3GlcNAc2). Another, smaller peak is at GU 6.6, which has lost only two Gal (2  ~0.8 GU); this is from the tri-antennary isomer, which has one a1-3 Gal and two a1-4 Gal.

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8.3.6. Reversed-Phase Liquid Chromatography Reversed-phase liquid chromatography (RPLC) on ODS columns is generally used in combination with other orthogonal methods for glycan analysis. Data for PA (2-aminopyridine)-labeled glycans is collected in the database GALAXY (www.glycoanalysis.info/ENG/ index.html) from three-dimensional mapping using ion-exchange, reversed-phase, and normal-phase separations. The retention of 2ABlabeled IgG glycans on RPLC can be related to families of glycans, that is, the presence or absence of a core fucose, a biscecting GlcNAc, or high mannose structures [15]. Further details can be obtained by coupling the RPLC to electrospray ionizationemass spectrometry (ESIeMS) and collecting fragmentation data in addition to mass composition data.

8.3.7. Porous Graphitic Carbon Porous graphitic carbon (PGC) is most often used as a sorbent for the separation of underivatized aliditol-type glycans obtained from release of O-glycans by reductive b elimination. PGC is usually coupled with ESIeMS for detection and fragmentation of glycans [16], because there is no clear correlation between retention and glycan structure.

8.4. MONOSACCHARIDE COMPOSITION ANALYSIS The International Conference on Harmonization guideline Q6B states that the carbohydrate content (neutral sugars, amino sugars, and sialic acids) should be determined for glycoprotein biopharmaceuticals. Monitoring the monosaccharide content of a biopharmaceutical during all stages of the product life cycle can help to identify any changes in glycosylation that can have a significant effect on the physiochemical or biological properties of a glycoprotein. The levels and types of sialic acids present should be determined, as two major sialics are found on N- and O-glycans in biopharmacuticals: N-acetyl-neuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Humans cannot synthesize Neu5Gc, and its presence on a biopharmaceutical can lead to adverse immune reactions, such as chronic inflammation [17]. Anti-Neu5Gc antibodies have been detected in normal human sera and can neutralize any Neu5Gc-containing biopharmaceutical, thereby lowering its efficacy. The choice of cell line can greatly influence the type of sialic acids present on a biopharmaceutical; for instance, 24% of the sialic acids on a mouse IgG are Neu5Gc compared to none on human IgG (Figure 8.6).

8.4. MONOSACCHARIDE COMPOSITION ANALYSIS

197

FIGURE 8.6 Comparison of Neu5Ac and Neu5Gc in human IgG and mouse IgG. For analytical conditions, see Figure 8.7.

Monosaccharides are released by acid hydrolysis then labeled with the flurophore to allow sensitive detection: 2-aminobenzoic acid (2-AA) for the neutral monosachharides, or 1,2-diamino-4,5-methylenedioxybenzene. 2HCl (DMB) for the sialic acids. Analysis by reversed-phase liquid chromatography provides reproducible quantitative results.

8.4.1. Hydrolysis of Monosaccharides The major monosaccharides that make up N-glycans and O-glycans are the neutral monosaccharides GlcNAc, GalNAc, Gal, Glc, Man, and Fuc, plus sialic acids. The neutral sugars can be hydrolyzed by incubation with 2 M trifluoroacetic acid (TFA) for 3 hr at 100 C. Harsher conditions may be required to completely remove N-acetylhexosamines that are attached directly to the protein backbone, so a stronger acid, such as 6 M HCl, can be used. However, the hexose sugars can degrade under these harsher conditions, so it is important to optimize the release conditions for

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8. GLYCANS AND MONOSACCHARIDES

individual glycoproteins. During hydrolysis, the N-acetyl groups on GlcNAc and GalNAc are hydrolyzed to glucosamine (GlcN) and galactosamine (GalN). Sialic acids should be released separately under milder conditions (2 M acetic acid for 2 hr at 80 C), as the conditions used to release neutral sugars destroy the sialic acids.

8.4.2. Labeling and Analysis of Monosaccharides The neutral monosaccharides are labeled (45 min at 80 C) with 2-AA, then analyzed by RPLC. The results of a typical analysis on a standard 3-mm particle, C18 column are shown in Figure 8.7 (30 min run time). Even faster run times (e.g., 6 min) can be obtained if 1.7-mm particle columns are used (Figure 8.8). Sialic acids are conjugated with DMB by an amination-cyclization reaction (3 hr at 50 C). When the sialic acid ring opens it exposes a ketone group next to the carboxylic acid, the DMB binds across these two adjacent ketone groups producing a fluorophore (other monosaccharides lack these adjacent ketone groups, so they are not fluorescently labeled). The results of a typical RPLC separation on a standard 3 mm, C18 column are shown in Figure 8.9 (30 min run time). Faster run times can be achieved using 1.7-mm particle columns (Figure 8.10).

FIGURE 8.7 Separation of 2AA-labeled monosaccharides by standard HPLC. Column: LudgerSep-R1, 3 mm particle size, 4.6  150 mm. Instrument: Waters Alliance 2795 with 2475 fluorescence detector. Solvent A: 0.2% butylamine, 0.5% phosphoric acid, 1% THF, in water (by volume). Solvent B: acetonitrile. Flow rate: 0.8 ml/min. Gradient: 0 to 7 min 96.5% A, 7 to 22 min 96.5 to 92.5% A, 22 to 23 min 92.5 to 50% A, 23 to 23.5 50% A (flow 1.2 ml/min), 23.5 to 29 min 50 to 96.5% A (1.2 ml/min), 29 to 30 min 96.5% A 0.8 ml/min. Temp: 30 C. Detection: lex ¼ 360 nm, lem 425 nm. Samples: (top trace) 2AA-labeled monosaccharide standards; (bottom trace) 2AA-labeled monosaccharides released from bovine fetuin glycoprotein.

8.4. MONOSACCHARIDE COMPOSITION ANALYSIS

199

FIGURE 8.8 Separation of 2AA-labeled monosaccharides by UPLC. Column: ACQUITY UPLC BEH C18, 1.7 mm particle size, 2.1  50 mm. Instrument: Waters ACQUITY UPLC H-Class with its UPLC-optimized fluorescence (FLR) detector. Solvent A: 0.2% butylamine, 0.5% phosphoric acid, 1% THF, in water (by volume). Solvent B: acetonitrile. Flow rate: 0.5 ml/min. Gradient: 0 to 0.5 min 100% A, 0.5 to 0.6 min 100 to 97.5% A, 0.6 to 3.6 min 97.5 to 90% A, 3.6 to 3.7 min 90 to 50% A, 3.7 to 5.7 min 50% A, 5.7 to 6 min 50 to 100% A. Temp: 30 C. Detection: lex ¼ 360 nm, lem 425 nm. Samples: (top trace) 2AA-labeled monosaccharide standards; (bottom trace) 2AA-labeled monosaccharides released from bovine fetuin glycoprotein.

FIGURE 8.9 Separation of DMB-labeled sialic acids by standard HPLC. Column: LudgerSep-R1, 3 mm particle size, 4.6150 mm. Instrument: Waters Alliance 2795 with 2475 fluorescence detector. Solvent A: methanol:acetonitrile:water (7:9:84 v/v). Flow rate: 0.5 ml/ min for 30 min. Temperature: 30 C. Detection: lex ¼ 373 nm, lem 448 nm. Samples: (top trace) DMB-labeled sialic acids Neu5Ac, Neu5Gc, Neu5,7Ac2, Neu5,Gc9Ac, Neu5, 9Ac2 and Neu 5,7, (8), 9Ac3Gc; (bottom traces) DMB-labeled quantitative standards Neu5Ac and Neu5Gc.

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8. GLYCANS AND MONOSACCHARIDES

The amounts of neutral or acidic monosaccharides can be quantified by comparison with standard curves for each sugar. Peak height is used rather than peak area for the neutral monosaccharides, because the GlcN and GalN sugars have a major peak followed by a minor epimer peak (1 mg/kg [33]

Precision (RSDwR)

Determine within-laboratory reproducibility (within-lab reproducibility is to be derived from ongoing QC)

 20% See [32] for details

Robustness

Can be derived from on-going method validationeverification through establishing average recovery and RSDwR

See [32] for details

Matrix effect

Comparison of response from solvent standards and matrix-matched standards

-

Source: Data from SANCO/10684/2009 (Appendix A) [32], SANCO 825/00 rev 8.1 [33].

Method validation has traditionally focused on quantitative aspects, and with the exception of the last few years, less attention has been paid to reliable identification. Confirmation of potential positives is a matter of concern due to the undesirable effects associated with erroneous

12.4. METHOD VALIDATION

327

identifications, that is, reporting either false positives or false negatives. Obviously, an analytical method must ensure the accurate quantification of analytes, but even more important is that the compound detected is correctly identified, especially when the maximum permitted levels are exceeded in the samples analyzed. Ideally, the identificationeconfirmation should be objective and safe, not depending on the subjective interpretation of the analyst. In PRA, the recent SANCO guidelines [33] can be applied. An interesting discussion on the identification and confirmation of chemical residues in food can be found elsewhere [34]. One of the most detailed criteria for the confirmation of contaminants comes from the European Commission Decision 2002/657/EC. This guideline applies to live animals and animal products [35], and confirmation is based on the accumulation of identification points (IPs). Any molecular spectrometric technique or combination of techniques can be used to obtain the minimum number of IPs necessary for the reliable identification of a compound (three or four IPs, depending on whether the compound is allowed or is banned). The number of IPs reached depends on the approach used, differentiating between MS, tandem MS, lowresolution (LR) and high resolution instruments. To qualify for the IPs reached, at least one ion ratio must be measured, and it must agree within specified tolerances. Moreover, the retention time (or relative retention time) of the analyte in the sample extract should correspond to that of the calibration solution, with a tolerance of 2.5% in LCeMS procedures. This criterion is a valuable tool for a rapid and easy comparison of MS techniques from the confirmation point of view. It is widely accepted that the acquisition of, at least, two SRM transitions in tandem MS, together with the retention time and the measurement of the ion intensity ratio, gives sufficient information for a safe confirmation of potential positives in samples. Typically, the most-sensitive transition is used for quantification and the other one for confirmation. However, the specificity of the transitions has to be considered as well, as it plays an important role in the confirmation process. In this sense, it is important to avoid the use of nonspecific transitions (due to common losses such as H2O, CO2, and HCl) to improve the confirmation [16]. Reporting false positives is unlikely, although possible; however, more risks exist of false negatives when one of the transitions is shared by a matrix and interference, as the ion ratio might be out of tolerance. As the quality of information is higher in HR MS more IPs are earned when using this type of analyzer in comparison to LR instruments. However, the mass accuracy derived from measurements using HR MS seems more important from a confirmatory point of view. Therefore, identification criteria based on mass accuracy (mass errors) are gaining popularity in organic contaminants research [36].

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12.5. ANALYSIS OF SAMPLES In addition to the validation of the analytical method, routine methods should include several controls to ensure the quality of the reported data [32,37]. The most usual approach consists of the injection of blank samples spiked at the LOQ and 10  LOQ levels, that is, quality controls (QC), in each batch of samples analyzed. For quality control compliance, a QC recovery range of 60e140% is used in routine multiresidue analysis [32]. Recoveries outside this range would require reanalysis of the sample batch. Results for samples that exceed MRL residue levels must be supported by individual recovery results in the same batch within the range of the mean recovery (70e120%)  2 RSD (relative standard deviation), at least for the confirmatory analyses. It is a requirement under ISO/IEC 17025 that laboratories determine and provide evidence on the uncertainty associated with analytical results. Measurement uncertainty is a quantitative indicator of the confidence in the analytical data. It describes the range around an experimental result within which the true value can be expected to lie with a defined probability (confidence level). Uncertainty can be estimated using an International Standards Organization (ISO) [38] or Eurachem approach [39]. Positive results, especially when residues are at or above the MRL or when unusual pesticides are detected, require additional confirmation. A reasonable approach when performing simultaneous LCeMS/MS analysis of many pesticides is the acquisition of only one SRM transition per compound, usually the most sensitive one [40]. In this analysis, detection and quantification of the compounds is carried out. To confirm a positive finding, a second analysis is made, including previously unused transitions for every compound detected. This approach requires, at least, two injections or the use of different confirmative methods depending on the analyte. Performing simultaneous quantification and reliable identification is another possibility widely applied, which requires the acquisition of at least two transitions per compound. In principle, a second analysis is not required, which might be considered an advantage. However, it might be seen also as a drawback, as reinjecting the sample implies a repetition of the analysis (normally on a different day), which minimizes the risk of sample contamination as it is manipulated twice independently. One should not ignore the possibility of contamination and, consequently, a second injection might be necessary or at least recommended in the case of positive results at noncompliant levels. According to Lehotay et al. [34], to support legal actions, at least two independent analyses are required for confirmation (at least one of them

12.5. ANALYSIS OF SAMPLES

329

should ideally involve MS detection after an analytical separation). However, in surveillance monitoring and data collection for nonenforcement reasons, a single analysis using MS detection should be satisfactory. Recently, Kmella´r et al [3] combined two parallel methods for the analysis of pesticides in fruit and vegetable samples using LCeMS/MS. Initially, a qualitative screening for 300 target pesticides was performed, using the most intensive transition for each pesticide; after that, presumed positive extracts were confirmed and quantified with a LCeMS/MS method optimized for 55 pesticides, by acquiring two transitions per compound. As an example, Figure 12.2 shows positive findings of hexythiazox and imidachloprid, in grape and red pepper samples, respectively, confirmed by the acquisition of two transitions, the agreement of the Q/q ratios, and retention times.

FIGURE 12.2

UPLCeMS/MS chromatograms corresponding to (a) a positive finding of imidachloprid in red pepper (0.32 mg/kg), (b) a positive finding of hexythiazox in grape (0.03 mg/kg), (c) 50 mg/l matrix-matched standard of imidachloprid, (d) 5 mg/l matrix-matched standard of hexythiazox; (Q) quantification transition, (q1) and (q2) confirmation transitions.

330

12. MULTIRESIDUE METHODS FOR PESTICIDES AND RELATED CONTAMINANTS

12.6. INDIVIDUAL METHODS FOR SPECIFIC COMPOUNDS Due to the high number of pesticides that might be present in food, the most useful approach for monitoring pesticides is the application of multiresidue methods able to simultaneously determine as many compounds as possible in one analysis. With the development of faster and more sensitive instruments, it is feasible to reasonably increase the number of target compounds up to 200e300 analytes in LCeMS/MS methods. Despite the advances made in instrumental and in sample preparation, individual methods are still needed, due to the special physicochemical characteristics of some pesticides. This occurs for phosphonic acid herbicides (glyphosate, glufosinate), quats (paraquat, diquat), the fungicide fosetil-Al, and the growth regulator ethephon, among others. Although some of these compounds can be determined by ion-exchange chromatography, this approach is not well suited to the electrospray interface typically used in LCeMS/MS, due to the use of buffers with high ionic strength. Single LCeMS/MS methods have been developed for residue analysis of very polar, non-QuECHERS amenable, pesticides in foods of plant origin. such as fruit, vegetables, or cereals, among others [41]. These methods use delicate and expensive analytical columns and are based on ionic-exchange chromatography. As an alternative, a derivatisation or ion-pair chromatography can be used for efficient separations. Ionpairing chromatography has been successfully used for the direct LCeMS/MS determination of ethefon [20] or fosetyl [21], which are lowmolecular-mass compounds that are present in anionic forms in the sample extracts. The use of the ion-pairing reagent tetrabutylammonium acetate (TBA) led to satisfactory results in fruit and vegetables at the low mg/kg level. The ion suppression produced by the continuous introduction of the ion-pairing reagent into the interface was minimized by adding TBA into the sample vial. Similarly, cyromazine and its metabolite melamine were determined by LCeMS/MS in chard samples using, in this case, tridecafluoroheptanoic acid (TFHA) to produce an ion pair with cationic analytes [19]. A similar approach was recently used for the determination of melamine in milk-based products [42]. The determination of the herbicide glyphosate at low residue levels is difficult, due to its ionic character, low volatility, low mass, and the lack of chemical groups that might facilitate its detection. Derivatization with FMOC has been used to facilitate its chromatographic retention, allowing the determination of glyphosate and its main metabolite AMPA in water and soil [17,18]. This approach could be applied to food samples as well.

12.7. LCeTOF MS IN THE FIELD OF PESTICIDE RESIDUE ANALYSIS

331

As briefly shown, LCeMS/MS is not only highly appropriate for development of MRMs, it is also one of the most efficient techniques for “difficult” pesticides that require specific analytical methodology.

12.7. LCeTOF MS IN THE FIELD OF PESTICIDE RESIDUE ANALYSIS Up to now, the use of time of flight (TOF) for quantification of organic pollutants has been quite limited [43e46], due to its lower sensitivity and linear dynamic range, compared to triple quadrupole in the SRM mode, as well as to the higher price of TOF instruments. However, the fullspectrum MS data, high mass resolution, and mass accuracy provided by TOF MS makes this technique especially useful for qualitative applications, such as screening and confirmation of pesticides. TOF instruments offer the possibility to investigate the presence of compounds in the samples once the analysis has been performed and MS data acquired, as the preselection of analytes is not required. Typically, extracted-ion chromatograms (XICs) at selected analyte m/z values are performed from full-scan accurate mass data with reasonable sensitivity, using narrow m/z windows (e.g., 0.02 Da). Identification of the compounds detected is based on accurate masses of the (de)protonated molecule and fragment ions when present, the compatibility of the fragment ion structures suggested by the chemical structure of the compound detected, the isotopic distribution, and retention time. A hybrid quadrupoleeTOF (QTOF) analyzer provides relevant additional information by obtaining product ion accurate mass spectra. QTOF MS/MS experiments are an excellent way to confirm potential positives revealed by, for example, TOF MS or QqQ analysis and are useful for elucidating the structures of (unknown) nontarget compounds [47]. An interesting feature of QTOF MS is the possibility of working under MSE mode [9]. MSE experiments involve the simultaneous acquisition of accurate mass data at low and high collision energy, which is feasible using a QTOF instrument. By applying low energy (LE) in the collision cell, fragmentation is minimized and the information obtained corresponds normally to nonfragmented ions (e.g., the (de)protonated molecule in ESI). On the contrary, at high collision energy (HE), fragmentation of the molecule is favored, resulting in more abundant fragments. Thus, both the (de)protonated molecule and fragment ion data are enabled in a single acquisition, without the need of selecting the precursor ion. MSE using an LCeQTOF MS instrument is a powerful tool for wide-scope screening. In the near future, a spectacular increase in QTOF MS applications, not only in the field of PRA but also for other organic pollutants is expected (for more details, see references [8,9]).

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12. MULTIRESIDUE METHODS FOR PESTICIDES AND RELATED CONTAMINANTS

An example of the application of MSE is shown in Figure 12.3. The detection of the fungicide quinoxyfen in a grape sample was made by the presence of a chromatographic peak at the m/z value of the protonated molecule at the expected retention time in the LE function (13.5 min, mass error 0.5 mDa). The identification of this fungicide was supported by the presence of four fragment ions in the HE spectra (all mass errors below 1.5 mDa). Chromatographic peaks in the corresponding XICs (for both the protonated molecule and fragment ions) were all at the same retention time. Structures suggested for the fragment ions and experimental mass errors are also shown in this figure. LCeTOF MS is an excellent technique to investigate the presence of pesticide metabolites in vegetables [48e50]. When the searched metabolites have already been reported in the literature, it is a simple operation to plot new XICs at their theoretical exact masses. Another strategy, based on the use of “fragmentationedegradation” relationships between the parent pesticide and its metabolites (assuming a common behavior in their fragmentation pathway), can be used [50]. This approach can be extended, assuming common fragmentation pathways, between metabolites. This allowed identifying an imazalil metabolite from the fragments observed for another metabolite detected in a lemon sample [10].

FIGURE 12.3 Detection and identification of the fungicide quinoxyfen in grape by UHPLCeQTOF (MSE mode). (a, top) LE spectra for the suspected compound in the sample; (a, bottom) HE spectra for the suspected compound in the sample and fragments suggested using MassFragment software; (b) XICs (20 mDa mass window) for the protonated molecule (LE and HE functions) and fragment ions (HE function).

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Retrospective analysis to search for compounds not included in the initial analysis is an attractive feature of TOF MS-based methods. Without additional injection of the samples, it is feasible to investigate the presence of other contaminants or metabolites. This has allowed the detection and identification of pharmaceutical metabolites in wastewater [51]. Obviously, this possibility is also available to other compounds, pesticides included, provided that they are compatible with the sample treatment and LCeMS analysis applied. A detailed comparison of the capabilities of LCeMS using QqQ, TOF, and QTOF for quantification, confirmation, and screening in the field of PRA is given in [45].

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[27] Botero-Coy AM, Marı´n JM, Iba´n˜ez M, Sancho JV, Herna´ndez F. Multi-residue determination of pesticides in tropical fruits using liquid chromatography/ tandem mass spectrometry. Anal Bioanal Chem 2012;402:2287e300. http://dx.doi.org/10.1007/ s00216-011-5431-3. [28] Afify AE-MMR, Mohamed MA, El-Gammal HA, Attallah FR. Multiresidue method of analysis for determination of 150 pesticides in grapes using quick and easy method (QuEChERS) and LC-MS/MS determination. J Food Agri Environ 2010;8:602e6. [29] Sancho JV, Pozo OJ, Lo´pez FJ, Herna´ndez F. Different quantitation approaches for xenobiotics in human urine samples by liquid chromatography/ electrospray tandem mass spectrometry. Rapid Comm Mass Spec 2002;16:639e45. [30] Kmella´r B, Fodor P, Pareja I, Ferrer C, Martı´nez-Uroz MA, Valverde A, et al. Validation and uncertainty study of a comprehensive list of 160 pesticide residues in multi-class vegetables by liquid chromatography-tandem mass spectrometry. J Chromatogr A 2008;1215:37e50. [31] Ferrer L, Thurman EM, Zweigenbaum JA. Screening and confirmation of 100 pesticides in food samples by liquid chromatography/tandem mass spectrometry. Rapid Comm Mass Spec 2007;21:3869e82. [32] Method validation and quality control procedures for pesticide residues analysis in food and feed. Document No. SANCO/10684/2009. SANCO, 2009. [33] Guidance document on pesticide residue analytical methods. SANCO/825/00 rev. 8.1, Directorate General Health and Consumer Protection, 16/11/2010. SANCO, 2009. [34] Lehotay SJ, Mastovska K, Amirav A, Fialkov AB, Martos PA, de Kok A, et al. Identification and confirmation of chemical residues in food by chromatography-mass spectrometry and other techniques. Trends Anal Chem 2008;27:1070e90. [35] European Commission. Decision 2002/657/EC of 12 August 2002 implementing Council Directive 96/23/EC, concerning the performance of analytical methods and the interpretation of results. [36] Herna´ndez F, Iba´n˜ez M, Sancho JV, Pozo OJ. Comparison of different mass spectrometric techniques combined with liquid chromatography for confirmation of pesticides in environmental water based on the use of identification points. Anal Chem 2004;76:4349e57. [37] Camino-Sa´nchez FJ, Zafra-Go´mez A, Oliver-Rodrı´guez B, Ballesteros O, Navalo´n A, Crovettob G, et al. UNE-EN ISO/IEC 17025:2005-accredited method for the determination of pesticide residues in fruit and vegetable samples by LC-MS/MS. Food Addit Contam 2010;27:1532e44. [38] Anonymous. Guide to the expression of uncertainty in measurement. Geneva, Switzerland: International Organisation for Standardization; 1995. ISBN: 92-67-10188-9. [39] EURACHEM/CITAC Guide quantifying uncertainty in analytical measurement, 2nd ed. Available at www.measurementuncertainty.org/mu/guide/index.html. ´ J, Sancho JV, Lo´pez FJ, Marı´n JM, Iba´n˜ez M. Strategies for [40] Herna´ndez F, Pozo O quantification and confirmation of multi-class polar pesticides and transformation products in water by LCeMS2 using triple quadrupole and hybrid quadrupole timeof-flight analyzers. Trends Anal Chem 2005;24:596e612. [41] Anastassiades M, Kolberg DL, Mack D, Sigalova I, Roux D, Fu¨gel D. Quick method for the analysis of residues of highly polar pesticides in foods of plant origin involving simultaneous extraction with methanol and LC-MS/MS determination, Version 5 (November 2010, Document History, see page 34). EU Reference Laboratories for Residues of Pesticides, 2010. [42] Iba´n˜ez M, Sancho JV, Herna´ndez F. Determination of melamine in milk-based products and other food and beverage products by ion-pair liquid chromatography-tandem mass spectrometry. Anal Chim Acta 2009;649:91e7.

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[43] Ferrer I, Thurman EM. Multi-residue method for the analysis of 101 pesticides and their degradates in food and water samples by liquid chromatography/time-of-flight mass spectrometry. J Chromatogr A 2007;1175:24e37. [44] Garcı´a-Reyes JF, Hernando MD, Ferrer C, Molina-Dı´az A, Ferna´ndez-Alba AR. Large scale pesticide multiresidue methods in food combining liquid chromatographyetimeof-flight mass spectrometry and tandem mass spectrometry. Anal Chem 2007;79:7308e23. [45] Grimalt S, Sancho JV, Pozo OJ, Hernandez F. Quantification, confirmation and screening capability of UHPLC coupled to triple quadrupole and hybrid quadrupole time-of-flight mass spectrometry in pesticide residue analysis. J Mass Spec 2010;45:421e36. [46] Garcı´a-Reyes JF, Gilbert-Lo´pez H, Molina-Dı´az A, Ferna´ndez-Alba AR. Determination of pesticide residues in fruit-based soft drinks. Anal Chem 2008;80:8966e74. ´ J, Iba´n˜ez M, Herna´ndez F. Potential of liquid chromatography/time[47] Sancho JV, Pozo O of-flight mass spectrometry for the determination of pesticides and transformation products in water. Anal Bioanal Chem 2006;386:987e97. [48] Herna´ndez F, Sancho JV, Iba´n˜ez M, Grimalt S. Investigation of pesticide metabolites in food and water by LCeTOF MS. Trends Anal Chem 2008;27:862e72. [49] Pico Y, Barcelo D. The expanding role of LC-MS in analyzing metabolites and degradation products of food contaminants. Trends Anal Chem 2008;27:821e35. [50] Garcı´a-Reyes JF, Molina-Dıaz A, Fernandez-Alba AR. Identification of pesticide transformation products in food by liquid chromatography/time-of-flight mass spectrometry via "fragmentation- degradation" relationships. Anal Chem 2007;9:307e21. [51] Herna´ndez F, Iba´n˜ez M, Gracia-Lor E, Sancho JV. Retrospective LCeQTOF-MS analysis searching for pharmaceutical metabolites in urban wastewater. J Sep Sci 2011;34:3517e26. http://dx.doi.org/10.1002/jssc.201100540.

C H A P T E R

13 Environmental Analysis: Persistent Organic Pollutants* L.C. Sander, M.M. Schantz, S.A. Wise Chemical Sciences Division, National Institute of Standards and Technology (NIST), Gaithersburg, MD O U T L I N E

*

13.1. Polycyclic Aromatic Hydrocarbons

341

13.2. Chlorinated Aromatic Compounds

346

13.3. Pesticides

347

13.4. Brominated Flame Retardants 13.4.1. BDEs 13.4.2. HBCDs and Brominated Cycloalkanes 13.4.3. TBBPA

352 353 356 359

13.5. Perfluoroalkyl Compounds 13.5.1. Water and Sediments 13.5.2. Biota 13.5.3. Human Source Materials

360 367 368 370

13.6. Reference Materials

372

13.7. Concluding Remarks

373

13.8. Disclaimer

375

References

375

Contribution of the U. S. Government. Not subject to copyright.

Liquid Chromatography: Applications http://dx.doi.org/10.1016/B978-0-12-415806-1.00013-9

337

Copyright Ó 2013 Elsevier Inc. All rights reserved.

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The term persistent organic pollutants (POPs) describes a broad range of anthropogenic compounds present in the environment. Persistent organic pollutants can be grouped into subclasses based on their original intended uses or chemical properties. In this chapter, five such groups of compounds are considered: polycyclic aromatic hydrocarbons (PAHs), chlorinated aromatic compounds, pesticides, brominated flame retardants (BFRs), and perfluoroalkyl and polyfluoroalkyl substances (PFASs) (see Figure 13.1). In 2001, the first meeting of the Stockholm Convention on Persistent Organic Pollutants was held, and 12 pesticides, industrial chemicals, and by-products were classified as POPs. In 2009, the fourth meeting of the conference was held, and nine additional chemicals were added to the list (see Table 13.1). The origins and uses (if any) of these compounds are largely unrelated, and yet, the compounds share several features. The main criteria of POPs as defined by the Stockholm Convention include: persistency, bioaccumulation, toxicity, and long-range environmental transport [1,2]. The polarity of POPs does not necessarily correlate with these properties nor are all POPs nonreactive. Compounds range from highly nonpolar (e.g., PAHs) to ionic (e.g., perfluoroalkyl acids, PFAAs) and may be subject to reactions in the environment and in biota; these transformation products may also meet the criteria for POPs. The biotransformation of xenobiotic compounds may involve, for example, hydrolysis, oxidation, or adduct formation reactions that increase the polarity of the metabolites to promote excretion. Other reaction processes occur in the environment (e.g., oxidation, photolysis) that transform the compounds to more polar forms. Because of the polarity of these transformation products, liquid chromatography is often better suited to their analysis than gas chromatography. Transformation products of POPs may exhibit similar or even greater toxicity than the parent compounds, and for these reasons, considerable interest exists in the measurement of POPs and their transformation products. Methods for the analysis of POPs have been developed based on gas chromatography (GC) and liquid chromatography (LC). GC offers a significant advantage for compatible analytes because of the highresolution separations that are inherent to the technique. Target compounds must have sufficient volatility and thermal stability for analysis by GC. LC is better suited to the analysis of polar or thermally labile compounds. Recently, Moriwaki reviewed the use of LC for PAHs, organochlorine compounds, and perfluorinated acids in environmental samples [3]. Probably the most widely utilized detection approach for environmental analysis is mass spectrometry (MS). Several LCeMS technologies have been used for POPs, including quadrupole, ion trap, and time-of-flight (TOF) mass analyzers; MS sources have included electrospray ionization (ESI), atmospheric pressure chemical ionization

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13. ENVIRONMENTAL ANALYSIS: PERSISTENT ORGANIC POLLUTANTS

Polycyclic aromatic hydrocarbons (PAHs)

Chrysene

Benz[a]anthracene

Triphenylene

Benzo[a]pyrene

Chlorinated aromatic compounds O

Cl Cl

Cl

Cl

Cl Cl

Cl

Cl

Cl

O

Cl

Cl

Cl

O

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)

Cl

O

Cl Cl

Cl

2,3,7,8-tetrachlorodibenzofuran

3,3',4,4'-Tetrachlorobiphenyl

3,3',4,4'-Tetrachlorodiphenylether

Pesticides Cl

CCl3

Cl Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl Cl

Cl

H3C

Cl

Cl

Cl

H3CS

O

CH3 N

N H

O

H

CH3

Cl

Cl

p,p’-DDT

Mirex

Aldrin

Aldicarb

Brominated flame retardants (BFRs) Br

Br

Br

Br

O

Br

Br

Br Br

Br

Br Br

Br

Br Br Br

Br Br

Br

Br

Br Br

Br

Br

Br Br

Br

Br

Br

Br Br

Polybrominated diphenyl ethers (PBDEs) (decabromodiphenylether,decaBDE shown)

Polybrominated diphenyls (PBBs) (decabromobiphenyl shown)

Decabromodiphenylethane (DBDPE)

Br Br Br Br

Br

Br

Br

Br

Br

Br

Br

Br

Tetrabromoethylcyclohexane (TBECH, α, β, γ, and δ stereoisomers)

Tetrabromoethylcyclooctane (TBCO,α, and β stereoisomers)

OH CH3

Br

Br

Br

Br

CH3

HO

Br

Hexabromocyclododecane (HBCD, α, β, γ stereoisomers)

Br

Tetrabromobisphenol A (TBBPA)

Perfluoroalkyl compounds (PFCs) F2 CF3

F2 F2

F2 F2

F2

F2 F2

COOH

Perfluoroocctanoic acid (PFOA)

CF3

F2 F2

F2 F2

F2 F2

CF3

-

F2

SO3

Perfluoroocctane sulfonate (PFOS)

CF3

F2

-

F2 F2

6-CF3-PFOS

SO3

F2

F2

CF3

F2

-

F2

SO3

F2

CF3 CF3

3,3-CF3-PFOS

FIGURE 13.1 Structures of selected persistent organic pollutants and related compounds.

