Novel holistic pharmacokinetic model applied to plasma and urine concentrations of 2,5-dihydroxybenzene sulphonate following administrations of calcium dobesilate and etamsylate to exercised horses.

Calcium dobesilate (CD) is a synthetic venoactive drug used in veterinary medicine to treat equine navicular disease. Etamsylate is a haemostatic agent used in horses for the treatment of exercise-induced pulmonary haemorrhage. Both etamsylate and CD dissociate in the circulatory system with 2,5-HBSA as the active drug. The aim of the research was to be able to provide detection time (DT) advice from pharmacokinetic (PK) studies in Thoroughbred horses to better inform trainers, and their veterinary surgeons, prescribing these substances for treatment of Thoroughbred racehorses. Two (pilot study) and six (final study) horses were given 28 and 9 repeated dose of CD (3 mg/kg BID) respectively. Two horses were each given a single intravenous (IV) dose of etamsylate (10 mg/kg). Plasma and urine 2,5-HBSA concentrations were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The CD pilot study revealed that steady state could be reached with a few days and that 2,5-HBSA in plasma and urine shows instability during storage at -20°C but appears stable at -80°C. A novel holistic non-linear mixed-effects three-compartmental PK model was developed that described both plasma and urine concentrations of 2,5-HBSA, from either CD or etamsylate administration. Typical values for 2,5-HBSA clearance and bioavailability were 2.0 mL/min/kg and 28% respectively. Using the parameters obtained from this PK model, in conjunction with methodology developed by Toutain, afforded a possible screening limit (SL) that can regulate for a DT of 3 days in urine; however, a corresponding SL in plasma would be below current levels of detection. However, it is the responsibility of the individual racing authorities to apply their own risk management with regard to SLs and DTs.

Re-evaluation of the pharmacokinetics of xylazine administered to Thoroughbred horses.

Xylazine is widely used worldwide as a short-acting sedative in general equine and racing practice. In the UK, although it has a legitimate use during training, equine anti-doping rules state it is a prohibited substance on race day. The aim of the study was to produce a detection time (DT) to better inform European veterinary surgeons so that xylazine can be used appropriately under regulatory rules. Previous publications have various limitations pertaining to analysis method, particularly for plasma and limited length of time of sample collection. In this study, pharmacokinetic data were produced for xylazine and 4-OH-xylazine in equine urine and plasma following a single intravenous xylazine dose of 0.4 mg/kg to six Thoroughbred horses. Pharmacokinetic parameters were generated from a 3-compartmental model with clearance = 15.8 ± 4.88 ml min-1  kg-1 , Vss = 1.44 ± 0.38 L/kg, terminal half-life = 29.8 ± 12.7 hr and a DT determined at 71 hr for the administration of xylazine (Chanazine® ) in plasma and urine. Urine screening should aim to detect the 4-OH-xylazine metabolite, which can act as an indicator for the xylazine plasma concentration. A DT of 72 hr has been agreed by the European Horserace Scientific Liaison Committee, to be implemented in June 2019.

The pharmacokinetics of orally administered butylscopolamine in greyhound dogs.

The oral tablet formulation of butylscopolamine, which is available without prescription, is commonly used by trainers of racing greyhounds to treat functional urethral obstruction. As medication control of butylscopolamine is therefore required for such use to ensure the integrity of greyhound racing, an administration study was performed in six greyhounds to determine the pharmacokinetics of orally administered butylscopolamine. A single dose of one 10 mg butylscopolamine tablet was administered orally to simulate this use in greyhound racing. Blood, urine and faeces were collected at regular intervals from the greyhounds for up to 9 days and butylscopolamine concentrations determined. There was some, but very limited, absorption of butylscopolamine, with rapid elimination from plasma with a mean half-life of 2 hr. Urine concentrations initially declined in a similar manner to the plasma pharmacokinetics but then entered a much longer half-life of approximately 50 hr. Faecal concentrations declined to very low levels between 48 and 120 hr. The use of orally administered butylscopolamine for functional urethral obstruction in greyhounds is unjustified due to this very limited drug absorption. Medication control of butylscopolamine's antispasmodic effect on the digestive tract is possible by setting screening limits based on the urinary and faecal drug levels as determined in this study.

