Testing for Prohibited Substances in Rural Agricultural Show Exhibits: A Twenty-Three Year Experience.

The administration of prohibited substances has been used in agricultural show competitions and animal racing industries to gain unfair competitive advantages. We report the first large prospectively designed descriptive study of drug testing in four species (n = 1,598) over a 23 year period. 4.7% of tested exhibits returned positive results. Commonly detected substances included legitimate veterinary therapeutics such as the sedative acepromazine and the non-steroidal anti-inflammatory phenylbutazone. Targeted testing was more likely to return a positive result than random screening (50 vs 4.7% respectively) although numbers in this targeted sample were small (n = 12). Random drug testing programs were successful in detecting the minority of exhibits using prohibited substances although a wide variety of drugs were found to be used. Further vigilance and research is required in an ever-changing competitive climate to remain at the forefront of detecting new medications in animal show competitions.

In vivo metabolism of the designer anabolic steroid hemapolin in the thoroughbred horse.

Hemapolin (2α,3α-epithio-17α-methyl-5α-androstan-17ß-ol) is a designer steroid that is an ingredient in several "dietary" and "nutritional" supplements available online. As an unusual chemical modification to the steroid A-ring could allow this compound to pass through antidoping screens undetected, the metabolism of hemapolin was investigated by an in vivo equine drug administration study coupled with GC-MS analysis. Following administration of synthetically prepared hemapolin to a thoroughbred horse, madol (17α-methyl-5α-androst-2-en-17ß-ol), reduced and dihydroxylated madol (17α-methyl-5α-androstane-2ß,3α,17ß-triol), and the isomeric enone metabolites 17ß-hydroxy-17α-methyl-5α-androst-3-en-2-one and 17ß-hydroxy-17α-methyl-5α-androst-2-en-4-one, were detected and confirmed in equine urine extracts by comparison with a library of synthetically derived reference materials. A number of additional madol derivatives derived from hydroxylation, dihydroxylation, and trihydroxylation were also detected but not fully identified by this approach. A yeast cell-based androgen receptor bioassay of available reference materials showed that hemapolin and many of the metabolites identified by this study were potent activators of the equine androgen receptor. This study reveals the metabolites resulting from the equine administration of the androgen hemapolin that can be incorporated into routine GC-MS antidoping screening and confirmation protocols to detect the illicit use of this agent in equine sports.

Monitoring dehydroepiandrosterone (DHEA) in the urine of Thoroughbred geldings for doping control purposes.

The use of testosterone and its pro-drugs, such as dehydroepiandrosterone (DHEA), is currently regulated in horseracing by the application of international testosterone thresholds. However, additional steroidomic approaches, such as steroid ratios, to distinguish overall adrenal stimulation from drug administrations and an equine biological passport for longitudinal steroid profiling of individual animals could be advantageous in equine doping testing. Thus, DHEA concentrations and related ratios (testosterone [T] to DHEA and DHEA to epitestosterone [E]) were assessed in the reference population by quantitative analysis of 200 post-race gelding urine samples using liquid chromatography-tandem mass spectrometry. DHEA concentrations ranged between 0.9 and 136.6 ng/mL (mean 12.8 ng/mL), T:DHEA ratios between 0.06 and 1.85 (mean 0.43), and DHEA:E ratios between 0.21 and 13.56 (mean 2.20). Based on the reference population statistical upper limits of 5.4 for T:DHEA ratio and 48.1 for DHEA:E ratio are proposed with a risk of 1 in 10 000 for a normal outlier exceeding the value. Analysis of post-administration urine samples collected following administrations of DHEA, Equi-Bolic® (a mix of DHEA and pregnenolone) and testosterone propionate to geldings showed that the upper limit for T:DHEA ratio was exceeded following testosterone propionate administration and DHEA:E ratio following DHEA administrations and thus these ratios could be used as additional biomarkers when determining the cause of an atypical testosterone concentration. Additionally, DHEA concentrations and ratios can be used as a starting point to establish reference ranges for an equine biological passport.

Application of testosterone to epitestosterone ratio to horse urine - a complementary approach to detect the administrations of testosterone and its pro-drugs in Thoroughbred geldings.

