Bioanalytical Separations, Volume 4 (Handbook of Analytical Separations)

Bioanalytical Separations H A N D B O O K OF A N A L Y T I C A L S E P A R A T I O N S Series Editor: ROGER M. SMITH ...

Author: Ian D. Wilson

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Bioanalytical Separations

H A N D B O O K OF A N A L Y T I C A L S E P A R A T I O N S Series Editor: ROGER M. SMITH

In this series:

Vol. 1: Separation Methods in Drug Synthesis and Purification Edited by K. Valk6 Vol. 2: Forensic Science Edited by M.J. Bogusz Vol. 3: Environmental Analysis Edited by W. Kleib6hmer Vol. 4: B ioanalytical Separations Edited by I.D. Wilson

HANDBOOK

OF

ANALYTICAL

SEPARATIONS

-

VOLUME

4

9 l y " tlcal Separations " B~oana

Edited

by

IAN D. WIL S ON AstraZeneca Macclesfield, U.K.

2003 ELSEVIER Amsterdam - Boston - Heidelberg - London - New York - Oxford - Paris San Diego - San Francisco - Singapore - Sydney - Tokyo

ELSEVIER SCIENCE B.V. Sara Burgerhartstraat 25 EO. Box 211, 1000 AE Amsterdam, The Netherlands 9 2003 Elsevier Science B.V. All rights reserved. This work is protected under copyright by Elsevier Science, and the following terms and conditions apply to its use: Photocopying Single photocopies of single chapters may be made for personal use as allowed by national copyright laws. Permission of the Publisher and payment of a fee is required for all other photocopying, including multiple or systematic copying, copying for advertising or promotional purposes, resale, and all forms of document delivery. Special rates are available for educational institutions that wish to make photocopies for non-profit educational classroom use. Permissions may be sought directly from Elsevier's Science & Technology Rights Department in Oxford, UK: phone: (+44) 1865 843830, fax: (+44) 1865 853333, e-mail: [email protected]. You may also complete your request on-line via the Elsevier Science homepage (http://www.elsevier.com), by selecting 'Customer Support' and then 'Obtaining Permissions'. In the USA, users may clear permissions and make payments through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA; phone: ( + 1) (978) 7508400, fax: ( + 1) (978) 7504744, and in the UK through the Copyright Licensing Agency Rapid Clearance Service (CLARCS), 90 Tottenham Court Road, London W1P 0LP, UK; phone: (+44) 207 631 5555; fax: (+44) 207 631 5500. Other countries may have a local reprographic rights agency for payments. Derivative Works Tables of contents may be reproduced for internal circulation, but permission of Elsevier Science is required for external resale or distribution of such material. Permission of the Publisher is required for all other derivative works, including compilations and translations. Electronic Storage or Usage Permission of the Publisher is required to store or use electronically any material contained in this work, including any chapter or part of a chapter. Except as outlined above, no part of this work may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without prior written permission of the Publisher. Address permissions requests to: Elsevier's Science & Technology Rights Department, at the phone, fax and email addresses noted above. 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. First edition 2003 Library of Congress Cataloging in Publication Data A catalog record from the Library of Congress has been applied for. British Library Cataloguing in Publication Data A catalogue record from the British Library has been applied for.

ISBN: 0-444-50658-6 ~) The paper used in this publication meets the requirements of ANSI/NISO Z39.48-1992 (Permanence of Paper). Printed in The Netherlands.

Preface "Nothing tends so much as to the advancement of knowledge, as the application of a new instrument. The native intellectual powers of men in different times are not so much the causes of the different success of their labour, as the peculiar nature of the means and artificial resources in their possession"

Sir Humphrey Davy, 1840 In the context of this volume "bioanalysis" has a very specific meaning. It is the analysis of drugs and their metabolites in biological fluids. There is probably no field which has benefited from the introduction of new instrumentation and ways of analysing samples as much as this type of analysis. Today the laboratories of pharmaceutical companies, and the contract houses that serve them, are filled with sophisticated HPLC-MSMS systems devoted to the analysis of compounds at concentrations unachievable by previous generations of analysts. Thus, detection and quantification at concentrations below 1 nanogram per millilitre are now commonplace, and the pace of innovation seems still to be increasing. The bulk of these advances in bioanalysis have resulted from the development and implementation of robust and sensitive HPLC-MS interfaces. The high capital cost of much of this instrumentation has also led to increasing pressures on improving the efficiency of method development and instrument usage. The application of these new HPLC-MS-based methods is covered in this volume in chapters on forensic bioanalysis and the role of this way of analysis in drug discovery. However, despite the success of such devices, all of the problems of bioanalysis have not been solved by their introduction, and there is a continuing need for sustained innovation. In particular the low concentrations, and the presence of large amounts of endogenous interferences in biological fluids and tissues, has meant that sample preparation techniques remain of prime importance to the bioanalyst seeking the highest sensitivities and specificity. This activity is reflected in several contributions on the theme of sample preparation. Chromatography is still important, even when the detector is as sensitive and specific as the mass spectrometer is claimed to be, and new phases of the types described in this volume, providing chiral separations or improvements in conventional chromatography, are always needed. In addition the linking of spectrometers other than mass spectrometers has been an important area of innovation, and this is reflected in contributions on HPLC-NMR and HPLC-ICPMS. Finally, it has always to be remembered that, because of the importance of bioanalytical data in the generation of regulatory submissions, such work is subject to

vi

Preface

very precise and challenging regulatory control. The needs of the regulators for bioanalytical separations are therefore also covered in the final contribution to the volume. I would like to thank all of the authors who have contributed to this work for their time, patience and expertise. The collected wisdom and scholarship that these chapters reflect have been a source of considerable pleasure and education to me as I have put the volume together. Ian D. Wilson

AstraZeneca Macclesfield, U.K.

vii

Contents P refa c e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 1.

1.1 1.2

1.3

1.4 1.5 1.6 1.7

New developments in integrated sample preparation for bioanalysis

M.W.J. van Hour, H.A.G. Niederl~inder, R.A. de Zeeuw and G.J. de Jong . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromatography-based extraction techniques . . . . . . . . . . . . . . . . 1.2.1 Solid-phase e x t r a c t i o n - gas chromatography . . . . . . . . . . . . 1.2.1.1 General aspects of SPE . . . . . . . . . . . . . . . . . . . 1.2.1.2 Applications of S P E - G C . . . . . . . . . . . . . . . . . . 1.2.1.3 Remarks regarding the applicability of S P E - G C . . . . . 1.2.2 Liquid c h r o m a t o g r a p h y - gas chromatography . . . . . . . . . . . 1.2.2.1 General aspects of L C - G C . . . . . . . . . . . . . . . . . 1.2.2.2 Applications of R P L C - G C . . . . . . . . . . . . . . . . . 1.2.2.3 Remarks regarding the applicability of R P L C - G C . . . . 1.2.3 Turbulent-flow chromatography . . . . . . . . . . . . . . . . . . . 1.2.3.1 General aspects of TFC . . . . . . . . . . . . . . . . . . 1.2.3.2 Applications of TFC . . . . . . . . . . . . . . . . . . . . 1.2.3.3 Remarks regarding the applicability of TFC . . . . . . . . Diffusion-based extraction techniques . . . . . . . . . . . . . . . . . . . . 1.3.1 Solid-phase microextraction . . . . . . . . . . . . . . . . . . . . . 1.3.1.1 General aspects of SPME . . . . . . . . . . . . . . . . . 1.3.1.2 Applications of S P M E - L C . . . . . . . . . . . . . . . . . 1.3.1.3 Applications of S P M E - M S . . . . . . . . . . . . . . . . 1.3.1.4 Remarks regarding the applicability of S P M E . . . . . . . 1.3.2 Membrane-based sample preparation techniques . . . . . . . . . . 1.3.2.1 General aspects of membrane-based techniques . . . . . . 1.3.2.2 Porous membrane techniques . . . . . . . . . . . . . . . 1.3.2.3 Non-porous membrane techniques . . . . . . . . . . . . . 1.3.2.4 Remarks regarding the applicability of membrane-based techniques . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . List of abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 1 4 4 4 5 7 7 7 9 11 12 12 13 14 16 16 16 18 24 24 26 26 28 32 35 35 38 38 39

viii

Contents

Chapter 2. Solid-phase extraction on molecularly imprinted polymers 2.1 2.2

2.3

2.4

2.5 2.6

Lars I. A n d e r s s o n and L e i f S c h w e i t z . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I m p r i n t preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1 R e m o v a l of t e m p l a t e m o l e c u l e s . . . . . . . . . . . . . . . . . . . 2.2.2 C h o i c e of t e m p l a t e . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3 F o r m a t of p o l y m e r . . . . . . . . . . . . . . . . . . . . . . . . . . M I S P E m e t h o d d e v e l o p m e n t strategies . . . . . . . . . . . . . . . . . . . 2.3.1 Non-specific adsorption . . . . . . . . . . . . . . . . . . . . . . . 2.3.2 S o l v e n t switch . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.3 E l u t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.4 T e m p l a t e b l e e d i n g . . . . . . . . . . . . . . . . . . . . . . . . . . S o l i d - p h a s e extraction applications . . . . . . . . . . . . . . . . . . . . . 2.4.1 O n - l i n e extraction s y s t e m s . . . . . . . . . . . . . . . . . . . . . . 2.4.2 Extraction systems with direct detection . . . . . . . . . . . . . . . 2.4.3 Off-line extraction s y s t e m s . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 3.

3.1 3.2 3.3

3.4 3.5 3.6 3.7 3.8

4.1 4.2

Techniques for sample preparation using solid-phase extraction

U w e Dieter Neue, C l a u d e R. Mallet, Ziling Lu, Y u n g - F o n g C h e n g and Jeffrey R. M a z z e o . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D e s c r i p t i o n of the sorbents . . . . . . . . . . . . . . . . . . . . . . . . . . Off-line M e t h o d s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1 1-D R e v e r s e d - p h a s e solid p h a s e extraction of biological s a m p l e s 3.3.2 2-D R e v e r s e d - p h a s e solid p h a s e extraction of biological s a m p l e s C a t i o n - e x c h a n g e solid p h a s e extraction . . . . . . . . . . . . . . . . . . . A n i o n - e x c h a n g e solid p h a s e extraction . . . . . . . . . . . . . . . . . . . On-line methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 4.

45 45 46 48 48 50 52 53 53 53 55 55 55 63 63 68 69

. .

73 73 74 75 76 77 82 84 85 89 89

Turbulent flow chromatography in bioanalysis

Tony E d g e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1 Band broadening processes . . . . . . . . . . . . . . . . . . . . . 4.2.2 Theoretical interpretation . . . . . . . . . . . . . . . . . . . . . . 4.2.3 D e s c r i p t i o n of the van D e e m t e r constants . . . . . . . . . . . . . . 4.2.3.1 A term . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.3.2 B t e r m . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.3.3 C term . . . . . . . . . . . . . . . . . . . . . . . . . . .

91 91 92 92 93 94 94 96 96

ix

Contents

4.2.4

4.3

4.4 4.5

D e v e l o p m e n t of turbulent flow c h r o m a t o g r a p h y m o d e l . . . . . . . 4.2.4.1 Definition of turbulent flow . . . . . . . . . . . . . . . . 4.2.4.2 Definition of turbulence . . . . . . . . . . . . . . . . . . 4.2.5 O v e r c o m i n g the p r o b l e m of pressure drop . . . . . . . . . . . . . . 4.2.6 Practical investigation . . . . . . . . . . . . . . . . . . . . . . . . 4.2.7 Mass transfer into pores . . . . . . . . . . . . . . . . . . . . . . . 4.2.8 C o m b i n i n g mass transfer and pressure drop . . . . . . . . . . . . . Applications of turbulent flow c h r o m a t o g r a p h y . . . . . . . . . . . . . . . 4.3.1 A p p l y i n g the m o d e l . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1.1 Single valve m e t h o d . . . . . . . . . . . . . . . . . . . . 4.3.1.2 Q u i c k elute m o d e . . . . . . . . . . . . . . . . . . . . . . 4.3.1.3 F o c u s m o d e . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.2 A p p l i c a t i o n areas . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.2.1 D r u g m e t a b o l i s m and p h a r m a c o k i n e t i c ( D M P K ) studies . 4.3.2.2 F o r e n s i c applications . . . . . . . . . . . . . . . . . . . . 4.3.3 Practical issues in bioanalytical T F C . . . . . . . . . . . . . . . . 4.3.3.1 Carryover . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.3.2 Pressure build up . . . . . . . . . . . . . . . . . . . . . . 4.3.3.3 Protein binding . . . . . . . . . . . . . . . . . . . . . . . 4.3.4 E n v i r o n m e n t a l applications of T F C . . . . . . . . . . . . . . . . . 4.3.5 Capillary turboflow c h r o m a t o g r a p h y . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 5. 5.1

5.2

97 99 102 102 106 107

110 111 111 112

112 115 115 115 121 123 124 124 125 125 126 127 127

Chiral bioanalysis

D.M. W a l l w o r t h and J.T. L e e . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.1 Scope and aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.2 The m e c h a n i s m of chiral r e c o g n i t i o n and choice of C S P . . . . . 5.1.3 M o b i l e p h a s e types . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.4 Direct versus indirect chiral separations . . . . . . . . . . . . . . 5.1.5 A c h i r a l - c h i r a l c o l u m n switching techniques . . . . . . . . . . . . 5.1.6 HPLC-MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.7 Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.8 Validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.9 Gas L i q u i d c h r o m a t o g r a p h y ( G L C ) . . . . . . . . . . . . . . . . 5.1.10 Capillary electrophoresis (CE) . . . . . . . . . . . . . . . . . . . 5.1.11 Supercritical fluid c h r o m a t o g r a p h y (SFC) . . . . . . . . . . . . . Chiral stationary phases . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.1 M a c r o m o l e c u l a r or p o l y m e r i c CSPs . . . . . . . . . . . . . . . . 5.2.2 Protein phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.3 Cyclodextrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.4 M a c r o c y c l i c antibiotics . . . . . . . . . . . . . . . . . . . . . . . 5.2.5 ~r-Complex C S P s . . . . . . . . . . . . . . . . . . . . . . . . . .

129 129 130 130 131 133 133 133 135 135 136 136 136 137 137 139 140 143 145

Contents 5.3

A p p l i c a t i o n s o f chiral H P L C in b i o a n a l y s i s . . . . . . . . . . . . . . . . .

147

5.3.1

[3-Adrenergic a g o n i s t s

. . . . . . . . . . . . . . . . . . . . . . .

147

5.3.2

[3-Adrenergic b l o c k e r s

. . . . . . . . . . . . . . . . . . . . . . .

150

5.3.3

Alcohol deterrent drugs . . . . . . . . . . . . . . . . . . . . . . .

153

5.3.4

A m i n o acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

153

5.3.5

A n a l g e s i c d r u g s (narcotics) . . . . . . . . . . . . . . . . . . . . .

155

5.3.6

Analgesic drugs (non-narcotic) . . . . . . . . . . . . . . . . . . .

156

5.3.7

A n e s t h e t i c drugs ( i n t r a v e n o u s ) . . . . . . . . . . . . . . . . . . .

157

5.3.8

Anorexic drugs . . . . . . . . . . . . . . . . . . . . . . . . . . .

157

5.3.9

Anthelmintic agents . . . . . . . . . . . . . . . . . . . . . . . . .

157

5.3.10

Antiarrhythmic agents

. . . . . . . . . . . . . . . . . . . . . . .

158

5.3.11

Antibacterial drugs . . . . . . . . . . . . . . . . . . . . . . . . .

160

5.3.12

Anticoagulants

. . . . . . . . . . . . . . . . . . . . . . . . . . .

160

5.3.13

Anticonvulsants . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.3.14

Antidepressants . . . . . . . . . . . . . . . . . . . . . . . . . . .

161 161

5.3.15

Antiemetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

164

5.3.16

Antifungals . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

164

5.3.17

Antihistamines

. . . . . . . . . . . . . . . . . . . . . . . . . . .

164

5.3.18

Antihyperlipoproteinemics . . . . . . . . . . . . . . . . . . . . .

165

5.3.19

Antihypertensives . . . . . . . . . . . . . . . . . . . . . . . . . .

166

5.3.20

Antiinflammatory drugs . . . . . . . . . . . . . . . . . . . . . . .

166

5.3.21

Antiischaemic drugs

. . . . . . . . . . . . . . . . . . . . . . . .

169

5.3.22

Antineoplastics

. . . . . . . . . . . . . . . . . . . . . . . . . . .

169

5.3.23

Antiparkinsonian agents

. . . . . . . . . . . . . . . . . . . . . .

169

5.3.24 5.3.25

Antipsychotic agents . . . . . . . . . . . . . . . . . . . . . . . . Antivirals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

171 171

5.3.26

Antiulcerative drugs . . . . . . . . . . . . . . . . . . . . . . . . .

171

5.3.27

Anxiolytics

. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

172

5.3.28 5.3.29

Biochemical markers . . . . . . . . . . . . . . . . . . . . . . . . Calcium channel blockers . . . . . . . . . . . . . . . . . . . . . . C h o l i n e s t e r a s e inhibitors . . . . . . . . . . . . . . . . . . . . . .

172 173 175

5.3.30 5.3.31

CNS Stimulants . . . . . . . . . . . . . . . . . . . . . . . . . . .

175

5.3.32

Gastroprokinetic agents . . . . . . . . . . . . . . . . . . . . . . .

176

5.3.33

Hallucinogenics . . . . . . . . . . . . . . . . . . . . . . . . . . .

176

5.3.34

H I V p r o t e a s e inhibitors . . . . . . . . . . . . . . . . . . . . . . .

177

5.3.35

Natriuretics . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

177

5.3.36

Leukotriene antagonists . . . . . . . . . . . . . . . . . . . . . . .

177

5.3.37

Mucolytics

. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

178

5.3.38

Radiosensitisers . . . . . . . . . . . . . . . . . . . . . . . . . . .

178

5.3.39

Sedative/hypnotics

178

5.3.40

Serotonin uptake inhibitors . . . . . . . . . . . . . . . . . . . . .

178

5.3.41

Thyromimetic agents . . . . . . . . . . . . . . . . . . . . . . . .

179

5.3.42

Vasodilators (cerebral)

. . . . . . . . . . . . . . . . . . . . . . .

179

5.3.43

Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

179

. . . . . . . . . . . . . . . . . . . . . . . . .

xi

Contents

5.4 5.5

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 6.

6.1

6.3 6.4 6.5

7.1 7.2

7.3

7.4 7.5

185 185 188 192 200 213 214

Immobilized enzyme reactors in liquid chromatography: On-line bioreactors for use in synthesis and drug discovery

Nektaria M a r k o g l o u and Irving W. Wainer . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immobilized enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.1 G e n e r a l a p p r o a c h e s . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.2 E n z y m e i m m o b i l i z a t i o n on c h r o m a t o g r a p h i c supports . . . . . . . 7.2.3. Effect of i m m o b i l i z a t i o n on e n z y m e stability . . . . . . . . . . . . 7.2.4 Effect of i m m o b i l i z a t i o n on e n z y m e kinetics . . . . . . . . . . . . 7.2.5 Effect of i m m o b i l i z a t i o n on the thermal stability of an e n z y m e . . 7.2.6 The effect of i m m o b i l i z a t i o n on the e n z y m e ' s r e s p o n s e to p H . . . On-line i m m o b i l i z e d e n z y m e reactors ( I M E R S ) . . . . . . . . . . . . . . 7.3.1 B i o c h r o m a t o g r a p h y . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.2 On-line I M E R s . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.2.1 On-line M i c h a e l i s - M e n t e n kinetics using an L C - I M E R format . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.2.2 A p p l i c a t i o n of I M E R s to on-line enantiospecific synthesis and purification . . . . . . . . . . . . . . . . . 7.3.2.3 O n - L i n e study of c o m p l e x biological systems using coupled IMERs . . . . . . . . . . . . . . . . . . . . . . . I m m o b i l i z e d e n z y m e s and I M E R s in drug discovery . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 8.

8.1 8.2

Method development in reversed-phase chromatography

U w e Dieter Neue, Eric S. G r u m b a c h , Jeff R. M a z z e o , K i m V a n Tran and Diane M. W a g r o w s k i - D i e h l . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.1 Tools for the m e a s u r e m e n t of selectivity differences and the quality of a separation . . . . . . . . . . . . . . . . . . . . . . . . 6.2.2 M e a s u r i n g selectivity differences . . . . . . . . . . . . . . . . . . M e t h o d d e v e l o p m e n t strategy . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 7.

180 180

215 215 216 217 217 218 219 220 222 222 222 224 225 226 228 233 233

Use of liquid chromatography-mass spectrometry in acute human toxicology

M.J. B o g u s z . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M e t h o d i c a l considerations . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2.1 O p t i m i z a t i o n of c h r o m a t o g r a p h i c analysis . . . . . . . . . . . . . . 8.2.2 Use of different ionization sources. Use of single- and triple q u a d r u p o l e instruments . . . . . . . . . . . . . . . . . . . . . . . . 8.2.3 Use of various mass analyzers . . . . . . . . . . . . . . . . . . . .

235 235 236 236 240 240

Contents

xii 8.3

8.4 8.5 8.6

Applications of L C - M S in clinical toxicological analysis . . . . . . . . . 8.3.1 Illicit drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.1.1 Opiate agonists . . . . . . . . . . . . . . . . . . . . . . . 8.3.1.2 A m p h e t a m i n e s . . . . . . . . . . . . . . . . . . . . . . . 8.3.1.3 C o c a i n e . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.1.4 C a n n a b i n o i d s . . . . . . . . . . . . . . . . . . . . . . . . 8.3.1.5 L S D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.2 T h e r a p e u t i c drugs . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.2.1 B e n z o d i a z e p i n e s . . . . . . . . . . . . . . . . . . . . . . 8.3.2.2 Antidepressants and antipsychotics . . . . . . . . . . . . 8.3.2.3 I m m u n o s u p r e s s a n t s and antineoplastic drugs . . . . . . . 8.3.2.4 Diuretics . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.2.5 Cardiac g l y c o s i d e s . . . . . . . . . . . . . . . . . . . . . 8.3.2.6 M u s c l e relaxants . . . . . . . . . . . . . . . . . . . . . . 8.3.2.7 Antidiabetics . . . . . . . . . . . . . . . . . . . . . . . . 8.3.2.8 H o r m o n e s . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.2.9 Other drugs of toxicological relevance . . . . . . . . . . . 8.3.3 E n v i r o n m e n t a l poisons and natural c o m p o u n d s . . . . . . . . . . . 8.3.3.1 Pesticides . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.3.2 Plant and bacterial toxins . . . . . . . . . . . . . . . . . 8.3.3.3 Inorganic c o m p o u n d s . . . . . . . . . . . . . . . . . . . . 8.3.4 S c r e e n i n g p r o c e d u r e s for multiple c o m p o u n d s . . . . . . . . . . . 8.3.4.1 G e n e r a l screening for various groups of drugs . . . . . . 8.3.4.2 G r o u p screening for substances b e l o n g i n g to the s a m e therapeutic class . . . . . . . . . . . . . . . . . . . . . . C o n c l u s i o n s and perspectives . . . . . . . . . . . . . . . . . . . . . . . . A b b r e v i a t i o n s used in the text . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

240 242 242 246 248 249 249 251 251 253 254 255 255 257 257 260 260 261 261 261 263 263 263 265 266 267 267

Chapter 9. HPLC-MS(MS) for bioanalysis in drug discovery and development 9.1 9.2

9.3

9.4 9.5

Brian Law . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T h e use of generic m e t h o d s . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.1 C o l u m n selection . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.2 E l u e n t selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.2.1 Organic modifier . . . . . . . . . . . . . . . . . . . . . . 9.2.2.2 Buffer and p H modifier . . . . . . . . . . . . . . . . . . 9.2.2.3 Buffer c o n c e n t r a t i o n . . . . . . . . . . . . . . . . . . . . 9.2.3 Effects of eluent p H and c o m p o u n d type . . . . . . . . . . . . . . S a m p l e p o o l i n g and cocktail dosing . . . . . . . . . . . . . . . . . . . . . 9.3.1 S a m p l e pooling . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3.2 Cocktail dosing . . . . . . . . . . . . . . . . . . . . . . . . . . . . Short c o l u m n H P L C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S a m p l e introduction onto H P L C . . . . . . . . . . . . . . . . . . . . . . .

271 271 272 273 277 277 278 278 280 281 281 283 283 287

xiii

Contents

9.6 9.7 9.8 9.9

The use of gradient elution with high flow rates . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 10.

10.1 10.2

10.3 10.4 10.5 10.6 10.7 10.8

10.9

10.10 10.11

11.1 11.2 11.3 11.4

Biomedical applications of directly-coupled chromatography-nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS)

John C. Lindon, Nigel J.C. Bailey, Jeremy K. Nicholson and Ian D. Wilson . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Technical developments in H P L C - N M R and H P L C - N M R - M S . . . . . 10.2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.2 The requirement for high dynamic range in N M R spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.3 Avoidance of compromised chromatographic resolution . . . . 10.2.4 The need for high N M R sensitivity . . . . . . . . . . . . . . . 10.2.5 Additional considerations for double coupling of N M R and MS to HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . Operational methods in H P L C - N M R and H P L C - N M R - M S . . . . . . Applications in combinatorial chemistry . . . . . . . . . . . . . . . . . Application to drug impurities . . . . . . . . . . . . . . . . . . . . . . Chiral H P L C - N M R and H P L C - C D for pharmaceutical mixtures . . . . Application to natural products . . . . . . . . . . . . . . . . . . . . . . Application to drug metabolism . . . . . . . . . . . . . . . . . . . . . . 10.8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.8.2 A summary of human metabolism studies . . . . . . . . . . . . 10.8.3 Animal metabolism studies of pharmaceuticals and model compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.8.4 Application to in vitro metabolism studies . . . . . . . . . . . 10.8.5 Application to drug metabolite reactivity . . . . . . . . . . . . Future developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.9.1 Automation and informatics . . . . . . . . . . . . . . . . . . . 10.9.2 Miniaturisation in separations coupled to N M R . . . . . . . . . 10.9.3 Hypernation . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 11.

288 291 291 291

293 293 294 294 294 295 296 296 298 300 301 302 304 306 306 307 310 316 316 321 321 322 323 325 325

Ultra-sensitive detection of radiolabelled drugs and their metabolites using accelerator mass spectrometry

Graham Lappin and Introduction . . . . . . . . . Instrumentation . . . . . . . Sample preparation . . . . . Data analysis . . . . . . . .

R. Colin . . . . . . . . . . . . . . . . . . . .

Garner . . . . . . . . . . . . . . . . . . . .

............... . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

331 331 333 335 336

xiv 11.5 11.6 11.7 11.8

Contents Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L i m i t s of detection and quantification . . . . . . . . . . . . . . . . . . . C o n c l u s i o n s and the future . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 12.

12.1

12.2

12.3

12.4 12.5 12.6

13.1 13.2

13.3

Biomedical applications of inductively coupled plasma mass spectrometry (ICP-MS) as an element specific detector for chromatographic separations

Fadi R. A b o u - S h a k r a . . . . . . . . . . . . . . . . . . . . . . . . A n i n t r o d u c t i o n to I C P - M S . . . . . . . . . . . . . . . . . . . . . . . . 12.1.1 Inductively c o u p l e d p l a s m a as an ion source . . . . . . . . . . . 12.1.2 Interfacing the I C P to a m a s s s p e c t r o m e t e r . . . . . . . . . . . . 12.1.3 T h e building blocks of an I C P - M S . . . . . . . . . . . . . . . . 12.1.4 A n a l y t i c a l capabilities of I C P - M S . . . . . . . . . . . . . . . . I C P - M S as an e l e m e n t specific detector for c h r o m a t o g r a p h i c separations 12.2.1 C o u p l i n g an H P L C to I C P - M S . . . . . . . . . . . . . . . . . . 12.2.2 C o u p l i n g G C to I C P - M S . . . . . . . . . . . . . . . . . . . . . 12.2.3 C o u p l i n g C E to I C P M S . . . . . . . . . . . . . . . . . . . . . . A p p l i c a t i o n s of I C P - M S in the b i o m e d i c a l field . . . . . . . . . . . . . . 12.3.1 D e t e c t i o n of m e t a b o l i t e s . . . . . . . . . . . . . . . . . . . . . . 12.3.2 P h o s p h o r y l a t i o n detection by I C P - M S . . . . . . . . . . . . . . 12.3.3 O t h e r applications . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 13.

337 341 345 347

351 351 351 354 354 358 359 359 360 361 362 362 364 366 368 370 371

Chromatography in a regulated environment

H.M. Hill . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R e g u l a t o r y issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.2.1 Regulatory environment . . . . . . . . . . . . . . . . . . . . . . 13.2.2 C o m p l i a n c e with G L P ? . . . . . . . . . . . . . . . . . . . . . . 13.2.3 I n s t r u m e n t qualification and validation . . . . . . . . . . . . . . B i o a n a l y t i c a l validation process . . . . . . . . . . . . . . . . . . . . . . 13.3.1 Full validation . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.3.1.1 P r e - s t u d y p h a s e . . . . . . . . . . . . . . . . . . . . . 13.3.2 A p p l i c a t i o n of a validated analytical m e t h o d . . . . . . . . . . . 13.3.2.1 S y s t e m suitability . . . . . . . . . . . . . . . . . . . 13.3.2.2 D i s p o s i t i o n of standards, Q C s and samples in a batch 13.3.2.3 C h r o m a t o g r a p h i c a c c e p t a n c e . . . . . . . . . . . . . . 13.3.2.4 R e i n t e g r a t i o n of c h r o m a t o g r a p h i c peaks . . . . . . . . 13.3.2.5 Standard curve a c c e p t a n c e . . . . . . . . . . . . . . . 13.3.2.6 Quality control a c c e p t a n c e criteria . . . . . . . . . . . 13.3.2.7 S a m p l e assay repeat criteria . . . . . . . . . . . . . .

373 373 375 376 376 378 378 379 379 392 393 394 395 396 397 397 397

Contents 13.3.3

XV

P o s t v a l i d a t i o n issues . . . . . . . . . . . . . . 13.3.3.1 M e t a b o l i t e s in safety testing ( M I S T ) 13.3.3.2 Cross validation . . . . . . . . . . . 13.3.3.3 M e t h o d transfer . . . . . . . . . . . . 13.3.3.4 Partial v a l i d a t i o n . . . . . . . . . . . 13.3.3.5

13.4 13.5

Limit assays

. . . . . . ......... . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . .

Study documentation . . . . . . . . . . . . . . . . . . . . . . . . . . Statistical c o n s i d e r a t i o n s . . . . . . . . . . . . . . . . . . . . . . . . 13.5.1 R a t i o n a l e b e h i n d the c o n s e n s u s statistics . . . . . . . . . . . 13.5.2 I n t e r b a t c h a n d i n t r a b a t c h p r e c i s i o n . . . . . . . . . . . . . . 13.5.3

. . . .

. . . .

398 398 400 400 401 402 403 404 404 405

Standard curves . . . . . . . . . . . . . . . . . . . . . . . . . .

406

T h e future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

406

13.6.1

Ethical implications . . . . . . . . . . . . . . . . . . . . . . . .

406

13.6.2

Instrumentation quantification and validation . . . . . . . . . . .

407

13.7

13.6.3 Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

407

13.8

13.7.1 Regulatory changes . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4O8 408

13.6

Subject index

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

407

413

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I.D. Wilson (Ed.), Bioanalytical Separations Handbook of Analytical Separations,Vol. 4 9 2003 Elsevier Science B.V.All rights reserved

CHAPTER 1

New developments in integrated sample preparation for bioanalysis M.W.J. van Hout l'*, H.A.G. NiederRinder ~, R.A. de Zeeuw 1 and G.J. de Jong 2 Department of Analytical Chemistry and Toxicology, University Centrefor Pharmacy, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands 2 Department of Biomedical Analysis, Faculty of Pharmacy, University of Utrecht, P.O. Box 80082, 3508 TB Utrecht, The Netherlands * Current address: Pharma Bio-Research Group B.V., Laboratories Assen, Westerbrink 3, 9405 BJ Assen, The Netherlands

1.1 INTRODUCTION Increasing knowledge of the working mechanisms of drugs has led to the development of very potent drugs. Hence, the administered dosages are small, and consequently, the concentration levels in biological fluids are decreasing. Furthermore, biological samples are very complex, because they contain many endogenous substances. Blood fluids, such as serum and plasma, represent an extra problem due to the presence of proteins. Protein binding may affect the extractability of the analytes. Deproteinisation techniques can help to overcome this problem. It may, however, also give rise to even more difficulties, since analytes can be co-precipitated with the proteins. Thus, sample pretreatment techniques are required that retain the analyte(s) of interest, at the same time efficiently removing the endogenous interferences. The most common systems exist of an extraction step prior to separation and detection. A considerable gain in sensitivity and selectivity can be obtained during the extraction, as the analytes of interest are usually concentrated and separated from the matrix. An ideal extraction method should be rapid, simple, inexpensive, and give reproducible and high recoveries without the possibility of degradation of the analytes. Furthermore, the extraction method should not generate large amounts of chemical waste [ 1]. Sample pretreatment used to be a long step in the analysis of biological samples. Since the numbers of samples to be analysed is increasing, very rapid, but still selective and sensitive systems are required. In modern systems using advanced sample handling, the separation step may be more time-consuming. However, with the introduction of short columns in liquid chromatography (LC) and the selectivity of the mass spectrometer (MS), throughput of samples is again more and more limited by the time References pp. 39-44

2

Chapter 1

required for sample pretreatment. This is especially the case in off-line systems, which may also require extensive manual work. Therefore, various systems have been developed in order to integrate sample pretreatment with the separation and detection technique (Fig. 1.1) [2].

Sample pretreatment

!i~,,~iiii!ii~ ~,~ ~i!~ii!ii~i

Separation/Detection

Off-line

At-line

On-line

In-line

Fig. 1.1. Schematic presentation of various integration methods of the sample pretreatment step with the separation and detection technique. (Reprinted from [2, modified], with permission from Elsevier Science).

New developments in integrated sample preparation for bioanalysis Basically, three possibilities have been proposed for integrated sample pretreatment in the analytical procedures, i.e. (1) at-line; (2) on-line; and (3) in-line. The at-line coupling involves sample preparation by a robotic device and an autoinjector to inject the extracts into the analytical instrument. No direct stream of liquid between extraction unit and analysing unit is present. Moreover, not the entire extract is transferred to the analysing instrument. Disadvantages as observed with off-line extractions, i.e. collection of the extract, evaporation and reconstitution, are not eliminated. An example of an at-line system is the 96-well plate design for solid-phase extraction (SPE). Samples can be extracted simultaneously, thus increasing the sample throughput, provided that the separation and detection can be performed very rapidly or by using simultaneous analytical instruments. With on-line systems, there is a direct transport of the entire extract to the analysing technique, and the latter is receiving the entire extract. Samples can be processed in series, i.e. samples are pretreated and analysed one after the other, or in parallel, in which one sample is being analysed while another is being extracted. The latter system offers a high sample throughput. A very prominent advantage of on-line systems is that some error-prone steps of the extraction procedure, such as evaporation and reconstitution are eliminated, hereby increasing precision and accuracy. In-line systems exist of sample pretreatment fully incorporated into the separation system, hereby creating a new device. In contrast to on-line procedures, application of in-line systems imply the direct injection of the sample into the analytical instruments. Various approaches for in-line SPE-capillary electrophoresis have been reported [2]. It should be noted that the differences in interfacing are often not as clear as mentioned above. For example, the extraction can be performed manually (off-line) or by robot (at-line), but the final step of the extraction, i.e. the desorption of the analytes may be performed on-line with the analytical step. Furthermore, dividing systems into on-line and in-line techniques is very disputable. These systems are usually closely related to each other and a distinct difference can often not be made. Therefore, in this chapter on-line and in-line systems will be considered as similar. The goal of this chapter is to show the current status of modem sample pretreatment techniques such as SPE, solid-phase microextraction (SPME) and membrane-based extraction systems, and to outline novel trends in the bioanalytical area with regard to integrated sample preparation. It will focus not only on pretreatment techniques integrated with chromatographic separation systems, but also on their direct coupling to MS. SPE was originally designed for off-line purposes [3-5], but is now routinely used in on-line systems with LC [6-9]. The combination of SPE on-line with gas chromatography (GC) is less common, especially in the bioanalytical field. The current state of SPE-GC will be discussed here. Since an LC column can also be used as cleanup prior to GC analysis [10-13], on-line LC-GC applications without any further sample pretreatment will also be presented. Turbulent-flow chromatography (TFC) is to a certain extent similar to SPE. The use of high flow-rates offers new possibilities for sample pretreatment [14-17]. Therefore, the current state in TFC will be presented. SPME was originally designed for the analysis of volatile compounds with GC [18-22]. However, nowadays SPME is also coupled with LC for analysis of less-volatile compounds. The applicability of these SPME-LC systems in bioanalysis will be shown. Membrane-based techniques are, like SPME, diffusion-based sample pretreatment References pp. 39-44

4

Chapter 1

techniques. Dialysis is a more mature membrane method for sample pretreatment [23-25]. However, non-porous membranes provide new challenges for clean-up of biological samples. Therefore, the focus of membrane-based techniques for sample clean-up will be on the latter type.

1.2 CHROMATOGRAPHY-BASED EXTRACTION TECHNIQUES 1.2.1 Solid-phase extraction - gas chromatography

1.2.1.1 General aspects of SPE

A very common and powerful sample clean-up and concentration technique is SPE. It was originally developed for off-line purposes, but due to the demand for speed and the growing numbers of samples, at-line (including 96-well designs [9,26,27]) and on-line systems, such as the Prospekt, have been developed for coupling with LC [6,7,28,29]. On-line SPE is a very attractive sample pretreatment technique since the entire process of activation, conditioning, extraction, washing, and elution takes place in an enclosed circuit, which eliminates error-prone steps like evaporation and reconstitution. Also, the entire eluate is usually injected into the analytical instrument. Therefore, better precision and sensitivity may be observed when compared to off-line SPE. The most common on-line coupling of SPE is with LC, since similar solvents are used and virtually no modifications have to be made to the instruments. As this technique has already evolved and matured, the on-line coupling of SPE-LC will not be discussed here in detail. Worth mentioning, however, is the growing interest for high-throughput systems based on short-column LC coupled with MS, or even direct coupling of SPE and MS [30-39]. In such systems the extraction and detection should offer both sensitivity and selectivity in order to be able to detect low quantities of analytes in biological fluids. It should be noted that many applications applying little or no separation prior to MS may have to deal with ion suppression effects [31,40-43], clearly showing that the SPE eluates are not always free of matrix compounds. Numerous applications on off-line SPE-GC have been reported for the analysis of biological samples, and various reviews have appeared to which the reader can be referred [3-5]. Also, at-line systems, e.g. PrepStation [44,45] and ASPEC [46,47], will not be considered here. With the latter systems the extraction is performed automatically, but the eluate is collected in vials and subsequently the eluate is, usually only partially, injected into the GC. Numerous applications have been reported about the usefulness of off-line SPE combined with gas chromatography for analysis of biological samples. However, on-line SPE-GC appears to be emanating as a rather unexplored, yet promising technique. The on-line coupling of SPE with GC implies injection of large volumes of solvent into the GC, thus requiring modification of the injection system. A number of interfaces have been proposed for this purpose [48], i.e.: (1) on-column injection; (2) loop-type injection; and (3) programmed temperature vaporiser (PTV). With on-column injection, solvent is introduced at a speed above the evaporation rate and at temperatures below the boiling point of the solvent, ensuring wetting of the retention gap [49]. Solvent is

New developments in integrated sample preparation for bioanalysis evaporated in the retention gap and eliminated via the solvent vapour exit (SVE). An extra retaining pre-column enables refocusing of the analyte prior to transfer to the actual analytical column. A second way to allow large-volume injection (LVI) is the loop-type interface, originally designed for on-line LC-GC [50]. Injection is performed by filling a loop and flushing the contents to the retention gap inside the GC, which is slightly above the boiling point of the solvent. Formation of vapour results in pressure build-up and prevents further penetration of the solvent into the retention gap. Finally, a PTV injector was designed by Vogt et al. [51,52]. The injector strongly resembles a conventional split/splitless injector. The main difference is injection of solvent at temperatures 30-40~ below its boiling point on a packed liner. The liner packing acts as a liquid reservoir. A high purge flow ensures evaporation of the solvent, while analytes are retained on the liner packing. After almost complete evaporation of the solvent, the analytes are thermally released from the packing and transferred to the GC column. The latter is still at low temperatures, allowing refocusing of the analytes.

