In force

Improving the long-term detection of testosterone and testosterone prohormone misuse in athletes

Principal investigator
M. Thevis
German Sport University
Year approved
Anabolic steroids

Project description

Code: 18C02MT 

Testosterone misuse still remains a challenging task for doping control laboratories as this steroid is produced naturally by each and everybody. As it is detectable in all urine samples, concentration-based thresholds have been established to uncover testosterone administration. As soon as these thresholds are exceeded, samples are forwarded to isotope ratio mass spectrometry determinations (IRMS) to elucidate the steroid´s source and to unambiguously differentiate between naturally elevated testosterone concentrations and doping.

Recently a promising new target analyte for IRMS determinations was reported, epiandrosterone (EPIA). This steroid enables to prolong the detection of a single testosterone or testosterone prohormone administration from 24 h to more than 100 h using IRMS. Unfortunately, EPIA is only excreted into urine in it sulfoconjugated form while all other steroids routinely employed in IRMS are excreted glucuronidated. To investigate EPIA an additional time consuming step in sample preparation is inevitable.

The aim of this study is to investigate the potential of two other possible long term markers in IRMS excreted glucuronidated and therefore avoiding the additional step. The so called “Epidiols” are known long term metabolites of epitestosterone and might also work for other steroid administrations. In a preliminary study employing dehydroepiandrosterone both Epidiols were remarkably influenced by the steroid administration supporting the hypothesis that these new markers will improve steroid detection by IRMS. To further substantiate this finding we are going to re-analyze other excretion studies after improving and validating the already existing method for IRMS determinations of both Epidiols.

Main Findings: 

A novel IRMS method for determination of carbon isotope ratios (CIR) of 5a- and 5bEpiD was developed and fully validated in line with WADA requirements encompassing limits of detection, linear range and absence of isotopic fractionation. By means of investigations on a reference population encompassing n=72 individuals it was possible to derive referece-based decision limits, which demonstrated that the novel method is fit-for-purpose. Several administration trials were investigated to elucidate the potential of both markers. For a single oral T administration, the effect on both 5a- and 5bEpiB was less pronounced compared to traditional markers and offreed merely short detection windows. After consecutive transdermal T-Gel administrations, especially 5bEpiD was significantly influenced while 5aEpiD shows similar CIR,s as 5a- and 5bDIOL. Unfortunately, the first sample after cessation of T-Gel administration was collected after 5 days, and here all CIR values had returnedd to natural abundance. The administration of a single oral dose of androstenedione resulted in a prolonged depletion of CIRs for 5a- and 5bEpiD compared to other target compunds, but the influence was less pronounced, i.e. the CIR values were not found to be influenced beyond the established reference-based decision limits.

While a direct application of 5a- and 5bEpiD as long-term markers for steroid administrations appears to offer limited added value, these additional target compounds may be beneficial in detecting multiple transdermal T-Gel administration even if so called low-doses are applied. Moreover, they could support the differentiation between single administrations (including inadvertent exposure) and multiple and therefore intended administrations. Further research will be necessary to elucidate this potential of 5a- and 5bEpiD.