title: Detection of 5α-androst-2-en-17-one and variants: Identification of main urinary metabolites in human urine samples by GC-MS and NMR.
abstract: 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 2α-hydroxy-5α-androst-3-en-17-one (M1) and 2β,3α-dihydroxy-5α-androstan-17-one (M2). A third metabolite, 3α,4β-dihydroxy-5α-androstan-17-one (M3) was also detected, however in lower amounts. The precursor steroids, 5α-androst-2-en-17-one (1) and 5α-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 2α,3α- and 3α,4α-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 β-glucuronidase and the chemical solvolysis, which may ease its identification. Copyright © 2016 John Wiley & Sons, Ltd.
authors.affiliation: INRS-Institut Armand Frappier, Laboratoire de controle du dopage, Laval, Quebec, Canada.
authors.forename: Christiane
authors.initials: C
authors.lastname: Ayotte
authors.affiliation: INRS-Institut Armand-Frappier, Laval, Quebec, Canada.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Sylvestre
authors.affiliation: Centre Hospitalier de l'Université Laval (CHUL), Endocrinologie et néphrologie, Québec, Quebec, Canada.
authors.forename: Alain
authors.initials: A
authors.lastname: Charlebois
authors.affiliation: Centre Hospitalier de l'Université Laval (CHUL), Endocrinologie et néphrologie, Québec, Quebec, Canada.
authors.forename: Donald
authors.initials: D
authors.lastname: Poirier
mesh_terms: D015068:17-Ketosteroids / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism / Q000652:urine*; D000736:Androstenes / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism / Q000652:urine*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004300:Doping in Sports; D004852:Epoxy Compounds / Q000737:chemistry*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D013256:Steroids / Q000737:chemistry* / Q000378:metabolism
chemical_list: D015068:17-Ketosteroids; D000736:Androstenes; D004852:Epoxy Compounds; D013256:Steroids; C531589:androst-3-en-17-one; C069740:androst-2-en-17-one
keywords: 5α-androst-2-en-17-one; GC-MS; NMR; doping; metabolites