1.2.1.2 Applications of SPE-GC A selective SPE-GC method was described by Farjam et al. [53], who coupled immunoaffinity sample pretreatment with GC. A column with immobilised antibodies was used for the extraction of [3-19-nortestosterone from urine. A reversed phase reconcentration column and a retention gap were used for interfacing the extraction and the GC. Desorption from the antibody-column was performed with methanol-water (95:5, v/v), and after subsequent dilution with water, the analytes were trapped on the reconcentration column. Elution from this column was performed with 75 p~l ethyl acetate. The high selectivity during trapping made it possible to analyse large urine samples (5-25 ml), with good sensitivity for all investigated steroid hormones (LOD about 0.1 ng/ml). The total analysis time was still 40 min, mainly due to the long GC analysis. Benzodiazepines were determined in plasma by on-line dialysis-SPE-GC [54]. Clean-up was based on performing dialysis for 7 min, and subsequently, the diffused analytes were trapped on a PLRP-S pre-column. After drying elution was performed with 275 p~l ethyl acetate, which was injected into the GC via a loop-type interface. The SPE step in this procedure was more a reconcentration step than an actual extraction process. Nonetheless, this on-line dialysis-SPE-GC system showed sufficient selectivity (Fig. 1.2). The authors claim that the benzodiazepines could be detected at therapeutic levels (5-25 ng/ml) and that extra selectivitity could be obtained by acidification of plasma prior to extraction (Fig. 1.2C and D), but these claims are not substantiated by their figures. The PTV injector is an interesting injection system, as it allows LVI and thus on-line LC-GC and SPE-GC. The possibilities of PTV-GC in combination with SPE for plasma samples have already been demonstrated [55]. Now that integrated, automatic instruments have become commercially available, on-line SPE-PTV/GC will be facilitated. Moreover, the PTV injector also offers possibilities for thermal desorption [10,56-60]. Thus, no solvent is introduced into the GC, hereby eliminating some difficulties observed with LVI-GC, such as introduction of large volumes of solvent and its evaporation within the GC system. However, the reports available so far all deal with References pp. 39-44

O",

E

E

50 mVI

QJ Z

15

Time (min)

20

25

Time (rain)

Fig. 1.2. On-line dialysis-SPE-GC-NPD of (A) untreated blank plasma, (B) untreated plasma spiked with 1 la,g ml -J of nitrazepam, (C) acidified blank plasma and (D) acidified plasma with 1 lu,g ml -~ of medazepam. (Reprinted from [54], with permission from Elsevier Science).

New developments in integrated sample preparation for bioanalysis environmental samples. Finally, the PTV injector has also been used for direct injection of plasma samples [61 ]. In this set-up, acidification and subsequent ultrafiltration were the only sample pretreatment steps. About 50 ~1 of the ultrafiltrate were injected onto a packed liner. No interference of the matrix was observed (see Fig. 1.3) and the chromatographic system was not damaged. The only drawbacks were occasional memory effects and the necessity to change the liner after 20 injections. The latter is probably due to the injection of ultrafiltrates which still contain some proteins, causing adsorption to the liner packing and GC column. The quantitation limit for ropivacaine was down to 300 pg/ml.

1.2.1.3 Remarks regarding the applicability of SPE-GC New devices have been developed for the coupling of miniaturised SPE with GC [62,63], enlarging the possibilities for incorporation of on-line SPE-GC into routine analysis. Until now, the number of applications of on-line SPE-GC in bioanalysis is very limited, in contrast to the numerous reports of this technique in the analysis of surface and drinking water [10-12,64,65]. This is probably due to the complexicity of biological matrices in comparison with water samples. Furthermore, various compounds in biomedical and pharmaceutical studies cannot be analysed with GC due to thermolability of the compounds. Nevertheless, the applicability of SPE-GC with LVI, and in particular the PTV injector, for biological samples seems worth further exploring.

1.2.2 Liquid chromatography - gas chromatography

1.2.2.1 General aspects of LC-GC A similar approach to SPE-GC is the coupling of LC on-line with GC in which the LC column functions as a sample pretreatment technique. Only the fractions of interest will be transferred to the GC (heart-cutting). The LC column is merely used for clean-up purposes and the GC column is used for the actual separation. As with on-line SPE-GC, on-line LC-GC also implies the introduction of relatively large liquid volumes into the GC, so that LVI must be used. As discussed with on-line SPE-GC, several approaches have been proposed in order to allow injections up to 1 ml into the GC. Nearly all online LC-GC applications involve normal phase (NP) LC, since the introduction of volatile elution solvents into the GC is more easily achieved than that of aqueous solvents [10]. However, direct analysis of biological, i.e. aqueous, samples in NPLC is not possible. Therefore, a separate sample pretreatment step, e.g. LLE or SPE, is always required. Consequently, no applications in the bioanalytical field have been reported for on-line NPLC-GC with direct injection of the sample. It is more common to use reversed phase (RP) LC in biomedical and pharmaceutical analysis. Coupling RPLC with GC implies introduction of large volumes of aqueous and ionogenic solutions into the GC. Water is very disadvantegous for GC analysis due to its high boiling point, high surface tension, poor wetting characteristics and aggressive hydrolytic reactivity, whereas non-volatile buffers (i.e. its ions) are also non-compatible

References pp. 39-44

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New developments in integrated sample preparation for bioanalysis with GC. Nonetheless, several techniques have been proposed for the analysis with online RPLC-GC [10-13,66-69]. Basically, they can be divided in two groups: (1) direct introduction of aqueous RPLC fractions using special GC-injection systems; and (2) phase-switching techniques, by which the analyte is transferred to an organic solvent prior to introduction into the GC. In the latter situation RPLC is coupled to GC via online LLE. Both approaches have found their way in environmental analyis [11-13], but the number of applications in bioanalysis is very limited. In a few cases RPLC has been coupled on-line with GC for the analysis of biological samples using an off-line sample preparation, i.e. LLE [70] or SPE [71] was applied prior to injection into the LC system.

1.2.2.2 Applications of RPLC-GC An example of the first approach, i.e. direct introduction, was described by Duquet and co-authors [72], who coupled ~RPLC on-line with GC using a aminopropyltriethoxysilane-deactivated retention gap. Diazepam was determined in urine by transferring only 2-~1 methanol-water (80:20 v/v) fractions from the LC into the GC. Goosens et al. [73-75] also applied a retention gap to transfer eluents from the RPLC to the GC for drug analysis. Up to 200 ~1 of eluent (acetonitrile-water) could be introduced into a Carbowax-deactivated retention gap by using an on-column interface and SVE [76]. The presence of remaining water after azeotropic evaporation was found to deteriorate the analysis. Thereofore, prior to introduction into the GC, addition of 10% acetonitrile to the LC eluent was performed resulting in an azeotropic acetonitrile-water mixture (84:16 v/v). By these means, the maximum amount of water remaining after evaporation was never exceeded. In order to inject ion-free fractions into the GC an anion-exchange micromembrane was inserted between the LC and GC parts [74]. Methanesulphonic acid was efficiently removed (99.9%) from the eluent acetonitrilewater, allowing the reproducible analysis of the potential drug eltoprazine, i.e. the coefficient of variation was found to be 3% (n = 5, 150 ~g/ml). A different approach, i.e. phase switching, was applied by Wessels et al. [77] and Ogorka et al. [78]. A phase switch was performed by using an LLE interface between the LC and GC part (loop-type interface). The set-up of this system is depicted in Fig. 1.4. Use of a GC-MS system allowed the identification of various unknown impurities in pharmaceutical products. Even though the instrumental set-up is rather complicated, reliable results were obtained for the quantitative determination of [3blockers in human serum and urine [79,80]. A total analysis time of 45 min. was required for the selective removal of the matrix compounds and efficient and repeatable LLE-GC analysis. Hy6tyl~inen et al. [81] applied a similar system to determine morphine and its analogues in urine. After LC separation, a phase switch was applied using an elevated temperature for the eluent and the extraction coil, which resulted in increased recoveries. After phase-switching, the analytes were derivatised on-line with N,O-bis(trimethylsilyl)trifluoroacetamide prior to GC-FID analysis. The total analysis time was less than 60 rain. As can be seen in Fig. 1.5, the LC clean-up procedure was effective as no matrix compounds were observed. Extra peaks in the chromatogram are due to the excess of derivatisation reagent. In principle this is a very powerful system. However, although the above studies show interesting approaches, the utility of the References pp. 39-44

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Fig. 1.4. Instrumentation for on-line coupled reversed phase LC-GC-MS. ( 9 John Wiley & Sons Inc. Reproduced from [77] with permission).


11

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o

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Fig. 1.5. GC chromatograms of (top) urine spiked with the opiates and (bottom) blank urine. Peaks: (1) dihydrocodeine, (2) codeine, (3) ethylmorphine, (4) morphine and (5) heroin. Concentration of the analytes was 3 ~g/ml. (Reprinted from [81], with permission from Elsevier Science).

resulting systems sometimes remain questionable. For example, in the final application of morphine and analogues in urine, it should be noted that the concentrations of the analytes are very high (3 ~g/ml), and the absence of interferences is not clearly demonstrated by Fig. 1.5. Furthermore, most of the analytes are metabolised prior to excretion into urine. It is thus more interesting to analyse the metabolites. Also, the analytes can be easily determined by SPE-HPLC-UV, allowing a faster and easier analysis without the requirement of phase switching and an extra derivatisation step.

1.2.2.3 Remarks regarding the applicability of RPLC-GC The results achieved with R P L C - G C are still not very remarkable, as analysis times are long and poor detection limits (> ~g/ml) are obtained. The possibility of LC-MS analysis without derivatisation and the availability of similar but better sample


12

Chapter 1

pretreatment techniques such as SPE will most certainly imply that RPLC-GC and NPLC-GC will not be applied on a routine basis.

1.2.3 Turbulent-flow chromatography 1.2.3.1 General aspects of TFC

Various techniques for SPE automation in combination with LC have been developed, e.g. ASPEC (Gilson), PrepStation (Hewlett Packard), Prospekt (Spark-Holland). Even though these systems greatly facilitate sample handling, the analysis time is usually still long. The analysis time can be significantly reduced when chromatography is performed with high flow-rates, e.g. under turbulent flow conditions. The latter was introduced by Quinn and Takarewski [82] in 1997 as a fast method for sample analysis. In this approach typical flow-rates of 3-5 ml/min are applied using a 1.0 mm i.d. column. These high flow-rates can be applied due to the low column back pressure associated with the use of large porous particles (typically 30-60 l~m) [14-17]. The solvent front profile is shaped like a plug rather than a parabolic profile as observed under laminar flow conditions. The high flow-rate and the plug flow profile increase the effective diffusion rates within the pores of the stationary phase. Consequently, the flow regime cannot be described by the Van Deemter equation. As a result, plate heights are significantly lower compared to predictions based on that equation. However, these conditions result in a considerable reduction of the chromatographic analysis time [14-17,83-86]. The typical TFC procedure, usually applied for the direct-injection analysis of crude plasma, basically consists of four stages, similar to conventional SPE: sample clean-up (extraction), analyte elution, LC separation and system re-equilibration [87]. It should be noted that the turbulent flow conditions can be used during each step of the procedure, but that some steps, e.g. the separation, can be performed using a laminar flow. During the purification step the analytes have to be separated from the matrix and should be retained by the stationary phase. Removal of plasma proteins from drugs is achieved by size exclusion (pore size 60,~) and slow diffusion of proteins into the pores. Since large particles are used, the technique also allows the use of large end-column frits (10-40 I~m) [ 15]. As a result, large protein molecules in the plasma sample can easily pass through these columns without clogging of the frits due to precipitation. Elution is performed using a steep gradient of organic solvent followed by chromatographic separation on a second column. Only limited separation is achieved on the analytical column due to the high percentage of organic solvent. Finally, the extraction and separation column are equilibrated with suitable solvents for subsequent analysis. A basic set-up of TFC is depicted in Fig. 1.6. As described above, TFC strongly resembles SPE, with the use of high flow-rates instead of normal flow-rates, and larger particles in the extraction column. As a consequence, the set-up for TFC (Fig. 1.6) is similar to that for SPE-LC systems. TFC is applicable for the analysis of drugs that bind to a high extent to proteins. In general, recoveries of 70-100% are obtained [87-89]. Only with very strongly bound drugs modification of the TFC procedure is required, e.g. low-flow sampling (0.5 ml/min) or acidification of the sample prior to extraction [15].

13

New developments in integrated sample preparation for bioanalysis From Pump B y

To Mass Spectrometer

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Pumps A Extraction Column

Configuration (a): Loading sample, extraction and equilibration

From Pump B ....

oMass

Spectrometer Analytical Column Autosampler

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Pumps A Extraction Column

Configuration (b): Eiution Fig. 1.6. Schematic representation of the flow path during the on-line extraction, elution and equilibration. ( 9 John Wiley & Sons Inc. Reproduced from [91, modified] with permission).

1.2.3.2 Applications of TFC The first application of TFC-MS for direct-injection analysis of plasma was reported by Ayrton et al. [85] in 1997. In successive years, more applications have been developed, mainly for the analysis of low molecular-weight drugs in plasma [14-16,83,84,86-94] and serum [14,95]. Nonetheless, Cass and co-authors [95] reported the successful determination of vancomycine, a 1448-Da peptide, in urine by means of a fully automated TFE-LC-MS/MS system with a detection limit of 1 ng/ml. Most applications of TFC focus on the determination of a single compound in plasma in a single run. Jemal et al. [92] reported the simultaneous determination of simvastatin


14

Chapter 1

and simvastatin acid in human plasma by direct-injection LC-MS/MS. The possibilities for multi-component determination in a single run were further explored by Wu and coauthors [15]. This study was set up for high-throughput pharmacokinetic screening using LC-MS/MS and a turbulent-flow column-switching system by which ten compounds had to be analysed simultaneously. The set-up of the system was similar to the design presented in Fig. 1.6. A 4-ml/min flow was used for 1 min. during which sampling and purification was performed. Then, the trapped analytes were eluted in a back-flush mode from the extraction column towards the analytical column using a flow of 0.4 ml/min. Elution was completed within two minutes. Flushing and equilibration of the extraction column was performed during the separation of the analytes on the analytical column. Good separation and peak shapes (Fig. 1.7) were achieved within a run time of 10 min. including the extraction time. A dynamic range of 1-2500 ng/ml was obtained, with a limit of quantitation (LOQ) of 1 ng/ml. Using the highest concentration, i.e. 2500ng/ml, a carry-over of 0.14+0.07% was observed. One extraction column could be used for 200-300 plasma sample injections without causing significant back-pressure increase. It should be noted, however, that the simultaneous analysis of the analytes in this study is of limited interest for pharmacokinetic screening. Instead, analytes and their metabolites or co-administered drugs should have been chosen as target compounds. A ternary-column system was introduced by Xia et al. [88] for high-throughput direct-injection analysis of plasma. Basically, the system consisted of two extraction columns in parallel and one analytical column. In this way, one column was equilibrated while on the other column the extraction of 10 txl plasma was performed. Thus, the equilibration step does not add extra time to the injection cycle time. The on-line purification step lasted for only 0.3 min. and the total run time was 1.6 min. The extraction recovery of the guanidine-type drug was > 95%. Using the sensitivity and selectivity of the mass spectrometer by operating it in the SRM mode, an LOQ of 1 ng/ ml was obtained. Good intraday and interday precision (< 6.6%) was achieved in the range of 3-1000 ng/ml. Ayrton and co-authors [90] applied ultra-high flow-rate capillary LC with MS/MS (SRM mode) for the direct determination of an isoquinoline drug in plasma. The extraction column had an internal diameter of 0.18 mm, thus allowing a flow-rate of 130 ~l/min for turbulent flow. Upon injection of only 2.5 Ixl plasma (diluted 1 : 1 with an aqueous standard solution), an LOQ of 0.5 ng/ml was obtained within a total run time of two minutes.

1.2.3.3 Remarks regarding the applicability of TFC Comparing the potentials of TFC with results obtained with LLE and automated 96-well SPE, it can be noted that similar results are obtained in terms of dynamic range, LOQ, accuracy and precision [15,83,85]. As turbulent flows reduce time-consuming steps, the speed of the system is superior to more conventional on-line SPE systems. The limited concentration potential is a disadvantage of TFC. Although this can be compensated by using capillary LC, the load capacity is then reduced, allowing less injection of the sample. The rate of solvent consumption of TFC is high in comparison to conventional LC. However, the total volumes per analytical run are similar for turbulent-flow LC-MS

TIC 5.05

7 I

Phentolamine

1

Haloperidol

K

4.22

1

Carbamazepine

I \ Temazepam

n

d 2.00 . 4 0 0 Fig. 1.7. Total ion chromatogram and MRM chromatograms of the LC-MS-MS assay for the 10 compounds with 100 )*.1 direct injection of a 250 nglml chimpanzee plasma standard. A 2.0 x 150 mm C18 column (Symmetry, Waters Corp.) was used as the analytical column, which was operated in the gradient mode with a flow rate of 0/4 mumin. (Reprinted from [ 1.51 Copyright 2000 Am. Chem. Soc.).



Puromycin

16

Chapter 1

and for SPE followed by conventional LC-MS [15]. Up till now, TFC is not rapidly expanding, probably due to the mainly size-exclusion-based principle for sample cleanup. As a result, virtually all applications are analyses of plasma or similar fluids, since the analytes of interest can be easily separated from proteins and other large matrix compounds. Handling a urine matrix is much more complicated as small drugs cannot be easily separated from small urine matrix components. Even though the speed is advantageous, TFC will probably not become a universal and widely applicable sample pretreatment technique. Some applications use turbulent flows only during the first step, i.e. purification, with subsequent separation on an LC column using laminar flows. Therefore, in such cases the term 'turbulent-flow chromatography' (TFC) should possibly be replaced by 'turbulent-flow extraction' (TFE), and TFC should only be used when the separation is performed under turbulent flow conditions. Furthermore, it should be noted that the sample pretreatment is actually LC modified into an SPE procedure. This can also be seen in Fig. 1.6, as the set-up for TFC is similar to SPE-LC. The technique might also be referred to as modified (ultra-high flow) LC-LC with subsequent detection. Another critical point was raised by Ayrton et al. [86]. Careful consideration of commonly used flows (3-5 ml/min) and particle sizes (30-60 txm) showed that the actual flows are not turbulent. Therefore, the term ultra-high flow-rate LC with direct sample injection might be preferred, as is also used now in other systems [34].

1.3 DIFFUSION-BASED EXTRACTION TECHNIQUES 1.3.1 Solid-phase microextraction 1.3.1.1 General aspects of SPME

At the introduction of SPME [96], it was designed for gas chromatographic analysis by direct sampling of liquids. SPME integrates sampling, extraction, concentration and sample introduction into a single solvent-free step [21,97]. Originally, SPME was performed with a modified syringe with a stainless steel needle in which a thinly-coated fused silica fiber (100 Ixm in diameter) could be moved up and down via the plunger [19,20]. The fiber is coated with a suitable stationary phase, which is usually a polymeric phase of 7- to 100-1xm thickness. The fiber is immersed into the sample (direct immersion SPME (DI/SPME)) or into the head-space of the sample (HS/SPME). The advantage of HS/SPME is that, because the fiber is not inserted into the sample itself, relatively dirty samples can be analysed while obtaining clean extracts. Another advantage is the relatively high speed, i.e. short equilibrium times in comparison to DI/ SPME. Furthermore, more aggressive sample preparation can be applied, e.g. extremely low or high pH values, without the risk of damaging the coating [21 ]. After equilibrium or a well-defined time, the fiber is transferred to undergo liquid desorption, usually followed by LC analysis. For GC analysis the analytes are thermally desorbed. For LC a special desorption chamber is used. SPME is an equilibrium technique and is based on the partition of the analyte between the stationary phase and the matrix. As a result, SPME is a non-exhaustive


17

extraction method. The amount of analyte extracted (n) is proportional to the initial sample concentration (Co) and sample volume (Vs), the volume of the stationary phase (Vf), and the fiber coating/sample distibution constant (Kf3 [22,98]: n = (Kf~•

Vf x V s x

Co)/(Kf~ •

Vf -[- Vs)

(1)

Extensive studies have been performed to describe the theoretical fundamentals of SPME [20,97,99,100,101]. Basically, the processes in SPME can be divided in thermodynamics and kinetics [20,102]. Because of the physicochemical properties of, for example, polydimethylsiloxane (PDMS, glass transition temperature-123 to -126~ [20,99]), which is the most commonly used phase in SPME, the extraction may be described as liquid-liquid extraction. Octanol-water partitioning coefficients (K~ may be used to estimate the extraction behaviour of an analyte towards PDMS, although it should be noted that octanol and PDMS differ severely with regard to chemical properties. PDMS is generally considered to be an absorptive phase [103], although adsorption effects may occur as well [100,104,105]. The diffusion coefficients of the analyte towards the coating and in the coating determine the time that is required to reach equilibrium. Agitation will increase the kinetic processes since diffusion in the sample is no longer limiting. In an agitated solution the diffusion towards the stationary phase through a static water layer around the fiber is the limiting process in DI/SPME [98,100,106]. Various factors can influence the extraction and desorption efficiency. If available, the coating of the fiber is usually chosen based on the principle 'like dissolves like'. Adjustment of the pH of the sample may affect the yield because non-ionised species are better absorbed by the commonly used fiber coatings. Addition of salt increases the ionic strength and favors salting-out, which often results in an increase of the yield. Increasing the sorption temperature has a dualistic effect. On the one hand, diffusion coefficients are higher at higher temperatures, thus leading to a decrease in time to reach equilibrium. On the other hand, higher temperatures lead to lower partition coefficients in the stationary phase, thus decreasing the extraction yield [99]. During desorption with an aqueous solvent, a pH shift may be applied to cause ionisation. Also, the addition of a suitable organic solvent may be helpful to speed up the desorption process [ 107]. All factors have been studied thoroughly [ 19,20,99,107] and will therefore not be discussed in detail here. Initially, a lot of work has been done on the environmental and pesticide residue analysis [108,109] with SPME-GC. Nowadays, many applications of SPME-GC with either HS/SPME or DI/SPME have been described in various fields, like the analysis of food [108,110,111], explosives [112], biological and pharmaceutical samples [18-20,108,113]. For blood samples, it is possible to determine the degree of proteinbinding of drugs, since SPME usually extracts the free fraction of the drug only [114,115]. Hence, the protein binding can easily be determined if the equilibrium between bound and free drug is not disturbed in the SPME procedure. Another possibility for the estimation of the degree of protein binding is to dilute the sample and determine the extracted amount for different dilution factors [ 116]. When, on the other hand, DI/SPME is applied to urine, matrix interference may be quite severe.


18

Chapter 1

In this chapter we will not focus on SPME-GC as it has already been extensively described elsewhere [18-22,108]. Instead, we will show the current status of the less common coupling of SPME to LC and the direct coupling of SPME to MS via an L C MS interface for the analysis of biological samples. Though the coupling of SPME with CE has been described [ 117-121 ], the application to biological samples has only been performed off-line [119] and will therefore not be discussed here.

1.3.1.2 Applications of SPME-LC In 1995 the first paper on SPME-LC appeared [122]. Basically, two formats of SPME have been coupled to LC, i.e. fiber-based SPME and in-tube SPME. Fiber-based SPME is similar to that applied for GC analysis. Only a specially designed LC desorption chamber [122,123], which acts as a chromatographic tee with a six-way valve, is required to allow desorption with solvent. This chamber can be simply interfaced with the LC. The extraction process is mainly passive though agitation and/or temperature elevation may be applied. With in-tube SPME the sample is aspirated and pushed back by means of a syringe through a capillary coated with the stationaire phase, thus creating an active extraction [124]. This aspiration-push back sequence can be repeated at will. The set-up (Fig. 1.8) is similar to open-tubular SPE and has been known for many years. An overview of bioanalytical applications using SPME-LC is presented in Table 1.1. Since the first application of fiber-based SPME combined with LC, some reports [107,127,130,131,135-137] have been made on the applicability to the field of bioanalysis. Volmer et al. [135] were the first to report the combination of SPME and LC-MS (SIM mode) in the bioanalytical field. The method was developed to analyse 11 corticosteriods and 2 steroid conjugates in urine. Sorption times of 10 to 60 min. were observed, and only 5 min. desorption was satisfactory with regard to carry-over. The detection limits were 4-25 ng/ml due to low yields (1.7-15%). Kumazawa and co-authors [137] described the determination of phenothiazines in whole blood and urine by fiber-based SPME and LC-MS/MS with electrospray ionisation, showing the problem of low yields versus the potential for selective and sensitive analysis. Before SPME was performed, whole blood was deproteinised by adding perchloric acid, centrifugated, and the pH was adjusted to about 8. With urine samples only pH adjustment was performed. A sorption time of 60 min. was applied, which ensured that equilibrium had been reached for all compounds, and subsequently, desorption was performed within 10 min. Even though the SIM mode was applied during single-MS analysis, severe matrix interference was observed for both urine and whole blood (Fig. 1.9a). Applying MS/MS and selected reaction monitoring (SRM) eliminated the visibly interfering matrix (Fig. 1.9b). The extraction yields were very low for whole blood (0.0002-0.12%), resulting in detection limits of 0.2-200 ng/ml. In the case of urine, better results were obtained, as less interference of matrix was observed. The yields were 2.6-39.8%, with detection limits of 4-22 pg/ml. Most probably, the low extraction efficiencies in blood are not due to the equilibrium nature of the extraction as claimed by the authors. It is more likely that a co-precipitation of proteins and bound drug due to acidifcation with perchloric acid occurred.

% t~

Dispenser

HPLC Pump t~ ~,,i.

Buffer tublnq

Stx-port valve

.-.........---~... Waste o0

/

I

Analytical column

INJECT position LOAD position

\ x"k

capillary

Adjustable capillary guide/depth gauge I Adjustable needle guide/depth gauge Vial retainer arm

\

/

,o

.

"% 9%

Solvent vial

Septum piercing needle Sample vial

,, *'~176176

Fig. 1.8. Instrumental setup of the new on-line SPME-HPLC interface based on an in-tube SPME capillary technique. A piece of GC column (in-tube SPME) hosts in the position of the former needle capillary. The aqueous sample is frequently aspirated from the sample vial through the GC column and dispensed back to the vial (INJECT position) by movement of the syringe. After the extraction step, the six-port valve is switched to the LOAD position for the desorption of the analytes from the in-tube SPME by flushing 100% methanol from another vial through the SPME capillary. The volume is transferred to the loop. After switching the Valco valve to the INJECT position, an isocratic separation using a mixture of 60:40 acetonitrile/water was performed. A detailed view of the in-tube SPME capillary is included at the left side of the figure. (Reprinted from [124]. Copyright 1997 Am. Chem. Soc.).

tO O TABLE 1.1 BIOANALYTICAL APPLICATIONS OF SPME-LC Compound

Sample

Type

System

Sorption; desorption time (min)a

Yield (%)a

LOD (ng/ml) a

Ref

Amphetamines Amphetamines, [3-blockers

Urine Urine Serum Urine Urine Urine Serum Urine Urine Urine Serum Ufine Serum Urine Urine Urine Urine Urine Urine Blood Urine

In-tube In-tube

LC-ESUMS LC-ESI/MS

11;4 11;4

81-98 c 0.7-16

0.38-0.82 0.1-1.2

[125] [126]

Fiber (DI) In-tube In-tube

~LC-UV p~LC-UV LC-ESUMS

180; 30

N.D. N.D. 3.0-9.2

3-40 N.D. 0.024-2.00

[127] [128] [1291

Fiber (DI) Fiber b (DI) In-tube

(tx)LC-ESI/MS LC-ECD LC-ESI/MS

180; 30 45; 2 x 5 11;4

LC-ESI/MS

10; 4

1-6 10 3-36 (LOQ) 9-43 (LOQ) 0.1-1.2

[130] [131] [1321

In-tube Fiber Fiber Fiber Fiber Fiber Fiber

ESI/MS LC-MS LC-ESI/MS LC-UV APCI/MS/MS LC-MS/MS

60; 2 15; 5 10; 6 45; 2 x 10 5;4 6O; 10

LC-ESI/MS

11;4

N.D. 45 88-110 c 70-109 c 84-113 c 71-11U N.D. 5-45 56-86 ~ 22 6.5 4-40 0.0002-0.120 60

Antidepressants (tricyclic) Antidepressants (tricyclic) Benzodiazepines Benzodiazepines Brombuterol + analogues [3-blockers [3-blockers Carnitines Corticosteroids + conjugates Flavonoids Lidocaine Lidocaine Phenothiazines Ranitidine

N.D.: no data available; DI: direct immersion. a: unless stated otherwise. b: molecularly imprinted polymer (MIP) coating. c: relative to aqueous samples.

(DI) (DI) (DI) (DI) (DI) (DI)

In-tube

10; 5 11;4

0.2-12.8 4-30 2.7-25.4 pg/ml 25 0.40 4-22 pg/ml 0.2-200 1.4

[1331 [134] [135] [136] [107]

[104] [137] [138]

%

Whole zoo

blood

extract

Urine

~,,.' ~ ; } 4 0

-1

9

Perazine 0

.

ZOO

Z

. . . m,'= ;36~

~ .

-

,

.

.

A

~

.

.

.

~...,.,,.~-~

.

.

.

.,- E - 0 3 _ . j . , ~ ~ , . 420

.

v-

. . . . .

i

,~'

Tnioridazilqe

~z t

o m/z :'f/c

100

--

,~

Prochloperazine

t

Thiethylperazine C lOG

.

.

.

.

.

.

.

.

.

.

.

.

~

.

~

-

.

0

.

m - -

,

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~

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s .... ..

--

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.

;. 4 ~ 0

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m/~

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m.l Z : 4 4 7

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, . . . . . . . . .

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100 ;

. . . . . . . . . .

6 : O0

~-.

9:00

. . . . . . . . . .

t 2 .?00

15 ;%0

Clospirazine

.....

]- m/z:44"/

---

, ....

Thioproperazine ....

......

T

11

, 9,~

~.__.

j~

J'.--

-

~'-.~::~

r '~ ~_~_ z. ~s2

~

,~-~

~.

I"~ ~

A

)

00~

'~

z ,sos

,oo1.,,,,, o

r':;'~'~,~

:,~~~~

" "~''~"~'--~----]

,~

-4~.A 9

_

~,~. ;~

~

xA

____

~,,

Perphenazine i/ll

..

Flup~ine

PUr.:44~

. . . . . . . . .

1''''~

i

~.~

E,C,4

9

Flup~bxol

Thioproperazine 0

'

Trifluoperazino /k~ _ _ ~ ...... -- . . . . ~ . - " " ' ~ --"

100

Clospirazine 0 1CO

""--- --

o '----,

m/z:438

-

. .

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P

1~176 ":~'~176~S~.-,.~-~,

o ] Flu~ine iCO

I

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ioridazme . .

- m.,'z : 374,

0

.

I00--a/=, 43S

_J

o ] Flupentixoi iCO

0 100

.

m/z-3"/l

C,

~ X . , . , _ . . . _ , . . _ ,

......

100 -

100

m/z:43~

iOO

mlz : 366

[,. %~ ~o'1":'~ , ~

m/z:408

] Tdflu~

D

F...,.,~3 3 6~ 8

Thiethylperaz

__

;434

o

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.

~. ~80

.,~

_J ~ L _ _ r-'-

~/z

E-~04

A P"~

m/z:400

a00

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extract n . / = : 34C

, . . . . . . . . . . . . . . .~.: .1.0 3 : 00

~ s Ar,.o, ,... z. ~9,

,

- -----A- ~---

/ ~

---=.,.r~

_

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i

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~;+0~

5.8 a

loo

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:100

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"" 15

:O0

A

Fig. 1.9. (A) SIM of S P M E - L C / M S for the 11 phenothiazine derivatives extracted from human whole blood and urine. The amounts of each drug spiked into 1 ml whole blood and urine were 0.5 ~g and 0.5 ng, respectively. The arrows show the locations where drug peaks should appear. The left vertical axes represent the relative percentage intensity of the peaks, the fight vertical axes show the absolute intensities of peaks recorded by the mass spectrometer and the horizontal axes indicate the LC running time (min).

~"

Urine extract

Whole blood extract IRIC-.-

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r

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0

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DA

o~ 3"~.::).(C'- 3"/L.60

0

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n~

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of

399.;0>

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. . . . . . . . . 404,70

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of 4C?.:0>

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of

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--

nf

.

100

. . . . . IRZ.',=> DA of 399. "/0> 4 0 0 . 7 0

.

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17%.4~,~

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of

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ga.

of

-

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k

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13'7170,,

g-02

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E+02

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?.'754

k

3")0.4;0-',.

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9Ir 3 .SSO

.oo

,,.,o-3~2"i:"

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o

|,02

0s

J•..

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0 ]Proper~azine

E*02 ? .8o1

I;.110

of' :339.60;"

Ioo - "~-'" ~

o

E * 0 4

.

o razJne 100

4

3.4)8

jl~ .

r

'~176

1

8..1

~t

ot

~

-- E * 0 1

L

tFlul~nli~ol o

........

.......

.......... k

]Tfifluopecazine

0 --" i00 I~1:C-> ~

.

....

0

100

100

. 5

rid~ine _

Z-02 I2.82S

: !- ' ' ~ " 3:00

FL.m

' . . . . .I S : O0

;'-; :

k

9 . . . . .l 9:00

.

~'--"

.....

12

I " "'" " " : O0

....

Is

Fig. 1.9. (B) SRM with SPME-LC/ES-MS/MS for the 11 phenothiazine derivatives extracted from human whole blood and urine. The amounts of each drug spiked into 1 ml of whole blood and urine were 0.5 ~g and 0.5 ng, respectively. Axes as in (A). ( 9 John Wiley & Sons Inc. Reproduced from [137] with permission).


23

With the introduction of in-tube SPME [124] it became possible to automate the analytical procedure, and consequently, a number of applications in the bioanalytical field have been reported [125,126,128,129,132,133,138]. Most applications show great similarity, and therefore only one application will be discussed here. The results of other in-tube SPME-LC studies are summarised in Table 1.1. Kataoka et al. [ 126] developed an automated in-tube SPME-LC-ESI/MS method for the simultaneous determination of 5 amphetamines and 9 [3-blockers in biological samples using single-MS (SIM mode). Applying a flow-rate of 100 l~l/min proved to be optimal [22,124], and varying the number of draw/eject cycles of 35-1~1 samples resulted in the extraction-time profile (Fig. 1.10). As can be seen, equilibrium was not reached after 20 cycles. However, when the extraction was stopped after 15 cycles (taking about 11 min.), excellent detection limits (0.1-1.2 ng/ml) and linearities (R> 0.998) were obtained. Similar effects of type of matrix were observed as with fiber-based SPME, i.e. extraction from urine resulted in higher yields than extraction from serum. Worth mentioning is the differences in extraction yield and kinetics of drugs within the same class. This phenomenon has also been observed with fiber-based SPME. This may be explained by differences in diffusion coefficients of the drugs, but this is not expected. Surprisingly, the optimal pH during sorption was 8.5, ensuring that all compounds (pK, 9-10) were protonated to a large extent. This is contrasting to most applications in which non-protonated analytes were more easily absorbed than the protonated species. An explanation of the use of this pH value might be that the capillary coating was stripped at pH 10, resulting in lower yields than with pH 8.5.

1,20E+07 --O--

1.00E+07

MA

--,.--MDMA --e--- Pi ndo|ol

8.00E+06

. ~

c:

Propranolol

;~-- hO~.butolol

o

~a 6.00E+06 t13

4.01~*'06 2.00E+06

0.00E+00 = 0

- .............

' 5

.

10

.

. 15

.

.

. 20

Number of draw/eject cycle

Fig. 1.10. Extraction-timeprofile of drugs with Omegawax 250 capillary. SPME conditions: drugs, 0.5 ~g/ ml; sample pH, 8.5 (Tris-HC1); draw/eject cycles, 15; draw/eject volume, 30 or 35 ~1; draw/eject rate, 100 i~l/min; desorption, mobile phase. AM = ( + )-amphetamine, MA = ( + )-methamphetamine, MDMA= (=)-3,4-methylenedioxymethamphetamine. ( 9 John Wiley & Sons Inc. Reproduced from [126] with permission).


24

Chapter 1

1.3.1.3 Applications of SPME-MS SPME is hardly ever used in high-throughput systems due to the relatively long sorption and desorption times. Even when SPME is followed by chromatographic separation, the sorption and desorption are usually the time-limiting steps. This also holds for fiberbased SPME systems and/or systems in which no separation is performed, i.e. the desorption device is directly coupled to the detector. P6rbs and co-authors [ 139] coupled HS/SPME directly to MS by thermal desorption and a short GC transfer-line for the determination of volatiles in cheese using only 10 min. sorption. A typical desorption profile of HS/SPME-MS is shown in Fig. 1.11. Orzechowska et al. [140] determined cocaine by direct coupling of HS/SPME with ion mobility spectrometry. Camitine, an essential factor in the fatty acid metabolism of organisms, was determined by SPMEESI/MS [134]. Severe matrix interference during detection and a long sorption time were the major drawbacks of this method. A possibility to enhance the throughput is to use non-equilibrium SPME, which is more commonly applied in in-tube SPME than in fiber-based SPME. Yet, such an approach may put extra pressure on the sensitivity and reproducibility of the method. Ai [141 ] described a theoretical model for fiber-based SPME allowing quantitation before equilibrium is reached. In a challenging practical application, Van Hout et al. [104] performed ultra-rapid non-equilibrium fiber-based SPME and coupled it directly to APCI/MS/MS for the determination of lidocaine in urine. Direct immersion of the 100 ~m PDMS fiber in urine was applied for 5 rain. Subsequently, the fiber was desorbed for 4 min., after which 1 min. analysis was performed. No matrix interference was observed. The method had not yet been optimised for quantitation, but initial results on repeatability and reproducibility looked promising. Even without an internal standard, coefficients of variation below 15% (LOQ) were observed at a level of 2 ng/ ml. The detection limit was 0.4 ng/ml and good linearity was observed. This approach clearly showed that the long equilibrium times of SPME are disadvantageous for rapid analysis, but that use of non-equilibrium SPME can help to overcome this problem.

1.3.1.4 Remarks regarding the applicability of SPME SPME has clearly demonstrated its utility for bioanalysis, and in particular for the analysis of urine samples. The number of applications for the analysis of plasma, serum and blood samples is more limited. So far, no reports have been found about ion suppression in SPME-LC-MS systems. This might be due to the clean extracts that can be obtained with SPME due to the low yields for both analytes and matrix compounds and differences in diffusion coefficients. However, it may also be due to the lack of attention for these effects. A major drawback of SPME is the small number of commercially available stationary phases, especially for fiber-based SPME, hereby limiting the choice for selectivity. The main commercially available coatings for fiber-based SPME are PDMS, polyacrylate and some mixed phases, e.g. PDMS/carboxen, PDMS/divinylbenzene (DVB), Carbowax/DVB and Carbowax/templated resin. From this list it is obvious that the choice for selective extraction is limited. Moreover, ion exchange phases are not available. For intube SPME, a piece of capillary GC column is commonly used, thus providing more


References pp. 39-44 Fig. 1.11. (a) Desorption peak of a sample of cru2 Camembert cheese obtained by SPME-MS. The extraction was carried out by placing an SPME Carboxed PDMS fiber in the headspace at 20°C for 10 min. (b) Average spectrum of 0-3 min. (Reprinted from [139]. Copyright 2001 Am. Chem. SOC.).

p3

VI

26

Chapter 1

diversity in stationary phases than in fiber-based SPME. In addition, phases for more polar analytes are available. Yet, with the development of more stationary phases for both fiber-based SPME [131,142-144] and in-tube SPME [132] the potential of SPME may be improved even further. The advantage of in-tube SPME over fiber-based SPME is the shorter sorption times, as can also be seen in Table 1.1. This is due to the fact that in-tube SPME is usually stopped before equilibrium is reached. However, as described above, non-equilibrium fiber-based SPME can be performed as well. With the latter, transfer of the fiber with the trapped analytes to the desorption chamber is required after sorption, which is not necessary with in-tube SPME. The latter system also allows the use of 100% organic solvent for desorption of the analytes. The use of high percentages of organic solvent is more complicated with fiber-based SPME, since the coating can be stripped from the silica. Samples containing particular matter should be filtered prior to extraction with intube SPME to prevent clogging of the capillary, which cannot cause problems in fiber-based SPME. With regard to automation, in-tube SPME has more to offer at this moment. Automation is more easily established with this system, since no transfer of the stationary phase to a desorption chamber is required. The availability of an autosampler for use with fiber-based SPME-LC is still being awaited [21]. Thus, both fiber-based and in-tube SPME have their own advantages and disadvantages. To some extent, the systems can be considered complementary and the best choice will depend on the sample and on the particular requirements of the individual applications. A recently introduced SPME device is the stir bar sorptive extraction [145], which has a larger volume of stationary phase than fiber-based SPME and in-tube SPME. Although it has not been applied for bioanalysis, it has already shown its potential for the coupling with LC and determination of (semi-) volatiles in aqueous samples.

