anabolic-androgenic steroids in horses

267 Pages·2015·5.66 MB·English

A -A S
NABOLIC NDROGENIC TEROIDS IN
H : N P
ORSES ATURAL RESENCE AND
U
NDERLYING BIOMECHANISMS

Anneleen Decloedt

Dissertation submitted in the fulfilment of the requirements
for the degree of Doctor of philosophy (PhD) in Veterinary Sciences,
Faculty of Veterinary Medicine, Ghent University
PROMOTER
Prof. dr. ir. Lynn Vanhaecke
Ghent University, Faculty of Veterinary Medicine
Department of Veterinary Public Health and Food Safety
Laboratory of Chemical Analysis

MEMBERS OF THE READING COMMITTEE
Prof. dr. James Scarth
HFL Sport Science, Cambridgeshire, United-Kingdom
Prof. dr. Peter Van Eenoo
Ghent University, DoCoLab, Zwijnaarde, Belgium
Prof. dr. Ann Van Soom
Ghent University, Faculty of Veterinary Medicine, Merelbeke, Belgium

MEMBERS OF THE EXAMINATION COMMITTEE
Dr. Ludovic Bailly-Chouriberry
Laboratoires des Courses Hippiques, Verrières-le-Buisson, France
Dr. Leen Van Ginkel
Wageningen University, RIKILT, Wageningen, The Netherlands
Prof. dr. Myriam Hesta
Ghent University, Faculty of Veterinary Medicine, Merelbeke, Belgium

This work was funded by the Fédération Nationale des Courses Françaises (via the Laboratoire
des Courses Hippiques) and executed at the Laboratory of Chemical Analysis (Faculty of
Veterinary Medicine, Ghent University, Merelbeke). The author and the promoter give the
authorisation to consult and to copy parts of this work for personal use only. Every other use is
subject to the copyright laws. Permission to reproduce any material contained in this work should
be obtained from the author.
“The universe is full of magic,
Just patiently waiting for our wits to grow sharper”

