J. Scarth FACULTEIT DIERGENEESKUNDE approved by EAEVE APPROACHESTO THE DETECTION OF STEROID ABUSE IN VETERINARY SPECIES APPROACHES TO THE DETECTION OF STEROID ABUSE IN VETERINARY SPECIES 2011 James Scarth Kaft.indd 1 28/02/2011 14:10:58 APPROACHES TO THE DETECTION OF STEROID ABUSE IN VETERINARY SPECIES James P. Scarth (Ghent University, Belgium and HFL Sport Science, UK) Thesis for submission in fulfilment of the requirements for the degree of Doctor (Ph.D) in Veterinary Sciences Promoter: Prof. Dr. H. De Brabander (Ghent University, Belgium) Co-promoter: Dr. J. Kay (University of Strathclyde, UK) Co-promoter: Dr. L. Vanhaecke (Ghent University, Belgium) Rector: Prof. Dr. P. Van Cauwenberge (Ghent University, Belgium) 1 of 331 TABLE OF CONTENTS ABBREVIATIONS 5 INTRODUCTION 7 PART 1: DETECTION OF ‘ENDOGENOUS’ STEROID ABUSE IN FOOD-PRODUCTION 27 Chapter 1: Presence, metabolism and detection of ‘endogenous’ steroid hormones in food producing animals 29 Chapter 2: Validation and application of an analytical biomarker approach for the detection of nandrolone abuse in the porcine 108 Chapter 3: Validation of analytical biomarker approaches for the detection of androgen, oestrogen and progestagen abuse in the bovine 133 PART 2: DETECTION OF ‘DESIGNER’ STEROID ABUSE IN ANIMAL SPORTS 163 Chapter 4: Steroid metabolism and detection in the equine 165 Chapter 5: Assessment of the applicability of in vitro technologies to study drug metabolism in the equine 197 Chapter 6: Metabolism of the ‘designer’ steroid estra-4,9-diene- 3,17-dione in the equine and comparison to human and canine 247 CHAPTER 7: GENERAL DISCUSSION 281 SUMMARY 315 SAMENVATTING 321 CURRICULUM VITAE 327 ACKNOWLEDGEMENTS 331 3 of 331 Abbreviations AAS anabolic-androgenic steroid. API atmospheric pressure ionization. BHA British Horseracing Authority. C18 steroid a steroid based on the estrane nucleus. C19 steroid a steroid based on the androstane nucleus. C21 steroid a steroid based on the pregnane nucleus. C24 steroid a steroid based on the cholane nucleus. C27 steroid a steroid based on the cholestane nucleus. CAD collision activated dissociation. CCα decision limit. CCβ detection capability. CRL European Community Reference Laboratory. CYP cytochrome P450. DHEA dehydroepiandrosterone. DMF dimethylformamide. DNA deoxyribonucleic acid. EI electron ionisation. EIA enzyme immunoassay. ELISA enzyme linked immunosorbent assay EPI enhanced product ion scan. ERC endogenous reference compound. EU European Union. FDA Food and Drug Administration. FEI Federation Equestre Internationale. FWHM full width at half maximum height. GC-C-IRMS gas chromatography combustion isotope ratio mass spectrometry. GC-MS gas chromatography-mass spectrometry. GC-MS/MS gas chromatography-tandem mass spectrometry. GBGB Greyhound Board of Great Britain. HCD higher-energy collision decomposition. HCl hydrochloric acid. HPLC high performance liquid chromatography. HR-LC-MS high resolution-liquid chromatography-mass spectrometry. IA Immunoassay. ICRAV International Conference of Racing Analysts and Veterinarians. IFHA International Federation of Horseracing Authorities. ILAC International Laboratory Accreditation Cooperation. IM intramuscular. 5 of 331 IUPAC International Union of Pure and Applied Chemistry. IV intravenous. LC-MS liquid chromatography-mass spectrometry. LC-MS/MS liquid chromatography-tandem mass spectrometry. LLOQ lower limit of quantification. LOD limit of detection. LOQ limit of quantification. LTQ linear trap quadrupole. MeOH methanol. MO methoxyamine. MO-TMS methoxyamine-trimethylsilyl. MRPL minimum required performance limit. MSTFA – N-Methyl-n (trimethylsilyl)-trifluoroacetamide. MTBSTFA – N (t-butyldimethylsilyl)-N-methyltrifluoroacetamide. m/z mass to charge ratio NAD+ nicotinamide adenine dinucleotide. NADPH nicotinamide adenine dinucleotide phosphate. NaOH sodium hydroxide. NMR nuclear magnetic resonance. ND not detected. NMP national monitoring programme. PTV programmable temperature vaporiser. QC quality control. qRT-PCR quantitative real time reverse transcriptase polymerases chain reaction technology RIA radio-immunoassay RSD relative standard deviation (also known as coefficient of variation). SCVPH Scientific Committee on Veterinary Measures relating to Public Health. SRM selected reaction monitoring. SPR surface plasmon resonance. THG tetrahydrogestrinone. TLC-FL thin layer chromatography-fluorescence. TBDMS tertiary-butyl,dimethyl-silyl TMS trimethylsilyl. TOF time of flight. u atomic mass unit ULOQ upper limit of quantification. UV ultraviolet. WADA World Anti-Doping Agency. 