Detection of methandienone and its metabolites in equine urine, plasma and hair following a multidose oral administration.

Methandienone is an anabolic-androgenic steroid that is prohibited in equine sports due to its potential performance enhancing properties. Metabolism and detection of methandienone in equine urine have been investigated comprehensively in literature; however, there is a limited knowledge about its metabolites in equine plasma and no information about its detection in equine hair. Following a multi-dose oral administration of methandienone to two Thoroughbred horses, 17-epimethandienone, methyltestosterone, two mono-hydroxylated, two di-hydroxylated and three 17α-methylandrostanetriol metabolites were detected in plasma. The majority of these were present as free analytes, whilst the mono-hydroxylated metabolites and one isomer of 17α-methylandrostanetriol were partially conjugated. Estimated peak concentrations of methandienone were 6,000 and 11,100 pg/ml; meanwhile, they were 25.4 and 40.5 pg/ml for methyltestosterone. The most abundant analyte in the post-administration plasma samples of both horses was the mono-hydroxylated metabolite; however, the parent compound provided the longest detection (up to 96 h). Screening analysis of hair enabled the detection of methandienone in mane hair samples only, for up to 3 months. Its mono- and di-hydroxylated metabolites were detected with greater peak responses for up to 6 months post-administration in both mane and tail samples, showing that these metabolites could be better analytical targets for hair analysis when administered orally. A follow-up methodology with an extensive wash procedure confirmed the presence of methandienone and its metabolites in a number of post-administration hair samples. Final wash samples were also analysed to assess the degree of internal incorporation (via bloodstream) against possible external deposition (via sweat/sebum).

Detection of FG-4592 and metabolites in equine plasma, urine and hair following oral administration.

FG-4592 is a hypoxia-inducible factor inhibitor that has been approved for therapeutic use in some countries. This class of compounds can increase the oxygen carrying capacity of the blood and thus have the potential to be used as performance enhancing agents in sports. The purpose of this study was to investigate the detection of FG-4592 and metabolites in equine plasma and mane hair following a multiple dose oral administration to two Thoroughbred racehorses, to identify the best analytical targets for doping control laboratories. Urine samples were also analysed, and the results compared to previously published urine data. Liquid chromatography-high resolution mass spectrometry was used for metabolite identification in urine and plasma. Liquid chromatography-tandem mass spectrometry was used for full sample analysis of urine, plasma and hair samples and generation of urine and plasma profiles. FG-4592 and a mono-hydroxylated metabolite were detected in plasma. FG-4592 was detected with the greatest abundance and gave the longest duration of detection, up to 312 h post-administration, and would be the recommended target in routine doping samples. FG-4592 was detected in all mane hair samples collected post-administration, up to 166 days following the final dose, showing extended detection can be achieved with this matrix. To the best of the authors' knowledge, this is the first report of FG-4592 and metabolites in equine plasma and hair samples. Urine results were consistent with the previously published data, with FG-4592 offering the best target for detection and longest detection periods.

Detection of the selective androgen receptor modulator S-23 and its metabolites in equine urine and plasma following oral administration.

S-23 is an arylpropionamide selective androgen receptor modulator that has been investigated in animal models for use as a male hormonal contraceptive but is not yet available therapeutically. S-23 is available alongside other selective androgen receptor modulators (SARMs) to purchase online via uncontrolled sites, sold as supplement products. It has been detected in several human doping cases, highlighting the importance of identifying the best analytical targets for equine doping control. The purpose of this study was to investigate the detection of S-23 and its phase I metabolites in equine urine and plasma following a multiple dose oral administration to two Thoroughbred racehorses. Liquid chromatography-high resolution mass spectrometry was used for metabolite identification, and liquid chromatography-tandem mass spectrometry was used for full sample analysis and generation of urine and plasma profiles. S-23 and seven phase I metabolites were observed in urine following enzyme hydrolysis and solvolysis. The most abundant analyte detected was the hydroxylated 4-amino-2-(trifluoromethyl)benzonitrile metabolite, which also allowed the longest duration of detection in urine from both horses, for up to 360 h following administration. The data suggest that this metabolite was likely to be highly conjugated with both sulphate and glucuronide moieties. In plasma, S-23 and two phase I metabolites were observed. S-23 was the most abundant analyte detected for both horses, allowing detection for up to 143 h post-administration. To the best of the authors' knowledge, this is the first report of S-23 and metabolites in equine urine and plasma samples.

