A simple, simultaneous and reliable HPLC method was developed for analysis of clozapine that is a widely prescribed drug especially for treatment of schizophrenia, but may have fatal toxicities in some cases. Its two metabolites are norclozapine and clozapine N-oxide in human plasma. Chromatographic conditions and detection parameters were adjusted in consequence of optimization study. An isocratic high-performance liquid chromatography method with ultraviolet detection at 220nm was utilized. Analytes are concentrated from plasma by liquid–liquid extraction with ethyl acetate, n-hexane and isopropylalcohol (80:15:5,v/v/v) which allows to obtain good extraction yields (>80%) for all analytes. Separation was performed on a C18 reversed-phase column using a mixture of acetonitrile and 62.4mM phosphate buffer (containing 0.3%triethylamine, pH4.5) at the ratio of 40:60(v/v). Method showed linearity with excellent correlation coefficients (r2>0.999) for each analyte. The relative standard deviations and relative standard errors calculated to present precision and accuracy between and withinday assay were less than 4% for low concentrations. The method was specific and sensitive with detection limits of 23.6μg/L, 19.3μg/L and 23.6μg/L for clozapine, norclozapine and clozapine N-oxide respectively. The procedure described is relatively simple, precise, and applicable for routine therapeutic drug monitoring especially in psychiatry clinics or in toxicology reference laboratories.

Keywords: Clozapine, Clozapine-N-oxide, Norclozapine, HPLC-UV, Optimization