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A simple and reliable method for analyzing the concentrations of clozapine and its biologically active metabolite, norclozapine, in human serum or plasma has been developed. This method is based on reversed-phase high-performance liquid chromatography (HPLC) with automated solid-phase extraction (SPE). For HPLC analysis, samples and standards are prepared with an ASPEC automatic sample preparator using 100 mg Bond-Elut C18 SPE columns. The HPLC assay is an isocratic method with a mobile phase of acetonitrile-methanol-10 mM dipotassium hydrogenphosphate, pH 3.7 (30:2:100, v/v/v) at a flow-rate of 1.5 ml/min with a C8 reversed-phase column. Detection is performed with a diode array detector set at 220 nm and with peak purity analyses at 210-365 nm. The absolute recovery varied from 85 and 95%. The intra-assay coefficients of variation (C.V.s) were from 4.2 to 8.0% and the inter-assay C.V.s were from 1.1 to 9.3% at therapeutic drug concentrations. The detection limit is 15 nmol/l. The method has been developed for use in a clinical laboratory for therapeutic drug monitoring. |
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