Detection of boldenone in the urine of female horses-ex vivo formation versus administration.

Boldenone is an anabolic-androgenic steroid (AAS) that is prohibited in equine sports. However, in certain situations, it is endogenous, potentially formed by the microbes in urine. An approach to the differentiation based on the detection of the biomarkers Δ1-progesterone, 20(S)-hydroxy-Δ1-progesterone and 20(S)-hydroxyprogesterone was assessed, and their concentrations were monitored in the urine of untreated female horses (n = 291) alongside boldenone, boldienone, testosterone and androstenedione. Using an ultra-sensitive analytical method, boldenone (256 ± 236 pg/mL, n = 290) and the biomarkers (Δ1-progesterone up to 57.6 pg/mL, n = 8; 20(S)-hydroxy-Δ1-progesterone 85.3 ± 181 pg/mL, n = 130; 20(S)-hydroxyprogesterone 43.5 ± 92.1 pg/mL, n = 158) were detected at low concentrations. The ex vivo production of Δ1-steroids was artificially induced following the storage of urine samples at room temperature for 7 days in order to assess the concentrations and ratios of the monitored steroids. The administration of inappropriately stored feed source also resulted in an increase in 20(S)-hydroxy-Δ1-progesterone concentrations and the biomarker ratios. Using the results from different datasets, an approach to differentiation was developed. In situations where the presence of boldenone exceeds a proposed action limit of 5 ng/mL, the presence of the biomarkers would be investigated. If Δ1-progesterone is above 50 pg/mL or if 20(S)-hydroxy-Δ1-progesterone is above 100 pg/mL with the ratio of 20(S)-hydroxy-Δ1-progesterone:20(S)-hydroxyprogesterone greater than 5:1, then this would indicate ex vivo transformation or consumption of altered feed rather than steroid administration. There remains a (small) possibility of a false negative result, but the model increases confidence that adverse analytical findings reported in female horses are caused by AAS administrations.

Presence and detection of endogenous steroids in the horse-A review.

Detection of doping with steroids that are also endogenous in the horse can be challenging, and a variety of approaches to distinguish exogenous administration from their natural presence are employed. Knowledge of endogenous concentrations of various steroids in different genders of horses (intact male, castrated male and female) and factors that could naturally affect them is beneficial for establishing ways for detection of their use. The current internationally adopted approaches include concentration-based thresholds in urine and plasma, steroid ratios in urine and targeting the administered intact steroid esters in plasma and hair. However, these have their limitations, and therefore, other strategies, such as additional biomarkers and steroid profiling based on longitudinal testing and multivariate analysis, have been investigated and could potentially improve detection of the use of endogenous steroids in horses. This paper aims to provide a comprehensive overview of the steroids (androgens, oestrogens and progestogens) that have been reported to be endogenous to horses in literature, their concentration ranges in different genders and factors potentially affecting them as well as current and possible future approaches to detect their use.

Equine metabolism of the selective androgen receptor modulator YK-11 in urine and plasma following oral administration.

