Inter-laboratory validation of two steroid profile markers with improved specificity (InterALMA)

Code: 20D09OP

In previous WADA funded projects (11A9RV, 12A13OP, 14A29OP) our research group discovered two endogenous steroid (EAAS) metabolites, namely, 6β-hydroxyandrosterone-3-glucuronide (6bOHA-3G), and 6β-hydroxyetiocholanolone-3-glucuronide (6bOHEtio-3G) particularly selective markers of the administration of testosterone. Small quantities of the compounds were synthesized and fully characterized. They showed to be resistant to enzymatic hydrolysis, a reason for its late discovery.

An analytical method has already been developed, fully validated and published for the direct analysis of those substances together with other regular steroid profile glucuronide markers. One way to improve the the application of the method would be to have access to labelled internal standards which will decrease the variability of the basal values.

These two new markers have proven to be sensitive to testosterone administration (oral, intramuscular and even transdermal, improving its detection window, as compared to the current T/E ratio. We are  currently testing their specificity to differentiate testosterone administration from ethanol consumption or the combination (WADA project ISF18D13OP). Preliminary results of this on-going project show that while TG/EG increases both with T and EtOH administration, the new markers increase specifically after T administration while they do not increase after EtOH administration. This differential behavior adds specificity and improves the steroid profile. 

The current project (inter-ALMA) aims at finally incorporating these new markers into a routine screening by performing an interlaboratory validation incorporating 5 different laboratories. For that purpose, analytical methodology, standards (already synthesize in the frame of the 2019 PCC project entitled “Essential reference materials to advance the long term detection of testosterone abuse”) and their deuterated analogues (synthesized in the frame of iner-ALMA) will be provided to the laboratories. The outcome after using common or comparable methodologies to analyze selected samples will be compared.