Detection of testosterone and/or its pro-drugs in the gelding is currently regulated by the application of an international threshold for urinary testosterone of 20 ng/mL. The use of steroid ratios may provide a useful supplementary approach to aid in differentiating between the administration of these steroids and unusual physiological conditions that may result in atypically high testosterone concentrations. In the current study, an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed to quantify testosterone (T) and epitestosterone (E). The method was used to analyze 200 post-race urine samples from geldings in order to generate the ratios for the reference population. Following statistical analysis of the data, an upper limit of 5 for T:E ratio in geldings is proposed. Samples collected from 15 geldings with atypical urinary testosterone concentrations (>15 ng/mL) but otherwise normal steroid profile, had T:E ratios within those observed for the reference population. The applicability of an upper T:E ratio to detect an administration was demonstrated by the analysis of a selection of incurred samples from testosterone propionate, dehydroepiandrosterone (DHEA), and a mixture of DHEA and pregnenolone (Equi-Bolic®) administrations. These produced testosterone concentrations above the threshold of 20 ng/mL, but also T:E ratios above the proposed limit of 5. In conclusion, consideration of the T:E ratio appears to be a valuable complementary aid to evaluate whether an atypical testosterone concentration could be caused by a natural biological outlier as opposed to the administration of these steroids. Copyright © 2016 John Wiley & Sons, Ltd.

In vivo and in vitro metabolism of the designer anabolic steroid furazadrol in thoroughbred racehorses.

Furazadrol ([1',2']isoxazolo[4',5':2,3]-5α-androstan-17ß-ol) is a designer anabolic androgenic steroid that is readily available via the internet. It contains an isoxazole fused to the steroid A-ring which offers metabolic stability and noteworthy anabolic activity raising concerns over the potential for abuse of this compound in equine sports. The metabolism of furazadrol was studied by in vivo and in vitro methods for the first time. Urinary furazadrol 17-sulfate and furazadrol 17-glucuronide metabolites were detected in vivo after a controlled administration and compared with synthetically-derived reference materials in order to confirm their identities. They were quantified to establish the excretion profile and a suitable limit of detection. Minor metabolites were also detected, including epifurazadrol, hydroxylated furazadrol, and hydroxylated and oxidised furazadrol, present as the sulfate and glucuronide conjugates. Phase II metabolites were subjected to enzymatic hydrolysis by Escherichia coli ß-glucuronidase and Pseudomonas aeruginosa arylsulfatase to further confirm the identity of the corresponding phase I metabolites. The metabolism profile was compared to the products obtained from an in vitro phase I metabolism study, with all but two of the minor in vivo phase I metabolites observed in the in vitro system. These investigations identify the key urinary metabolites of furazadrol following oral administration, which can be incorporated into anti-doping screening and confirmation procedures.

A stereochemical examination of the equine metabolism of 17alpha-methyltestosterone.

An investigation was conducted into the stereochemistry of the equine urinary metabolites of 17alpha-methyltestosterone observed after oral administration. Standards of the complete range of C3/C5/C16 stereoisomeric 17alpha-methylandrostane-3,17beta-diols, 17alpha-methylandrostane-3,16,17beta-triols and 17alpha-hydroxymethylandrostane-3,17beta-diols were purchased or synthesised, and were used to unequivocally identify the absolute structures of the metabolites. Phase I metabolism was found to involve combinations of Delta(4)-3-ketone reduction with both 5alpha,3beta- and 5beta,3alpha-stereochemistry, hydroxylation at C16 with both 16alpha- and 16beta-stereochemistry and hydroxylation of the 17alpha-methyl substituent. Phase II metabolism involved mainly sulfation with a lesser degree of beta-glucuronidation.

Detection of 17alpha-hydroxyprogesterone caproate in equine plasma by gas chromatography/tandem mass spectrometry.

A method was developed for the analysis of the synthetic progestin 17alpha-hydroxyprogesterone caproate in equine plasma following its administration by intramuscular injection. The method employed a reversed-phase solid-phase extraction followed by enol-trimethylsilylation and analysis by gas chromatography/tandem mass spectrometry. The intact ester was detectable in the plasma for up to 2 weeks after a single therapeutic dose, and was found to be stable in equine whole blood for at least 2 months.

Analysis of anabolic steroids in the horse: development of a generic ELISA for the screening of 17alpha-alkyl anabolic steroid metabolites.