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13. ENVIRONMENTAL ANALYSIS: PERSISTENT ORGANIC POLLUTANTS

TABLE 13.1 Compounds Designated as POPs by the First (2001) and Fourth (2009) Meetings of the Stockholm Convention on Persistent Organic Pollutants [1] Compound

Annex

INITIAL COMPOUNDS Aldrin

A

Chlordane

A

DDT

B

Dieldrin

A

Endrin

A

Heptachlor

A

Hexachlorobenzene

A

Mirex

A

Toxaphene

A

Polychlorinated biphenyls (PCBs)

A and C

Polychlorinated dibenzo-p-dioxins

C

Polychlorinated dibenzofurans

C

ADDITIONAL COMPOUNDS alpha Hexachlorocyclohexane

A

beta Hexachlorocyclohexane

A

Chlordecone

A

Hexabromobiphenyl

A

Hexabromodiphenyl ether and heptabromodiphenyl ether

A

Lindane

A

Pentachlorobenzene

A and C

Perfluorooctane sulfonic acid (and salts)

B

Tetrabromodiphenyl ether and pentabromodiphenyl ether

A

Note: The initial group of 12 compounds was supplemented with 9 additional compounds (or groups of compounds). Annexes define actions that must be taken by the signatories to the agreement. Annex A chemicals are to be eliminated (production and use); Annex B chemicals are restricted (with specific exemptions); Annex C chemicals require measures for reduction of unintentional release.

(APCI), atmospheric pressure photoionization (APPI), and direct electron ionization (EI). Sancho et al. [4] discuss the advantages and limitations of the various mass-analyzer technologies, and Anacleto et al. [5] describes the applications of various MS sources.

13.1. POLYCYCLIC AROMATIC HYDROCARBONS

341

This chapter provides an overview of some of the LC methods used in the determination of persistent organic contaminants in environmentally relevant samples and associated measurement issues. Emphasis has been placed on compounds and groups of compounds included in the Stockholm Convention, including PAHs, chlorinated aromatic compounds, pesticides, BFRs, and PFAAs, with a focus on research carried out since 2000. Purely GC-based methods have been excluded from this discussion, as have reports that emphasize sample cleanup and processing research. Excellent reviews of these topics have been published previously [6e13].

13.1. POLYCYCLIC AROMATIC HYDROCARBONS Environmental sources of polycyclic aromatic hydrocarbons originate from incomplete combustion or pyrolysis of organic matter. Both natural sources (e.g., geothermal processes and forest fires) and human-made sources (combustion of fossil fuels and wood, industrial processes) contribute to environmental contamination. It has been estimated that approximately 90% of PAH emissions are anthropogenic [14]. PAHs are usually produced in complex, isomeric mixtures [15], and they exhibit all the properties usually attributed to POPs. For example, the compounds accumulate in the food web; they are persistent in the environment and are transported to remote locations; and they have detrimental effects on animal life. PAHs are known carcinogens and mutagens; however, the level of activity is highly variable among different compounds. A primary motivating factor in the determination of individual PAHs is the differences in toxicity attributed to specific PAH isomers. Both GC and LC methods are widely utilized in the determination of PAHs. Wise, Sander, and May [16] describe the advantages and limitations of LC-based methods for PAHs, and compare the results of LC fluorescence (LCeFL) with GCeMS measurements used in the certification of natural matrix standard reference materials (SRMs). GC with mass spectrometric detection offers the potential for high-resolution and selective separations of complex matrix samples. GC separations of PAHs are limited, however, by coelution of certain isomers with methylpolysiloxane stationary phases (e.g., chrysene and triphenylene [228 u]; benzo[b]fluoranthene, benzo[j]fluoranthene, and benzo[k]fluoranthene [252 u]; and dibenz[a,c]anthracene and dibenz[a,h]anthracene [278 u]). GC separations of isomers have been achieved by use of liquid crystalline stationary phases and other phases in combination with optimized methods. LC methods for PAHs are inherently less efficient than GC methods, but certain LC columns offer enhanced selectivity toward PAH isomers, enabling separations. These isomer separations are based on molecular

342

13. ENVIRONMENTAL ANALYSIS: PERSISTENT ORGANIC POLLUTANTS

shape of the solute, and this property of the LC column has been termed shape selectivity or shape recognition. Wise et al. [17] demonstrated that solute retention for shape-selective LC columns is correlated with the relative dimensions of PAH molecules. They defined a molecular descriptor, the length-to-breadth ratio (L/B) as the ratio of the length and width of a box drawn to enclose the van der Waal radii of the atoms of the molecule. Orientation of this construct is important; better correlations of L/B result for boxes drawn to achieve maximum values for L/B. Column selection is crucial for achieving shape-selective separations of PAHs. The majority of all LC separations are carried out on octadecylsilane (C18) columns; however, the properties of these columns vary significantly, based on the approaches used in the synthesis of the stationary phase. Most commercial C18 columns are prepared using monofunctional octadecylsilanes, which result in single siloxane-bond linkages between the C18 ligand and the silica surface, with bonding densities of 3.5 mmol/m2 and less. Less commonly, trifunctional octadecylsilane reagents are reacted with silica in the presence of water to produce “polymeric” C18 stationary phases with bonding densities of approximately 5 mmol/m2. Polymeric C18 columns exhibit enhanced shape recognition toward PAH isomers that is often dramatic (see Figure 13.2). Manufacturers of polymeric C18 columns sometimes designate these products as “PAH columns” to indicate the intended applications. Theoretical and practical aspects of shape selectivity have been studied in detail in our laboratory [18]. LC methods for the determination of PAHs in environmental samples are often based on fluorescence detection. Many, but not all, PAHs exhibit native fluorescence properties, and because fluorescence is relatively uncommon for other constituents in environmental samples, highly selective detection methods are possible. Selectivity is further enhanced through the use of time-programmed changes in the excitation and emission wavelengths, optimized to maximize detection sensitivity for specific analytes. Conditions can also be tuned to reduce the responses of potential interferences. The U.S. Environmental Protection Agency (EPA) published an early application for the determination of PAHs by LCefluorescence [19]. EPA Method 610 is based on the use of a specific polymeric C18 column to resolve 16 high-priority pollutant PAHs in aqueous effluents, with subsequent fluorescence detection. Wise developed LCeFL methods that were applied to complex matrix samples [20]. Orthogonal LCeFL and GCeMS methods were used in the certification of environmentally relevant samples that included shale oil [21,22], petroleum crude oil [20], coal tar [23e25], urban dust [25e27], diesel particulate matter [25], marine sediment [28,29] and mussel tissue [26,30e32]. Separate LCeFL analyses of SRM 1941 marine sediment and SRM 1974 mussel tissue are illustrated in Figure 13.3.

343

13.1. POLYCYCLIC AROMATIC HYDROCARBONS

(a)

(b)

0

5

10

15

20 Minutes

25

30

35

FIGURE 13.2 Separation of PAH isomers (302 u) with a monomeric C18 column (upper chromatogram) and a polymeric C18 column (lower chromatogram). Source: Adapted from Reference [213].

Whereas GC methods for PAHs are commonly based on mass spectrometric detection with electron ionization, MS detection is less common for LC methods for PAHs. Poor ionization efficiencies reportedly result from the application of traditional MS sources (e.g., particle beam EI and ESI) due to the relatively high ionization energies (IEs) for PAHs

344

13. ENVIRONMENTAL ANALYSIS: PERSISTENT ORGANIC POLLUTANTS

Marine sediment (SRM 1941)

λ1 λ2 λ3 λ4 λ5 λ6 λ7 λ8

IS-3

IS-1

λ1 0

= = = = = = = =

249/362 250/400 285/450 333/390 285/385 406/440 296/405 300/500

nm nm nm nm nm nm nm nm

IS-2

λ2 λ3 λ4

λ5

10

λ6

λ7

20

λ8 30

40

Time (min)

Mussel tissue (SRM 1974) IS-2

IS-1

IS-3

λ1 0

λ2 λ3 λ4 10

λ5

λ6 20

λ7 30

λ8 40

Time (min)

FIGURE 13.3

RPLC analysis of SRM 1941 marine sediment and SRM 1974 mussel tissue using wavelength programmed fluorescence detection. Chromatographic conditions: Vydac 201TP column; mobile-phase linear gradient from 50% acetonitrile in water to 100% acetonitrile, over 50 min at 1.5 ml/min. Source: From Reference [20].

(e.g., z7 eV to 8 eV for the 16 high-priority pollutant PAHs) [33]. Fragmentation is limited, due to the stability of the fused ring structure of PAHs. Improved detection sensitivity has been demonstrated for APCI sources operated in the positive ion mode, under carefully optimized conditions [5,34,35]. A newer approach to the MS detection of PAHs is given by dopantassisted (DA) APPI [36,37]. In this technique, a photodopant (e.g., toluene

13.1. POLYCYCLIC AROMATIC HYDROCARBONS

345

or anisole) is introduced to the APPI source through postcolumn infusion. The dopant is ionized by 10-eV photons from the krypton lamp, and this energy is transferred to the analytes to assist ionization. Itoh, Aoyagi, and Yarita [38] investigated the effectiveness of various dopant reagents toward the detection of PAHs and concluded that mixtures of toluene and anisole provided the broadest improvements in detection limits for the 16 high-priority pollutant PAHs. In general, DA APPI has been reported to provide detection limits that are roughly comparable to fluorescence detection [39] and GCeMS; however, detector responses among these methods are highly variable for specific PAHs. LCeDA APPI/MS has been used for the determination of PAHs in fresh-water sediments [39], shrimp [40], milk [35], mussels [41], air particulates, and coal tar samples [34]. Ding, Ashley, and Watson [42] determined PAHs in mainstream cigarette smoke by LCeDA APPIeMS/ MS. Quantification was based on either (M•þ) or ([M þ H]þ) species, and confirmation of component identity was made from an analysis of precursor and product ions. Smoker et al. [40] also utilized tandem mass spectrometry for confirmation. In many cases, fragmentation was limited and product ions differed from the precursors by only 2 mass units. LC methods for PAH transformation products have also been developed based on mass spectrometric detection. Oxygenated PAHs (OPAHs) are formed during incomplete combustion of organic matter and through photo-oxidation and gas-phase reactions of aerosols with ozone, hydroxyl radicals, and nitrogen oxides [43]. OPAHs are also formed through metabolism of PAHs, and are measured in biofluids as markers of PAH exposure [44]. Examples of OPAHs include hydroxyl, keto, and quinone derivatives of PAHs. These compounds are significantly more polar than PAH hydrocarbons, and some OPAHs, particularly quinones, are not amenable to fluorescence detection (i.e., reduction of diketones to hydroxyl PAH may be required for fluorescence detection). The polarity of OPAHs does increase their compatibility with APCI and ESI sources, and LCeMS and LCeMS/MS methods have been published. Galceran and Moyano [45] evaluated ESI and APCI sources operated in positive and negative modes, for application to hydroxyl PAH and PAH quinones. Different mechanisms of ionization were proposed for the sources and modes of operation, but similar limits of detection were possible with either source. Tandem mass spectrometry is routinely applied to OPAHs in preference to single MS detection, to permit analyte confirmation. However, Zhu et al. [46] developed a very rapid LCeAPCIeMS method for the metabolites of benzo[a]pyrene, based on a UHPLC separation. Quantitation of eight BaP metabolites was possible in less than 10 min, for extracts of fish tissue. Sagredo et al. [47] also used LCeESIeMS, to study epimerization of BaP tetrols extracted from rat blood.

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13. ENVIRONMENTAL ANALYSIS: PERSISTENT ORGANIC POLLUTANTS

A number of LC tandem MS methods have been reported for OPAHs in environmental samples and biological fluids. Ramsauer et al. [48] and Andreoli et al. [49] describe LCeESIeMS/MS methods for the determination of PAH metabolites in human urine. Andreoli et al. specifically targeted naphthalene metabolites for study for occupationally exposed workers, while Ramsauer et al. studied a broader range of hydroxylated PAHs in urine from smokers and nonsmokers. Levels of hydroxyl PAHs in smokers urine were also measured by Jacob, Wilson, and Benowitz [50] by LCeAPCIeMS/MS; however, this method utilized a derivitization step with pentafluorobenzyl bromide to increase sensitivity. LCeAPCIeMS/ MS was employed by Fan et al. [51] to determine 1-hydroxypyrene and 3-hydroxybenzo[a]pyrene in urine. OPAHs have been determined in atmospheric aerosols and air particulate matter by LCeAPCIeMS/MS methods [43,52,53]. Delhomme, Millet, and Herckes [52] concluded that diketones were best detected in the negative mode, whereas better responses for ketones and pyrones were obtained with operation in the positive mode. Lintelmann et al. [43] determined OPAHs in samples from high-volume air filters, using LCeAPCIeMS/MS operated in the positive mode. The method was optimized and validated using SRM 1649a urban dust and SRM 1650a diesel particulate matter.

13.2. CHLORINATED AROMATIC COMPOUNDS Several unrelated groups of chlorinated aromatic compounds can be considered together based on their similar composition and toxicity: polychlorinated biphenyl congeners (PCBs), dioxins and furans, chloronaphthalenes, and chlorodiphenyl ethers (see Figure 13.1). Most of these compounds are included in the Stockholm Convention Agreements of 2001 and 2008, and their production is reduced or eliminated in most parts of the world. However, the compounds are highly persistent in the environment and exhibit other properties associated with POPs. Because chlorinated aromatic compounds are nonpolar and stable at elevated temperature, gas chromatography is the preferred approach for analysis. Numerous reviews have been published that detail GC methods of analysis for PCBs, dioxins, and other chlorinated aromatic compounds in environmental samples [6,9,54]. The use of liquid chromatography for the analysis of chlorinated aromatic compounds is rarely reported. An exception exists for LC methods used for sample cleanup. The complexity of environmental samples often necessitates the fractionation of sample extracts, which are then analyzed separately by GC-based methods. These LC fractionation methods have been developed based on size-exclusion chromatography

13.3. PESTICIDES

347

(for removal of lipids) and normal-phase chromatography with silica or aminopropyl columns (for class separation of PAHs, pesticides, and PCBs). An interesting application was reported by Haglund et al. [55] for the fractionation of PCB congeners based on the degree of ortho substitution. A 2-(1-pyrenyl)ethyldimethylsilylated column was used with a hexane mobile phase to resolve ortho and nonortho congeners. This processing step enables the determination of nonortho (planar) PCB congeners by GC, which otherwise are subject to interferences from coeluting ortho PCB congeners. This approach has been utilized in the determination of ortho and nonortho PCBs in water and pine wood extracts [56], fish oil [57], sediments and soils [58], and sewage sludge and soil samples [59]. In addition to PCB congeners, Martinez-Cored et al. [56] developed a method based on a pyrenyl-silica column that resolved dibenzo-p-dioxins and polychlorinated dibenzofurans in a separate fraction. A similar separation has been reported for ortho and nonortho PCB congeners with activated carbon [60] and porous graphitic carbon [61]. Because better separations of PCB congeners are routinely achieved by GC, LC methods are rarely reported. However, in a recent example, Olsovska et al. [62] describe a UHPLC method for PCB mixtures. Three columns with sub-2-mm diameter particles were compared: C18 (1.5 mm), BEH C18 (1.7 mm), and BEH Phenyl (1.7 mm). Better overall selectivity and separations were achieved with the C18 column (see Figure 13.4). It was suggested that the LC method could be applied to specialized applications, such as PCB biodegradation studies.

13.3. PESTICIDES The determination of pesticides (also known as plant protection products) and related compounds in environmental samples represents a significant challenge for the analyst. One estimate [63] places the number of current active pesticides at 881, and these compounds span a broad range of chemical properties. Classical organochlorine (OC) pesticides are stable, highly persistent environmental contaminants; the preferred method of analysis is gas chromatography with mass spectrometry, electron capture, or flame-ionization detection [9,13,64]. Contemporary use pesticides are typically more polar and less stable than OC pesticides, and as such, LC may be more suitable for their analysis. Over the past decade, advances in mass spectrometry have permitted development of robust methods of analysis for polar and ionic pesticides by various LCeMS related technologies. Measurement of pesticide levels in the range of parts per billion or parts per trillion (i.e., 10 9 g/g or 10 12 g/g) are now possible. The preferred approach for these compounds is LCeMS/MS with APCI or ESI. Pesticide transformation

348

13. ENVIRONMENTAL ANALYSIS: PERSISTENT ORGANIC POLLUTANTS

FIGURE 13.4

Separation of PCB congeners by UHPLC with three sub-2-mm particle diameter columns. Peak identifications are PCB designations. Source: From Reference [62]; reproduced by permission of Elsevier.

products (TPs) are a subclass of pesticides; these compounds are typically formed through photodegradation, oxidation, hydrolysis, or biotransformation. Transformation products are more polar than the parent compounds and are also best characterized by using LCeMS methodologies. The use of pesticides is highly regulated in most countries [65]. The World Health Organization includes numerous pesticides and

13.3. PESTICIDES

349

transformation products in its lists of chemical hazards in drinking water [66]. In the European Union (EU), a number of regulations affect the selection, application, handling, and disposal of pesticides, with the goal of reducing pesticide levels in the environment and the food web and ultimately reducing the adverse impact on human health. In 1992, European Commission (EC) Directive 91/414/EEC was initiated, which requires approval for use of any plant protection products. Pesticides not approved are banned or restricted from use in the EU. In June 2007, the Registration, Evaluation, and Authorization of Chemicals Program (REACH) became effective, and pesticides not previously registered under 91/414/EEC became subject to this directive [67]. This regulation is broader in scope, with application to all hazardous industrial chemicals. The reduction of contaminants in coastal and inland waters is a goal of Water Framework Directive (WFD) 2000/60/EC, which further affects pesticide use. For water intended for human consumption, a limit of 0.1 mg/l for individual pesticides has been set, with a 0.5 mg/l maximum limit for total pesticides. In the United States, the Environmental Protection Agency publishes methods for the determination of pesticides and other contaminants in water and environmental samples [68], while the Food and Drug Administration (FDA) and the U.S. Department of Agriculture (USDA) share responsibility for pesticides in food. The determination of pesticides in environmental matrices is the topic of several recent reviews. In separate efforts, Lacorte and Fernandez-Alba [69], Sancho et al. [4], and Hernandez et al. [70] discuss the advantages of the use of time-of-flight mass spectrometry for the analysis of pesticides and pesticide transformation products in food and environmental samples. Pico and Barcelo [71] review efforts to study metabolites and degradation products of pesticides and veterinary drugs in food. An emphasis was placed on the advantages and limitations of the various mass analyzers used in applications. For example, high-accuracy mass analyzers offered a significant advantage for the identification of unknown metabolites but with the limitation of lower sensitivity compared with triple quadrupole (QqQ) MS. Petrovic et al. [72] include polar pesticides in their review of LC methods for emerging environmental contaminants. John, Worek, and Thiermann [73] provide an extensive review of LCeMS methods for organophosphorus compounds (OPCs), which include pesticides, flame retardants, and plasticizers. Because OPCs are often ionic, hydrophilic, and thermally labile, LC separation approaches are preferred to GC approaches. The authors give in-depth discussions of approaches for sample processing and the application of various massspectrometry technologies to OPC analysis. Hernandez, Sancho, and Pozo [74] review the application of LCeMS technologies to the determination of pesticides in biological fluids. Sancho et al. [4] classify methods based on their end-use application: (a) sample screening for the presence of selected

350

13. ENVIRONMENTAL ANALYSIS: PERSISTENT ORGANIC POLLUTANTS

pesticides of interest, (b) quantitative analysis, (c) identity confirmation for suspected analyte(s), and (d) identity elucidation for unknown analytes. The applicability of time-of-flight, quadrupole, and ion-trap MS technologies to these applications is discussed. Numerous efforts have been directed toward the development of methods for multiresidue pesticide screening and quantitation. Nogueira, Sandra, and Sandra [75] describe a single-quadrupole LCeMS method for screening 12 neutral pesticides in water samples, which include N-methyl carbamates, phenylureas, and triazines. Detection limits of 0.5 ng/l to 3.0 ng/l were obtained for most of the pesticides studied, with relative standard deviations (RSDs) below 4.9%. Kampioti et al. [76] developed a method for the determination of up to 20 pesticides from different classes, selected on the basis of EU regulations and current usage. On-line solid-phase extraction (SPE) was used for water samples, coupled with reversed-phase LCeESIeMS/MS. Two selected reaction monitoring transitions were monitored per compound. Method repeatability ranged from 2.0 % to 12.1% RSD. Famiglini et al. [77] describe a novel method for simultaneous analysis of organochlorine pesticides and phenoxy acids in water samples, without derivitization. The inclusion of nonpolar OC pesticides in the multicomponent method was achieved through application of a direct-EI interface [78] to a mass selective detector (MSD). Graphitized carbon black was used for solidphase extraction enrichment of the polar and nonpolar pesticides. Famiglini et al. [79] also describe a method exclusively devoted to OC pesticide analysis. Whereas most multiresidue methods employ SPE for sample enrichment, Grulick and Alder [80] utilized direct sample injection for the determination of 300 pesticides in water samples. A conventional reversed-phase LC separation was developed with gradient elution using a C18 column with a run time of approximately 23 min. The LC was coupled to a triple quadrupole mass spectrometer with electrospray ionization in the positive ion mode. Two selected reaction monitoring transitions were collected for each analyte by means of repeated analyses (i.e., two sample injections). Because interfering signals were noted for many of these transitions, both the LC separation and confirmatory transitions were essential for correct analyte identification. Kovalczuk et al. [81] developed a high-throughput method for multiresidue pesticide analysis in food matrices based on ultra high-performance liquid chromatography (UHPLC) with coupling to tandem mass spectrometry. Separation of 17 polar pesticides was achieved in under 8 min (see Figure 13.5). The authors compared UHPLC and conventional LC approaches, and they observed higher separation efficiencies with the LC method. Faster separations and better limits of quantitation were possible with the UHPLC method, however. A different approach for multiresidue

13.3. PESTICIDES

351

FIGURE 13.5

UHPLCeMS/MS separation of an apple extract, fortified with 17 polar pesticides. Peak identification: (1) carbendazim, (2) thiabendazole, (3) carbofuran, (4) carbaryl, (5) linuron, (6) methiocarb, (7) epoxiconazole, (8) flusilazole, (9) diflubenzuron, (10) tebuconazole, (11) imazalil, (12) propiconazole, (13) triflumuron, (14) bitertanol, (15) prochloraz, (16) teflubenzuron, (17) flufenoxuron. Source: From Reference [81], reproduced by permission of Elsevier.

pesticide screening was taken by Garcia-Reyes et al. [82]. Reversed-phase liquid chromatography (RPLC) was coupled with time-of-flight mass spectrometry to provide accurate mass spectra over the range 50 to 1000 m/z, for each of the LC peaks. Automated identification of target pesticides was based on preliminary matches with retention and m/z data, with accurate mass confirmation for positive results. Quantitation was then undertaken by a separate LCeESIeMS/MS linear ion-trap method (QqQLIT). Two SRM transitions were collected for additional confirmation. The methodology was initially developed for application to 100 target pesticides, and future expansion to 400 pesticide residues is planned. Methods have also been developed for specific classes of pesticides. N-methylcarbamates are systemic insecticides characterized by moderate polarity and relatively low thermal stability. For these reasons, LC methods are more suitable than GC methods for the analysis of environmental samples. In the case of aldicarb, metabolic pathways involve oxidative conversion to aldicarb sulfoxide and aldicarb sulfone. These transformation products are reportedly more toxic and persistent than the parent compound [83] and are important to monitor along with the parent compound. One of the first LC methods for the determination of carbamate pesticides and their transformation products is based on LC

352

13. ENVIRONMENTAL ANALYSIS: PERSISTENT ORGANIC POLLUTANTS

with postcolumn hydrolysis and derivitization with fluorescence detection [84,85]. This method exhibits high selectivity and sensitivity but lacks confirmation for positive results. Nunes et al. [83] developed an LCeAPCIeMS method for the determination of aldicarb, aldicarb sulfoxide, and aldicarb sulfone that permitted confirmation of the target analytes. El Arache, Sabbah, and Morizur [86] developed an LCeESIeMS/MS method for phenyl-N-methylcarbamates and hydrolysis products. Carbamates were determined in the positive ion mode for protonated molecular ions [M þ H]þ, whereas transformation products were determined in the negative mode for [M e H]- ions. Organophosphorus pesticides constitute about one third of the total pesticides used globally [87]. As a class, organophosphorus pesticides consist of a P¼O or P¼S group with a readily hydrolysable leaving group and two organo substituents with greater stability. Organophosphorus pesticides are neurotoxins, and toxicity results from acetylcholinesterase inhibition. Both LC and GC methods are applicable to the analysis of these compounds, with the usual caveat that certain compounds may be too polar or too thermally labile for GC analysis. LC methods frequently utilize reversed-phase separations, with or without ion-pairing reagents, coupled with single or triple quadrupole mass spectrometry (see reference [73] and the references therein).

13.4. BROMINATED FLAME RETARDANTS Brominated flame retardants are commercially manufactured compounds used in the production of polymers (e.g., plastics, electronic circuit boards, building materials, cushioning foams, and textiles) to reduce flammability. First produced in the early 1970s, BFR usage has grown steadily, and estimates of current usage of all types of BFRs exceed 200 kilotons (i.e., 1.8  108 kg) worldwide. BFRs constitute a diverse array of compounds that include brominated aliphatics, aromatics, and cycloaliphatics (see Figure 13.1). To date, 75 BFRs have been produced; currently three compoundsddecaBDE, tetrabromobisphenol A (TBBPA), and hexabromocyclododecane (HBCD or HBCDD)dconstitute the bulk of commercial BFR production [2]. Other commonly used BFRs include BDEs (also designated PBDEs), decabromodiphenylethane (DBDPE or deBDethane), tetrabromoethylcyclohexane (TBECH), and tetrabromocyclooctane (TBCO). Bromine radicals released during thermal decomposition of BFRs act as efficient electron scavengers to inhibit combustion. Other flame retardants that utilize chlorination or phosphorylation to inhibit combustion have been developed; however, current usage of these materials is insignificant compared with brominated reagents. Methodologies used for the analysis

13.4. BROMINATED FLAME RETARDANTS

353

of BFRs have been extensively reviewed [10,13,88e90]. Wang and Li list numerous other BFR reviews with different focus topics [90]. BFRs can be categorized as additive or reactive materials. Additive BFRs are incorporated in the polymer as a physical mixture and are not chemically bonded. Reactive BFRs are selected to copolymerize with other polymer constituents and are typically more resistant to leaching into the environment than polymers formulated with additive BFRs.

13.4.1. BDEs BDEs were the first widely used BFRs, available as three technical mixtures with different degrees of bromination. PentaBDE consists of a mixture of BDE-99/47/100/153/154 congeners; octaBDE consists of a mixture of BDE-183/197/196/207 congeners, and decaBDE consists of BDE-209/206. The production of polybrominated biphenyls (PBBs), pentaBDE, and octaBDE technical mixtures has been discontinued due to bans or restrictive measures imposed by the EU, the EPA, and state governments in the United States [91,92]. Of the three BDE technical mixtures, only decaBDE remains in production. Decabromodiphenylethane was introduced in the mid-1980s as an alternative to decaBDE, and its usage is expected to increase as decaBDE is phased out. Because products containing penta-, octa-, and decaBDE BFRs are in existence, measurement of the various BDE congeners is still relevant. The analysis of BDEs has been reviewed by Stapleton [10]; Kierkegaard, Sellstrom, and McLachlan [93]; Wang and Li [90]; and Covaci et al. [88]. Because BDEs have similar physical and chemical properties to polychlorinated biphenyl congeners (and, in fact, the same naming conventions are used for BDEs as PCBs), similar methods have also been used for their analysis. With the exception of the higher brominated BDEs (i.e., certain octa-, nona-, and decaBDEs), gas chromatographyemass spectrometry (GCeMS) and related techniques are the methods of choice for determination of BDEs from complex samples [6,94]. Decabromodiphenyl ether, BDE-209, is thermally unstable and is observed to debrominate during analysis by GC; consequently, LC methods of analysis are preferred. LC methods are also preferred for BDE metabolites and transformation products, which are often sufficiently polar to require derivitization for analysis by GC; derivitization is not required for LC analysis. Debrauwer et al. [95] studied various atmospheric pressure ionization techniques applied to the LCeMS analysis of BDEs and TBBPA, including electrospray ionization (EI), atmospheric pressure chemical ionization, and atmospheric pressure photoionization (APPI), under

354

13. ENVIRONMENTAL ANALYSIS: PERSISTENT ORGANIC POLLUTANTS

positive and negative ionization conditions. The authors found that polar degradation products of TBBPA were efficiently ionized by negative-ion APPI, especially for mobile phase conditions with reduced water content. APPI was also applied to the detection of BDE congeners, and useful results were obtained in both positive-ion and negative-ion modes. Lagalante and Oswald [96] utilized negative-ion APPI tandem mass spectrometry to determine BDEs in a house dust standard reference material (SRM 2585). Eight BDEs of primary interest to the U.S. EPA were resolved using a C18 reversed-phase gradient elution method. BDE levels agreed with NIST certified values at the 95% confidence interval for all congeners determined. Li et al. developed extraction methods for water analysis of BDEs based on liquideliquid microextraction [97] and single-drop microextraction [98] that specifically targeted BDE-209. Samples were subsequently analyzed by LCeUV with detection at 226 nm. LCeUV methods have also been developed for screening polymeric waste materials (such as television and computer housings) prior to recycling [99e101]. The characterization of BFRs in potentially recyclable materials is important since EU directive 2002/96/EC (WEEE) [92] restricts the use or reuse of BDEs and polybrominated biphenyls. Schlummer et al. [101] compared LCeUV and APCI LCeMS methods of detection for screening BDEs in plastics. LCeUV detection was presented as a cost-effective alternative to LCeMS for the routine screening required by EU directives. Linsinger et al. [102] discuss the development of BFR certified reference materials (CRMs) prepared by fortifying polyethylene and polypropylene with penta-BDE, octa-BDE, deca-BDE, and decabrominated biphenyl. Certification of these materials was performed by an intercomparison of measurements from 16 laboratories that used GC based methods. Bierla et al. [103] developed a novel approach to the analysis of BDEs using LC with inductively coupled plasma mass spectrometry (ICP MS) for the detection of heteroatom-bearing compounds (see Figure 13.6). Polyatom interferences were eliminated by use of a collision cell. High specificity for brominated compounds was achieved in complex matrix samples. BDE transformation products, such as hydroxylated BDEs, offer similar analytical challenges to BDEs. Certain hydroxylated BDEs exhibit structural similarities to thyroxine and estrogen and may produce toxic effects [104,105]. LC approaches are typically preferred over GC approaches, since derivitization steps are not required. Lupton et al. [106], and Kato et al. [107] each developed LCeMS/MS methods for determination of OH-BDEs, based on APCI or ESI operated in the negative-ion mode. Lai et al. [108] employed UHPLC with ESIeMS/MS to separate nine OHBDEs in under 7 min (see Figure 13.7). The method was validated using spiked rat plasma samples and applied to a pharmacokinetic study of 6-OH-BDE-47 in rats dosed with this compound. Chang et al. [109] also

13.4. BROMINATED FLAME RETARDANTS

355

FIGURE 13.6 LCeICP MS chromatograms of BDE standards. Peak identification: (1) BDE 47, (2) BDE 85, (3) BDE 100, (4) BDE 138, (5) BDE 155, (6) BDE 201, (7) BDE 206, (8) BDE 207 and BDE 208. Source: From Reference [103]; reproduced by permission of The Royal Society of Chemistry.

