Long-term detection of methyltestosterone administration: unambiguous metabolite identification in a controlled trial

Code: 241C05MP

Recently, the success of the integration of new long-term markers for anabolic steroids such as metandienone greatly improved the detectability of their misuse in sports. Tetrahydromethyltestosterone-sulfate (THMTS) was proposed as a promising long-term marker for methyltestosterone administration, however, reference material for direct analysis of the intact conjugates is not available for all diastereomers. Reportedly, the longest detectable diastereomer was tentatively assigned as 3α5α17epiTHMTS after solvolysis using GCMS and literature reported Kovac indices for diastereomer allocation. Our preliminary data from intact sulfoconjugate analysis suggest 3α5βTHMTS instead. It remains open until now weather this difference is due to potential epimerization by solvolysis, sample pre-treatment or interindividual variability. Additionally, A-Ring reduced 17β-hydroxymethyl-13-ene (20OHNorTHMT) metabolites have not yet been consequently investigated after methyltestosterone administration. Analogous metabolites resulted in drastic extension of the detection window after Oral Turinabol administration.

Within the project the analysis of tetrahydromethyltestosterone (THMT) diastereomers as classically monitored metabolites of methyltestosterone, A-Ring reduced 17β-hydroxymethyl-13-ene metabolites (20OHNorTHMT), as well as intact sulfoconjugates, will be included in the analysis to evaluate the best suited metabolites after an administration of methyltestosterone.

Therefore, methods will be optimized for sensitive and selective detection using the various diastereomeric sets of target analytes. Furthermore, the suitability of different possibilities for sample preparation will be evaluated specially focusing on the sulfoconjugates. Their diastereomeric structure will unambiguously be confirmed by direct analysis of the intact conjugates and comparison with references of all diastereomers. Full characterization of the relevant THMTS diastereomers with LC-HRMS and NMR will be provided after chemical synthesis and purification. Including postadministration samples from six volunteers will help for investigation of interindividual variabilities of metabolite excretions.

Main findings

164 antibodies were tested regarding their sensitivity in comparison with clone AE7A5 EPO antibody. Out of 70 polyclonal and 94 monoclonal antibodies a clear favourite was identified. It surpassed clone AE7A5 not only by sensitivity but also by specificity. The antibody is a recombinant rabbit monoclonal antibody (clone HL1794) and is worldwide available from the manufacturer (Genetex) as well as large international distributors (e.g. Abcam, ThermoFisher Scientific, Fisher Scientific). The antibody was fully validated for urine using an HRP-labelled and cross-reactivity minimized secondary antibody. It allowed the detection of all ERAs (rEPO, NESP, EPO-Fc, CERA) down to at least 0.1 MRPL of TD2024EPO. Similar results were obtained serum samples. The antibody did also not bind to proteins known to be nonspecifically bound by clone AE7A5 (ZAG, TrxR, NSE, NNE, enolase). Conclusions: Anti-EPO antibody clone HL1794 outperformed the current detection antibody clone AE7A5 regarding sensitivity and specificity. Thus, it is a very promising antibody, which will further improve detection of ERA-doping.