1.3.2 Membrane-based sample preparation techniques 1.3.2.1 General aspects of membrane-based techniques As an alternative to SPE and/or LLE, membranes may be used as a sample preparation technique [23,25,146-153]. When using the latter, it is essential to differentiate between porous and non-porous membranes. Sample pretreatment with porous membranes is based on the principle of size exclusion to differentiate between substances, whereas non-porous membranes utilise the difference in partition coefficients of substances, thus being an actual extraction technique. An overview of the various techniques is given in Table 1.2. The porous membrane techniques (PMTs) drew major attention in the late 1980s and early 1990s, whereas the applicability of non-porous membranes for sample pretreatment in the biomedical field is being explored more recently. In PMTs, the liquids on each side are physically connected through pores. Transport through the membranes is based on size-exclusion, i.e. sufficiently small molecules can permeate through the pores, whereas larger molecules cannot. This can result in an efficient clean-up from large matrix molecules, but no distinction can be made between small molecules. The latter is only possible to some extent with electrodialysis, for which an ion-exchange membrane is used. Now, large molecules and molecules with a


27

TABLE 1.2 OVERVIEWOF MEMBRANE-BASEDSAMPLEPREPARATIONTECHNIQUES Driving force

Mainly combined with

Dialysis Porous Electrodialysis P o r o u s

Concentrationdifference Potentialdifference

LC CE

Filtration Membrane extraction

Pressure difference Concentration difference

LC LC, GC, CE

Technique

Membrane Principle type Size-exclusion Size-exclusion and selective ion transport Porous Size-exclusion Non-porous Differencein partition coefficient

given charge will be excluded. Strictly taken, PMT is not an extraction technique, but a filtration process. Extensive descriptions of the principles of porous-membrane techniques were given by Van de Merbel [23] and co-authors [24,25], and will therefore not be discussed here. Non-porous membrane techniques (NPMTs) employ an organic or polymeric (solid or liquid) layer, placed between two other liquid phases. The analyte must actually be extracted from the donor phase, dissolve into the membrane in order to be able to pass through, and then be released in the acceptor phase. The behavior of the analytes largely depends on partition coefficients between the different parts of the membrane system. Only analytes that are easily extracted from the donor phase and, in addition, are easily released from the membrane into the acceptor phase will be transported. Thus, the separation is based on the same principles as LLE with back-extraction. It is thus possible to separate molecules of similar size, yet with different physicochemical properties [146,152]. The non-porous membrane technique can be subdivided into four main groups: (I) Supported liquid membrane extraction (SLME), (II) Microporous membrane liquid-liquid extraction (MMLLE), (III) Polymeric membrane extraction (PME) and (IV) Membrane extraction with a sorbent interface (MESI). SLME is the most widely used non-porous membrane technique [146], but various applications of MMLLE, PME and MESI have been reported as well [23,25,146-150,152]. It should be noted that MMLLE is considered to be NPMT, even though a membrane with micropores is used. All NPMTs utilise a membrane unit constructed from two blocks of inert material with a machined groove in each. A membrane is placed between the blocks and the total unit is clamped together. Hence, two flow channels are formed, one being the donor channel, the other being the acceptor channel. In principle, SLME utilises a pH shift between the donor phase, in which the analyte is uncharged, and the acceptor phase, in which the analyte is protonated, thus ensuring that no back-extraction in the (organic) membrane can occur. MMLLE is performed with organic solvent as the acceptor phase in the micropores of the organic membrane, and can therefore be compared with a single liquid extraction. MMLLE is mainly used for the analysis of hydrophobic compounds that cannot be extracted from an organic membrane into an aqueous acceptor solvent,


28

Chapter 1

as is the case with SLME. PME is similar to SLME, with the exception that a polymeric membrane is used. Due to this membrane it is also possible to use organic solvent in the donor and/or acceptor phase. However, the composition of the membrane is fixed, limiting further chemical tuning. Furthermore, low diffusion coefficients and slow mass transfer may lead to slow extraction. MESI differs from the previous techniques in that a solid polymeric membrane is used. MESI was mainly developed for the combination with GC, thus in order to use a gaseous acceptor phase [146,148,152], while the donor phase is aqueous or gaseous. Obviously, MESI works best for the analysis of volatile and relatively non-polar compounds [152]. Most applications of MESI are in the environmental field for the analysis of aqueous samples [146,154-156]. Both PMTs and NPMTs usually use the terms efficiency and/or enrichment. The efficiency is defined as the ratio between number of moles input to the system during the extraction and the amount collected in the acceptor, and can be directly measured [ 150]. Efficiency should not be confused with the term recovery, which is commonly used with extraction techniques. Recovery is (or should be) relatively constant under the selected conditions, and should, therefore, not affect the accuracy of the system if the response is corrected. With membrane-based techniques, the efficiency is usually not allowed to become 100%, because of time dependence. It is obvious that efficiency may be sacrificed for speed if sensitivity is not of major concern. As a result, efficiency is not always constant, as the time for sample preparation can be varied. Moreover, various factors, e.g. the composition of the donor phase, acceptor phase, the membrane and the sample, can affect the efficiency of the system. The most often observed side effects, i.e. binding to matrix proteins and adsorption to the membrane, with consequent carry-over, have been described in various studies and these effects will be pointed out later in this chapter. Besides efficiency the term enrichment is also often mentioned, especially with PMTs. Enrichment is the accumulated amount of analyte in the acceptor phase during a given time. In membrane techniques the efficiency decreases with increasing donorflow. Contrary, the enrichment increases with increasing donor-flow (Fig. 1.12). At very low donor-flows the enrichment is close to zero, as the extracted analyte is diluted immediately in the acceptor phase. With high donor-flows, the efficiency is decreasing due to incomplete diffusion of the analyte into the acceptor phase, but the enrichment is increasing with increasing donor-flow. However, high donor-flows imply large consumption of sample, and can therefore only be applied if sufficient sample is available [ 146,150].

1.3.2.2 Porous membrane techniques As shown in Table 1.2, three major types of PMTs can be distinguished. Although a porous membrane is also used in microdialysis, the latter is a rather different technique, being mainly applied for in vivo studies [157-162] and will therefore not be discussed here. The applications of on-line filtration are limited to fermentation broths [23]. Electrodialysis has been coupled on-line to CE for the determination of inositol phosphates in plasma [163-165]. Only one report is available for the on-line coupling with LC [164]. Despite a high efficiency (95%), ephedrine could only be determined in serum three times before the membrane faltered. The studies on electrodialysis were merely explorative and no applications in routine analysis have been reported yet. The

New developments in integrated sample preparation for bioanalysis 1

.

.

.

.

.

.

.

0.8

29

120 100 8O

0.6 W

60 0.4

40

0.2

20

0 0

0.05

r

0.1

0 0.15

Fig. 1.12. Extractionefficiency E and enrichment factor Ee (arbitrary units) as functions of the reduced flow parameter % Note: q~ is the volumetric flow divided by the membrane area. (Reprinted from [150], with permission from Elsevier Science).

most common PMT is dialysis, employing a cellulose-based membrane and an aqueous donor and acceptor phase. Though some reports have been made about the on-line coupling of dialysis with GC [54,166] and CE [2,167,168], most bioanalytical applications couple dialysis on-line with LC. An overview of the results obtained with the latter system is presented in Table 1.3. The major disadvantages of dialysis are the typically low efficiency and the long dialysis time. Several approaches have been applied for concentration, e.g. automated sequential trace enrichment of dialysates (ASTED) [25,176,202] using a reversed phase enrichment column. Selective trapping has also been performed [177,182]. Furthermore, the use of membranes might result in adsorption of the analytes by the membrane [ 182]. Covering of the active sites by adding a surfactant can minimise adsorption [ 192,196]. The binding of analytes to macromolecules in the matrix such as proteins has an even more pronounced effect on the dialysis efficiency. Only the unbound fraction can diffuse through the membrane. As a consequence, the efficiency may be substantially lower than with aqueous solutions. This will especially be the case for compounds that bind to a high degree to proteins. This allows the determination the free fraction of analyte [174,176,185,188]. A number of ways have been suggested to release the bound drug from the protein. The simplest way to reduce protein binding is to dilute the sample. Another possibility is to change the pH of the sample, hereby changing the structure of the macromolecule and/or the charge on the analytes, causing the release of the analytes of interest [174-176,184,187,192]. Denaturation of the proteins can be performed by addition of a strong acid. However, this may also result in loss of bound drug that coprecipitates with proteins. A more selective and elegant strategy for releasing the analytes from the proteins is the addition of competing agents or displacers for the protein binding sites [24,174,176,180,187], e.g. fatty acids with appropriate chain


TABLE 1.3 APPLICATIONS OF DIALYSIS ON-LINE WITH LC IN BIOANALYSIS Year

C ompound

S ample

1985 1985 1986

Amino acids Barbiturates Enoximone + metabolite

Serum Serum Serum

1986

Salicylic acid

Serum

1987

Mitomycin C

1987

Anticonsulvants Theophylline Corticoids Phenobarbitone Phenytoin Phenylbutazone Theophylline Warfarin Azidothymidine Des-enkephalin~/-endorphin Oxytetracycline

Plasma Urine Serum

1988 1988

1990 1990 1991 1991 1992

Oxolinic acid Flumequine Iopentol

1992

Pholcodine

1992 1992 1993

Benzodiazepines Rogletimide Antiviral drug* + metabolite#

Detection

Dialysis time (min)

UV Salicylate electrode UV UV

Efficiency (%)

LOD a

Ref

2 30 17 48 86-105

6 nM N.D. 10 15 N.D.

[1691 [1701 [171]

25

1

[173]

50

50-170

[174]

[172]

Serum Plasma Serum

UV UV

3 12

85-90 b 68

30 nM N.D.

[1751 [1761

Plasma Plasma

UV Flu

17 15

40 25

20 10

[1771 [178]

Whole blood Plasma Whole blood plasma

UV

7.3

60

50

[1791

UV

7.3

60-69 b

50

[180]

UV

7.15

500 c

[1811

Flu

8.25

50 47 60

40

[182]

UV MS/MS UV

7.6 14

Q u i c k Elute M o d e Elute

e l e t a ~ ~e w a u e

tl~Vt~)

i

....

Fig. 4.13. The three basic steps to using the Quick Elute method.

~i~~

Re-equilibration

~J~

Turbulentflow chromatography in bioanalysis

ll5

of TFC to a wider range of analytes. Ensuring that the cleaning stage is thorough will reduce carryover as well as increasing the sensitivity of the technique.

4.3.1.3 Focus mode To achieve chromatographic resolution another method was developed which allowed for a separation of isobaric compounds or reduction in ion suppression effects. There are four simple steps to using this particular technique. The first step is exactly the same as with the quick elute mode, sample is loaded onto the column under turbulent flow conditions which allows separation of the two basic components, namely that of matrix and analytes. The second stage is to transfer the analytes onto a second column, using an organic solvent plug. Both pumps are running aqueous mobile phases and so the result is that the analytes will be retained on the second column as the solvent plug is diluted in the internal 'T'. Careful selection of the flow rates will ensure that the analytes are focused at the top of the column, ready for the third stage. The third stage is to elute the compounds of interest from the top of the second column into the detector. A laminar flow gradient is used here to ensure that optimal separation is obtained. Laminar flow is used as a consequence of the detector requirements. Whilst the compounds are eluting the turboflow column is being cleaned and the sample loop refilled with organic solvent. Finally, the system is re-equilibrated ready for the next injection.

4.3.2 Application areas

Turbulent flow chromatography coupled to tandem mass spectrometry has been used in a wide range of applications from analysis of pharmacokinetic samples, to forensic applications and environmental samples, providing fast, sensitive and specific analysis for the determination of compounds in a range of matrices. The following section will highlight some of these application areas.

4.3.2.1 Drug metabolism and pharmacokinetic (DMPK) studies The first publications based on turbulent flow chromatography, after the original issuing of the patent, applied to the analysis of biological fluids in a commercial environment, were by Ayrton [46,47]. The technique was successfully used in the analysis of a novel pyridine-isoquinolone compound and its deuterated analogue, under development by Glaxo-Wellcome at the time of the research. The compound was found to be strongly protein bound, in excess of 95%. Using a single valve technique, the authors were able to demonstrate that accurate and precise data could be obtained over the range 5-1000 ng/ml with a 10 ~1 sample of plasma spiked at the appropriate levels. The flow was split prior to the mass spectrometer by 10:1, which obviously affects sensitivity, but also reduces possible fouling of the mass spectrometer source. It was noted that using a silica-based column that there was no appreciable pressure rise with an overnight run of 100 samples, with the injection volume raised to 50 ~1 (comprising