YK-11 is a steroidal selective androgen receptor modulator, a compound class prohibited in both equine racing and human sports because of their potentially performance enhancing properties. YK-11 is easily accessible via internet-based supplement vendors making this compound a possible candidate for doping; however, its phases I and II metabolism has not yet been reported in the horse. The purpose of this study was to investigate the in vivo metabolites of YK-11 in urine and plasma following oral administration with three daily doses of 50 mg to two Thoroughbred horses. In vitro incubations with equine liver microsomes/S9 were also performed for use as metabolite reference materials; however, this resulted in the formation of 79 metabolites with little overlap with the in vivo metabolism. In plasma, parent YK-11 and seven phase I metabolites were detected, with five of them also observed in vitro. They were present nonconjugated in plasma, with one metabolite also indicating some glucuronide conjugation. In urine, 11 phase I metabolites were observed, with four of them also observed in vitro and six of them also detected in plasma. Nine metabolites were excreted non-conjugated in urine, with two of them also indicating some sulfate conjugation. Two minor metabolites were detected solely as sulfate conjugates. The most abundant analytes in urine were a mono-O-demethylated breakdown product and di-O-demethylated YK-11. The most abundant analytes in plasma were two isomers of the breakdown product with an additional hydroxylation reaction, which also provided the longest detection time in both matrices.

Detection of the growth hormone secretagogue MK-0677 in equine hair following oral administration.

MK-0677 (ibutamoren) is an orally active non-peptide growth hormone secretagogue that binds to the ghrelin receptor stimulating the secretion of endogenous growth hormone. It is one of the most prevalent performance-enhancing compounds currently available online and is potentially subject to abuse both in human and equine sports. The aim of the current study was to investigate whether it could be detected in equine hair following oral administration of MK-0677 mesylate to a Thoroughbred racehorse. MK-0677 and its O-dealkylated metabolite were extracted using an existing method for prohibited substances in equine hair and analysed by liquid chromatography tandem mass spectrometry. This enabled the detection of MK-0677 in all hair samples collected, up to 209 days in mane and 358 days in tail. A follow-up methodology with an extensive wash procedure was carried out for selected hair samples, which unambiguously verified the presence of MK-0677. Wash criteria to differentiate between internal incorporation (via bloodstream) and external deposition (via sweat and sebum) was also assessed and indicated internal incorporation for the samples collected at later time points (≥52 days) and a combination of internal incorporation and external deposition for hair samples collected at the earlier time point (2 days).

In vitro metabolism of the REV-ERB agonist SR-9009 and subsequent detection of metabolites in associated routine equine plasma and urine doping control samples.

SR-9009 is a synthetic compound widely available to purchase online as 'supplement' products due to its potential performance-enhancing effects, presenting a significant threat with regard to doping control in sport. In vitro metabolism with equine liver microsomes was performed to identify potential targets for detection of SR-9009. Six metabolites were identified, with the most abundant consisting of N-dealkylated metabolites (M1-M3). The addition of the identified metabolites to high-resolution accurate mass databases resulted in a positive finding for the N-dealkylated metabolite M1 of SR-9009 in an associated plasma and urine doping sample. Liquid chromatography-high-resolution mass spectrometry was used to verify the presence of the N-dealkylated metabolite (M1) in both matrices, with a low concentration of the parent compound and additional N-desalkyl metabolites (M2 and M3) detected in the plasma sample as supporting evidence of administration. To the best of the authors' knowledge, this is the first report of an adverse analytical finding in an equine sample for SR-9009 or its metabolites in equine doping control.

Differentiation of boldenone administration from ex vivo transformation in the urine of castrated male horses.

