Solid-phase extraction and high-performance liquid chromatographic analysis of clozapine and norclozapine in human...

A rapid high-performance liquid chromatographic method for the simultaneous quantitation of plasma clozapine and its principal metabolite, norclozapine, was developed using a Spherisorb C8 5-micron reverse-phase column and an acetonitrile/potassium phosphate buffer/triethylamine mobile phase. Clozapine and norclozapine were extracted from plasma on solid-phase reusable microcolumns. Absolute recovery of clozapine, norclozapine, and the internal standard, flurazepam, was quantitative. The mean intraassay coefficient of variation was better than 6% for clozapine and norclozapine at therapeutic concentrations. Based on a plasma sample volume of 200 microliters, the limit of accurate quantitation of both clozapine and norclozapine was approximately 25 micrograms/L (coefficients of variation = 11.4% and 8.4%, respectively). The method has been used over the past 2 years for single-dose pharmacokinetic studies and for monitoring patients receiving clozapine therapy.