Development of a GC/C/IRMS method – Confirmation of a novel steroid profiling approach in doping control

In doping control, an athlete can only be convicted with the misuse with endogenous steroids like testosterone (T), if abnormal values of steroid metabolites and steroid ratios are observed and if the subsequent analysis with isotope ratios mass spectrometry (IRMS) confirms the presence of exogenously administered androgens. In this work, we compare the results of a novel steroid profiling approach with the performance an in-house developed IRMS method. The developed IRMS has the advantage over other methods to be relatively short in time and with target compounds androsterone, etiocholanolone, 5β-androstane 3α,17β-diol and 5α-androstane 3α,17β-diol. Pregnanediol was used as an endogenous reference compound (ERC).Reference limits for the IRMS values were established and applied as decision limits for the evaluation of excretion urine from administration with oral T, T-gel, dihydrotestosterone (DHT) – gel and dehydroepiandrosterone (DHEA). Results indicated the importance of both androstanediols as important IRMS markers where relative values compared to an ERC (Δδ13C) yielded better detection accuracy than absolute δ13C-values. The detection times of all administered endogenous steroids were evaluated using the proposed thresholds.The results of traditional steroid profiling and a new approach based upon minor steroid metabolites monitoring introduced in a longitudinal framework were evaluated with IRMS. With traditional steroid profiling methods, 95% of the atypical samples could be confirmed whereas an additional 74% of IRMS confirmed was provided by a new biomarkers strategy.These results prove that the other steroid profiling strategies can improve the efficiency in detection of misuse with endogenous steroids.

► An IRMS confirmation method was developed and validated for the purpose of anti-doping control.
► Population based reference limits for delta-values are established.
► We evaluated the results of administration studies conducted with T, DHT and DHEA.
► We compared the diagnostic performance of alternative steroid profiling methods with that of the confirmatory IRMS analysis.