TABLE OF CONTENTS
T C
ABLE OF ONTENTS
Chapter I – General Introduction 1
1. Steroids 3
1.1 Chemical structure
1.2 (Steroid) hormones and their role in the endocrine system
1.3 Biosynthesis of steroid hormones
1.4 Anabolic-androgenic steroids (AAS)
1.5 Synthesis and absorption of the steroid precursor cholesterol
2. Anabolic steroid abuse 12
2.1 Scientific history of anabolic steroid abuse
2.2 Residue-analysis in farm animals: regulations
2.3 Doping regulations: humans and horses
2.4 Sex steroids as registered drugs
3. Endogenous steroids 17
3.1 Reference ranges for endogenous steroids in humans
3.2 Reference ranges for endogenous steroids in horses
3.3 Hormonal disorders in horses
4. Phytosterols 22
4.1 Biological role and chemical structure
4.2 Link to anabolic steroids
5. Equine digestive tract 26
5.1 Anatomical overview
5.2 Diet
6. Analytical instrumentation 29
6.1 History
6.2 Phase I and II metabolites
6.3 UHPLC-MS/MS
6.4 GC-C-IRMS
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7. Aims 36
Chapter II – Development and validation of a UHPLC-MS/MS method to
quantify low levels of anabolic-androgenic steroids naturally present in urine
of untreated horses 47
1. Abstract 49
2. Introduction 50
3. Material and methods 53
3.1 Chemicals and reagents
3.2 Sample collection and extraction
3.3 Hydrolysis of the glucuro- and sulfo-conjugated compounds
3.4 Solid phase extraction, washing, and solvolysis
3.5 UHPLC-MS/MS analysis
3.6 Standard addition approach and data analysis
3.7 Quality assurance: validation of the analytical method
4. Results and discussion 59
4.1 Validation study
4.1.1 Specificity
4.1.2 Selectivity
4.1.3 Linearity
4.1.4 Precision
4.1.5 Trueness
4.1.6 Stability
4.1.7 Limit of detection and quantification
4.2 Urine screening of untreated horses
5. Conclusion 69
5.1 Executive summary
Chapter III – Optimisation of phytosterol extraction and analysis from
feed by D-Optimal design and UHPLC-MS/MS 75
1. Abstract 77
2. Introduction 78
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3. Material and methods 81
3.1 LC-MS2 reagents and chemicals
3.2 Feed and food samples
3.3 Extraction protocol optimisation
3.4 UHPLC-Triple quadrupole MS/MS analysis
3.5 Data analysis and quality assurance of the analytical method:
validation according to EU Council Decision 2002/657 and
AOAC guidelines
3.6 Quantitative analysis of different feed and food samples
4. Results and discussion 86
4.1 Extraction protocol optimisation
4.2 Data analysis and quality assurance of the analytical method: validation
according to EU Council Decision 2002/657 and AOAC guidelines
4.2.1 Specificity
4.2.2 Selectivity
4.2.3 Calibration curves
4.2.4 Trueness: recovery
4.2.5 Precision
4.2.6 Limit of detection and quantification
4.3 Quantitative analysis of different food and feed samples
5. Conclusion 99
5.1 Highlights
Chapter IV – In vitro simulation of the equine hindgut as a tool to study
the influence of phytosterol consumption on the excretion of anabolic-
androgenic steroids 103
1. Abstract 105
2. Introduction 106
3. Material and methods 109
3.1 LC-MS2 reagents and chemicals
3.2 In vitro digestion: batch incubations
3.2.1 Buffers and broths
3.2.2 Fecal inoculum
3.2.3 Digestion protocol and sampling
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3.2.4 Validation of the in vitro digestion protocol
3.2.5 In vitro digestion supplements
3.2.6 Bacterial strains
3.3 Extraction of in vitro digestion and urine samples
3.3.1 Hydrolysis
3.3.2 Solid phase extraction (SPE), liquid-liquid extraction and solvolysis
3.3.3 Pooling and reconstitution
3.4 UHPLC-triple quadrupole MS/MS analysis
3.5 Quantification: data analysis and quality assurance of the analytical method
4. Results 118
4.1 Quantification: data analysis and quality assurance of the analytical method
4.2 In vitro digestion: proof of principle
4.3 Blank in vitro digestion
4.4 In vitro digestions with added steroid precursors
4.5 In vitro digestions with added phytosterols or herbal supplements
5. Discussion 129
6. Conclusion 133
Chapter V – Moldy feed, a possible explanation for the excretion of
anabolic-androgenic steroids in horses? 141
1. Abstract 143
2. Introduction 144
3. Material and methods 147
3.1 LC-MS/MS reagents and chemicals
3.2 Buffers, broths and feed samples
3.3 Aerobic incubation of moldy feed
3.4 Aerobic incubation of a commercial grain mix with Mycobacterium sp.
3.5 Activity of extracellular enzymes excreted by corn-related molds
3.6 In vitro digestion of moldy feed
3.7 UHPLC-Triple Quadrupole MS/MS analysis
3.8 Data analysis, quantitative and qualitative assurance of the analytical method
4. Results 155
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4.1 Aerobic incubation of moldy feed
4.2 Aerobic incubation of a commercial grain mix with Mycobacterium sp.
4.3 Activity of extracellular enzymes excreted by corn-related molds
4.4 In vitro digestion of moldy feed
5. Discussion 159
6. Conclusion 164
Chapter VI – Influence of glucocorticoid treatment on the excretion of
anabolic-androgenic steroids: in vitro simulations and in vivo case study
171
1. Abstract 173
2. Introduction 174
3. Material and methods 176
3.1 In vivo experimental set-up
3.2 In vitro simulation of the equine hindgut: biotransformation of glucocorticoids
3.3 LC-MS reagents and chemicals
3.4 Extraction of urine and in vitro digestion samples
3.4.1 Hydrolysis
3.4.2 Solid Phase Extraction (SPE)
3.4.3 Pooling and reconstitution
3.5 Analytical methods (UHPLC-MS/MS and UHPLC-Orbitrap-HRMS)
3.6 Quantification
4. Results 182
4.1 Urinary profile after intra-articular administration of betamethasone
4.2 In vitro digestion: biotransformation of glucocorticoids
5. Discussion 184
Chapter VII – General Discussion and Future Perspectives 191
1. Positioning of the research 193
1.1 Economical relevance of horse sports and racing
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TABLE OF CONTENTS
1.2 Anabolic steroid abuse: back to the future?
1.3 Endogenous AAS: complicating doping analysis
1.4 Analytical methods to detect (endogenous) steroid abuse
2. Scientific contributions and research findings 198
2.1 Development and validation of a UHPLC-MS/MS method to quantify low
levels of AAS naturally present in urine of untreated horses
2.2 Extraction optimisation, development and validation of a UHPLC-MS/MS
method for the detection and quantification of phytosterols in feed
2.3 Validation of an in vitro digestion simulation of the equine hindgut as a tool to
study the influence of phytosterol consumption on the excretion of anabolic-
androgenic steroids in horses
2.4 Moldy feed, a possible explanation for the detection of anabolic-androgenic
steroids in horses?
2.5 Influence of glucorticoid treatment on the excretion of anabolic-androgenic
steroids in equine urine
3. Future perspectives 211
3.1 Endogenous steroid formation
3.2 Dietary contamination and transformation
3.3 Phytosterol-enriched feed consumption: in vivo
3.4 Mashes and silage
3.5 Influence of glucocorticoid treatment on the excretion of AAS
3.6 Equine Biological Passport
Summary 225
Samenvatting 231
CV 237
Dankwoord 247

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