13C/12C ratios of endogenous urinary steroids investigated for doping control purposes.

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 (13)C/(12)C ratio of a target substance (androsterone, a testosterone metabolite) was compared to the (13)C/(12)C ratio of an endogenous reference compound (11beta-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 (13)C/(12)C ratios of androsterone and 11beta-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 (13)C/(12)C 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 (13)C/(12)C ratios, no significant change in the difference of (13)C/(12)C (11beta-hydroxyandrosterone) minus (13)C/(12)C (androsterone) could be observed.