Mass Spectrometry in Sports Drug Testing: Characterization of Prohibited Substances and Doping Control Analytical Assays (Wiley - Interscience Series on Mass Spectrometry) [1 ed.] 0470413271, 9780470413272, 0470626623, 9780470626627

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MASS SPECTROMETRY IN SPORTS DRUG TESTING Characterization of Prohibited Substances and Doping Control Analytical Assays

Mario Thevis German Sport University Cologne, Center for Preventive Doping Research— Institute of Biochemistry, Cologne, Germany

A JOHN WILEY & SONS, INC., PUBLICATION

MASS SPECTROMETRY IN SPORTS DRUG TESTING

WILEY-INTERSCIENCE SERIES IN MASS SPECTROMETRY Series Editors Dominic M. Desiderio Departments of Neurology and Biochemistry University of Tennessee Health Science Center Nico M. M. Nibbering Vrije Universiteit Amsterdam, The Netherlands A complete list of the titles in this series appears at the end of this volume.

MASS SPECTROMETRY IN SPORTS DRUG TESTING Characterization of Prohibited Substances and Doping Control Analytical Assays

Mario Thevis German Sport University Cologne, Center for Preventive Doping Research— Institute of Biochemistry, Cologne, Germany

A JOHN WILEY & SONS, INC., PUBLICATION

Copyright © 2010 by John Wiley & Sons, Inc. All rights reserved Published by John Wiley & Sons, Inc., Hoboken, New Jersey Published simultaneously in Canada No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4470, or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) 748-6011, fax (201) 748-6008, or online at http://www.wiley.com/go/permission. Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. For general information on our other products and services or for technical support, please contact our Customer Care Department within the United States at (800) 762-2974, outside the United States at (317) 572-3993 or fax (317) 572-4002. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic formats. For more information about Wiley products, visit our web site at www.wiley.com. Library of Congress Cataloging-in-Publication Data: Thevis, Mario, 1973– Mass spectrometry in sports drug testing : characterization of prohibited substances and doping control analytical assays / Mario Thevis. p. ; cm. Includes bibliographical references and index. ISBN 978-0-470-41327-2 (cloth) 1. Doping in sports–Diagnosis. 2. Mass spectrometry. 3. Drugs–Analysis. I. Title. [DNLM: 1. Doping in Sports. 2. Spectrum Analysis–methods. 3. Substance Abuse Detection–methods. QT 261 T418m 2010] RC1230.T54 2010 362.29–dc22 2009052128 Printed in Singapore 10 9 8 7 6 5 4 3 2 1

For Jan and Linus

CONTENTS

Preface Acknowledgments 1

History of Sports Drug Testing

xi xiii 1

1.1 Historical Attempts of Artificial Performance Enhancement 1 1.2 Background and Rationale of Doping Controls 2 1.2.1 Cheating 3 1.2.2 Health Issues 3 1.2.3 Ethical Issues 3 1.3 Early Detection Methods: Possibilities and Limitations of Assays Without Mass Spectrometry 4 1.3.1 First Applications Using Chemical and Biological Approaches in Horse Doping Control 4 1.3.2 First Applications Using Chemical Approaches in Human Doping Control 8 1.4 Introduction of Mass Spectrometry to Doping Control Analysis 15 1.4.1 First Approaches and Adverse Analytical Findings 16 1.4.2 Progression of Analytical Methods 21 References 24 2

Mass Spectrometry and the List of Prohibited Substances and Methods of Doping

44

2.1 Criteria for the Mass Spectrometric Identification of Prohibited Compounds 44 2.1.1 Low Molecular Weight Analytes 45 2.1.2 High Molecular Weight Analytes 46 vii

viii

CONTENTS

2.2 Modern Mass Spectrometers in Doping Controls: Advantages and Disadvantages of Available Techniques 48 2.2.1 Ionization Techniques in Routine Doping Controls 48 2.2.2 Mass Analyzers in Routine Doping Controls 53 References 60 3

Structure Characterization of Low Molecular Weight Target Analytes—Electron Ionization

