Characterization of equine urinary metabolites of selective androgen receptor modulators (SARMs) S1, S4 and S22 for doping control purposes

Abstract

Selective androgen receptor modulators, SARMs, constitute a class of compounds with anabolic properties but with few androgenic side-effects. This makes them possible substances of abuse and the World Anti-Doping Agency (WADA) has banned the entire class of substances. There have been several cases of illicit use of aryl propionamide SARMs in human sports and in 2013, 13 cases were reported. These substances have been found to be extensively metabolized in humans, making detection of metabolites necessary for doping control. SARMs are also of great interest to equine doping control, but the in vivo metabolite pattern and thus possible analytical targets have not been previously studied in this species. In this study, the urinary metabolites of the SARMs S1, S4, and S22 in horses were studied after intravenous injection, using ultra high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-QToF-MS). Eight different metabolites were found for SARM S1, nine for SARM S4, and seven for SARM S22. The equine urinary metabolite profiles differed significantly from those of humans. The parent compounds were only detected for SARMs S4 and S22 and only at the first sampling time point at 3 h post administration, making them unsuitable as target compounds. For all three SARMs tested, the metabolite yielding the highest response had undergone amide hydrolysis, hydroxylation and sulfonation. The resulting phase II metabolites (4-nitro-3-trifluoro-methyl-phenylamine sulfate for SARMs S1 and S4 and 4-cyano-3-trifluoro-methyl-phenylamine sulfate for SARM S22) are proposed as analytical targets for use in equine doping control.

Investigation of the selective androgen receptor modulators S1, S4 and S22 and their metabolites in equine plasma using high‐resolution mass spectrometry

“…wileyonlinelibrary.com/journal/rcm urinary metabolites. [14] The extracted ion chromatograms of the proposed metabolites were also compared to those of the sample collected before administration. To study the retention time and mass stability of the system, a standard solution containing meloxicam and leucine enkephalin was analyzed before and after each batch of study samples and evaluated based on retention time and mass accuracy.…”

“…Over the time period of the qualitative analyses, the maximum mass error was <2 ppm and the variation in retention time was ±0.03 min for the two compounds. The metabolites were denoted in accordance with Hansson et al, [14] in which the metabolites that had undergone the same biotransformation reactions were given the same number.…”

“…For all metabolites found in plasma, the retention times and product ions matched the corresponding metabolites in urine. [14] However, for the minor metabolites, fewer fragments were detected in plasma compared to urine due to the lower concentrations in the first matrix.…”

“…[7] Furthermore, the identification of metabolites also corroborates that a substance has passed through a metabolic system and that an adverse finding is not caused by contamination of the sample. The metabolism of different SARMs of the arylpropionamide class has been studied in vitro in a variety of systems such as human liver microsomes, [8] equine liver microsomes, [9] bovine liver microsomes [10] and fungal incubates, [11] as well as in vivo in both human [12,13] and equine [14] urine. The three aforementioned in vivo studies showed that the SARMs were metabolized rapidly and extensively.…”

Challenges in detecting substances for equine anti‐doping

“…[66,67] SARMs are also of great importance in equine doping control, and some studies concerning the in vivo metabolism pattern has not been referred in this species. [68] Hansson et al [69,70] described the urinary metabolites of the SARMs S1, S4, and S22 in horses after intravenous injection using UHPLCquadrupole Time-of-Flight-MS (UHPLC-QToF-MS), reporting a significant number of metabolites for each of the above substances. The equine urinary metabolite profiles differed from those of humans.…”