FIGURE 13.7 SRM chromatograms from the UHPLCeESIeMS/MS analysis of OHBDEs at 20 ng/ml and the internal standard at 100 ng/ml: (1) 3,5-DBP, (2) 3’-OH-BDE-7, (3) 6’-OH-BDE-17, (4) 4-OH-BDE-17, (5) 2’-OH-BDE-28, (6) 4’-OH-BDE-49, (7) 6-OHBDE- 47, (8) 2’-OH-BDE-68. Source: From Reference [108], reproduced by permission of Elsevier.

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13. ENVIRONMENTAL ANALYSIS: PERSISTENT ORGANIC POLLUTANTS

developed an LCeESIeMS/MS method for phenolic compounds including OH-BDEs; however, this method required sample derivitization with dansyl chloride prior to analysis. Interestingly, OH-BDEs and other polar brominated compounds are produced naturally by marine sponges, mussels, and algae [88]. These compounds are referred to as brominated natural compounds (BNCs); LC methods and measurements for BNCs has been reviewed by Covaci et al. [88].

13.4.2. HBCDs and Brominated Cycloalkanes Restrictions placed on the use of BDEs by EU legislation has led to increased utilization of alternative BFRs [91,92]. HBCDs are widely used as an additive BFR in polystyrene foams, building materials, textiles, and furniture, and over the period 1990 to 2000, the quantities of HBCD used worldwide doubled. Aspects of HBCDs have been reviewed by Covaci et al. [88], and Morris et al. [110]. HBCD is prepared by bromination of cis-trans-trans-1,5,9-cyclododecatriene. Technical mixtures are composed primarily of three diastereomers (a, b, g), originally designated by the order of elution in RPLC. g-HBCD is the most abundant isomer in the technical mixture, with 75% to 89% mass fraction, a-HBCD is typically 10% to 13% mass fraction, and b-HBCD is 1% to 12% mass fraction. Trace levels of two additional diastereomers (d, ε) may also be present. Resolution of the three HBCD diastereomers is easily achieved by reversed-phase LC on C18 columns, often in less than 15 min. The usual elution order for the diastereomers is a-, b-, g-HBCD; however, an alternate elution order (i.e., a-, g-, b-HBCD) was reported by Dodder et al. [111] for shape-selective columns operated with methanolic mobile phases (see Figure 13.8). The reversed elution order for b-HBCD and g-HBCD provides the basis for the development of orthogonal methods that may help eliminate matrix interferences. Yu et al. [112] also studied the influence of mobile phase composition on HBCD diastereomer selectivity and show that MS sensitivity was also affected by this parameter. Because HBCDs lack chromaphores, detection is primarily by mass spectrometry. Although APCI would be expected to yield better ionization efficiency than ESI. based on the nonpolar characteristics of HBCD, the opposite trend is observed. Budkowski and Tomy [113] compared negative-ion ESI with APCI and found only weak intensities for m/z 642 for APCI, compared with stronger responses for m/z 640.6 (i.e., [M e H]-) for ESI. Morris et al [110] examined positive and negative ESI modes and found that negative ESI was more sensitive. Ross and Wong [114] further studied the application of APPI for the detection of HBCDs using a photoionizable dopant (1,4-dibromobutane) in the mobile phase. Lower detection limits were observed with the addition of the dopant, and APPI

13.4. BROMINATED FLAME RETARDANTS

357

FIGURE 13.8 MRM chromatograms of atmospheric particle-phase samples on (a) Eclipse C18 column and (b) Carotenoid C30 column. The mobile-phase composition is 90% methanol/10% water (volume fractions). Source: From Reference [111], reproduced by permission of Elsevier.

was less affected by matrix interferences than ESI. Galindo-Iranzo et al. [115] increased the sensitivity of a negative-ion ESI method through chloride adduct generation. Ammonium chloride was added to the mobile phase, and the resulting [M þ Cl]þ ions were fragmented to [M e H]- ions using tandem mass spectrometry. A 14-fold improvement in the limit of detection was reported.

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13. ENVIRONMENTAL ANALYSIS: PERSISTENT ORGANIC POLLUTANTS

The issue of matrix effects that result in signal suppression (or possibly, enhancement) is a cause for concern, in that analyses may be subject to bias. Tomy et al. [116] report one of the first applications of isotopically labeled internal standards to the determination of HBCD diastereomers in complex matrix samples. The use of 13C- and 2Hlabeled HBCD isomers, in principle, should reduce measurement bias, since quantitation is based on the ratio of coeluting HBCD species (i.e., the analyte and the internal standard) rather than absolute responses of the analytes. Analyte response may vary with instrument conditions and matrix effects; however, the ratio remains constant. The use of isotopically labeled internal standards for the individual diastereomers is now common practice for BFRs [117]. HBCD isomers have been determined by LCeMS/MS methods in diverse sample types, including estuary [94] lake, river, and snow water samples [118]; styrenic polymers [119]; marine sediments [120]; fish [121], marine mammal tissues [122e124]; seaweed [125]; foods [126]; and human biological matrices [124,127]. Because the substituted carbon atoms in HBCD are chiral, enantiomeric forms of each diastereomer exist; 16 stereoisomers are theoretically possible [128]. Biotransformation of the stereoisomers can potentially alter the enantiomeric ratios [129], and methods for the determination of six individual enantiomers have been developed. Gomara et al. [130] studied the influence of mobile phase modifiers, gradient conditions, column temperature, and flow rates to achieve baseline resolution of the prevalent HBCD enantiomers, using a permethylated b-cyclodextrin column (see Figure 13.9). In this work, spiked meat, fish, and butter samples were analyzed to assess detection limits and matrix effects. Ko¨ppen et al. [131] determined HBCD enantiomers in fish samples obtained from fish farms in Etnefjorden, Norway. Levels of HBCDs were detectable in the fish, and the enantiomeric ratios were observed to differ among different fish species. Several recent reports describe the measurement of HBCD enantiomers in marine mammals [122], fish [130,131], human serum[132], and, marine sediment [133]. In certain cases where non-racemic enantiomeric fractions were observed, biotransformations have been attributed to cytochrome P450 metabolism. Two additional brominated cycloalkanes are currently used as additive BFRs. 1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane (TBECH) and 1,2,5,6 tetrabromocyclooctane (TBCO) are produced for use in textiles, paints, and plastics. TBECH can exist as four diastereomers (designated a, b, g, and d), and TBCO can exist as two diastereomers. Chiral separations of TBECH and TBCO enantiomers have not yet been reported. These BFRs exhibit some of the same analytical challenges as HBCDs, and LCeMS approaches offer the most promise for the development of robust methods.

13.4. BROMINATED FLAME RETARDANTS

359

FIGURE 13.9

Separation of HBCD enantiomers using a chiral permethylated betacyclodextrin LC column. Source: From Reference [130], reproduced by permission of Elsevier.

Zhou et al. [134] evaluated three APIeMS techniques: ESI, APCI, and APPI. ESI operated in the negative-ion mode was found to offer the most sensitive response for g- and d-TBECH [g- and d-TBECH þ Cl]-; for a- and b-TBECH, APCI gave the best response. Baseline resolution of the six TBECH and TBCO diastereomers was demonstrated using a 1.8-mm particle diameter UHPLC C18 column.

13.4.3. TBBPA Tetrabromobisphenol A (4,4’-isopropylidenebis(2,6-dibromophenol); TBBPA) is the most widely used BFR in terms of production quantities [2]. It is used as both a reactive and an additive BFR in a variety of polymers, epoxy resins, and adhesives and, in particular, is a constituent in printed circuit boards at levels up to 34% (mass fraction). The acute toxicity of TBBPA is relatively low; however, concern for its potential as an endocrine disruptor exists. TBBPA shares structural similarities to thyroxine (T4), and the compound competitively binds to the thyroid hormone transport protein transthyretin [135]. Analysis by gas chromatography requires derivitization, and LC methods are preferred to reduce sample processing. Biotransformation of TBBPA

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through debromination results in lesser brominated analogs; these compounds are reportedly strong agonists for estrogen-receptor mediated gene expression [136]. Chu, Haffner, and Letcher [137] developed an LCeESIeMS/MS method for the determination of TBBPA and lesser brominated analogs in sediments and wastewateresludge samples. Sample recovery and matrix interference effects were largely compensated by the use of an isotopically labeled internal standard. Saint-Louis and Pelletier [138] studied ionsuppression effects for the same types of matrices and found that ion suppression was more severe for sewage sludge samples than for marine sediment extracts. Guerra et al. [139] developed an LC linear ion-trap MS/MS method to measure TBBPA and BDEs at ultratrace levels with limits of detection below 1 pg. The authors compared selected reaction monitoring with enhanced product-ion (EPI) MS approaches and observed slight improvements in sensitivity with the EPI method. These methods were then applied to the analysis of sludge and sediment samples [140]. Bacaloni et al. [141] developed a LCeAPPIeMS/MS method for both TBBPA and BDEs in water samples. Matrix effects were not observed for these samples, and quantitation was based on external calibration. Morris et al. [142] measured TBBPA and HBCDs in aquatic samples by LCeESIeMS, using an isotopically labeled internal standard for TBBPA determinations. Tollba¨ck [143] developed a rapid LCeESIeMS method for the determination of TBBPA in air samples, also based on the use of an isotopically labeled internal standard. TBBPA could be determined at levels 30 to 40 times lower with ESI than with APCI. Hayama et al. [144] describe a method for determination of TBBPA in human serum samples by LCeESIeMS/MS, also based on an internal standard calibration approach.

13.5. PERFLUOROALKYL COMPOUNDS Perfluoroalkyl and polyfluoroalkyl substances (PFASs) are persistent organic pollutants with nearly ubiquitous occurrence in environmental samples. PFASs have anthropogenic origins; over the period 1951 to 2004, it has been estimated that up to 7300 tons (i.e., 6.6  106 kg) of PFASs have been released into the environment [145]. PFASs are used in the manufacture of industrial materials and consumer products. The compounds are characterized by a nonpolar fluorinated hydrocarbon chain with a polar end group, and they exhibit both lipophobic and hydrophobic properties. Principal applications include uses as surfactants, nonstick coatings, and stain repellants. The compounds are also precursors in the synthesis of fluoroelastomers and fluoropolymers and are used in the

13.5. PERFLUOROALKYL COMPOUNDS

361

production of textiles, carpets, and grease-resistant packaging for food. Individual PFASs differ in alkyl chain length and in the functionality of the end group. Three subcategories are commonly used to classify PFASs: perfluoroalkyl sulfonic acids (PFSAs), perfluoroalkylcarboxylic acids (PFCAs), and fluorotelomer alcohols (FTOHs). Historically, PFSAs and PFCAs have been prepared by electrochemical fluorination, whereas fluorotelomer alcohols are synthesized by telomerization [146]. The electrochemical fluorination process results primarily in straight chain homologs, but branched chain isomers are known to be present. Branched chain isomers can also be synthesized through telomerization synthesis with the use of branched chain telogen precursors. FTOHs have an even number of carbon atoms and contain a nonfluorinated ethylene group adjacent to the polar end group. Pathways for the conversion of FTOHs to PFCAs in the environment have been proposed [147,148]. Perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) are the two most commonly measured PFASs; however, analytical methods have been developed to determine other, shorter and longer chain PFSAs and PFCAs (see Table 13.2 for a list of common PFASs and abbreviations). Recommended terminology and abbreviations have been described by Buck et al, [146]. These compounds have been determined in a wide range of samples types, which include human biological fluids, fish and other marine organisms, water samples, and food. Advances in analytical instrumentation that occurred over the past decade now permit the determination of PFAAs in environmental samples at pg/g levels [149,150]. A survey of PFOS and PFOA levels reported for various samples is provided in Table 13.3. Health risks associated with PFASs are not yet well established; however the assessment of potential risks is the subject of numerous studies [151e153]. Known or suspected risks include chronic toxicity, carcinogenic activity, endocrine effects, and bioaccumulationebiomagnification. PFASs are included in the National Health and Nutrition Examination Survey (NHANES) conducted by the Centers for Disease Control and Prevention (CDC) [154], and the National Toxicology Program has included various PFASs in their assessment of toxicity, carcinogenicity, and persistence in human blood [155]. Regulations that affect the use and environmental release of PFASs vary for specific compounds and with the regulating authority. For example, the production of PFOS was voluntarily discontinued by the 3M Company in 2000 to 2002, and current use of this material is restricted in the United States [156], Canada, and the EU; however, production and use of PFOS is increasing in China. Recently, PFOS has been added to the list of restricted use compounds (Annex B) in the Stockholm Convention on Persistent Organic Pollutants [157], and the

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TABLE 13.2

Names, Acronyms, MS Fragmentation Data for Fluorinated Compounds

Name

Acronym

Precursor ion (m/z)

Product ions (m/z)

Perfluorobutanesulfonic acid

PFBS

299

80, 99

Perfluorohexanesulfonic acid

PFHxS

399

80, 99

Perfluoroheptanesulfonic acid

PFHpS

449

80

Perfluorooctanesulfonic acid

PFOS

499

80, 99, 130, 280

Perfluorodecanesulfonic acid

PFDS

599

Perfluorobutanoic acid

PFBA

213

169

Perfluoropentanoic acid

PFPeA

263

219

Perfluorohexanoic acid

PFHxA

313

269, 119

Perfluoroheptanoic acid

PFHpA

363

319, 169

Perfluorooctanoic acid

PFOA

413

369, 169, 219

Perfluorononanoic acid

PFNA

463

419, 219, 169

Perfluordecanoic acid

PFDA

513

469, 219, 269, 119

Perfluoroundecanoic acid

PFUnA

563

519, 269, 219

Perfluorododecanoic acid

PFDoA

613

569, 169, 319

Perfluorotridecanoic acid

PFTriA

663

619

Perfluorotetradecanoic acid

PFTA

713

669

Perfluorohexadecanoic acid

PFHxA

813

769

Perfluorooctadecanoic acid

PFOcA

913

869

Perfluorooctane sulfonamide

PFOSA

498

78

n-Methylperfluorooctane sulfonamide

MeFOSA

512

n-Ethyl perfluorooctane sulfonamide

EtFOSA

526

SULFONIC ACIDS

CARBOXYLIC ACIDS

SULFONAMIDES

169

Source: Data is compiled from references [174,188,190,200].

European Food Safety Authority (EFSA) has established a tolerable daily intakes for PFOS and PFOA [158]. Methods for the determination of PFAAs in environmental analysis have been reviewed by Villagrasa, de Alda, and Barcelo [145], de Voogt

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TABLE 13.3 Survey of PFOS and PFOA Levels in Various Samples Sample type

PFOS

PFOA

Unitsa

Reference

ANIMAL SAMPLES Bivalve (soft tissue)

3.62e4.55

ng/g dw

[184]

Bivalve (shell)

0.62e0.71

ng/g dw

[184]

Fish, bluegill

1.22e428

ng/g ww

[190]

Fish, sardine

0.73e132

ng/g ww

[188]

Fish, young hake

1.25e3.54

0.21e1.03

ng/g

[192]

Insect larvae

20e144

1.3e4.3

ng/g ww

[188]

Liver, beaver

38.7

0.28

ng/g ww

[174]

Liver, polar bear

263e6340

255.0

803.5 > 113.0

DTX-1

Neg

817.5 > 255.0

817.5 > 113.0

YTX

Neg

1141.5 > 1061.7

1141.5 > 855.5

45 OH-YTX

Neg

1157.5 > 1077.7

1157.5 > 871.5

HomoYTX

Neg

1155.5 > 1075.5

1155.5 > 869.5

45 OH-HomoYTX

Neg

1171.5 > 1091.5

1171.5 > 869.5

PTX-1

Pos

892.5 > 821.5

892.5 > 213.2

PTX-2

Pos

876.5 > 823.4

876.5 > 213.2

AZA-1

Pos

842.5 > 824.5

842.5 > 806.5

AZA-2

Pos

856.5 > 838.5

856.5 > 820.5

AZA-3

Pos

828.5 > 810.5

828.5 > 792.5

Notes: The mobile phase is composed of water (A) and ACNewater (95:5) (B), both containing 50 mM formic acid and 2 mM ammonium formate. The mass spectrometry (MS/MS) transitions for the toxins monitored officially in the Europe Union (EU) are also indicated. Column: BDS-Hypersil C8, 50 mm  2 mm, 3 mm particle size. Flow: 0.2 ml/min,

424

15. ANALYSIS OF NATURAL TOXINS

ion mode, and in Figure 15.1(b), the analysis of OA, DTX-1, and DTX-2 was in the MRM negative ion mode. In brief, chromatographic separation was performed by gradient elution in a 3-mm Hypersil-BDS-C8 column (50 mm  2 mm) with a temperature of 25 C and a flow rate of 0.2 ml/min. The transitions suitable for each toxin are also presented in Table 15.7. This technique has been evaluated and considered a useful method [52,58]. A short, narrow-bore column packed with a 3-mm Hypersil-BDSC8 phase is one of the most widely used columns capable of separating a wide range of toxins using a rapid gradient [41,61]. The method was also applied in the new technologies, ultraperformance liquid chromatography with tandem mass spectrometry detection (UPLCeMS/MS), and more than 20 analogs were separated in only 6.6 min [62]. Another advantage is that acidic conditions facilitate good separation of acidic OA analogs by suppressing ionization of the carboxyl groups and preventing deleterious ion-exchange interactions with residual silanol groups in the stationary phase [63]. Nevertheless, the chromatography of compounds included in the YTX group can be problematic under acidic conditions [52]. Recently, we observed that the quantification of OA, DTX-1, and DTX-2 can be affected by several parameters, such as sample solvent, MS detection method, mobile phase solvent brands and equipment [64]. For example, when OA, DTX-1, and DTX-2 were quantified by MS detection methods that include different numbers of monitored compounds, the toxin amounts were increased or decreased. This was checked with two MS methods: one included only the specific transitions for dinophysistoxins (4 transitions) and the other included the transitions for 6 lipophilic toxins (a total of 10). Quantities analyzed by LCeMS/MS using the MS method with 10 transitions were considerably lower than those obtained by a method with 4 transitions. The underestimations were up to 40% for OA, 39% for DTX-1, and 37% for DTX-2. This means that the number of transitions included in the MS methods, not fixed in the reference method [54], affects the toxin quantification. Since standards for all toxins are not available, the calibration curve of one toxin standard is often used to quantify other toxins from the same group, assuming a given ionizationconversion factor. It was shown, however, that it is incorrect to assume that analogs from the same toxin group provide an equimolar response by MS/MS tandem detection and, therefore, in the absence of suitable quantification data, should be considered unreliable [64]. Definitely, the LCeMS/MS method is a useful tool to show the toxin profile or to characterize new toxins. However, to protect public health and food safety, it should be carefully used under controlled conditions. A recent validation study has proven that results improve greatly by reducing the number of variable parameters [65].

15.4. SAXITOXIN AND ANALOGS

425

15.4. SAXITOXIN AND ANALOGS Paralytic shellfish poisoning is common worldwide and the most lethal form of phycotoxin intoxication. Toxin levels can be extremely high, with up to 650 mg/kg flesh detected in mussels from the coast of Mexico (E. Cacho, personal communication). PSP toxins are a group of more than 21 tetrahydropurines, all of them chemical analogs of Saxitoxin (STX), the first described [66]. They can be classified into three subgroups: Carbamate (STX, neoSTX, and gonyautoxins (GTX1-4)), N-sulfocarbamoyl (GTX5-6, C1-4), and Decarbamoyl (dcSTX, dcneoSTX, dcGTX1-4). These are usually quantified by a semiquantitative mouse bioassay [67], which is the reference method internationally accepted in monitoring programs. Chemical methods used to determine PSP toxins are fluorimetric assays, high-pressure liquid chromatography with fluorimetric detection (either precolumn or postcolumn oxidation), liquid chromatographyemass spectrometry (LCeMS), and capillary electrophoresis methods. The HPLC methods are widely used to quantify PSP toxins present in seafood samples, but they are also useful to provide the PSP profile, because chromatographic methods are identification methods as well. These toxins have only a weak chromophore group, and they must be modified before detection: When they are oxidized in an alkaline solution, a purine is formed that becomes fluorescent at acidic pH levels. This reaction can be performed either precolumn or postcolumn. Bates and Rapoport [68] first studied the oxidative alkaline conditions of PSP toxins to get fluorescent compounds; then Buckley, Oshima, and Shimizu [69] added them to their method and established the basis of the HPLC method for these compounds with postcolumn reaction. In 1984, Oshima et al. [70] proposed a method based on alkaline oxidation to produce highly fluorescent derivatives but with problems to separate toxins such as GTX1, GTX4, and GTX3, GTX5. Afterward, they described a method able to separate almost all the PSP toxins with three eluents according to the basicity of the three groups of toxins (Group I: C1eC4; group II: GTX1,4, GTX5 (B1) and GTX6 (B2), dc-GTX1,4; group III: NEO, dcSTX and STX) [71]. In a later work, Oshima [72] improved the method, which included a cleanup procedure for raw extracts. The main disadvantage is that it is time consuming. Among the group of precolumn methods, the Lawrence and Menard method of 1991 should be highlighted [73]. Its main disadvantage is the inability to easily distinguish among different toxin groups with similar toxicity. Following this, Lawrence, Menard, and Cleroux [74] modified the chromatographic conditions to reduce the analysis time and improve performance. This method was validated by the AOAC [67] through a collaborative process and adopted as an AOAC official method. It is based on the precolumn oxidation of PSP toxins with

426

15. ANALYSIS OF NATURAL TOXINS

hydrogen peroxide and sodium periodate followed by fluorimetric detection. It was validated for the determination of STX, NEO, GTX2,3, GTX1,4, dcSTX, GTX5 (B1), C1,2, and C3,4 in molluscs (mussels, clams, oysters, and scallops) [67]. The validation was further extended to two more toxins: dcNEO and dcGTX2,3 [75]. A quantitative radio-receptor assay has been recently validated for this group [76].

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[73] Lawrence JF, Menard C. Liquid chromatographic determination of paralytic shellfish poisons in shellfish after prechromatographic oxidation. J AOAC 1991;74:1006e12. [74] Lawrence JF, Menard C, Cleroux C. Evaluation of prechromatographic oxidation for liquid chromatographic determination of paralytic shellfish poisons in shellfish. J AOAC Int 1995;78:514e20. [75] Turner AD, Norton DM, Hatfield RG, Morris S, Reese AR, Algoet M, et al. Refinement and extension of AOAC Method 2005.06 to include additional toxins in mussels: singlelaboratory validation. J AOAC Int 2009;92:190e207. [76] Van Dolah FM, Leighfield TA, Doucette GJ, Bean L, Niedzwiadek B, Rawn DF. Singlelaboratory validation of the microplate receptor binding assay for paralytic shellfish toxins in shellfish. J AOAC Int 2009;92:1705e13. [77] Chang PK, Ehrlich KC, Fujii I. Cyclopiazonic acid biosynthesis of aspergillus flavus and aspergillus oryzae. Toxins 2009;1:74e99. [78] Dorner JW. Mycotoxins in food: methods of analysis. In: Nollet LML, editor. Handbook of food analysis, New York: Marcel and Dekker; 1996. p. 1089e146. [79] Hsieh HY, Shyu CL, Liao CW, Lee RJ, Lee MR, Vickroy TW, Choua CC. Liquid chromatography incorporating ultraviolet and electrochemical analyses for dual detection of zeranol and zearalenone metabolites in mouldy grains. J Sci Food Agri. http://dx. doi.org/10.1002/jsfa.4687, (wileyonlinelibrary.com); 2011. [80] Maragos CM, Kim EK. Detection of zearalenone and related metabolites by fluorescence polarization immunoassay. J Food Protect 2004;67:1039e43. [81] Pires OR, Sebben A, Schawartz EF, Morales RAV, Bloch C, Schwartz CA. Further report of the occurrence of tetrodotoxin and new analogues in the Anuran family Brachycephalidae. Toxicon 2005;45:73e9.

C H A P T E R

16 Liquid Chromatography in the Pharmaceutical Industry R. Szucs*,y,**, C. Brunelli*, F. Lestremau*, M. Hanna-Brown*,yy *

Analytical R&D, Pfizer Global R&D, Sandwich, Kent, UK Pfizer Analytical Research Centre, Ghent University, Ghent, Belgium ** Irish Separation Sciences Cluster, Dublin City University, Dublin, Ireland yy Warwick Centre for Analytical Science, University of Warwick, UK y

O U T L I N E 16.1. The Role of Separation Science in Pharmaceutical Drug Development

432

16.2. Increasing Chromatographic Resolution 433 16.2.1. High Resolution Liquid Chromatography through Increased Efficiency 434 16.2.2. High Resolution Liquid Chromatography through Increased Selectivity 439 16.3. Chromatographic Method Development: RPLC 16.3.1. Required Method Performance 16.3.2. Selection of the Stationary Phase, pH, and Organic Solvent 16.3.3. Optimization of Temperature and Gradient 16.3.4. Optimization of the Mobile Phase pH

444 445 445 447 448

Acknowledgments

452

References

453

Liquid Chromatography: Applications http://dx.doi.org/10.1016/B978-0-12-415806-1.00016-4

431

Copyright Ó 2013 Elsevier Inc. All rights reserved.

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16. LIQUID CHROMATOGRAPHY IN THE PHARMACEUTICAL INDUSTRY

16.1. THE ROLE OF SEPARATION SCIENCE IN PHARMACEUTICAL DRUG DEVELOPMENT Pharmaceutical drug development is a lengthy, high-risk process [1] that, for the purpose of describing applications of separation science, can be divided into three distinct phases: drug discovery, drug development, and filingepostfiling activities. Although analytical chemistry and, in particular, separation science play a pivotal role in all these phases, in this contribution we focus on applications related to the drug development phase. This phase typically starts with the nomination of a drug candidate, which is subsequently taken into various phases of clinical testing where apart from efficacy, safety, dosage, side effects, and long-term adverse reactions are studied [1]. The primary focus of analytical scientists supporting drug development is to provide evidence of the safety of medicines administered to patients and volunteers during these trials. This is typically achieved through the use of state-of-the-art analytical technology, of which various forms of separation science techniques, often combined with spectroscopic detection techniques, represent a significant majority. The data generated from these experiments binds the knowledge confirming what are the chemicals that have been synthesized and to what purity, confirming what impurities are present and how safe these are, establishing safe limits for each impurity in the drug substance and in drug product for manufacturing sites to adhere to around the world and also confirming how stable the drug substance or drug product is in order to establish a shelf life and optimum packaging. The overarching goal of the analytical chemist is to provide evidence to the regulators of the drug’s safety and quality. To do this, analytical chemists provide support to synthetic chemists, who focus on the development, optimization, and scale-up of synthetic routes that generate the drug substance in the most economically viable way while minimizing its negative environmental impact. In doing so, analytical chemists need to establish impurity control strategies; define specifications for starting materials, reagents, and finished product (drug substance); and develop reliable testing procedures for in-process controls, impurity profiling, and assays for the synthetic intermediates and drug substance. Despite a surge in applications of spectroscopic techniques to support the development of synthetic processes, chromatography retains its number one position as the preferred technique, given its versatility (various modes and detector hyphenation options) and propensity for samples derived from complex matrices and samples with constituents of unknown chemical structure. Dosage form development and optimization, essential for required pharmacokinetic performance and long-term stability, of the finished drug product also requires analytical support, and here again, chromatography

16.2. INCREASING CHROMATOGRAPHIC RESOLUTION

433

is usually selected for its ability to deal with samples containing a wide diversity of components with different physicochemical properties, such as small organic molecules and polymeric excipients. The early versions of the synthetic processes are often prone to generation of a relatively large number of structurally known or unknown processrelated impurities (PRIs), observed in isolated synthetic intermediates or the final drug substance. They can originate from starting materials, be a byproduct of synthetic steps, or products of chemical degradation. The origin of these impurities as well as their impact on the quality of the final product need to be investigated and understood to establish a suitable control strategy. Chemically, these PRIs are typically related to compounds purposefully generated during the synthesis; in the most extreme cases, they can be optical isomers, positional isomers, diastereomers, or multimers of desired products. All these components need to be separated and quantified to establish their impact on the quality and safety of the final product. This can be a challenging task as these compounds often possess very similar physicochemical properties, and their separation requires chromatographic techniques that provide very high resolution. It is often necessary to use multiple techniques combined with a series of detection techniques to increase confidence that all potential PRIs are separated and detected. In the first part of this chapter, we focus on multiple ways of enhancing chromatographic resolution. In the later stages of the drug development phase, where synthetic processes have been adequately optimized with sources of PRIs at best eliminated or at least minimized and the stability of the final drug product has been optimized through formulation development, the complexity of chromatographic separation is usually significantly reduced. During this stage of development, the manufacturing processes are being qualified, which leads to the generation of a very large numbers of samples. The emphasis now shifts from resolution to the speed, ruggedness, and robustness of the analytical techniques. The second part of this chapter provides an overview of useful tools and techniques that may be applied in such a setting. In the final part of this chapter, we focus on novel trends in chromatographic method development related to the analytical quality by design initiative (AQbD). We provide a real life example of several practical steps taken in the method development workflow.

16.2. INCREASING CHROMATOGRAPHIC RESOLUTION Chromatographic resolution in liquid chromatography (LC) is determined by three factors [2]: efficiency (N), selectivity (a), and a retention

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factor (k). In this section, we focus on demonstrating how efficiency and selectivity can be used to develop high resolution methods to separate complex mixtures. The retention factor (an intrinsic property of any analyte and dependent on its physicochemical properties) can also be used to enhance resolution and is described in the final part of this chapter.

