New Insights into the Metabolism of Methyltestosterone and Metandienone: Detection of Novel A-Ring Reduced Metabolites

Metandienone and methyltestosterone are orally active anabolic-androgenic steroids with a 17 alpha-methyl structure that are prohibited in sports but are frequently detected in anti-doping analysis. Following the previously reported detection of long-term metabolites with a 17 xi-hydroxymethyl-17 xi-methyl-18-nor-5 xi-androst-13-en-3 xi-ol structure in the chlorinated metandienone analog dehydrochloromethyltestosterone ("oral turinabol"), in this study we investigated the formation of similar metabolites of metandienone and 17 alpha-methyltestosterone with a rearranged D-ring and a fully reduced A-ring. Using a semi-targeted approach including the synthesis of reference compounds, two diastereomeric substances, viz. 17 alpha-hydroxymethyl-17 beta-methyl-18-nor-5 beta-androst-13-en-3 alpha-ol and its 5 alpha-analog, were identified following an administration of methyltestosterone. In post-administration urines of metandienone, only the 5 beta-metabolite was detected. Additionally, 3 alpha,5 beta-tetrahydro-epi-methyltestosterone was identified in the urines of both administrations besides the classical metabolites included in the screening procedures. Besides their applicability for anti-doping analysis, the results provide new insights into the metabolism of 17 alpha-methyl steroids with respect to the order of reductions in the A-ring, the participation of different enzymes, and alterations to the D-ring.