DETERMINATION OF CLOZAPINE AND ITS MAJOR METABOLITES IN HUMAN PLASMA AND RED-BLOOD-CELLS BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH ULTRAVIOLET ABSORBENCY DETECTION

An isocratic high-performance liquid chromatographic (HPLC) method wit h UV absorbance detection is described for the quantification of cloza pine '-methyl)piperazino-5H-dibenzo[b,e]-1,4-diazepine) and its two ma jor metabolites in plasma and red blood cells (RBCs). The method invol ves sample clean-up by liquid-liquid extraction with ethyl acetate. Th e organic phase was back-extracted with 0.1 M hydrochloric acid. Loxap ine served as the internal standard. The analytes were separated by HP LC on a Kromasil Ultrabase C-18 analytical column (5 mu m particle siz e; 250X4.6 mm I.D.) using acetonitrile-phosphate buffer pH 7.0 (48:52, v/v) as eluent and were measured by UV absorbance detection at 254 nm . The limits of quantitation were 20 ng/ml for clozapine and N-desmeth ylclozapine and 30 ng/ml for clozapine N-oxide. Recovery from plasma o r RBCs proved to be higher than 62%. Precision, expressed as % C.V., w as in the range 0.6-15%. Accuracy ranged from 96 to 105%. The method's ability to quantify clozapine;md two major metabolites simultaneously with precision, accuracy and sensitivity makes it useful in therapeut ic drug monitoring.