16.2.1. High Resolution Liquid Chromatography through Increased Efficiency Efficiency in liquid chromatography is directly proportional to the column length (L) and indirectly proportional to the particle diameter (dp) [3,4]. Resolution can therefore be increased either by using a longer column or a smaller particle size. Column Length The pressure drop across the column is directly proportional to the column length and the viscosity (h) of the mobile phase [4]: DP ¼

f0 u0 hL d2p

where f0 is the flow resistance factor and u0 is the linear velocity (0 denotes an unretained component). Since the pressure drop is limited by the instrumentation (e.g., 400 bar), it is not possible to increase the column length beyond a certain value without a corresponding reduction in the mobile phase viscosity, while keeping the linear velocity and particle size constant. Mobile phase viscosity can be reduced by operating the LC at elevated temperatures. Indeed, efficiencies of ca. 200,000 theoretical plates were achieved by coupling eight 25-cm columns, creating a total column length of 2 meters [3], as shown in Figure 16.1. The practical consequences of such increased efficiency are twofold. First, assuming the range of retention factors for a mixture between 0.3 and 12 and the required resolution between any two peaks is a minimum of 1.0, the theoretical peak capacity (np) [5] ! pffiffiffiffi 1 þ klast N  ln þ1 np ¼ 1 þ kfirst 4RS is increased from approximately 90 to 260 for a single 25-cm column or eight 25-cm columns coupled together, respectively. A required resolution RS ¼ 1.0 was used in this calculation, because such a value ensures an ability for the peak to be detected, hence, its presence in the sample will be known and action can be taken to optimize the resolution further, should that be required. To have a 90% probability of being able to resolve each

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FIGURE 16.1 Chromatogram of the test mixture showing how nearly 200.000 theoreticxal plates can be achieved using a 2-m column. Isocratic elution acetonitrile/ water (40/60), 1 ml/min, 80  C, Column 8  25 cm  4.6 mm, Zorbax SB-C18, 5mm. Source: Reproduced with permission from Ó2007 Elsevier B.V.

component of a mixture, the theoretical peak capacity needs to be exceeded by a factor of 20 [6]; therefore, only around 15 components will be detected with the high resolution method. As the average synthetic process typically generates 15e25 PRIs, further method optimization, even with the high resolution method, is usually required. One such extreme example is shown in Figure 16.2, where a sample related to a dual therapy (where two individual pharmaceutical actives, each for a different therapeutic effect, are combined in a single formulation) was analyzed with a high resolution method. The two pharmaceutical

FIGURE 16.2 Chromatogram of a resolution mixture from a dual-therapy product (mixture of two pharmaceutical actives spiked with all related PRIs). Gradient elution, mobile phase A: 20 mM ammonium acetate pH 5.8; mobile phase B: acetonitrile. Gradient time 0 minutes, 10% B; time 130 minutes, 90% B. (Other conditions as in Figure 16.1.)

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actives in this example had very different physicochemical properties, one being acidic and the other basic. Additionally, as both pharmaceutical actives are synthesized in separate processes, each brings a series of PRIs specific to its synthesis. As can be seen from Figure 16.2, practically all components of the complex mixture were baseline separated except the diastereomeric pair eluting at ca. 100 minutes. Complete baseline separation of this pair requires an alternative separation mode. This example demonstrates that high resolution methodology is an important tool, which increases the confidence that all components of the mixture are detected and can be further controlled, thus ensuring patient safety. A second practical consequence of this high resolution method is that it can readily replace lengthy method development activities, as demonstrated in the following example. In this example, a drug substance, being monitored under various stability conditions to enable a shelf-life assignment and depth of understanding around potential degradation products such that appropriate storage and packaging conditions could be assigned, exhibited increased levels of a poorly resolved diastereomic pair using the RPLC stability-indicating method. Significant effort to optimize this method to achieve baseline separation was ultimately unsuccessful, as shown in Figure 16.3(a). Transferring the method to a high resolution format with minor optimization of other experimental conditions led to a near baseline separation in a short time, Figure 16.3(b). This demonstrates the power of this technology as a substitute for method development.

(a)

(b)

FIGURE 16.3 Chromatographic separation of diastereomers generated during an ICH stability study. (a) Original stability indicating method failed attempt to separate diastereomeric pair: Gradient elution, mobile phase A, 10-mM KH2PO4 pH 2.0; mobile phase B, acetonitrile. Gradient time 0 minutes, 17% B; time 17 minutes, 22.5% B; time 25 minutes; 22.5% B; time 35 minutes, 29% B; time 46.5 minutes, 33% B; time 60 minutes, 53% B; time 80 minutes, 95% B. Column 15 cm  4.6 mm, XBridge Shield RP18, 3.5 mm. 1 ml/min, 48 C. (b) High resolution method. Gradient elution, mobile phase A, 0.1% formic acid; mobile phase B,e acetonitrile. Gradient time 0 minutes, 15% B; time 68 minutes, 26% B; run time 80 minutes, Column 2  10 cm  2.1 mm; ACQUITY UPLC BEH Shield RP18, 1.7 mm; 0.45 ml/min, 80 C.

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16.2. INCREASING CHROMATOGRAPHIC RESOLUTION

As chromatographic retention is directly proportional to the column length, one obvious drawback of the high resolution methods is the long analysis time. This can be partially compensated by operating at higher flow rates, as the minimum of the plate-height curves moves to a higher optimal linear flow rate while maintaining the same efficiency [4]. In this way, approximately 100.000 theoretical plates were generated in a conventional analysis time frame of 35e40 minutes using four 25-cm columns coupled together (Figure 16.4). When working at an elevated temperature, one must be aware not only of the reduced retention times but also of the impact of the temperature on the chromatographic elution order. While under extreme circumstances, a complete reversal of elution order has been observed [7], these thermodynamic effects (which are largely unpredictable) can have both a positive and negative impact on the resolution, as demonstrated in Figure 16.5. Although the retention time overall was reduced for both samples, retention times of certain mixture components were affected more than others, which created multiple peak overlaps and crossovers, Figure 16.6. One final note worth mentioning about high resolution methods is a practical tip regarding the necessity to preheat the mobile phase. As can be seen from Figure 16.7, up to a 15% loss of theoretical efficiency was observed when operating a LC at 80 C without a preheater. The column temperature was controlled with a Polaratherm Series 9000 oven

(a) 18000

22000

5

25000

10

22000 15

20

20000 25

30

min

(b) 66000 71000 91000 81000 5

10

15

20

75000 25

30

35 min

FIGURE 16.4 Chromatograms comparing the separation efficiency and analysis times between a conventional and high resolution method with enhanced flow rate. Conditions for both analyses: Isocratic elution acetonitrile/water (40/60). (a) Column 25 cm  4.6 mm, Zorbax SB-C18, 5 mm, flow 1 ml/min, 30 C. (b) Column 4  25 cm  4.6 mm, Zorbax SB-C18, 5 mm, flow 2 ml/min, 80 C.

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(b)

(a)

FIGURE 16.5 Effect of temperature on chromatographic elution order. Gradient elution, mobile phase (a), 0.1% formic acid; mobile phase (b), acetonitrile. Gradient time 0 minutes, 10% B; time 35 minutes, 90% B. Column 15 cm  4.6 mm, XBridge Shield RP18, 5 mm. 1 ml/min.

FIGURE 16.6

Effect of temperature on elution order. Denotation and other conditions

as in Figure 16.5.

equipped with a mobile phase preheater (SelerityTechnologies, Salt Lake City, Utah). The preheater temperature was set equal to the oven temperature and the effluent temperature was controlled at a temperature close to ambient temperature (30 C). Particle Size It can be seen from a simplified height equivalent to a theoretical plate (HETP) as function of linear velocity of the mobile phase (u) curve (Heu plot) [8], HETP ¼ 2ldp þ

d2p 2gDm B þ fðkÞ u ¼ A þ þ Cu Dm u u

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439

FIGURE 16.7 Graph showing the loss in efficiency when operating an LC at high temperatures without a mobile phase preheater module.

that the first term (A), which describes the eddy diffusion, and the third term (C), which mainly describes the mass transfer resistance in the mobile phase, are both strongly dependent on the particle size of the column packing dp. Therefore, any reduction in particle size should result in a lower HETP and “flatter” Heu plots. This was experimentally verified by several authors, for example, [8]. There are at least two practical limitations to reducing the particle size, one being the pressure limit of the instrumentation and the other the thermal effects related to generation of frictional heat, as discussed in [9]. Despite the inevitable loss of efficiency observed due to the frictional heating, in excess of 70,000 theoretical plates were generated at temperatures close to ambient (40 C) when using five coupled 1.7-mm particle columns [8] (Figure 16.8). It was also reported that the maximum number of plates is strongly influenced by the properties of the analyte [10], and therefore, caution must be exercised when drawing conclusions on the theoretical limits in LC performance achievable for a particular class of compounds.

16.2.2. High Resolution Liquid Chromatography through Increased Selectivity An obvious disadvantage of achieving high resolution methodology through efficiency lies in the fact that chromatographic resolution is only

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FIGURE 16.8 High efficiency analysis of a test sample using five coupled Acquity 1.7-mm columns at near ambient temperature (40oC). Analysis conditions: Total column length, 450 mm; flow rate, 0.17 ml/min; (system) pressure, 999 bar; temperature, 40 C (Polaratherm); detection, 210 nm (20 Hz). Mobile phase: 30/70 acetonitrile/water. Peak identification: 1 ¼ uracil, 2 ¼ caffeine, 3 ¼ pyridine, 4 ¼ aniline, 5 ¼ phenol, 6 ¼ acetophenone, 7 ¼ benzene. Source: Reproduced with permission from Ó2006 Elsevier B.V.

proportional to the square root of the efficiency. In other words, to increase the resolution from 1 (partially resolved) to 2 (baseline resolved) requires a fourfold increase in efficiency. As can be seen from the previous section, this is not a trivial task. The retention factor also offers only a limited contribution to the resolution after the optimal k values have been established [2]. While changes in retention factors can be induced via influencing the physicochemical properties of analytes, such as LogD, ionization, hydrogen bonding, and so forth, chromatographic methods developed in such a way almost inevitably suffer from robustness issues. This is because close control of some method parameters, such as pH or ionic strength of the mobile phase, for example, can be subject to environmental factors (e.g., ambient temperature) but also variability in lab practices, which are difficult to control. By far, the most efficient and ultimately most robust way of achieving chromatographic resolution is through the specific selectivity offered by

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stationary phase chemistry. Today’s market is effectively flooded with hundreds of stationary phases, each offering subtly different interactions with markers used to assess selectivity and classify chromatographic columns [11]. Availability of such a large and diverse array of stationary phase chemistries is, in general terms, a positive situation, as it allows scientists to select combinations of phases, typically 2e5, that cover the most different separation mechanisms across the selectivity “map” of phases available at fixed conditions, hence, increasing the probability that all components of a mixture will be detected by at least one of the methods if utilized in a screening procedure. One way to select phases that are very different from each other, that is, offer the most orthogonal separation mechanisms is based on the concept of practical peak capacity [12]. Indeed, most pharmaceutical companies use some sort of column screen as an integral part of new product development, in order to develop an understanding of process related impurities or as a starting point for further method development [13]. In our laboratories, we developed a system capable of running four orthogonal RPLC methods in parallel, as schematically shown in Figure 16.9. The system consists of four independent chromatographic lines, each one running a different RPLC method. These four methods were carefully selected, based on maximal practical peak capacity [12], which takes into account method orthogonality as well as each individual method’s peak capacities. The mass spectrometer in combination with photodiode-array detection

Autosampler with 4 injection loops Pump 1

Column 1

DAD 1

Mobile phase 1

Pump 2

Column 2

TOF-MS

DAD 2

Mobile phase 2

MUXTM Pump 3

Column 3

DAD 3

Mobile phase 3

Pump 4

Column 4

DAD 4

Mobile phase 4

FIGURE 16.9 Schematic representation of the parallel orthogonal screening system employed in our labs.

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16. LIQUID CHROMATOGRAPHY IN THE PHARMACEUTICAL INDUSTRY Method 1

A

Main component B

Method 2

Main component A+B

Method 3

Main component

A B

Method 4 A

Main component B 5

10

15

20

25

30 Time (minutes)

FIGURE 16.10 Example output from the parallel orthogonal screening system. See the text for details.

(DAD) is used to assign tentative identities to all sample components based on automated software [14] that combines mass spectral information with UV responses. An example of the output is shown in Figure 16.10. It is obvious that, due to the difference in separation mechanism, the elution order for this sample mixture is changing, so each method provides unique additional information about the impurity profile. For example, the impurity labeled as A is well separated from everything else in the sample by methods 1, 3, and 4 but elutes under the main band in method 2. Similarly, the impurity labeled as B elutes under the main band in methods 2 and 3 but is only partially separated from the main band in method 1 and baseline separated from everything else in the sample by method 4. Our parallel system has been in operation since 2000, but with the advent of UHPLC, it is now becoming obsolete and gradually replaced by a sequential screening system that screens four stationary phases at different pH and mobile phase compositions. This system is described in more detail and with relevant examples in the next section. Orthogonal screening systems such as the one outlined here essentially serve dual purposes. In the early stages of drug development, these systems provide confidence that all impurities or degradation products, which are formed during the synthetic process or by purposefully degrading the drug substance, are detected. To understand how well such a system performs, we have randomly selected 30 compounds from our databases of retention times, and from these, we created all possible combinations of mixtures containing between 2 and

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443

FIGURE 16.11 The probability (%) of all components in a sample being “detectable” when using up to six orthogonal methods.

10 components. Assuming an average peak width, we then calculated two things: 1. The probability of being able to detect all components when using up to six orthogonal methods (i.e., resolution between all components is not the target here, instead detectability of each component is sought). The results of this simulation are shown in Figure 16.11. 2. The probability of one method successfully being able to baseline resolve all components in the mixture when using up to six orthogonal methods. The results of this simulation are shown in Figure 16.12. It can be concluded from the first simulation (Figure 16.11.) that ,when using the three most-orthogonal methods, there is a 90% probability that, if the number of components in the sample does not exceed seven, each mixture component will be detectable. This is acceptable for most of the early development samples; however, more complex samples require all six methods. Another conclusion can be drawn from this simplified simulation. Figure 16.12 shows the probability (%) of being able to achieve baseline resolution among all mixture components using at least one of the six orthogonal methods. It can be seen that, for those more complex mixtures with five or more components, the probability of success drops fairly rapidly, even if all six orthogonal methods are used. This demonstrates the second purpose for which these orthogonal screens

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FIGURE 16.12

Simulation showing the probability (%) of at least one method offering baseline resolution of all components in a sample mixture when employing up to six orthogonal methods.

can be used: They are suitable for identification of a starting point for a future method development or optimization, as is discussed in the next section.

16.3. CHROMATOGRAPHIC METHOD DEVELOPMENT: RPLC Chromatographic analysis represents a large proportion of the analytical testing carried out to support drug development activities as well as quality control in manufacturing. The chromatographic method life cycle typically consists of three steps: method development, method validation, and method application. This process is continuously applied in parallel with product development, as the changes in the synthetic process and formulation composition usually trigger the need for method development or optimization. Chromatographic method development is often carried out in an ad-hoc fashion, driven by personal preferences or past successes. Although ultimately successful, such an approach leads to a large volume of waste, where waste can be substituted by any combination of the following: waste of time, human, and hardware resources; unused data, solvents, and other chemicals potentially harmful to the

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environment. The method development process usually ends when the predefined resolution criteria are met and the method is validated in a one-parameter-at-a-time fashion. In this chapter, we describe an alternative approach to method development, which is based on systematic application of a predefined workflow in combination with software that enables extrapolation of a few carefully designed experiments and therefore allows users to select experimental conditions that provide the more robust solution. The systematic approach also enables building a knowledge base that can be used to reduce the complexity of future method development challenges.

16.3.1. Required Method Performance Every method design and development process should start with a clear definition and understanding of the requirements for method performance, that is, what is the minimal tolerable resolution; what accuracy, precision, and sensitivity must be achieved; as well as a clear understanding of the attribute to be measured and the scope and context of the intended method application. For example, the target performance criteria for a method to measure trace level impurities versus an assay or identification method requires different performance criteria as does the intended purpose of the methoddthat is, whether it is to be used as an inprocess control during manufacturing or as a finished product release method. Equally, whether the sample matrix is a formulated drug product, drug substance, or biological fluid also affects the method development process focus areas. Although it is not always possible, the chemical identity of all sample components requiring qualitative or quantitative measurement is useful information. The next stage of the process is to assess the most appropriate measurement technique to meet the performance requirements while satisfying business demands such as cost and time efficiency, ease of use, and requirements for sample preparation. Once a suitable measurement technique has been selected, a process of method development must be initiated. Within our laboratories, for RPLC, we employ a screening process to get to the most robust separation conditions in the shortest amount of time. The following details each step of the screening process.

16.3.2. Selection of the Stationary Phase, pH, and Organic Solvent With the ultimate objective of maximum method robustness and ruggedness in mind, selection of the appropriate stationary phase is

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probably the most critical factor in chromatographic method development. Identification of the stationary phase that provides optimal selectivity for all components that need to be separated can be a lengthy and complex task. The complexity originates from the fact that the current market is flooded with profuse numbers of stationary phases from a multitude of suppliers, many of which offer only marginal differences in selectivity. To the best of our knowledge, there is no reliable knowledge based system to guide analytical chemists in making an informed decision, so the selection of the stationary phase is often, at best, based on personal preferences and experience or, at worst, based on what columns are lurking in the analytical laboratory cupboards and drawers. The vast majority of pharmaceutical compounds are ionic in character (acids or bases) and therefore selection of an appropriate mobile phase pH is also extremely important. It is a strong conviction of the authors of this contribution that mobile phase pH should be selected based on the physicochemical properties of the compounds that require separation rather than used as an optimization parameter. This is because selectivity differences induced through optimization of pH are often susceptible to human errors (adjustment of the pH of the stationary phase) or environmental factors, such as laboratory temperature. The pH dependence of the octanolewater distribution coefficient (LogD) can be used as a suitable approximation of how chromatographic retention is affected by changes in pH. LogD values can be calculated, for example, using the ACD Labs software (ACD/LC Simulator, Advanced Chemistry Developments Inc.) as shown in Figure 16.13. In this figure, the slopes and multiple crossovers of log P vs. pH lines close to neutral pH indicates a strong susceptibility of chromatographic retention and resolution to small changes in pH. On the other hand, the small plateau area around pH 10 suggests this is a potentially acceptable and robust mobile phase pH area to yield good resolution and relative independence of retention and resolution from small changes in pH. To rationalize the selection of stationary phases and limit the choice, nearly all pharmaceutical companies have developed screening procedures that include three to five stationary phases, at least three pHs, and also different types of organic modifiers, which also affect selectivity [13]. Table 16.1 shows the conditions applied in method development screens in our laboratories. Results obtained from these screens are evaluated together with the predicted LogD values; and the combination of stationary phase, mobile phase pH, and organic modifier that provide the best overall resolution and peak shape are selected for the next experiment, in which the temperature and gradient elution profile are optimized. On some occasions, especially when the complexity of the sample is high, this first screening step does not provide satisfactory results.

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FIGURE 16.13

Example plot of log D versus mobile phase pH for a pharmaceutical sample comprising 13 components.

In those cases, secondary column screens or alternative elution modes (SFC, HILIC) are applied.

16.3.3. Optimization of Temperature and Gradient The stationary phase, organic modifier, and pH selected from the first screening sequence are taken into a second screening sequence, wherein temperature and gradient elution (mobile phase strength) are optimized. This sequence comprises six experiments, examples of which are shown in Table 16.2. Three temperatures (low, medium, and high, e.g., 30 C, 48 C, and 60 C) are applied in combination with two linear gradients, differing in gradient slope (e.g., 45 minutes and 15 minutes). Chromatograms resulting from this screening sequence are then imported into software (e.g., ACD Labs LC Simulator), which perform a linear extrapolation from the two experimental points of the gradient elution data and a nonlinear extrapolation from the three experimental points of the temperature data. The output is displayed in a resolution map format (Figure 16.14), allowing analysts to optimize temperature and gradient elution profile in silico while maintaining the required resolution of all components in the sample. This approach also enables analysts to select suitable temperature and gradient profiles, which also ensure maximum

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TABLE 16.1 Screening Experimental Conditions Used for the First Sequence: Initial Selection of Stationary Phase, pH, and Organic Modifier Stationary phase

Organic modifier

Aqueous buffer/pH

Waters Acquity BEH C18 1.7 mm dp

Acetonitrile

0.1% v/v Formic acid, pH ¼ 3.0 10 mM Ammonium acetate, pH ¼ 6.5 0.1% v/v Ammonium hydroxide, pH ¼ 10.0

Methanol

0.1% v/v Formic acid, pH ¼ 3.0 10 mM Ammonium acetate, pH ¼ 6.5 0.1% v/v Ammonium hydroxide, pH ¼ 10.0

Waters Acquity BEH HSS T3 1.8 mm dp

Acetonitrile

0.1% v/v Formic acid, pH ¼ 3.0 10 mM Ammonium acetate, pH ¼ 6.5 0.1% v/v Ammonium hydroxide, pH ¼ 10.0

Waters Acquity BEH Shield RP18 1.7 mm dp

Acetonitrile

0.1% v/v Formic acid, pH ¼ 3.0 10 mM Ammonium acetate, pH ¼ 6.5 0.1% v/v Ammonium hydroxide, pH ¼ 10.0

Waters Acquity BEH Phenyl 1.7 mm dp

Acetonitrile

0.1% v/v Formic acid, pH ¼ 3.0 10 mM Ammonium acetate, pH ¼ 6.5 0.1% v/v Ammonium hydroxide, pH ¼ 10.0

Methanol

0.1% v/v Formic acid, pH ¼ 3.0 10 mM Ammonium acetate, pH ¼ 6.5 0.1% v/v Ammonium hydroxide, pH ¼ 10.0

Note: Gradient: time 0 min - organic content 5%, time 20 min ¼ organic content 95%, Flow rate 0.4 ml/min. All columns 2.1  100 mm.

method robustness and ruggedness, that is, where overall resolution of all components remain adequate with respect to changes in temperature and organic modifier content of the mobile phase.

16.3.4. Optimization of the Mobile Phase pH To ensure that, after optimization of the temperature and content of the organic modifier, the pH selected in the first screening sequence is

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TABLE 16.2 Example of the Second Screening Sequence Used to Optimize the Temperature and Gradient Elution Experiment

Temperature 

Gradient profile

1

Low (e.g., 30 C)

Time ¼ 0 min; organic content ¼ 5% Time ¼ 15 min; organic content ¼ 95%

2

Low (e.g., 30 C)

Time ¼ 0 min; organic content ¼ 5% Time ¼ 45 min; organic content ¼ 95%

3

Medium (e.g., 48 C)

Time ¼ 0 min; organic content ¼ 5% Time ¼ 15 min; organic content ¼ 95%

4

Medium (e.g., 48 C)

Time ¼ 0 min; organic content ¼ 5% Time ¼ 45 min; organic content ¼ 95%

5

High (e.g., 65 C)

Time ¼ 0 min; organic content ¼ 5% Time ¼ 15 min; organic content ¼ 95%

6

High (e.g., 65 C)

Time ¼ 0 min; organic content ¼ 5% Time ¼ 45 min; organic content ¼ 95%

Note: The stationary phase and mobile phase pH were selected from the first screening.

still suitable, that is,the overall resolution is not strongly affected by minor changes in pH, we recommend performing an additional screening experiment, in which the selected pH is changed, typically within 1.0 pH unit. This is done in five experiments (þ1.0, þ0.5, 0, e0.5, e1.0 pH units, a narrower range is also possible), and the optimal pH is selected, as shown in Figure 16.15. Figure 16.16 shows the agreement between the simulated chromatogram and the “real” chromatogram obtained at optimal conditions. selected using the described method development workflow. Once a suitable and robust set of separation method conditions have been achieved through the steps outlined already, a full assessment of the entire method must ensue. This involves taking the method and mapping it out as a process, starting at sampling through sample preparation to chromatographic method, detection and reporting of data, with each step defined as a “focus area.” Each focus area can then be examined systematically with respect to the potential variables within that area that could contribute to the method’s success or failure. Each potential variable can be scored and classified as being “controllableefixed” and not requiring experimentation to optimize or, alternatively, as potential “optimizable” variables. The most highly ranking variables (those with the highest risk of causing method failure) in this exercise are ideally investigated through one-factor-at-a-time or multivariate experimental design experiments, so that a depth of understanding is achieved around the impact of each variable and any interdependent relationships. This exercise also should yield a set of method ranges across which the method

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95 90

Column Temperature, °C

85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 8

16

24

32

40

48

56

64

72

80

88

96

4.35 4.17 3.99 3.81 3.62 3.44 3.26 3.08 2.90 2.72 2.54 2.36 2.17 1.99 1.81 1.63 1.45 1.27 1.09 0.91 0.72 0.54 0.36 0.18 0.00

Solvent B, % 15 15 15

240000 220000 200000 180000

777

160000

555

140000

333

444 888

120000 222

100000

10 10 10

80000

14 13 13 14 13

999

60000

666 12 12 12

40000 20000

111 1

2

3

4

5

6

7

8

9

10

11 min

FIGURE 16.14

Example output of the optimization of the temperature and gradient profile. Top: Resolution map showing the most robust method conditions (dark). Bottom: Predicted chromatogram.

has been demonstrated to be robust; for example, for sample preparation conditions, ranges may include pH of diluents or sample extraction time, while for the chromatographic separation, step ranges may include gradient step time ranges, pH, temperature, injection volume, or mobile phase buffer ionic strength. Once the ranges have been defined, it is important to assess whether the predefined method performance criteria, especially relating to accuracy and precision, are met over the total method range surface. This final evaluation of the method is typically

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FIGURE 16.15 Example plot of chromatographic resolution versus pH; selection of optimal mobile phase pH after temperature and gradient profile optimization.

referred to as method verification. If any of these method verification experiments prove the method to be out of compliance with the predefined method performance criteria, then the method ranges must be reset (tightened) and the new ranges verified. At this stage, method validation at the intended method set point should be performed and is typically done so using ICH (International Conference on Harmonization) Q2(R1) guidelines [15]. If the preceding process has been adhered to, then there should be an extremely low risk that ICH method validation will fail. Further, the depth of knowledge and understanding around the method is an aid to method transfer activities and should ultimately facilitate long term reliability of the method in routine use in a manufacturing environment. Finally, it is noteworthy that the long term reliability and transferability of a method is usually directly proportional to the level of understanding around the method and how this understanding is captured and communicated to the receiving laboratory. We cannot stress how important it is to appropriately capture the knowledge around the rationale for selection of each method parameter, as well as being specific in the details around seemingly fundamental method details. We have found it particularly valuable to perform method walk-troughs with our receiving laboratories and, based on the conversations about each step of the method process, agree on the exact wording in the method descriptions.

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FIGURE 16.16 Agreement between the predicted and experimentally verified chromatogram obtained at optimal experimental conditions. Top: Predicted chromatogram, Bottom: Experimentally obtained chromatogram.

If careful attention is paid to these basic fundamentals, then the frequency of method transfer failures throughout the life cycle of the method can be reduced.

Acknowledgments The authors express their gratitude to colleagues at the Pfizer Analytical Research Centre, University of Ghent in Belgium, especially Prof. Pat Sandra and Prof. Frederic Lynen for their invaluable input. We also acknowledge the input of many colleagues at Pfizer Analytical R&D in Sandwich, UK, and Groton, USA, for their input into developing the method development workflow and for providing examples.

REFERENCES

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References [1] PhRMA pharmaceutical industry profile. Increased length and complexity of the http://www. research and development process. Available at, politicsandthelifesciences.org/Biosecurity_course_folder/readings/phrma.pdf, 2003 (accessed 28.02.11). [2] Schoenmakers PJ. Optimisation of chromatographic selectivity, Chapter 1. Amsterdam: Elsevier; 1986. [3] Lestremau F, Cooper A, Szucs R, David F, Sandra P. High-efficiency liquid chromatography on conventional columns and instrumentation by using temperature as a variable, I. Experiments with 25 cm  4.6 mm I.D., 5 mm ODS columns. J Chromatogr A 2006;1109:191e6. [4] Lestremau F, de Viliers A, Lynen F, Cooper A, Szucs R, Sandra P. High efficiency liquid chromatography on conventional columns and instrumentation by using temperature as a variable. Kinetic plots and experimental verification. J Chromatogr A 2007;1138: 120e31. [5] Giddings JC. Maximum number of components resolvable by gel filtration and other elution chromatographic methods. Anal Chem 1967;39:1027e8. [6] Davis JM, Giddings JC. Statistical theory of component overlap in multicomponent chromatograms. Anal Chem 1983;55:418. [7] VanHoenacker G, Sandra P. High temperature liquid chromatography and liquid chromatography-mass spectroscopy analysis of octylphenol ethoxylates on different stationary phases. J Chromatogr A 2005;1082(2):193e202. [8] de Villiers A, Lestremau F, Szucs R, Gelebart S, David F, Sandra P. Evaluation of ultra performance liquid chromatography, part I. Possibilities and limitations. J Chromatogr A 2006;1127:60e9. [9] de Villiers A, Lauer H, Szucs R, Goodall S, Sandra P. Influence of frictional heating on temperature gradients in ultra-high-pressure liquid chromatography on 2.1 mm I.D. columns. J Chromatogra A 2006;1113:84e91. [10] de Villiers A, Lynen F, Sandra P. Effect of analyte properties on the kinetic performance of liquid chromatographic separations. J Chromatogr A 2009;1216:3431e42. [11] Lesellier E, West C. Description and comparison of chromatographic tests and chemometric methods for packed column classification. J Chromatogr A 2007;1158:320e60. [12] Liu Z, Patterson Jr DG, Lee ML. Geometric approach to factor analysis for the estimation of orthogonality and practical peak capacity in comprehensive two-dimensional separations. Anal Chem 1995;67:3840e5. [13] Dumaret M, Sneyers R, Janssens W, Somers I, VanderHeyden Y. Drug impurity profiling: method optimization on dissimilar chromatographic systems, part I: pH optimization of the aqueous phase. Anal Chim Acta 2009;656:85e92. [14] Xue G, Bendick AD, Chen R, Sekulic SS. Automated peak tracking for comprehensive impurity profiling in orthogonal liquid chromatographic separation using mass spectrometric detection. J Chromatogr A 2004;1050:159e71. [15] ICH, Q2(R1). Validation of analytical procedures: Text and methodology. Available at, www.ich.org/products/guidelines/quality/article/quality-guidelines.html, 1997 (accessed 10.08.12).

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C H A P T E R

17 Determination of Veterinary Drug Residues in Foods by Liquid ChromatographyeMass Spectrometry: Basic and Cutting-Edge Applications M.D. Marazuela*, S. Bogialliy *

Department of Analytical Chemistry, Faculty of Chemistry, Universidad Complutense de Madrid, Spain y Department of Chemistry, University of Padua, Padova, Italy O U T L I N E 17.1. Introduction 17.1.1. Veterinary Residues 17.1.2. Regulatory Aspects in the European Union

456 456 457

17.2. Options in Veterinary Residue Analysis using LCeMS 17.2.1. Sample Preparation Issues 17.2.2. LCeTandem Mass Spectrometry (LCeMS/MS) 17.2.3. LCeHigh Resolution Mass Spectrometry

459 460 461 467

17.3. Conclusions

471

References

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Liquid Chromatography: Applications http://dx.doi.org/10.1016/B978-0-12-415806-1.00017-6

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Copyright Ó 2013 Elsevier Inc. All rights reserved.

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17.1. INTRODUCTION 17.1.1. Veterinary Residues Veterinary drugs are a broad group of pharmaceuticals exhibiting many different chemical and therapeutic properties [1]. The use of veterinary medicinal products in food-producing animals may leave residues of the parent compounds, their metabolites, or conjugates in food products derived from treated animals. The presence of veterinary drug residues in food continues to occupy a high position in European consumer’s food concern rankings, as a potential health risk [1,2]. For instance, there is a growing concern to the increased cases of allergies and antimicrobial-resistant microorganisms through excessive use of veterinary antibiotics. Other drugs (e.g., diethylstilbestrol, nitrofurans, and chloramphenicol) have been banned, due to their demonstrated carcinogenicity. International regulatory bodies, such as the Food and Drug Administration (FDA) in the United States and the European Union (EU) have established strict regulations and controls of the use of veterinary drugs to guarantee consumer protection. Therefore, European member states are obliged to draft national residue monitoring plans to evaluate the level of veterinary drug residues in animalderived foods. The last report published by the European Food Safety Authority (EFSA) referring to residue control in the European Union, indicated that 0.32% of the targeted samples were noncompliant for antibacterials, anabolic agents, and prohibited substances, among other veterinary drugs [3]. The correct implementation of surveillance and residue monitoring programs involves the development of robust and sensitive analytical methods to provide control authorities with effective tools. Since veterinary drugs are generally quite polar and thermolabile compounds, liquid chromatography coupled to mass spectrometry (LCeMS) has become the predominant methodology for determination of these substances in foods [4]. Nowadays, two major trends are observed in the field of veterinary drug residue analysis [5e9]: (a) “new” matrices are gaining interest for the detection of banned substances (e.g., hair, thyroid, retina) and authorized drugs (e.g., eggs, honey, fish and animal feed); (b) methods for residue analysis are moving from targeted analytical approaches that detect and quantify residues of the administered compounds or their metabolites toward MS based screening methods that allow, in a single analysis, the detection of target residue, as well as retrospective analysis for the identification of unknowns.