6 of 331 Introduction INTRODUCTION Steroid structure and pharmacology If one were to ask a member of the public what images the word ‘steroid’ conjured into their imagination, the use of anabolic-androgenic steroids (AASs) in athletics or bodybuilding would no doubt rank near the top of the list. However, steroids have a range of structures and pharmacological actions that reach far beyond the anabolic effects of AASs. The term ‘steroid’ itself refers to any compound possessing the basic perhydrocyclopentanophenanthrene nucleus (Figure 1) (Makin, 1995). A) B) Figure 1 – A) the perhydrocyclopentanophenanthrene nucleus, on which all steroids are based and B) cholesterol as an example. Each carbon is assigned a number and the four hydrocarbon rings are numbered A-D, as shown. The nomenclature of this class of compounds is complex and large arrays of different systems are used. These include; the official International Union of Pure and Applied Chemistry (IUPAC) recommended systematic nomenclature (IUPAC, 2010), a range of ‘trivial’ or ‘common’ names and those of some proprietary preparations. Additionally, many organisations use their own nomenclature (for example the company Steraloids). However, these often deviate from the IUPAC recommendations. The choice of how to 7 of 331 Introduction best name a steroid in a particular situation is, therefore, dependent on a number of factors. If one were to always use only the IUPAC systematic name, then this could make the text difficult to read for a non-expert. However, inappropriate over-use of trivial names does not always give enough information in order to inform the reader. Therefore, a combination of systematic and trivial names is often employed as a pragmatic compromise (such as described by Makin et al. 1995) and this will be used in the current text. As an example of the different ways of naming a steroid, some options for testosterone are given below: Trivial name: testosterone. IUPAC systematic name: 17β-hydroxy-androst-4-en-3-one. Proprietary example (containing testosterone esters): Sustanon. When depicted in the orientation shown in Figure 1, substituents on the steroid backbone may protrude below or above the plane of the paper and are drawn as such using either a dashed or solid wedge respectively (indicating the stereochemistry α and β respectively). Hydrogens in positions 8, 9, 10, 13 and 14 (when present) take β, α, β, β, and α orientation respectively in all steroids discussed in this manuscript so their stereochemistry will not be shown in any of the subsequent diagrams. A substituent in position 5 may take either the α or β form, so hydrogens in this position will always be labelled. A wavy line indicates that stereochemistry is unspecified. In order to aid in the systematic naming of steroids, a number of different hydrocarbon backbones are specified for use by IUPAC. These differ in the number and orientation of carbons, which range from the 17-carbon (C17) gonane nucleus to the 27-carbon (C27) cholestane nucleus (on which cholesterol is based). The range of steroid backbones used in systematic nomenclature is shown in Figure 2. In this text, when describing the trivial name for the oestrogens, the English version will be used (as opposed to the USA use of estrogens). However, when systematically naming steroids that are based on the estrane nucleus, the ‘o’ will not be used (in accordance with IUPAC guidelines). No endogenous and very few exogenous steroids are based on the gonane nucleus. The oestrogens and nandrolone (17β-hydroxy-estr-4-en-3-one) are based on the estrane nucleus. The majority of androgens are based on the androstane nucleus and the majority of progestagens and corticosteroids are based on the pregnane nucleus. Most 8 of 331 Introduction of the bile acids are based on the cholane nucleus and sterols such as cholesterol are based on the cholestane nucleus. Gonane (C17) Estrane (C18) Androstane (C19) Pregnane (C21) Cholane (C24) Cholestane (C27) Figure 2 – the range of hydrocarbon backbones used in steroid nomenclature. 9 of 331 Introduction While many steroids are known to be endogenous (discussed further in chapter 1), a wide range of exogenous steroid structures have been synthesized by chemists over the years in order to optimise their biological properties. Pharmacologically, steroids possess a range of activities far more diverse than their seemingly similar structures may suggest. The following discussion considers the major effects of different steroid classes in mammals. There are some subtle differences between various species, but these will not be considered here since it is only a general overview. Cholesterol (cholest-5-en-3β-ol – Figure 1) is derived from dietary intake, but is also synthesized in the body. Cholesterol acts to regulate the fluidity of cell membranes and is the precursor to the endogenous androgens, oestrogens, progestagens, corticosteroids, vitamin D, the bile acids and, in certain