Identification and characterization of in vitro phase I and reactive metabolites of masitinib using a LC-MS/MS method: bioactivation pathway elucidation

Masitinib is a selective tyrosine kinase inhibitor (TKI). It is currently registered in Europe for the treatment of mast cell tumors in dogs. The current study reports the identification and characterization of fourteen phase I metabolites of masitinib by reversed phase liquid chromatography triple quadrupole mass spectrometry (LC-QqQ-MS). Phase I metabolic reactions were reduction, demethylation, hydroxylation, oxidation and N-oxide formation. Structures of the proposed phase I metabolites showed high lability to form reactive metabolites. So incubation was performed in the presence of 1.0 mM GSH or 1.0 mM KCN to check for reactive metabolites. No GSH adduct was found, while eight cyano adduct structures were determined based on full MS scan and MS2 scan data for each metabolite. Interestingly, a literature review showed no previous studies have been made on the in vitro metabolism of masitinib or detailed structural identification of the formed metabolites.