In vitro and in vivo studies of androst-4-ene-3,6,17-trione in horses by gas chromatography-mass spectrometry

This paper describes the application of gas chromatography-mass spectrometry (GC-MS) for in vitro and in vivo studies of 6-OXO in horses, with a special aim to identify the most appropriate target metabolite to be monitored for controlling the administration of 6-OXO in racehorses. In vitro studies of 6-OXO were performed using horse liver microsomes. The major biotransformation observed was reduction of one keto group at the C3 or C6 positions. Three in vitro metabolites, namely 6 alpha-hydroxyandrost-4-ene-3,17-dione (M1), 3 alpha-hydroxyandrost-4-ene-6,17-dione (M2a) and 3 beta-hydroxyandrost-4-ene-6,17-dione (M2b) were identified. For the in vivo studies, two thoroughbred geldings were each administered orally with 500 mg of androst-4-ene-3,6,17-trione (5 capsules of 6-OXO (R)) by stomach tubing. The results revealed that 6-OXO was extensively metabolized. The three in vitro metabolites (M1, M2a and M2b) identified earlier were all detected in post-administration urine samples. In addition, seven other urinary metabolites, derived from a further reduction of either one of the remaining keto groups or one of the remaining keto groups and the olefin group, were identified. These metabolites included 6 alpha,17 beta-dihydroxyandrost-4-en-3-one (M3a), 6,17-dihydroxyandrost-4-en-3-one (M3b and M3c), 3 beta,6 beta-dihydroxyandrost-4-en-17-one (M4a), 3,6-dihydroxyandrost-4-en-17-one (M4b), 3,6-dihydroxyandrostan-17-one (M5) and 3,17-dihydroxyandrostan-6-one (M6). The longest detection time observed in urine was up to 46 h for the M6 metabolite. For blood samples, the peak 6-OXO plasma concentration was observed 1 h post administration. Plasma 6-OXO decreased rapidly and was not detectable 12 h post administration. Copyright (C) 2009 John Wiley & Sons, Ltd.