Boldenone is an anabolic-androgenic steroid that is prohibited in equine sports. However, in certain situations, it is endogenous or is believed to be formed by microbes in urine, and therefore, an approach for the differentiation is required. Following the identification of Δ1-progesterone and 20(S)-hydroxy-Δ1-progesterone as potential biomarkers of microbial activity, the presence of six steroids was investigated in the postrace urine of castrated male horses (geldings, n = 158). In line with endogenous findings from several other species when ultrasensitive methods are employed, boldenone was detected at low concentrations in all urine samples (27.0-1330 pg/ml). Furthermore, testosterone and androstenedione were detected in 157 samples (≤12,400 and 944 pg/ml, respectively), boldienone in two samples (≤22.0 pg/ml) and 20(S)-hydroxy-Δ1-progesterone in 20 samples (≤66.0 pg/ml). Δ1-Progesterone was not detected in any population samples analysed on arrival at the laboratory. The ex vivo transformation of boldienone, boldenone, androstenedione, Δ1-progesterone and 20(S)-hydroxy-Δ1-progesterone was induced following the storage of urine samples at room temperature for 7 days but not after refrigeration. Because the administration of inappropriately stored feed sources also resulted in an increase in 20(S)-hydroxy-Δ1-progesterone concentrations, a biomarker approach to distinguish steroid administrations was proposed. In situations where the presence of boldenone would exceed a proposed action limit, the presence of Δ1-progesterone and 20(S)-hydroxy-Δ1-progesterone would be investigated. If either Δ1-progesterone or 20(S)-hydroxy-Δ1-progesterone would exceed 50 and 100 pg/ml, respectively, for instance, then this would indicate ex vivo transformation or consumption of altered feed rather than steroid administration.

Equine metabolism of the growth hormone secretagogue MK-0677 in vitro and in urine and plasma following oral administration.

Ibutamoren mesylate, or MK-0677, is an orally active, nonpeptide growth hormone secretagogue that has been developed to stimulate excretion of endogenous growth hormone. It has been evaluated for the treatment of a range of clinical conditions but is not available therapeutically. Nonetheless, MK-0677 is widely available to purchase online, sold as 'supplement' products. The mode of action and relative ease of purchase make MK-0677 a potential threat with regard to sports doping. The aim of this study was to investigate the metabolism of MK0677 in the horse following in vitro incubation and oral administration to two Thoroughbred racehorses, in order to identify the most appropriate analytical targets for doping control laboratories. Liquid chromatography high resolution mass spectrometry was used for metabolite identification, and subsequently, liquid chromatography-tandem mass spectrometry was used to generate full metabolite profiles for post-administration urine and plasma samples. Fourteen phase I metabolites were identified in vitro; 13 of these were subsequently detected in urine and nine in plasma collected post-administration, alongside the parent compound in both matrices. In both urine and plasma, the longest duration of detection was observed for an O-dealkylated metabolite of MK-0677, and therefore, this would be the best target for the detection of MK-0677 administration. MK-0677 and the O-dealkylated metabolite were found to be excreted largely unconjugated in urine and plasma.

In vitro and in vivo metabolism of the anabolic-androgenic steroid oxandrolone in the horse.

Oxandrolone is an anabolic-androgenic steroid with favourable anabolic to androgenic ratio, making it an effective anabolic agent with less androgenic side effects. Although its metabolism has been studied in humans, its phase I and II metabolism has not been previously reported in the horse. The purpose of this study was to investigate the in vitro metabolism of oxandrolone (using both equine liver microsomes and S9) and in vivo metabolism following oral administration (three daily doses of 50 mg of oxandrolone to a single Thoroughbred horse), using both gas and liquid chromatography-mass spectrometry techniques. The in vitro phase I transformations observed included 16-hydroxylated (two epimers), 17-methyl-hydroxylated and 16-keto metabolites. In addition to parent oxandrolone and these hydroxylated metabolites, the 17-epimer and a 17,17-dimethyl-18-norandrost-13-ene analogue were detected in biological samples following the administration. 16-keto-oxandrolone was only observed in urine. The 16- and 17-methyl-hydroxylated oxandrolone metabolites were predominantly excreted as sulfate conjugates in urine, whereas parent oxandrolone, its epimer and 17,17-dimethyl-18-norandrost-13-ene derivative were found predominantly in the unconjugated urine fraction. The most abundant analyte detected in both plasma and urine was parent oxandrolone. However, the longest detection period using the developed analytical method was provided by 17-hydroxymethyl-oxandrolone in both matrices. The results of this study provided knowledge of how best to detect the use of oxandrolone in regulatory samples.

Identification of equine in vitro metabolites of seven non-steroidal selective androgen receptor modulators for doping control purposes.

