AbstractRetention, separation selectivity and system efficiency of selected basic psychotropic drugs (clozapine, aripiprazole, vortioxetine and zolpidem) and drug metabolites (desmethylclozapine, clozapine N-oxide and dehydroaripiprazole) on Hydro RP, Phenyl-Hexyl and Polar RP columns were studied. Mobile phases containing methanol or acetonitrile as organic modifiers, acetate buffer at pH 3.5 and addition of diethylamine (DEA) as a silanol blocker were applied. Significant differences in the retention, peak shapes and systems’ efficiency of the investigated compounds were obtained depending on the tested chemically bonded stationary phases with various ligands. Based on the obtained results the Phenyl-Hexyl column was selected for analysis of the drugs and their metabolites in human serum and saliva samples. Solid phase extraction (SPE) was applied for sample pre-treatment. The best SPE-HPLC-DAD procedure was used for simultaneous analysis of clozapine, aripiprazole and their metabolites in body fluids. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was applied for confirmation of the presence of the investigated compounds in biological samples. The lower limit of quantification (LLOQ) of clozapine obtained using the proposed method was 10 ng/mL. The validated method for determining the presence of clozapine and its main metabolite was successfully applied in therapeutic drug monitoring.