This study investigated the main metabolites of the compound via in vitro incubation with human liver microsomes (HLM) and detection by LC-HRMS for doping control. Six Phase I metabolite types—namely, 17-epimerization, hydroxylation, C3-keto reduction, 18-nor formation, reduction, and demethylation—and five Phase II metabolites—mainly glucuronides—were identified, indicating extensive HLM metabolism.

ABSTRACT

Methyldienolone, a synthetic anabolic androgenic steroid (AAS), has been banned in sports by the World Anti-Doping Agency (WADA) because of its performance-enhancing properties. This study aimed to investigate the main metabolites using in vitro incubation with human liver microsomes (HLM) and to detect them through liquid chromatography-high-resolution mass spectrometry (LC-HRMS) for doping control purposes. A total of six groups of Phase I metabolites, including 17-epimerization, hydroxylation, C3-keto reduction, 18-nor modifications, reduction, and demethylation, as well as five different Phase II metabolites, such as glucuronide conjugates, were characterized, indicating extensive metabolism by HLM. Structural characterization of these metabolites was improved through derivatization with methoxylamine and hydroxylamine, which enabled their detection with higher sensitivity by LC-HRMS. These novel metabolites provide new insights into the metabolism of methyldienolone and may contribute to antidoping analysis. The synthesis of reference materials is necessary to confirm the structure of the proposed metabolites in the future.