AbstractThe reactions involved in the metabolic pathways of SR9009 were characterized by liquid chromatography–mass spectrometry (LC–MS) to identify the most appropriate marker(s) of use. The effects of gender, genetic polymorphism, and drug–drug interaction on the metabolic profile of SR9009 were also evaluated. In vitro approaches were based on the use of human liver microsomes and cytochrome P450 isoforms. Sample preparation included an enzymatic hydrolysis (performed only for the phase II investigation) followed by liquid–liquid extraction. The chromatographic separation was carried out using a reverse‐phase column; detection was performed by either a triple‐quadrupole or a time‐of‐flight system in positive electrospray ionization and different acquisition modes. In the presence of human liver microsomes, SR9009 was biotransformed to 13 metabolites by CYP3A4, CYP3A5, CYP2C19, and CYP2D6 isoenzymes. The reactions included hydroxylation, de‐alkylation, oxidation, and combinations thereof, the de‐alkylated metabolites being the most abundant. Once formed the mentioned metabolites underwent glucuronidation. Concerning the effects of gender, genetic polymorphism, and drug–drug interaction on the metabolic profile of SR9009, our observation have shown the following: (a) No significant alterations were measured between female and male, (b) significant differences were registered using either the CYP2D6 or CYP2C19 allelic variants, and finally (c) significant alterations were registered in the presence of ketoconazole, miconazole, fluoxetine, nefazodone and paroxetine; moderate variation were instead registered with fluconazole, itraconazole, gestodene, and levonorgestrel. This observation put in evidence the importance to take into account both genetic polymorphism and drug–drug interaction to select the most appropriate marker(s) of use in doping analysis.