Evaluation of Different Detection and Identification Methods of Intact Tetrahydro‐Methyltestosterone Sulfate Metabolites in Doping Control

Tetrahydro sulfate metabolites are well-established long-term biomarkers of methyltestosterone use that can be detected intact by LC-MS/MS. However, their identification using product ion spectra under collision-induced dissociation conditions in negative ion mode is an analytical challenge under the provisions of the WADA TD2023IDCR. In this study, six out of eight potential tetrahydro-methyltestosterone sulfate metabolites were microscale-synthesized to facilitate both the structural elucidation of the detected metabolites and the development of direct identification methods. Their identification was based on their GC-MS/(MS) analysis after TMS derivatization or LC-HRMS/(MS) analysis following derivatization of the free 17β-hydroxy group with carbonyldiimidazole (CDI), producing 17β-OH-imidazole carbamate derivatives. The resulting derivatives were detectable in both negative and positive ion modes, enabling their identification through characteristic product ion spectra. Urine samples from two 17α-methyltestosterone excretion studies were analyzed using these methods, and detection/identification time windows of intact sulfate metabolites were estimated under TD2023IDCR and compared with those obtained from GC-MS/(MS) analysis of the glucuronide fraction after hydrolysis. Overall, the inclusion of the tetrahydro-methyltestosterone sulfate metabolites significantly extends the detection time window for methyltestosterone abuse. Still, the established identification time window was similar to, or shorter than, that derived from the glucuronide fraction analysis.