Simultaneous determination of clozapine, norclozapine and clozapine-N-oxide in human plasma by high-performance liquid chromatography with ultraviolet detection

A reversed-phase high-performance liquid chromatographic (HPLC) method for the simultaneous determination of clozapine and its two major metabolites, norclozapine and clozapine-N-oxide in human plasma has been developed and v alidated. The isocratic HPLC assay uses a mobile phase consisting of an ace tonitril-buffered aqueous solution containing 146 muL of triethylamine and 200 muL of 85% phosphoric acid, adjusted to pH 3.3 with 10% potassiumhydrox ide solution (400:600, v/v) at a flow-rate of 0.8 ml/min and a Lichrospher 100 RP-18 reversed-phase column and UV detection at 215 nm. Doxepine was us ed as the internal standard. Mean recoveries for clozapine, norclozapine, c lozapine-N-oxide and doxepine were 95%, 98%, 96% and 94% respectively, wher eas the respective mean repeatability coefficients of variation were 3.4%, 2.7%, 4.3% and 0.9%. Reproducibility coefficients of variation were 1.3%, 1 .8%, 3.6% and 0.5%, respectively. The mean correlation coefficient for the linear calibration curve (n = 2) for clozapine and norclozapine at a concen tration range of 100-1600 ng/mL was 0.9998 and 0.9997, respectively; for cl ozapine-N-oxide (20-200 ng/mL) it was found to be 0.9986, The lower limits of quantitation were 12.5 ng/mL, 10 ng/mL and 12.5 ng/mL for clozapine, nor clozapine and clozapine-N-oxide, respectively. Copyright (C) 2001 John Wile y & Sons, Ltd.