Focus Mode Load

v.o,,. , . ~

~~~~":~

Transfer

9~"

! -

~~,~

! I

~.

,.oz~

I [

'h! %%

%

-

Aqucousm~il,~ ph~c

Elute

~,~~-:~

refocused

Aqueous rrmhil~ph~.~

Re-equilibration

i~ ~ ~ ....J ~.............

linear ~rldi

l

....

ch Aquco~ r,~c~d~ilc ld~.~

Fig. 4.14. The four basic steps to using the Focus Mode method.

4~


117

25 ~l of plasma and 25 p~l internal standard mix) and also that there was no significant fouling of the mass spectrometer interface, a common problem with protein precipitation. The total run time was quoted as 2.5 minutes. Chassaing et al. [48] developed a TFC-MS-MS method which was successfully used and validated for the determination of three basic compounds, doxazosin, CP-122,288 and dofetilide in dog plasma in a 96-well format. Chassaing used a quick elute configuration, utilising 2 Rheodyne 6 port switching valves as well as two standard Agilent 1100 HPLC pumps. The extraction column was a Cohesive turboflow column (C 18, 50 mm x 1 mm 50 ~m average particle diameter). Samples were prepared by adding 200 p~L of a solution of internal standard, made in 90% water, 10% methanol) to the 200 ~L plasma. These were prepared in a 96 well plate format, centrifuged at 2000g for 1 hour at 4~ prior to injection. This process ensured that no particulates reached the system, increasing the column lifetime. The assay showed an excellent linearity across the range of 5 to 500 ng/ml for all compounds. Studies also showed that the analysis was repeatable over a period of time as the interday assay results clearly show. To ensure the validity of the data generated using TFC, 22 real samples from 2 studies were re-analysed using an old solid phase extraction sample pre-treatment followed by analysis of the extract using HPLC-MS-MS. The experiments showed that there was excellent agreement between the plasma concentrations measured by TFC-MS-MS and those measured with the SPE-LC-MS-MS method (see Fig. 4.15). Although most TFC is employed as a precursor to detection by MS or MS-MS, Kennedy [49] used TFC coupled to UV detection to determine ciprofibrate in human plasma. Ciprofibrate is a fibric acid derivative hypolipidaemic agent, used for the treatment of primary hyperlipidaemias resistant to appropriate dietary management including hypercholesterolaemia, hypertriglyceridaemia and combined hyperlipidaemias. A range of concentrations between 1 and 30 ~g/mL were determined. Human plasma was initially diluted with an equal volume of 0.1M hydrochloric acid, with 40 ~L of the resulting mixture being injected onto the turbo flow system. A single valve method was employed for the analysis with the total analysis time being 2 minutes. It was noted that a further 1.3 minutes was spent waiting for the autosampler to prepare for the subsequent injection. The reported data showed excellent linearity over the specified range, with no observable carryover for this particular compound. Calculation of the limit of quantification (LOQ) using the guidelines of the International Conference on Harmonisation (ICH) gave a value of 58 ng/mL. The reported validation data was as good as if not better than data generated using a SPE method. The columns lasted in excess of two hundred injections of diluted plasma, however it was noted that using the developed procedures the columns were not used to their full extent. Kennedy also reported that the performance of the column did not deteriorate with increasing accumulation of backpressure. Zimmer [50], using a quick elute method, demonstrated that TFC could be successfully applied to the analysis of two internal compounds named as compound A and compound B, over a dynamic range of 1 to 500 ng/mL. The validation data presented clearly showed that this technique could be successfully employed in the routine analysis of biological fluids. Cross-validation of the results using data generated References pp. 127-128

TABLE 4.1 INTRA- AND INTER-DAY IMPRECISION AND INACCURACY OBTAINED FROM CHASSAING [49] Intra-day

(ng/ml)

Inter-day

Mean observed concentration (ng/ml)

n

SD

CV (%)

Inaccuracy (%)

Mean observed concentration (ng/ml)

5.50 16.18 249.23 399.12

7 7 7 7

0.36 0.94 6.34 4.94

6.51 5.84 2.54 1.24

9.98 7.86 -0.31 -0.22

5.21 15.72 248.48 399.47

5.20 14.79 240.84 380.24

7 7 7 7

0.23 0.37 3.60 5.78

4.44 2.53 1.50 1.52

4.06 -1.39 -3.66 -4.94

5.32 14.20 241.70 384.58

7 7 7 7

0.96 1.31 5.33 11.8

18.08 9.22 2.20 3.06

6.48 -5.33 -3.32 -3.86

SD

CV (%)

Accuracy (%)

20 21 21 21

0.68 1.30 5.31 8.39

13.1 8.29 2.14 2.10

4.28 4.80 -0.61 -0.13

5.02 14.73 238.50 376.11

20 21 21 21

0.48 0.59 3.96 10.1

9.63 4.01 1.66 2.68

0.37 -1.77 -4.60 -5.97

4.87 14.51 237.48 383.78

20 21 21 21

0.33 0.86 5.82 6.00

6.81 5.96 2.45 1.56

-2.56 -3.29 -5.01 -4.06

Doxazosin level 5 15 25O 4OO CP-122,288 level 5 15 250 400 Dofetilide level 5 15 250 400

4~

Turbulent flow chromatography in bioanalysis 1000 -

o

119

TFC

100

g~

o

lO

0

I

1

I

I

I

2

4

6

8

10

time (hour)

Fig. 4.15. Cross validation data for real study samples.

by a liquid-liquid extraction technique gave very good correlation for both the drags analysed, less than 5% standard deviation for 42 samples for both compounds. One of the most significant findings of the research was the dramatic reduction in analysis time, for one batch of 96 samples, the sample analysis time was reduced to less than a third of the original liquid-liquid extraction method (16 hours). It was noted by the author that this could be further reduced, by reducing the autosampler cycle time. This could be achieved either by reducing the number of washes or more effectively by having the autosampler perform the majority of its cycle during the previous analysis. Jemal [51 ] used a single valve method for the analysis of a variety of compounds with mass spectrometry as the detection system. The initial sample was diluted with an equal amount of an aqueous solution of the internal standard, with 50 b~l of the resulting mixture injected onto the system. Samples were placed in 2 mm vials, vortexed and then centrifuged to allow separation of any particulates within the sample. Two quick elute methods were described for the analysis of compounds in rat plasma and in human plasma. The total analysis time was 4 minutes, with 1 minute for loading of the sample and elution of the matrix, 2 minutes for the elution of the drug, and one minute for reequilibration of the system ready for the next injection. The second method used an analytical column in-line with the extraction column during the elution step to provide chromatographic resolution between the two isomeric compounds. This method had a total run time of 5 minutes with slightly longer loading and elution steps than the previous method. The results presented are very good over the specified range (1-2000 ng/mL), with accuracy ( < 10% across the range) and precision ( < 4% at the lower limit of quantification) (LLOQ) well within regulatory levels. Wu et al. [52] were able to analyse up to 10 compounds simultaneously from cassette dosing experiments using a single valve method with two columns, extraction column and an analytical column. In this study two pumps were used, one to load the sample onto the extraction column, the other to elute the compounds onto the mass spectrometer. Comparisons with on-line SPE revealed that separation efficiency and, dynamic range, accuracy and precision were comparable between the two methods over References pp. 127-128

120

Chapter 4

the range investigated, 1-1000 ng/mL. These studies revealed that pressure build-up using on-line extraction columns under laminar conditions was a problem with the columns failing after 80-100 injections. Using turbulent flow chromatography, with the specially developed columns gave substantially better column lifetimes Injection of 100 ~1 of sample plus internal standard (approximately 85% plasma) was used in the study. A tandem mass spectrometer was used as the detection, with a gradient applied across the extraction column after the initial extraction step had been finished to elute the compounds of interest from the column to the detector. It was found that turbulent flow chromatography was applicable to poorly water-soluble compounds as well as highly protein bound compounds. Brignol et al. [53] investigated the analysis of terbinafine, an antifungal agent, from human and pig plasma. Two procedures were utilised an off-line protein precipitation method followed by LC-MS-MS and the on-line technique. The off-line analysis was able to reach a LLOQ of 0.0678 ng/mL in human plasma, with acceptable precision and accuracy values. Pre-concentration of the precipitated plasma meant that the limit of quantification was lower than using an on-line technique, however it also meant that more plasma was required, which is not always desirable. The dynamic range was from the LLOQ to 89.9 ng/mL. Using the off-line technique recoveries varied across the range from 53.4% to over 70%. A focus method was used for the analysis using TFC. Although the limit of quantitation was higher (0.117 ng/mL), primarily since only 50 ~L was used as opposed to 200 ~L, the precision values were better than quoted for the off-line technique (ranging from 1.15% to 6.68%). Also, since the sample did not have to blown down and then reconstituted in a suitable mobile phase, the sample preparation time was substantially less. Taxol is widely known for its therapeutic effect in the treatment of some cancers. Dongen [54] developed a method for the analysis of this compound in human plasma. Example chromatograms are shown in Fig. 4.16, with the statistics from the validation study shown in Table 4.2. Within most industries there are pressures to produce data more quickly, and using turbulent flow chromatography, Dongen was able to reduce method development time down to two days. Other authors have very generic methods, which require virtually no method development, and they become applicable to a wide range of compounds. Using such a genetic method, Hermann [55] was able to analyse over 1000 different compounds. Samples were prepared by taking 100 p~L of plasma and adding 200 p~L of acetonitrile containing an internal standard. The sample was then centrifuged for ten minutes at 10,000 rpm and the supernatant placed into an autosampler vial. The samples were then analysed using the generic focus method. To illustrate the applicability of the TFC approach, six compounds, with a large range of hydrophobicites, were chosen and analysed in rat plasma. The chromatogram for each compound at 5 ng/mL in rat plasma is shown in Fig. 4.17a. All the compounds have good sensitivities, high recoveries (> 90%) and excellent peak shapes. As well as urine and plasma, a variety of other different matrices have been used in TFC, including brain homogenate, undiluted plasma, and microsome mixtures. There was no significant difference found between the various matrices, Fig. 4.17b. Currently


121 intensity: 2759 cps

217

100" 90" 80' 70' 60' 50' 40' 30' 20' 10' 0

' 161 0:21

Ik

261 0:41

361 1:02

401 1:22

501 1:43

66f" 761 2:03 2:24

-'-

801 2:44

A

. . . .1 ~ . . . . . 901 1 0 0 1 1101 Scan 3:05 3:25 3:46 Time

.t,_..,~,~.

Fig. 4.16. Chromatogramsof 10 ng/mL of taxol spiked in human plasma.

the technology is being refined to allow the analysis of whole blood with no sample pretreatment.

4.3.2.2 Forensic applications Grant [56] developed an assay for the analysis of cortisol and a metabolite in urine. Cortisol is a naturally occurring hormone which influences metabolism, inflammation, electrolyte and water balance. Their synthetic derivatives are used therapeutically for their anti-inflammatory and immunosuppressive actions. They are used in certain sports to improve the performances of the athletes (euphoria, motor activity). The ability to distinguish between the active components and some of the metabolites formed within the body is important for the accurate determination of the amount of initial drug taken. A focus method was employed which allowed urine samples to be prepared and analysed in less than five minutes. The data presented in Table 4.2 shows that cortisol

TABLE 4.2 RESULTS OBTAINED BY GRANT [57] FOR THE ANALYSIS OF CORTISOL OVER THE RANGE 0.25 ng/ml-250 ng/mL Concentration/ng/mL 0.25 25 100 250


Average response (n = 3)

%RSD

2.74E2 1.96E4 7.86E4 2.33E5

13.7 7.4 5.2 8.9

Chapter 4

122

Deprenyl 300~ :1

~ 2ooo.

2

~

" Time

4|

2

5'

;

~

.......

Time(rain)

(rain)

20001800"

Haloperidol

K252a

1600~., 1400~ " 6000 ,..., :~" 1200" "~ ~

v

I000-

..~"~ 4500

81)03000 600-

J~

400I

200" i

0 _ 2

.

.

9

4

2

s

Time(rain) Time

(rain)

4000~

CEP-1347 300OO"

2

1000~

0-

0 2

3

4

5

2

Time(rain)

~ 3

4

Time(rain)

Fig. 4.17. (a) Example chromatograms of 6 compounds, all at 5 ng/mL, with differing hydrophobicites all

analysed directly from rat plasma using one standard method.

can be detected precisely over the 0.25 to 250 ng/mL range. This approach has also been used for a wide range of opiate analysis in urine. In particular the focus method has been successfully used in the analysis of codeine, dihydrocodeine, morphine, and heroin. As a comparison, the previous approach to analysing these types of compounds is using an automated solid phase extraction technique followed by blowing the subsequent extract down, then reconstitution in a suitable mobile phase and injection onto a GC-MS. HPLC-MS can be used but sample preparation is still required before the sample can be analysed. The whole sample preparation time takes about 30 minutes

Turbulent flow chromatography in bioanalysis 7oooo-

123

Plasma

Urine

7OOOO" 6OOO0" 5O0OO

4OOOO

~ 4oooo-~

j

3oooo. 2OO0010000-

lOOOO-

~2

;

~

J

o

-

2 Tin~

Time (mln)

?0000"

7OOOO"

Brain

(mill)

Intestine

6OOOO5~000,

5OOO0

I=1.,1(}000.

40ooo

21)00010000-

10000-

0-

0-

2

2

Time (min)

Time (mill)

CSF 300OO

9~

20000

10000"

o

. 2

.

.

.

.

.

" ;

, 4

5

Time (rain)

Fig. 4.17. (b) 50ng/mL K252a in rat plasma, urine, brain homogenate, liver homogenate, intestinal perfusates and cerebral spinal fluid.

with the analysis time being about 10 minutes. The sample preparation can be parallelised, substantially reducing sample preparation time but the samples still have to be analysed after they have been prepared.

4.3.3 Practical issues in bioanalytical TFC During the development of turbulent flow chromatography to the analysis of biological samples, there have been several issues that have been raised that gave analysts cause


124

Chapter 4

for concern regarding the technique. Some of the major issues are discussed in the next section, with solutions to them supplied. 4.3.3.1 Carryover

Carryover is a problem that has been reported by some authors, although it is compound dependent. The investigations by Chassaing [48] revealed that there was substantial carryover, more than 1%, for the compounds being investigated. Several sources for the carryover were determined including the autosampler, the column, and the valves. The majority of the observed carryover, about 40%, was from the autosampler. It was discovered that overfilling the sample loop on the autosampler resulted in a gradual build up of proteins on the porous rotor seal of the autosampler. A partial loopfill, and a careful rinse procedure of both the syringe and the injection valve, substantially reduced carryover, by almost a third, to acceptable levels. Carryover attributed to the column was initially identified as being due to an ion pairing mechanism rather than a pH mechanism. Thus, the addition of 0.01% TFA was sufficient to remove the carryover. However, Chassaing noted that to make the column cleaning more genetic a range of different solvents were used in the column cleaning step. In particular a high pH cleaning step was also added (0.1% NH3, pH 8), as well as a strongly elutropic organic solvent such as THF/acetonitrile. The final source of carry-over appeared to be the rotor seals, and the Tefzel stators in the Rheodyne six port valves. Use of one valve reduced the carryover slightly, but compromised the overall performance of the system. Instead a series of valve switching was incorporated to ensure that the various channels were cleaned thoroughly. This resulted in an increase in the time of analysis but meant that the method became genetic and applicable to a wide range of compounds. The use of multiple valves results in a greater potential for contamination of the system, resulting in carryover. There are several mechanisms to alleviate this problem. The approach taken by Chassaing has proven successful, but it is possible to reduce carryover from the valves by changing the material of the rotors and stators. Grant et al. [57] have demonstrated that use of PAEK substantially reduces carryover. 4.3.3.2 Pressure build up

Although using a turbulent mobile phase has been shown to improve the lifetime of a column when performing bioanalysis, it was discovered that centrifugation [43] of the sample prior to analysis improved the longevity of the column substantially. It is now standard procedure to centrifuge all biological samples prior to analysis with turbulent flow chromatography. This has been shown to remove excess particulates from the sample, which cause the premature column failure. Another source found for the premature failure of columns was the storage temperature. Work performed by Takarewski et al. [43] clearly demonstrates that storing the samples at 5~ as opposed to room temperature does affect the lifetime of the columns since the samples deteriorate at the higher temperatures quicker than at the lower temperature.

Turbulentflow chromatography in bioanalysis

125

4.3.3.3 Protein binding A significant finding from the work of Chassaing et al. [48] was that although the three compounds analysed were highly protein bound (>90%), there was no significant reduction in analyte response when comparing an injection of analyte spiked into plasma with that of an identical concentration in aqueous solution. Acidification of the load solvent ensures that the proteins are softly denatured and that the analyte under investigation can be trapped by the turboflow column. This has also been observed by other authors [51,52].

4.3.4 Environmental applications of TFC Work performed at the University of Leipzig [58,59] has shown that substantial benefits can be achieved with the use of turbulent flow chromatography in the analysis of a range of water samples, including river and lake samples. Traditional methods of water analysis for organic contaminants, such as pesticides and herbicides, involves a lengthy analysis procedure with many different steps. Turbulent flow chromatography has allowed the analysis of environmental samples without the need of complex sample preparation. In the method developed it is possible to determine concentrations of contaminants in river water for all of the compounds listed between 1-125 ng/litre in an analysis time of about 15 minutes. TFC has been successfully coupled to other analytical techniques to obtain greater sensitivity within the environmental field. Using the turboflow column as an on-line

500000 -

R2 = 0.9865

a 2 = 0.9844

R2 = 0.999

R2 = 0.9765

400000- R 2 = 0 . 9 9 8 9

m "~ r == 300000

R2

9 Isoproturon

R2 = 0.999

600000 - R 2 = 0 . 9 9 9 2

9Diuron 9Chlortoluron x Simazin Atrazin

R2 = 0.993

9Terbutylazin

= 0.9986

+ Prometn]n

R2 = 0.9993 =E 5n, 2O0000 " R 2 = 0 . 9 9 9 5

. Chlorfenvinphos - Chlorpyrifos 9Alachlor

100000

9Tdfluralin

0

20

40

60

80

100

120

140

C o n c e n t r a t i o n (spiked) [ngll]

Fig. 4.18. Calibrationdata for 12 compounds extracted from clean water, comparable data was also obtained for fiver water samples.


126

Chapter 4

solid phase extraction cartridge allows larger sample volumes to be injected, which is critical for analysis of pollutants within an aqueous media. Typically the detection limits required for organic pollutants in water are in the range of pg/mL, a factor of 1000 lower than typically required for bioanalysis. In order to obtain such sensitivity solid phase extraction or liquid/liquid extraction has been used to pre-concentrate the sample volume from several hundred millilitres to a few hundred microlitres. The use of turboflow chromatography allows the sample to be loaded onto the extraction column substantially faster than with solid phase extraction and since all the sample is being analysed, less initial sample is required. Results from Asperger et al. [58] have shown that this approach can yield high recoveries, and very low limits of detection. Using a single valve method these authors were able to detect l pg/mL of a pesticide mixture spiked into clean water, using an initial sampling volume of 10mL. However, this technique is not applicable to non-polar compounds such as PAHs, chlorobenzenes, and chloronitrobenzenes, as these compounds do not ionise well using the LC-MS interface. Large volume injection using a programmable temperature vaporizing (PTV) injector for GC-MS is another technique that has also been utilised to increase the sensitivity of the analytical technique for GC analysis. It relies on the compounds of interest being dissolved in a more volatile solvent, typically the eluant from solid phase extraction. The analytes are trapped onto a sorbent, whilst most of the solvent is evaporated before the compounds of interest are swept into the column. This is becoming a routine tool within environmental laboratories. It is however, possible to couple this technology to TFC. Results from Bahl et al. [59] show that coupling of the two techniques is feasible with data being presented on apolar compounds not applicable for direct analysis using TFC-LC-MS.

4.3.5 Capillary turboflow chromatography Ayrton et al. [60] noted that using smaller ID columns would reduce the solvent consumption. Using specially prepared 180 ~m diameter column, packed with 30 ~m particles, enabled flow rates to be used that allowed direct introduction into the mass spectrometer with no requirement to split the flow. This results in a lowering of the detection limits, and validation data was presented showing concentrations from 0.5 ng/ ml to 500 ng/ml with only 5 p~l sample injected (as with the previous work the sample was diluted with internal standard 1 : 1, equivalent to 2.5 ~1 injection of neat plasma). Capillary turbulent flow chromatography has also been applied to the analysis of methadone and its metabolites from serum. Souverain et al. [61] used a single valve technique with 180 ~m i.d. column packed with 30 ~zm particles. The linearity was shown to be good, as was the limit of quantification which was reported as 10 ng/mL using a single quadrupole mass spectrometer as the detector. The LOQ of the method was found to be improved by the addition of a second analytical column, which allowed for some focusing of the analytes and also allowed for chromatographic separation of the various components.


127

4.4 CONCLUSIONS There is a substantial amount of evidence to indicate that TFC is applicable for the analysis of a wide range of compounds. The lack of sample preparation means that method development time is substantially reduced in comparison to standard approaches. It is a technique that is geared towards high throughput laboratories that have a sample throughput problem. However, it is not a panacea, just as with other sample preparation techniques such as SPE, protein precipitation and liquid/liquid extraction there will be compounds that will be difficult to analyse, however, as a front line approach to bio-analysis, it is probably the quickest and easiest to use. A fuller understanding of the mechanism of turbulent flow chromatography is being developed, and research groups are coupling the predictive power of computational fluid dynamics with classical chromatographic theory. This will give a greater insight into the effects of fluid dynamics on a chromatography system, which will help in the design of superior stationary phases. It may lead to a situation where laminar flow chromatography becomes redundant, and the only form of chromatography is based around chaotic fluid dynamics.

4.5 REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

H.M. Quinn and J.J. Takarewski, International Patent Number WO 97/16724. J.J. van Deemter, EJ. Zuiderweg and A. Klinkenberg, Chem. Eng. Sci., 5 (1956) 271. G. Guiochon, Anal. Chem., 52 (1980) 2002. J.H. Knox and M. Saleem, J. Chromatogr. Soc., 7 (1969) 614. R.A. Hartwick and D.D. Dezaro, in: E Kucera (Ed.), Microcolumn High Performance Liquid Chromatography, Elsevier, Amsterdam, 1984. J.H. Knox, J. Chem. Soc., (1961) 433. J.C. Giddings, Unified Separation Science, Wiley-InterScience publications, 1991, p. 283. J.C. Giddings, Anal. Chem., 34 (1962) 1186. J.H. Knox, J. Chromatogr., A, 831 (1999) 3. J.H. Knox and R.EW. Scott, J. Chromatogr., 282 (1983) 297. G.I Taylor, Proc. Royal Soc., A219 (1953) 186. J.C. Giddings, Anal. Chem., 35 (1963) 1338. J.J. van Deemter, 2nd Informal Symposium of the Gas Chromatography Discussion Group Cambridge, 1957, (quoted in Gas Chromatography, J.H. Purnell (Ed.) Wiley London, 1962, p. 128.). J.C. Giddings, Dynamics of Chromatography - part 1, Marcel Dekker, New York, London, 1965. M. Golay, Gas Chromatography, D. Desty (Ed.), 1958, Butterworth, London, p. 35. R.B. Bird, W.E. Steward and E.N. Lightfoot, Transport Phenomena, Wiley, New York 1960. R. Aris, Proc. Roy. Soc., A235 (1956) 67. L.J. Tichacek, C.H. Barkelew and T. Baron, A.I.Ch.E.J., 3,439, (1957). R. Aris, Proc. Roy. Soc., A252 (1959) 538. T. Pretorius and T.W. Smuts, Anal. Chem., 38 (1966) 1. J.C. Giddings, W.A. Manwaring and M.N. Myers, Science, 154 (1966) 146. C.J. Oberhauser, A.E. Niggebrugge, D. Lachance, J.J. Takarewski, M.M. Pegram and H.M. Quinn, LCGC. 18 (2000) 716. H.A. Kusch, J.M. Ottino and D.M. Shannon, Ind. Eng. Chem. Res., 28 (1989) 302. J. Baldyga, J.R. Bourne and S.J. Hearne, Chem. Eng. Sci., 52 (1997) 457. M.C. Jones, R.D. Nassimbene, J.D. Wolfe and N.V. Fredrick, Chem. Eng. Sci., 51 (1996) 1009. G. Taylor, Proc. Roy. Soc., A219 (1953) 186.

128 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61

Chapter 4 A.A. Barres, Chem. Eng. Sci., 5 (1997) 807. O. Reynolds, Roy. Soc. Phil. Trans., 35 (1883) 84. C.O. Bennett and J.E. Myers, "Momentum, Heat, and Mass Transfer", 3rd Ed., McGraw-Hill, NY (1982) pp. 202-209. E Moin and J. Kim, Scientific American, 276 (1997) 62-68. J. Baldyga and J.R. Bourne, Chem. Eng. Commun., 28 (1984) 231. S. Ergun, Chem. Eng. Prog., 48 (1952) 89. F.A.L. Dullen, Chem. Eng. J., 10 (1975) 1. I.E Macdonald, M.S. E1-Sayed, K. Mow and EA.L. Dullen, Ind. Eng. Chem. Fundam., 18 (1979) 199. T. Farkas, G. Zhong and G. Guichon, J. Chrom. A., 849 (1999) 35. J.C. Giddings, Unified Separation Science, J.Wiley & Sons (1991) p. 65. EC. Blake, Trans. Amer. Soc. Chem. Engrs., 14 (1922) 415. EC. Carman, Trans. Inst. Chem. Eng., 15 (1937) 150. S.P. Burke and W.B. Plummer, Ind. Eng. Chem., 20 (1928) 196. A. Edge, 11th Nordic Mass Spectrometry Conference, Loen, Norway, 18-21 Aug. 2001. A.E. Fick, Annalen der Physik (Leipzig), 170 (1855) 59. EW. Atkins, Physical Chemistry 2nd Edition, Oxford University Press, p. 879. J. Takarewski, D. Mageira and H.M. Quinn, 47th ASMS Conference, Dallas, Texas, 1999. B. Biddlecombe, 25th Annual meeting British Mass Spectrometry Society, Cambridge, U.K., 19-20 Dec 2001. W.D. van Dongen, R. Ramaker, F. van Schalk, B. Ooms and E. Koster. 25th Annual meeting British Mass Spectrometry Society, Cambridge, U.K., 19-20 Dec 2001. J. Ayrton, Rapid Commun. Mass Spec., 11 (1997) 1953. J. Ayrton, G.J. Dear, W.J. Leavens, D.N. Mallet and R.S. Plumb. Rapid Commun. Mass Spectrom., 11 (1999) 1953. C. Chassaing, P. Macrae, E Wright, A. Harper, J. Luckwell, K. Saunders and R. Venn, Chromatographia, 53 (2001) 122. W.N. Kennedy, H.M. Quinn, J.J. Takarewski, C.J. Oberhauser and D.J. Malpas, J. Chromatogr. Submitted. D.Zimmer, V. Packard, W. Czembor and C. Muller, J. Chrom. A., 854 (1999) 22. M. Jemal and W. Yuan-Qing, Rapid Commun Mass Spectrom., 12 (1998) 1389. J-T. Wu, H. Zeng, M. Qian, B.L. Brogdon and S.E. Unger, Anal. Chem., 72 (2000) 61. N. Brignol, R. Bakhtiar, L. Dou, T. Majumdar and F.L.S. Tse, Rapid Commun. Mass Spectrom., 14 (2000) 141. W.D. van Dongen, TFC user group meeting, Holland, Oct. 2001,. J.L. Herman, Rapid Comm. Mass Spectrom., 16 (2002) 1-6. R. Grant, 15th IMSC, Barcelona, Aug. 2000. R. Grant, C. Cameroon, S. Mackenzie and M. Young, Rapid Commun. Mass Spectrom., 16 (2002) 1785-1792. Asperger, J. Efer and W. Engewald, 10th Symposium on Handling of Environmental and Biological Samples in Chromatography, April 2001, Mainz, Germany. K. Bahl, J. Efer, A. Asperger and W. Engewald, HTC 2002, Bruges, Belgium. J. Ayrton, G.J. Dear, W.J. Leavens, D.N. Mallet and R.S. Plumb, Rapid Commun. Mass Spectrom., 13 (1999) 1657. S. Souverain, D. Ortelli, S. Rudaz, J.L. Veuthey and E. Varesio, J.E 17th Montreux symposium on LCMS, SFC-MS, CE-MS, and MS-MS, Switzerland, Nov. 2000.

I.D. Wilson (Ed.), Bioanalytical Separations Handbook of Analytical Separations,Vol. 4 9 2003 Elsevier Science B.V. All rights reserved

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CHAPTER 5

Chiral bioanalysis D.M. Wallworth and J.T. Lee Advanced Separation Technologies Ltd, Blake Street, Congleton, U.K.

5.1 INTRODUCTION Although the importance of enantiomeric drugs and the science of analytical chiral chromatography have become well established [1,2], new challenges have presented themselves in the last few years as single enantiomeric drugs have progressed through the development stages into pre-clinical and clinical studies. Chiral analysis of biological samples introduces new demands not previously experienced in chiral chromatography. In the past, achieving baseline separation of two enantiomers within 30-40 minutes under any mobile phase conditions possible was acceptable, if not considered quite an achievement. For bioanalysis, not only is a good enantioseparation now required, but also speed becomes far more critical. For the high throughput of many hundreds of samples under the commercial time pressures of a clinical trial, every minute saved is crucial. Mobile phase choice also becomes more significant because of the resulting requirements of sample preparation, including its speed and ability to be automated. Aqueous chiral methods are often preferred over normal phase systems, allowing the possibility of simple or on-line sample preparation techniques, and for reasons of better solubility of polar compounds, the use of less costly solvents and, most importantly, easier interfacing of HPLC with tandem mass spectrometry detection (HPLC-MS-MS). In addition, lower therapeutic levels have increased the demands on sensitivity and on the chromatography itself. There is an increasing drive to select either single isomer or racemate early in the development process. Early screening usually defines the pharmacokinetic profile, but there is always the possibility of in vivo chiral inversion (prochiral to chiral, chiral to achiral, chiral to diastereomer, chiral to chiral) when administering a single enantiomer. Potential enantioselective absorption, distribution, metabolism and excretion define a range of bioanalytical studies to be completed. For these studies, HPLC-MS-MS is increasingly being used for optimum selectivity and sensitivity, especially where there is the possible presence of chiral metabolites that may need to be monitored. References pp. 180-184

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5.1.1 Scope and aim This review covers the developments in chiral bioanalysis since 1995, focusing mainly on the techniques used in HPLC but also providing a brief introduction to bioanalytical applications in GC, CE and SFC. It will include a review of the principle types of chiral stationary phases (CSPs), their mechanism of action, typical mobile phase choices and applicability. A method of choice must be sufficiently robust to withstand large numbers of biological samples. It must be capable of method transfer to other research laboratories, such as a contract laboratory facility, as the drug development process progresses. New chemical entity submissions to the FDA and other organisations around the world also need to incorporate validated data using freely available methods. Direct HPLC methods (rather than the use of achiral stationary phases with chiral additives or derivatisation of the solute with chiral reagents) are generally preferred in pharmaceutical research. It is mainly for these reasons that HPLC (and in particular HPLC-MS-MS) will be a main focus for this chapter. It is also for these reasons that the chapter will only include commercially available and proven technologies. It is not intended as a comprehensive review of all CSPs currently available for all types of applications.

5.1.2 The mechanism of chiral recognition and choice of CSP In nature, chiral recognition by amino acids, proteins, enzymes, receptors is commonplace. This capability has been utilised for chiral discrimination in HPLC or GLC, by utilising such selectors in a CSE The protein phases such as Chiral-AGP or the amino acid w-acid/base phases are two examples. Many CSPs initially developed from natural products have been modified to increase robustness and applicability to chiral bioanalysis. Often, more than one CSP may appear to be suitable for a particular chiral assay and the choice may well be made on peak shape (for best sensitivity), elution order (for very low enantiomeric ratios in a single enantiomer racemisation study) or retention times for speed. The basic concept of a chiral interaction in HPLC is the formation of a dynamic diastereomeric complex between the CSP and the analyte. The complex forms by utilising one or more of different types of interactions such as hydrogen bonding, -rr-w interactions, inclusion complexing, dipole-dipole and ionic interactions, to name just a few. Some types of interaction are specific to certain CSPs and some are specific to certain mobile phases. Many CSPs operate under several different types of interaction and most also have the capability for non-chiral interactions. It is often possible to look for useful points of interaction between the CSP and the racemate and then to choose the best CSP and mobile phase to enhance that effect, aiming to get the maximum strength for the differential chiral interactions of the two enantiomers. Non-chiral interactions need to be kept at a minimum but are generally useful for anchoring the racemate. Along with lowered solubility and lowered mass transfer, they are often responsible for peak tailing observed in chiral HPLC. In practice, automated and genetic screening techniques are frequently used to determine the CSP of choice from a range that includes those best for the compound

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131

TABLE 5.1 CHIRAL INTERACTIONSBETWEENCSP AND SOLUTE Interaction

Mobile Phase Type for the Interaction to Dominate

Ionic Ion-dipole interactions Hydrogen bonding Dipole-dipole Steric interactions Hydrophobic/inclusion interactions ~r-w interaction

Reversed-phase, polar organic mode Reversed-phase Polar organic mode, normal phase, reversed-phase Normal phase, polar organic mode Normal phase, polar organic mode Reversed-phase Normal phase

class of interest, or those best for the mobile phase of choice (for reasons of solubility or detection). Chiral method development kits are also available from many chiral column suppliers (for example, Regis, Advanced Separation Technologies, Akzo Nobel) for speed in determining the optimum CSE An indication only of the most suitable CSP is often the result of such a screen, optimisation being conducted on the single (often longer) column separately. Table 5.1 shows the types of chiral interactions that occur in many CSPs and in which mobile phase they are most enhanced. If the functional groups close to the chiral (stereogenic) centre have specific interaction capabilities (such as w-deficiency), this may well influence the initial choice of CSE There have been many attempts over the years to classify and characterise the wealth of stationary phases that are available, but perhaps the simplest is to categorise CSPs in terms of their main interaction. Table 5.2 provides details of the most commonly used types of CSP, their main mode of interaction and commercial notation.

5.1.3 Mobile phase types Reversed-phase solvent systems are generally composed of an aqueous portion, which may or may not be buffered, and a miscible organic portion. A typical response is to observe an increase in retention and resolution as the aqueous portion is increased, although this may also depend on pH. For the macrocyclic antibiotic and cyclodextrin phases, however, an increase in retention and resolution has been observed [3] at both high and low compositions of the organic component. Typical chiral interactions in reversed-phase chromatography are generally ionic, hydrogen bonding and inclusion complexation. The usual components for normal phase eluants are hexane (or isohexane, heptane) with 2-propanol (or ethanol, isobutanol) as the lesser component. As the concentration of the polar component is increased, retention and resolution decrease. Predominant interactions are in this case -rr-~r interactions and hydrogen bonding. The polar organic mode is a novel mobile phase originally developed by Armstrong for use with the cyclodextrin type CSPs. It was shown [4] to be highly advantageous for obtaining efficient separations on these phases and has subsequently References pp. 180-184

Chapter 5

132 TABLE 5.2 PRINCIPAL COMMERCIALLY AVAILABLE CHIRAL STATIONARY PHASES Chiral Stationary Phase Type

Commercial CSP

Typical Mobile Phase Conditions

Charge transfer (Pirkle TM type) phases (Hbonding, w-complex)

Regis Kromasil Sumichiral Chirex

NP (some RP)

Cyclodextrins (H-bonding, inclusion) Cyclodextrin Derivatives (as above, plus w-complex for Cyclobond SN, RN, DMP)

CYCLOBOND TM ChiraDexTM NucleodexTM

RP and POM NP, RP and POM

Chiral Polymers (H-bonding, hydrophobic, plus w-complexation or ionic interaction where indicated) 1. Proteins: o~-acid glycoprotein bovine serum albumin human serum albumin ovamucoid cellobiohydrolase 2. Cellulose/Amylose (w-complexation) 3. Cellulose/Amylose, Aqueous

CHIRAL-AGPT M ResolvosilTM CHIRAL-HSAT M Ultron ES-OVM CHIRAL-CBHT M Chiralcel and ChiralpakTM

RP RP RP RP RP NP Only RP Only

Astec CLC Nucleosil Chiral 1 Chiralpak WH/WM/WE

Aqueous Cu complex

Crownpak CR

Aqueous perchloric acid

CHIROBIOTIC T M

NP, RP and POM

Ligand Exchange (ionic complex)

Crown Ethers (ionic)

Macrocyclic Glycopeptides (multiple)

been used extensively with the macrocyclic antibiotics. It is composed of methanol (or acetonitrile/methanol mixtures when used with cyclodextrin CSPs) with added acid and base and is therefore essentially anhydrous, providing long term stability for most of these CSPs. It is simply the ratio of acid to base that mainly controls enantioselectivity, with ionic interactions playing a role when the polar organic mode is used with the macrocyclic antibiotics. One main advantage of this mobile phase is that there are few requirements of the analyte for separation to be effective: there must be at least two hydrogen bonding functional groups on the chiral molecule, only one of which must be on or near the stereogenic centre. This low requirement means that this mobile phase system has the potential for very broad applicability in chiral HPLC. Typical functional groups for hydrogen bonding are: Halogen: I > Br > C1 > F Amine: 3 ~ ~ 2 ~ > 1~ Carbonyl: -COOH, -CHO, -C=O, -COOR Sulpho-, phospho- and hydroxyl groups Methoxy groups

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The organic solvent primarily controls the retention and the ratio of acid to base controls the resolution. The type of acid and base can also be chosen for interfacing with HPLCMS, and it is in this area that its use is seen to be growing strongly. The polar organic mode operates through hydrogen bonding, dipole-dipole interactions and/or ionic interactions (when used with the macrocyclic antibiotic CSPs). Whilst this mobile phase is essentially a 'normal phase' solvent, it is most commonly known as the 'polar organic mode', or 'polar ionic mode' when used with the macrocyclic antibiotic CSPs. More recently, a modified version of the polar organic mode has been used with the cellulosic and amylosic CSPs, although typically either acid or base are added, rather than both

[5]. 5.1.4 Direct vs. indirect chiral separations Alternative methods for chiral separations include the use of derivatisation techniques whereby a chiral derivatisation reagent is used to form a diastereomer of the chiral solute. An achiral (frequently a C18) column is then used for the separation since diastereomers now have different physical properties that can be utilised in an HPLC method. Whilst this is a feasible technique, it tends not to be practical for several reasons. Firstly, it relies on optimum chiral purity for the derivatisation reagent, on an assumption that each enantiomer will react with the reagent to an identical extent, that the derivatisation reagent is highly selective and has non-detectable or non-interfering by-products, and that the reaction conditions are simple and fast enough for high throughput clinical assays. In most cases, the increased validation issues alone are sufficient to discount achiral methods as a method of choice. In addition, indirect methods have largely been superseded by technological improvements in stationary phases, but are always an alternative where no direct method can be found.

5.1.5 Achiral-chiral column switching techniques There are many instances of methods involving achiral-chiral column switching techniques being incorporated into biological assays. Where the chiral drug has active chiral metabolites, all solutes of interest will need to be quantified. Often it is necessary to combine the separating capabilities of both traditional reversed-phase and chiral stationary phases to achieve this. This can be accomplished either on-line (where a column switching valve directs the fractions containing chiral moieties onto a coupled chiral column), or off-line (collecting fractions for the drug and any active chiral metabolites and evaporating to dryness before injecting onto the CSP in a separate assay). Instances of the former are prevalent in the literature, despite the increased difficulties of optimisation and choice of compatible mobile phases.

5.1.6 HPLC-MS Technological advances over the past five years have led to a rapidly increasing use of MS detection in drug research. Sensitivity and specificity are often increased over alternative methods of detection and where characterisation of the molecule is required, References pp. 180-184

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MS is particularly attractive. When combined with HPLC, MS has gained in popularity for the trace analysis of drugs and their metabolites in biological fluids. Whilst there are a large number of pharmaceutical applications already developed using normal phase solvents, it is sometimes beneficial to re-develop such methods to reversed-phase ones or to take additional safety precautions when drug development requires a bioassay and the high sensitivity of HPLC-MS. Utilisation of normal phase methods have a potential for an unstable ion current and poorer sensitivity in HPLC-MS and combined with high voltage electrospray ion source, it presents a potential explosion hazard. Post-column mixing with an aqueous solvent can be used to circumvent these problems [6,7]. It is generally considered that the rigorous use of nitrogen as nebulising and solvent drying gas, plus the post-column addition of water are essential for the safe use of normal phase solvents in HPLC-MS. If an alternative method is available that utilises volatile organic buffers, especially with high organic modifier content, then this may be preferred. Electrospray ionisation (ESI) and atmospheric pressure chemical ionisation (APCI) are two of the most successful HPLC-MS interfaces, producing little or no fragmentation of the solute. ESI does have some limitations in its low tolerance of salts, detergents and inorganic buffers which give signal suppression. It is also concentration sensitive such that the best results are achieved at relatively low flow rates, requiring the use of narrow bore (typically 2 mm internal diameter) columns, or the use of standard (4.6 mm internal diameter) columns with post-column split flow, allowing a portion of the eluent to be transferred to the MS interface. Recent new technologies such as turboassisted ion-spray (TSIP) allow for flow rates that are typically 1.0 to 1.5 ml/min. APCI offers a wider linear dynamic range than ESI and is relatively more tolerant of higher buffer concentrations, providing best sensitivity by the addition of an electrolyte. Typically, no sensitivity gains are achieved with lower internal diameter columns or lower flow rates for APCI. In designing a chiral assay for HPLC-MS, it is important that inorganic buffers and high aqueous mobile phases are avoided as these can potentially lead to ion suppression. Where unavoidable (as in the protein phases), success with HPLC-MS can be achieved by changing buffer type, and ammonium acetate has been successfully used in many cases. When using flammable solvents such as hexane in normal phase assays, safety measures are required when using ESI or APCI interfaces. In a recent study of verapamil, sotalol, doxazosin and oxybutynin using API-MS-MS [8], a 2 mm internal diameter Chiralcel AD CSP was used successfully in normal phase mode using hexane, 2-propanol and diethylamine as mobile phase. The heated nebuliser system of the SCIEX system used nitrogen as both the nebuliser and auxiliary gas to displace any oxygen and post-column addition of an aqueous reagent provided the ion concentrations required to sustain ionisation in the API source and to dilute out the hexane. A mixture of 25% 0.025 M aqueous ammonium acetate and 75% 2-propanol was found to be miscible with mobile phases of up to 95% hexane, without compromising chromatographic resolution. The column flow rates were maintained below 0.2 ml/min and the reagent added at a ratio of 4:1 to the mobile phase. Although this technique potentially introduces sensitivity problems by virtue of dilution, it has been used in many other assays such as the normal phase separation of omeprazole [9] and Org4428 (an antidepressant) [ 10].

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135

For reversed-phase chiral HPLC/MS bioanalysis, API-compatible solvents such as methanol, acetonitrile and ethanol enhance the formation of ions and assist in the nebulisation and desolvation processes to help achieve optimum sensitivity. Of the reversed-phase CSPs available, the macrocyclic antibiotics appear to be extremely broad ranging and versatile and to be cost-effective for HPLC-MS bio-analysis [ 11 ], with no separation deterioration observed for a plasma assay of methylphenidate after approximately 2500 injections. Other similar examples have been reported [e.g. 12,13]. Although electrospray is well suited to polar conditions, it shows poor sensitivity in the presence of triethylamine (TEA) [ 14]. Plasmaspray ionisation HPLC-MS interfaces, however, can use TEA to advantage. Suprofen and p-chlorowarfarin were used [14] to demonstrate the use of a Cyclobond I 2000 RN column in the polar organic mode in conjunction with MS detection as a viable method for the analysis of those compounds having no UV chromophore, or having UV chromophores absorbing below the UV cutoff of this mobile phase (ca. 240 nm).

5.1.7 Temperature It is well documented that a reduction in the temperature at which chiral chromatography is performed generally increases enantioselectivity since in the majority of cases chiral separations are enthalpy dominated. However, there have been a few cases reported where elution order is entropy-governed and therefore is temperature dependant such that there is a 'crossover' point where elution order reversal occurs. In a study on the chiral separation of sotalol on Chiral-CBH [ 15] it was found that the (R)-enantiomer eluted before the (S)- at 5-15~ but that the isomers eluted in the opposite order at 40~ The crossover point occurred between 25 and 30~ and was found to be mobile phase dependant. Although relatively rare, there have been other reports of this phenomenon, e.g. for R,S prominal on a cyclodextrin column [16], for mosapride on Chiral-AGP [ 17] and for various compounds on Chiralcel OD [18]. These examples emphasise the need for temperature control in chiral HPLC.

5.1.8 Validation For bioanalytical methods, there are many different issues associated with validation. It is important that the measured concentrations of each enantiomer replicates the in vivo levels and is not affected by in vitro degradation or interconversion. This encompasses a wide range of issues from sample collection (the effect of anti-coagulants and/or acidification on enantiomeric ratios), sample storage, sample preparation (any effect of protein precipitation or extraction), and the effect of an achiral-chiral column switching technique or the effect of derivatisation, if used. The success of the enantiomeric separation in terms of degree of resolution will also affect calibration, limits of quantification (LOQ) and linearity of the method. All of the above are highlighted in a useful review article [ 19]. See also Hill, this volume. References pp. 180-184

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5.1.9 Gas liquid chromatography (GLC) Although just 25 references for chiral bioanalysis by GLC were published for the period covered by this review, by far the majority were determinations of small molecules such as biomarkers, or of drugs of abuse. Because of the inherent sensitivity of GLC detection systems, chiral GLC provides high sensitivity, often to ng or pg concentrations. It therefore is useful as an alternative detector for use with LC methods by heart cutting the analyte of interest, evaporating to dryness and re-injecting onto a GLC system. In general, however, most drug molecules are insufficiently volatile to enable direct GLC methods, requiring derivatisation to acquire a degree of volatility. GLC often also requires more complex sample preparation. For these reasons, and the rapid growth in the use of highly sensitive H P L C - M S - M S methods, chiral GLC is not frequently used in pharmaceutical research and so will not be covered further here.

5.1.10 Capillary electrophoresis (CE) Method development in CE is often to be fast, it is a versatile technique and consumable costs are low. Even so, there is a reluctance to use the technique for bioanalysis, perhaps for reasons of available expertise, generally lower sensitivity or for difficulties experienced in method transfer. A comprehensive and valuable review of the use of CE in bioanalysis has recently been published and offers excellent coverage of the topic

[2o]. 5.1.11 Supercritical fluid chromatography (SFC) As chiral HPLC has matured, interest in the use of SFC has been renewed for this area. SFC offers several advantages over HPLC, such as the lower pressure drop across the column, allowing for increased flow rates, and the possible use of multiple columns or column coupling. Supercritical CO2 has a polarity similar to that of pentane, so must be used in conjunction with a polar modifier such as methanol, acetonitrile or dichloromethane. Acidic and basic modifiers can also be added. For this reason, it is generally considered as possible replacement for normal phase chiral applications [21 ]. Samples that require aqueous conditions are not generally amenable to SFC. SFC can be performed using either a capillary column (5-100 Ixm i.d.) or a packed column (designated pcSFC), using columns designed for HPLC. Whilst many CSPs have been used successfully in pcSFC, including celluloses, charge transfer, cyclodextrins [22] and macrocyclic antibiotics [23], there is no guarantee that a separation that works well by HPLC will also do so in SFC. However, sharper peaks, faster equilibration times and reduced analyses times have been reported [21], making it seemingly ideal for bioanalysis. In a study of a range of 44 different drug types, 70% separated on Chiralpak AD, 66% on Chiralcel OD, 50% on Chirobiotic T, and 40% on Chirobiotic V [24]. However, no true clinical applications were found at this time. It is possible that the introduction of new instrumentation, pcSFC may well find its place in many laboratories over the next few years.

137

Chiral bioanalysis