70

3.1 Stimulants 70 3.2 Narcotics 81 3.3 Anabolic Androgenic Steroids 87 3.3.1 Unsaturated 3-Keto-Steroids 88 3.3.2 α,β-Saturated Ketosteroids 92 3.3.3 3-Keto-1,4-Diene, 3-Keto-4,6-Diene, and 3-Keto-4,9(11)-Diene Steroids 95 3.3.4 Steroid Derivatization 95 3.4 Selective Androgen Receptor Modulators (SARMs) 99 3.4.1 2-Quinolinone-Based SARMs 100 3.4.2 Hydroxybicyclic Hydantoin-Derived SARMs 100 3.5 Diuretics 104 3.5.1 Thiazide-Derived Drugs 104 3.5.2 Benzoic Acid-Derived Loop Diuretics 106 3.5.3 Potassium-Sparing Diuretics 106 3.6 β2-Agonists 110 3.7 β-Receptor Blocking Agents 114 3.8 Calcium-Channel Modulators (RYCALS) 118 3.9 Carbohydrate-Based Agents 122 3.9.1 Mannitol 122 3.9.2 Glycerol 125 3.9.3 Hydroxyethyl Starch and Dextran 127 References 130 4

Structure Characterization of Low Molecular Weight Target Analytes: Electrospray Ionization 4.1 Stimulants 149 4.2 Narcotics 155

148

CONTENTS

ix

4.3 Anabolic Androgenic Steroids 159 4.3.1 α,β-Saturated 3-Keto-Steroids 159 4.3.2 3-Keto-4-ene and 3-Keto-1-ene Steroids 164 4.3.3 3-Keto-1,4-diene Steroids 165 4.3.4 3-Keto-4,6-diene Steroids 167 4.3.5 3-Keto-4,9-diene Steroids 167 4.3.6 3-Keto-4,9,11-triene Steroids 167 4.3.7 Stanozolol 168 4.4 Selective Androgen Receptor Modulators (SARMs) 171 4.4.1 Arylpropionamide-Derived SARMs 173 4.4.2 Hydroxybicyclic Hydantoin-Derived SARMs 174 4.4.3 2-Quinolinone-Derived SARMs 178 4.4.4 Tetrahydroquinoline-Derived SARMs 178 4.5 Diuretics 181 4.5.1 Thiazide-Derived Drugs 181 4.5.2 Benzoic Acid-Derived Loop Diuretics 182 4.5.3 Potassium-Sparing Diuretics 186 4.6 β2-Agonists 187 4.7 Calcium-Channel Modulators (RYCALS) 191 4.8 Peroxisome-Proliferator Activated Receptor-δ (PPARδ) And Adenosine Monophosphate Activated Protein Kinase (AMPK) Agonists 193 4.9 Hypoxia-Inducible Factor (HIF)-Stabilizers And Sirtuin Activators 198 4.9.1 HIF-Stabilizers 198 4.9.2 Sirtuin Activators 202 4.10 β-Receptor Blocking Agents 207 4.11 Glucuronic Acid and Sulfate Conjugates of Target Analytes 212 References 214 5

Structure Characterization of High Molecular Weight Target Analytes: Electrospray Ionization 5.1 5.2 5.3 5.4 5.5 5.6 5.7

Human Chorionic Gonadotrophin (hCG) 225 Erythropoietins (EPO) 227 Synacthen 230 Insulins 233 Hemoglobin-Based Oxygen Carriers (HBOCs) 237 Human Growth Hormone (hGH) 242 Sermorelin (Geref) 249

225

x

CONTENTS

5.8 Insulin-Like Growth Factor-1 (IGF-1) 251 5.9 Gonadorelin (LHRH) 255 References 256 6

Modern Mass Spectrometry-Based Analytical Assays

275

6.1 GC-MS and Isotope Ratio Mass Spectrometry 276 6.1.1 Stimulants/Narcotics 276 6.1.2 Anabolic Androgenic Steroids 285 6.1.3 Diuretics 303 6.1.4 β2-Agonists and β-Receptor Blocking Agents 306 6.1.5 Carbohydrate-Based Agents 307 6.2 LC-MS/MS 310 6.2.1 Stimulants/narcotics/β2-Agonists/β-Receptor Blocking Agents 311 6.2.2 Anabolic Androgenic Steroids/Corticosteroids/ Hormone Antagonists and Modulators 313 6.2.3 Masking Agents (Including Diuretics) 315 6.2.4 Peptide Hormones 50% 25% to 50%