Identification and quantification of metabolites common to 17α-methyltestosterone and mestanolone in horse urine

Anabolic steroids with the 17α-methyl,17β-hydroxyl group, which were developed as oral formulations for therapeutic purposes, have been abused in the field of human sports. These anabolic steroids are also used to enhance racing performance in racehorses. In humans, structurally related 17α-methyltestosterone (MTS) and mestanolone (MSL), which are anabolic steroids with the 17α-methyl,17β-hydroxyl group, have metabolites in common. The purpose of this study was to determine metabolites common to these two steroids in horses, which may serve as readily available screening targets for the doping test of these steroids in racehorses.Urine sample collected after administering MTS and MSL to horses was treated to obtain unconjugated steroid, glucuronide, and sulfate fractions. The fractions were subjected to gas chromatography/mass spectrometry (GC/MS), and 17α-methyl-5α-androstan-3β,17β-diol, 17α-hydroxymethyl-5α-androstan-3β,17β-diol, 17α-methyl-5α-androstan-3β,16β,17β-triol, and 17α-methyl-5α-androstan-3β,16α,17β-triol were detected as the common metabolites by comparison with synthesized reference standards. The urinary concentrations of these metabolites after dosing were determined by GC/MS. 17α-Methyl-5α-androstan-3β,16β,17β-triol was mainly detected in the sulfate fractions of urine samples after administration. This compound was consistently detected for the longest time in the urine samples after dosing with both steroids. The results suggest that 17α-methyl-5α-androstan-3β,16β,17β-triol is a very useful screening target for the doping test of MTS and MSL in racehorses.