Development and validation of an HPLC method for the simultaneous determination of clozapine and desmethylclozapine in plasma of schizophrenic patients

Summary A high-performance liquid chromatographic method with amperometric detection has been developed for the determination of levels of clozapine (CLZ) and its active metabolite N-desmethylclozapine (DMC) in human plasma. The analysis was performed on a 5 μm C8 reversed phase column (150×4.6 mm i.d.), with acetonitrile-phosphate buffer (pH 3.5), as the mobile phase. The detection voltage was +800 mV and the cell and column temperature were 50°C. Linear responses were obtained between 2 ng mL⁻¹ and 100 ng mL⁻¹. Absolute recovery for both clozapine and desmethylclozapine exceeded 88% and the detection limit was 1 ng mL⁻¹. Repeatability, intermediate precision and accuracy were satisfactory. The method, which is rapid, sensitive and selective, has been applied to therapeutic drug monitoring in schizophrenic patients following administration of Leponex^? tablets. In 21 patients in steady state at a mean daily clozapine dosage of 358 mg (ranging from 150 to 500 mg day⁻¹), clozapine levels averaged 379 ng mL⁻¹ (ranging from 102 to 818 ng mL⁻¹) and DMC levels averaged 233 ng mL⁻¹ (ranging from 70 to 540 ng mL⁻¹). The method requires only a very small amount of plasma (100 μL), and thus it is suitable for pharmacokinetic studies, as well as for therapeutic drug monitoring.