Evaluation of two glucuronides resistant to enzymatic hydrolysis as markers of testosterone oral administration

Abstract

Testosterone (T) has traditionally been the most commonly reported doping agent by doping control laboratories. The screening of T misuse is performed by the quantification of six endogenous androgenic steroids and the ratio T/E included in the Athlete Biological Passport (ABP). The inclusion of additional metabolites can improve the screening capabilities of ABP. In this study, the potential of 3α-glucuronide-6β-hydroxyandrosterone (6OH-Andros3G) and 3α-glucuronide-6β-hydroxyetiocholanolone (6OH-Etio3G) as markers of T oral administration was evaluated. These glucuronides have been shown to be resistant to enzymatic hydrolysis and their quantification by means of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was reported as the only way to obtain feasible results. Urine samples were collected from five volunteers before and after the oral administration of 40mg of T undecanoate and were analyzed by a LC-MS/MS method recently developed. Concentration of 6OH-Andros3G and 6OH-Etio3G compounds and those of the glucuronides of T (TG), epitestosterone (EG), androsterone and etiocholanolone were established and different concentration ratios were calculated. The detection windows (DWs) for the T administration obtained by each selected ratio were compared to the one of TG/EG. The results showed that four out of the nine tested markers presented DWs much larger for all volunteers than those obtained by the World Anti-Doping Agency established T/E marker or other alternative markers. The 6OH-Andros3G/EG, 6OH-Etio3G/EG, 6OH-Andros3G/TG and 6OH-Etio3G/TG markers were able to identify the T abuse up to 96h after the administration, extending our detection capability for the misuse up to 84h more than the classic marker. The importance of these markers was also highlighted by their prolonged capacity to detect the T misuse in the case of one volunteer whose TG/EG barely exceeded his individual threshold. As a consequence, the four markers presented in this study seem to have an exceptional potential as biomarkers of T oral administration.

Evaluation of markers out of the steroid profile for the screening of testosterone misuse. Part I: Transdermal administration

“…All calculated basal values were in agreement with the population ranges previously reported for these compounds . As previously reported, both M‐T/E and L‐T/E groups showed similar levels of cysteinyl markers .…”

“…Similarly, for the evaluation of the glucuronoconjugated metabolites, ratios between the concentrations of both 6OH‐Andro3G and 6OH‐Etio3G with either TG or EG were selected, since they have been reported for the screening of the T oral administration …”

“…Additionally, the ratios Δ 1 ‐AED/Δ 15 ‐AD, Δ 6 ‐AED/Δ 15 ‐AD and Δ 6 ‐T/Δ 15 ‐AD managed to detect the administration of a single transdermal dose of T gel while the classic T/E marker failed to do so . Moreover, 2 glucuronoconjugated metabolites of T, 3α‐glucuronide‐6®‐hydroxyandrosterone (6OH‐Andros3G) and 3α‐glucuronide‐6®‐hydroxyetiocholanolone (6OH‐Etio3G), which are resistant to enzymatic hydrolysis were recently evaluated after oral administration of T undecanoate . The urinary concentrations of both glucuronides, showed a dramatic increase after the administration.…”

Direct quantitation of endogenous steroid sulfates in human urine by liquid chromatography‐electrospray tandem mass spectrometry

“…Consequently, an important research activity was developed in the recent years to look for additional markers, [5][6][7][8][9][10][11][12][13][14] including other endogenous metabolites excreted in the glucuronide fraction, 5,6 metabolites excreted as conjugates with cysteine, 7,8 or glucuronide metabolites resistant to enzymatic hydrolysis. Consequently, an important research activity was developed in the recent years to look for additional markers, [5][6][7][8][9][10][11][12][13][14] including other endogenous metabolites excreted in the glucuronide fraction, 5,6 metabolites excreted as conjugates with cysteine, 7,8 or glucuronide metabolites resistant to enzymatic hydrolysis.…”

Evaluation of markers out of the steroid profile for the screening of testosterone misuse. Part II: Intramuscular administration

“…In summary, the data obtained in the 2 parts of this study together with the previously published data for other administration routes provide the most comprehensive insight about the usefulness of cysteinyl metabolites, resistant glucuronoconjugates, and 1‐CPG in the screening of T misuse.…”