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17.1.2. Regulatory Aspects in the European Union According to Council Directive 96/23/EC, the EU member states have to adopt and implement national residue monitoring plans for specific groups of residues [10]. Two main groups of pharmacologically active substances must be monitored, as summarized in Table 17.1 [10,11]: • Group A comprises forbidden substances (~83 in 2010), such as hormones and b-agonists, among others. TABLE 17.1 Banned and Authorized Veterinary Drugs in Food-Producing Animals GROUP A. BANNED SUBSTANCES A.1. Hormones Stilbenes and derivatives Thyreostats Steroids Resorcylic acid lactones including zeranol A.2. b-agonists A.3. Others Nitrofurans (including furazolidone) Dimetridazole Metronidazole Ronidazole Chloramphenicol Dapsone Chlorpromazine GROUP B. LICENSED VETERINARY DRUGS B.1. Antibacterials b-Lactams Tetracyclines Macrolides Aminoglycosides Sulfonamides and trimethropim Quinolones B.2. Other veterinary drugs Anthelmintics Anticoccidiostats, including nitroimidazoles Carbamates and pyrethroids Carbodox and olaquindox Sedatives Nonsteroidal anti-inflamatory drugs (NSAIDs) Other pharmacologically active substances

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• Group B refers to authorized veterinary drugs for which maximum residue limits (MRLs) have been fixed in different animal products and animal species and other contaminants. Commission Decision 2002/657/EC [12] defines the performance criteria for analytical residue methods and is probably the key document of legislation to be consulted by residue control laboratories. This reference document introduces the concept of the minimum required performance limit (MRPL) for compounds belonging to Group A; it corresponds to the minimum content of an analyte in a sample that has to be detected and confirmed, and it constitutes the lowest level that can be reliably considered “nonzero.” The decision limit (CCa) and detection capability (CCb) replaced the formerly used limits of detection (LODs) and quantification (LOQs) [13]. CCa is defined as “the concentration at and above which it can be concluded with an error probability of a that a sample is non-compliant (positive).” Thus, a reflects the rate of false positive results. CCb is defined as “the smallest content of the substance that may be detected, identified and/or quantified in a sample with an error probability of b” which therefore, reveals a false negative result. Decision 2002/657/EC introduces criteria for confirmatory analysis, based on the so called identification points (IPs). Then, for confirmation of Group A substances, a minimum of four IPs are required, whereas for compounds listed in Group B the minimum number of IPs is set to three for a satisfactory confirmation of a compounds’ identity. Although the cited document still accepts detection techniques like diode-array (DAD) and fluorimetric detection (FLD) as possible confirmatory techniques, the confirmation of veterinary residues in food is performed nowadays by LC coupled to different MS detection systems. The system of IPs relies on the identification power of the different mass analyzers. For instance, a low resolution mass spectrometer (e.g., triple quadrupole, QqQ or ion trap IT), provides 1.0 IP for the precursor ion and 1.5 IPs for each product ion. By contrast, high resolution mass spectrometers (HRMS; resolution 20,000 fwhm, full width at half maximum) provide 2.0 IPs for the precursor ion and 2.5 IPs for each product ion, which means that (2 þ 2.5n) IPs can be acquired when working in the product ion scan mode. In addition to IPs, the retention time of the suspected peak has to correspond to the measured retention time of the relative standard, and the area ratio between the selected ion traces has to be equal in the sample and in the standard [12]. The current Decision 2002/657/EC, however, has not been revised since its publication. The evolution in analytical instrumentation, in particular, the introduction of fast chromatography and HRMS analyzers, accompanied by recent European regulatory changes demands an update

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of the current criteria for identification in residue analysis [14]. Additional and more detailed criteria for the use of mass accuracy and mass resolution have to be implemented to define clearly the requirements for a reliable confirmation with LCeHRMS technologies.

17.2. OPTIONS IN VETERINARY RESIDUE ANALYSIS USING LCeMS A detail discussion on LCeMS techniques would be outside the scope of the present chapter; therefore we focus on the possibilities and limitations of the current LCeMS methods applied to veterinary residue analysis. Hence, LCeMS methods for monitoring veterinary residues in food samples can be split into two categories: (a) quantitative confirmatory methods for target compounds and (b) screening methods for the identification of target, nontarget, or unknown compounds (Figure 17.1).

FIGURE 17.1 LCeMS based analytical strategies for the determination of veterinary drug residues in food. Acronyms: LCeMS/MS: liquid chromatographyetandem mass spectrometry; LCeMSn: liquid chromatographyemultidimensional mass spectrometry; QqQ: triple quadrupole; MRM: multiple reaction monitoring; IT: ion-trap analyzer; LIT: linear ion trap; LCeHRMS: liquid chromatographyehigh-resolution mass spectrometry; TOF: time of flight; QTOF: quadrupoleetime of flight.

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17.2.1. Sample Preparation Issues In residue analysis, sample preparation is often the bottleneck [15]. Several reviews devoted to this topic have been published in recent years [16e18]. Although the high selectivity of the MS systems can overcome problems related to the presence of interfering compounds extracted from such complex matrices, nevertheless, qualitative and quantitative analysis are severely affected by the unwelcome “matrix effect” frequently occurring when using electrospray (ESI)-MS sources. Therefore, the complexity of the food matrices and the legal residue limits (on the order of ng g 1), generally impose adequate sample preparation before LCeMS determination. Typical sample preparation steps include homogenization, extraction (liquideliquid extraction, LLE, or instrumental based techniques), cleanup (usually by solid-phase extraction, SPE), and concentration of extracts. Sometimes, derivatization has to be incorporated into sample preparation (e.g., release of bound residues or deconjugation). For quantitative analysis, the preparation of adequate calibration standards also may be a key aspect: in some cases, matrix-matched standards or the standard additions method may be necessary, as well as the use of suitable internal standards (e.g., isotopically labeled compounds) [19]. Matrix-matched calibration is now preferred, as it is the best compromise in terms of speed and cost of analysis, taking into consideration the features of the MS analyzers. However, current trends in sample preparation for veterinary residues have evolved toward (a) reduction or elimination of organic solvents, (b) process automation, and (c) more generic extraction procedures for multiclass analysis. Thus, protein precipitation followed by ultrafiltration or SPE with polymeric cartridges (e.g., HLB), “dilute-and-shoot,” or QuEChERS methodologies [17,20] are being used. Briefly, “dilute and shoot” is the simplest preparation strategy when designing multiclass methods [21,22]. Although it allows high sample throughput, dilution of “crude” extracts can reduce matrix effects to a certain degree, which might cause MS source contamination problems and lower sensitivity. On the other hand, the QuEChERS (quick, easy, cheap, effective, rugged, and safe) method, originally developed for pesticide analysis [23], has been also applied to the determination of multiclass veterinary drugs in different food commodities [24e27]. The conventional QuEChERS strategy applies acetonitrile extraction, followed by removal of water and proteins by salting out with sodium chloride and magnesium sulphate. Afterwards, dispersive SPE (deSPE) is usually applied. The QuEChERS approach has many advantages, as it is simple and quick, reduces the

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amount of organic solvents, and provides moderate to high recoveries of analytes covering a wide polarity range. However, although these sample preparation procedures for multiclass determination offer many advantages, such as simplicity, high sample throughput, and reduced costs, compromises have to be made when developing analytical methods (e.g., lack of sensitivity or accuracy, significant matrix effects due to the absence of cleanup, or higher instrumentation maintenance, among others).

17.2.2. LCeTandem Mass Spectrometry (LCeMS/MS) Basic Principles Conventional reversed-phase LC has been the most common choice in veterinary drug analysis. However, some veterinary drugs exhibit properties that make difficult their separation with traditional C18 columns. That is the case of many antibacterials (e.g., tetracyclines, aminoglycosides, macrolides, and fluoroquinolones) that either form chelates with metallic column impurities form chelates on the column with metallic impurities or uncovered silanolic residues or are poorly retained on the C18 columns because of their high polarity (e.g., aminoglycosides). In other situations, the separation of isomeric and tautomeric forms (e.g., tetracyclines and glucocorticoids) is possible by a careful optimization of the chromatographic conditions. These specific problems have been partly solved with the use of high purity columns, column washing with chelating agents [28], addition of ion-pairing agents to the mobile phase, and alternative stationary phases, like hydrophilic interaction liquid chromatography (HILIC) [29] or porous graphitized carbon (PGC) columns [30]. The ongoing development of LC has opened new strategies for veterinary drug analysis. For instance, replacing conventional LC with ultraperformance liquid chromatography (UHPLC), using sub-2-mm particles, has led to shorter analysis times (typically from ~20e40 min to ~5e10 min) and increased resolution and sensitivity, although the chromatographic separation has to be optimized, because sometimes, the fast gradient used in UHPLC promotes matrix effects [31]. Coupling LC with tandem mass analyzers (typically, triple quadrupole) has become, so far, the most widely used technique for confirming and quantifying veterinary residues in food samples. QqQ is the workhorse in target compound analysis, because it displays outstanding capabilities in terms of sensitivity, selectivity, and linear range, when working in a multireaction monitoring mode (MRM), by selecting a precursor ion to product ion transitions [32,33]. According to the IPs criteria discussed in Section 17.1.2, confirmation of a regulated compound is assured by

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selection of at least two MRM transitions (in the correct ion ratios and with a signal-to-noise ratio S/N > 3). Generally, the transition with the worst S/N is used for confirmation purpose, while quantitative analysis is carried out with the best one, to increase the precision of the results. The major limitation of QqQ working in the MRM mode is that a complete mass spectrum cannot be obtained, so it is difficult to detect the presence of metabolites or nontarget compounds. This concern is important when metabolites are explicitly included in the list of the regulated substances, (e.g., benzoimidazoles, carprofen, ceftiofur, and florfenicol) [11]. Conversely, the classical tridimensional ion trap analyzer has been used for this purpose [34], because it provides high sensitivity in full-scan mode and has the ability to perform multiple-stage fragmentations (MSn), but it does not have adequate sensitivity and reliability to perform quantitative trace analysis. On the other hand, the new fast-switching QqQ analyzers enable up to hundreds of time-scheduled specific MRM transitions to be conducted in a single LCeMS/MS run, which allows multiresidue determinations. However, optimization of acquisition parameters (e.g., selection of the precursor ion, collision energy, and product ion) is required for every single compound, resulting in time consuming method setup. Tandem MS is not limited to combining a single type of mass analyzers. Thus, hybrid systems, such as quadrupoleeion trap (QIT) or quadrupolee linear ion trap (QLIT) can partially overcome the limitations of the QqQ and IT [35,36]. However, the widespread use of QqQ is principally due to its easier operating performance, better robustness for routine analysis, and relatively low cost [32,33]. Applications Several hundreds of papers have been published on target analysis of veterinary drugs in food using LCeMS/MS. Table 17.2 shows some representative examples with their main features and drawbacks. These procedures result in steps appropriate for confirmatory analysis that follows a positive screening assay, indicating at least the class of drugs tested. The sample preparation protocols, as well as the experimental LCeMS conditions have to be adapted for each class of veterinary drugs. The traditional approach combines LLE with aqueous [37e39] or organic solutions [40,41], followed by off-line SPE cleanup using C18 [41], hydrophilic-lipophilic balance (HLB) [37], silica [40], NH2 [40], or polymeric-sulfonate [42] sorbents. Several LCeMS/MS methods are considered advanced or innovative procedures, because they are partially or fully automated [43e47], multiclass [48,49], or employ easy sample preparation [24,25,48,50e52], UHPLC [49,53e55], or alternative LC columns, like HILIC [56,57] or PGC

TABLE 17.2 Summary of Representative LCeMS/MS Methods for the Determination of Target Veterinary Drug Residues in Food Samples Tandem MS system

QuEChERS extraction with acidified ACNeEDTA þ MgSO4 and NaAc

C18 (UHPLC)

Animal tissues

QuEChERS extraction with acidified ACN þ Na2SO4, followed by deSPE using an NH2 sorbent. Additional strong cation-exchange cleanup was required for determination of nitroimidazoles

38 drugs Antibacterials, malachite green, imidazoles

Fish tissues

Monensin

Milk

Matrix

Sample preparation

Ref.

18 drugs Antibacterials, anthelmintics

Milk

QqQ

Fast analysis (100 ppb). Not suitable for European tolerance levels.

[34]

QuEChERS extraction with acidified ACNeEDTA þ MgSO4

C18 (UHPLC)

QqQ and QLIT

QqQ is one order of magnitude more sensitive than QLIT, less dependent on matrix effects and more accurate.

[35]

(Continued)

463

Comments

17.2. OPTIONS IN VETERINARY RESIDUE ANALYSIS USING LCeMS

Stationary phase

Compounds

TABLE 17.2 Summary of Representative LCeMS/MS Methods for the Determination of Target Veterinary Drug Residues in Food Samplesdcont’d

Dilution in phosphate buffer. Extraction with ACN followed by deproteinization with TCA and three subsequent LLE steps adopted for the different classes of drugs

C18

Animal tissues

Enzymatic digestion, extraction with methanol and cleanup with hexane and SPE combining C18 and NH2 sorbents

10 drugs b-lactams

Milk

8 drugs Glucocorticoids

Animal tissues

Sample preparation

Comments

Ref.

42 drugs Antibacterials

Honey

QLIT

Rather complex sample preparation. Relatively poor precision.

[36]

22 drugs Anabolic steroids

C18

QqQ

Extensive sample preparation. Quantitative analysis failed because of a limited linearity in matrix.

[40]

Centrifugation and further cleanup with on-line SPE C18

C12

QqQ

Automatic sample preparation. LOD lower than 1.5 ng ml-1.

[45]

PLE extraction with hexaneeethyl acetate (1:1, v/v) at 50 C and 1000 psi

High-purity C18

QqQ

Fast sample preparation without cleanup. Separation of the two isomers betamethasone and dexamethasone Performance fit-forpurpose for low concentrations of corticosteroids.

[46]

17. DETERMINATION OF VETERINARY DRUG RESIDUES IN FOODS

Tandem MS system

Matrix

464

Stationary phase

Compounds

PHWE extraction at 70 C and 1500 psi,

C18

QqQ

Fast sample preparation without cleanup. Environmentally friendly method.

[47]

17 drugs Antibacterials

Honey

Extraction with aqueous EDTA under mild acidic conditions (pH 4.0) followed by cleanup with HLB cartridges

C18 (UHPLC)

QqQ

Fast chromatographic run 100 drugs All relevant families

Muscle, kidney, liver, fish, and honey

Deproteinization with ACNeEDTAeammonium sulphateesuccinic buffer. Centrifugation, evaporation, dilution, and cleanup with Evolute ABN cartridges

UHPLCeOrbitrap

Multiresidue and multimatrix method. Superior analytical performance than a previous TOF-based method

[73]

Acronyms: QTOF: quadrupole time-of-flight; TOF: time-of-flight; UHPLC: ultraperformance liquid chromatography; NSAIDs: nonsteroidal anti-inflammatory agents; MRL: Maximum residue limit.

17. DETERMINATION OF VETERINARY DRUG RESIDUES IN FOODS

Compounds

17.3. CONCLUSIONS

471

In another work [73], the better analytical performance of an UHPLCeOrbitrap method over a previously published TOF-based method is demonstrated [69] for the determination of more than 100 veterinary drugs in difficult matrices (muscle, kidney, liver, fish, and honey). These improvements are attributed to the combination of a more effective sample preparation with the higher resolution (50,000 versus 12,000 fwhm) and superior mass stability of the Orbitrap analyzer over the previously used TOF instrument.

17.3. CONCLUSIONS Over the past decade, LCeMS methods for the control of veterinary residues in foods have changed significantly. Two types of approaches can be clearly distinguished: target oriented procedures (mainly based on LCeQqQ) and screening procedures (based on LC coupled to accurate mass full-scan detectors, such as TOF, QTOF, and Orbitrap). A traditional scheme involving off-line extraction and cleanup, separation on a C18 column, and determination with QqQeMS in MRM mode can cover almost all confirmatory target analysis of regulated veterinary drugs according to EU specifications. Former methods devoted to the analysis of specific classes of drugs are being progressively replaced by multiresidue screening procedures. Regarding sample preparation, generic methods for the simultaneous extraction of various classes of veterinary drugs are preferred over more specific extraction procedures. The development of such advanced methods is rather time consuming and difficult, because a number of parameters have to be carefully optimized to obtain efficient processes in the extraction, purification, separation, and determination of a large variety of compounds. Nevertheless, the resulting protocols are very useful for laboratories devoted to routine food safety control that have to analyze a huge amount of samples. Whereas LCeMS/MS is an ideal tool if a clearly defined set of analytes has to be confirmed and quantified in a given sample, HRMS analyzers offer the highest degree of certainty in analyte identification (of both target and unknown compounds) as well as real multiresidue capabilities, even with retrospective analysis. However, some limitations, such as the lack of identification libraries, need to be addressed to allow fully functionality of this technology. In summary, LCeMS/MS and high resolution LCeMS are considered powerful, complementary techniques that can cover the majority of current challenges related to the determination of veterinary residues in animal-derived foods.

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References [1] Turnipseed SB, Andersen WC. Veterinary drug residues. Comp Anal Chem 2008;51: 307e38. [2] Verbeke W, Frewer LJ, Scholderer J, De Brabander HF. Why consumers behave as they do with respect to food safety and risk information. Anal Chim Acta 2007;586:2e7. [3] European Food Safety Authority. Report for 2009 on the results from the monitoring medicinal product residues and other substances in live animals and animal products. Supporting Publications 2011;158:1e70. [4] De Brabander HF, Noppe H, Verheyden K, Vanden Bussche J, Wille K, Okerman L, et al. Residue analysis: future trends from a historical perspective. J Chromatogr A 2009;1216:7964e76. [5] Stolker AAM, Zuidema T, Nielen MWF. Residue analysis of veterinary drugs and growth promoting agents. Trends Anal Chem 2007;26(10):967e79. [6] Regal P, Nebot C, Va´zquez BI, Cepeda A, Fente CA. Determination of the hormonal growth promoter 17a-methyltestosterone in food-producing animals: bovine hair analysis by HPLCeMS/MS. Meat Scie 2010;84:196e201. [7] Grataco´s-Cubarsı´ M, Castellari M, Valero A, Garcı´a-Regueiro JA. Hair analysis for veterinary drug monitoring in livestock production. J Chromatogr B 2006;834:14e25. [8] Boscher A, Guignard C, Pellet T, Hoffman L, Bohn T. Development of a multi-class method for the quantification of veterinary drug residues in feedingstuffs by liquid chromatographyetandem mass spectrometry. J Chromatogr A 2010;1217:6394e404. [9] Peters RJB, Stolker AAM, Mol JGJ, Lommen A, Lyris E, Angelis Y, et al. Screening in veterinary drug analysis and sports doping control based on full-scan, accurate-mass spectrometry. Trends Anal Chem 2011;29(11):1250e68. [10] Council Directive 96/23/EC, on measures to monitor certain substances and residues thereof in live animals and products. Off J Eur Comm 1990;L 125:10e32. [11] Commission Regulation 37/2010/EC of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin. Off J Eur Union 2010;L15:1e72. [12] Commission Decision 2002/657/EC of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. Off J Eur Union 2002;L221:8e35. [13] Kaufmann A. Validation of multiresidue methods for veterinary drug residues; related problems and possible solutions. Anal Chim Acta 2009;637(1e2):144e55. [14] Vanhaecke L, Gowik P, Le Bizec B, Van Ginkel L, Bichon E, Blokland M, et al. European analytical criteria: past, present and future. J AOAC Int 2011;94:360e72. [15] De Brabander HF, Naden Bussche J, Verbeke W, Vanhaecke L. The economics of residue analysis. Trends Anal Chem 2011;30(7):1088e94. [16] Ridgway K, Lalljie SPD, Smith RM. Sample preparation techniques for the determination of trace residues and contaminants in foods. J Chromatogr A 2007;1153:36e53. [17] Kinsella B, O’Mahony J, Malone E, Moloney M, Cantwell H, Furey A, et al. Current trends in sample preparation for growth promoter and veterinary drug residue analysis. J Chromatogr A 2009;1216:7977e8015. [18] Marazuela MD, Bogialli S. A review of novel strategies of sample preparation for the determination of antibacterial residues in foodstuffs using liquid chromatographybased methods. Anal Chim Acta 2009;645:5e17. [19] Trufelli H, Palma P, Famiglini G. An overview of matrix effects in liquid chromatography-mass spectrometry. Mass Spec Rev 2011;30(3):491e509. [20] Moreno-Bondi MC, Marazuela MD, Herranz S, Rodrı´guez E. An overview of sample preparation procedures for LCeMS multiclass antibiotic determination in environmental and food samples. Anal Bioanal Chem 2009;395(4):921e46.

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[38] Zhu WX, Yang JZ, Wei W, Liu YF, Zhang SS. Simultaneous determination of 13 aminoglycoside residues in foods of animal origin by liquid chromatographyeelectrospray ionization tandem mass spectrometry with two consecutive solid phase extraction steps. J Chromatogr A 2008;1207:29e37. [39] Wang J. Analysis of macrolide antibiotics, using liquid chromatographyemass spectrometry, in food, biological and environmental matrices. Mass Spec Rev 2009;28:50e92. [40] Blasco C, Van Poucke C, Van Peteghem C. Analysis of meat samples for anabolic steroids residues by liquid chromatography/tandem mass spectrometry. J Chromatogr A 2007;1154:230e9. [41] Ortelli D, Edder P, Corvi C. Analysis of chloramphenicol residues in honey by liquid chromatographyetandem mass spectrometry. Chromatographia 2004;59:61e4. [42] Zhang S, Liu Z, Guo X, Cheng L, Wang Z, Shen Z. Simultaneous determination and confirmation of chloramphenicol, thiamphenicol, florfenicol and florfenicol amine in chicken muscle by liquid chromatographyetandem mass spectrometry. J Chromatogr B 2008;875:399e404. [43] Babin Y, Fortier S. A high-throughput analytical method for determination of aminoglycosides in veal tissues by liquid chromatography/tandem mass spectrometry with automated clean-up. J AOAC Int 2007;90:1418e26. [44] Mottier P, Hammel YA, Gremaud E, Guy PA. Quantitative high-throughput analysis of 16 (fluoro)quinolones in honey using automated extraction by turbulent flow chromatography coupled to liquid chromatographetandem mass spectrometry. J Agri Food Chem 2008;56:35e43. [45] Kantiani L, Farre M, Sibum M, Postigo C, de Alda ML, Barcelo D. Fully Automated analysis of b-lactams in bovine milk by online solid phase extractioneliquid chromatographyeelectrosprayetandem mass spectrometry. Anal Chem 2009;81: 4285e95. [46] Chen D, Tao Y, Liu Z, Zhang H, Liu Z, Wang Y, et al. Development of a liquid chromatographyetandem mass spectrometry with pressurized liquid extraction for determination of glucocorticoid residues in edible tissues. J Chromatogr B 2011;879: 174e80. [47] Carretero V, Blasco C, Pico´ Y. Multi-class determination of antimicrobials in meat by pressurized liquid extraction and liquid chromatographyetandem mass spectrometry. J Chromatogr A 2008;1209:162e73. [48] Mol HJG, Plaza-Bolan˜os P, Zomer P, de Rijk TC, Stolker AAM, Mulder PPM. Toward a generic extraction method for simultaneous determination of pesticides, mycotoxins, plant toxins, and veterinary drugs in feed and food matrices. Anal Chem 2008;80: 9450e9. [49] Martinez Vidal JL, Aguilera-Luiz MM, Romero-Gonzalez R, Garrido Frenich A. Multiclass analysis of antibiotic residues in honey by ultra performance liquid chromatographyetandem mass spectrometry. J Agri Food Chem 2009;57:1760e7. [50] Fagerquist CK, Lightfield AR, Lehotay SJ. Confirmatory and quantitative analysis of b-lactam antibiotics in bovine kidney tissue by dispersive solid-phase extraction and liquid chromatographyetandem mass spectrometry. Anal Chem 2005;77:1473e82. [51] Mastovska MK, Lightfield AR. Streamlining methodology for the multiresidue analysis of b-lactam antibiotics in bovine kidney using liquid chromatographyetandem mass spectrometry. J Chromatogr A 2008;1202:118e23. [52] Nicolich RS, Werneck-Barroso E, Sı´poli-Marques SA. Food safety evaluation: detection and confirmation of chloramphenicol in milk by high performance liquid chromatographyetandem mass spectrometry. Anal Chim Acta 2006;565:97e102. [53] Wang J, Leung D. Analyses of macrolide antibiotic residues in eggs, raw milk, and honey using both ultra-performance liquid chromatography/quadrupole time-of-flight

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[69] Kaufmann A, Butcher P, Maden K, Widmer M. Quantitative multiresidue method for about 100 veterinary drugs in different meat matrices by sub 2mm particulate high performance liquid chromatography coupled to time of flight mass spectrometry. J Chromatogr A 2008;1194:66e79. [70] Peters RJB, Bolck YJC, Rutgers P, Stolker AAM, Nielen MWF. Multiresidue screening of veterinary drugs in egg, fish and meat using high resolution liquid chromatography accurate mass time of flight spectrometry. J Chromatogr A 2009;1216:8206e16. [71] Turnipseed SB, Storey JM, Clark SB, Miller KE. Analysis of veterinary drugs and metabolites in milk using quadrupole time-of-flight liquid chromatography mass spectrometry. J Agri Food Chem 2011;59:7569e81. [72] Kaufmann A, Butcher P, Maden K, Walker S, Widmer M. Quantification of anthelmintic drug residues in milk and muscle tissues by liquid chromatography coupled to tandem mass spectrometry. Talanta 2011;85:991e1000. [73] Kaufmann A, Butcher P, Maden K, Walker S, Widmer M. Development of an improved high resolution mass spectrometry based multiresidue methods for veterinary drugs in various food matrices. Anal Chim Acta 2011;700:86e94.

C H A P T E R

18 Analysis of Vitamins by Liquid Chromatography A. Gentili, F. Caretti Department of Chemistry, Faculty of Mathematical, Physical and Natural Sciences, University of Rome La Sapienza, Italy O U T L I N E 18.1. Introduction

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18.2. Liquid Chromatographic Determination of Water-Soluble Vitamins 18.2.1. Vitamin B1 18.2.2. Vitamin B2 18.2.3. Vitamin B3 18.2.4. Vitamin B5 18.2.5. Vitamin B6 18.2.6. Vitamin B8 18.2.7. Vitamin B9 18.2.8. Vitamin B12 18.2.9. Vitamin C

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18.3. Liquid Chromatographic Determination of Fat-Soluble Vitamins 18.3.1. Vitamin A 18.3.2. Vitamin D 18.3.3. Vitamin E 18.3.4. Vitamin K

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Liquid Chromatography: Applications http://dx.doi.org/10.1016/B978-0-12-415806-1.00018-8

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18.1. INTRODUCTION Vitamins are essential micronutrients, varying widely in chemical structure, biological activity, and physicochemical properties [1]. Owing to this heterogeneity, the classification as water-soluble (B-complex, C) and fat-soluble (A, D, E, K) is based on their solubility characteristics. On the whole, 13 groups are recognized in human nutrition (the B-complex groups together the vitamins B1, B2, B3, B5, B6, B8, B9, and B12) and each of them is composed of several biologically active forms, known as vitamers, which differ in structure, biopotency, and stability. Due to their relevance in the human physiology, the requirement of accurate data on forms and concentrations of vitamins naturally occurring in foods has become more stringent in recent years. Likewise, the determination of the forms added to fortified foods and supplements needs reliable analytical procedures. Although liquid chromatography (LC) is the ideal technique for vitamin quantitative analysis, many of the current international methods are based on microbiological assays [1e3]; some of them are outdated, time consuming, expensive, and characterized by a high measurement uncertainty. On the contrary, the scientific literature has continuously presented new LC-based procedures suitable for the individual and simultaneous vitamin analysis. At present, also the European Committee for Standardization (CEN) and the Association of Official Analytical Chemists (AOAC) International consider LC as the first choice to determine the vitamins B1, B2, B6, C [1,2] and the fat-soluble vitamins [1,3]. For the other water-soluble vitamins, the published LC methods have to be tested collaboratively before they can be applied as official methods. Before performing a LC analysis, it is advisable to adopt some preventive measures to restrain losses due to vitamin instability. The most important factors that lead to inactivation are light, air, temperature, pH, trace metals, and ionic strength. Since the majority of the vitamins are photosensitive, the use of low actinic amber glassware and subdued light is recommended for the whole duration of the analysis. Another precaution that cannot be disregarded is the addition of a proper antioxidant to the solvents employed both for the preparation of standard solutions and extraction procedures. When vitamin determination is applied to food and biological matrices, further problems have to be tackled: (a) (inter- and intragroup) chemical heterogeneity (b) their presence at trace levels in the real samples; (c) matrix complexity; and (d) interactions with other matrix constituents, such as polysaccharides (food), proteins, and lipids (food and biological samples). With regard to the first point, the subtle structural differences among vitamers of a specific group may make their chromatographic separation difficult; another facet pertains to the

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unavailability and cost of standards for homologs. In consequence, it is preferred to analyze each vitamin singly, applying extraction conditions able to free all bound forms (by means of acidic, alkaline, or enzymatic digestion) or to convert the several forms into a more stable form (see B9, B12 determination as examples). In this way, it is possible to increase the concentration of the single-target homolog in the final extract, to simplify the analysis and hold down expenditure for the purchase of standards.

18.2. LIQUID CHROMATOGRAPHIC DETERMINATION OF WATER-SOLUBLE VITAMINS The choice of the LC mode for the analysis of water-soluble vitamins depends on the extraction procedure employed and the vitamin form to be quantified (Figure 18.1). The most popular LC modes are normal-phase (NP), reversed-phase (RP), ion-pair RP, ion-suppression RP, and ionexchange chromatography.

18.2.1. Vitamin B1 Vitamin B1 [1] exists in nature both in free (thiamin) and esterified form (thiamin monophosphate, diphosphate, and triphosphate), while thiamin hydrochloride is used as a supplement [4]. To evaluate the total content of vitamin B1 in a food, extraction usually consists of an acid hydrolysis (0.1 M HCl in a water bath at 100 C or in an autoclave at 121 C) followed by an enzymatic digestion (diastases possessing a phosphatase activity) [1,2,5,6]. The acid treatment frees protein-bound forms and converts starch into soluble sugars. The enzymatic treatment may require several hours (on average 3 hr) of incubation for complete dephosphorylation of the thiamin esters. RP and ion-pair RP chromatography are the most common forms of LC used for the free thiamin determination [1,5e10]. Highly deactivated columns contain the phenomenon of peak broadening and tailing, due to the interaction of a vitamin-B1 basic site with silanol groups on the stationary phase. Polarity and low molecular weight are responsible for poor retention on RP columns; mobile phases containing high percentages of water and a suitable ion-pair agent (for example alkyl sulphonates) are expedients that both improve the peak shape and increase retention of the vitamin. Owing to its low molar absorptivity, the use of UV detection is mainly indicated for the analysis of fortified foods containing high concentrations of thiamin [1,7]. Low content of endogenous vitamin and high quantities of interfering substances in an extract require

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18. ANALYSIS OF VITAMINS BY LIQUID CHROMATOGRAPHY

FIGURE 18.1 Names and structures of the water-soluble vitamins naturally occurring in foods. (a) Vitamins B1, B2, B3, and B5; (b) vitamins B6, B8, sand B9; (c) B12. C.