Selective androgen receptor modulators, SARMs, are a large class of compounds developed to provide therapeutic anabolic effects with minimal androgenic side effects. A wide range of these compounds are available to purchase online and thus provide the potential for abuse in sports. Knowledge of the metabolism of these compounds is essential to aid their detection in doping control samples. In vitro models allow a quick, cost-effective response where administration studies are yet to be carried out. In this study, the equine phase I metabolism of the non-steroidal SARMs GSK2881078, LGD-2226, LGD-3303, PF-06260414, ACP-105, RAD-140 and S-23 was investigated using equine liver microsomes. Liquid chromatography coupled to a QExactive Orbitrap mass spectrometer allowed identification of metabolites with high resolution and mass accuracy. Three metabolites were identified for both GSK2881078 and LGD-2226, four for LGD-3303 and RAD-140, five for PF-06260414, twelve for ACP-105 and ten for S-23. The equine metabolism of GSK-2881078, LGD-2226, LGD-3303 and PF-06260414 is reported for the first time. Although the equine metabolism of ACP-105, RAD-140 and S-23 has previously been reported, the results obtained in this study have been compared with published data.

Equine metabolism of the selective androgen receptor modulator AC-262536 in vitro and in urine, plasma and hair following oral administration.

AC-262536 is one of a number of selective androgen receptor modulators that are being developed by the pharmaceutical industry for treatment of a range of clinical conditions including androgen replacement therapy. Though not available therapeutically, selective androgen receptor modulators are widely available to purchase online as (illegal) supplement products. The growth- and bone-promoting effects, along with fewer associated negative side effects compared with anabolic-androgenic steroids, make these compounds a significant threat with regard to doping control in sport. The aim of this study was to investigate the metabolism of AC-262536 in the horse following in vitro incubation and oral administration to two Thoroughbred horses, in order to identify the most appropriate analytical targets for doping control laboratories. Urine, plasma and hair samples were collected and analysed for parent drug and metabolites. Liquid chromatography-high-resolution mass spectrometry was used for in vitro metabolite identification and in urine and plasma samples. Nine phase I metabolites were identified in vitro; four of these were subsequently detected in urine and three in plasma, alongside the parent compound in both matrices. In both urine and plasma samples, the longest detection window was observed for an epimer of the parent compound, which is suggested as the best target for detection of AC-262536 administration. AC-262536 and metabolites were found to be primarily glucuronide conjugates in both urine and plasma. Liquid chromatography-tandem mass spectrometry analysis of post-administration hair samples indicated incorporation of parent AC-262536 into the hair following oral administration. No metabolites were detected in the hair.

Investigation of the metabolism of the selective androgen receptor modulator LGD-4033 in equine urine, plasma and hair following oral administration.

LGD-4033 is one of a number of selective androgen receptor modulators (SARMs) that are being developed by the pharmaceutical industry to provide the therapeutic benefits of anabolic androgenic steroids, without the less desirable side effects. Though not available therapeutically, SARMs are available for purchase online as supplement products. The potential for performance enhancing effects associated with these products makes them a significant concern with regards to doping control in sports. The purpose of this study was to investigate the metabolism of LGD-4033 in the horse following oral administration, in order to identify the most appropriate analytical targets for doping control laboratories. LGD-4033 was orally administered to two Thoroughbred horses and urine, plasma and hair samples were collected and analysed for parent drug and metabolites. LC-HRMS was used for metabolite identification in urine and plasma. Eight metabolites were detected in urine, five of which were excreted only as phase II conjugates, with the longest detection time being observed for di- and tri-hydroxylated metabolites. The parent compound could only be detected in urine in the conjugated fraction. Seven metabolites were detected in plasma along with the parent compound where mono-hydroxylated metabolites provided the longest duration of detection. Preliminary investigations with hair samples using LC-MS/MS analysis indicated the presence of trace amounts of the parent compound and one of the mono-hydroxylated metabolites. In vitro incubation of LGD-4033 with equine liver microsomes was also performed for comparison, yielding 11 phase I metabolites. All of the metabolites observed in vivo were also observed in vitro.