Due to the potential for misuse of a wide range of anabolic steroids in horse racing, a screening test to detect multiple compounds, via a common class of metabolites, would be a valuable forensic tool. An enzyme-linked immunosorbent assay (ELISA) has been developed to detect 17alpha-alkyl anabolic steroid metabolites in equine urine. 16beta-Hydroxymestanolone (16beta,17beta-dihydroxy-17alpha-methyl-5alpha-androstan-3-one) was synthesised in six steps from commercially available epiandrosterone (3beta-hydroxy-5alpha-androstan-17-one). Polyclonal antibodies were raised in sheep, employing mestanolone (17beta-hydroxy-17alpha-methyl-5alpha-androstan-3-one) or 16beta-hydroxymestanolone conjugated to human serum albumin, via a 3-carboxymethyloxime linker, as antigens. Antibody cross-reactivities were determined by assessing the ability of a library of 54 representative steroids to competitively bind the antibodies. Antibodies raised against 16beta-hydroxymestanolone showed excellent cross-reactivities for all of the 16beta,17beta-dihydroxy-17alpha-methyl steroids analysed and an ELISA has been developed to detect these steroid metabolites. Using this 16beta-hydroxymestanolone assay, urine samples from horses administered with stanozolol (17alpha-methyl-pyrazolo[4',3':2,3]-5alpha-androstan-17beta-ol), were analysed raw, following beta-glucuronidase hydrolysis, and following solid-phase extraction (SPE) procedures. The suppressed absorbances observed were consistent with detection of the metabolite 16beta-hydroxystanozolol. Positive screening results were confirmed by comparison with standard LCMS analyses. Antibodies raised against mestanolone were also used to develop an ELISA and this was used to detect metabolites retaining the parent D-ring structure following methandriol (17alpha-methylandrost-5-ene-3beta,17beta-diol) administration. The ELISA methods developed have application as primary screening tools for detection of new and known anabolic steroid metabolites.

The detection of modafinil and its major metabolite in equine urine by liquid chromatography/mass spectrometry.

A method has been developed for the detection of modafinil and its major metabolite, modafinil acid, in equine urine by solid-phase extraction and positive ion electrospray ionisation liquid chromatography/mass spectrometry. The method has been applied to the analysis of equine urine samples obtained after the oral administration of modafinil. Modafinil acid was the major component in the urine, and was detected up to 4 days post-administration. Unchanged modafinil was present at substantially lower concentrations, and was detected for only 24 hours.

Direct detection of boldenone sulfate and glucuronide conjugates in horse urine by ion trap liquid chromatography-mass spectrometry.

A study of the equine phase II metabolism of the anabolic agent boldenone is reported. Boldenone sulfate, boldenone glucuronide and their C17-epimers were synthesised as reference standards in our lab and a method was developed for their detection in a horse urine matrix. Solid phase extraction was used to purify the analytes, which were then detected by ion trap LC/MS. Negative and positive ionisation mode MS(2) were used for the detection of sulfate and glucuronide conjugates, respectively. Boldenone sulfate and 17-epiboldenone glucuronide were detected as the major and minor phase II metabolites, respectively, in horse urine samples collected following the administration of boldenone undecylenate by intramuscular injection.

Detection of stanozolol and its metabolites in equine urine by liquid chromatography-electrospray ionization ion trap mass spectrometry.

The equine phase I and phase II metabolism of the synthetic anabolic steroid stanozolol was investigated following its administration by intramuscular injection to a thoroughbred gelding. The major phase I biotransformations were hydroxylation at C16 and one other site, while phase II metabolism in the form of sulfate and beta-glucuronide conjugation was extensive. An analytical procedure was developed for the detection of stanozolol and its metabolites in equine urine using solid phase extraction, acid solvolysis of phase II conjugates and analysis by positive ion electrospray ionization ion trap LC-MS.

The detection of piroxicam, tenoxicam and their metabolites in equine urine by electrospray ionisation ion trap mass spectrometry.

An investigation has been conducted into the metabolism and urinary excretion of orally administered piroxicam and tenoxicam in the horse. The major component detected in urine after the administration of piroxicam was 5'-hydroxypiroxicam, which was detectable up to 24 h post-administration. Unchanged piroxicam was present only as a minor component. In contrast, unchanged tenoxicam was the major component observed after the administration of tenoxicam, being detectable for 72 h post-administration, while 5'-hydroxytenoxicam was a minor component. Phase II beta-glucuronide conjugation in each case was found to be negligible. The ion trap mass spectral characteristics of piroxicam, tenoxicam, 5'-hydroxypiroxicam and 5'-hydroxytenoxicam under electrospray ionisation conditions were examined in some detail.