18.2. LIQUID CHROMATOGRAPHIC DETERMINATION

FIGURE 18.1 (Continued).

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18. ANALYSIS OF VITAMINS BY LIQUID CHROMATOGRAPHY

FIGURE 18.1 (Continued).

a more sensitive and selective detector [8e10]. Fluorescence detection can be used, provided the thiamin is oxidased to thiochrome by pre- or postcolumn reaction with alkaline hexacyanoferrate(III). Precolumn derivatization is more often used, but the postcolumn one is convenient for routine analysis and to eliminate the problems of reducing sugars, produced during acid hydrolysis and competing with thiamin for the oxidizing agent [10]. Liquid chromatographic methods for thiamin determination in foodstuffs and other matrices are reviewed by Lynch and Young [11].

18.2.2. Vitamin B2 Vitamin B2 [1] is a generic term indicating a group of compounds characterized by equal biological activity: riboflavin, riboflavin-5’-phosphate (FMN, flavin mononucleotide), and riboflavin-5’-adenosyldisphosphate (FAD, flavin adenine dinucleotide). In animal tissue, FMN and FAD are coenzymes bound tightly but not covalently to the corresponding apoenzymes. Forms for food fortification are FMN and riboflavin hydrochloride [4]. An extraction protocol, analogous to that described for vitamin B1, permits measuring all flavins, both the endogenous ones and those used as supplements [1,6,8,10]: acidic hydrolysis promotes the release of the protein-bound forms and converts FAD to FMN; the succeeding enzymatic digestion (takadiastase, amylase, acid phosphatase, claradiastase) is used for dephosphorylating FMN and hydrolyzing starch. RP and ion-pair RP chromatography on C18 stationary phases with UVeVis [12,13] or fluorescence detection [6,8,10] are the LC methods most frequently employed; other works report separations on C8 [14] and amide-C16 columns [15]. Free flavins in aqueous solution exhibit an

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intense yellowish-green fluorescence (450 nm excitation/522 nm emission) due to their 3-imino group, whereas protein-bound forms do not fluoresce, since their interaction is established through this functional group [1]. The UVeVis spectrum of riboflavin shows four bands centered at 223, 266, 373, and 445 nm [1]. The use of a 254-nm fixed-wavelength absorbance detector is common [12], but the detection at 446 nm is less susceptible to interferences [13].

18.2.3. Vitamin B3 Two forms of vitamin B3, also known as niacin, are found in food [1,2]: nicotinic acid and nicotinamide. In living tissues, nicotinamide is a moiety of the coenzymes nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP); in meat, it is found free because of the postmortem hydrolysis of NAD [1]. Nicotinamide is also a form used for food fortification [4]. Nicotinic acid is the prevalent vitamer in mature cereal grains; nevertheless, it is unavailable due to its linkage to a number of polysaccharides (niacytin) and polypeptides (niacinogen) [1]. The determination of “total” (free plus bound) or “free” (bioavailable) niacin is based on well established extraction procedures [1,2,16]: Total niacin is released from the food matrix by autoclaving at 121 C with alkali [17] or 1e2.5 N mineral acid [18]; free niacin is isolated by boiling with 0.1 N mineral acid for 1 hr [19] or incubating with NAD glycohydrolase (NADase) at 37 C for 18 hr [20]. Most studies used absorbance detection at 254 to 264 nm for both B3 vitamers [17e19,21]. Their absorptivity is affected by pH: in an acidic solution, it is higher and lmax remains almost unchanged at 261 nm [1]. UV detection is convenient but not very sensitive or selective and the interfering compounds have to be removed by a cleanup step. To improve detection limits and selectivity without having recourse to the purification of hydrolysates, some researchers [20,22,23] propose an LC method based on the UV irradiation of the postcolumn effluent in the presence of H2O2 and Cu2þ to obtain fluorescent compounds (280 nm excitation/380 nm emission). When the extraction procedure is applied to the determination of total niacin, nicotinic acid is the only B3 vitamer to be monitored by LC, because all nicotinamide is converted into the acid form. Anion exchange chromatography [18] is applied less frequently than RP chromatography under ion-suppression and ion-pairing conditions [17,19]. Van Niekerk et al. [24] assembled a two-dimensional chromatography system, provided by C18 and anion-exchange columns, to increase the selectivity of the UV detection at 254 nm.

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Recently, electrospray (ESI) ionization in the positive ion mode was utilized to reduce interference problems due to matrix coextracted materials, detecting icotinic acid in food materials [25] and both B3 vitamers in roasted coffee [26], selectively.

18.2.4. Vitamin B5 Vitamin B5 occurs in three biologically active forms in foods [1]: pantothenic acid, coenzyme A (CoA), and acyl carrier protein (ACP). Calcium or sodium pantothenate are the forms generally used as supplements in infant formula [4]. The total quantification of vitamin B5 requires the release of pantothenic acid from CoA and ACP. Since it consists of pantoic acid linked through an amide linkage to b-alanine, chemical hydrolysis cannot be used. The only alternative to free pantothenic acid from CoA is the digestion with a number of enzymes (pepsin, alkaline phosphatase, pantetheinase); nevertheless, this treatment is unable to release the vitamin from ACP [27,28]. For the extraction of free pantothenic acid from milk and calcium pantothenate from infant formula an acidic deproteination is often used, followed by centrifugation and filtration [29,30]. Ion-suppression RP (TFA, formic acid, phosphate buffer) on C18 [27e29] and C8 columns [30] is the commonly used chromatographic mode. The poor selectivity and sensitivity of UV detection (a very weak absorbance at 204 nm due to the carbonyl group) makes LCeUV unsuitable to determine the low concentration of vitamin B5 in nonformulated foods. Some researchers have overcome these problems using multiwavelength UV detection (at 200, 205, and 240 nm) [30], fluorimetric detection (postcolumn derivatization of b-alanine with ortho-phthaldialdehyde in the presence of 2-mercaptoethanol) [27], and mass spectrometry with electrospray ionization [28,31]. The last solution allows the measurement of endogenous pantothenic acid in starch-containing foods [28], achieving a limit of quantitation (LOQ) adequate to quantify vitamin B5 contents greater than 0.024 mg/100 mg. Also, Pakin et al. [27] suggested a method that is sensitive and applicable to the determination of free and total pantothenic acid in any foodstuff, but it is probably too complex to be utilized for routine analyses.

18.2.5. Vitamin B6 Six vitamers of B6, having equivalent biopotency, are found in nature [1,16]: pyridoxine or pyridoxol, pyridoxal, pyridoxamine, and their 5’-phosphate esters; acid pyridoxic and pyridoxine-glucoside are inactive forms occurring in plant tissues. Regulation 1925/2006/EC cites

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pyridoxine hydrochloride, pyridoxine 5’-phosphate, and pyridoxine dipalmitate as the utilizable forms to enrich foods [4]. The preferred technique for vitamin B6 assay is LC, due to its high selectivity, which permits the simultaneous separation and quantitation of the several homologs. Different extraction protocols can be applied prior to LC analysis to estimate the total or bioavailable vitamin B6 [1,2,16]. For routine analysis, the approach used is the hydrolysis of the conjugated forms; in this way, chromatography is limited to pyridoxine, pyridoxamine, and pyridoxal, and the quantitation results are comparable to those obtained by microbiological assay. Traditional methods using acidic media and high temperature (0.1 N HCl, 121 C) denature proteins, disintegrate the food matrix, and hydrolyse phosphorylated and glycosilated forms completely [16,32]; the inconvenience may be an overestimation of bioavailable vitamin. A selective extraction, carried out at room temperature with a deproteinizing agent (sulfosalicylic acid, trichloroacetic acid, metaphosphoric acid, and perchloric acid), frees pyridoxal hydrolyzing Schiff bases formed with food proteins, preserves all vitamers (free, phosphorylated, and glycosylated) and allows their individual quantification; in this case, the chromatographic separation of all homologs is more complex [33e35]. The two most common approaches are based on a combination of chemical hydrolysis [36,37] or deproteination [38,39] with enzymatic digestion. The latter is usually performed with acid phosphatase [38] or takadiastase [36]; b-glucosidase is indispensable to determine bioavailable vitamin B6 in plant foods [37]. Ndaw et al. [32] tested a mixture of enzymes (a-amylase, papain, acid phosphatase) to release, in a single step, the bound forms of vitamins B1, B2, and B6; the acid hydrolysis proved to be superfluous, due to the presence of protease. Usually, the individual and simultaneous separation of free [32,36,38,39] and conjugated B6 [33e35] vitamers is carried out by means of RP chromatography on C18 columns with acidic mobile phases. These methods utilize the native fluorescence of B6 vitamers [1,32e39], increasing the weak intensity of phosphate esters at low pH by means of postcolumn derivatization with sodium hydrogensulphite.

18.2.6. Vitamin B8 The only biologically active form of vitamin B8 is D-(þ)-biotin, a unique steroisomer found in nature among the eight isomers theoretically possible [1,40]. In animal and plant tissues, most biotin is covalently bound to a lysine residue, free (D-biocytin) or belonging to biotindependent enzymes through an amide attachment. Biotin is also the form used for food enrichment [4].

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Extraction of total biotin is obtained by acidic hydrolysis (2e3 M HCl at 100 C or 1e3 M H2SO4 by autoclaving at 121 C) that breaks bonds with proteins and totally converts D-biocytin into D-biotin [41,42]. Possible losses of vitamin depend on both the acid concentration and the duration of autoclaving. Enzymatic digestion with papain for 18 hr leaves biocytin intact and allows the determination of available biotin (biotin plus biocytin); takadiastase is added for starchy foods, such as cereals [42]. Owing to the low concentration and the absence of a strong chromophore, few RP HPLC methods for the endogenous vitamin B8 analysis in foods have been published [1,2]. Most of them [41,42] take advantage of avidin, the egg-white protein, labeled with a fluorescent marker (fluorescein 5-isothiocyanate) as a postcolumn derivatizing agent; its fluorescence is enhanced on binding to biotin and biocytin, its specific ligands. A LC tandem mass spectrometry (MS) method [42], using an ESI source in the positive ion mode and biotin-d6 as internal standard, was able to determine biotin in foods at low concentrations after hydrolysis with sulfuric acid and enzymatic digestion with papain. The whole procedure was reliable and faster than the microbiological assay.

18.2.7. Vitamin B9 Folate and folacin [1] are generic terms referred to a group of compounds with vitamin B9 activity, including folic acid (pteroylglutamic acid), dihydrofolic acid, tetrahydrofolic acid (H4folic acid), 5-formyl-H4folic acid, and 5-methyl-H4folic acid. Folic acid is not considered a natural physiological form, and is the chemical form used as a food supplement [4]. All folates exist in nature at low levels and predominantly as polyglutamates containing no more than seven glutamate residues with a g-peptide linkage. Even if the theoretical number of folates approaches 150, in nature about 50 are observed. Multiplicity, instability, and low concentrations of folates in animal and plant tissues have constituted an obstacle to the development of LC methods for their determination [1,2,43]. For the same reasons, sample preparation and purification are key steps carried out according to a well established protocol [2,44]. During extraction, a number of precautions should be taken to avoid the pH-dependent interconversion of some species, oxidative losses (adding antioxidants, such as ascorbate and 2-mercaptoethanol), and thermal degradation [45]. Folates are released from the food matrix by autoclaving the food sample in a buffered aqueous medium: particles are broken up, starch is gelatinized, folatebinding proteins are denaturated as well as enzymes catalyzing folate

18.2. LIQUID CHROMATOGRAPHIC DETERMINATION

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degradation or interconversion. The autoclaved sample is submitted to a tri-enzyme treatment [44]. The first digestion is performed with protease to free the protein-bound folates definitively. The second uses a-amilase to release the starch-bound folates. Finally, the third treatment, carried out with the folic acid conjugase, deconjugates the polyglutamates to the monoglutamate forms. A cleanup step on solid-phase extraction (SPE) cartridges is often employed to remove interfering compounds co-extracted with folates from the real matrix. The most used stationary phases include strong anion-exchange (SAX) materials and affinity chromatographic sorbents with immobilized folate-binding protein. Both provide high recoveries, but only the affinity column purifies and concentrates the extracts efficiently. LC with UV detection (at 280 or 290 nm) is used for the analysis of foods with low concentrations of folates [46,47], after tri-enzyme digestion followed by cleanup on the affinity sorbent to concentrate the extracts by about 10-fold [46]. Nevertheless, fluorimetric detection is more efficient at determining naturally occurring folates [48] as well as ESIeMS that, combined with isotope dilution, allows accurate quantification [49,50]. Ion-suppression RP chromatography is the most suitable mode for coupling with both detectors [48]. In fact, folates show native fluorescence (288 nm excitation/353e356 nm emission) that is enhanced in an acidic medium (mobile phase at pH 2.3 using phosphate buffer) for the reduced forms but not for folic acid; the solution adopted for the latter is to produce a fluorescent pterin fragment through its postcolumn oxidative cleavage [51]. Acidic mobile phases support ESI ionization of folates, but the phosphate buffer has to be substituted by the more volatile formic acid [49,53e55]. Ion-pair RP chromatography is carried out at neutral or basic pH, but it requires acidification of the column effluent to make fluorescence detection possible [52].

18.2.8. Vitamin B12 Vitamin B12 [1] is a term that generically describes a group of cobaltcontaining organometallic compounds with antipernicious anaemia activity, known as cobalamins,. The major forms occurring in foods of animal origin include 5’-deoxyadenosylcobalamin (coenzyme B12) and methylcobalamin, two coenzymes covalently bound to their apoenzymes; hydroxocobalamin is their photooxidation product. The general extractive protocol [56] applied for determining total vitamin B12 is used to release the protein-bound vitamers and to convert all natural cobalamins into a single and stable form, known as cyanocobalamin. To this end, Heudi et al. [57] dissolved a food sample in a buffered solution (pH 4) containing sodium cyanide (at 100 C for 35 min) then digested it with pepsin (at 37 C for 3 hr).

488

18. ANALYSIS OF VITAMINS BY LIQUID CHROMATOGRAPHY

Determination of vitamin B12 by LCeUV is difficult to perform in nonsupplemented foodstuffs because of the very low concentrations of the vitamin and the poor sensitivity and selectivity of the detection system employed. These problems were overcome using an immunoaffinity column and monitoring the vitamin at 361 nm [58]. The extractive procedure of Liebiedzi nska et al. [7] was based on an enzymatic digestion with aamilase and papain after autoclaving salmon samples at 121 C in the presence of cyanide; cyanocobalamin was monitored by an electrochemical detector. Fluorescence detection [59] can be used after chemical or enzymatic hydrolysis, indispensable to release a-ribazole, a characteristic fluorescent fragment of vitamin B12. Nevertheless, the latter may occur in foods as a vitamin B12 metabolite; therefore, it is essential to proceed with extensive purification of the extracts before carrying out the hydrolysis. LCeMS using electrospray ionization was successfully applied to the analysis of vitamin B12 in some fortified foods [60] and cultivated mushrooms [61]. It might constitute a promising tool for the cobalamin analysis since it requires neither a derivatizion step nor an accurate cleanup procedure.

18.2.9. Vitamin C L-ascorbic acid (AA) and L-dehydroascorbic acid (DHAA) are the two main C vitamers occurring in nature [1]. In food analysis, the valuation of the vitamin C total content should account for both forms, since DHAA is readily reduced to AA in the animal body. D-isoascorbic acid (D-IAA), also known as erythorbic acid or D-araboascorbic acid, has analogous reductive properties but only 5% of the antiscorbutic activity of L-AA; this epimer is a by-product of vitamin C, and is approved within the European Community as an antioxidant additive [62]. The capability of LC to distinguish the two ascorbic acid isomers and their primary oxidation products is very useful for analyzing processed foods. Forms used for supplementation are AA, sodium-, calcium-, or potassium-L-ascorbate and L-ascorbyl 6-palmitate [4]. AA is susceptible to both chemical and enzymatic oxidation [1]. Chemical oxidation is catalyzed by minerals, such as Cu(II) and Fe(III), in the presence of oxygen at a pH-dependent rate; enzymatic oxidation by the ascorbate oxidase occurring in plant tissues. Light and heat are other factors that promote its degradation. For these reasons, the extraction procedure should be designed to stabilize the vitamin; for example, metaphosphoric acid [63e66] denatures proteins, inactivates enzymes, provides a medium below pH 4 (degradation rate is minimal at pH 2), and inhibits metal catalysis, whereas EDTA [67,68] chelates the minerals efficiently.

18.3. LIQUID CHROMATOGRAPHIC DETERMINATION

489

Several LC methods have been proposed for vitamin C analysis [1,62e73]. The good selectivity of silica-based aminopropyl columns in separating homologues of vitamin C is probably due to the hydrogen bonding between hydroxyl protons of vitamin C with the neutral amino group of the stationary phase rather than to differences in pKa values of the vitamers [64,69]. A disadvantage is the short lifetime of this kind of stationary phase because of the possible reaction of the amine group with the carbonyl group of reducing sugars, or other compounds, to form Schiff bases. Other modes are ion exclusion (poorly selective) [70], RP with and without ion suppression [63,65,71], and RP ion-pair chromatography [72,73]. The problem with RP packings is that ascorbic acid is only weakly retained and, therefore, requires the addition of a cationic ion-pair agent to the mobile phase. To this end, several primary, secondary, and tertiary amines have been employed as relatively hydrophobic modifying reagents to obtain sharp, well-defined peaks; tetrabutylammonium has been the most used [1]. The UV (254 nm) [65,66] and amperometric detection (a potential of þ0.7 V is often chosen using either a platinum or glassy carbon electrode) [70,71] have been utilized for the direct monitoring of AA, while its fluorimetric detection requires chemical derivatization [64]. DHAA has a weak molar absorptivity and it is electrochemically inactive. A precolumn reduction to AA by using cystein or dithiotreitol makes possible absorbance detection but not electrochemical (high noise) detection [74]. Recently, LCeMS with electrospray ionization in the negative ion mode [75,76] was used for the analysis of vitamin C in several food commodities; the main advantage was the simultaneous determination of AA and DHAA with no need for oxidationereduction or derivatization steps [76].

18.3. LIQUID CHROMATOGRAPHIC DETERMINATION OF FAT-SOLUBLE VITAMINS Fat-soluble vitamins occur in the lipid fraction of foods [1], composed mainly of triglycerides and partly of sterols, phospholipids, and other lipoidal constituents. These substances, having solubility analogous to those of the fat-soluble vitamins, complicate the isolation of fat-soluble vitamins and constitute a source of interference during the following analysis [1,3]. Hot saponification [1,3,77e79] is the most effective tool for removing the majority of fatty material, hydrolyzing ester linkages of glycerides, phospholipids, esterified sterols, and carotenols. Moreover, alkaline hydrolysis frees bound forms of vitamins (for instance, esterified and protein-bound forms) and degrades chlorophylls in water-soluble products. Also gelatine of the vitamin premix, added to supplemented

490

18. ANALYSIS OF VITAMINS BY LIQUID CHROMATOGRAPHY

foods, is dissolved. Starch-containing products, such as breakfast cereals, are digested first with takadiastase and then saponified to avoid the formation of undissolved particles [78]. Normally, saponification is carried out with a mixture of ethanol and 50% (w/v) aqueous KOH solution in the presence of an antioxidant (pyrogallol, ascorbic acid, butylated hydroxytoluene) for 30 min at about 80 C [1,3]. The unsaponifiable fraction (fat-soluble vitamins, carotenoids, sterols, etc.) is extracted from the alkaline digest by liquideliquid extraction using a water-immiscible organic solvent (hexane, petroleum ether, petroleum ether: diethyl ether at 1:1, v/v). Hexane has the advantage of providing extracts that do not contain soaps and are nearly neutral; nevertheless, when used, it is advisable maintaining the percentage of ethanol below 40% (v/v) to avoid losses of slightly polar vitamins, such as retinols and tocopherols. Saponification can be used for vitamins A, D, and E, while it is not suitable for K vitamers, which are quickly decomposed in alkaline media at high temperature [1,3]. Alcoholysis [1] is milder (ambient temperature) and more rapid (2 min) than saponification. Methanol reacts with KOH to form potassium methoxide, which, in turn, converts glycerides to methyl esters and soaps. It has been used to determine vitamin A palmitate in nonfat milk and vitamin D in whole milk [80]. Enzymatic hydrolysis [81,82] with lipase (from Candida rugosa or from porcine pancrease) is an alternative procedure to remove glycerides in vitamin K determinations. Addition of papain (from Carica Papaya) aids the digestion of meat and foods of animal origin. The hydrolysate is first alkalinized (potassium carbonate in ethanol) to precipitate fatty acids as soaps then extracted with a water immiscible organic solvent (hexane or pentane). It has also been used in combination with supercritical fluid extraction (SFE) [83]. Also, direct solvent extraction [84] is used for the isolation of vitamers susceptible to degradation in alkaline media (retinyl esters, vitamers K), but it is ineffectual for removing interfering fatty substances. In some cases, ultrasonication has been employed to break up the lipoproteic complex encapsulating fat-soluble vitamins [85]. Several LC modes have been used for the separation of fat-soluble vitamins. The choice depends on the vitamin forms to be determined, the nature of the food matrix, and the sample treatment. Adsorption chromatography has two main advantages: (a) geometric and positional isomers are generally resolved on silica stationary phases [86,87]; (b) relatively high loads of lipoidal material can be tolerated by this type of column. The latter feature allows the direct injection of extracts obtained by means of direct liquid extraction [88] or sample dissolution in hexane (e.g., vitamin E from oils) [89], when the recourse to saponification is not essential for the analyte isolation. In these cases, fluorescence detection is

18.3. LIQUID CHROMATOGRAPHIC DETERMINATION

491

preferred to absorption detection, which may reveal intrusive peaks of lipid origin. The majority of LC separations of fat-soluble vitamins are based on RP chromatography on C18 columns, but the use of a triacontyl stationary phase (C30) is becoming more common, due to its superior shape selectivity [90,91]. Shape discrimination with C30 columns improves at subambient temperature, permitting resolution of geometric and positional isomers partly or fully, depending on the mobile phase composition. Nevertheless, C30 columns are less efficient and the chromatographic peaks broader than those obtained with C18 columns. Nonaqueous reversed-phase (NARP) chromatography [92] has been employed for the separation of fat-soluble vitamins [93] and carotenoids [94]. This chromatographic technique uses either C18 columns with a high carbon loading (20%) or C30 columns and low polarity mobile phases. A typical NARP mobile phase consists of a polar solvent (e.g., acetonitrile), and a solvent of lower polarity (e.g., dicloromethane) to solubilize analytes and adjust the mobile phase strength. The good chromatographic selectivity is due to the small difference in polarity between the mobile and stationary phases.

18.3.1. Vitamin A Vitamin A-active compounds [1] are present in foods of animal origin as retinoids (retinol, retinyl esters, retinylaldehyde, retinoic acid) and in those of plant origin as carotenoids (only carotenoids with one unsubstituted b-ionone ring and with an 11-carbon polyene chain at least are provitamins A). Retinyl palmitate is the main form used as a food supplement [4]. Food sample pretreatment may consist of either (a) saponification to quantify the free forms (retinol or xanthophylls may occur free or esterified in foods) [95,96] or (b) direct extraction to determine the unaltered A vitamers [84,88]. Alkaline hydrolysis is also an expedient to simplify the vitamin A analysis, since retinol is the only form to be quantified; nevertheless, due to its sensitivity to light and oxygen, it is important to prevent photo-oxidation by inclusion of a antioxidant (ascorbic acid, hydroquinone, or pyrogallol). A drawback of hot saponification is the generation of artifacts, such as geometric isomers of retinol and carotenoids [97]. Retinol and its esters can be monitored by UV (325 nm) [88,96] and fluorescence (324e328 nm excitation/470 nm emission) detection [98]; the latter has the advantage that b-carotene does not interfere with the vitamin A determination even if there is coelution, while the disadvantage is a more limited linear dynamic range. RP chromatography on a C18 column with

492

18. ANALYSIS OF VITAMINS BY LIQUID CHROMATOGRAPHY

semiaqueous mobile phases is the most often used mode for vitamin A analysis [3,100,101], but adsorption chromatography on silica is the more efficient approach for separating geometric isomers of retinol [86,96]. Carotenoids show a characteristic three peak spectrum in the UVeVis region [99]. The absorption maxima of all-trans b-carotene, main provitamin A, occur at 428, 453, and 478 nm (in hexane or ethanol); its cis isomers may be identified according to the following indications: (a) a small hypsochromic shift of lmax (usually 2e6 nm for mono-cis, 10 nm for di-cis, and 50 nm for poli-cis isomers); (b) an ipochromic effect; (c) a reduction in fine structure; and (d) the appearance of a cis-peak in the near-UV range, between 330 and 350 nm. RP chromatography [102] is preferred to NP for the determination of provitamins A, because many carotenoids can be monitored within the same chromatographic run: the xanthophylls are eluted early, while the carotenes require strong mobile phases (little or no water) for their displacement. Carotenes and their cis isomers are poorly resolved on monomeric C18 phases, while their separation on polymeric C18 or C30 phases depends on the organic modifier and gradient elution conditions [103]. In addition, the C30 sorbent provides the highest selectivity [90,91] at low temperature (19 C) because of the improved alignment with the alkyl chains; only under these conditions can lutein and zeaxanthin (structural isomers) be adequately separated. Lately, LCeMS analysis of carotenoids and retinoids has been reported for food matrices such as fish eggs [104] and infant formula [105,106]. Atmospheric pressure chemical ionization (APCI) is the method of choice for their detection in the positive ion mode: Retinol gives an intense dehydrated pseudomolecular ion, [MHeH2O]þ, at m/z 269; retinyl esters are fragmented in the ion source producing [MHefatty acideH2O]þ ions (i.e., retinol dehydrated at m/z 269); carotenoids generate several ions such as [M þ H]þ, [M]þ•, and [MeH]þ. The ESI interface is better for the ionization of xanthophylls when semiaqueous mobile phases are used for their separation.

18.3.2. Vitamin D Vitamin D is the name given to a series of compounds with antirachitic activity [1]: cholecalciferol (vitamin D3) is present in foods of animal origin, whereas ergocalciferol (vitamin D2) is produced by plants, fungi, and yeast (Figure 18.2). In animal organisms, vitamin D is metabolized to its biologically active forms, 25-hydroxy- and 1a,25-dihydroxycholcalciferol. The extraction of vitamin D from fatty foods necessitates alkaline hydrolysis [1,3,85]. Thermal isomerization of vitamin D to previtamin D during hot saponification entails losses of 10e20% making its quantification difficult. Overnight cold saponification (prolonged digestion at

18.3. LIQUID CHROMATOGRAPHIC DETERMINATION

493

room temperature) is not affected by this problem and, moreover, demands less operator attention and yields greater recovery than the hot saponification. Cleanup or fractionation procedures [1,16,85,106e109] include sterol precipitation in a digitonin solution stored at e20 C overnight, solid-phase extraction, gel permeation chromatography, and LC on a semipreparative scale. These steps are essential (a) to remove the excessive amounts of sterols, which might alter the retention time of the vitamin and interfere with its UV detection (265 nm in ethanol or hexane); (b) to achieve an adequate enrichment factor of the final extract as vitamin D occurs at very low concentrations in nature. SPE is more advantageous than the other methods; the most used sorbent is C18, but polar sorbents, such as silica, aminopropyl silica, and Florisil, are also efficient at removing sterols, carotenoids, vitamin E, and other interfering material. An alternative procedure to perform a reliable quantitative analysis of total vitamin D is the conversion of both D vitamers to isotachisterol [110],

FIGURE 18.2 Names and structures of the fat-soluble vitamins and pro-vitamins A carotenoid naturally occurring in foods. (a) Vitamins A and D; (b) vitamins E and K.b

494

18. ANALYSIS OF VITAMINS BY LIQUID CHROMATOGRAPHY

FIGURE 18.2 (Continued).

a compound more stable to heat and light; moreover, the absorption maximum at 301 nm and the high extinction coefficient contribute to improve selectivity and sensitivity. Ultraviolet and electrochemical detectors are the most widely used for the quantification of vitamin D. The UV detector has the

18.3. LIQUID CHROMATOGRAPHIC DETERMINATION

495

advantage that all vitamin-D-active compounds absorb within the range of 250e265 nm [1,106,107], the wavelength 280 nm has also been used to reduce interference problems [111]. The highest sensitivity, selectivity, and linear dynamic range of the electrochemical detector [109] allows the analysis of samples containing low concentrations of vitamin D and the simplification of sample treatment. APCI is advantageous for vitamin D detection in milk products [78,112,113], due to its identification power in complex matrices with low concentrations of cholecalciferol. The high sensitivity and selectivity of the LCeMS technique allows simplification and improvement of the reliability of the analysis of endogenous vitamin D contents in foods [114,115]. NP chromatography [1,85] has the advantage of tolerating relatively heavy loads of fatty material and separating vitamin D from its hydroxylated metabolites; nevertheless, it cannot resolve vitamins D2 and D3. Hexane containing a small percentage (less than 5% v/v) of a more polar solvent (isopropanol, dichloromethane, or ethyl acetate) is the most used mobile phase [85]. RP columns having a high carbon loading [107e109] can differentiate vitamin D2 from D3, making possible the use of one homolog as an internal standard for the other [107] as well as facilitating the separation of their hydroxylated metabolites [108].

18.3.3. Vitamin E Vitamin E has eight biologically active forms [1]: four tocopherols (saturated isoprenoid side chain) and four tocotrienols (unsaturated isoprenoid side chain), designed as a-, b-, g-, and d-according to the number and position of methyl groups on the chromanol ring. Of these, a-tocopherol is the most important (plant sources) and active. During sample treatment, vitamin E must be protected from light and oxygen (flushing with nitrogen and adding an antioxidant) to quantify its actual content. Except for its quantification in oils, which can be performed by direct injection onto a NP column, of a sample diluted with hexane or mobile phase [89] and hot saponification is required for all other foods [77e79]; nevertheless, alkaline conditions (volumes of alkali and ethanol, time, and temperature) must be carefully balanced to avoid losses [77]. The highest recovery is obtained when the digestion is completed under reflux conditions [1]. NP chromatography [87,116] completely resolves the eight homologues, which elute in order of increasing polarity: a-tocopherol, a-tocotrienol, b-tocopherol, g-tocopherol, b-tocotrienol, g-tocotrienol, d-tocopherol, and d-tocotrienol. The decreasing number of methyl groups and the unsaturation in the side chain make these compounds more polar and therefore

496

18. ANALYSIS OF VITAMINS BY LIQUID CHROMATOGRAPHY

more retentive. Separation of b- and g- positional isomers is due to the diverse interactions that the methyl groups establish with the silanol groups of the silica stationary phase. RP chromatography [116] is less effective for separating the E vitamers; in fact, b- and g- positional isomers coelute on C18 columns, but a complete separation is achieved on C30 columns [117]. Tocopherols and tocotrienols show a low intensity of absorption between 292 and 298 nm in ethanol [1], and fluorescence detection (290e296 nm excitation/330 nm emission) [89,118] is preferred to analyze vitamin E in complex food matrices. Polar solvents, such as diethyl ether and alcohols, provide a strong flourescence response, and their inclusion in the hexane mobile phase improves the detection limits achievable in NP chromatography [1]. The first description of LCeMS analysis dates back to 1998 [119]; tocopherols and carotenoids were detected by ESI as silver ion adducts, after postcolumn treatment with acetone containing silver perchlorate. In spite of the low detection limits, the poor solubility of this salt in organic solvents causes contamination problems and makes this ionization technique, unsuitable for routine analysis. ESI in the negative ion mode has been used [120] for the quantification of tocopherols and tocotrienols after pressurized liquid (solvent) extraction (PLE) with methanol at 50 C and 110 bar from cereal samples. Nevertheless, Lanina et al. [121], comparing the performances of the ESI and APCI sources in both ionization modes, observed that the positive ion mode is less efficient than the negative ion mode because of the signal dispersion between [M þ H]þ and [M]•þ; the authors chose APCI in the negative ion mode for the detection of tocopherols in sunflower oil and milk due to the larger dynamic range and lower detection limits. Positive ion APCI was instead the preferred ionization mode of other researchers [122,123].

