Detection of 5α-androst-2-en-17-one and variants: Identification of main urinary metabolites in human urine samples by GC-MS and NMR

Two steroids were identified in a supplement named D-2 following the detection of unknown compounds during the routine testing of an athlete's sample. The main glucuroconjugated metabolites were isolated from this urine by high performance liquid chromatography (HPLC) following enzymatic hydrolysis and identified by gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) analyses as being 2 alpha-hydroxy-5 alpha-androst-3-en-17-one (M1) and 2 beta,3 alpha-dihydroxy-5 alpha-androstan-17-one (M2). A third metabolite, 3 alpha,4 beta-dihydroxy-5 alpha-androstan-17-one (M3) was also detected, however in lower amounts. The precursor steroids, 5 alpha-androst-2-en-17-one (1) and 5 alpha-androst-3-en-17-one (2) were present in the first D-2 products offered on the Internet. Later, the corresponding 17-hydroxyl compounds were offered as such or as esters (acetate, cypionate) in different relative ratios. Both M2 and M3 were synthesized from the trans-diaxial hydrolysis of the corresponding 2 alpha,3 alpha- and 3 alpha,4 alpha-epoxides (3). These were excreted in the hours following the controlled administration of the commercial product called D-2 R to a male volunteer and were also produced from the incubation of 1 and 2 with S9 liver fractions. Some preparations contain predominantly the alkene in C-2 and, therefore, an efficient detection method must include both primary metabolites M1 and M2. The latter was found equally in the fractions extracted following the enzymatic hydrolysis with beta-glucuronidase and the chemical solvolysis, which may ease its identification. Copyright (C) 2016 John Wiley & Sons, Ltd.