Methandriol(3b) [HRP]

The use of anabolic steroids was banned by the International Olympic Committee for the first time at the Olympic Games in Montreal in 1976. Since that time the misuse of anabolic steroids by athletes has been controlled by analysis of urine extracts by gas chromatography-mass spectrometry (GC-MS). The excreted steroids or their metabolites, or both, are isolated from urine by XAD-2 adsorption, enzymatic hydrolysis of conjugated excreted metabolites with beta-glucuronidase from Escherichia coli, liquid-liquid extraction with diethyl ether, and converted into trimethylsilyl (TMS) derivatives. The confirmation of an anabolic steroid misuse is based on comparison of the electron impact ionization (EI) mass spectrum and GC retention time of the isolated steroid and/or its metabolite with the EI mass spectrum and GC retention time of authentic reference substances. For this purpose excretion studies with the most common anabolic steroids were performed and the main excreted metabolites were synthesized for bolasterone, boldenone, 4-chlorodehydromethyltestosterone, clostebol, drostanolone, fluoxymesterone, formebolone, mestanolone, mesterolone, metandienone, methandriol, metenolone, methyltestosterone, nandrolone, norethandrolone, oxandrolone and stanozolol. The metabolism of anabolic steroids, the synthesis of their main metabolites, their GC retention and El mass spectra as TMS derivatives are discussed.

Due to the potential for misuse of a wide range of anabolic steroids in horse racing, a screening test to detect multiple compounds, via a common class of metabolites, would be a valuable forensic tool. An enzyme-linked immunosorbent assay (ELISA) has been developed to detect 17 alpha-alkyl anabolic steroid metabolites in equine urine. 16 beta-Hydroxymestanolone (16 beta,17 beta-dihydroxy- 17 alpha-methyl-5 alpha-androstan-3-one) was synthesised in six steps from commercially available epiandrosterone (3 beta-hydroxy-5 alpha-androstan-17-one). Polyclonal antibodies were raised in sheep, employing mestanolone (17 beta-hydroxy-17 alpha-methyl-5 alpha-androstan-3-one) or 16 beta-hydroxymestanolone conjugated to human serum albumin, via a 3-carboxymethyloxime linker, as antigens. Antibody cross-reactivities were determined by assessing the ability of a library of 54 representative steroids to competitively bind the antibodies. Antibodies raised against 16 beta-hydroxymestanolone showed excellent cross-reactivities for all of the 16 beta, 17 beta-dihydroxy-17 alpha-methyl steroids analysed and an ELISA has been developed to detect these steroid metabolites. Using this 16 beta-hydroxymestanolone assay, urine samples from horses administered with stanozolol (17 alpha-methyl-pyrazolo[4',3':2,3]-5 alpha-androstan-17 beta-ol), were analysed raw, following beta-glucuronidase hydrolysis, and following solid-phase extraction (SPE) procedures. The suppressed absorbances observed were consistent with detection of the metabolite 16 beta-hydroxystanozolol. Positive screening results were confirmed by comparison with standard LCMS analyses. Antibodies raised against mestanolone were also used to develop an ELISA and this was used to detect metabolites retaining the parent D-ring structure following methandriol (17 alpha-methylandrost-5-ene-3 beta,17 beta-diol) administration. The ELISA methods developed have application as primary screening tools for detection of new and known anabolic steroid metabolites. (c) 2005 Elsevier Ltd. All rights reserved.