Identification and characterization of the human urinary metabolites of trimetazidine using liquid chromatography high resolution mass spectrometry, an anti-doping perspective

Trimetazidine (TMZ) is a clinically effective anti-angina and anti-ischemic metabolic drug used to treat chest pain. It can also improve the exercise capacity of athletes and has been classified by the World Anti-Doping Agency (WADA) as a cardiac metabolism modulator (S4.5C); thus, it has been banned in-competition and out of-competition. To date, phase I metabolism is main metabolic pattern of TMZ, and a total of 11 phase I metabolites were reported. In this study, TMZ metabolic profiles were comprehensively studied. Five healthy volunteers (two males and three females) took TMZ tablets and their urine samples were collected over one month. The sample were cleaned via solid-phase extraction (SPE) without enzymatic hydrolysis, and the urine extracts were analyzed via Quadrupole-Orbitrap liquid chromatography-mass spectrometry (Quadrupole-Orbitrap LCMS/MS) with accurate mass measurements. Trimetazidine and 35 metabolites were detected. The chemical structures of 29 new phase II metabolites were elucidated especially base on their fragmentation patterns and accurate molecular formula determination. Six previously reported TMZ metabolites were also found. Nine metabolic pathways, namely, demethylation, glucuronidation, sulfonation, C-oxidation, N-oxidation, N-acetylation, N-formylation, N-methylation, hydroxylation and combinations, were provided, among which glucuronidation, one of the main metabolic pathways, has not been previously reported. The detection times of the 35 metabolites were compared with that of the parent drug, and the results showed the potential of two new biomarkers M8 and M9, identified as TMZ-N-glucuronide and O-demethyl-N-methyl-TMZ-O-glucuronide, respectively, for monitoring TMZ misuse.