18.3.4. Vitamin K The K homologues [1] are a family of 2-methyl-1,4-naphthoquinones possessing cofactor activity for g-glutamylcarboxylase and differ in the side chain attached at C3. Phylloquinone (vitamin K1) has a phytyl side chain and occurs in green plants. Vitamin K2 includes a group of compounds synthesized by bacteria and characterized by a polyisoprene side chain; each of them is designated menaquinone-n (MK-n, with n from 4 to 13) according to the number of isoprenyl units. Menadione (vitamin K3), menadiol (vitamin K4), and vitamin K1(25) are synthetic forms [1,4]. Owing to their instability to alkali, hot saponification is unworkable. Enzymatic hydrolysis [81,82,124,125] and direct solvent extraction [124e127] are the most-common techniques employed for extraction.

18.4. MULTIVITAMIN METHODS

497

Liquid-phase reductive extraction with zinc chloride in an acid medium was tested to extract K vitamers as acetonitrile soluble hydroquinones from dairy products [125,126]. Acid hydrolysis [125] proved advantageous in isolating long chain menaquinones from cheese, provided the digestion time (10 min) was short. Semipreparative LC [125,127,128] and SPE [126] have sometimes been employed as a cleanup and concentration step after solvent extraction. Matrix solid-phase dispersion (MSPD) followed by PLE with ethyl acetate at 50 C and 1500 psi [129] and SFE using carbon dioxide at 8000 psi and 60 C [130] are fast, alternative procedures for extracting phylloquinone. Phylloquinone and menaquinones exhibit a UV spectrum characteristic of the naphthoquinone ring (five absorption maxima at 242, 248, 260, 269, and 325 nm), but UV detection is seldom used [131], due to its poor sensitivity and selectivity. Most methods employ fluorescence detection (238e244 nm excitation/418e430 nm emission) after a postcolumn reduction with a methanolic solution containing zinc chloride, sodium acetate, and acetic acid in a reactor packed with zinc metal powder [81,82,124e126]; this LC method is sufficiently sensitive for the determination of low concentrations of menaquinones in animal products [81,125]. Electrochemical detection is an alternative for the determination of phylloquinone in foods of animal and plant origin [127,128]. The K homologs are separated on C18 columns [81,125e128,] which cannot resolve their geometric isomers; however, this separation is achieved on C30 columns [82,124] and on silica columns [131]. Due to the lipophilic nature of the long chain menaquinones, NARP chromatography is well suited to their separation and determination.

18.4. MULTIVITAMIN METHODS Chromatographic techniques are suitable for quantitative multianalyte determinations. In particular, LC is the technique of choice for the direct analysis of polar, nonvolatile, and heat-sensitive compounds, such as water-soluble vitamins (see Figure 18.3 for an example); moreover, having no molecular weight limitations, it can be used for the separation of cobalamins, polyglutamates, FAD, and CoA. LC is also the most common technique used for the concurrent analysis of fat-soluble vitamins and provitamin A carotenoids (see Figure 18.4 for an example). The key step of a multivitamin method is the development of a simultaneous and quantitative extraction procedure. The intra- and intergroup heterogeneity of water-soluble vitamins makes it difficult to realize this goal. The application of an acid treatment, to hydrolyze the bound forms, can be used for simultaneous extraction of B1, B2, B3, B6, B8,

498

18. ANALYSIS OF VITAMINS BY LIQUID CHROMATOGRAPHY

FIGURE 18.3 Extracted ion current profiles of the water-soluble vitamins detected in a green kiwi extract (see reference [138] for the details). The LCeSRM (standard reference material) chromatogram was acquired by a high-flow ESI source (TurboIonSpray source). Each analyte was identified on the basis of the retention time, the two selected SRM transitions and their relative abundance. Only the most intense SRM ion current is reported in the figure.

18.4. MULTIVITAMIN METHODS

499

FIGURE 18.4 Extracted ion current profiles of the fat-soluble vitamins detected in a green kiwi extract. This is an example of NARP chromatography (see Table 18.1, reference [137], for the details): a nonaqueos mobile phase was chosen as the best compromise between chromatographic resolution and support to positive APCI ionization of analytes.

B12, and C vitamins [132,133], but it is not appropriate for B5 and B9, which are sensitive to low pH [133]. Since the heterogeneity of the fatsoluble vitamins is less marked, it was simpler to develop an extraction protocol suitable for isolating more than one vitamin from the same

500

18. ANALYSIS OF VITAMINS BY LIQUID CHROMATOGRAPHY

matrix. Hot saponification is the most used extraction procedure [78,107,134e136], especially for foods with a high fat content. It is indicated for the majority of fat-soluble vitamins and carotenoids but causes a rapid decomposition of K vitamers. MSPD was tested as an alternative technique for isolating both fat-soluble [137] and water-soluble vitamins [138] from plant foods. The UVediode array [132,134e136,139,140] and fluorescence detection [141,142] have been used to develop multivitamin LC methods, which, however, remain limited to a few analytes responding to the same detection system and extracted quantitatively with the same procedure. Moreover, by means of a UV detector, other difficulties are represented by the absence of a strong chromophoric group in some vitamins (pantothenic acid and biotin), which absorb with modest sensitivity in the low UV region only, where the selectivity is scarce (absorption of interfering compounds). A promising detector for the development of multivitamin LC methods is the mass spectrometer, which overcomes problems due to poor chromatographic resolution by extracting the characteristic ions associated with target analytes. The ESI interface is the most efficient at promoting the ionization of polar substances, such as water-soluble vitamins, which, provided with acidic or basic groups, can be deprotonated or protonated. Moreover, ESI can ionize high molecular-weight compounds and produce multicharged pseudomolecular ions, such as [M þ nH]nþ or [MenH]ne. This feature is useful in the detection of cobalamins that give intense mono- and double-charged pseudomolecular ions [138]. Since all watersoluble vitamins respond in the positive ion mode, this has been chosen for the development of multivitamin methods [138,143e145]. Similarly, the APCI interface operating in the positive ion mode represents the best compromise [78,137,145] for the simultaneous detection of fat-soluble vitamins and carotenoids. The use of a mass spectrometer as chromatographic detector offers a great advantage in vitamin analysis: the possibility of simplifying the extraction procedure. The selectivity of the LCeMS technique reduces problems due to intrusive peaks from matrix components, while its sensitivity (ng or pg injected for real samples) allows the direct injection of an extract, eliminating the concentration step and the exposure to heat (most water-soluble vitamins posses low thermal stability). Sample preparation time is reduced as well as the duration of exposition to air and light (most vitamins and carotenoids are susceptible to these factors). Few analytical methods have been proposed for multivitamin determination in dietary supplements and food products by LC. The most emblematic multivitamin methods published in the last 10 years are summarized in Table 18.1.

TABLE 18.1

LC Methods for the Simultaneous Determination of Vitamins

Analytes

Matrix

Analytical technique

Separative conditions

Extraction procedure

Method performance Recovery (%)

Limits

Ref

B1: 91e103% B2: 88e103% B3: 93e100% B6: 90e107%

LODs in the range of 0.02 to 0.07 g/100 g

[141]

In the range of 0.003 to 0.038 mg/ml

[132]

WATER-SOLUBLE VITAMINS B1 (thiamin) B2 (riboflavin, flavin mononucleotide), B3 (nicotinamide) B6 (pyridoxal, pyridoxine)

Infant formulas, milk-based infant formula

HPLCeUVeFL (a) UV detection for: B3 l ¼ 258 nm FL detection for: B2 (0e5 min), lex/lem ¼ 450/510 nm B6 (5e20 min), lex/lem ¼ 290/390 nm (b) UV detection for: B1 l¼254 nm

Luna Prodigy ODS(3) column (150  4.6 mm; 5 mm), flow rate 2 ml/min. Isocratic elution with a wateremethanol mobile phase containing sodium dioctylsulfosuccinate and formic acid; pH adjusted to 2.8 for (a) B2, B3, and B6 or to 4.4 (b) for B1 determination.

TCA was added to reconstituted powdered sample (6.0 g) or liquid sample (30 ml); the solution was shaken for 15 min, and an aliquot was filtered and injected for LC determination.

B1 (thiamin), B2 (riboflavin), B3 (nicotinamide, nicotinic acid), B6 (pyridoxine, pyridoxal), B9 (folic acid) B12 (cyanocobalamin)

Milk- and soy-based infant formulas, cereal and fruit-based products

HPLCeDAD l ¼ 266 nm for B1, B2, B3, B9; l ¼ 326 nm for B6 l ¼ 361 nm for B12

RPeamide C16 column (250  4.6 mm; 5 mm), flow rate 1 ml/min. Gradient elution with 10 mM potassium dihydrogenphosphate pH ¼ 6 and acetonitrile.

Sample (5e10 g) was Higher hydrolyzed with 0.1 M than 90%. HCl (90 C, 30 min), cooled, adjusted to pH 4 with sodium acetate, and incubated with takadiastase (50 C, 2 hr). TCA was added (90 C, 10 min); sample was cooled, its pH adjusted to 6 with KOH, and diluted to known volume with mobilephase buffer. Aliquots were centrifuged, filtered, and injected into the LC column.

(Continued)

TABLE 18.1 LC Methods for the Simultaneous Determination of Vitaminsdcont’d

Analytes

Matrix

Analytical technique

B1 (thiamin), B2 (riboflavin), B3 (nicotinamide, nicotinic acid), B5 (pantothenic acid), B6 (pyridoxine, pyridoxal, pyridoxamine), B9 (folic acid)

Italian pasta and fortified pasta

(a) HPLCeUV B3 l ¼ 260 nm; folic acid l ¼ 280 nm (b) HPLCeFL derivatized B1 lex/lem ¼ 366/435 nm; B2 lex/lem ¼ 422/522 nm (c) HPLCeESI(+)eQqQc and HPLCeAPCI(+) eQqQ; 1 SRM transition. Separate acquisition for B9 and B5.

Method performance

Separative conditions

Extraction procedure

For (a) and (b): Supelcosil C18 (250  4.6 mm; 5 mm) Isocratic elution with: 60:40 (v/v) methanolesodium acetate buffer (pH 4.5) for B1 and B2; a mobile phase containing sodium acetate buffer, acetic acid, and sodium 1-heptanesulfonate monohydrate for B3; 640 mL of a sodium acetate and sodium sulfate (pH 5.3) mixture + 360 ml of acetonitrile for B9; For (c): Discovery RP-Amide C16 column (150  4.6 mm; 5 mm). Gradient elution with aqueous ammonium formate (pH ¼ 3.75) and methanol. Flow-rate of 0.75 ml/min.

For B1, B2, B3, and B6, Higher than 90% a ground sample (1 g) was  autoclaved with HCl (120 C, 30 min), cooled, diluted to known volume with aqueous ammonium acetate, vortexed, and centrifuged. The filtered supernatant was injected onto the LC column. For B5, a ground sample (4 g), was treated with acetate buffer (pH ¼ 5.6) and autoclaved (121 C, 15 min),. cooled, diluted to known volume with aqueous ammonium formate, vortexed, and centrifuged. The filtered supernatant was injected onto the LC column. For B9, ground sample (2.5 g) was mixed with a sodium phosphateesodium citrate/ ascorbate buffer (pH ¼ 8), heated, cooled, and incubated with papain and di-a-amylase (40 C, 2 hr). The solution was diluted to known volume with aqueous ammonium formate, vortexed, and centrifuged.

Recovery (%)

Limits

Ref

[133] LODs for LCeESIeMS/MS in the range of 0.5 to 5 mg/l; LODs for LCeAPCIe MS/MS in the range 0.5 to 2.7 mg/l.

B1 (thiamin), B2 (riboflavin), B3 (nicotinamide), B5 (pantothenic acid), B6 (pyridoxine), B9 (folic acid), B12 (cyanocobalamin), C (ascorbic acid)

Infant formulas, infant milk, vitaminenriched milk

HPLCeDADeFL FL detection for: B2 lex/ lem ¼ 400/520 nm B6 lex/ lem ¼ 290/410 nm UV detection for: B1 l ¼ 245 nm, B3 l ¼ 261 nm, B5 l ¼ 195 nm, B9 and C l ¼ 282 nm, B12 l ¼ 370 nm

B5 (pantothenic acid), B8 (biotin), B9 (folic acid), B12 (cyanocobalamin)

Fortified milk and rice powders

UPLCeESI(+)-QqQ 2 SRM transitions. IS: methotrexate

The supernatant was filtered and injected into the LCeMS system. For LCeUV analysis, the extract was loaded onto a preconditioned SAX cartridge. The cartridge was washed with a sodium sulfate and sodium chloride solution and adjusted to pH ¼ 5.3 with acetic acid. Elution with 1 ml of the washing solution (adjusted pH to 2.5 with TFA). Only for B5 Spherisorb ODS-2 C18 column To a reconstituted, homogenized solid sample (250  4.6 mm; (95-98%) and (0.5 g) or liquid sample (5.0 B12 (95-104%). 3 mm) thermostatted at 40 C. g), a precipitation solution Gradient elution with an aqueous phosphate buffer (pH (containing zinc acetate, ¼ 2.95) and methanol, at a flow phosphotungstic polyhydrated, and glacial rate of 1 ml/min. acetic acid in water) was added. The mixture was vortexed, centrifuged, filtered, and injected into the LC column. BEH C18 column (100  2.1 mm; 1.7 mm) thermostatted at 35 C. Gradient elution with 0.1% aqueous formic acid and 0.1% formic acid in acetonitrile, at a flow rate of 0.2 ml/min.

In the range of Sample (1 g) spiked with IS and aqueous ammonium 85e105%. acetate added. After magnetic agitation and ultrasonic bath extraction, 10 ml of chloroform were added. The solution was shaked again, centrifuged, filtered, and finally injected for UPLCeMS/MS analysis.

CCas in the range of 0.0030.580 mg/kg. CCbs in the range of 0.0050.950 mg/kg.

[142]

[143] Instrumental LODs in the range of 0.005 to 0.03 mg/l.

(Continued)

TABLE 18.1

LC Methods for the Simultaneous Determination of Vitaminsdcont’d Separative conditions

Extraction procedure

Analytes

Matrix

Analytical technique

B1 (thiamin), B2 (riboflavin), B3 (nicotinic acid, nicotinamide), B5 (pantothenic acid), B6 (pyridoxine, pyridoxal, pyridoxamine, pyridoxal 5’-phosphate, pyridoxamine 5’phosphate), B8 (biotin), B9 (folic acid), B12 (cyanocobalamin), C (ascorbic acid)

Tomato pulp, green and golden kiwi, maize flour

HPLCeESI(+)eQqQ 2 SRM transitions.

Alltima C18 column (250  4.6 mm; 5 mm). Gradient elution with 5 mM aqueous formic acid and 5 mM formic acid in acetonitrile at a flow-rate of 1 ml/min.

B1 (thiamin), B2 (riboflavin), B3 (nicotinamide), B5 (pantothenic acid), B6 (pyridoxine) B8 (biotin) B9 (folic acid)

NIST SRM 1849 infanteadult nutritional formula

HPLCeESI(+)eQqQ 1 SRM transition. IDMS. Isotopically labeled vitamins: 4,5,4-methyl[13C3]-thiamine chloride; 4,5-bis(hydroxymethyl)[13C4]-pyridoxine hydrochloride; 2,4,5,6[2H4]-nicotinamide; calcium pantothenate-

Reversed-phase LC: Synergi HydroRP column (250  2 mm; 4 mm). Gradient elution with: (a) 0.1% aqueous formic acid and 0.1% formic acid in acetonitrile or (b) aqueous formic acideammonium formate buffer (pH ¼ .7) and 0.1% formic acid in acetonitrile.

Method performance Recovery (%)

Limits

Ref

Homogenized sample (2 g), was mixed with BHT as antioxidant and, for tomato and kiwifruits, with diatomaceous earth (1 g). Extraction cartridge: first 0.5 g of C18 was introduced in a 6-ml syringlike glass tube, then the sample. Teflon frits placed above and below the sorbentefood matrix bed. Elution with 14 ml of ethanolewater (50:50, v/v) solution, and 100 ml injected into the LCeMS/MS system.

Maize flour: higher than 70%, with the exception of vitamin C (19%), pyridoxal-5’phosphate (40%), and B9 (40 %). Tomato pulp higher than 64%, except vitamin C (41%). Kiwi higher than 73%, except for nicotinamide (30%)

LODs in the range [138] of 0.68 to 239 ng/g; LOD of vitamin C was 30.22 mg/g/

Sample (0.2-0.5g) was spiked with labeled vitamins and extracted with a 0.1 M pH¼2 phosphate buffer. Centrifugation, filtration and injection into the LC system.

d

d

[144]

[13C15 6 N2]; biotin-2H2; 13 15 [ C4 N2]-riboflavin; folic acid-[13C5]/

HILIC: ZICeHILIC column (150  2 mm; 3.5 mm). Gradient elution with aqueous formic acideammonium formate buffer (pH ¼ 3.7) and 0.025% formic acid in acetonitrile. In the range of 98e104%

B2 (riboflavin), B3 (nicotinic acid), B5 (pantothenic acid), B9 (folic acid), C (ascorbic acid)

Honey

HPLCeUV B3 and C l ¼ 254 nm; B2, B5 and B9 l ¼ 210 nm.

Alltima C18 column (250  4.6 mm; 5 mm). Gradient elution with 0.025% aqueous trifluoroacetic acid and acetonitrile, at a flow rate of 1 ml/min.

Reconstituted, homogenized sample (10 g) was treated with NaOH and phosphate buffer (pH ¼ 5.5). The solution was diluted to known volume with water, filtered and injected into the LCeUV system.

B1 (thiamin), B2 (riboflavin), B3 (nicotinamide), B5 (panthothenic acid), B6 (pyridoxine)

NIST SRM 3280 multivitamine multielement tablets and SRM 1849 infanteadult nutritional formula

HPLCeESI(+)eMS, acquisition in SIM mode. IDMS. Isotopically labeled vitamins: nicotinamide[2H4]; thiamine chloride[13C3]; calcium pantothenate monohydrate-[13C3,15N]; pyridoxine hydrochloride[13C4].

Cadenza CD-C18 column (250  4.6 mm; 3 mm), thermostatted at 22 C. Gradient elution, with aqueous ammonium formate pH ¼ 4 and methanol, at a flow rate of 0.8 ml/min.

Sample (2 g) was treated with d extraction solution (1% aqueous acetic acid), placed on vortex and ultrasonic bath. Acetonitrile added and placed at e20 C overnight. Centrifuged, filtered, and injected into the LCeMS system.

LODs: B2, B3 0.25 mg/kg; B5 0.58 mg/kg; B9 0.15 mg/kg; C 0.1 mg/kg

[140]

d

[145]

(Continued)

TABLE 18.1

Analytes

LC Methods for the Simultaneous Determination of Vitaminsdcont’d

Matrix

Method performance

Separative conditions

Extraction procedure

HPLCeAPCI(+)eMS Acquisition in SIM mode IS: 5,7-dimethyltocol, ergocalciferol.

Nucleosil 100-5 column (250 mm  4.6; 5 mm). Isocratic elution with hexaneedioxine2propanol (96.7:3:0.3, v/v/v) at a flow rate of 1.45 ml/min.

Saponification (85 C, Higher than 96%. 30 min, reflux) of reconstituted sample (30 g) (for starch-containing products incubation at 45 C for 30 min with takadiastase) with ethanolic KOH, Na2S, and sodium acetate. Addition of sodium 1-pentanesulfonate to cooled sample, dilution to known volume with water, and SPE extraction (Chromabond XTR). Elution with hexane, evaporation to dryness and residue dissolved in the mobile phase, filtered, and injected into the LC column.

LODs in the range [78] of 0.08 and 1.4 ng injected.

HPLC-DAD l ¼ 326 nm for retinoids l ¼ 294 nm for tocopherols l ¼ 450 nm for carotenoids IS: b-cryptoxanthin

Spheri-5 ODS column (220  4.6 mm; 5 mm). Isocratic elution with acetonitrileemethylene chlorideemethanol (70:20:10, v/v/v) at a flow rate of 1.3ml/min. Gradient elution to confirm lutein and zeaxanthin, not resolved under isocratic conditions.

Individual forms: an aliquot (200 ml) was heated (85 C) and mixed; IS was added to 1ml sample before deproteinization with ethanol. Two extractions with a 0.01% BHT hexaneemethylene chloride (5:1, v/v) solution (ultrasonic bath, 5 min)

[134] LOQs < 0.03 mmol/l for retinol, retinyl acetate, and retinyl palmitate; 0.02e0.04 mmol/l for lycopene, a- and b-carotene, b-cryptoxanthin, lutein, and zeaxanthin.

Analytical technique

Recovery (%)

Limits

Ref

FAT-SOLUBLE VITAMINS A (retinol, 13 cis-retinol, 3-dehydroretinol) D (cholecalciferol) E (a-tocopherol)

Milk-based infant formula, infant cereals. SRM/RM 1846 milk.

A (retinol, retinyl acetate, Natural and fortified milk retinyl palmitate, bproducts carotene, a-carotene) E (a-tocopherol, g-tocopherol, tocopheryl acetate) Carotenoids (lutein, lycopene)

Liquideliquid extraction: recoveries of retinyl palmitate and a-tocopherol were >85%. Alkaline hydrolysis: recoveries of retinyl palmitate

A (retinyl acetate) E (tocopheryl acetate) D (cholecalciferol) K (phylloquinone)

Fortified milk, powdered milk.

HPLCeDAD l ¼ 230 nm for K, D, and E l ¼ 280 nm for A, E, D, and K l ¼ 300 nm for K, D, and E

Microsorb C18 column (250  4.6 mm; 5 mm) thermostatted at 30 C. Isocratic elution with a 3% (w/v) sodium dodecylsulphate aqueous solution pH ¼ 7 (phosphate buffer) with the presence of 15% (v/v) butyl alcohol. Flow rate of 2 ml/min.

and a-tocopheryl followed by centrifugation. Pooled organic phases were acetate were 95%. evaporated, reconstituted, filtered and injected into the LC system. Total A and E content: 1 ml sample was added of IS and pyrogallic acid and submitted to alkaline hydrolysis with methanolic KOH, vortexed, and placed in an ultrasonic bath (45 C, 15 min). Cooled sample was extracted twice with acqueous 5% NaCl, followed by isopropanol and hexaneemethylene (5:1, v/v). Organic phases were pooled, washed with water until pH < 7, evaporated, reconstituted, filtered, and injected into the LC system.

1000 bar), the separation efficiency has steadily increased with ultra high-pressure liquid chromatography (UHPLC) using multiple core-shell columns (Nth z 100,000) being the current state of the art. Such developments allow for either 1 min analyses with medium resolution or >1 hr separations with extremely high resolution. Not surprisingly then, UHPLC is the cornerstone of TCM QC. It first started with determining one or two marker compounds but evolved to the simultaneous quantitation of preferably all known bioactive constituents. This is the topic of Section 19.2.1. However, sometimes, the mode of action of a TCM has not been clarified; then, an overall image of preferably all constituents relative to a standard is the best solution. This is highlighted in Section 19.2.2. Combining two separation mechanisms or hyphenation with multiple detectors can increase the information content. Finally, LC can also be hyphenated with postcolumn (bio) chemical assays for faster identification of leads and examples are presented in Section 19.2.3.

19.2.1. Multicompound Quantitation Having many active synergistic constituents (a multicomponent character) interacting with multiple receptors in the human body is seen as a major advantage of TCMs and pivotal for the overall therapeutic effect [2]. However, the multicomponent character is also a major obstacle for the quantitation of components, identification of structures, explanation of the mode of action, and elucidating metabolic pathways. To date, hundreds of papers report on the simultaneous determination of multiple components in crude materia medica or finished TCMs [10,12e20]. Developing a quantitative multicompound assay is a challenging task, especially if several analytes from different compound classes need to be simultaneously analyzed. The validation may take many months. Electrospray ionizationemass spectrometry (ESIeMS) as a detector is becoming more common, as it combines high sensitivity with high selectivity. In our view, a state-of-the-art assay should fulfill as many of the following requirements as possible: • Simplicity, that is, using standard equipment, fast sample pretreatment, and low personnel input. • Green character, that is, using small volumes of nonhalogenated organic solvents. • Purity of reference substances determined by quantitative nuclear magnetic resonance (NMR) [21]. • Extraction efficiency of all analytes  95%.

522

19. APPLICATIONS OF LIQUID CHROMATOGRAPHY IN THE QUALITY

• Accuracy (absolute recovery after spiking) between 95% and 105%. • Relative recovery of entire analytical process  90%. • Interday precision for all analytes (expressed as relative standard deviation, RSD) < 5%. • Intermediate precision for all analytes < 10%. • Preferably, baseline resolution of all analytes, peak purity  98%. • Fast LC separation, preferably < 30 min. • Selective detection. • Linear calibration curves, r2  0.998. • Sample stability during LC analysis  98%. • Limit of quantification (LOQ) less than 10% of lowest analyte concentrations in plant or drug. • Easy quantitation (internal or external standardization). To date no published method meets all these demands. However, a good procedure is that reported by Fan et al., who simultaneously quantified hydroxysafflor yellow A, anhydrosafflor yellow B, five kaempferol glycosides, and 6-hydroxyapigenin 6-O-glucoside-7-Oglucuronide, together with two other active compounds, guanosine and syringin, in Carthamus tinctorius by LCeDAD (photodiode-array detector) [22]. The optimized extraction (water, 60  C, 45 min) was simple, green, and fairly selective, as no additional sample preparation other than filtration proved necessary. The 10 active compounds were separated on a 5 mm 250  4.6 mm C18 column in 70 min. Thus, the chromatography was rather traditional, that is, slow and solvent intensive. All compounds characterized by UV, NMR, MS, and HPLC showed good linearity (r2  0.9989) and the recoveries at three spiking levels varied from 94.8% to 105.4%. Repeatability and intermediate precision varied between 0.95e3.4% and 0.9e4.65%, respectively. Peak purity and sample stability were also checked. The quantitative analysis of those 10 components in 46 batches of C. tinctorius samples of different origin was carried out. The total amounts of 10 components varied from 4.88 to 37.55 mg/g, that is, a sevenfold variation. The concentration of the main active constituent, hydroxysafflor yellow A, varied almost ninefold. These results indicate that comprehensive QC of C. tinctorius is absolutely necessary. Another example is the simultaneous determination by LCeMSn of seven active compounds in a plasma sample collected after oral administration of a Zi-Shen pill (ZSP): xanthone glycosides (neomangiferin and mangiferin), timosaponins (timosaponin E1, timosaponin B-II and timosaponin B), and two alkaloids (palmatine and berberine) [23]. Ginsenoside Re (for xanthones and timosaponins) and tetrahydroberberine (for alkaloids) were chosen as internal standards (IS) for MS quantitation. Sample preparation consisted of only protein

19.2. LIQUID CHROMATOGRAPHIC ANALYSIS

523

precipitation with acetonitrile, centrifugation, and subsequent dilution with water. For the two xanthones, this was apparently not enough, as for neomangiferin and mangiferin, ~40% ion suppression and ~70% ion enhancement, respectively, were observed. Other procedures, such as liquideliquid extraction (LLE), were tried but gave unsatisfactory recoveries for the timosaponins. This shows that it is not easy and perhaps not desirable to aim at a quantitative procedure for multiple chemical classes. Chromatographic separation was achieved on a 3.5 mm 150 mm  2.1 mm Agilent Zorbax SB-C18 column in 9 min at a flow rate of 0.25 ml/min, which is both fast and economical. The MS detection was carried out either in negative (for xanthones and timosaponins) or positive ion (alkaloids) mode with multiple reaction monitoring (MRM). This method shows high selectivity and sensitivity and fair precision (3.8e9.2% interday) and accuracy ( 6.9e7.0% bias). The linear range was 5e1000 ng/ml for mangiferin; 0.5e100 ng/ml for neomangiferin, tinosaponin E1, timosaponin B-II, and timosaponin B; 0.05e10 ng/ml for palmatine and berberine. The limit of quantitation was 0.2 ng/ml for mangiferin; 0.5 ng/ml for neomangiferin, timosaponin E1, timosaponin B-II, and timosaponin B; and 0.05 ng/ml for palmatine and berberine. Extraction recoveries were greater than 80% for all analytes, with the exception of palmatine (range 73.9e77.5%) and berberine (range 64.7e69. 1%). Figure 19.1 shows the representative chromatograms of blank plasma and plasma obtained 4 hr after oral administration of ZSP. The concentrationetime profiles and pharmacokinetic parameters of these seven constituents in rats could be obtained with this method. These two figures are a bit low, again raising the question whether it is always wise to aim at a multiclass quantitation.

19.2.2. High Resolution Separation and Fingerprinting Ideally, LC separates mixtures into individual constituents. However, as the technique evolved and separation performance increased, in several cases it was discovered that isolated components previously thought to be pure were actually combinations of coeluting species. This means that one-dimensional LC cannot always give a satisfactory separation of each analyte. This fact, together with the need to evaluate complex samples like TCMs, has triggered the development of multiple hyphenation. Two main routes are used to resolve more peaks and produce “fingerprints”: Either LC is combined with other chromatographic systems (two-dimensional separation methods, see the next section) [24e26] or LC is coupled with a detector capable of providing spectroscopic data, that is, DAD, MS, or NMR (the section after that) [27e31].

524

19. APPLICATIONS OF LIQUID CHROMATOGRAPHY IN THE QUALITY

FIGURE 19.1 Representative MRM chromatograms of (I) berberine, (II, IS) tetrahydroberberine, (III) palmatine, (IV) mangiferin, (V) neomangiferin, (VI) timosaponin B, (VII) timosaponin B-II, (VIII) timosaponin E1, and (IX, IS) ginsenoside Re. Left: blank plasma sample; right: plasma sample collected from a rat 4 hr after oral administration of ZSP extract at a dose of 1.94 g/kg. Source: Reprinted with permission from [23]. Copyright (2010) Elsevier.

