High performance liquid chromatography coupled with triple-quadrupole mass spectrometry was applied in the determination of in vitro metabolism products of selected antibiotic drugs (cefotaxime, ciprofloxacin, fluconazole, gentamicin, clindamycin, linezolid, and metronidazole). The analytes were separated on a reversed phase C18 column, with acetonitrile and 0.1 % aqueous formic acid as the mobile phase. Tandem mass spectrometry with positive electrospray ionization was used to facilitate the structural characterization of the potential metabolites. Metabolism studies on human liver microsomes were performed via cytochromes P450 (phase I) and via NADPH/UDP-glucuronosyltransferase (phase II) mediated reactions. LC-MS/MS experiments allowed potential metabolite peaks, including sum formulae suggestions, to be identified; high resolution MS/MS experiments led to the identification of various oxidative and reductive modifications of target compounds in phase I biotransformation, and conjugation products with glucuronic acid in phase II reactions. A total of 11 potential metabolites and their proposed structures were characterized during the incubation of human liver microsomes by comparing their retention times and spectral patterns with those of the parent drug. Dehydrogenation and reactions of side chains such as hydroxylation and hydrolysis of ester bonds constituted the major metabolic pathways. Finally, LC-MS/MS spectrometry was revealed to be a suitable analytical tool to procure a feasible analytical base for the envisioned in vivo experiments.

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