In order to detect the misuse of endogenous anabolic steroids such as testosterone by athletes a total of n = 1734 suspicious urine samples were investigated by gas chromatography/combustion/isotope ratio mass spectrometry throughout the years 2005, 2006 and 2007. The ¹³C/¹²C ratio of a target substance (androsterone, a testosterone metabolite) was compared to the ¹³C/¹²C ratio of an endogenous reference compound (11β-hydroxyandrosterone). N = 1340 samples were investigated due to elevated testosterone/epitestosterone ratios, with n = 87 (6.5%) exceptional findings regarding their isotopic ratios. An additional n = 164 samples were investigated because of elevated dehydroepiandrosterone concentrations, with n = 2 (1.2%) exceptional findings. The remainder were subjected to isotope ratio analysis because of elevated androsterone levels or because this was requested by sports federations. Significant differences between female and male samples were found for the ¹³C/¹²C ratios of androsterone and 11β-hydroxyandrosterone but not for samples taken in or out of competition. A further n = 645 samples originating from other World Anti-Doping Agency accredited laboratories, mainly throughout Europe as well as South America, South Africa and Southeast Asia, were investigated. The ¹³C/¹²C ratios of the urinary steroids differ significantly for each geographical region, reflecting the dietary status of the individuals. The system stability over time has been tested by repeated injections of a standard solution and repeated processing of frozen stored blank urine. Despite a drift over time in absolute ¹³C/¹²C ratios, no significant change in the difference of ¹³C/¹²C (11β-hydroxyandrosterone) minus ¹³C/¹²C (androsterone) could be observed. Copyright © 2009 John Wiley & Sons, Ltd. Copyright © 2009 John Wiley & Sons, Ltd.

On the one hand, 19-norandrosterone (NA) is the most abundant metabolite of the synthetic anabolic steroid 19-nortestosterone and related prohormones. On the other hand, small amounts are biosynthesized by pregnant women and further evidence exists for physiological origin of this compound. The World Anti-Doping Agency (WADA) formerly introduced threshold concentrations of 2 or 5 ng of NA per ml of urine to discriminate 19-nortestosterone abuse from biosynthetic origin. Recent findings showed however, that formation of NA resulting in concentrations in the range of the threshold levels might be due to demethylation of androsterone in urine, and the WADA 2006 Prohibited List has defined NA as endogenous steroid. To elucidate the endogenous or exogenous origin of NA, ¹³C/¹²C-analysis is the method of choice since synthetic 19-nortestosterone is derived from C₃-plants by partial synthesis and shows δ¹³C_VPDB-values of around −28‰. Endogenous steroids are less depleted in ¹³C due to a dietary mixture of C₃- and C₄-plants. An extensive cleanup based on two high performance liquid chromatography cleanup steps was applied to quality control and doping control samples, which contained NA in concentrations down to 2 ng per ml of urine. ¹³C/¹²C-ratios of NA, androsterone and etiocholanolone were measured by gas chromatography/combustion/isotope ratio mass spectrometry. By comparing δ¹³C_VPDB-values of androsterone as endogenous reference compound with NA, the origin of NA in doping control samples was determined as either endogenous or exogenous.