Fingerprinting analysis is considered an effective method to control the quality of herbal drugs, including TCMs, because it provides a better overall picture of the content and the relative concentration of constituents. In the specific chromatographic patterns, multiple compounds in TCMs can be recognized and allow for conclusions regarding the product0 s “phytoequivalence” [32]. The World Health Organization accepts fingerprint analysis as a methodology for the assessment of natural products [33]. LC is the most popular analytical technique for the fingerprinting analysis of TCMs. LC hyphenated techniques, with the help of data recognition methods, such as similarity analysis, exploratory data analysis, clustering, pattern recognition, and multivariate calibration, have been introduced to fingerprinting research to obtain more chemical information on TCMs [7,8,11]. Multidimensional LC for TCM Analysis The attractiveness of two-dimensional LC is its potential to significantly improve the separation power. The peak capacity for two-dimensional separations is defined as a linear combination of the peak capacities in both

19.2. LIQUID CHROMATOGRAPHIC ANALYSIS

525

separation dimensions. For example, when combining orthogonal LC modes with peak capacities of 50 and 100, theoretically, the resulting twodimensional capacity would be 5000 [34]. In reality, the two-dimensional LC performance is less, as columns are never 100% orthogonal and peaks are never evenly distributed over the entire separation space [35,36]. Problems related to two-dimensional LC include instrumental complexity, long run times, incompatibility of first- and second-dimension eluents, required data processing, and sometimes poor reproducibility. Nonetheless, a number of two-dimensional LC methods have been developed for the analysis of TCMs, and some are listed in Table 19.1. From Table 19.1, it follows that mostly commercial columns with different separation mechanisms have been combined, such as strong cation-exchange (SCX) chromatography  reversed-phase (RP) liquid chromatography [40,41] or RP  normal-phase (NP) liquid chromatography (-CN column) [38]. Also, a number of tailor-made columns immobilized with biological macromolecules have been used in the first dimension for TCMs analysis [25,43,45]. Due to a specific interaction with these bio-macromolecules, such separations may provide significantly more information on components. In most of the cases, the two columns have been combined off-line, which is simpler. However, a few are truly comprehensive (LC  LC). An example of the latter is the method developed by Wang et al. for the frequently used TCM Longdan Xiegan Decoction (LXD). In the first dimension, an immobilized liposome chromatography (ILC) column was used, and in the second dimension, a reversed-phase column coupled with DAD and atmospheric pressure chemical ionization (APCI)eMS [25]. The separation on the first dimension column was accomplished in 70 min, and equal volumes were collected at 10 min intervals. The separation on the C18 column was completed in 10 min to match the interval. A modulation time of 10 min should be considered too long, and some of the separation obtained in the first dimension might have been lost again. Nevertheless, with this method, more than 50 components in the ethyl acetate fraction of an LXD extract were separated. Figure 19.2 shows the contour plot of the ethyl acetate fraction of the LXD extract. Among them, eight flavonoids (3e10) and two iridoids (1 and 2) were identified by their UV and mass spectra. The first dimension retention of this two-dimensional LC system reflects interaction of analytes with membranes, a first prerequisite to penetrate membranes in vivo. As such it can be regarded as a kind of biological fingerprinting of a complex TCM. A disadvantage of this column is its moderate chromatographic efficiency. Fingerprinting and Chemometric Analysis Fingerprint chromatograms obtained by LC coupled to one or more detectors can be considered as characteristic profiles that together reflect

526

TABLE 19.1

Examples of Two-Dimensional LC for Analysis of TCMs Separation conditions

Detection

Purpose

Ref.

Ligusticum chuanxiong and Angelica sinensis

Comprehensive, Kromasil-CN (5 mm, 150 mm  4.6 mm)  Chromonolith Speed ROD (50 mm  4.6 mm)

DADeAPCIeMS

Separation and identification of compounds

[37]

Swertia franchetiana

Off-line, Intersil CN-3 (5 mm, 250 mm  4.6 mm)  XTerra MS C18 (5 mm, 150 mm  2.1 mm)

UVeESIeMS

Separation and identification of compounds

[38]

Dracaena cochinchinensis xylem

Off-line, Ultimate XB-CN (5 mm, 250 mm  4.6 mm)  Ultimate XB-C18 (5 mm, 250 mm  4.6 mm)

UV

Separation and determination of compounds

[39]

Lonicera caprifolium

Off-line, Kromasil-SCX (5 mm, ˚ , 150 mm  4.6 mm)  120 A ˚, Kromasil-ODS (5 mm, 120 A 150 mm  4.6 mm)

DADeAPCIeMS; MALDIeTOFeMS

Isolation and identification of compounds

[40]

Coptis chinensis

Comprehensive, SCX PolySEA column (3 mm, 150  0.3 mm)  Magic C18 AQ, (5 mm, 50 mm  0.3 mm)

UV

Separation and analysis of compounds

[41]

Corydalis yanhusuo

Off-line, XTerra Prep MS C18 OBDTM (5 mm, 100 mm  19 mm)  XTerra Prep MS C18 OBDTM column (5 mm, 100 mm  19 mm)

UV and MS- triggered collection

Isolation and purification of alkaloids

[42]

19. APPLICATIONS OF LIQUID CHROMATOGRAPHY IN THE QUALITY

TCMs

Off-line, Click oligo(ethylene glycol prep column (10e20 mm, 300 mm  70 mm)  XTerra Prep MS C18 OBDTM column (5 mm, 100 mm  19 mm)

UVeQTOFeMS

Purification of compounds

[43]

Isatis tinctoria

Off-line, XTerra MS C18 (5 mm, 150 mm  50 mm)  homemade polarcopolymerized C18 (5 mm, 150 mm  10 mm)

UVeMS

Purification of polar compounds

[44]

Longdan Xiegan decoction

Comprehensive, immobilized liposome chromatography ˚ , 150 mm column (5 mm, 300 A  4.6 mm)  C18 (100 mm  4.6 mm)

APCIeMS

Screening of membranepermeable compounds

[25]

Rheum palmatum

Silica-bonded HSA column ˚ , 150 mm  (5 mm, 300 A 4.6 mm)  Chromonolith Speed ROD (50 mm  4.6 mm)

DADeAPCIeMS

Analysis of biological fingerprinting

[45]

19.2. LIQUID CHROMATOGRAPHIC ANALYSIS

Dalbergia odorifera

527

528

19. APPLICATIONS OF LIQUID CHROMATOGRAPHY IN THE QUALITY

FIGURE 19.2

Two-dimensional chromatogram of the LC 3 LCeUV separation of a 5 ml injection of a Longdan Xiegan decoction. Peak assignment: (1) geniposide; (2) gentiopicroside; (3) oroxylin A-7-O-glucuronide; (4) wogonoside; (5) 7-O-b-D-glucuronopyranosylchrysin; (6) baicalin; (7) ononin; (8) liquiritin apioside; (9) 30 ,40 -dihydroxy5,6-dimethoxyflavone 7-O-glucoside; (10) liquiritin. Chromatographic conditions. ILC column (first dimension.): isocratic elution with 10 mM ammonium acetate solution (pH 6.8) at 0.05 ml/ min; C18 column (second dimension): linear gradient elution from 10% ACN to 70% ACN in 7 min, 7e10 min 10% ACN (re-equilibration) at 2.0 ml/min; detection wavelength, 210 nm. Modulation time: 10 min. Source: Reprinted with permission from [25]. Copyright (2009) Elsevier.

the complex chemical composition of the TCM sample. A typical example is the LCeDAD fingerprinting for QC of Qingkailing injections. This TCM shows good efficacy in case of blood circulation diseases, phlogistic disease, virosis, and some inexplicable fevers [26]. Fingerprints (Figure 19.3) of retention time versus UV response at multiple wavelengths (derived from a time versus wavelength contour plot) are used for a quality evaluation of injections produced by different manufacturers by means of principal component analysis (PCA). In comparison with a simple LCeUV profile at one wavelength, these combined fingerprints were more informative. A total of 14 Qingkailing injection samples (marked as 1e14) were collected from two TCMs manufacturers and a mixed sample of 11e14

19.2. LIQUID CHROMATOGRAPHIC ANALYSIS

529

FIGURE 19.3 Fingerprints of Qingkailing injection at various wavelengths as obtained from a time versus wavelength contour plot. (A) So-called max plot; (BeE) fixed wavelength chromatographic fingerprints; (F) chromatogram of standard chemicals at 254 nm. (1) uridine; (2) adenosine; (3) chlorogenic acid; (4) caffeic acid; (5) geniposide; (6) baicalin. Source: Reprinted with permission from [26]. Copyright (2005) Elsevier.

(from manufacturer B) in equal proportions (named authentic sample, AUS) were evaluated by this method. After performing PCA on the data of the fingerprints, the scores plot of PC1 versus PC2 showed that there were two distinctly different types of samples from manufacturer A, and these were both well separated from those of B. This suggests that not only were the products from manufacturer B different from those from manufacturer A but also that A sold two distinctly different types of Qingkailing injections, the difference being caused by their different preparation procedures. Fingerprinting Analysis in Chinese Pharmacopoeia 2010 The Chinese Pharmacopoeia is the book most involved with the QC of TCMs. It contains monographs (analytical protocols) for many materia medica (crude products) and finished drugs. As a supplementary technique in addition to marker compound quantitation (Section 19.2.1), official chromatographic fingerprinting protocols and the so-called specific chromatogram technique are included in the 2010 version. The LC fingerprintings of Shuanghuanglian injection, five solid oral dosage forms (Guizhi fuling, Tianshu, Yaotongning and Nuodikang capsules, and

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19. APPLICATIONS OF LIQUID CHROMATOGRAPHY IN THE QUALITY

Fufang Danshen Diwan) and six extracts (Notoginseng root extract, triol saponins; Notoginseng root and stem extract, total saponins; Salvia root and stem extract, total phenolic acids; Zedoary turmeric oil; and Centella extract) and the so-called LC-specific chromatograms of two solid oral dosage forms and six extracts have been standardized [33]. The major difference between fingerprints and specific chromatograms is the evaluation method used after obtaining the chromatogram. In the case of fingerprinting chromatograms, a number of specified characteristic peaks are used to evaluate the similarity to a reference sample by chemometric methods and software. The relative retention time (RTT) and relative peak area (RPA) of characteristic peaks versus a reference peak are used as evaluation parameters. The similarity analysis is based on the correlation coefficients (r) of the evaluation parameters of the sample fingerprint and the fingerprint of a reference sample, as supplied by the State Food and Drug Administration (SFDA) of China. The similarity ranges from 0% to 100% with 0% meaning no similarity between the evaluated fingerprinting and the reference fingerprint. For a regular product, the similarity (evaluation index) should be higher than 90%. For example, a fingerprint of Fufang Danshen Diwan is obtained by means of UHPLC with an Acquity HSS T3 1.8 mm 100  2.1 mm column in combination with a 10 min gradient at 0.4 ml/min at 40  C. Mobile phase A is water containing 0.02% H3PO4, B is watereacetonitrile (20:80, v/v) containing 0.02% H3PO4. The detection wavelength is 280 nm. Eight characteristic (common) peaks in the sample according to the reference fingerprint (Figure 19.4) recorded in the Pharmacopoeia are evaluated by chemometric software (reference peak: salvianic acid A (peak 1)) [33]. Because standardized reference samples are not always available for reference fingerprinting, the so-called specific chromatogram method also is used to discriminate between high- and low-quality TCMs. The specific chromatogram approach uses only the RTT of several characteristic peaks versus a reference peak to evaluate the quality similarity of TCMs. In this case, no software is used. The relative deviation

FIGURE 19.4 Reference fingerprint HPLCeUV profile of Fufang Danshen Diwan as occurring in the Chinese Pharmacopoeia 2010 [33]; (1) salvianic acid (reference compound).

19.2. LIQUID CHROMATOGRAPHIC ANALYSIS

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(RD) of the retention times should be less than 5% of the specified values listed in the Pharmacopoeia. For example, the specific chromatogram of ginseng root extract is obtained by using a linear gradient of water with 0.1% H3PO4 (A) to acetonitrile (B) on a C18 column with UV detection at 203 nm. There are seven characteristic peaks: ginsenosides Rg1, Re, Rf, Rb1, Rc, Rb2, and Rd in the chromatogram. According to the Pharmacopeia, the specified values of the characteristic peaks Rf, Rb1, Rc, Rb2, and Rd relative to the reference peak Rd are 0.84, 0.91, 0.93, 0.95, and 1.00, respectively. This means that, if the retention time of Rd is 60 min, the peak with retention time from 47.88 min to 52.92 min (0.84  60 min  2.52 min) can be considered as ginsenoside Rf [33]. Although it is a positive QC development that fingerprinting methods have been introduced in the Chinese Pharmacopoeia 2010, at the same time it should be remarked that the techniques and evaluation are still somewhat primitive. Neither of the two proposed methods takes the absolute concentration of constituents into consideration. The specific chromatogram method also does not take the relative concentration into account. From a quality control point of view, it is obvious that differences in absolute as well as relative concentrations influence the quality of a TCM. A good LC fingerprint can provide this information and enables discrimination between samples. Therefore, improved chemometric evaluation protocols are necessary.

19.2.3. On-Line Liquid Chromatographic Mining of Active Compounds Finding new leads in TCMs for new drugs remains high on the agenda. To date the pharmaceutical industry is facing an unprecedented challenge: more funds are invested, but fewer new drugs are generated. This predicament is partially attributed to the limitations of the current onedrugeone-target paradigm in drug discovery [46]. Therefore, more attention is given to multicomponent therapeutics, which incorporate two or more active ingredients in one drug to hit multiple targets, such as TCMs. It is furthermore a fact that, in the past 30 years, around half the new drugs that came to the market were natural products, natural products derivatives, or synthetic analogs of natural products. For drug screening, the hit rate in natural products libraries (0.3%) is significantly higher than in combinatorial and synthetic compound libraries (0.001%). When TCM libraries are used, the hit rate can be as high as 0.5% and the time to market is shorter [47,48]. As a result, the overall costs of development can be significantly reduced. Therefore, knowing which compounds are responsible for the beneficial effects of TCMs is indispensable for finding new leads, elucidating the mode of action, and indirectly for achieving better QC. For the screening

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of TCMs for active compounds, pharmacological models involving experimental animals, organ and tissue models, cellular models, receptors, and enzymes have been employed. However, off-line screening of extracts does not provide information on individual compounds and sometimes suffers from false positive or negative results. To solve this problem, various approaches have been proposed. In biological fingerprinting analysis (BFA), a TCM is incubated with the target macrobiomolecule, and LC fingerprints before and after the interaction are compared. A decrease is indicative of an interaction [49,50]. In bioaffinity chromatography, drug targets, such as enzymes, antibodies, liposomes, DNA, or a cell membrane, are immobilized on a stationary phase and any bioactive components are chromatographically retarded due to their specific interactions (see the next section). The third and most advanced option is LC-based on-line screening (or high resolution screening) [51], in which separated analytes react post column in an assay. This allows for the direct pinpointing of active constituents in complex mixtures (see the section after next). Examples are screening for the presence of enzyme inhibitors [52] or antioxidants [53]. Bioaffinity Chromatography To screen for bioactive components in a complex matrix by means of affinity chromatography, usually frontal affinity chromatography (FAC) with mass spectrometric detection is used [54e57]. An example of FAC in the TCMs field is the screening of the extract of Phyllanthus urinaria for interaction with immobilized polyclonal antibodies against “compound A” [54]. Compound A is a strong inhibitor of hepatitis C virus (HCV) NS3 protease; hence, each clone of the antibody contains some similarity with the active site of the protease. The Phyllanthus extract containing potential ligands was continuously infused through the column. The order of elution of compounds depends on their affinities for the receptor with the compound showing the strongest interaction eluting last (Figure 19.5). Brevifolin, brevifolin carboxylic acid, corilagin, ellagic acid, and phyllanthusiin U in the TCM showed activity and their high inhibitory activities could be confirmed in off-line analyses. Liquid Chromatographic High Resolution Screening Coupling LC with a postcolumn reactor is a powerful method for the fast individual screening of active components in complex mixtures [58e63]. Due to the superior separation power of LC, no tedious isolation steps are necessary. High resolution screening consists of LC analysis, reaction of analytes with target solution (e.g., an enzyme) in a first reaction coil, and interaction of a reporter ligand or substrate with the target in a second coil (Figure 19.6). Sometimes, the two coils are combined. Full integration of the bioassay into the LC flow can be viewed as the ultimate

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FIGURE 19.5 Frontal affinity selected ion chromatograms of each compound in a Phyllanthus urinaria extract. The infusion flow rate was 5 ml/min. ESIeTOFeMS was used to measure the concentration of each compound: (3) brevifolin, (4) brevifolin carboxylic acid, (11) corilagin, (5) ellagic acid; (13) phyllanthusiin U. Source: Reprinted with permission from [54]. Copyright (2003) American Chemical Society.

achievement in interfacing bioactivity and chromatography. Parallel spectrometric detectors (MS, DAD, or NMR) provide chemical characterization. De Jong et al. developed an acetylcholinesterase (AChE) assay for the screening of AChE inhibitors in natural extracts after LCeMSn [64]. The effluent of the LC column was mixed with AChE solution in the first reaction coil, allowing putative inhibitors to bind to AChE. The substrate solution (30 mM acetylcholine dissolved in 97.5% 10 mM ammonium bicarbonate, pH 7.8, and 2.5% methanol) was delivered by a superloop, and allowed to react in the second reaction coil. In the absence of an inhibitor, the substrate was converted to acetic acid and product (choline).

FIGURE 19.6 Principle of high resolution screening as used in the detection of acetylcholineesterase inhibitors in a Narcissus extract. Source: Reprinted with permission from [64]. Copyright (2006) Elsevier.

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The reactants were subsequently analyzed by ESIeMS. A 10 mM uracil solution was used as a system monitoring compound (SMC) to detect any ion suppression. A significant decrease in the substrate and a matching increase in the product trace were observed by starting the AChE pump. A positive peak in the substrate trace (Ach) and a negative peak in the product trace (Ch) matched the peak observed in the galanthamine

FIGURE 19.7 Coupling of a gradient LC system to an MS-based AChE inhibitory assay. Trace A, m/z 288 (galanthamine in Narcissus extract); Trace B, system monitoring compound (SMC) detected at m/z 113; Trace C, product trace (choline) m/z 104; Trace D, substrate trace (acetylcholine) m/z 146; Trace E, total ion current (TIC). Source: Reprinted with permission from [64]. Copyright (2006) Elsevier.

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(AChE inhibitor) trace (Figure 19.7). Therefore, this method could successfully detect galanthamine as the active AChE-inhibiting peak in the extract of Narcissus cv “Bridal Crown” bulbs. Narcissus species are used in TCMs [65].

19.3. CONCLUSIONS From the examples presented in the previous sections, it is evident that LC was, is, and will remain in the foreseeable future the key technique for quality control, lead identification, and metabolomics of herbs and phytopharmaceuticals in general and TCMs in particular. The main advantage of LC over other chromatographic techniques, like gas chromatography (GC), thin-layer chromatography (TLC), centrifugal partition chromatography (CPC), and capillary electrophoresis (CE), is its universal scope in terms of analytes, high separation power, reproducibility, sensitive, and flexible detection (refractive index, UV, ELSD, fluorescence, electrochemical, MS, NMR) and the ease of hyphenation (bioassays). However, this is not equal to stating that everything is perfect and no more progress or development can be expected. What are currently the shortcomings in TCMs analysis by LC? Which developments can we expect in the coming years? In general, the LC, as applied to plant analysis, is still rather traditional, traditional meaning here 60 min analyses on 5 mm 250  4.6 mm columns at 1 ml/min. Even though TCM samples are often complex, both time and solvent use can and will decrease through the application of smaller diameter, shorter columns with smaller particles (UHPLC). Short ~2.5 mm core shell LC columns might even provide UHPLC resolution while running on standard 400 bar LC equipment. Modern LC column technology will make the analyses also “greener”, another aspect that will receive more attention as analytical chemists also are responsible for a better environment. However, as greener usually also means cheaper, money is another incentive. Still another aspect that deserves continued attention is proper validation. Validation is tedious and expensive but, if neglected, the outcome is meaningless. Key issues that should always be checked, and also for TCMs analyses, are the absolute purity of reference substances, extraction efficiency, absolute and relative recovery, and precision. We expect further improvements in the validation of TCM analyses in the coming years. Proper data processing (chemometrics) requires more consideration. For instance, currently the Chinese Pharmacopoeia uses only relative retention times and sometimes relative ratios (i.e., qualitative information) to evaluate LC profiles. However, the absolute concentrations of components are clearly also of importance for the quality of TCMs. Thus,

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the Pharmacopoeia methodology (chemometric evaluation software) should be adapted in such a way that also takes into account such facets. With LCeDADeMS becoming more affordable, multidetector fingerprints also become a more common and proper, integrated evaluation of the two chromatograms for QC, which again requires smart software. The same holds for the application of multidimensional chromatography. LCLC will become more common but the huge amount of sometimes four-dimensional data (time  time  concentration  mass spectrum) require not only automated software for evaluation but also software for converting hundreds of individual chromatograms into a four-dimensional space. On the other hand, greater complexity is not always better and carries a bigger risk of increased downtime and black box analyses, so it is good to think carefully if LCLC is really needed for QC. TCMs are a fruitful library for new drug leads, therefore, the mining of TCMs is on the rise. In Section 19.2.3, various LC approaches that allow directly obtaining bioactivity and spectroscopic data on individual compounds are highlighted. Certainly, high resolution screening (HRS) will expand in the coming 10 years, but it is good to realize that on-line assays also have disadvantages. Slow or large assays requiring, for example, bacteria or whole experimental animals cannot be hyphenated to LC, and the organic modifier necessary for RPLC is difficult to combine with fragile enzymes. Finally, especially for TCMs, HRS has the intrinsic drawback that it provides no information on synergism. Thus small scale high-throughput off-line assays, such as those that, after parking an entire HPLC run in a 96 or 384 well plate, will remain popular as well. Often, the used assay provides the first information on the mode of action of active TCMs constituents. In turn, there will be a spin-off towards QC. If the pharmacologically active constituents are known, they should be included in multicompound LC quantitations. A lot of development is expected in this field in the coming decennium. The last remark to make is that, no matter how fantastic the sample pretreatment and the ensuing LCeMS separation and quantitation, QC controls only information; that is, it can only reject a poor drug and not remedy it. A good drug starts with correctly identified, high-quality plants, properly grown, harvested, and stored (GAP ¼ good agricultural practice) that are blended and processed in the right fashion (GMP ¼ good manufacturing practice).

Acknowledgments We thank Wageningen University for partial funding of this research via a graduate student fellowship to Y.S. as well as the National “863” Research Foundation of China (2010AA023001) and the national Natural Science Foundation of China (20927005, 21275049) for financial support.

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C H A P T E R

20 Analysis of Neurotransmitters and Their Metabolites by Liquid Chromatography K.E. Bosse, J.A. Birbeck, B.D. Newman, T.A. Mathews Department of Chemistry, Wayne State University, Detroit, Michigan O U T L I N E 20.1. Introduction 20.1.1. Analytical Considerations for Microdialysis 20.1.2. Liquid Chromatography Methods for Microdialysis and Tissue Content

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20.5. Purines: Adenosine Triphosphate and Adenosine

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20.8. Multianalyte Monitoring of Neurotransmitters from Diverse Classes

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20.9. Clinical Applications of Microdialysis Sampling and Liquid Chromatographic-Based Analysis 20.9.1. Cerebral Ischemia 20.9.2. Pharmacokinetic Analysis of Brain Cancer Therapies

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References

596

20.1. INTRODUCTION A fundamental objective of bioanalytical chemistry is to quantify biological molecules in their native environments. This goal is challenging because implantation of a sensor or device inherently perturbs the endogenous environment (i.e., tissue damage) and can lead to fouling of the surface of the sensor/electrode. In-vitro models, such as cell lines or liposomes, offer viable alternatives that circumvent some complications associated with complex in-vivo systems. However, without native complexity, crucial findings regarding the inherent regulation of and contextual interactions between biomolecules remain undiscovered. Therefore, it is imperative that sampling techniques and instrumental approaches are developed that demonstrate efficacy permissible for invivo monitoring. The brain is composed of numerous neurotransmitters, neuromodulator proteins, and small molecules that are essential to transmit electrical signals across neurons via chemical communication. This neuronal cross-talk governs the execution of a wide variety of functions, including movement, mood, thoughts, and involuntary actions (e.g., digestion and breathing) [1]. Chemical signaling molecules typically belong to one of the following classes of neurotransmitters: biogenic amines (monoamines), amino acids, peptides, purines, or other small molecules, such as nitric oxide [2]. Analytical methods have provided valuable information regarding the diverse roles of neuroactive molecules to maintain physiological function in “normal, healthy” controls and dysregulation of these systems in “disease” states. Several methods are available for the observation of brain chemistry, including positronemission topography (PET), magnetic-resonance imaging (MRI or functional MRI), fast-scan cyclic voltammetry (FSCV), biosensors, and sampling methods like microdialysis. Microdialysis has the distinctive ability to sample multiple analytes at their equilibrium concentration for basal level estimations through coupling to separation techniques, such as high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) and detections systems, such as electrochemical, fluorescence, or mass spectrometry (MS). The chemical specificity imparted by these analytical techniques enable researchers to accurately analyze multiple biochemical molecules simultaneously, at extremely low

20.1. INTRODUCTION

543

FIGURE 20.1 Representative setup for in-vivo microdialysis using an LC method.

concentrations, over long periods of time, and without loss of fluid. While the multianalyte capabilities of analytical systems coupled to microdialysis offer powerful advantages, multiple analyte monitoring often leads to poor temporal resolution, not suitable for dynamic measurements. Microdialysis, as illustrated in Figure 20.1, is one of the most popular techniques for sampling and analyzing biological molecules from living tissue. Microdialysis techniques have been employed in many fields, including pharmacokinetics, toxicology, bioprocessing, and neuroscience [3]. Dialysis utilizes a semi-permeable membrane that allows endogenous biological molecules to diffuse down their concentration gradient into the probe in order to sample fluid from the surrounding environment (Figure 20.2). Numerous microdialysis probes are available that are amenable for sampling structurally diverse analytes from a variety of tissue types [3]. The most common type of microdialysis probe for brain tissue sampling is a concentric probe, which contains two tubes placed inside each other. The inner tube contains the exogenous perfusate that flows into the brain. The perfusate matches the invivo matrix chemical composition of the fluid in the brain (artificial cerebral spinal fluid; aCSF) in the absence of neurotransmitter analytes. The outer tube allows analytes, including neurotransmitters, to diffuse

544

20. ANALYSIS OF NEUROTRANSMITTERS AND THEIR METABOLITES

FIGURE 20.2 Sampling from a concentric microdialysis probe. The left side represents the microdialysis probe, while the right side represents the magnified image of the tip of the microdialysis probe. The microdialysis probe is continuously perfused with artificial cerebral spinal fluid (aCSF) at a low flow rate through the inlet. At the microdialysis tip is a concentration gradient for the neurotransmitters of interest. The probe is devoid of neurotransmitters, leading to a concentration gradient between the probe and the extracellular space. Small molecules in the extracellular space, typically less than 6000 Daltons, easily diffuse across the microdialysis membrane, while larger molecules, such as proteins, are excluded. Once small molecules are inside the probe, they are swept away and collected for either on- or off-line analysis.

down their concentration gradient into the probe for collection in dialysate. The probe has an internal diameter (i.d.) of approximately 250e350 mm and the membrane material is often composed of polycarbonate or polyether to specifically exclude macromolecules [3,4]. Excluding large molecular-weight molecules minimizes the need for sample preparation, involving centrifugation or protein precipitation, prior to analyzing the samples by affording a means of intrinsic sample purification. While in-house fabrication of dialysis probes can reduce the cost of these experiments and is used to design probes for specific applications, a majority of studies use probes purchased from vendors, due to the broad commercial availability and difficulty in fabricating miniaturized probes for mouse studies (Table 20.1).

TABLE 20.1 Concentric Microdialysis Probes Membrane material

Membrane length (mm)

Membrane diameter (mm)

Molecular weight cutoff (kDa)

CMA/Microdialysis http://www.microdialysis.com/us/home 800-232-2380

Cuprophane (CMA/7 and 11)

1e2 (CMA/7) 1e4 (CMA/11)

0.24

6

Polyarylethersulphone (PAES)

1e4

0.5

10 or 20

Eicom http://www.eicom-usa.com/1.html 888-680-7775

Artificial cellulose “cuprophan” (CX-I)

0.5e10

0.22

50

BASi http://www.basinc.com/index.php 800-845-4246

Polyacrylonitrile (PAN): “BR style”

2 or 4

0.32

30

Cellulosic: “MBR style”

1, 2, or 4

0.22

38

Polyacrylonitrile (PAN)

1e6

0.34

45e50

Regenerated cellulose (RC)

1e6

0.216

13 or 18

Custom build your probe

Customer Specify 2e5

N.S.

13 or 18

Brainlink http://www.brainlink.nl/index.php?pag¼1 [email protected] Plastics One http://www.plastics1.com/PCR/PreclinicalResearch-Components.php 540-772-1166

20.1. INTRODUCTION

Vendor

(Continued)

545

546

Concentric Microdialysis Probesdcont’d Membrane length (mm)

Membrane diameter (mm)

Molecular weight cutoff (kDa)

PES (MAB 2) Requires push-pull of pump

1e4

0.6

35

PES or cuprophane (MAB 4)

1e4

0.2

6

PE (MAB 5)

1e4

0.3

3 MDa

PES (MAB 6)

1e4

0.6

15

Dry PES (MAB 8)

1 or 2

Dry PES (MAB 9)

1, 2, 3, or 4

0.6

6

Polyacrylonitrile (PAN)

Custom

0.36

20

Vendor

Membrane material

Microbiotech http://www.microbiotech.se/index.htm [email protected]

Synaptec http://synaptechnology.com/index.html 248-210-8703 N.S. ¼ Not specified.

6

20. ANALYSIS OF NEUROTRANSMITTERS AND THEIR METABOLITES

TABLE 20.1

20.1. INTRODUCTION

547

Analysis of dialysate samples is the same whether collection is performed on- or off-line. On-line sample collection is used to automate this process for microliter volumes (typically < 20 ml) through direct delivery of the microdialysis sample to the analytical system for separation and detection. Automated dialysate collection minimizes the impact of issues arising from handling microliter volumes, such as sample loss, evaporation, degradation, and surface tension [5]. Further, real-time feedback of experimental results is achieved through continuously collecting the dialysate sample. A challenge with on-line sampling is that the temporal resolution of sample collection is dependent on the analysis time of the chromatographic and detection system employed. Taking into account these complications with on-line sampling, many microdialysis studies have collected samples off-line, which are frozen and analyzed at a later point in time. With off-line collection of microdialysate, the temporal resolution of analyte sampling is not affected by the analysis method time, which is beneficial in cases were lengthy derivatization or long separation time is required. Often simultaneous, multiprobe dialysis experiments performed in a single animal are collected off-line to maximize sample throughput. Limitations to off-line collection of dialysis samples involve the handling of microliter samples, which can be difficult to evaluate due to surface tension or evaporation, and the chemical stability of the analyte of interest for later analysis [5].

20.1.1. Analytical Considerations for Microdialysis The analytical criteria for on-line microdialysis sampling is more stringent than off-line sampling, but in either case the overarching objective is to rapidly collect and analyze microliter samples that typically have subnanomolar concentrations of the neurotransmitter of interest. Optimizing the microdialysis sampling method requires consideration of the following analytical parameters: (a) analyte recovery determined by perfusion flow rate, (b) temporal resolution of microdialysis sampling, and (c) figures of merit, such as sensitivity, limit of detection, linearity of calibration curves, and linear dynamic range. Analyte Recovery Determined by Perfusion Flow Rate An inherent advantage of using in-vivo microdialysis as the sampling method is that the animal serves as its own control. The microdialysis experiment starts with the collection of baseline samples to determine baseline resting neurotransmitter levels. When there is less than ~10% variability in baseline neurotransmitter concentration, the effect of various pharmacological and behavioral manipulations on the

548

20. ANALYSIS OF NEUROTRANSMITTERS AND THEIR METABOLITES

levels of the neurotransmitter of interest can be evaluated over time. Experiment-induced changes in neurotransmitter levels are either reported as an absolute value (concentration) or relative (percent) change compared to baseline values. Neurochemical measurements from in-vivo microdialysis are often uncorrected for recovery, because it is assumed that the probe performs consistently throughout the duration of the experiment. One of the most important issues to consider when making microdialysis measurements is the recovery of the analyte from the dialysis probe and the numerous factors that can influence recovery. The factor that the experimenter has the most control over is perfusion flow rate, which can regulate percent recovery, sample volume, and throughput capabilities related to the temporal resolution of the method. Employing a low perfusion flow rate (