Bioformation of boldenone and related precursors/metabolites in equine feces and urine, with relevance to doping control.

Boldenone (1-dehydrotestosterone) is an exogenous anabolic-androgenic steroid (AAS) but is also known to be endogenous in the entire male horse and potentially formed by microbes in voided urine, the gastrointestinal tract, or feed resulting in its detection in urine samples. In this study, equine fecal and urine samples were incubated in the presence of selected stable isotope labeled AAS precursors to investigate whether microbial activity could result in 1-dehydrogenation, in particular the formation of boldenone. Fecal matter was initially selected for investigation because of its high microbial activity, which could help to identify potential 1-dehydrogenated biomarkers that might also be present in low quantities in urine. Fecal incubations displayed Δ1-dehydrogenase activity, as evidenced by the use of isotope labeled precursors to show the formation of boldenone and boldione from testosterone and androstenedione, as well as the formation of Δ1-progesterone and boldione from progesterone. Unlabeled forms were also produced in unspiked fecal samples with Δ1-progesterone being identified for the first time. Subsequent incubation of urine samples with the labeled AAS precursors demonstrated that Δ1-dehydrogenase activity can also occur in this matrix. In all urine samples where labeled boldenone or boldione were detected, labeled Δ1-progesterone was also detected. Δ1-progesterone was not detected any non-incubated urine samples or following an administration of boldenone undecylenate to one mare/filly. Δ1-progesterone appears to be a candidate for further investigation as a suitable biomarker to help evaluate whether boldenone present in a urine sample may have arisen due to microbial activity rather than by its exogenous administration.

Elucidation of the biosynthetic pathways of boldenone in the equine testis.

Boldenone is an anabolic-androgenic steroid that is prohibited in equine sports. Urine from the uncastrated male horse contains boldenone that is thought to be of endogenous origin and thus a threshold ('cut-off') concentration has been adopted internationally for free and conjugated boldenone to help distinguish cases of doping from its natural production. The testis is likely to be a source of boldenone. Qualitative analysis was performed on extracts of equine testicular homogenates (n = 3 horses) incubated non-spiked and in the presence of its potential precursors using liquid chromatography tandem mass spectrometry (LC-MS/MS) and LC high resolution mass spectrometry (LC-HRMS). Samples were analysed both underivatised and derivatised to increase the certainty of identification. In addition to previously reported endogenous steroids, analysis of non-spiked testicular tissue samples demonstrated the presence of boldenone and boldienone at trace levels in the equine testis. Incubation of homogenates with deuterium or carbon isotope labelled testosterone and androstenedione resulted in the matching stable isotope analogues of boldenone and boldienone being formed. Additionally, deuterium and carbon labelled 2-hydroxyandrostenedione was detected, raising the possibility that this steroid is a biosynthetic intermediate. In conclusion, boldenone and boldienone are naturally present in the equine testis, with the biosynthesis of these steroids arising from the conversion of testosterone and androstenedione. However, additional work employing larger numbers of animals, further enzyme kinetic experiments and pure reference standards for 2-OH androstenedione isomers would be required to better characterize the pathways involved in these transformations.

Important considerations for the utilisation of methanolysis in steroid analysis.

The effective analysis of anabolic-androgenic steroids in urine usually requires a suitable deconjugation method for the analysis of phase II metabolites such as sulphates and glucuronides. Acid hydrolysis using methanolysis is one adopted method of deconjugation that efficiently and rapidly cleaves both sulphates and glucuronides contemporaneously. The formation of artefactual by-products is a known disadvantage of this harsh method. However, the possible promotion of deuterium-hydrogen exchange of isotopically labelled internal standards has received little attention in the literature. This report demonstrates a complete deuterium-hydrogen exchange from deuterium labelled D9 -progesterone to progesterone driven by the acidic conditions of the methanolysis. The likely mechanisms of this exchange reaction are postulated, and the results compared to other deuterated steroids. This finding highlights the importance for careful consideration when selecting labelled internal standards in a conjunction with methanolysis.