Development and validation of simultaneous quantification of total and free clozapine and its two metabolites in human plasma using ultra high-performance liquid chromatography coupled to tandem mass spectrometry

Several recent pharmacokinetic studies of clozapine (CLZ) and its metabolites have reported that plasma CLZ concentrations are associated with both efficacy and adverse effects, suggesting the usefulness of therapeutic drug monitoring. Although several quantification methods for total and free drug concentrations have been established, a simultaneous quantification method for total and free concentrations of CLZ and its metabolites using ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) has not been developed. In this study, we aimed to develop a simultaneous quantitative measurement method for wide ranges of plasma concentrations of total and free CLZ and its two major metabolites, N-desmethyl CLZ (NDC) and CLZ N-oxide (CNO), using UHPLC-MS/MS. Plasma samples were prepared by solid phase extraction, and the free fraction was obtained by ultrafiltration. This method meets the validation requirements of the U.S. Food and Drug Administration. The quantification method demonstrated good linearity over wide calibration ranges for total CLZ (10–1000 ng/mL), total NDC and CNO (40–4000 ng/mL), free CLZ (2–2000 ng/mL), and free NDC and CNO (0.8–800 ng/mL). Ultrafiltration recovery rates for free CLZ, NDC, and CNO were approximately 60.0 %, 65.4 %, and 72.8 %, respectively. The total drug recovery rates ranged from 91.4 % to 107.5 %, and the free drug recovery rates ranged from 88.5 % to 117.1 %. Furthermore, we successfully measured total and free CLZ, NDC, and CNO concentrations in plasma samples of 12 patients with schizophrenia treated with CLZ. We have successfully developed and validated a method for quantitative measurement of total and free CLZ, NDC, and CNO concentrations in plasma.