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 17 alpha-methylandrostanetriol metabolites were detected in plasma. The majority of these were present as free analytes, whilst the mono-hydroxylated metabolites and one isomer of 17 alpha-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). Metabolism and detection of methandienone in urine are well established in horses, but there is limited knowledge about its metabolites in plasma and no information about its detection in hair. Following an oral administration, nine Phase I metabolites were observed in plasma, and the longest detection time was achieved by targeting the parent compound and the mono-hydroxylated metabolite. In hair, the parent compound and hydroxylated metabolites were detected and the mono-hydroxylated offered the longest detection time.image