Metabolic studies of mesterolone in horses

Mesterolone (1alpha-methyl-5alpha-androstan-17beta-ol-3-one) is a synthetic anabolic androgenic steroid (AAS) with reported abuses in human sports. As for other AAS, mesterolone is also a potential doping agent in equine sports. Metabolic studies on mesterolone have been reported for humans, whereas little is known about its metabolic fate in horses. This paper describes the studies of both the in vitro and in vivo metabolism of mesterolone in racehorses with an objective to identify the most appropriate target metabolites for detecting mesterolone administration. In vitro biotransformation studies of mesterolone were performed by incubating the steroid with horse liver microsomes. Metabolites in the incubation mixture were isolated by liquid-liquid extraction and analysed by gas chromatography-mass spectrometry (GC-MS) after acylation or silylation. Five metabolites (M1-M5) were detected. They were 1alpha-methyl-5alpha-androstan-3alpha-ol-17-one (M1), 1alpha-methyl-5alpha-androstan-3beta-ol-17-one (M2), 1alpha-methyl-5alpha-androstane-3alpha,17beta-diol (M3), 1alpha-methyl-5alpha-androstane-3beta,17beta-diol (M4), and 1alpha-methyl-5alpha-androstane-3,17-dione (M5). Of these in vitro metabolites, M1, M3, M4 and M5 were confirmed using authentic reference standards. M2 was tentatively identified by mass spectral comparison to M1. For the in vivo metabolic studies, Proviron (20 tablets x 25 mg of mesterolone) was administered orally to two thoroughbred geldings. Pre- and post-administration urine samples were collected for analysis. Free and conjugated metabolites were isolated using solid-phase extraction and analysed by GC-MS as described for the in vitro studies. The results revealed that mesterolone was extensively metabolised and the parent drug was not detected in urine. Three metabolites detected in the in vitro studies, namely M1, M2 and M4, were also detected in post-administration urine samples. In addition, two stereoisomers each of 1alpha-methyl-5alpha-androstane-3,17alpha-diol (M6 and M7) and 1alpha-methyl-5alpha-androstane-3,16-diol-17-one (M8 and M9), and an 18-hydroxylated metabolite 1alpha-methyl-5alpha-androstane-3,18-diol-17-one (M10) were also detected. The metabolic pathway for mesterolone is postulated. These studies have shown that metabolites M8, M9 and M10 could be used as potential screening targets for controlling the misuse of mesterolone in horses.