5.2 C H I R A L STATIONARY PHASES 5.2.1 Macromolecular or polymeric CSPs Cellulose and amylose are the most common of naturally occurring optically active polymers and as such offer great potential as chiral selectors. In their natural state, however, they are not practical as CSPs. Once the hydroxyl groups of the glucose moieties are derivatised, and they are immobilised on to a silica support, they become very valuable phases. As early as 1973, Hesse and Hagel demonstrated the efficacy of crystalline cellulose triacetate [25]. It was later shown that the chiral selectivity would not be lost by dissolving the polysaccharide in organic solvents [26] and coating onto silica gel. Since the enantioselectivity of these phases is strongly influenced by their tertiary structure [27], it is difficult to predict chiral recognition purely from the structure of the glucose monomer unit. Chiral recognition appears to be higher when the glucose hydroxyl group is derivatised with an aromatic group to form the benzoate, especially if the aromatic group has alkyl or other electron donating functional groups. This has led to a wide range of CSP's varying by the type of derivative (Fig. 5.1). The resulting increase in carbonyl oxygen electron density plays a significant role in enantioselectivity through increased participation in dipole-dipole interactions with a carbonyl group, or in hydrogen bonding with an amine or hydroxyl on the analyte. The effect is most o~

Amylose derivatives

Cellulose derivatives

o

R,:

.,, CHIRALCEL OA

RI:

CHIRALCEL OO CHIRALCEL OD.R CHIRALCEL OD-RH

RI:

o

CHIRAI.CEL OB

.~ Rt: ~ ,

O CHIRALCEL OJ

CHIRALCEL OJ-R ~ 0

c.,

"':

N

CHIRALPAK AD CHIRALPAK AD-RH

R=:

o

CH~ CHIRALPAK AS

CHIRALPAKAS-RH

CHIRALCELOC CHIRAt.CELOK Fig. 5.1. Commerciallyavailable cellulosic and amylosic derivatised chiral stationary phases. References pp. 180-184

138

Chapter 5

marked for alkyl substituted phenylcarbamate derivatives. The position of the phenyl functional group is also critical in that it will affect the formation of an ordered tertiary structure. The resulting interactions- hydrogen bonding, 7r-Tr stacking and dipoledipole interactions, are more effective under normal phase conditions. The structures of the phenylcarbamate cellulosic phases have been determined [28] and shown to have a chiral helical groove with the polar carbamate residues lying along the main chain. If a racemate can enter this groove, chiral selectivity can be accomplished. Chiralcel OD, prepared from tris(3,5-dimethylphenylcarbamate) cellulose, is one of the most successful of the cellulosic range of CSPs. It is stable in mixtures of hexane and 2-propanol where it offers a wide range of chiral separations: out of a range of 510 compounds, 62% were resolved on this CSP [28]. Its amylose equivalent also exhibits high enantioselectivity for a wide range of structures. Of all of the amylose derivatives formed, it appears that the tris(3,5-dimethylphenylcarbamate), Chiralcel AD, is the most useful CSP and is complementary to the cellulosic form. Together with the tris(4methylbenzoate) cellulose CSP (OJ) and tris((5)-l-phenylethylcarbamate) (AS), these form the most effective columns out of the range. They have good capacity in normal phase, and are often used for preparative HPLC. These CSPs, however, are not covalently attached to the silica support, but are coated on to a wide pore silica that has first been silanised [29]. Consequently, some caution must be exercised with types of mobile phase solvents used. In normal phase separations, trifluoroacetate (TFA) or diethylamine (DEA) are often added to improve peak shape and it is general practice to dedicate a column for the use of acid/base modifiers because of their strong affinity for the stationary phase. Columns are stored in hexane after use. Since there are no ionic functional groups to utilise for enantiomeric interactions with charged analytes, reversed-phase conditions have been little used in the past. However, the addition of a counter ion to an aqueous solvent system can effectively neutralise an ionised analyte to enable its separation on polysaccharide phases under reversed-phase conditions. Specially prepared versions of the cellulosic and amylosic phases were introduced to enable the use of reversed-phase solvents. For Chiralcel OD-RH, OJ-R and Chiralpak AD-RH (R- designates a reversed-phase format, H - designates a higher performance (5 txm) version), this enables their easier use in bioanlysis. Chiralcel O D R can be operated in 100% acetonitrile, 100% methanol, 100% ethanol and 0-100% water, maintaining the pH between 2 to 7 for maximum column life. Enantioselectivity is controlled through the choice of buffer salt and its pH, maintaining analyte neutrality. A charged analyte may also have a high degree of solvation, limiting its direct interaction with this type of CSP [30], resulting in poor separation. For neutral compounds, a simple water-organic modifier mix can be used and no beneficial effect is generally seen when buffers, acids or bases are added [30]. In general, acetonitrile exhibits a lower retention time than methanol: there is no rule for choosing one over the other in optimising peak shape. For acidic analytes, ion suppression is effected at pHs between 2 and 4, while basic compounds often require the addition of a suitable counter ion, since high pH ion suppression is not possible for silica gel supports. The concentration and type of ion pair reagent are critical for cellulosic CSPs. Generally, the following series of counter ions decreases in retentivity [31 ] in the order:

Chiral bioanalysis

139

PF66> BF44> C104 > SCN > I > NO~ > H2PO4 > Br > C1 > AcOIn contrast, the amylose phases show no such effect and require basic pH rather than added counter ions to effect a separation under reversed-phase conditions. Although it is expected that hydrogen bonding plays a lesser role in reversed-phase separations, many molecules separate under both reversed- and normal phase conditions. Of a series of propanolol analogues [32], approximately 70% separated on the polysaccharide CSPs in both normal and reversed-phase modes, although only 10% showed any reversal of elution order in going from one to the other. Examples of the use of Chiralcel OD-R for sample enantiomeric drugs such as benzoin and indapamide has been reported [33]. Neutral molecules were separated with water-acetontrile mixtures, while acidic and basic compounds required the addition of 0.05% trifluoroacetic acid to the mobile phase. Resolutions varying from 1.78 to 7.51 were obtained, indicating their applicability to bioanalysis, although such assays were not included in this study.

5.2.2 Protein phases Bonded protein phases are employed widely for bioanalysis and are used in reversedphase mode only, using a combination of aqueous buffers and small amounts of organic solvents. Method development tends to be fast, but because of the large size of the chiral selector, protein-based CSPs have the lowest capacity of all CSPs available- a feature that fortunately does not affect their use in bioanalysis. Although all proteins are by their nature stereoselective, only a few have been discovered to have enantioselectivity over a wide range of solutes. Of these, ~ - a c i d glycoprotein (AGP), is the most broad ranging and is an extremely stable protein. It can tolerate organic solvents in high concentrations and can be used over a reasonably wide range of pH without the protein being denatured. The protein is immobilised by a crosslinking covalent attachment to surface modified silica. The carbohydrate portion of AGP contains 14 sialic acid residues, making the protein very acidic in character: when used over the pH range of 4 to 7, it retains a negative charge. Both amines and weak acids separate well on this phase. In most cases, the enantioselectivity for acidic compounds increases with decreasing pH as the charge on the AGP protein and the consequential repulsion decrease. For amines, a pH range of 6-7 is used, with organic modifiers such as 2-propanol or acetonitrile being used to control retention. Neutral analytes also show enantioselectivity that is affected strongly by changing pH. Hydrogen bonding, hydrophobic interactions and steric interactions with the tertiary structure of the AGP also play roles. It is thought that the amount and concentration of the organic modifier effect a separation by competing with the solute for the hydrophobic portions of the protein molecule, or with hydrogen bonding sites, depending on the nature of the organic solvent. It is also known that the concentration of the organic modifier will induce a temporary change in the tertiary structure of the protein, making some interacting sites more, or less, available. For some separations, the addition of a charged modifier, such as N,N-dimethyloctylamine or octanoic acid, is References pp. 180-184

140

Chapter 5

necessary for enantioselectivity. Once a protein column has been used with such charged modifiers, however, its structure and selectivity may be permanently altered and therefore a column needs to be dedicated for this purpose. Commercially known as Chiral-AGP, this CSP has been used extensively for bioanalysis, both alone and when coupled with a reversed-phase column. Although phosphate buffer is used routinely, quite often this can be replaced with ammonium acetate for interfacing with HPLC-MS detection. Of the other protein phases available, cellobiohydrolase (available as Chiral-CBH), human serum albumin (Chiral-HSA and Hypersil HSA), bovine serum albumin (Resolvosil BSA) and ovomucoid (Ultron ES-OVM) all have their place in bioanalysis. Cellobiohydrolase is a very stable enzyme that separates a variety of basic solutes when immobilised onto silica, while HSA is primarily used for the separation of weak and strong hydrophilic acids and zwitterionic solutes. However, cellobiohydrolase is easily poisoned by metals [34] and must therefore be used with a pre-column and with EDTA in the mobile phase to prevent deactivation. Ovomucoid, purified from chicken egg white, provides enantioselectivity for amines and carboxylic acids principally and is similar in behaviour to AGE A review of other, less commonly used, protein phases is available [35].

5.2.3 Cyclodextrins Cyclodextrins are cyclic oligomers of ct-(1,4)-linked glucose, produced by the enzymatic coupling of glucose units following the action of cyclodextrin glycosyltransferase on starch. They form into crystalline, homogeneous toroidal structures of different molecular sizes. Although many forms are known, it is the 6, 7 and 8 glucose unit structures (denoted et, [3 and ~/, respectively) that have proved useful for chiral HPLC. In aqueous or hydro-organic solutions, cyclodextrins are known to form hostguest complexes with a variety of molecules, ions and solvents [36]. The internal cavity of the cyclodextrin molecule is composed of glycoside oxygens and methylene hydrogens giving it an apolar character, whilst the surface is hydrophilic as a result of the 2,3 and 6-position hydroxyl groups. As a consequence, non-polar molecules (or the most hydrophobic portions of a molecule) tend to prefer to reside inside the cavity where they can bind through dipole-dipole interactions, hydrogen bonding or London dispersion forces with the outer edge. The orientation of the aromatic ring is selective due to the electron sharing of the aromatic methylene groups with those of the glucoside oxygens. The other prominent characteristic of the cyclodextrin phases is that the cyclodextrin ring provides a chiral environment of its own. [3-cyclodextrin, for example, has 35 stereogenic centres. Based on the evidence of x-ray crystallographic data, the [3 and ~/structures appear to have quite rigid and inflexible structures and are stable to a wide variety of aqueous and organic solvents. In contrast, the bonds of the oL-cyclodextrin appear to be weaker and capable of stretching, a fact that can be utilised by using high aqueous buffer conditions to encourage deformation of the cavity to enable inclusion of a molecule. Typically, substituted phenyl, naphthyl and biphenyl tings can be separated on [3-

Chiral bioanalysis

141

cyclodextrin, smaller molecules on [3, and molecules with three to five rings in their structure are best separated on ~/-cyclodextrin. Some of the primary (6-position) hydroxyl groups have been used to covalently bond the cyclodextrin to the surface of silica or other media. When this was first accomplished in the early 1980s [37], it was, along with the protein phases, among the first of the commercially available reversed-phase CSPs. In determining whether a separation is possible in the reversed-phase mode, the most hydrophobic portion of the molecule would preferentially have halogen, nitrate, sulphate, phosphate or hydroxyl (depending on the pH) functional groups for inclusion to dominate. Following inclusion of this part of the molecule, the aim is for the part of the molecule containing the chiral centre to interact with rim of the cavity (Fig. 5.2). Carbonyl, carboxyl and amine moieties prefer to hydrogen bond, so these will interact with the mouth of the cavity in preference to inclusion. Enantioselectivity is then optimised by varying buffer type and concentration, pH, organic modifier, flow rate and temperature. The secondary hydroxyl groups can be derivatised selectively, generally using those in position 2 first and then in position 3, changing the physical and chemical properties of the cyclodextrin and extending the separation capabilities. Fig. 5.3 shows those derivatives that have been produced commercially. One of the derivatives, the naphthylethyl carbamate, introduces ~vcomplexing capabilities through the aromatic ring and can therefore also be used very effectively with normal-phase solvents (when interactions are surface phenomena only), as well as in the reversed-phase mode where inclusion complexing predominates. Each of the six derivative types incorporates different possible mechanisms and are therefore capable of different ranges of applications. All of them have in common the fact that they can be used in both reversed-phase and in polar solvents with different mechanisms and consequential increased opportunities for enantioseparation: in the case of the naphthylethyl carbamate and 3,5-dimethyl phenylcarbamate, normal phase chromatography is additional to this list. Changing back and forth from each solvent system does not damage the column, provided buffers are washed out correctly and intermediate, miscible solvents are used in the case of normal phase.

NIl 2

A H NH. "-,

H

o/

Fig. 5.2. Inclusioncomplexingschematic for the enantioselectivemechanismon cyclodextrinCSPs. References pp. 180-184

Chapter 5

142

i

Silica Gel

CYCLOBOND I 2000 SUFFIX

R=

DM (dknethylated)

OCH 3

AC**

COCH3

(acetylated)

OH

SP or RSP (hydroxypropyl ether)

I

~CH2CHCH 3

'S

CH3 ~CONHCH

CH3 ~CONH--~ CH3

RNorSN (naphthylethyt carbarrete)

DMP (3,5-dimethylphenyl c a rba fret e)

Fig. 5.3. Commercially available derivatised cyclodextrin chiral stationary phases.

The usefulness of the cyclodextrin range of CSPs has been further extended by using the polar organic mode (described in section 5.1.3), when the interaction of the solute with the CSP occurs with the mouth of the cavity (inclusion does not occur). For the cyclodextrin-based CSPs, a non-hydrogen bonding, polar-organic solvent (such as acetonitrile) is used as the main component of the mobile phase. The acetonitrile tends to occupy the cyclodextrin cavity resulting in an emphasis on hydrogen bonding interactions between the solute and the hydroxyl groups at the mouth of the cyclodextrin cavity, or with the appended carbamate, acetate, or hydroxypropyl functional groups. A hydrogen bonding solvent (such as methanol) can be added to decrease the retention of highly retained compounds. In addition, small amounts of glacial acetic acid and anhydrous triethylamine are added to control protonation of the analyte. It is the ratio

Chiral bioanalysis

143

of these that is used to effect the separation. An effective ratio of acid to base (when using the AcOH-TEA combination) has been found in the range 4:1 to 1:4, the typical average being 1.5 : 1.0 and the concentrations used vary over the range 0.002% to 2.5%. This ratio range changes to 2:1 to 5 : 1 when acetic acid and ammonia are used (for MS compatibility). Method development techniques generally start with a 9 5 : 5 : 0 . 3 : 0 . 2 acetonitrile-methanol-glacial acetic acid-triethylamine composition, and optimisation achieved by varying the acid/base ratio and/or organic composition. The ratio of acetonitrile to methanol can also be adjusted to optimise retention (affecting resolution also, but to a much lesser extent). This mobile phase has not only led to a wider range of applications, but has produced highly efficient, faster separations not previously possible on these phases. It also has other advantages. Compounds that exist as hydrochloride salts can be separated in this mobile phase but not generally in normal phase. When using achiral-chiral coupled column systems, fractions from the reversed-phase column can be switched directly into the cyclodextrin CSE The demands on the structure of the analyte for potential chiral separation are also much lower than with other CSPs in different mobile phases. Cyclodextrin CSPs were first introduced commercially under the Cyclobond TM nomenclature, Cyclobond I being the [3 cyclodextrin, Cyclobond II the ~/and Cyclobond III the ot version. The derivatives are named as in Fig. 5.3. Other available CSPs include Nucleodex| available in oL, [3 and ~/native cyclodextrin and in a permethylated version, where all of the secondary hydroxyl groups have been methylated, so that this phase operates via inclusion and proton acceptance. The ChiraDex| range offers a native [3 and ~/cyclodextrin phase and differs in the choice of more polar spacer arm used to attach the cyclodextrin to the silica media, giving it different characteristics to other cyclodextrin technologies.

5.2.4 Macrocyclic antibiotics Macrocyclic antibiotics are the newest class of chiral selectors but have already proved to be invaluable for a wide range of applications in chiral HPLC, SFC and CE, and especially in bioanalysis. There are several closely related oligophenolic glycopeptides (Fig. 5.4). Of these, Vancomycin, Teicoplanin and Ristocetin (denoted Chirobiotic V, T and R, respectively) have been used for a wide variety of pharmaceutical applications [38]. Vancomycin is an amphoteric glycopeptide containing 18 chiral centres surrounding three shallow inclusion pockets, bridged by five aromatic ring structures. Hydrogen donor and acceptor sites lie close to the ring structures, and two sugar moieties are positioned at the edge of the structure. Twenty chiral centres are present in teicoplanin, together with four inclusion pockets, seven aromatic rings, three sugar molecules and a multitude of hydrogen donor and acceptor sites. Uniquely, one of the sugars is a glucosamine that has a N-acyl hydrocarbon chain, making teicoplanin considerably more surface active than the other glycopeptides. Ristocetin was the latest to be developed and is the most complex. It has 38 chiral centres, four pockets and six sugar molecules. The peptide chain and additional ionisable groups give this structure the complexity and diversity to separate a wide range of analytes. All of the three References pp. 180-184

CHIROBIOTIC V (Vancomycin)

C H I R O B I O T I C R (Ristocetin A)

H 3C

.o

NH~ ~

HO HO ~ _ ~ _

"~~, n~ ,r - - O ~ o

,o,..< ~'o

o~-""h'-u-,,--.L

HO

"

~:_.j

E

c ~

OH

3

HO "~- ~

HO OH

~o

.o.

~,

O

.N

O

,

~'O

~ ~

OH

CH

-o ~ -"y-"-

3

OH

NH 2 HOHOH~

CHIROBIOTIC T (Teicoplanin)

CH~cH O

~..~OH

--.o .o

-H

OH

o

OH

CH 3

o.

o.

O

~~"~--7~

HO

o,. ~O..o

~o, ~ N " ~ ~

HO ~

c.~o. o.

~

OH

CHIROBIOTIC TAG (Teicoplanin Aglycone)

,~j~

,~

CI

O

H HO~,~ W

NH 2

..

H O

CI

CI

o

~'--"~-- OH

H

O

B H N

O C i

O

H H I

O I

.-NH 2

~N H

HO ~

O OH

H

~'~0 ~

CH~)H

o~~

HO

H

~

Fig. 5.4. Commercially available macrocyclic glycopeptide chiral stationary phases.

L~

Chiral bioanalysis

145

glycopeptides are covalently bonded to silica using multiple linkages, making them extremely stable for both analytical and preparative LC applications. Each of the three CSPs operate in three different solvent modes - reversed-phase, polar organic and normal p h a s e - and enantioselectivity can be different in each mode. Strong interactions are available from w-~r complexation, hydrogen bonding and ionic interactions. Dipole-dipole interactions are less strong and inclusion or steric interactions weak. The mobile phase can therefore be chosen to enhance the most favourable interaction for enantioselectivity. By using intermediate solvents (such as ethanol when moving from the polar organic to normal phase), a single column can be switched between all three solvents systems without any detrimental effect. The three CSPs also appear to be complementary in that an increase in selectivity is often seen when moving from one column to another, using the same mobile phase. In the reversed-phase mode, typical solvent systems comprise a buffer with methanol, tetrahydrofuran or (occasionally) acetonitrile. Interestingly, enantioselectivity has been observed in both high aqueous and high organic content. Like the cyclodextrin columns, the macrocyclic antibiotics can also be used in the polar organic mode. In this case, 100% methanol is used in combination with acid and base. Again, it is the acid/base ratio that governs enantioselectivity: retention is controlled by changing the total concentration of the acid and base. Like the cyclodextrin columns, the choice of acid and base for the polar organic mode can be varied, such that volatile ones can be used for bioanalysis with HPLC-MS. Fast HPLC-MS bioanalysis using single salts such as ammonium acetate or trifluoroacetate have also been achieved (Table 5.3 is a summary of recent HPLC-MS clinical studies using the polar organic mode). A recent development in this type of CSP is the formation of the aglycone derivative of the teicoplanin bonded phase [39]. Having removed the sugar molecules, the resulting Chirobiotic TAG column displays especially enhanced c~ values for native, synthetic and some N-blocked amino acids. Interestingly, neutral molecules, such as oxazolidinones, hydantoins and benzodiazepines often separate well on the Chirobiotic TAG CSP in 100% of a single polar organic solvent.

5.2.5 ~-Complex CSPs Some of the earliest phases of development for chiral chromatography were based on the principle of 'charge transfer'. The chiral selector in these types contains either a wacid or w-base, aiming for interaction with a w-basic or w-acidic analyte, respectively. Other simultaneous interactions must also be present for enantioselectivity to occur and these include hydrogen bonding, steric repulsion and/or dipole-dipole interactions, with hydrogen bonding taking precedence [40]. Since these interactions are strongest in normal phase, solvents systems for enantioselectivity are often hexane-ispropanol mixtures. The length and geometry of the tether connecting the chiral selector to the silica surface is found to be critical [40]. These CSPs are the simplest to understand mechanistically, but can be highly specific to certain compound classes. A large array of ~r-acid or ~r-base type systems have been developed since the pioneering work of Pirkle [41] that started with the bonding of the 3,5-dinitrobenzoyl References pp. 180-184

4~

TABLE 5.3 S U M M A R Y OF H P L C / M S C L I N I C A L STUDIES IN THE POLAR O R G A N I C M O D E Flow rate

tR enantiomers

100 MeOH/0.03% ATFA*

1.2 ml/min

5.0, 10.4 min


1.0 ml/min

6.1, 7.2 min

50 • 4.6 mm


1.0 ml/min

6.2, 6.8 min

50 z 4.6 mm


1.0 ml/min

1.1, 1.6 min 7.0, 7.8 min 3.2, 3.7 min (metabolite at 2.0)

Compound

Column

Column size

Mobile phase

Ritalinic acid [ 188]

CHIROBIOTIC T

50 • 4.6 mm

Methylphenidate [ 171 ]

CHIROBIOTIC V

150 • 4.6 mm

Fluoxetine [ 188]

CHIROBIOTIC V

Nicardepine [ 188]

CHIROBIOTIC V

Metoprolol [ 188]

CHIROBIOTIC T

150 • 4.6 mm


1.2 ml/min

Salbutamol [45]

CHIROBIOTIC T

250 • 4.6 mm

100/0.5/0.1 MeOH/AcOH/NH4OH

2.0 ml/min

* ATFA is ammonium trifluoroacetate.

r

Chiral bioanalysis

147

derivatives of some amino acids, noteably phenylglycine (denoted DNPBG). The only shortcoming of these types was the need to have an analyte structure with a complementary ~r-acid or w-base functionality, often making the CSP very specific for certain classes of structures. If it did not have such a functionality, derivatisation would be necessary. The results of the degree of enantioselectivity achieved with a wide range of phases developed were used to prepare 'reciprocal' phases and to model the interactions occurring [41]. In addition, chiral selectors have been designed for certain 'target' molecules of interest. For example, the [3-GEM-1 CSP (Regis) was originally designed for the separation of non-steroidal anti-inflammatory drugs (NSAIDs), but required that the carboxylic acid group be converted to the anilide derivative. Subsequent research led to the Whelk O-1 CSP (Regis) that resolved underivatised NSAIDs and also a wide range of other molecular types [40]. This was the first of a series of [3-complex CSPs that have been designed to contain both [3-acid and [3-base groups. Consequently, they are the most broad ranging of this type of chiral phase. More recently, two CSPs based on tartaric acid derivatives that have been polymerised and crosslinked have been developed (Kromosil CHI, Eka Nobel). The retention and selectivity are mainly dependant on the hydrogen bonding capabilities of the analyte and so are mostly used in normal phase solvents. They are advantageous for preparative chiral HPLC, and no reports to date have been noted for their use in bioanalysis. Table 5.4 shows the variety of [3-complex CSPs that are available commercially. Application areas for enantioselectivity have not been added for reasons of complexity but are available from individual manufacturers data. 5.3 A P P L I C A T I O N S OF C H I R A L H P L C IN BIOANALYSIS

5.3.1 [~-Adrenergic agonists The enantiomers of terbutaline in urine samples have been determined by utilising achiral-chiral column-switching techniques [42]. Following silica SPE extraction, endogenous compounds were separated from terbutaline and betaxolol (internal standard) on a silica HPLC column. A column-switching valve equipped with a silica pre-column enabled concentration of the terbutaline fraction before its normal phase chiral separation on a Sumichiral OA-4900 CSE Detection was by fluorescence (excitation 276 and emission at 306 nm) provided an assay sensitivity of 0.3 ng/ml with linearity from 1 to 250 ng/ml. A Chirobiotic T column was evaluated for the enantiomeric resolution of clenbuterol in the method development study looking at the side effects of the drug [43]. Liquidliquid extraction and an internal standard of practolol were used in the method. The CSP was used in the polar ionic mode with a mobile phase of methanol-acetonitrile-acetic acid-triethylamine (70:30:0.3:0.2 v/v/v/v). The enantiomers of both clenbuterol and the internal standard were separated in under 13 minutes: it was noted that there was no variation in peak retention times over the 1000 samples of the study. Clenbuterol has also been separated on the w-donor column, Chirex 3022 [44]. Normal phase conditions provided a resolution of 4.2 in 15 minutes, and a LOQ of 0.1 nmol by way of UV detection at 254 nm. References pp. 180-184

TABLE 5.4 RANGE OF w-COMPLEX CSPs AVAILABLE Bonding chemistry

Type

Commercial name

1-(3,5-dinitrobenzamido)-tetrahydrophenanthrene 1-(3,5-dinitrobenzamido)-tetrahydrophenanthrene

w-acceptor/w-donor rr-acceptor/w-donor

R,R- and S,S-Whelk-01 (Regis) R,R- and S,S-Whelk-02 (uses trifunctional bonding

3,5-dintrobenzoyl-diphenylethylenediamine 3,5-dintrobenzoyl- 1,2-diaminocyclohexane 3-(3,5-dintrobenzamido)-4-phenyl-[3-1actam Dimethyl N-3,5-dintrobenzoyl-oL-amino-2,2-dimethyl-4-pentenylphosphonate N-3,5-dintrobenzoyl-3-amino-3-phenyl-2-( 1,1-dimethyl)-propanoate N-(1-naphthyl)leucine 3,5-Dinitrobenzoylphenylglycine

w-acceptor/w-donor w-acceptor/w-donor w-acceptor w-acceptor w-acceptor w-donor w-acceptor

3,5-Dinitrobenzoylleucine 1R,3R-chrysanthemate-R-phenylglycine 3,5-Dinitrobenzoyl-R-naphthylglycine 3,5-Dinitroanaline-S-valine 3,5-Dinitroanaline-S-tert-leucine 3,5-Dinitroanaline-R-phenylglycine S- 1-(ot-naphthyl)ethylamine-S-valine R- 1-(o~-naphthyl)ethylamine-S-valine S- 1-(a-naphthyl)ethylamine-S-proline R- 1-(ot-naphthyl)ethylamine-S-proline S- 1-(ot-naphthyl)ethylamine-S-tert-leucine R- 1-(oL-naphthyl)ethylamine-S-tert-leucine S- 1-(ot-naphthyl)ethylamine-S-indoline-2-carboxylic acid R- 1-(ot-naphthyl)ethylamine-S-indoline-2-carboxylic acid O,O'-bis(3,5-dimethylbenzoyl)-N,N'-diallyl-L-tartardiamide O,O'-bis(4-tert-butylbenzoyl)-N,N'-diallyl-L-tartardiamide

w-acceptor w-acceptor w-acceptor w-acceptor w-acceptor w-donor w-donor w-donor w-donor w-donor w-donor w-donor w-donor w-donor w-donor

chemistry for > stability to hydrolysis) (Regis) ULMO (Regis) DACH-DNB (Regis) Pirkle 1-J (Regis) R- and S-a-Burke 2 (Regis) R,R- and S,S-[3-GEM 1 (Regis) D- and L-Naphthylleucine (Regis) D- and L-Phenylglycine (Regis) Chirex 3001, Sumichiral OA-2000 D- and L-Leucine (Regis) Sumichiral OA-2200 Chirex 3005 Chirex 3010, Sumichiral OA-3100 Chirex 3011, Sumichiral OA-3200 Chirex 3012, Sumichiral OA-3300 Chirex 3014, Sumichiral OA-4000 Sumichiral OA-4100 Chirex 3017, Sumichiral OA-4400 Chirex 3018, Sumichiral OA-4500 Chirex 3019, Sumichiral OA-4600 Chirex 3020, Sumichiral OA-4700 Sumichiral OA-4800 Chirex 3022, Sumichiral OA-4900 Kromasil CHI-DMB Kromasil CHI-TBB

t~

Chiral bioanalysis

149

Salbutamol (albuterol) is a safe and effective short-acting bronchodilator, considered one of the best of its type. It is administered as the racemate, even though it is wellknown that its clinical activity is mainly associated with the (-)-R isomer. Whilst many methods have been developed, many suffer from lack of sensitivity and so can be suitable only for defining the pharmacokinetics of salbutamol after oral and intravenous dosing [45]. These authors described a method that was sufficiently sensitive for inhaled doses using H P L C - M S - M S and on-line robotic 96-well plate extraction. A total assay time of less than 5 minutes was achieved for the simultaneous separation of the racemic parent drug and both enantiomers of the 4-O-sulphate metabolite in plasma and urine (Fig. 5.5). A Chirobiotic T column in an HPLC-MS compatible polar ionic mode solvent of methanol, acetic acid and ammonia ( 1 0 0 0 : 5 : 1 ) was used for the assay, ^)

;-$allmmmol

250000

m/z 24.0 -,+ 1411

2OOOOO 15OOOO

I00000

R .

.

.

.

.

.

.

.

-

. . . . . . .

.

.

.

.

.

B) ~243

.

. . . . .

~:

;-

:

.

+

,]-SaPmm~ol

--~15|

l .k

.

_

,

.

i

_

.

_

,

_

li|

. . . . .

R

C) m/z 342 --+244

1.0

2.0

3.0 T~m

4.0

5.0

(mi~)

Fig. 5.5. HPLC-MS/MS SRM chromatograms of parent drug and its O-sulphate metabolite on Chirobiotic T column resulting from a 50 ~1 injection of extracted urine following inhalation of racemic Salbutamol [45]. References pp. 180-184

150

Chapter 5

splitting 200 I~1 of the column effluent to the ionspray interface. This avoided the difficulty of using normal phase solvents with APCI. A sensitivity of 25 pg/ml was achieved and the method was applied to some 4000 samples. A similar method was developed by Fried et al. using fluorescence detection at 230 excitation and 310 nm emission, enabled concentrations down to 125 pg/ml to be detected from a 1 ml plasma sample [46]. In this case, a mobile phase of methanol, acetonitrile, acetic acid and diethylamine provided a 13-minute assay time. During the cross-validation study, an interfering endogenous substance was noted in both human and canine plasma. To accommodate this, the separation was further optimised by reducing the amount of methanol by 10%. This increased resolution from 1.5 to 1.8 although the enantioselectivity and k' were largely unchanged. The method was applied to samples from a single-dose inhalation of racemic salbutamol and also to a canine inhalation study of the single R-(-)-enantiomer. No chiral inversion of the R-(-)-isomer was noted in the dog. A further method for albuterol has been developed using a Chiral-AGP column [47] but the authors reported only an 80% resolution. Salbutamol had been previously assayed with clenbuterol in plasma and urine using a normal phase method on the w-donor, Chirex 3022 [48]. This separation, when combined with fluorescence detection, provided an LOQ 10-fold less than the current HPLC-MS methods for the parent drug enantiomers. Chiral-AGP was the method of choice for the determination in urine of the R,R and S,S isomers of formoterol, a long acting [3-adrenoceptor agonist [49]. Complete pharmacokinetic data had previously been lacking due to the absence of a sensitive chiral method. Electrochemical detection was used with this method for single inhalation doses in human studies, facilitated by using a mobile phase of 2-propanol and phosphate buffer with added 1 mM KC1 and EDTA (Fig. 5.6). The limits of detection for the R,R and S,S isomers were 60 and 75 pmol/L respectively.

5.3.2 [3-Adrenergic blockers An achiral-chiral column-switching technique was used for the quantification of metoprolol in human urine [50]. A silica HPLC column was first used to separate metoprolol and the internal standard from interfering urinary compounds with the analytes then switched on to the chiral column via a silica trap column. Chiralcel OD in normal phase mode and fluorescence detection provided a limit of quantification of 25 ng/ml for each enantiomer. Direct and indirect methods for the determination of metoprolol were compared for the R(+) and S(-) enantiomers in human plasma [51]. The direct method used a Chiralpak AD (using solid-phase sample preparation) or a Chiralcel OD-H column, while the indirect method used derivatisation with S-(-)-menthyl chloroformate followed by separation of the resulting diastereomers on a reversed-phase C8 column. It was found that the direct, Chiralpak AD, method provided the highest sensitivity, although it did not measure the o~-hydroxy metabolites. A further study [52] did achieve this, enabling the separation of the parent drug and the four enantiomers of the othydroxy metabolites in 13 minutes (Fig. 5.7). A Chirobiotic T column in the polar ionic

Chiral bioanalysis

151

RR

$s

IS

r

Inject

Fig. 5.6. Separation of the RR and SS enantiomers of formoterol and its diastereomer as internal standard on Chiral-AGP using electrochemical detection ( + 0.63V vs. Ag/AgC1) [49].

mode was used. Unusually, the mobile phase also contained dichloromethane (to which this CSP is stable), added to assist in the separation of the closely eluting metabolite isomers. The analytes were detection by fluorescence and the lower limit of quantification was repoted as 0.5 ng/ml for metoprolol and 1.0 ng/ml for oL-hydroxy metoprolol. A fully automated assay for oxprenolol in human plasma was developed that utilised on-line dialysis (using an ASTED system) through a cellulose acetate membrane for sample preparation [53]. Since the [3-adrenergic blocking activity of this compound is mainly dependant on the S-(-) enantiomer, it was considered important to measure the plasma concentrations of the individual enantiomers. Dialysis was followed by a precolumn clean-up and trace enrichment: injection onto a Chiralcel OD-R column in a mobile phase of acetonitrile and phosphate buffer containing sodium perchlorate provided an assay time of about 12 minutes. The influence of perchlorate concentration on the separation was studied and it was found that increased concentrations, up to a limit of 0.45 M, gave rise to an improvement in enantioselectivity and resolution. It was postulated that this was due to an ion-paring effect. References pp. 180-184

Fig. 5.7. Typical chromatograms of standards and unknown from a clinical assay for metoprolol and a-hydroxymetoprolol on Chirobiotic T in the polar ionic mode [52].( I ) blank, (2) standard containing 2 nglml each isomer, (3) standard containing 50 nglml each isomer, (4) post dose sample from a typical patient.

Chapter 5

Chiral bioanalysis

153

The enantiomers of atenolol were separated using a Chiral-CBH column equipped with a silica pre-column, added to compress the peaks and improve sensitivity [54]. The mobile phase comprised 2-propanol, 50 ~M cellobiose and 50 ~M EDTA and the method was validated for use with CSE A sensitive assay for all four enantiomers of labetolol was developed using a Chirex 3022 ~r-donor CSP in normal phase with fluorescence detection [55]. The retention times were 33, 37, 43 and 51 minutes and each was linear over a concentration range of 2.5 to 125 ng/ml. Differing ratios of the enantiomers were observed in plasma compared to urine: a ratio of 41 : 25 : 25 : 9 was found for the S,S (inactive), S,R, R,S and R,R (active) enantiomers, respectively in plasma ( 5 8 : 1 4 : 4 : 2 4 for urine), even though the active R,R was only 2% of the total dosage. Pindolol was determined in human serum using a Chiralcel OD-R CSP in a mobile phase of acetonitrile and perchlorate [56]. Fluorescence detection at 310 nm (excitation 270 nm) enabled detection limits of 1.2 and 21 ng/ml for the R and S enantiomers in serum, and 4.3 and 76 ng/ml for the same in urine. The widely used blocker, propanolol, has been determined in plasma and urine using racemic alprenolol as internal standard [57]. It was confirmed that the concentrations of the enantiomers were dose dependant and that the concentrations of the (S)-isomer were always higher than those of the (R)-form. The method used Cyclobond I 2000 in the polar organic mode.

5.3.3 Alcohol deterrent drugs The knowledge that disulfiram binds selectively to the single free cysteine on HSA was used to develop a method for the analysis of this drug in plasma on a Chiral-HSA CSE Low concentrations of disulfiram as a mobile phase modifier improved the performance of this CSP for disulfiram and a range of co-administered drugs, and was useful for studying their interactions [58].

5.3.4 Amino acids The concentrations of amino acids in blood serum are known to be influenced by hepatic diseases. Whilst extensive studies have previously looked at the levels of L-amino acids, a recent study [59] looked at the levels of D-serine as a measure of hepatic function, since nutritional serine is thought to be metabolised by D-amino acid oxidase, especially in the liver and kidney. Total serine was measured as its benzofurazan derivative using achiral, ODS chromatography, collecting the NBD-serine fraction and re-chromatographing on a Sumichiral OA-4700, a w-donor Pirkle-type CSE The method was used to measure serum concentrations of D-serine in liver transplant patients who had been suffering from biliary atresia: it was found that D-serine levels in blood serum gradually decreased with the recovery of hepatic function. In the past, many amino acid determinations in biological samples have been carried out utilising derivatisation techniques, principally to provide enhanced detection. One References pp. 180-184

Chapter 5

154

such study [60], used the fluorescent chiral tagging reagent, R(-)- or S(+)-4(3-isothiocyanatopyrrolidin- 1-yl)-7-(N,N-dimethylaminosulphonyl)-2,1,3-benzoxadiazole, to study the amounts of both hydrophilic and hydrophobic D-amino acids in urine. The resulting diastereomeric compounds could be separated on a C18 column and detected by fluorescence at 5 5 0 n m (excitation at 460nm) with identification by ESI-MS. The separation of amino acids for bioanalysis without derivatisation on a Chirobiotic T column relies on a free COOH on the amino acid which interacts ionically with a primary amine on the CSE A comprehensive study of methods suitable for a wide range of amino acids, including enantiomers, positional isomers and proteinogenic chiral amino acids such as DOPA and ot-ABA was carried out [61]. An isocratic method in combination with the high specificity of H P L C - M S - M S was used to increase the number of underivatised amino acids that could be separated simultaneously (Fig. 5.8). Post-column addition of acetic acid was used to increase the MS response, enabling sensitivities that were 10 to 100-fold greater than achieved by fluorescence detection. Although the method was developed for the analysis of meteorite samples, it was

I u

_

!

Fig. 5.8. Simultaneous isocratic analysis of 15 underivatised amino acids by HPLC-ICP/MS/MS on Chirobiotic T [61].

Chiral bioanalysis

155

reported that the developed method would be suitable for therapeutic amino acids and peptides currently under investigation for cancer and schizophrenia.

5.3.5 Analgesic drugs (narcotics) Methadone is a central acting analgesic with a high affinity for Ix-opiod receptors that has been used to treat opiate dependence and also cancer pain. Although used therapeutically as the racemic mixture, the R-(-) isomer is about 25-50 times more potent an analgesic than its S-( + ) counterpart, due to both pharmacodynamic effects and to enenatioselective pharmacokinetics. A method was reported [62] that enabled the simultaneous assay of the enantiomers of the parent compound, its major metabolite, 2-ethylidine-l,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and an internal standard (Estazolam) for the first time on Cyclobond I 2000 RSE Previous methods were available on Chiral AGP and on Chiralcel OJ but they were reported to give overlap for the metabolites. More importantly, the method reported no interference from most common drugs of abuse or commonly administered compounds such as the benzodiazepines and caffeine. The assay utilised a reversed-phase mobile phase of acetonitrile/0.08% triethylamine acetate buffer (pH 4.5). Linearity for both serum and urine over a 0.05 to 2.0 ixg/ml concentration range was observed, with an intra-day and inter-day precision of

cr

1

.

.

83,840

259/213

SIL (R)-

0.00

.

2.00

4.s Time (min)

SlL (S)-kt 6.00

Fig. 5.12. HPLC/MS/MS chromatographic profiles for ketoprofen and stable isotope labelled (SIL) ketoprofen on Chirex 3005. A: Human plasma spiked with 50 ng/ml SIL ketoprofen; B: Calibration standard containing 0.1 ng/ml ketoprofen and 50 ng/ml SIL ketoprofen [ 131 ].

A column switching technique was used for a study of the potential for chiral inversion of a morpholine derivative, 2-(S)-[3,5-bis(trifluoromethyl)benzyloxyl]4-[3-(5-oxo-lH,4H-1,2,4-triazolo)methyl]-3-(S)-phenylmorpholine, having 2 chiral centres [137]. The achiral separation was timed to switch a segment from 26 to 38 minutes directly on to the chiral column, a Chiralcel OD-H and a mobile phase of nhexane-methyl-t-butyl ether-methanol was used to separate the 4 enantiomers over a 50-minute period. UV at 210 nm was used to monitor the separation. The study showed that there was no in vivo inversion at either of the chiral centres in dogs. Chiralpak AD was the CSP of choice for the resolution of sulindac, using hexaneethanol containing 0.05% TFA [138]. Urinary samples were injected either directly or after dilution onto an ODS column and the endogenous component-free eluate collected for analysis by the chiral method. The method was used in a study of urinary excretion of free and conjugated sulindac in human volunteers.

Chiral bioanalysis

169

5.3.21 Antiischaemic drugs Column switching techniques were used for the clean up of gerbil plasma samples for subsequent analysis of a potential antiischaemic agent, dichlorophenyl ketoamino acid, on a CrownPak CR ( + ) CSP [139]. Following C18 SPE extraction, the samples were loaded on to a cyano HPLC column. The fraction eluting between 13 and 40 minutes was switched onto the CSP and the enantiomers eluted with a mobile phase of perchlorate and methanol. Detection at 257 nm UV gave an LOQ of 20 ng/ml.

5.3.22 Antineoplastics The four enantiomers of a potent inhibitor of human leukocyte elastase were determined in plasma [140 After liquid-liquid extraction, a normal phase separation of the enantiomers was carried out on Chiralcel OD-H. Fluorescence detection provided an LOQ of 50 ng/ml, compared with 10 ng/ml for an achiral method carried out on the same samples. Rogletimide has effective antitumor activity in cases of breast carcinoma, and exhibits fewer side effects than its analogous predecessor, aminoglutethamide. A sensitive and stereospecific assay for its enantiomers was developed and validated using a Chiralcel OJ-R column, using aminoglutethamide as internal standard and UV detection at 257 n m [ 141]. When optimising the mobile phase composition 80:20 (v/v) sodium perchlorate-acetonitrile was found to give the best peak shape and retention (6-8 minutes) for the enantiomers. The LOQ was 100 ng/ml for each enantiomer, identical to a corresponding method developed using CE [ 142]. Cyclophosphamide in serum was separated on a Chiral-AGP equipped with a C1 guard column: detection at 195 nm gave an LOQ of 1.25 mg/ml [143].

5.3.23 Antiparkinsonian agents The increasing use of LC/MS using cyclodextrin phases is exemplified by the separation of the anti-parkinsonian drug, trihexylphenidyl (THP) [144] (Fig. 5.13). THP belongs to a group of synthetic anticholinergic drugs that compete with the neurotransmitter acetylcholine at its receptor sites. It does, however, show a range of side effects. While achiral analysis employed GC methods, a chiral HPLC method was required. HPLCESI-MS detection was used because of poor UV absorption. Because this molecule has an aromatic group and two hydrogen bonding groups (one on the stereogenic centre), it was considered suitable for the polar organic mode. A native cyclodextrin column, Cyclobond 1 2000, was used in a 2.0 mm internal diameter format. The optimal mobile phase was found to be 9 5 : 1 : 0 . 5 : 0 . 3 (v/v/v/v) acetonitrile-methanol-acetic acidtriethylamine. It was found that the ratio of acid to base had a dramatic effect on both resolution and retention and this was used in the optimisation process. Calibration curves showed good linearity over the range of 1.3 to 132.3 ng/ml, and also at THP concentrations 48 times higher than the maximum therapeutic plasma levels of 55 ng/ ml. References pp. 180-184

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Chiral bioanalysis

171

5.3.24 Antipsychotic agents Remoxipride is a benzamide that has demonstrated stereoselective affinity for central dopamine D2-receptors from the S-(-) configuration. The first direct chiral HPLC method for the enantiomers in human plasma was reported [145] and used to investigate the systemic inversion of administered S-(-) remoxipride in healthy human volunteers. A Chiralcel OD-R CSP was used in a mobile phase of acetonitrile with potassium hexafluorophosphate at pH 3.5 and detection made at 214 nm. Plasma samples underwent extensive liquid-liquid extraction, enabling a sensitivity of 20 ng/ml, with a precision of 10.6 and 10.4% for the R-( + )- and S-(-)-enantiomers, respectively. It was noted that despite the fact that the column required conditioning with unextracted standards, after storage in methanol for two years, the column exhibited the same retention times, peak response and resolution in a subsequent study. The R-( + ) isomer is inactive, and showed higher toxicity than its counterpart but was not found in any of the plasma samples in this study. The four enantiomers of amisulpride, a benzamide derivative, were separated on a Chiralpak AS in hexane/ethanol with added diethylamine [ 146] and the method applied to pharmacokinetic studies in human plasma.

5.3.25 Antivirals A series of synthetic homo-isoflavonoids, chloro-substituted rac-3-benzylchroman4-ones, were studied for possible enantiomeric anti-rhinovirus activity [ 147]. Two CSPs were compared for their analysis. The paper reports the separation of the enantiomers of 3-(4-chlorobenzyl)chroman-2-one and of its 6-chloro derivate on an R,R Whelk-01 column. A further chlorinated derivative that was unresolved on the Whelk column, was partially separated on a Chiralcel OD-H CSP.

5.3.26 Antiulcerative drugs The pharmacokinetics of pantoprazole, a long-acting proton pump inhibitor was investigated in rats [148]. Gradient elution using a mobile phase of sodium perchlorate and acetonitrile was used to separate the enantiomers on a Chiralcel OJ-R CSE Detection at 290 nm was sufficient to show that serum concentrations of both enantiomers declined rapidly with time. Chiral inversion was noted for intravenous feeding of the racemate but not for feeding of the single enantiomer in rats. In an earlier method, gradient elution was used to separate the enantiomers of pantoprazole sodium sesquihydrate on a Chiralcel OJ-R: a gradient of acetonitrile/perchlorate was used, increasing to 100% acetonitrile over 15 minutes at 40~ [149]. The column was backflushed with the starting mobile phase at the end of the cycle. Chemometric techniques were used for the optimisation of the chiral separation of omeprazole and its metabolites on Chiral-AGE providing a separation within 15 minutes [150] Column temperature and acetonitrile concentration were found to be the most important variables. Detection was by UV at 302 nm. References pp. 180-184

172

Chapter 5

Good retention, resolution and stability were reported over 400 runs of a human serum assay of lansoprazole using Chiral-AGP as the CSP [151]. A mobile phase of 2-propanol and phosphate buffer at pH 7 combined with UV detection at 283 nm were used.

5.3.27 Anxiolytics A combination of UV and circular dichroism detectors was used in a pharmacokinetic study of lorazepam in plasma [152]. The separation of the enantiomers was compared for both a Chiralcel OD column in normal phase and on a Shodex [3-cyclodextrin column under reversed-phase conditions. UV detection provided a sensitivity limit of 3 ng/ml and a circular dichroism (CD) detector was used for identification. Reversal of elution order was observed between the two methods (R- eluting before S- for the cyclodextrin method). In a previous study [ 153], a method developed on ChiralPak AS using hexane-2-propanol-ethanol was developed a tested with spiked plasma. Three analogues of the 5-HT receptor agonist, 8-hydroxy-(di-n-propylamino)tetralin, were synthesised and screened for pharmacological activity as potential orally-active CNS drugs [154]. A method was developed for all the analogues on Chiral-AGP and factorial design techniques used to determine the importance on selectivity of all variables. This was used to support an in vitro metabolism and pharmacokinetic study, in which the analyst replaced the guard column and reconditioned the analytical column every 40-50 samples. Interestingly, sensitivity was found to double by the application of a short ammonium acetate gradient instead of the original isocratic phosphate buffer method. A chiral method for diazepam, and its chiral and achiral metabolites in spiked plasma was developed using reversed-phase conditions and a Chiralcel OD-R CSP [ 155]. It was reported to give good recoveries and detection limits (25 ng/ml at 210 nm).

5.3.28 Biochemical markers For the determination of D- and L-lactate in rat serum, it was necessary to derivatise with 4-(N,N-dimethylaminosulphonyl)-7-piperazino-2,1,3-benzoxadiazole in the presence of triphenylphosphine and 2.2'-dipyridyl disulphide in acetonitrile for 40 minutes, followed by acetylation of the hydroxyl group of the lactate. The resulting derivatives were separated on a Chiralcel OD-RH column in methanol and detected by fluorescence at 560 nm (excitation at 450 nm) [156]. The levels of D- and L-lactic acid were determined from calf serum by using a ligand exchange technique [157]. Ultrafiltrates of the sample were injected onto a short (50 x 4.6 mm) ChiralPak MA column using acetonitrile and 2 mM CuSO4 as mobile phase. The acid was detected at 236 nm. 2-Hydroxyglutaric acid is a chiral polar aliphatic dicarboxylic acid excreted in very small amounts in mammalian urine. D-2-hydroxyglutaric aciduria and L-2-hydroxyglutaric aciduria are two distinct inherited metabolic diseases: accurate diagnosis of the

Chiral bioanalysis

173

disease relies on the determination of the configuration of the enantiomer excreted in excess in urine. The two enantiomers have been separated using a Chirobiotic R CSP interfaced with an ESI source, with detection in negative ion mode MS/MS [ 158]. Using a mobile phase of triethylamine acetate (pH 7.0) and methanol (9:1), almost baseline separation of the two enantiomers was achieved in less than 6 minutes. Glyceric acid is another marker whose increased excretion is an indicator for two other metabolic diseases, D- and L-glyceric aciduria. The separation of the enantiomers was achieved using a narrow bore Chirobiotic R CSP in reversed-phase mode interfaced to MS/MS with an ESI source in the negative ion mode [159]. A flow rate of 0.3 ml/min enabled direct connection to the MS without a split. The isomers were separated at 3.6 and 4.5 minutes and the method used for confirmation of the disease in three patients. Pipecolic acid is an important biochemical marker for the diagnosis of peroxisomal disorders. In this case, a Chirobiotic T CSP interfaced directly to HPLC-MS/MS in a simple mobile phase of methanol/water, the L-enantiomer eluting before the D- (11.7 minutes) at 7 minutes [160]. The method was validated using an internal standard of phenylalanine and showed a linear range of 0.5 to 80 Ixmol~.

