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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. [ABSTRACT FROM AUTHOR]

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). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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., (© 2024 John Wiley & Sons, Ltd.)