Metabolism of anabolic steroids in man: synthesis and use of reference substances for identification of anabolic steroid metabolites

Abstract

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 β-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.