5.3.29 Calcium channel blockers 4-Aryl-1,4-dihydropyridines of the nifedipine type are the most studied class (Fig. 5.14) of organic calcium channel modulators and have been used extensively for the treatment of cardiovascular diseases such as hypertension, cardiac arrythmias or angina. A detailed structure-activity study [161] established that calcium channel modulation is dependant on the absolute configuration C4 (R- or S-configuration), where the orientation of the C4-aryl group acts as a 'molecular switch' between antagonist and agonist activity. Due to these opposing pharmacological effects, the development of single enantiomers is critical. A study [162] was carried out of 29 different dihydropyridine structures having the important structural features of biologically active analogues. In all but one case, baseline resolution was achieved (R > 1.26) on at least one of the eight CSPs selected. A strong influence of the substitution on the C4 group was noted for Chiralcel OD-H in normal phase and this CSP separated 82% of the dihydropyridines. The cyclodextrin types, as exemplified by ChiraDex, separated 76%, and was dependant on the bulkiness of the aromatic moiety. The most selective for this type of heterocycle was found to be Chirobiotic T and Chirobiotic V with 92% of the structures separated, mostly at low k' values. Interestingly, the two that did not separate were conformationally restricted dihydropyridines that readily separated on the other columns. The other commercial column tested was the w-acidic Whelk-01 and this separated 42% of the racemates. One of the most often used calcium channel blocking agents, nicardepine, also has potent oral vasodilating activity. A sensitive UV assay was developed [ 163] to determine the ( + ) enantiomer, which was found to be approximately three times more potent that the (-) isomer. Sensitivity in previous methods had been hampered by broad peaks. This method utilised a w-donor Sumichiral OA-4500 column in the normal phase solvent hexane-l,2-dichloroethane-ethanol-trifluroacetic acid. A rapid combined solid phase References pp. 180-184

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CH3OOC,~COOCH2CH 1 p~g on column) in positive ion mode. However, with acidic analytes such as ibuprofen and its metabolites, ion suppression was complete and, even at high sample concentrations, MS data could not be obtained. Formic acid was found to provide a suitable compromise between the needs of MS on the one hand and NMR spectroscopy on the other. The single proton of formic acid, which has a sharp, readily-suppressable NMR singlet near ~8.5, gives minimal interference in the resulting NMR spectra and enables MS data to be acquired for acidic analytes. There are two ways to configure the NMR and mass spectrometers, either in parallel or in series. As NMR spectroscopy is a relatively insensitive technique, large volumes and high concentrations of analytes are used wherever possible to compensate and to reduce analysis time. As a result, generally 4.6 mm HPLC columns are used to avoid problems of overloading. This means flow rates of the order of 0.5-1.0 ml/min are used which meets the requirements of the NMR spectrometer without compromising the chromatography. Such high flow rates can easily be accommodated by modern mass spectrometers. However, MS is concentration dependent, as opposed to mass sensitive, and also destructive. Operating the NMR spectrometer and the MS in parallel, and thus splitting the flow such that a minor fraction goes to the MS, has little effect on sensitivity but enables the bulk of the peak of interest to be collected for further testing if required. It also has benefits such as enhancing source lifetime allowing the mass spectrometer to be operated at optimum sensitivity for longer. If the flow is split prior to the NMR spectrometer, with the length of the capillary to the MS adjusted such that the analyte peak is detected by the MS as it fills the NMR flow cell, the MS can be used to supplement the UV data to direct NMR experiments. Further, splitting in this manner enables the use of stop-flow NMR with minimum degradation of the integrity of the chromatography [ 13]. Running in series, i.e. with the sample destructive MS after the non-destructive NMR spectrometer, allows for the completion of all NMR experiments whether on-flow or stop flow before MS analysis begins, but introduces the possibility of peak dispersion before MS analysis for any peaks trapped between the NMR spectrometer and MS when the flow is stopped. Series operation also causes the NMR flow cell and its connections to be operated at higher pressures than they were designed for, with the consequent possibility that leaks are more likely. Series operation also fails to take advantage of the mass spectrometry ability to flag up peaks of interest quickly.


298

Chapter

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NMR spectrometer ~:~4 l~ . . . . . ' :: ~=:~ ~'t ~!~. . . . .

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Given the strength of the magnetic field surrounding an NMR magnet, there is obviously the potential for interference with the operation of the mass spectrometer. There is also the potential for the presence of the MS to interfere with the operation of the NMR spectrometer. Some experiments have been carried out to investigate this in the situation where the mass spectrometer was sited at the ten gauss line with its axis radial to the NMR. Varying the angle of the mass spectrometer to the NMR had no effect on either the data from the mass spectrometer or the NMR. The HPLC, NMR spectrometer and MS were situated approximately at the comers of an equilateral triangle but this was largely dictated by the size of the laboratory and the position of the supplies to the mass spectrometer. Nevertheless, the mass scale needed to be recalibrated and there may also have been a drop in low mass sensitivity (below m/z= 150) but this was not quantified. No effect on the operation of the NMR spectrometer with the MS located in this position was observed [13]. Since these studies were reported, the advent of shielded superconducting NMR magnets has alleviated the potential problems considerably. A schematic of a typical arrangement for combining HPLC with both NMR spectroscopy and mass spectrometry is shown in Fig. 10.1.

10.3 OPERATIONAL METHODS IN H P L C - N M R AND H P L C - N M R - M S The five main options which can be employed for HPLC-NMR using either isocratic or gradient elution are continuous-flow, stop-flow, "time-sliced" stop-flow, peak collection

Biomedical applications of directly-coupled chromatography

299

into capillary loops for post-chromatographic analysis and automatic peak detection with UV-detected triggered NMR acquisition. The simplest of these is continuous-flow detection, but this is usually only practical when using ~H or 19F NMR spectroscopy for detection unless isotopically enriched compounds are available. However, there are a few examples of HPLC-NMR studies using 2H and 3~p NMR detection in the drug metabolism field [14,15]. Where continuous-flow NMR detection is used for gradient elution, the NMR resonance positions of the solvent peaks shift with the changing solvent composition. For effective solvent suppression, these solvent resonance frequencies must be determined as the chromatographic run proceeds. If the retention times of the analytes are known, or there is an efficient method for their detection on-line, such as UV, MS or radioactivity, stop-flow HPLC-NMR spectroscopy becomes a viable option. In the stop-flow technique, all the usual techniques available for high-resolution NMR can be used. In particular, these include valuable techniques for structure determination such as 2-dimensional NMR experiments, which provide correlation between NMR resonances based on mutual spin-spin coupling such as correlation spectroscopy (COSY) or total correlation spectroscopy (TOCSY) [ 11 ]. In practice, it is possible to acquire NMR data on a number of peaks in a chromatogram using a series of stops during elution without on-column diffusion causing an unacceptable loss of chromatographic resolution. There are two further special categories of stop-flow experiment. Firstly fractions eluting from the column can be stored in capillary loops for later off-line NMR study ("peak picking"). Secondly, the flow can be halted at short intervals during the passage of the eluting peak through the NMR flow cell ("time-slicing") in a manner analogous to the use of a diode-array UV detector to obtain spectra from various portions of the peak. This allows chromatographic peak purity to be estimated. Time-slicing is most useful where the separation is poor, or where the compounds under study have weak/no UV chromophores making it difficult to determine the retention times. Stop-flow acquisition may also be performed using pre-concentration of analytes, effected by column trapping. Griffiths and Horton [16] constructed a system whereby analytes were concentrated on a small chromatographic column. The analytes were then re-eluted using a stronger solvent which resulted in a more concentrated peak for subsequent analysis by NMR. Fully automated analysis is also an option wherein the samples are placed in an autosampler and predefined HPLC-NMR experiments are performed. The software allows automatic detection of UV peaks in the chromatogram based on predetermined time-windows or peak intensities. The successful detection of each UV peak triggers the system to stop the flow at an appropriate time to isolate the peak in the NMR flow probe. Then data relating to the peak (intensity, retention time) are transferred to the NMR host computer and used to define the parameters for the automatically-acquired NMR spectrum. This automatic NMR operation includes field homogeneity optimisation, setting and optimisation of all NMR acquisition parameters and the predefinition of the resultant signal-to-noise ratio required in the spectrum. The measurement of 2-dimensional NMR spectra can also be performed. With currently available


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commercial software, the automated run can be halted at any time with reversion to manual control if desired. Despite the apparent difficulties of performing multiple stopped flow analysis, a process which seems to defy much chromatographic "common sense" it is not usually necessary to make any compromises in a desired chromatographic procedure to accommodate the various types of HPLC-NMR experiment. It is also noteworthy that the powerful structural elucidation capabilities of NMR spectroscopy often ensure that complete chromatographic separation is not necessary for full characterisation of the peak. In the case of HPLC-NMR-MS experiments there are some additional considerations. So far, the principal MS ionisation method used has been electrospray in either positive or negative ion mode (using either single quadrupole or ion-trap mass spectrometers) and this puts further constraints on the chromatographic solvent systems as outlined earlier. Often in HPLC-NMR studies the initial chromatographic separation has been developed off-line from the NMR using non-deuterated solvents. It is not always simply a matter of replacing non-deuterated solvents with deuterated solvents to reproduce this chromatography for HPLC-NMR or HPLC-NMR-MS as this can give rise to changes in retention times. For this reason it is standard practice to run an initial chromatographic run with a small amount of the sample to check retention time data etc., and then scale up the amount injected for stop-flow NMR, when optimum conditions have been established. We have found that during the initial run it is often possible to acquire a great deal of valuable MS data which can then be used to guide the selection of peaks for study by NMR spectroscopy. Ideally this also allows the second run to be acquired while mixing the eluent just prior to the mass spectrometer with a non-deuterated solvent to back-exchange the deuterium atoms in exchangeable situations (e.g. NH and OH groups) for hydrogens. In this way, if these initial data cannot be readily understood, the number of exchangeable hydrogens in any compound can be counted as it elutes [12]. If this is to be performed successfully however, sufficient time must be allowed for back exchange to occur. As alluded to above, the double hyphenation of NMR and MS to HPLC [12,17-19] brings some additional benefits, particularly, when the analyte reaches the MS before the NMR spectrometer. Thus, MS can be used to search for particular diagnostic groups or fragments (e.g. an increase in m/z of 16 for hydroxylated metabolites, or 196 for a glucuronide) and such data can then be used to direct NMR spectroscopy to particular peaks in the chromatogram [20].

10.4 APPLICATIONS IN COMBINATORIAL C H E M I S T R Y Characterisation of the structure and conformation of small biologically active molecules is part of the standard approach to lead generation in drug design studies. In particular, it is now possible to automatically synthesise many thousands of small molecules and then rapidly measure their effects in a given pharmacological test system. The potential power of such techniques comes from the immense number of compounds which can be generated and screened for activity. Two studies have evaluated HPLC-


301

NMR in the field, one based on a mixture of 27 closely related tripeptides [21] and the other on two separate mixtures of four aromatic compounds and three pentapeptides [22]. In the first case, the tripeptide application comprised a synthetic mixture of the 27 combinations of the tripeptide formed from alanine (A), methionine (M) and tyrosine (Y), as the C-terminal amide, H2N. CH(R~). CONH. CH(R2). CONH. CH(R3). CONH 2 where R1, R2 and R3 take all combinations of A, M and Y [21]. Based on chemical shifts and peak multiplicities, the on-flow HPLC-NMR characterisation of the majority of the components in the mixture of 27 tripeptides was achieved and it demonstrated that this approach is likely to be an effective method for compound mixtures. The elution positions of all of the alanyl-containing peptides were determined with the exception of A - M - M - N H 2 which may have co-eluted with another peptide or may have been synthesised in a much smaller quantity. The only other tripeptides for which assignments were not obtained were the MYz-NH 2 isomers and two of the three M2Y-NH2 isomers. These elute towards the end of the gradient run and may not be as well resolved under these HPLC conditions. Additionally with changes in the relative chemical shifts of the solvent signals, the intensities of the non-Nterminal oL-CH protons and the methionyl [3-methylene signals from these peptides may have been reduced by the effects of the solvent suppression irradiation of the water and acetonitrile resonances respectively. With further optimisation of the elution conditions, it is possible that all 27 analytes could have been resolved and characterised.

10.5 APPLICATION TO DRUG IMPURITIES

The manufacture of a drug, and its quality control, is regulated by a variety of national authorities. As well as the need to demonstrate drug efficacy, there is also a strong emphasis on the characterisation of the purity of final drug substances and it is necessary to obtain full characterisation and identification of any impurities at the level of > 0.1% of the UV peak area using HPLC analysis [23]. In order to characterise such impurities, it is usually necessary to isolate individual components by preparative HPLC. This work is often time consuming and expensive and yet may not give conclusive identification. Furthermore it is possible for the impurities to be degraded during sample extraction and purification. There is, therefore, a considerable need to develop and validate new methods for determining product purity. One of the first published real applications of HPLC-NMR was concerned with the identification of an impurity in a synthetic drug precursor [24] and a number of examples are now in the literature including characterisation of impurities in a bulk batch of fluticasone propionate [25] an anti-inflammatory drug used for the treatment of the underlying inflammatory component of asthma, a GART inhibitor AG2034 [26] and the degradation products of a HIV protease inhibitor [27]. Directly-coupled HPLC-NMR and HPLC-NMR-MS can greatly enhance the ability to characterise impurities in a pharmaceutical product. As such, this approach is potentially significant as a general tool for purity analyses and would be expected to be important in speeding up production chemistry processes and for regulatory affairs. A


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combination of HPLC-NMR and HPLC-MS has been used to study the degradation products of an antifungal drug candidate [28]. Both techniques contributed complementary structural information that aided both the structure elucidation and proposed pathway of formation for the degradation products. An impurity in the synthetic drug naftopidil has been identified using HPLC-NMR spectroscopy as 1-naphthylglycerol [29]. Also, the autoxidation and photodegradation of ethynylestradiol has been studied using the same approach [30]. In another study, Mistry et al. showed that HPLC-NMR spectroscopy could detect and characterise impurities below the 0.1% peak level [25], the relevant limit for submissions to regulatory authorities. However, NMR spectra on the peaks which were at a level of 0.2% or less of the parent drug, required considerable and time-consuming data acquisition to achieve acceptable signal-to-noise ratios. Although this appears to involve a high cost in NMR analysis time it would be justified in cases such as that found with fluticasone propionate where there are few alternative analytical approaches. It may, therefore, be beneficial to concentrate the impurities, if stable, before the HPLCNMR by the application of solid phase extraction chromatography, column switching or concentration/enrichment of the impurities by preparative HPLC. Such techniques can be feasible because of the availability of large amounts of sample from drug production batches. The technique could also be applicable to the investigation of formulated drug substances where impurities often appear as a result of the drug substance reacting with the formulation compound or on samples from degradation studies. These types of adduct can sometimes be unstable during sample extraction and purification. This area is of considerable practical importance in the pharmaceutical industry, and although applications have been reviewed recently [31 ], it is worthy of further study.

10.6 CHIRAL H P L C - N M R AND H P L C - C D FOR PHARMACEUTICAL MIXTURES

Many pharmaceutical products are chiral molecules either as single isomers or more commonly as racemic mixtures. In addition, many formulated products are mixtures of active compounds together with a number of additives such as excipients. For chiral molecules, the pressure to develop single isomer forms as therapeutics in preference to racemic mixtures arises from the fact that one enantiomer is usually more biologically active than the other and also that enantiomers can have very different toxicity profiles. Chiral HPLC on-line with NMR has been performed to demonstrate the application of chiral HPLC-NMR spectroscopy to the separation and characterisation of different isomers present in a drug substance employing, as an example, atracurium besylate, a neuromuscular blocking agent used widely in surgery [32]. Atracurium besylate, (2,2 '-(3,11 -dioxo-4,10-dioxatrideca-methylene)-bis- (2-methyl- 1,2,3,4-tetrahydropapaverinium benzenesulfonate), is prepared from racemic 1,2,3,4-tetrahydropapaverine and has four chiral centres. However, because of the symmetry of the molecule, atracurium has 10 distinct species with the structure given in Fig. 10.2 where the configuration at

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C1 can be R or S. Since the final stage of synthesis is quaternization at N2, the isomers have been distinguished, simply for convenience, by the configuration of the substituents at the C1-N2 bond, such that when a tetrahydroisoquinoline residue has the benzyl group at C1 and, arbitrarily, the 3,11-dioxo-4,10-dioxatridecamethylene chain is in a cis configuration, this is called a "cis" residue. In achiral media, different NMR spectra are expected for each of the four types of enantiomeric pairs and for the two meso compounds. In general because of the synthetic approach, the ratio of cis to trans residues is about three which, assuming that quaternization at one tetrahydroisoquinoline residue does not affect quaternization at the other, leads to proportions of cis-cis, cis-trans and trans-trans isomers in the ratio of 11:6: 1. The preference for cis residues has been proved using NOE NMR measurements and by x-ray crystallography on related substances. The ~H NMR chemical shifts, principally of the H8 proton, can be affected not only by whether the C1-N2 configuration in the residue is cis or trans but also by the configuration of the remote tetrahydroisoquinoline unit. In summary therefore, after on-line chiral HPLC separation, NMR spectroscopy has been used to characterise compounds in terms of the cis and trans isomers and to identify the racemic pairs on the basis of their identical NMR spectra. In addition, HPLC-CD was used to identify the absolute configuration of the enantiomers based on the known CD spectrum of R-laudanosine hydrochloride, a closely related molecule. The resulting chromatograms using both UV and CD detection are shown in Fig. 10.3. In this instance HPLC-NMR spectroscopy was useful for identifying the isomeric configuration at the C1-N2 bond, for identifying the enantiomeric pairs of compounds and for distinguishing them from the meso forms. The HPLC-CD experiments were complementary in that, whilst unable to distinguish the C1-N2 isomers (cis or trans), it was possible to determine the absolute stereochemistry at C1 at each tetrahydroisoquinoline residue as either R/R, S/S or R/S based on the sign of the CD response at a chosen wavelength. A consistent finding was that the S isomers eluted before the R isomers and the trans forms eluted before the cis forms. By these means a full characterization of all of the 10 isomers of atracurium was achieved.

10.7 A P P L I C A T I O N TO NATURAL PRODUCTS Natural products have been and remain a rich source of leads for the pharmaceutical industry and many marketed drugs are either natural products or are modifications of such substances. Hence considerable effort is spent in isolating and characterising chemicals from natural sources which can be tested in a variety of biological screens. Often it is necessary to carry out laborious extraction and purification steps and the advent of directly-coupled HPLC-NMR has been explored as an alternative technique for natural product identification. To evaluate the approach, a model mixture of nine aporphine alkaloids has been used to specifically test the loop storage mode of operation of HPLC-NMR [33]. The first application of HPLC-NMR to natural products was in 1994 on the photochemical reactivity of azadirachtin [34]. The technique has begun to assume


305

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greater prominence in for the identification of natural products, particularly when used in combination with other hyphenated techniques such as H P L C - M S - M S . This area of research has been reviewed by Wolfender [35-37] who has reported numerous applications in this area including the characterisation of polyphenols and bitter components from Gentianaceae species [38], the assignment of stereochemistry at a double bond in a new secoiridoid glycoside, seemannoside [39], antifungal materials from the African plant Swertia calycina [38,39], compounds from the Leguminosae family [40], prenylated flavanones from dichloromethane extracts of Monotes engleri [41], naphthoquinones from Cordia linnaei [42], pyrrolizidine alkaloids from Senecio species [43] and antioxidant compounds from the leaves of Orophea enneandra [44]. Bringmann et al. have identified new naphthylisoquinoline alkaloids from a root extract of Ancistrocladus likoko using directly-coupled H P L C - N M R spectroscopy [45]. H P L C - N M R was also used to determine products from coupling reactions catalysed by enzymes isolated from Ancistrocladus as well as Triphyophyllum species [46] and to


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characterise ecdysteroid components in Silene otites [47]. Other studies on natural products using, amongst other techniques, HPLC-NMR include the characterisation of vitamin derivatives [48], saponins from Bacopa monniera Wettst [49], antibacterial sesquiterpene lactones from an extract from Vernonia fastigiata [50]. HPLC-NMR-MS has been used to identify components from Hypericum perforatum L. [51], quercetin glycosides and phloretin glycosides from apple peel [52] and ecdysteroids from Silene otites [53]. Natural products research is also an area where the combined application of HPLCCD and HPLC-NMR has proved advantageous. Bringmann et al. [54] demonstrated the principle by obtaining the structures of chiral compounds in crude plant mixtures from Habropetalum dawei. A more recent paper from the same group demonstrates the application of the 'LC-MS/LC-NMR/LC-CD triad' to the screening of crude plant extracts [55]. In particular, novel alkaloids were identified from extracts of Ancistrocladus griffithii. In addition to applications in phytochemistry, other natural products have been studied. Aptamine was identified as the active component from a marine sponge, Aaptos sp. [56]. The compound was identified by HPLC-NMR spectroscopy from a crude organic extract. Sandvoss et al. used HPLC-NMR-MS to study compounds from the starfish Asterias rubens [57,58]. Previously unreported asterosaponins were identified from subfractionated extracts. The relative complexity of the structures clearly demonstrated the potential of having both MS and NMR available in one HPLC run. Microbial production of secondary metabolites is also an important source of novel therapeutic agents. However, the physiological and biochemical factors that determine the onset of production of a specific secondary metabolite in a particular species are incompletely understood. Generally, a range of analytical techniques, often elaborate, time-consuming and involving extensive sample pre-treatment, have to be developed in order to monitor the details of the metabolic changes and substrate consumption that accompany secondary metabolite production. To provide rapid multi-parametric information about the microbial fermentation process, JH HPLC-NMR has been applied to characterise microbial metabolites directly in the broth supernatants from a wild-type strain of S. citricolor [59]. This species produces aristeromycin, the carbocyclic analogue of adenosine, a secondary metabolite with antibiotic properties. The 600 MHz ~H NMR spectrum of the broth was particularly complex in the chemical shift region between g4.2 and g3.4, since it contains major sugar signals obscuring minor metabolites. Hence an HPLC method with on-line ~H NMR detection was employed to physically separate components which gave signals in this region. HPLC-NMR has also been used to identify biotransformation products of warfarin in cultures of Streptomyces rimosus [60]. 10.8 APPLICATION TO DRUG METABOLISM 10.8.1 Introduction By far, the largest body of work to date using HPLC-NMR and HPLC-NMR-MS is in the field of drug metabolism where the methodology has been used extensively for the


307

identification of metabolites in studies from clinical trials involving human subjects, the investigation of model drugs in animals in vivo and also through the use of in vitro systems such as liver microsome incubations. The results in the literature are summarised briefly below and one example, the identification of the metabolites of 2-bromo-4-trifluoromethylaniline found in rat urine [61], is given in somewhat more detail since it encapsulates many of the different aspects which comprise HPLC-NMRMS and thus serves to illustrate the various modes of operation of the use of the technology.

10.8.2 A summary of human metabolism studies

Antipyrine has been extensively employed as a probe to investigate the induction of drug metabolism. In man, the main metabolites are conjugates of norantipyrine, 4-hydroxyantipyrine and 3-hydroxymethylantipyrine. Stop-flow HPLC-NMR, on a 500 MHz NMR spectrometer, was used to analyse urine samples obtained from a human volunteer following the oral administration of antipyrine [62]. This experiment enabled the unambiguous determination of the structures of the major antipyrine metabolites, rapidly and without any pre-treatment (other than preconcentration by freeze-drying) of the sample. By this means, it was possible to identify the ether glucuronides of 4-hydroxyantipyrine, norantipyrine-glucuronide and 4-hydroxyantipyrine itself, the latter either excreted as such or produced by degradation of the glucuronide. Norantipyrine tautomerises to give the 5-enol and it is the O-glucuronide which is formed in preference to the corresponding N-glucuronide In addition, a minor component showing signals for both olefinic hydrogen and glucuronide proton resonances was detected, and this probably corresponds to 3-hydroxymethylantipyrine glucuronide. Ibuprofen is a widely used non-steroidal anti-inflammatory drug (NSAID) which is subject to extensive metabolism, via both Phase I (hydroxylation and oxidation) and Phase II (glucuronidation) pathways. The principal Phase I metabolites are hydroxy and carboxy oxidation products. In man, the metabolites of ibuprofen are rapidly excreted in the urine following administration of normal therapeutic doses to a healthy volunteer. The use of HPLC-NMR on urine samples obtained following a normal therapeutic dose of the compound (400 mg) formed the first application of HPLC-NMR to the study of drug metabolism. This work employed gradient elution on freeze-dried urine [63]. From a continuous-flow HPLC-NMR run it could be seen that three ~H NMR signals around ~5.6 were assignable to anomeric protons from glucuronide conjugates in the samples. Confirmation of this identification was obtained via stop-flow measurements. This allowed identification of the glucuronide of the side chain hydroxylated metabolite of ibuprofen, the glucuronide of ibuprofen itself and the glucuronide of the diacid metabolite. Also identified were unconjugated hydroxy-ibuprofen, and the side chain oxidised diacid metabolite. The 2-D TOCSY spectrum (with double solvent suppression) in stop-flow mode was useful in confirming the proposed structures in that the cross peaks due to the glucuronide spin system, and those for the methyl-methine spin system were readily visible. More recently, a comprehensive study employing HPLCReferences pp. 325-329

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NMR-MS has been used to confirm the identities of these metabolites [64], and in addition identify a number of additional minor components. A similar application of HPLC-NMR in drug metabolism was the study of the metabolic fate of racemic flurbiprofen ([_+ ]-2-(2-fluoro-4-biphenylyl)propionic acid) [65] which, in the same way as seen for ibuprofen is converted to a number of Phase I and Phase II metabolites. The principal metabolite is 4'-hydroxy-flurbiprofen and 3',4'-dihydroxy flurbiprofen is a minor metabolite, but all metabolites are excreted mainly as the glucuronide conjugates. The presence of fluorine in a drug molecule enables selective detection of drug metabolites for compounds such as flurbiprofen. Apart from fluoride ion, usually present at low concentration in biofluids and giving a single broad resonance, there are virtually no endogenous fluorine-containing compounds and hence 19F NMR spectroscopy provides a rapid diagnostic method for assessing the number and level of metabolites of fluorine-containing drugs in body fluids. The 600 MHz ~H NMR spectrum for a urine sample from a human volunteer, following ingestion of 200 mg of flurbiprofen was complex, showing a multiplicity of signals from endogenous metabolites as well as those due to flurbiprofen-related compounds. This complexity precluded any detailed structural or quantitative analysis demonstrating the limitations of 1H NMR in this case. The corresponding resolution-enhanced 19F NMR spectrum (with ~H decoupling) indicated the presence of four major fluorine containing species. The chemical shifts of the major fluorinated metabolites were all suggestive of modifications to the drug distant to the fluorine-containing phenyl ring whilst a total of some 24 separate fluorinated components were detected in this spectrum altogether. The pseudo-2-D contour plot for the continuous-flow HPLC-NMR experiment, with 19F detection showed four J9F resonances, present as two pairs at retention times of 30.5 and 36.6 min. Of these, the peaks eluting at 30.5 min. corresponded in chemical shift to the largest resonances seen in the ~F NMR spectrum of the whole urine whilst those eluting at 36.6 min. corresponded to the remaining major components. The pairing of the chromatographic peaks was due to the glucuronides being present as diastereoisomers as a result of the conjugation of [3-D-glucuronic acid with the R and S isomers of flurbiprofen or its metabolites. Interestingly the intensities of the signals for the two diastereoisomers in each pair of resonances were not equal indicating either differential excretion or metabolism of the two isomers of the racemic drug or inversion of the chiral centre in vivo. The experiment was repeated using stop-flow JH NMR detection at 600 MHz at the appropriate retention times for the 19F NMR-detected metabolites. The resulting 1H NMR spectra were consistent with the [3-D-glucuronic acid conjugate of the hydroxylated metabolite eluting at 30.5 min. and flurbiprofen glucuronide at 36.6 min. Using the "time slicing" technique, the IH NMR spectrum was obtained for this peak after elution for a further 30 s. This latter spectrum is of only one of the diastereoisomers of flurbiprofen-[3-D-glucuronide, revealing the inhomogeneity of the chromatographic peak. Further studies with ~H NMR in stop-flow mode enabled a further, minor, flurbiprofen metabolite to be identified as the free 4'-hydroxyflurbiprofen. Another fluorinated drug candidate, the HIV-1 reverse transcriptase inhibitor BW935U83 has also recently been studied using hyphenated HPLC-NMR methods


309

[66]. In this study, the presence of both fluorine and chlorine atoms provided complementary markers beneficial for NMR and MS detection respectively. By obtaining 19F NMR spectra and on-flow HPLC-~9F NMR pseudo-2D spectra, it was possible to determine the number and location of metabolites. This was particularly useful in this case as not all compounds of interest had UV chromophores and would thus have been missed using conventional detection methods. A combination of NMR and MS data acquired simultaneously using HPLC-1H NMR-MS then allowed structural elucidation. Paracetamol, or acetaminophen, is one of the most widely studied of all xenobiotics and its metabolic fate is well documented. A number of ~H NMR studies of paracetamol metabolism in man have been conducted, and the major metabolites, namely, the phenolic ether glucuronide, the sulfate and the product with N-acetylcysteinyl substituted at position C3, together with paracetamol itself were all detected and quantified in urine [67,68]. More recently HPLC-NMR and HPLC-NMR-MS has been used to characterise these metabolites in biofluids including urine [69]. It has also been shown that paracetamol can undergo deacetylation-reacetylation in vivo (futile deacetylation). This was first shown in the rat (see Section 8.3) but a human study has also been undertaken, showing that for the glucuronide metabolite there is about 1% futile deacetylation and for the sulfate metabolite the figure is about 2% [70]. Whilst most HPLC-NMR studies have been undertaken on urine drug metabolites are often also present in other biofluids such as blood plasma (albeit at low concentration). Blood plasma is, physico-chemically, a more complex biofluid than urine, with high concentrations of proteins and lipoproteins with multiphasic elements. The low molecular weight substances present may also bind to the plasma proteins resulting in complications for analysis by ~H NMR methods. However, the determination of drug metabolites in human blood plasma by HPLC-NMR has been demonstrated in a study of plasma from dialysis patients suffering from chronic renal failure [69]. The 750 MHz ~H NMR spectra of plasma samples from these subjects indicated the presence of a paracetamol-like metabolite which subsequently, using ~H HPLC-NMR at 600 MHz, was identified as paracetamol glucuronide. This is a somewhat unusual example as, in most cases, drug metabolites are rapidly eliminated from the plasma. However, for patients with renal failure the reduced ability of the subject to eliminate these compounds results in a build up in the plasma Tolfenamic acid is another NSAID which undergoes oxidation in vivo with the metabolites being conjugated with [3-D-glucuronic acid. This metabolism has been investigated using 800 MHz 1H HPLC-NMR spectroscopy of human urine following oral administration of the drug to a volunteer [71 ]. The stop-flow approach was used and a number of glucuronide conjugates were identified. These included those of the parent compound and of compounds with both methyl group and ring hydroxylation. The NSAID naproxen has also been studied using hyphenated methods, particularly with respect to phase II conjugation [72]. HPLC-MS and HPLC-NMR spectroscopic approaches were employed alongside each other. The combination of on-flow and stopped-flow HPLC-NMR, together with HPLC-MS data allowed determination of the metabolite structures. This paper also reported on work undertaken to study the effects References pp. 325-329

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of buffers on the chromatographic separations. Both phosphate and TFA buffers, often used in HPLC-NMR are impractical in HPLC-MS (or HPLC-NMR-MS) experiments due to ion suppression problems. The authors therefore studied the applicability of ammonium formate as a buffer for both systems resulting in effective ionisation for the MS. GW524W91 was intended for use as an anti-HIV infection agent. The human metabolite profile has been determined via a combination of methods including 19F NMR of human urine [73] and urine from a clinical trial has also recently been analysed by HPLC-~H NMR spectroscopy at 500 MHz [74]. In this work the stop-flow technique was used to examine each UV-absorbing peak in the chromatogram. ~H NMR spectroscopy was used to detect the presence of the characteristic doublet resonance (at a chemical shift near ~8) due to the nucleoside base proton. This approach enabled the identification of unchanged GW524W91 as the most abundant of the compound-related material in the sample. In addition, the de-aminated compound, and one of the diastereomeric sulfoxides were also observed. In the case of the glucuronide, identification was possible from the characteristic anomeric proton chemical shift appearing at ~4.51 and spectral assignment was confirmed via the use of a IH-1H 2-D TOCSY experiment, obtained by overnight data acquisition in stop-flow mode. For GW524W91 itself, it was also possible, due to the large amounts of material present in the sample, to obtain a ~H-~3C HMQC spectrum [11 ] from an overnight experiment.

10.8.3 Animal metabolism studies of pharmaceuticals and model compounds In the rat, paracetamol and its metabolites are excreted via the bile as well as in urine. HPLC-NMR has been used to analyse bile from cannulated animals dosed orally with paracetamol using reversed-phase chromatography with gradient elution. The major paracetamol metabolite present in the bile was the phenolic glucuronide with smaller quantities of the sulphate also present. Using HPLC-NMR, characteristic spectra for both of these compounds were obtained without difficulty, using the stop-flow technique [69]. Employing the same HPLC conditions used for the analysis of bile, ~H HPLCNMR spectra of the urine from the paracetamol-dosed rats were obtained, confirming the presence of the sulfate and glucuronide conjugates and paracetamol. The Nacetylcysteinyl conjugate was also detected and a ~H NMR spectrum obtained by stop-flow analysis. This spectrum required about 50 min. to collect and still gave a poor signal-noise ratio as expected for such a minor metabolite. In this type of application it is often necessary to compromise on the data acquisition regime, both in terms of achieved signal-noise ratio and in the spectral digital resolution. This may be the result of the need to preserve sample viability, or because the time scale of events being monitored does not permit long acquisition times. In such instances, advantages, in terms of information content, may be more readily obtained if data are subject to further processing after acquisition and so the maximum entropy technique [75] was applied to the NMR spectrum from the N-acetylcysteinyl metabolite of paracetamol and the optimum lineshape (Lorentzian) and linewidth were both obtained by maximising the probability value of the derived solution. The result clearly showed


311

the loss of symmetry of the phenyl ring as a result of the formation of the Nacetylcysteinyl adduct with substitution meta to the N-acetyl of paracetamol confirmed by the ~H chemical shifts of the remaining aromatic protons. The non-equivalent methylene and the methine resonances of the cysteine moiety are also present in the spectrum, with chemical shifts consistent with S-substitution. Furthermore, signals could be observed for the N-acetyl protons, albeit somewhat attenuated by the acetonitrile solvent signal irradiation. It has recently been shown using NMR spectroscopy in conjunction with isotopelabelling studies that there is a significant degree of deacetylation-reacetylation (futile deacetylation) of paracetamol metabolites in vivo in the rat [76]. If this also occurs in humans, then it may help to explain the observed incidence of nephrotoxicity of paracetamol in that the process would result in levels of the potent nephrotoxin 4-aminophenol in vivo. Confirmation of the levels of futile deacetylation in individual metabolites of isotopically-labelled paracetamol in man has been achieved using directly-coupled HPLC-NMR spectroscopy at 600 MHz. In this study a solid phase extract of a 0-4 h urine after dosing with paracetamol-d3 was separated using HPLC with a methanol-water gradient elution. Methanol was used instead of the more usual acetonitrile to avoid the large methyl NMR resonance of the latter which would obscure any observation of transacetylated products. Good ~H NMR spectra were obtained from the sulfate and glucuronide conjugates of paracetamol-d3 and quantification of the level of transacetylated products for both of these metabolites was obtained by integration of the observed acetyl peak relative to the peaks from the aromatic protons. In man, it was shown that paracetamol glucuronide underwent transacetylation to an extent of 1% and for the sulfate conjugate the level was 2% [70]. This compares with results for the sulfate in the rat of about 10% [76]. Phenacetin was once in widespread use as an analgesic but, after being implicated as a cause of kidney toxicity, it was withdrawn from the market. Recently, the rat metabolism of phenacetin has been re-investigated using HPLC-NMR and HPLCNMR-MS [77]. This approach showed that the compound is metabolised principally to paracetamol with subsequent conjugation producing paracetamol glucuronide and paracetamol sulfate. N-hydroxyparacetamol was also tentatively identified. Using the same HPLC-NMR approach, the level of futile deacetylation in phenacetin in the rat was much higher than for paracetamol. This has been quantified in the major metabolites, paracetamol sulfate and paracetamol glucuronide, as 30% and 36% respectively using HPLC-NMR spectroscopy. The level of futile deacetylation for paracetamol and a further, tentatively assigned, metabolite, N-hydroxyparacetamol sulfate, was quantified at 32% [77]. The compound DPC423 is extensively metabolised in animals and man and this process has been investigated in detail using a combination of techniques including HPLC-NMR spectroscopy. A number of novel metabolic pathways were identified, including glutamate conjugation [78]. This has been investigated further using a series of analogues of DPC423 to investigate the transfer of glutamate to a drug benzylamine moiety [79]. The pathway has also been studied in relation to the metabolism of paracetamol [80]. References pp. 325-329

312

Chapter 10

There is a number of other HPLC-NMR and HPLC-NMR-MS studies which include the characterisation of xenobiotic metabolites. The metabolites of the potential antipsychotic agent iloperidone have been elucidated. Identification of metabolites in biological fluids from rats, dogs and humans was achieved using HPLC-MS-MS and, for bile in particular, HPLC-NMR was used to identify a number of structures [81]. Metabolites of the multidrug resistance inhibitor LY335979 have been characterised using HPLC-NMR from rat bile and from human liver microsome incubations. An Noxide metabolite was produced from oxidation of a quinoline nitrogen and, in addition, three glucuronide metabolites were identified, formed by conjugation after oxidation in the quinoline ring [82]. Metabolites of the potential anti-thrombotic drug, roxifiban, have been identified [83]. In another study, the metabolites of trifluoroperazine in the rat have been identified using HPLC-NMR spectroscopy [84]. The implementation of HPLC-NMR-MS, connected in series, has been demonstrated and used to identify metabolites of the non-nucleoside HIV reverse transcriptase inhibitor, GW420867. Again the major substance proved to be glucuronide conjugate of a ring-hydroxylated derivative of the compound [85]. Finally, the use of ion-exchange chromatography coupled with MS and NMR spectroscopy has been used to identify a novel metabolite of GW273629 [86]. The metabolic fate and urinary excretion of 2-bromo-4-trifluoromethylaniline has been studied in the rat using 19F NMR spectroscopy and directly-coupled H P L C - N M R MS [61 ]. The 19F NMR spectrum of whole rat urine collected 0-8 hours after i.p. dosing with 50 m g . k g -j of the compound is shown in Fig. 10.4 indicating the number and relative levels of fluorinated molecules in the urine. It was clear that there was very little of the parent compound in the urine from addition of authentic material. After a solidphase extraction step, HPLC-NMR and HPLC-NMR-MS experiments were carried out to identify the three most abundant species seen in Fig. 10.4. Thus, Fig. 10.5 shows the continuous-flow 19F NMR-detected chromatogram as a contour plot and this gives the retention times of the major fluorine-containing species. Subsequent H P L C - N M R - M S experiments with X9Fand ~H NMR spectroscopic detection and negative ion electrospray MS at these retention times demonstrated that the major metabolite (labelled A in Fig. 10.7) was 2-amino-3-bromo-5-trifluoromethylphenylsulfate accounting for 23% of the dose being excreted in the 0-8 hour urine. The spectra corresponding to this metabolite are shown in Fig. 10.6, with Fig. 10.6(a) being the 19F NMR spectrum, Fig. 10.6(b) showing the ~H NMR spectrum with the expected meta-coupled aromatic protons and Fig. 10.6(c) being the negative ion electrospray mass spectrum of the fully deuterated molecule. A similar approach was used to identify Peak B in Fig. 10.4 as 2-bromo4-trifluoromethylphenylhydroxylamine-N-glucuronide (7% of the dose) and Peak C in Fig. 10.4 as 2-amino-3-bromo-5-trifluoromethylphenylglucuronide (1.4% of the dose). In addition MS could be used to detect and identify a number of minor metabolites below the NMR detection limit [61 ]. Similar studies have also been carried out on 2-chloro-4-trifluoromethylaniline [87] and 3-methyl-4-trifluoromethylaniline and its acetanilide [88], whilst HPLC-NMR and HPLC-MS studies have also been performed on 2,3,5,6-tetrafluoro-4-trifluoromethylfluoroaniline [89], and a range of HPLC-NMR-MS and HPLC-ICPMS-TOFMS

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studies have been performed on 4-bromoaniline [90]. H P L C - N M R - M S with concomitant radioactivity detection has been used to investigate the metabolism of ~3C-acetyl labelled [3-blocker practolol [91 ]. Whilst the bulk of the reported studies have involved the use of either tH or 19FH P L C - N M R , some limited work has been done using other nuclei. Thus, some preliminary work has been carried out to determine the possibility of using 2H NMR spectroscopic detection in HPLC-NMR. To this end, the metabolism of dimethylformamide-dT, DMF-d7, in the rat was investigated using 2H NMR spectroscopy [92]. 2H NMR detection of the deuterated metabolites from D M F - d 7 in rat urine was attempted in continuous-flow H P L C - N M R [14]. These experiments were carried out at a 2H observation frequency of 92.1 MHz (which corresponds to ~H at 600 MHz). Based on chemical shifts and the known metabolism of DMF-d7, the parent compound was the major drug-related material present. The next two most abundant species had very similar retention times, with each containing one type of methyl group. One of these arises from dimethylamine-d6 whilst the other was one rotational form of Nhydroxymethyl-N-methylformamide-d6. In addition, one peak was seen which was detected in the 0-8 h urine only and which was not then assigned. This compound also shows a formyl deuteron resonance and possibly arises from a demethylated product such as N-methylformamide-d4. It is clear that 2H NMR spectroscopy is not likely to be of major use in drug metabolism, but because the 2H nucleus has a rapid NMR relaxation time, data can be acquired rapidly and for small molecules the linewidths are reasonably sharp. This means that for equal numbers of nuclei (i.e. the same level of isotopic substitution), it has approximately the same sensitivity as ~3C NMR spectroscopy although the chemical shift range and hence spectral dispersion is much higher for the latter. As well as deuterium 3~p NMR spectroscopy offers useful possibilities for H P L C NMR even though relatively few drugs contain phosphorus. Probably the most


315

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316

Chapter 10

important class are those related to cyclophosphamide which are used in chemotherapy. The presence of phosphorus in such a molecule does provide the opportunity for using 31p NMR as a specific method for detection of metabolites. The toxicity of the anticancer drug ifosfamide has been studied using extracts from the urine of patients on ifosfamide therapy [15] and this study was combined with investigations of ifosfamide metabolism in the rat. In this case, using urine samples which had been freeze-dried and reconstituted at a 12-fold concentration, it was possible to locate the drug-related substances using 3~P-detected HPLC-NMR in the continuous flow mode and subsequently to use stop-flow methods to characterise the metabolites using ~H NMR. Although 31p NMR spectroscopy is much less sensitive than ~H or 19F NMR spectroscopy, it proved useful for the identification of the chromatographic retention times of phosphorus-containing species in this study, particularly since the metabolites of ifosfamide have poor UV absorption characteristics. Under the HPLC conditions used, the parent drug and three metabolites were identified, ifosforamide mustard, 4-hydroxyifosfamide and 2-de(chloroethyl)ifosfamide.

10.8.4 Application to in vitro metabolism studies In vitro techniques, such as the use of tissue slices and cell suspensions, are being used

increasingly to obtain insights into the metabolism of new compounds in both animals and man. Directly coupled 750 MHz HPLC-~H NMR spectroscopy has been applied to the characterisation of low level metabolites of 3-nitro-2-(2-fluorophenoxy)pyridine and 3-amino-2-(2-fluorophenoxy)pyridine produced by rat microsomes [93]. In stop-flow HPLC-NMR mode, the direct injection of microsomal extracts enabled the separation and characterisation of minor metabolites. Unequivocal identification of the metabolites was achieved without the use of radiolabel or synthetic standards. GW1370U87, 1-ethyl-phenoxathiin-10,10-dioxide, was intended for use as a monoamine oxidase-A inhibitor and its metabolism has been studied in human liver microsomes [94]. The supernatant from the cells was collected, concentrated and examined using 600 MHz ~H HPLC-NMR in stop-flow mode. The UV-detected chromatogram was uncomplicated with most, but not all, of the UV peaks being due to GW1370U87 or its metabolites. Chromatography was stopped at the top of each peak and a IH NMR spectrum obtained. In all, six GW1370U87-related HPLC peaks were characterised. In addition, the metabolism of the multi-drug resistance inhibitor LY335979 [82] and 7-ethoxycoumarin [95] have been studied in human liver microsome incubations.

