Addressing recent challeges in IRMS – Development of a method applicable to 1- androstene steroid 6-alph-OH androstenedione and androstarienedione

Code: 20A07MT

All doping agents that may also be produced endogenously, i.e. as part of the human metabolism, necessitate further investigations in order to differentiate between a doping rule violation and endogenous production. Testosterone is the prime examples for this procedue and in many cases, only the determination of carbon isotope ratios (CIR) allows for an unambiguous discrimination between the endogenous production of elevated amounts of testosterone and the administration of this doping agent. A similar approach has been established for boldenone, a so called pseudo-endogenous compound, which is not produced by everyone but can occasionally be found in urine specimens. In such cases, the determination of CIR is the best solution to identify the source of these steroids.

Recently, the World Anti-Doping Agency (WADA) has identified several new pseudo-endogenous steroids that should be addressed with caution by doping control laboratories and strongly recommended the use of CIR to identify the origin of these agents. For the majority of compounds (1-androstenediol, 1-testosterone, 1-androstenedione, 1-androsterone and androstatrienedione), no isotope ratio mass spectrometry-based methods exist, so the aim of this research project is the development and validation of these approaches. For the steroid 66α-hydroxy-androstenedione, a method has already been published, but is currently not fully validated according to the recently established WADA guidelines. Therefore, this compound will also be included in this study.

Following method optimization and validation, EQAS and/or excretion study samples already present in our laboratory will be analyzed as proof-of-concept together with samples derived from the routine screening showing low concentration of the steroids under investigation.

Main findings

Our study confirms that the ratio 6OH-Etio-3G/EG can complement the results obtained by T/E for testosterone administration. The ratio 6OH-Etio-3G/EG was found to be subject to a high intraindividual vatiation, therefore making the selection of a threshold based on its variance very difficult. On one hand, a proper isotopically labeleled internal standard Is still needed to reduce the analytical variability. On the other, there must be a circadian variabllity that would require adding other markers to compensate for it. Finally, both T/E and 6OH-Etio-3G/EG were not able to differentiate between alcohol and testosterone administrations under the mild conditions used (transdermal testosterone and low ethanol dosage).