10.8.5 Application to drug metabolite reactivity Many drugs containing carboxylate groups form [3-1-O-acyl glucuronides as major metabolites. Such ester glucuronides are potentially reactive due to the susceptibility of the acyl group to nucleophilic reactions and they can undergo hydrolysis, acyl migration


317

and covalent adduct formation. The acyl migration reactions result in positional isomers and anomers as shown below and these may be reactive towards serum proteins with toxicological consequences. The acyl group migrates successively to the 2-, 3- and 4-hydroxyl groups of the glucuronic acid moiety, thereby allowing the formation of both oL- and [3-anomers of the positional isomers (see Fig. 10.7). Synthetic fluorobenzoic acid and trifluoromethylbenzoic acid glucuronide conjugates were chosen as model compounds of carboxylate group-containing drugs and an HPLC method has been developed for the simultaneous determination of the 1-, 2-, 3- and 4-positional isomers of the acyl glucuronides, and their c~- and [3-anomers for 2-, 3- and 4-fluorobenzoic acids together with the aglycones formed via hydrolysis. A typical result is shown in Fig. 10.8 which depicts the continuous flow 750 MHz ~H HPLCNMR characterisation of the glucuronides from an equilibrium mixture of transacylated glucuronides of 4-fluorobenzoic acid, measured in the continuous flow mode [96]. The ~H NMR frequency is on the horizontal axis and the chromatographic retention time is on the vertical axis. Each of the glucuronide isomers is eluted separately and can be identified from its NMR spectrum. It has been noted that in general the elution order of transacylated glucuronides is [3-4-O-acyl-, oL-4-O-acyl-, oL-3-O-acyl-, [3-3-O-acyl-, [32-O-acyl- and oL-2-O-acyl- irrespective of the nature of the carboxylic acid-containing moiety. This directly-coupled HPLC-NMR method has been used to investigate the acyl migration kinetics of individual isomers of 2-, 3- and 4-fluoro-, and 2- and 3-trifluoromethylbenzoyl-D-glucopyranuronic acid separated from an equilibrium mixture of the [3-1-O-acyl isomer, the e~- and [3-2-O-acyl isomers, the oL- and [3-3-O-acyl isomers and the oL- and [3-4-O-acyl isomers at pH 7.4 and 25~ [96-99]. Both continuous-flow HPLC-NMR at 750 MHz and stop-flow methods have been used. For detailed kinetic studies, each isomer was separated using reversed-phase HPLC and then led into an NMR flow probe in a 600 MHz NMR spectrometer. The flow was stopped and sequential ~H NMR spectra collected, thus allowing the direct observation of the appearance of the glucuronide positional isomers of that particular glucuronide isomer which had been isolated. This is illustrated in Fig. 10.9 which shows the build-up of other products following the introduction of the [3-4-O-acyl-glucuronide of 2-fluorobenzoic acid into the NMR probe after HPLC separation. The rate constants for the decomposition of the various isomers were determined and the acyl migration reactions were simulated using a mathematical model of the kinetics of the glucuronide rearrangement (incorporating 9 first-order rate constants determining acyl migration reactions and 6 first-order rate constants describing the mutarotation of the 2-, 3- and 4-positional isomers. The acyl migration of the glucuronide metabolite of the model drug 6,11 dihydro11-oxo-dibenz (b,e) oxepin-2-acetic acid has also been investigated in pH 7.4 buffer using and urine directly-coupled 600 and 750MHz stop-flow HPLC-~H NMR spectroscopy [100,101]. Other substances studies using this approach include the glucuronide of a novel retinoid known as CD271 [102], the glucuronides of enantiomeric 2-phenyl propionic acids [103] and S-naproxen glucuronide [104,105]. More recently, the presence of the oL-1-O-acyl isomer has been confirmed demonstrating that the back reaction from the oL-2-O-acyl isomer is possible. This is References pp. 325-329

318

Chapter 10 glucuronic acid

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"~


321

reflected in the reaction scheme shown in Fig. 10.7 and increases the number of kinetic parameters to be determined [ 106]. It is clear that directly-coupled HPLC-NMR spectroscopy offers a unique analytical approach to obtain structural information of inter-converting compounds in a complex mixture of isomers. This method will be of value in the elucidation of the reactivity of drug glucuronides in terms of acyl migration and enable an investigation of the potential for protein binding. Furthermore, this HPLC-NMR approach to the study of glucuronide acyl migration reactions allows unique kinetic information to be obtained relating to glucuronide reactivity and this approach will be useful in future structureactivity studies on the toxicity of drug ester glucuronides. The metabolism of paracetamol proceeds through a reactive intermediate known as NAPQI which can react with glutathione at the 2, 3, or ipso carbons. This reaction has been investigated using directly-coupled HPLC-NMR at 500 MHz [107]. The two reactants, (NAPQI and glutathione, GSH) were mixed and the product mixture separated using directly-coupled HPLC-NMR. In fact all three isomers were shown to be produced with the ipso derivative being most abundant and the 2'-isomer the least. By holding the ipso-NAPQI-GSH adduct in the flow probe of the NMR spectrometer for one hour the rate at which it decomposed to the other isomers and other species could be monitored.

10.9 FUTURE DEVELOPMENTS 10.9.1 Automation and informatics

While this review demonstrates the wide applicability of HPLC-NMR achieved thus far, it is possible to identify areas where rapid advances are to be expected that will further increase the utility of HPLC-NMR. One area for example, is automation, where the ability for improved analysis times in coupled separation-NMR techniques will be a major factor in enabling the more widespread application of the technology. Indeed, it is not difficult to envisage a time when HPLC-NMR combined with in-line HPLC-MS will, automatically, provide the complete qualitative and quantitative metabolic fate of xenobiotics in a single chromatographic run. In order to analyse and extract all information from the huge amounts of data likely to be generated from such systems, novel methods of data analysis are also required. Pattern recognition for example has been combined with flow injection NMR spectroscopy to provide analysis of high throughput biochemical screening [108]. Principal components analysis (PCA) has also recently been applied to HPLC-NMR to differentiate between regio-isomers in a less than optimal HPLC separation [109]. By applying PCA to the on-flow pseudo-2D HPLC-NMR spectrum, it was possible to determine the number and identity of 3 unresolved isomers. In another study, ~H NMR spectra of rat urine samples from subpopulations of normal laboratory rats showed characteristic spectral differences when analysed using PCA. This statistical approach allowed the direction of HPLC-NMR experiments for identification of the endogenous species responsible for the differences [110].


322

Chapter 10

Other new technical developments are also occurring which, in the foreseeable future, will provide greatly increased NMR sensitivity, reduced solvent usage or additional analytical information. These developments include the use of higher magnetic field strengths and hence observation frequencies. In addition the development of NMR probes and preamplifiers cooled with cryogenic liquids will provide lower detection limits and higher sensitivities to a degree surpassing any arising from increases in magnetic field [ 111 ]. It is also possible to envisage new areas of application for HPLC-NMR-MS, and for example recently HPLC-NMR spectroscopy was used to separate and characterise lipoproteins fractions from human blood plasma [112].

10.9.2 Miniaturisation in separations coupled to NMR Miniaturisation of analytical equipment is occurring with respect to both the NMR detection systems, and the initial chromatographic mode. The improvement of methods for the rapid analysis of multiple samples has led to the development of NMR probes containing multiple detection coils [113]. By placing up to four coils within the same cell, such 'Multiplex NMR' significantly reduces sample analysis time. Different approaches exist for the detection of each sample, and more importantly, the elimination of cross-talk [ 114,115]. Microcoil probes have also been demonstrated for use in HPLC-NMR applications [ 116]. The union of capillary HPLC with microcoil NMR has been demonstrated for the detection of terpenoids. With a 1.1 ~1 observation volume, it was possible to detect 37 ng of oL-pinene [117]. This approach with its consequent low solvent usage allows fully deuterated NMR solvents to be employed at reasonable cost and alleviates the solvent suppression problems referred to earlier. It has been found that detection of low nanogram quantities of material can be achieved in 3-4 minutes under stop-flow conditions and 2-dimensional NMR spectra are therefore possible also [116,118]. It is expected that new eluent systems for HPLC which are advantageous for NMR and MS detection will be developed and some preliminary results have been given on the use of superheated D20 in this respect [ 119,120]. Capillary electrophoresis (CE) coupled to NMR has been shown to be a very powerful addition to the armoury of analytical methods. The technique is very simple experimentally, with all that is required being a length of fused silica capillary with an optical window to enable detection, a detector (UV, fluorescence or mass spectrometry), a high voltage source, two electrode assemblies and buffer solutions in suitable reservoirs. The technique has been shown to provide very high separation efficiencies but the small injection volume (a few nl) means that high sensitivity can only be achieved if concentrations of the analyte in the sample are high. The use of NMR spectroscopy for detection in CE has been demonstrated [121-124]. This has an active volume of ~-5 nl and limits of detection using ~H NMR in the ng range for acquisition periods of the order of 1 minute [125]. The limit of detection in concentration terms is about that of HPLC-NMR but in mass terms represents about two orders of magnitude less.


323

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Fig. 10.10. 600 MHz on-flow CEC-NMR spectrum of paracetamol metabolites separated from an extract of human urine. The use of CE-NMR, and the related technique CEC-NMR at an observation frequency of 600 MHz, has been applied to the detection and characterisation of paracetamol metabolites found in human urine [126,127], a crude synthesised dinucleotide and its related by-products [ 128], and aspartame and caffeine [129]. As an illustration, the on-flow CEC-NMR spectrum of paracetamol metabolites from a human urine extract sample is shown in Fig. 10.10. The sample was a solid phase extract of urine from a human volunteer after administration of 500 mg of paracetamol. The 600 MHz NMR spectrum was acquired with 8 scans per row (i.e. about 10 s acquisition time) and the contours seen correspond to the two major metabolites, namely the glucuronide and sulfate conjugates of paracetamol. Also visible is the spectrum of the endogenous species hippurate. Extracted individual rows corresponding to these three substances from this on-flow NMR-detected separation are shown in Fig. 10.11. This on-flow detection is of about 300 ng of paracetamol glucuronide [ 126,127]. A more recent application is the use of capillary isotachophoresis (cITP) prior to NMR detection [130]. cITP is a sample focusing method. It uses a leading and terminating electrolyte, where the leading electrolyte (with a high electrophoretic mobility) forms the front zone and the terminating electrolyte (with a low electrophoretic mobility) the rear zone, with the sample in between the zones. When an electric field is applied, the components separate into discrete bands, with the sample components focused as a function of the ion concentration of the leading electrolyte. The work demonstrated a 100-fold increase in NMR signal-to-noise ratio when comparing non-focused samples with those that were focused using cITE

10.9.3 Hypernation Further hyphenation of other spectroscopic techniques for analyte identification ('hypernation') such as infra-red spectroscopy has recently been demonstrated [131].


Chapter 10

324

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ppm

Fig. 10.11. Single rows extracted from the on-flow chromatogram shown in Fig. 10.10. The identification of the two paracetamol metabolites and the endogenous substance hippurate are as shown. For the paracetamol metabolites, H2/6 are the aromatic protons ortho to the N-acetyl group, H3/5 are meta. H1 '-H5' denote the protons of the glucuronide ring and NCH3 denotes the N-acetyl resonance. For hippurate the numbering scheme has the side-chain at C 1.

H P L C - U V - I R - N M R - M S was employed for the analysis of a mixture of NSAIDs, allowing almost complete structural characterisation to be performed [132]. A time of flight (TOF) mass spectrometer was used in this system enabling accurate mass (and therefore atomic composition) to be obtained. While such a system undoubtedly provides a large amount of structural information however, the degree of complexity of such a system increases rapidly with the addition of each new technique. It is necessary, for example, to ensure solvents (and modifiers) are compatible with all techniques [ 133]. A slightly modified system using flow injection rather than HPLC along with UV, IR, N M R and MS has also recently been reported [134], and the same system coupled to superheated water chromatography has been applied to the analysis of a mixture of pharmaceuticals [135] and a series of plant extracts [136]. In addition, it is expected that publications on the use of other types of mass spectrometric detection in H P L C - N M R - M S , such as time-of-flight (TOF) and ion-


325

cyclotron r e s o n a n c e MS, w h i c h allow accurate masses and h e n c e e m p i r i c a l m o l e c u l a r f o r m u l a e to be d e t e r m i n e d , will be f o r t h c o m i n g

10.10 CONCLUSIONS

The progress that has b e e n m a d e in the coupling of H P L C and related techniques with N M R s p e c t r o s c o p y over the last decade has b e e n very rapid. The n u m e r o u s applications of the t e c h n i q u e in b i o m e d i c a l analysis are e v i d e n c e of its utility and the n u m b e r of reports of the use of H P L C - N M R in this area continues to increase m a r k i n g the transition of H P L C - N M R f r o m a research technique to a routine analytical m e t h o d o l o g y . F u r t h e r d e v e l o p m e n t s leading to the use of m u l t i p l e h y p h e n a t i o n (hypernation) with H P L C - N M R - M S and h i g h e r concatenations have also b e g u n to generate applications. With further t e c h n o l o g i c a l advances in the areas of miniaturised flow probes, for use with capillary separations, and c r y o p r o b e s leading to l o w e r sample r e q u i r e m e n t s the usefulness of N M R c o u p l e d to separations will u n d o u b t e d l y increase.

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Ultra-sensitive detection of radiolabelled drugs and their metabolites using accelerator mass spectrometry Graham Lappin and R. Colin Garner Xceleron Ltd., York Biocentre, York, YOIO 5NY, U.K.

11.1 I N T R O D U C T I O N Our current understanding of the metabolism of endogenous and xenobiotic compounds has come in the main from the use of molecules labelled with radioisotopes. The compound under investigation is synthesised so that selected atoms in the molecule are enriched with the desired radioisotope. The resulting radiolabelled compound, which is chemically identical to its non-radioactive counterpart, is then administered to a test system and radioactivity is followed over time. This enables the passage of the compound and its metabolites to be distinguished from the myriad of endogenous substances. Test systems have ranged from isolated organelles, cells, plants, animals, soils, water and ecosystems. Radiotracer studies are an essential part of the registration dossier for pharmaceuticals, pesticides and some veterinary products and the techniques used today are essentially the same as those used by Hans Krebs to elucidate the TCA cycle in the 1950s. Radiotracer studies in animals, otherwise known as absorption, distribution, metabolism and excretion (ADME) studies typically follow the radioactive compound and its metabolites through organs, tissues, blood, bile, urine and faeces. For pharmaceuticals, radiotracer studies can be performed in volunteers or in patients (with their informed consent) although for ease of collection samples for analysis are limited to blood and excreta and, rarely, biopsy samples. A number of radioisotopes have been used in ADME studies but since most pharmaceuticals are organic, the most frequently used tracer is 14C, a low energy [3emitter. Central to the conduct of ADME studies are methods for the detection and quantification of radioactivity. For [3-emitters, the most commonly used methods of detection are liquid scintillation counting (LSC) or radioluminography. In LSC, the radioactive sample is dissolved in a scintillation cocktail where the kinetic energy of the References pp. 347-349

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[3-particles is converted into bursts of photons which are detected by a photomultiplier. For a review of radiotracer LSC see [1 ]. The main drawback of LSC is one of sensitivity. A [3-particle is emitted when a 14C atom decays to ~4N. The frequency of decay is dependent on the half-life (tl/2) of the radioisotope; the longer the t~/2, the fewer decay events occur in any given time. For ~4C, with a tl/2 of 5730 years, only 0.012% of the atoms decay in a year and therefore to measure one disintegration per minute (dpm) by LSC, 4.3 • 109 ~4C atoms have to be present in the sample (see Section 11.4). Because of this inherent lack of analytical sensitivity, situations can arise where LSC is inadequate and a more sensitive method of detection is required. Examples of such situations are listed below. (i) There may be constraints on the maximum attainable specific activity of the test compound due to the route of synthesis. This can be a particular problem with products such as proteins or secondary metabolites produced during fermentations ie those molecules derived from biological sources. (ii) The specific activity of the test compound may be limited by autoradiolysis. (iii) The amount of radioactivity that can be administered to a test system may be limited by the physical dose that can be given. If the dose is low, so is the amount of radioactivity. (iv) The amount of radioactivity that can be administered to humans is limited by international regulations that control human radiation exposures particularly for volunteer studies where the individual gains no therapeutic benefit. (v) Biological samples may contain low levels of radioactivity because of poor absorption or low bioavailability. (vi) For certain sample preparations such as protein, DNA or isolated cell types, uptake of radioactivity may be very low. (vii) Problems arising from any of the above may be exacerbated if radiochromatographic analysis is required. In the case of (iv), the amount of radioactivity that can be administered to human volunteers per annum, according to the World Health Organisation (WHO) guidelines, is limited to 1 mSv, but more commonly 0.5 mSv is used [2]. This value does vary from country to country with some countries such as Japan not permitting radioactive studies in human volunteers. The Sievert (Sv) is a measure of radiation exposure (the integral of dose and duration of exposure) which is caused by the ionising radiation and can be determined on a compound specific basis from dosimetry studies performed in animals (typically pigmented rats). The radioactive dose is a product of the energy of the radioemission, the time resident in the body and the susceptibility of certain tissues to radiation damage, particularly the GI tract in the case of oral dosing [3]. Each tissue is graded by a factor relating to the likelihood of DNA in that tissue becoming mutated; the gonads have a higher susceptibility factor than the brain for example. Typically, a 0.5 mSv dose in the human is equivalent to 1.5-3 MBq, or about 20 KBq/kg body weight. This is about 100-fold less than the dose that can be used in laboratory animal studies. Moreover, for drugs with long plasma half-lives, or those that bind to protein or melanin, the residence time in the body can be prolonged and so the amount of radioactivity that can be administered may have to be significantly reduced. Further to

Ultra-sensitive detection of radiolabelled drugs

333

these difficulties, subjects receiving the maximum allowable dose cannot participate in further radiolabelled experiments for 1 year. This puts severe restrictions on radiolabelled cross-over studies. The regulatory perspective of using radiolabels in human studies is given in [4]. In 1977 two papers appeared, coincidentally, in the journal Science, reporting the development of the accelerator mass spectrometer [5,6]. Accelerator mass spectrometry (AMS) measures the number of 12C, ~3C and ~4C atoms in a sample; the result of AMS sample analysis is expressed as an isotope ratio. AMS therefore, does not rely on the detection of infrequent decay events to detect 14C and consequently it is about a million times more sensitive than LSC. AMS was originally developed for carbon dating and it was some 13 years later that the first papers reporting its use for biomedical research were published (26A1 measurements [7] and DNA binding [8]). At the time of writing there are about 40 AMS facilities in the World, mostly involved in environmental, archaeological or geological research. There is only one company dedicated to commercial biomedical AMS analysis in existence (Xceleron Ltd, York U.K.) [9].

11.2 INSTRUMENTATION Figure 11.1 shows a schematic of a typical AMS. The sample, which has to be specially prepared (see Section 11.3) is placed into a caesium "sputter" ion source (A). The ion source is held under vacuum at about 10-6 Yorr, whilst the vacuum in the rest of the instrument is around 10-9 Torr. On bombardment with caesium vapour, atoms within the sample form negative ions that are extracted by a series of plates held at several thousand volts more positive than the ion source. The negative ion beam enters an injection magnet (B) where ions are selected depending upon their mass to charge ratio (m/z). For carbon analysis the injection magnet resolves to 1 m/z allowing the 12C-, ~3Cand ~4C-ions to pass as a series of pulses in sequence by virtue of the electric field being 'bounced' to permit only the selected atoms through. At this stage, molecular ions within the defined mass range are also present, such as 91 and ~2CH~ and these also pass through the injection magnet. The pulsed ion beam passes into a tandem electrostatic Van de Graaff particle accelerator (C) where the negative ions "drop" through a very high potential difference towards a positive terminal held at typically 1-5 million volts. The charge is delivered by accelerator chains, which are surrounded with pressurised SF 6 as an insulating gas to prevent sparking from the high voltage terminal. As the ions travel towards the terminal they attain high velocities, dependant upon the voltage of the accelerator. At 5 MV, their velocity is about 3% c (9,000 km/s). The highenergy ion beam is focused to collide with argon gas molecules or a thin carbon foil, 0.02-0.05 txm thick (D) in a collision cell. This has the effect of stripping the outer valency electrons and, with the loss of electrons, the charge on the ions switches from negative to positive. The extent of the electron stripping depends upon the energy of the ions, which is dependent upon the voltage of the accelerator. At 1 MV volts, the most abundant ions are C ~+ and C 2+ at about 40% each. There is insufficient energy at this voltage to form ions with higher charges above about 5% abundance. At 5 MV, over References pp. 347-349

334

Chapter 11

i

Fig. 11.1. Schematic of an accelerator mass spectrometer. Key: A Negative ion sputter source B Injection magnet C Tandem electrostatic Van de Graft particle accelerator D Electron stripper E High energy analysing magnet F Faraday cups G Quadrupole H Electrostatic cylindrical analyser I Gas ionisation detector

60% of the carbon ions are C 4* and about 30% are C 3*, ions with lower charges are unstable at this energy. Molecular charged ions such as ~3CH- and ~2CH2 do not survive the electron stripping process and are converted to atomic species. The positive ion beam is repelled by the positive high terminal voltage and exits the accelerator with a velocity of about 6.4% c (19,300 km/s) at 5 MV. The term tandem accelerator is derived from this two-stage "pull-push" effect. The ion beam passes into a high-energy analysing magnet (E) where ~2C, ~3C and 14C are separated according to their mass momentum charge state ratio. For maximum sensitivity the analysing magnet is tuned to the most abundant ions (C 4* at 5 MV). ~2C and ~3C are relatively abundant compared to ~4C and their current can be measured with Faraday cups (F). The ~4C beam is focused by a quadrupole (G) and electrostatic cylindrical analyser (H) and the atoms are counted in a gas ionisation detector (I). The instrument measures the 14C/12C/13C ratio and thereby the amount of ~4C per mg carbon (see Section 11.4). Along with carbon ions, other atomic species can also be formed in the AMS that can be potentially interfering; these are known as isobars. Whereas molecular species such as 13CH- and 12CH~ are destroyed during ion stripping, isobars are transmitted all the


335

way through to the gas detector. The principal isobar formed alongside carbon is believed to be lithium ((7Li2)4+) although its origin is somewhat open to question [9]. Although the lithium isobar enters the gas detector, at the very high energies involved in AMS (ca 20-100 MeV) it can be separated from 14C and eliminated from the counting procedure [10]. 14N is not an interfering isobar of ~4C as negative nitrogen ions decay in about 5 • 10-~4 s and so do not reach the accelerator. Prior to the development of AMS, cyclotron accelerators were used for isotope separation. These instruments however, were based on positive ion sources and so ~4N caused major interference with 14C. In addition to carbon, a range of other isotopes can be measured using AMS. The isotope must form a stable negative ion; the instrument must operate at sufficiently high voltages for the electron stripping process to occur for that particular isotope and the energy of the resulting ions must be sufficient to separate the analyte isotope from any isobars. Instruments operating at 5 MV can be used to analyse a range of isotopes including 4~Ca, 3~'C1,3H, 57Fe and 26A1 amongst many others [reviewed in 11]. Although there are important biomedical applications involving these isotopes, this article will concentrate on the use 14C.

11.3 SAMPLE PREPARATION At the present time, there are limited ways in which samples can be introduced into the AMS ion source. Carbon can be introduced as CO2 gas [12] but more commonly samples for ~4C analysis are placed in the ion source in the form of graphite [13]. The chemistry behind sample preparation is shown schematically in Fig. 11.2. A sample of the biological material is placed in a glass tube containing copper oxide, which is sealed under vacuum and heated at 900~ for 2 hours, where the carbon in the sample is oxidised to CO2. The sample tube is connected to one arm of a Y-manifold. To the other arm is attached a glass graphitisation tube containing titanium hydride and zinc powder as reductants and cobalt as a catalyst. The sample tube is dipped into dry ice/isopropanol and the graphitisation tube into a bath of liquid nitrogen. The CO2 in the sample tube

Carbon in biological sample CuO2

9~176176 r

Cu

Oxidation

CO2 500~ withCo Catalyst

H2from Till2 + Zn

H20

Reduction

Graphite Fig. 11.2. A schematic of the graphitisation process.


Chapter 11

336

is cryogenically transferred to the graphitisation tube, which is heat-sealed under vacuum. The tube is heated at 500-550~ for about 10 hours to reduce CO2 to graphite. The graphite/cobalt mix is taken out of the tube and pressed into hollow cathodes before being placed into the AMS ion source. The minimum sample size for AMS is governed by the amount that can be physically graphitised and pressed into the sample cathodes. Practically, this is between 0.5 and 2 mg graphite. The size of the biological sample taken for analysis should therefore be sufficient to generate this amount of carbon following the graphitisation process. Blood is about 10% w/v carbon, plasma is about 4% w/v and urine about 1% w/v and so the sample size taken for analysis is adjusted accordingly. If the biological sample is not of sufficient size to produce an appropriate amount of carbon, then it can be mixed with carbon carrier (see Section 11.6). Currently there are no direct LC-AMS interfaces available. For HPLC analysis, the eluate is collected as series of fractions, the mobile phase solvent is removed and carbon carrier added to each fraction prior to oxidation and reduction to graphite. A similar process has been described for GC, whereby compounds eluted from a GC were cold trapped [14]. A direct sample interface is being developed [15]. Further direct, or semidirect, sample introduction techniques are considered a priority for the development of AMS technology (see Section 11.7).

11.4 DATA ANALYSIS

AMS instruments for ~4C analysis have traditionally been used for carbon dating and the commercial software tends to be biased to this application. In particular, one of the output values is a percentage of modem carbon (pMC). This term is somewhat confusing for the biomedical researcher and so it warrants a brief explanation. All living entities contain ~4C in equilibrium with the natural abundance in the atmosphere. A level of J4C referred to as "100% modem" (100pMC) corresponds to 1 ~4C atom per 1.18 • 10 ~2 atoms of carbon, or 97.6 attomole ~4C per mg carbon. AMS standards with precisely known pMC values are available as instrument checks and to normalise data if necessary. The two most widely used are standard oxalic acid from the U.S. National Institute of Standards Technology (NIST) and a crop of sugar harvested in Australia in the 1960s and certified by the Australian National University (ANU). The NIST oxalic acid standard has a pMC of 95 and ANU sugar has a pMC of 150.61 (the latter standard was harvested during a period where radioactive fallout from atomic weapons was still relatively high, hence the pMC value is > 100. At the time of writing, the atmosphere has a pMC of about 110). As stated in Section 11.1, the number of decay events for any radioisotope is governed by its t~/2. The longer the tl/2, the fewer decay events occur in any given time. The number of decay events per minute for a given amount of radioisotope is defined by equation (1).a dN

~ This equation is derived from the fundamental equation defining half life: - - = - K N . dt

Ultra-sensitive detection of radiolabelled drugs In 2 dpm=--xN t~/2

337 (1)

where In 2 is the natural log of 2 (0.6932), tl/2 is the half-life in minutes and N is the number of atoms of the radioisotope, calculated by multiplying the number of moles by Avrogadro's number (6.0225 x 1023). From equation (1), 1 mole ~4C, with a tl/2 of 5730 years (3.0138 x 10 9 minutes) equals 1.3852 z 10 j4 dpm. b Thus, 1 dpm equals 7.2192 z 10-~5 mole, which equals 4.3477 z 10 9 atoms. As stated above, 100 pMC is equivalent to 97.6 attomole ~C per mg carbon. When converted to units of radioactivity this is equal to 13.56 dpm per g carbon. It is important to understand that AMS provides a dpm value per g carbon based on the isotope ratio and not absolute values such as dpm per g of biological sample. This is a fundamental difference in the output of AMS compared to LSC. AMS measures numbers of atoms and not radioactive decay events. It is only to provide units familiar to biomedical researchers that the isotope ratio is converted to dpm values. To calculate dpm per g sample, the proportion of carbon in the sample must be known and this is measured using a suitable C,H,N analyser.

11.5 APPLICATIONS There are a number of published accounts where results from LSC and AMS were directly compared and correlation coefficients of 0.999 were achieved [16-18]. The upper limit for LSC is hundreds of thousands or millions of dpm, whereas the upper limit for the AMS gas detector, before it becomes saturated, is around 50 dpm, and ideally no more than 10 dpm. In order to compare LSC and AMS values therefore samples measured by LSC need to be diluted up to 1000 fold prior to AMS analysis. There are a number of reviews on the application of AMS in biomedical research [9,18-28]. The first published accounts of biomedical AMS examined the covalent binding of animal carcinogens to DNA. Prior to the advent of AMS, detecting the presence of radiolabelled adducts on macromolecules such as DNA was very challenging. Even using potent carcinogens, only a very small proportion of an administered radiolabelled compound forms adducts [29] and hence the isolated DNA has a very low specific activity. The first results using AMS to detect radioactive adducts on DNA showed that the limit of detection was in the region of 1 adduct per 10 ~ bases, an order of magnitude greater sensitivity than 32P-post labelling [8]. The macromolecular binding of heterocyclic amines, such as those formed in the cooking of protein-rich foods have been studied [reviewed in 30]. In many cases, dietary relevant doses were administered and tissues showing pathologies were analysed in rodents and humans [8,30-36]. The macromolecular binding of benzene at doses as low as 700 pg/ kg, (118 kBq/kg) was successfully investigated and showed detection levels in pg The maximum theoretical specific activity of a compound containing one J4C per molecule, is therefore 1.3852 z 10j4 dpm/mole or 2.3 GBq/mmole.

b


338

Chapter 11

benzene equivalents/g [37,38]. DNA binding of trichloroethylene [39], benzo[a]pyrene [36,40] tamoxifen and toremifene [41,42] have also similarly been investigated. In such studies, 6 adducts in 1012 nucleotides were routinely measured. The potential for (~4C)nicotine to form adducts with protein and DNA in mice at a level equivalent to a single cigarette (125 Ixg/kg bodyweight) has also been investigated [43]. For DNA adducts to be chemically characterised, isolated DNA is hydrolysed (enzymatically or by acid) and the individual nucleotides or nucleosides are separated chromatographically. Hydrolysed adducted DNA has been analysed by HPLC-AMS and adducts detected down to the fg level [31,34,39,41]. Typically, nucleotides were separated using a MVC-18 column (250 x 4.6 mm, 3 txm particle size) eluted with 0.1% (v/v) trifluoracetic acid (solvent A) and acetonitrile (solvent B) at 1 mL/min, using a gradient of 10% to 35% B over 35 min. In addition to studying adduct formation, the metabolism of the heterocyclic amines, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) have been studied in rodents and humans [33,44,45,46]. Following the administration of ~4C-PhIP at levels as low as 50 ng/kg, the distribution of PhIP and its metabolites was followed in tissues and the milk of female rats, as well as distribution of radioactivity in the tissues of the pups [47]. Although MeIQx and PhIP are potential carcinogens, because the human dose administered was as low as 300 ng/kg, a level lower than that encountered from everyday exposure to eating cooked meat, it was ethically possible to dose volunteers. Urine from PhIP-dosed mice and humans analysed by HPLC-AMS showed that there were marked differences in the metabolic profiles between species. The principal metabolite in the mouse was 4'-PhIP-sulphate whereas in the human the principal metabolite was the N-OH-PhIP-N2-glucuronide. Typically, metabolites were separated on a C18 250x4.6 mm column (3 txm particle size) eluted with 0.1% (v/v) trifluoracetic acid (solvent A) and acetonitrile (solvent B) at 1 mL/min, using a gradient of 5% to 30% B over 30 min. Knowledge of the action of potential carcinogens and other toxic chemicals have been largely based on animal models due to the ethical constraints of administering such substances to humans. With the advent of AMS however, it is possible to administer extremely small doses of compound, below the levels that would be considered to have harmful effects. It is now possible therefore, to directly compare carcinogen metabolism in humans with that of laboratory animals and challenge some of the risk assumptions made on the basis of animal models alone. In order to study the metabolism of certain endogenous compounds, it is necessary to monitor turn-over for prolonged periods of time. Prior to the advent of AMS, it was very difficult to do this in humans as the long exposure times placed severe constraints on the amounts of radioactivity that could be dosed. Using AMS however, the long term metabolism of fat was investigated by the analysis of exhaled ~4CO2 over a 320 day period after administering as little as 74 kBq ~4C-triolein precursor [48]. Folic acid metabolism has been studied in human volunteers following the ingestion of just 3.7 kBq pteroyl-[~4C(U)]-glutamic acid for up to 202 days. The limits of detection were 0.4, 0.04 and 0.12 fmol J4C-folate/mL in plasma, urine and faeces respectively [49,50]. The metabolism of ~4C-[3-carotene was studied in an adult volunteer following the


339

administration of 7.4 kBq [51]. Radioprofiling of plasma by HPLC, fraction collection and AMS allowed the kinetics of retinyl acids, retinol and [3-carotene to be followed over 202 days. Compounds were separated using an XDB-C18 column (3 x 150 mm, 3-5 ~m particle size) fitted with a guard column with the same packing. The column was eluted isocratically with acetonitrile/methanol/2-propanal with 0.1% ammonium acetate (49:21:30 v/v/v) at 1 mL/min. The dermal absorption of atrazine was studied in human volunteers [52]. Atrazine was administered in doses down to 167 ~g, 239 kBq in the form of a skin patch. Urine was analysed by HPLC-AMS after injecting approximately 10 dpm on column and showed a complex series of metabolites, six of which were tentatively identified on the basis of co-elution with standards. Metabolites were separated using a YMC 303 S-5 120A ODS-AQ column and guard column (250x 4.6 mm) eluted with 0.1% (v/v) aqueous acetic acid (solvent A) and 0.1% acetic acid (v/v) in acetonitrile (solvent B) at 1 mL/min. The gradient was 100% A to 100% B over 55 min. The absorption kinetics of trichloroethylene through human skin, measured in vitro, has also been studied using very small ~4C-labelled doses, followed by AMS analysis [53]. More recently, AMS has been applied to the study of pharmaceutical metabolism, primarily at our Centre. A pharmaceutical under development was dosed to four human volunteers using a radioactive dose of 1.85 kBq per subject [54]. Rates of excretion were investigated in urine and faeces, along with plasma pharmacokinetics. Metabolite profiles were studied by HPLC-AMS in plasma and extracts of faeces. At the Cm,x, plasma contained just 2 dpm/mL. To put the dose of 1.85 kBq per subject into context, the average human contains about 3.7 kBq naturally occurring ~4C [27]. This study was therefore conducted using essentially what was a non-radioactive dose. In another study, how the particle size of formulated Daflon 500 | effected absorption was investigated in 12 human volunteers administered just 925 Bq [55]. This radioactive dose was so low, that conventional tabletting methods were used for formulation. AMS was used to measure the ~4C-content of urine and faeces and quantitative recoveries of radioactivity were achieved. An ADME study was performed with (~4C)-GI1817771 on six male volunteers using a dose of 121 Bq per subject, equivalent to a radioactive exposure of 0.06 ~Sv [56]. Radioactivity was measured in serum, urine and faeces. Approximately 99.2% of the administered dose was recovered in faeces over the five day study period. Four healthy male volunteers were dosed with 1.85 kBq Rl15777, a farnesyl transferase inhibitor and plasma and excreta were analysed by AMS [57]. Urine was analysed by HPLC-AMS before and after treatment with [3-glucuronidase. The pharmacokinetic data are shown in Fig. 11.3 and the chromatogram for urine is shown in Fig. 11.4. Metabolites were separated using a Luna C-18 column (250x 4.6 mm, 5 ~m particle size) eluted with ammonium acetate buffer (pH 5.5)/methanol/acetonitrile (10:10:80 v/v/v). The gradient was 100% A to 40% A over 5 min., held for 15 min. then to 20% A over 10 min. The flow rate started at 1 mL/min, for 10 min. then at 0.5 mL/min. Fractions were collected every 60 seconds. The maximum level of radioactivity in the urine chromatograph was ca 3.3 dpm/fraction. One of the first human experiments conducted in the development of a new pharmaceutical is a dose escalation and tolerability study. In one such study, a small References pp. 347-349

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i- + -

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Fig. 11.3. A typical pharmacokinetics curve generated from plasma samples analysed by AMS. Note that at C . . . . Reproduced with permission [57].

the samples contain less than 2.5 dpm/mL.

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Fig. 11.4. HPLC-AMSchromatogramof urine metabolites before and after treatment with [3-glucuronidase. Reproduced with permission [57]. dose of 14C-labelled compound (1.85-18.5 kBq) was administered along with an unlabelled drug, which represented an exposure of less than 10 IxSv [58,59]. The concentration of radioactivity at the Cmax was less than 1 dpm/mL but by injecting just 0.35 dpm on the HPLC column a complex series of metabolites was revealed (Fig. 11.5). Metabolites were separated using a C18 (20 x 3.9mm 5 Ixm particle size) column eluted with 20 mM aqueous ammonium acetate, adjusted to pH 4.6 with acetic acid (solvent A) and acetonitrile (solvent B). The gradient was 95% A to 0% A over 85 min. The flow rate was 1 mL/min, and the column temperature was 40~ The studies described in [58,59] required regulatory approval from the Swiss authorities in order to administer the radioactive dose to humans. In some other countries it is feasible to administer these very low levels of radioactivity to humans without regulatory approval, thus negating the need for dosimetry studies. This opens up the possibility of combining early human experiments with the radiolabelled study thereby avoiding volunteer recruitment, specifically for this purpose, at a later stage. For certain life-saving pharmaceuticals such as cancer and HIV treatments, the first human studies are in patients. Given that there is already one account of using conventional tabletting machines to incorporate very low levels of '4C [55], this opens the possibility of conducting ADME studies with patients at this very early stage. 11.6 L I M I T S OF D E T E C T I O N AND QUANTIFICATION Since AMS counts individual atoms, it can be argued that the absolute limit of detection (LOD) for the technique is essentially zero. In order to obtain a statistically significant References pp. 347-349

Chapter I I


343

count however, about one thousand atoms are counted, which puts the statistical limit of detection in the zeptomole range (zepto = 10-2~). In practice, the LOD for AMS, as for any analytical technique, is defined by the signal to noise ratio. The "signal" depends upon the amount of J4C from isotopically enriched drug and the "noise" depends upon the amount of endogenous ~4C. Since the ratio of ~4C to ~2C is fixed in all living entities (see Section 11.4) then the amount of endogenous ~4C increases proportionally with the total amount of carbon in the sample. Some biological samples (eg faeces) contain a higher proportion of carbon than others (eg serum) and so the LOD in faeces is somewhat higher than in serum. There are a number of ways of reducing the amount of endogenous carbon in a sample, whilst not significantly lowering the contribution of ~4C from the drug. For example, proteins can be precipitated by the addition of water-miscible organic solvents such as acetonitrile. Protein is then removed by centrifugation. Although it is possible to graphitise faeces directly, the amount of endogenous ~4C can be significantly reduced, thus improving the LOD, by including a solvent extraction step in the analysis. This technique does of course, rely on quantitative extraction efficiencies. Aliquots of the solvent extracts are dried to remove the solvent and then graphitised for AMS analysis. The LOQ for a series of matrices was recently determined [56] and are shown in Table 11.1. The LOQ in this case was defined as five times the standard deviation of the background. Note that the LOQ for serum was improved nearly 100-fold following solvent extraction. Experiments undertaken by the authors, whereby plasma was spiked with decreasing amounts of radioactivity demonstrated that, as a general guide, 0.1 dpm/mL above background could be reliably quantified and 0.06 dpm/mL could be reliably detected (Fig. 11.6). Probably the most efficient method of separating endogenous carbon from that of the drug is HPLC. Under ideal conditions, the drug and its metabolites are eluted from the HPLC column in a highly pure form and endogenous carbon is removed entirely, leaving only drug related J2C and ~4C. LOD's for HPLC are therefore, in theory, extremely low. In practice, the amount of carbon contained in a HPLC fraction is too low to be handled in the graphitisation process and therefore "carrier carbon" has to be

TABLE 11.1 LOQs FOR AMS DETERMINED IN A NUMBER OF BIOLOGICALMATRICES. LOQ IS DEFINEDAS FIVE TIMES THE STANDARD DEVIATION OF THE BACKGROUND VALUE. TBT - GLYCEROL TRIBUTANOATE. LP- LIQUID PARAFFIN Sample type Faeces Urine Serum Serum extract HPLC faction using TBT HPLC faction using LP


LOQ (dpm/mL) 0.86 0.20 0.43 0.005 0.036 0.018

0.5000 0.4500 0.4000

0.3500 0.3000 J

"~ 0.2500 "0

0.2000 0.1500 0.1000 0.0500

0.0000

0

10

20

30

40

50

60

Percent dilution Fig. 11.6. A calibration line generated from the serial dilution of plasma. The LOQ in this case was 0.1 dpm (3 x SD) and the LOD was 0.06 dpm above background. (Plasma was used for dilution thus maintaining a constant carbon background for each data point). Error bars are standard error.

~


345

added to bring the quantity to a manageable amount (Section 11.3). The apparent dichotomy of first removing background carbon and then adding it back again is overcome by using ~4C-depleted carriers originating from immensely old carbon sources from the petrochemicals industry. One such commonly used carrier is glycerol tributanoate (TBT). TBT however, still contains some ~4C and where the lowest limits of detection are required substances such as liquid paraffin are used as the carrier. The LOQ for HPLC fractions shown in Table 11.1 compares those using TBT and liquid paraffin carriers. Under certain circumstances the use of carbon carrier can lead to errors. Providing the actual amount of carbon in the sample is vanishingly small, It can be assumed to be zero and only the carbon in the carrier is taken into account when calculating the dpm/mL value from the isotope ratio. This is potentially a source of error, as the sample will always make some contribution to the carbon level, albeit potentially very small. As a general rule therefore, where the carbon content is measurable by a conventional C,H,N analyser it should be taken into account. Only if it is below the sensitivity of the analyser should it be assumed to be zero, in which case the error is very minor and probably within the precision of the measurement and can be ignored. Lower LODs than those shown in Table 11.1 have been achieved using HPLC-AMS (unpublished data). As little as 0.024 dpm of a plasma extract was injected onto a HPLC. Fractions were collected, liquid paraffin added as a carbon carrier, graphitised and analysed by AMS. The resulting chromatogram can be seen in Fig. 11.7. The LOQ was estimated at 0.0008 dpm/fraction (0.0016 dpm/mL) based on 3 times the standard deviation for the background. The peak eluting at 40-41 minutes was shown to be endogenous ~4C eluting from the column and was not drug related. The chromatogram in Fig. 11.7 illustrates that when operating at such low levels of detection, endogenous carbon coincidentally eluting from the column can be misleading and control samples (ie pre-dose) should always be run alongside samples so that these spurious peaks can be eliminated. The sensitivity of AMS is both its biggest advantage and its biggest drawback as even minute amounts of contamination can result in anomalous results. If experiments are conducted in laboratories where "conventional" studies are performed, contamination is a real danger. The AMS laboratory has to have controlled entry where "high" levels of radioactivity are excluded. To ameliorate this difficulty, some companies have opted to build special facilities for conducting AMS work [56].

11.7 CONCLUSIONS AND THE FUTURE Although several groups are working on interfaces which will allow the direct or semidirect introduction of biological sample into the AMS instrument [ 12], no such interface is readily available at the time of writing. The current technique of sample preparation by graphitisation, is labour intensive. We are developing methods to permit samples to be directly inserted into the ion source thus eliminating the graphitisation process. In contrast to the sample preparation time, AMS run times per sample can be as short as 100 seconds and still give good precision. AMS is the most sensitive analytical method References pp. 347-349

C~

Background peak

0.0035 0.0030 i

c o

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21

26

31

36

41

Retention time (min) Fig. 11.7. HPLC-AMS chromatogram produced from 0.024 dpm injected on column. The peak eluting at eluting from the column.

ca

41 min. is not drug related but background carbon

r~


347

ever developed and a range of studies, particularly in humans, which were previously impossible, can now be performed. Regulatory authorities are increasingly aware of the technique and are becoming less likely to grant exemptions from performing certain studies on the grounds that it is not feasible to detect ~4C-tracer at very low levels. Up to the present time, AMS has been used largely as a problem-solving tool but there is increasing interest in using the technology strategically as part of the drug registration process. It is possible to combine radiolabelled studies in humans with other volunteer studies, thus saving time and cost. With AMS, radiolabelled studies can be performed with patients and even with children [60]. There is also great potential for acquiring data on human metabolism at a much earlier stage which can assist in compound selection or in the selection of the most appropriate species for toxicology studies. The textbook approach to radiotracer studies, particularly those involving humans has been overturned by the arrival of AMS in biomedical research. The use of AMS has the potential to eliminate the need for dosimetry studies altogether and to free human ADME work from the regulatory constraints that surround the use of radiolabelling. The extreme sensitivity of AMS allows researchers to venture into new territories, following the radiotracer into individual cell types, or binding to biomolecules and receptors. The applications for AMS in biomedical research have, in reality, only just started.

11.8 R E F E R E N C E S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

M.E L'Annunziata and M.J. Kessler, in: M.E L'Annunziata (Ed.), Handbook of Radioactivity Analysis, Academic Press, New York, 1998. International Commision on Radiological Protection in Biomedical Research. ICRP Publication 62 Ann. ICRP 22(3) (1992) 1-18. G.W. Dolphn and I.S. Eve, Health Physics, 12 (1966) 163. J.G. Dain, J.M. Collins and W.T. Robinson, Pharmaceut. Res., 11 (6) (1994) 925. C.L. Bennett, R.E Beukens, M.R. Glover, H.E. Gove, R.B. Liebert, A.E. Litherland, K. H. Purser and W.E. Sondheim, Science, 198 (1977) 508.. D.E. Nelson, R.G. Korteling and W.R. Stott, Science, 198 (1977) 507.. J. Barker, J.E Day, T.W. Aitken, T.R. Charlesworth and R.C. Cunningham, Nucl. Instr. Meth. Phys. Res., B, 52 (1990) 540. K.W. Turtletaub, J.S. Felton, B.L. Gledhill, J.S. Vogel, J.R. Southon, M.W. Caffee, R. C. Finkel, D.E. Nelson, I.D. Proctor and J.C. Davis, Proc. Natl. Acad. Sci. USA, 87 (1990) 5288. J. Barker and R.C. Garner, Rapid Commun. Mass Spec., 13 (1999) 285. U. Zoppi, M. Suter and H-A. Synal, Nucl. Instr. Meth. Phys. Res., B, 89 (1994) 262. M. Hotchkis, D. Fink, C. Tuniz and S. Vogt, Appl. Radiat. and Isotopes, 53 (2000) 31. D.A. Mucciarone and R.B. Dunbar, J. Sed. Petrol., 62 (1992) 731. J.S. Vogel, Radiocarbon, 34 (1992) 344. L.A. Currie, T.I. Eglinton, B.A. Benner Jr andA. Pearson, Nucl. Instr. Meth. Phys. Res., B, 123 (1997) 475. D.J.W. Mous, W. Fokker, R van den Broek, R. Koopmans, C.B. Ramsey and R.E.M. Hedges, Radiocarbon, 40 (1998) Abstract 35. R.C. Garner, J. Barker, C. Flavell, J.V. Garner, M. Whattam, G.C. Young, N. Cussans, S. Jezequel and D. Leong, J. Pharm. Biomed. Anal., 24 (2000) 197. S.D.Gilman, S.J. Gee and B.D.Hammock, J.S. Vogel, K. Haack, A. Buchholz, P.H.T. Freeman, R.C. Wester, X. Hui and H.I. Maibach, Anal. Chem., 70 (1998) 3463. B. Kaye, R.C. Garner, R.J. Mauthe, S.E H.T. Freeman and K.W. Turteltaub, J. Pharm. Biomed. Anal., 16 (1997) 541.

348 19 20 21 22 23 24 25 26 27 28

29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

46 47 48 49 50

Chapter 11 J.S. Vogel and K.W. Turteltaub, Nucl. Instr. Meth. Phys. Res., B, 92 (1994) 445. J.S. Vogel, K.W. Turteltaub, R. Finkel and D.E. Nelson, Anal. Chem., 67 (1995) 353A. S.E H.T. Freeman and J.S. Vogel, Int. J. Mass Spectrom. Ion Processes, 143 (1995) 247. C. Tuniz, J.R. Bird, D. Fink and G.E Herzog, Accelerator Mass Spectrometry Ultrasensitive Analysis for Global Science, CRC Press, 1998. B.C. Cupid and R.C. Garner, Accelerator Mass Spectrometry - A new tool for drug metabolism studies, in: Gooderham, N. (Ed,), Drug Metabolism: Towards the New Millennium, IOS Press (1998). R.C. Garner, European Pharmaceutical Contractor, Technomark Consulting Services November 1999. R.C. Garner and D. Leong, Nucl. Instr. Meth. Phys. Res., B, 172 (2000) 892. R.C. Garner, Curr. Drug Metab., 1 (2000) 205. K.W. Turteltaub and J.S. Vogel, Curr. Pharmaceut. Design, 6 (2000) 991. R.C. Garner, Ultrasensitive analysis of isotopes in drug discovery and development using accelerator mass spectroscopy, in: U. Pleiss and R Voges (Eds.), Synthesis and Applications of Isotopically Labelled Compounds, Vol. 7, John Wiley & Sons Ltd., 2001 W.K. Lutz, Mutation Res., 65 (1979) 289. K.W. Turteltaub, J.S. Vogel, C.E. Frantz and E. Fultz, IARC Sci. Publ., 124 (1993) 293. K.W. Turteltaub, R.J. Mauthe, K.H. Dingley, J.S. Vogel, C.E. Frantz, R.C. Garner and N. Shen, Anal. Res., 376 (1997) 243. T. Su, W. He, J. Gu, T.W. Lipinskas and X. Ding, Amer. Soc. Pharmacol. Exp. Ther., 26 (1998) 825. K.W. Turteltaub, K.H. Dingley, K.D.Curtis, M.A. Malfatti, R.J. Turesky, R.C. Garner, J.S. Felton and N.E Lang, Cancer Lett., 143 (1999) 149. R.J. Mauthe, K.H. Dingley, S.H. Leveson, S.E H.T. Freeman, R.J. Turesky, R.C. Garner and K.W. Turteltaub, Int. J. Cancer, 80 (1999) 539. K.H. Dingley, K.D. Curtis, S. Nowell, J.S. Felton, N.E Lang and K.W. Turteltaub, Cancer Epidemiol. Biomarkers Prev., 8 (1999) 507. T.J. Lightfoot, J.M. Coxhead, B.C. Cupid, S. Nicholson and R.C. Garner, Mutation Res., 4721 (2000) 119. M.R. Creek, C. Mani, J.S. Vogel and K.W. Turtletaub, Carcinogenesis, 18 (1997) 2421. C. Mani, S. Freeman, D.O. Nelson, J.S. Vogel and K.W. Turteltaub, Toxicol. Appl. Pharmacol., 159 (1999) 83. A. Kautiainen, J.S. Vogel and K.W. Turteltaub, Chem. Biol. Interactions, 106 (1997) 109. R. Goldman, B.W. Day, T.A. Carver, R.J. Mauthe, K.W. Turteltaub and EG. Shields, Chem. Biol. Interactions, 126 (2000) 171. E.A. Martin, E Carthew, I.N.H. White, R.T. Heydon, M. Gaskell, R.J. Mauthe, K.W. Turteltaub and L.L. Smith, Carcinogenesis, 18 (1997) 2209. I.N.H. White, E.A. Martin, R.J. Mauthe, J.S. Vogel, K.W. Turteltaub and L.L. Smith, Chem. Biol. Interactions, 106 (1997) 149. C. Mani and K.W. Turteltaub, Drug Met. Rev., 32 (supp. 2) (2000) abstract 129 from the 10th North American ISSX Meeting, Indianapolis, USA. K.W. Turteltaub, J.S. Vogel, C.E. Frantz and N. Shen, Cancer Res., 52 (1992) 4682. R.C. Garner, T.J. Lightfoot, B.C. Cupid, D. Russell, J.M. Coxhead, W. Kutschera, A. Priller, W. Rom, E Steier, D.J. Alexander, S.H. Leveson, K.H. Dingley, R.J. Mauthe and K.W. Turteltaub, Cancer Lett., (1999) 161. N.P. Lang, S. Nowell, M.A. Malfatti, K.S. Kulp, M.G. Knize, C. Davis, J. Massengill, S. Williams, S. MacLeod, K.H. Dingley, J.S. Felton and K.W. Turteltaub, Cancer Lett., 143 (1999) 135. R.J. Mauthe, E.G. Snyderwine, A. Ghoshal, S.EH.T. Freeman and K.W. Turteltaub, Carcinogenesis, 19 (1998) 919. K. Stenstr6m, S. Leide-Svegborn, B. Erlandsson, R. Hellborg, S. Mattsson, L-E. Nilsson, B. Nosslin, G. Skog and A. Wiebert, Appl. Radiat. Isot., 47 (1996)417. B.A. Buchholz, A. Arjomand, S.R. Dueker, ED. Schneider, A.J. Clifford and J.S. Vogel, Anal. Biochem., 269 (1999) 348. A.J. Clifford, A. Arjomand, S.R. Dueker, ED. Schneider, B.A. Buchholz and J.S. Vogel, Mathematical Modeling in Experimental Nutrition, Clifford and Mtiller (eds) Plenum Press New York 1998.

Ultra-sensitive detection of radiolabelled drugs 51 52 53 54 55 56 57 58 59 60

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S.R. Dueker, Y. Lin, B.A. Buchholz, ED. Schneider, M.W. Lame, H.J. Segall, J.S. Vogel and A.J. Clifford, J. Lipid Res., 41 (2000) 1790. B.A. Buchholz, E. Fultz, K.W. Haak, J.S. Vogel, S.D. Gilman, S.J. Gee, B.D.Hammock, X. Hui, R.C. Wester and H.I. Maibach, Anal. Chem., 71 (1999) 3519. K.T. Bogen, G.A. Keating, S. Meissner and J.S. Vogel, J. Expos. Anal. Environ. Epidemiol., 8 (1998) 253. J. Dain, J. Warsheski, R.C. Garner and V. Fischer, Drug Metabolism Reviews 32 (supp. 2) (2000) abstract 23 from the 10th North American ISSX Meeting, Indianapolis, USA. R.C. Garner, J.V. Garner, S. Gregory, M. Whattam, A. Calam and D. Leong, J. Pharm. Sci., 91 (2002) 32. G. Young, W. Ellis, J. Ayrton, E. Hussey and B. Adamkiewicz, Xenobiotica, 31 (2001) 619. R.C. Garner, I. Goris, A.A.E. Laenen, E. Vanhoutte, W. Meuldermans, S. Gregory, J.V. Garner, D. Leong, M. Whattam, A. Calam and C.A.W. Snel, Drug Metab. and Disposition 30 (2002) 823-830. E Waldmeier and C. Garner, Drug Met. Rev., 133 (supp. 1) (2001) abstract 239 from the 6th International ISSX Meeting (2000), Munich, Germany. E Waldmeier and R.C. Garner, J. Labelled Comp. Radiopharm., 44 (2001) 973. S. Leide-Svegborn, K. Stenstrom, M. Olofsson, S. Mattsson, L-E. Nilsson, B. Nosslin, K. Pau, L. Johansson, B. Erlandsson, R. Hellborg and G. Skog, Europ. J. Nucl. Med., 26 (1999) 573.


I.D. Wilson (Ed.), Bioanalytical Separations Handbook of Analytical Separations, Vol. 4 9 2003 Elsevier Science B.V. All rights reserved

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Biomedical applications of inductively coupled plasma mass spectrometry (ICP-MS) as an element specific detector for chromatographic separations Fadi R. A b o u - S h a k r a GV Instruments, Crewe Road, Manchester M23 9BE, U.K.

12.1 AN I N T R O D U C T I O N TO I C P - M S Inductively coupled plasma mass spectrometry (ICP-MS) is an instrumental analytical technique based on the use of a high temperature ionisation source (ICP) coupled to a mass spectrometer. The technique evolved from the work of Gray and Houk in the early 1980s [ 1]. Since then it has rapidly matured into the technique of choice for routine ultra trace-element analysis. This is mainly due to its advantages in terms of detection limits, relative freedom from interference and speed of analysis.

12.1.1 Inductively coupled plasma as an ion source

Inductively coupled plasmas were developed in the early 1960s [2]. They are flame-like discharges that can reach up to 10,000 K in temperature and are formed in a stream of argon, though other gases have been used, flowing through an electromagnetic field. Typically in an ICP the gas flows into three concentric tubes. These tubes are assembled together in what is commonly referred to as the plasma torch (Fig. 12.1). The flowing gas in the outer tube, which is typically in the range of 13-17 L/min, is often referred to as the cool gas and its primary role is to ensure that the high temperature of the plasma does not melt the torch. In addition to this primary function, the cool gas also plays a role in giving the plasma its distinct shape. The flowing gas in the central tube, which is usually in the range of 1 L/min, is commonly referred to as the plasma gas and is actually the gas that gets ionised to form the plasma. Finally, the gas flowing in the References pp. 370-371

352

Chapter 12

/

z

/

. / \,

/

Fig. 12.1. A schematic of the drawing of an ICP torch. innermost ring is called the carrier gas and is used to punch the plasma whilst carrying an aerosol or particulate form of the sample to be analysed. As shown in Fig. 12.1, one end of the torch is encircled by a coil. A radio-frequency current flows through the coil to create an electromagnetic field in which the argon flows. To light the plasma a high voltage discharge (spark) is passed through the argon. When passing through the electromagnetic field, electrons from the discharge gain energy. Some of these electrons transfer this energy through collisions to the argon atoms in the gas thereby ionising them. This will create sets of ion-electron pairs that are in turn energised in the presence of the electromagnetic field and are made to participate in this cascading method of energy transfer from the coil to the gas. The outcome of this cascade is the formation of a steady-state plasma that is maintained as long as the radio-frequency current is upheld at a sufficient intensity and the gas flow is preserved. The fireball-like argon plasma is characterised by a bluish white emission which is a combination of emissions from the line spectrum of atomic argon and the continuous spectrum of ion-electron recombinations taking place within the plasma. When formed, the plasma has the shape of a prolate spheroid, and the rapid expansion and acceleration of the argon gas inside it makes it difficult for sample introduction. However, by punching a central channel inside the plasma, its shape is changed into an annular "doughnut" form with the outer "plasma" gas virtually shielding the central "carrier" gas and with very little mixing taking place between the two. As a result, samples can be introduced in a gaseous or aerosol form along this central channel without significantly disturbing the plasma or changing its composition. However, during its stay in this channel, the sample will be efficiently desolvated, atomised, excited and ionised. Hence any chemical species injected into the plasma will be broken down into its constituting elements which are then ionised independently of their original form, but depending mainly on their ionisation energies as predicted by the Saha Equation (see Appendix 1). In fact, most elements in the periodic table will be efficiently converted into singly charged ions. As shown in Fig. 12.2 the efficiency of ionisation, decays as the ionisation energy of the element approaches that of argon (15.8 eV). Hence, elements such as F, Ne and He are so poorly ionised that the technique cannot be used for direct trace level determinations of these elements.

% 120.0%

...........................................................................................................................................................................................................................

r~

"..,I

100.0%

Xx x@ x

,..q

X XX

80.0%

X X

I::

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X X

60.0%

X

X

40.0%

X

_.

X X

20.0% !

X X ................ ,

0.0% 5.0

6.0

, ................. 7.0

, 8.0

...... , ......... 9.0

, ........ 10.0

X X . . . . . X ..... ~

, .......... 11.0

12.0

13.0

14.0

X

............

15.C

IE (eV)

Fig. 12.2. A representation of the efficiency of ionisation of elements in an ICP as a function of their ionisation energy. L~h

354

Chapter 12

12.1.2 Interfacing the ICP to a mass spectrometer

Mass spectrometers require a low operating pressure in order to avoid high voltage discharges and to ensure that the mean free path of the ions is long enough to avoid collisions with the background atmosphere in the system. The transfer of ions from the ICE which operates at atmospheric pressure, into a mass spectrometer was the focal point of early research into the feasibility of the technique. In most modern ICP-MS systems, the plasma is interfaced to the mass spectrometer via a series of vacuum chambers confined within extraction cones with small apertures. The design in terms of shape and aperture size of these cones plays an important role in defining the analytical performance of the system. Common to all ICP-MS systems is the presence of the socalled sampler and skimmer cones. The former is the interface between the ICP and the first vacuum chamber. This cone is subject to an intense heat as the plasma impinges on its surface and is therefore in need of efficient cooling. As a result, these cones are usually made of a material that has good resistance to heat and is a good thermal conductor. The most popular material used for the design of these cones is nickel, although dependent on the application of interest A1, Cu and Pt have been used for this purpose. As the plasma makes contact with the cooled surface of the sampler cone, it cools down rapidly forming a boundary layer between the two. In that layer, ions tend to recombine forming a plethora of molecular species, such as ArC1 + and ArO+, that are not normally observed in bench chemistry. It is crucial that the ions entering the first vacuum chamber are sampled from the central channel of the plasma and not this boundary layer. Hence the aperture of the sampler cone has to be larger than a critical diameter (usually 0.4 mm), but must not be too large as to admit gases from outside the central channel of the ICE Once inside the first vacuum chamber, the sampled gas, which at this stage consists of a mixture of ions, atoms and electrons, expands rapidly. The role of the skimmer cone is to ensure that a representative population of this mixture is extracted into the next stage. In order to achieve this, skimmer cones are designed to have sharper edges and smaller apertures than sampler cones (see Fig. 12.3).

12.1.3 The building blocks of an I C P - M S

Although the ICP and the interface arguably play the central role in ICP-MS, research investigating and characterising different areas of the technique are still being carried out. Figure 12.4 shows a schematic of the building blocks of an ICP-MS. Sample introduction is a much-researched area and often called the "Achilles heel" of ICP-MS [3]. When dealing with liquid samples, it is crucial that the sample introduction unit reproducibly delivers to the ICP a representative portion of the sample. Typically, the sample introduction unit consists of a pump, a nebuliser and a spray chamber. The pump ensures that a constant flow of the sample reaches the nebuliser and is also used to pump out any sample waste from the spray chamber. The nebuliser is used to convert the stream of liquid sample into a fine aerosol and the spray chamber acts as a trap that

Biomedical applications of inductively coupled plasma mass spectrometry

355

Load Coil u

I......... ................................. i Optics /

/

//

L

~/ i

Skimmer

/

........,

"'. Sampler

Torch

I i.i ................... ............ j

To pump Fig. 12.3. Schematic of an ICP-MS interface, the sampled region of the plasma expands behind the sampler whilst a representative portion flows through the skimmer and to the ion optics region.

filters out large droplets. Obviously, it is desirable that the sample introduction unit delivers a very large proportion of the introduced sample to the plasma. However, most nebuliser/spray chamber systems have a very poor efficiency of sample delivery (ca. 2%). This is mainly due to the wide spectrum of droplet sizes delivered by the nebuliser. However, the ICP does not tolerate the disturbances caused by large droplets. Furthermore, since the residence time of the aerosol in the plasma is independent of the aerosol droplet size, the products of small desolvated droplets will have more time to atomise and ionise than those from large droplets (because larger droplets take longer

Sample Intoduction

i ......................................................................................

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~1 Sample In.-J | Nebuliser .....................

i ~-Spray Chamber .....Waste out Turbomolecular pump

Turbomolecular pump

!

IRotary Pump I Fig. 12.4. A schematic of the building blocks of an ICP-MS.


356

Chapter 12

to desolvate in the plasma). Hence, the plasma is more efficient when handling small aerosol droplets. Therefore, the spray chamber is designed to ensure that only the finest of the aerosol droplets produced by the nebuliser ( < 10 Ixm) reach the ICE which in turn means that most of the sample is lost to waste. Improving the efficiency of the sample introduction system has been achieved through the use of efficient nebulisation techniques such as the use of ultrasonic transducers to shatter the aerosol. With proper design, these so-called ultrasonic nebulisers could provide a nearly 100% efficiency. However, it was observed that with such a high transfer rate, the solvent loading of the ICP becomes excessive and causes changes to the plasma composition/characteristics. This problem was overcome by introducing a desolvating stage prior to the aerosol reaching the plasma. Another approach to improving efficiency is the use of low sample flow rates. High efficiency low flow nebulisers have been used extensively in ICP-MS during the past few years. They enabled the coupling of sample flow rates as low as 10 txl/min. The efficiency of these nebulisers in producing an aerosol with small size droplets is extremely high, however, since the sample consumption rate is low, there is little overloading of the plasma or indeed gain in sensitivity when using these nebulisers. Apart from efficiency, another important analytical issue strongly associated with the sample introduction, is stability. In order to achieve high quality analytical data, instruments must be stable both in the short term and the long term. Short term instabilities are often associated with the pulsating nature of nebulisers. The spray chamber's secondary role is to smooth out these pulses. A typical short-term stability figure for ICP-MS is < 2% relative standard deviation (RSD) over 10 minutes. Longterm instabilities are on the other hand associated with partial blockages of the nebulisers and changes in the physical parameters affecting the performance of the spray chamber namely, temperature and surface wetting. Nebuliser blockages can be overcome by the careful choice of the type of nebuliser suitable for the application. For example, when dealing with clean samples or solvents, standard concentric nebulisers are often the right choice since it is possible to match these nebulisers to the required flow rate. With samples or solvents containing high levels of salts, more salt tolerant nebulisers are recommended. These nebulisers are widely commercially available and the most popular of them are the cross flow nebulisers and the Burgener nebulisers. Finally when dealing with high levels of suspended particulates, V-groove type nebulisers offer the ability to handle these samples or solvents but often at the expense of a 30-50% reduction in sensitivity. Thermally isolating the spray chamber and maintaining it at a constant temperature has now become a standard practice in order to avoid temperature associated long-term drift. This can be achieved either by using a thermoelectric "Peltier" cooling device to control the temperature or by "jacketing" the spray chamber with a continuous flow of water or a cooling fluid flowing from a chiller/recirculator unit. In addition to minimising drift, this approach can be used to selectively reduce the plasma loading from volatile solvents such as alcohol by maintaining the spray chamber temperature between -5~ and - 10~ The choice of material for the manufacture of spray chambers plays an important role in ensuring their compatibility with various solvents and the stability of their


357

performance. Most common spray chambers are made of quartz. However, inert spray chambers made of perfluoroalkoxy polymers (PFA) or polypropylene are also widely used. As described in Sections 12.1.1 and 12.1.2, the aerosol passing through the spray chamber, is ionised in the ICP and introduced into the vacuum through the interface. After the interface, the ions are guided through a set of ion lenses into the mass spectrometer region. These traditionally electrostatic lenses are designed to steer the ions without changing the composition of the ions beam exiting the skimmer. The magnitude of this undertaking is often underestimated. The difficulty arises from the fact that, as the positive ions are focused, electrons exit the ion beam rendering it rich in positively charged ions that tend to repel each other (columbic effects). Different approaches have been adopted with some success by different commercial ICP-MS manufacturers and they vary from accelerating the ion beam [4] to reducing the ion current by off axis extraction of the ions. Most of these approaches however, involve the use of an increased number of lenses. Although this can complicate the issue of instrument tuning, automated optimisation is often offered since these lenses are all computer controlled. A secondary role of the ion optics is to prevent neutral species and photons from reaching the detectors, since this will cause an elevated background and, as such, poorer detection limits. Different configurations that address this issue are currently in use. They include the use of either photon stops or off axis lenses. More recently, reaction/collision cells have been used as ion guides in ICP-MS. They offer more tolerance for the ions energy spread and interactions than electrostatic lenses. Furthermore, these cells offer the potential of improving detection limits by reducing interferences from other species. The origins of these interferences will be discussed in Section 12.1.4. The most popular form of mass filters used in ICP-MS are quadrupoles. They offer speed of analysis, robustness, stability and ease of use. Other mass filters used include magnetic sector, time-of-flight and ion trap. Each of these mass filters offer advantages and disadvantages over quadrupole systems. Magnetic sector ICP-MS systems offer the potential of resolving interferences from overlapping peaks. However, they tend to be larger and more expensive that quadrupole systems. Time of flight (TOF) ICP-MS offer the potential of detecting whole spectra instantaneously, which could be useful when handling short transient bursts of signals (e.g. ablation of inclusion fluids in rocks). However, the current TOF-ICP-MS systems tend to have lower sensitivity than quadrupole ICP-MS and are yet to be widely accepted in the market [5]. Ion traps are the most recent addition to the ICP-MS family [6]. They offer the capabilities of isolating, storing and, if required, conducting ion-molecule reactions on the ions of interest. The greatest advantage of ion trap ICP-MS is that high efficiency can be obtained from continuous weak beams, through the use of long accumulation times. This advantage is however, not very beneficial when dealing with short transient signals. The main disadvantage of ion trap ICP-MS is that the trap has a limited storage capacity (ca. 106 ions). This is a major limitation for ICP-MS since large beams of Ar +, O +, and other molecular argon ions are often encountered. One means of overcoming this limitation is the use of larger ion traps and the rapid elimination of the abundant ions References pp. 370-371

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either by applying a resonant frequency to eject them or by reacting them out with an appropriate reaction gas.

12.1.4 Analytical capabilities of ICP-MS There are a number of analytical features that distinguishes ICP-MS from other analytical techniques. It is a very sensitive instrumental technique for elemental determination with detection limits that compare favourably with, and in most cases exceed those of, other leading techniques for ultra trace element analysis namely graphite furnace atomic absorption (GF-AAS) and neutron activation analysis (NAA). However, unlike GF-AAS, ICP-MS is a multi-element technique that can handle a continuous flow of samples, and unlike NAA, ICP-MS is rapid with total determinations taking only a few minutes whilst in some cases NAA analysis can take months. In terms of speed of analysis, ICP-MS has a similar sample throughput as other rapid analytical techniques such as atomic absorption spectroscopy (AAS) or inductively coupled plasma optical emission spectroscopy (ICP-OES), but outdoes both techniques by nearly three orders of magnitude on detection limits. Typical detection limits for ICP-MS are in the low to sub ng/L range. This applies for most of the elements in the periodic table with the exception of halogens where the high ionisation energy of these elements makes them difficult to ionise in the ICP. Other analytical characteristics to consider when evaluating the technique are interferences and robustness. Interferences can be generally classified as spectroscopic and non-spectroscopic interferences. Spectroscopic interferences are due to overlapping signals. These overlaps can be either due to peaks that share the same nominal mass or due to tailing from adjacent peaks. Peaks that share the same nominal mass with the ion of interest can be due to either elemental ions or to ionised molecular species. An example of elemental ion interferences is the overlap of 5aFe+ and 58Ni+ and an example of molecular ion interferences is the overlap of 56Fe+ and 4~ Whilst the origin of the interfering elemental ions is in most cases elements in the sample that are ionised in the plasma, molecular ions are usually due to recombinations taking place in the interface region (since as described earlier the plasma cools down rapidly in that region) and involving the major components of the sample such as O, N, C combining either with each other or with Ar from the plasma gas. Identifying the presences of these interferences is normally straightforward since these interferences tend to have their own isotopic patterns which, when present, distort the isotopic pattern of the element of interest. Overcoming these interferences can be achieved either by using an isotope which is free of interference (e.g. in the case of the iron interference on 58Ni+, 6~ can be used) or by adopting a mathematical correction that takes into account the isotopic distribution of the interfering species to correct for its contribution (e.g. in the case of the 5~Fe+ interference on 58Ni+, the contribution from 58Fe+ can be calculated as the Signal for 56Fe+ multiplied by the ratio of the abundances of 5SFe/56Fe). In extreme cases, where there is no appropriate alternative isotope to use and where the magnitude of interference is so much larger than the signal of interest such that the uncertainty from the interference correction can lead to erratic results, high resolution ICP-MS


359

(HR-ICP-MS) can be used to physically separate the two peaks. Typically the separation power of an HR-ICP-MS system is reported as the ratio of the mass of the ion of interest over the mass difference between the two ions, and its working range is from 400 up to 10,000. The higher the resolution figure the better the separation. However, this is done at the expense of sensitivity. For example in the case of 58Fe+ interference on 58Ni+, the mass difference between the two ions is 5 7 . 9 3 5 3 4 7 9 - 57.9332805 = 0.002067 and the required resolution to separate them is 57.9353479/0.002067 = 28,023, which is outside the range of a HR-ICP-MS. Whereas the resolution required to separate 56Fe+ and 4~ is 55.9349421/0.022356 = 2502, which is well within the capabilities of the system. A more recent approach for addressing these spectroscopic interferences is the use of collision/reaction cell instruments where one of the interfering species is reacted out [7,8]. For example, with a reaction cell instrument, the addition of hydrogen in the cell will react out the ArO § ions to form ArO neutrals and H +, H 3+ species which do not interfere with 56Fe+. Tailing from adjacent peaks is dependent on the abundance sensitivity of the system. Most ICPMS instruments have an abundance sensitivity of < 10-6 on the low mass side and < 10-7 on the high mass side. In other words, if the Signal of 56Fe+ is 100,000,000 counts per second (cps), there will be < 100 cps contribution from this signal of at mass 55 and < 10 cps at mass 57. This problem is not often a major issue with ICP-MS. However, if needed, either H R - I C P - M S or collision/reaction cell ICP-MS can be used to overcome it. Non-spectroscopic interferences are usually manifested in the form of a response change for the element of interest as a result of influence from the sample matrix or some of its components. Typical forms of non-spectroscopic interferences observed in ICP-MS are: (a) reduction in sensitivity as a result of partial blockages of the nebuliser or the cones; (b) changes in sensitivity due to changes in nebulisation characteristics as the matrix/solvent changes; (c) Signal suppression from high levels of easily ionisable elements in the sample (e.g. > 1000 ppm Na) which can increase the electron density in the central channel of the plasma, thereby lowering the ionisation efficiency (see Appendix 1); and (d) Selective loss of sensitivity for light elements in the presence of high levels of heavy elements (e.g. > 100 ppm U) due to columbic repulsion in the interface and the early elements of the ion optics. The magnitude of these effects does vary with different designs but in general, careful selection of the nebulisation system and cone apertures will minimise them dramatically. Nebuliser blockages can be eliminated by selecting more tolerant nebulisers whist the effect of the solvent change is often non observable [9].

12.2 ICP-MS AS AN ELEMENT SPECIFIC DETECTOR FOR CHROMATOGRAPHIC SEPARATIONS 12.2.1 Coupling an HPLC to ICP-MS Coupling HLPC to ICP-MS is relatively simple to achieve. The flow rates from HPLC systems, which can vary between 1 ixl/min up to 2-3 ml/min, are directly compatible References pp. 370-371

360

Chapter 12

with the sample introduction flow rates in ICP-MS. As such HPLC-ICP-MS especially ion chromatography (IC) ICP-MS was used as a tool for identifying elemental species since as far back as the late 1980s [10]. However, it rapidly became obvious after the initial euphoria that HPLC-ICP-MS as an integrated technique cannot be effected by simply linking the outlet of the LC to the inlet of the ICE Considerations related mainly to the mobile phases in use had to be taken seriously. The high levels of salts used in most IC mobile phases caused blockages at the nebuliser tip and cone apertures. Furthermore, mobile phases consisting of a high percentage of an organic solvent (such as those used mainly in reversed phase HPLC) cause rapid blockages of the cones due to the deposition of carbon. In order to address the issue of salt, on line dilution as well as exchange membranes, whereby Na, K and Ca ions are exchanged with hydrogen ions, were used. With regard to handling organic solvents, the problem arises from the fact that carbon has a relatively high ionisation energy (11.3 eV), which as a result leads to a lower ionisation efficiency in the plasma. Hence with high levels of organic solvents there will be a large population of carbon atoms in the plasma. These atoms tend to deposit on the apertures of the sampler and skimmer cones, thereby blocking them. In order to address this problem, the levels of organic solvents reaching the plasma have to be reduced. This is achieved by using low flow rates (< 200 txl/min) and desolvating the aerosol generated by the nebuliser prior to it reaching the plasma through cooling the spray chamber to sub-zero temperatures (typically -5~ In addition, oxygen is bled through the plasma in order to assist in burning the excess carbon. However, this process can change the electrical impedance of the ICE Therefore, unless the RF circuitry of the instrument is designed to cope with this impedance shift, the plasma will extinguish. Purpose made desolvating nebulisers are currently commercially available and are often used to couple HPLC to ICP-MS. With these nebulisers, the generated aerosol, is first heated, then in one case partially desolvated through passing the hot mixture of gas and aerosol into a thermally controlled chamber kept at sub-zero temperatures, and finally desolvated through passing the mixture in a heated semi porous membrane with a counter flow of Ar on the outside. Although, these nebulisers provide an ideal way for linking HPLC to ICP-MS, volatile species can be lost through the membrane.

12.2.2 Coupling GC to ICP-MS GC-ICP-MS has potentially superior detection power to HPLC-ICP-MS. This is mainly because, GC has a superior resolution, and the low gas flows from the GC can be directly injected into the plasma thereby providing a nearly 100% efficiency in terms of the transport of the analytes. In their paper in 1986, Van Loon et al., were the first to report on the coupling of a GC to ICP-MS for the purpose of detecting organotin compounds [11]. This was quickly followed by a number of papers by various groups. With the advance of capillary GC, Kim et al. reported on the use of a heated fused silica capillary transfer line to affect the GC-ICP-MS coupling [12]. Although commercial GC-ICP-MS transfer lines are currently available, most researchers developed their own in-house systems [ 13,14]. The designs are basically simple with a deactivated but uncoated fused


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silica running inside a heated tube that extends between the GC and the tip of injector in the plasma torch. Heated argon is made to flow inside the heated transfer line around the fused silica. This makes up the flow required to punch the plasma and also ensure uniform heating of the fused silica. Some of the limitations observed with current GC-ICP-MS transfer lines were discussed recently by Glindemann et al. [15]. They related to signal repeatability, a potential carbon deposit from high organic solvents, and the limitations of current designs to handle high boiling analytes. The authors proposed a newer interface, based on rapid dilution of the effluent gases with hot argon and solvent peak elimination by periodic flow reversal in the transfer line. Using this interface, the authors could separate C26 n-paraffin (412~ at only 140~ temperature in the transfer line (Fig. 12.5).

12.2.3 Coupling CE to ICPMS Olesik et al. first reported on the coupling of CE to ICP-MS in 1995 [ 16]. It was clear from their work that the interface must, in addition to providing a stable electrical current across the capillary, prevent the introduction of a laminar flow through the capillary. The issue arises from the special nebulisers needed to handle the low flows of CE. These nebulisers tend to be self-aspirating. This will create a suction effect that causes a laminar flow through the capillary and compromises the efficiency of the electrophoretic separation.


362

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Most CE-ICP-MS publications to-date focus on the design of the CE interface. The most popular interface is based on a micro-concentric nebulizer, and uses a coaxial electrolyte sheath flow (make-up) to establish the electrical connection to the analyte. Recently Cetac technologies (Omaha, USA) released a commercial CE interface that employs a micro-concentric nebuliser and with the nebuliser uptake rates minimised to allow for minimal dilution (make up flow rate of < 10 ~l/min) without affecting the separation. In addition to these interfaces, direct injection nebulisers (DIN) have also been investigated as a possible CE-ICP-MS interfacing tool [17]. Although these nebulisers can offer nearly 100% efficiency in terms of sample delivery, their performance is offset by their high cost. A more affordable version of these nebulisers called the direct injection high efficiency nebuliser (DIHEN), has recently been made commercially available (J.E. Meinhard Associates, Inc., Santa Ana, CA, USA). These nebulisers connect directly to the back of the plasma and are capable of handling extremely low flows (< 10 pA/min) whilst maintaining a very efficient sample delivery rate. Majidi et al. reported that using this type of nebuliser reduced peak broadening and resulted in sharp symmetrical peaks and improved signal to noise performance [18]. Similarly, Bendahl el al [19] used DIHEN to separate Se species by CE-ICP-MS and reported detection limits in the range of 25-125 fg (0.3-1.5 fmol) of selenium.

12.3 APPLICATIONS OF ICP-MS IN THE BIOMEDICAL FIELD 12.3.1 Detection of metabolites

The full characterization of the chemical, pharmacological and toxicological properties of any biologically active substance, including its metabolites, is a prerequisite that most current regulatory authorities demand. Hence, the detection and identification of impurities or metabolites structurally related to these substances are of the utmost importance. ICP-MS as an element specific detector can play a major role in this process. Traditionally, the determination of the metabolic fate of drugs and other xenobiotics in experimental animals and man required the radiochemical labelling of compounds. In the absence of a suitable label, the detection of metabolites can be a difficult exercise, especially if they are significantly different in structure from the parent. Furthermore, even when the detection and identification of metabolites is possible using traditional MS and UV detectors, quantification is more challenging as the MS and UV response of the metabolites may significantly differ from that of the parent. Since a large number of these biologically active materials contain heteroatoms, the specificity of ICP-MS for detection of those atoms, coupled with its detection power leads to much simpler chromatograms and rapid identification of the retention times of the compounds of interest. Knowing these retention times, it is possible then to use molecular mass spectrometry such as electrospray ionization mass spectrometry (ESIMS) in order to identify them. Furthermore, the sensitivity of ICP-MS is element specific but compound independent therefore, by knowing the concentration of one


363

compound in the detected chromatogram, it is possible to accurately calculate the concentration of the rest of the detected species. This information will be invaluable for "mass balance" studies where the amount of starting material is compared to the total amount of detected impurities, degradation products or metabolites found together with the remaining starting material. In the case of substances bereft of heteroatoms, tags can be used to provide these atoms. ICP-MS can then be used to detect and quantify the complexes containing these tags. A number of papers have recently reported the use of ICP-MS for tracing the metabolic fate of xenobiotics and drugs. In a short communication, Nicholson et al. investigated the fate of 4-bromoaniline in the urine of rats [20]. Their results highlighted the potential of the technique as a simple, rapid and specific method for the detection of metabolites. This paper was rapidly followed be a series of investigations from the group profiling Dichlofenac (using C1 and Sulfur detection) [21] and 2-bromo4-trifluoromethylaniline [22,23]. In that later work, parallel analysis of the chromatographic eluent was carried out using ICP-MS, ESI-MS (with a time of flight mass analyser) and diode array detection. This combination proved to be a powerful tool for immediate identification and quantification of the metabolites (see Fig. 12.6). A similar approach was adopted by the group of Marshall et al. in their investigation of bradykinin metabolism in human and rat plasma [24,25]. Bradykinin is a bioactive peptide produced from the actions of kininogenases on kininogens and exerts its role by activating B2 receptors. However, in order to detect bradykinin by ICP-MS, the peptide had to be labelled with an appropriate heteroatom. The group adopted what they called a "labelled ligand" approach in which bromine was used as a tag. The structures of bradykinin and its tagged product are shown in Fig. 12.7. Another interesting work on metabolite profiling by HPLC-ICP-MS was carried out by Duckett et al. [26]. The

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364

Chapter 12

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authors investigated the profile produced by the earthworm Eisenia veneta following exposure to 2-fluoro-4-iodoaniline. The ICP-MS profile was produced by tracing the 127I signal, and the authors used a post column injection of a known concentration of iodine to work out the amount of metabolite found in the various parts of the worm as a percentage of the initial dose of the drug. Hywel Evans et al. [27], used ICP-MS to detect impurities in Cimetidine based upon monitoring the presence of sulphur in the eluents following chromatographic separation of a solution containing the drug. Their data indicated the presence of compounds that were not detected in earlier studies using ESI-MS. The higher detection power of the ICP-MS is also highlighted in a recent work by Smith et al. [28]. In this work, blood samples from animals treated with a platinum-based anticancer drug were analysed for the presence of the drug and its biologically active compounds. This study provided a direct comparison between ICP-MS and triple quadrupole atmospheric pressure ionisation mass spectrometry (API-MS-MS). Although API-MS-MS is acknowledged as the standard technique for conducting such investigations, the result of the study showed that ICP-MS did out-perform it in terms of quantification limits (0.1 ng/mL for HPLC-ICP-MS compared to 5 ng/mL for the HPLC-MSMS method) and linear dynamic range (over five orders of magnitude for HPLC-ICP-MS compared to typically three orders of magnitude for the HPLC-MSMS method. Axelsson et al. [29] reported on the potential of the technique for general organic compounds related to the pharmaceutical industry. The paper discussed a variety of compounds including iodine containing X-ray contrast substances and gadolinium metal complexes. Metal complexes can be used as tags for specific antibodies that can then target antigens of interest and make them detectable using ICP-MS. In addition, the paper discussed the detection of phospholipids using phosphorus as the target element. Furthermore, the paper addressed the issue of phosphorylation which is again a major potential application field of ICP-MS.

12.3.2 Phosphorylation detection by ICP-MS Phosphorylation is a key process in the regulation of protein activity and has long been appreciated as an essential mechanism for the control of cellular function [30]. Phosphorylation reactions are associated with numerous biological processes and as such they attract at lot of interest from biomedical researchers. This research does not just aim at identifying the presence of phosphorylation but also at pinpointing the exact position of the phosphorylated amino acid. Typical analytical techniques used for this purpose involve the digestion of proteins followed by HPLC-MS-MS and the tracing of

365


the indicative PO3 ion at mass 79 that the phosphorylated peptides yield under collisionally induced dissociation. Alternatively, proteins are separated using gel electrophoresis, and this step is followed either by the analysis of the gel using MALDIMS or by excising specific spots and analyzing them using HPLC-MS-MS. However, with both techniques, the ionization efficiency is compound-dependent, resulting, for example, in reduced ionization efficiency for phosphopeptides compared to unmodified peptides. As a result, information about the phosphorylation status of a protein using these methods is only qualitative and large phosphopeptides with poor fragmentation characteristics (mol wt > 2500) may escape their recognition as such. Other techniques involve the incorporation and subsequent detection of radioactive phosphorus (e.g. 32p, 33p) or the use of anti-phosphopeptide antibodies, but again each technique does suffer from specific limitations. Axelsson et al. [29] used HPLC-ICP-MS to distinguish between mono-phosphorylated and di-phosphorylated peptides. Their results showed that the technique was highly selective since no non-phosphorylated peptides were detected whilst it was easy to identify mono- and di-phosphorylated peptides that were at similar concentrations. Furthermore, a tryptic digest of [3-caesin was analysed using the technique and the resulting chromatogram as compared to UV detection is shown in Fig. 12.8. The detection capability for the HPLC-ICP-MS method in this study was however limited 100-

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366

Chapter 12

due to background interferences on mass 31 from NOH and NO species, that resulted in a relatively elevated baseline. The use of high resolution ICP-MS which can provide a mass spectrometric separation between phosphorus and the above mentioned ions, as well as the use of reaction cell-based ICP-MS, which offers the potential to selectively react some of the ions extracted from the ICP have led to an improvement in the detection power of the technique. Wind et al. [31 ] used capillary LC coupled to high resolution ICP-MS to detected low levels of P in a mixture of synthetic phosphopeptides, a tryptic digest of activated MAP kinase and protein kinase A catalytic subunit. The data using this technique compared well with LC-ESI-MS. The same author then published work using both phosphorus and sulfur detection for accurately determining the degree of phosphorylation for cysteine- and methionine-containing proteins and peptides [32]. Marshall et al. [33] used a laser ablation ICP-MS to investigate phosphorylation. In this system shots from a UV-laser (213 nm) were used to ablate fine spots (ca. 120 ~m in diameters) from the surface of gels and gel blots and the ablated material was swept directly into the ICP-MS. The authors reported some success with the gel blots being able to easily measure low pico-molar levels of [3-caesin. However, with electrophoresis gels, the authors reported the presence of a very high baseline that affected the analytical performance of the technique

12.3.3 Other applications ICP-MS has also been coupled with chromatographic separation to evaluate the biological effects of some metal/semi metal elements that enter the human body through either the food chain, environmental activities or a combination of both. One element that attracted such an attention is arsenic. In nature, arsenic is often found in sulphidic ores in the form of metal oxides. However, the commercial use of arsenic compounds as arsenical pesticides, herbicides and crop desiccants and the use of arsenic as an additive to livestock feed, particularly for poultry has raised its environmental levels and exposed humans to relatively higher levels of this element. HPLC-ICP-MS has been used to investigate the fate of arsenic in humans through determining the distribution of arsenic species in human/animal urine [34], and hair [35]. The technique has also been applied for the analysis of water [36] and seafood products [37]. More recently arsenic trioxide, a well-known carcinogen, has been used for the treatment of leukemia in humans [38]. This report agrees fully with data from our laboratory where we observed only very small amounts of dimethylarsinic acid (DMA) in the urine of control subjects from china, whereas the urine of patients from the same region treated for leukemia showed very high levels of DMA as well as monomethylarsonic acid (MMA) and inorganic arsenic(V) ions (Fig. 12.9). Furthermore, in order to highlight the role of diet, we show in Fig. 12.9 a chromatogram of the urine from a control subject on a rich seafood diet. The chromatogram shows extremely high levels of arsenobetaine, which is rich in various types of fish. Other forms of arsenic that could be taken through diet are arsenosugars and arsenocholine, which exists at very high levels in seaweeds [39].

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368

Chapter 12

reported on the speciation of Se in urine and quote detection limits in the range of 30-80 pg/ml (1.5-4 pg on column) [41 ]. These values are an order of magnitude lower than the best previously reported data of 50-218 pg by Lafuente et al. [42]. More recently, Gammelgaard et al. [43], used a traditional ICP-MS coupled to a low flow ion pairing chromatographic system and managed to obtain detection limits of 800-1700pg/ml. However, since with this chromatographic system the injection volume is only 3 txl as compared to 50 txl by Marchante-Gayon, the absolute detection limits (pg on column) of the two methods are comparable. Therefore, one could safely assume that using this ion pairing method coupled to a collision/reaction cell instrument, the detection limits of Se will be pushed even lower making the potential of detecting low femtomolar concentrations of these compounds a real possibility. Toxic organic species of tin and mercury have also been analysed by ICP-MS using GC separations. Organotin compounds, are introduced into the environment through their use as antifouling paints on ships and also as wood preservatives, fungicides, biocides, and polymer additives. Detection of these species was reported in the very first GC-ICP-MS paper by Van Loon et al. [11]. More recently, Wahlen [44] published a comparison between GC-ICP-MS and HPLC-ICP-MS for the analysis of tin species. The paper concluded that whilst GC-ICP-MS is a more sensitive technique (3 pg of tributyltin detection limits by HPLC-ICP-MS compared to 0.03 pg by GC-ICP-MS), complex sample preparation procedures meant that HPLC-ICP-MS is a cheaper and much easier technique to use for the analysis of large sample batches. Human exposure to mercury is mainly due to the accumulation of methyl mercury in fish. In humans, methyl mercury exhibits neurotoxic effects, hence there is an interest in monitoring its levels especially at source, that is in fish. A number of scientists have used GC-ICP-MS for this purpose with typical detection limits reported to be in the region 0.02-0.08 pg [45,46]. Finally, mixed mode HPLC has been coupled to ICP-MS in order to identify some of the protein-bound trace elements in human serum [47]. The separation allowed proteins/large size molecules to elute in the first 3 minutes whilst small molecules such as free ions or amino acids eluted at times > 3.5 min. Based on the data shown in Fig. 12.10, the authors concluded that Cu, Zn, and Fe were bound to proteins such as albumin ceruloplasmin and transferrin. Se, Mg, Ca, Sr, Ba and I, on the other hand could be found in both proteins and small molecules, whilst Mo, Br, and C1 were only observed as free ions or bound to small molecules. This technique could be a valuable tool as a rapid screening method for the distribution of trace elements in biological fluids.

12.4 SUMMARY Inductively coupled plasma mass spectrometry is a powerful analytical technique that is recently finding popularity as an element specific detector for chromatographic separations. This has opened the door for novel research in the biomedical field investigating the metabolic fate of drugs, phosphorylation reactions, the role of essential trace elements in human health, and the mechanisms in which toxic elements affect the

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Separation Methods in Drug Synthesis and Purification (Handbook of Analytical Separations)

Principles of Chemical Separations with Environmental Applications

Membrane Separations Technology: Principles and Applications

Chiral Separations by Capillary Electrophoresis (Chromatographic Science)

Separations and reactions in organic supramolecular chemistry

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Handbook of Analytical Techniques, 2 Volume Set

Chiral Separations by Liquid Chromatography (Acs Symposium Series)

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Chiral Separations by Liquid Chromatography: Theory and Applications

Handbook of Stable Isotope Analytical Techniques, Volume I (Handbook of Stable Isotope Analytical Techniques)

Chiral Separations by Liquid Chromatography and Related Technologies

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Bioanalytical Separations, Volume 4 (Handbook of Analytical Separations)

Protein Separations' Handbook Collection

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Centrifugal Separations in Biotechnology

Membrane Separations in Biotechnology

Chiral Separations Methods and Protocols

Chiral Separations: Methods and Protocols

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Chiral Separations Liquid Chromatography

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