Development and Validation of a Candidate Reference Measurement Procedure for Plasma Clozapine Quantification Using ID-LC–MS/MS

Abstract

We developed a candidate reference measurement procedure (cRMP) based on isotope dilution liquid chromatography–tandem mass spectrometry (ID-LC–MS/MS) for the accurate quantification of clozapine (CLO) in human plasma. After systematic optimization of chromatographic separation conditions for CLO, we evaluated various sample pretreatment methods. The analytical performance of the cRMP was rigorously validated, encompassing specificity, recovery, precision, linearity, limit of quantitation (LoQ), limit of detection (LoD), carryover, stability, and method comparison. Measurement uncertainties were assessed in accordance with GUM, taking CLO purity, balance weighing, calibration curve, measurement imprecision, recovery, and carryover into account. Under optimized conditions, CLO showed effective separation from potential interferents in human plasma. The method exhibited excellent linearity (R² = 0.9988) across a concentration range of 5.65–1693.51 ng/g. The total coefficients of variation (CVs) were 2.04%, 0.97%, and 0.65% at concentrations of 24.53, 98.06, and 987.02 ng/g, respectively. Average recoveries ranged from 97.80% to 99.28%, with a LoQ of 2.73 ng/g and a LoD of 0.91 ng/g. The expanded measurement uncertainties (U) were 1.3 ng/g (k = 2) at 24.53 ng/g, 3.8 ng/g (k = 2) at 98.06 ng/g, and 35.3 ng/g (k = 2) at 987.02 ng/g. The developed cRMP for CLO quantification demonstrates excellent specificity, accuracy, precision, and traceability, meeting all the criteria for cRMPs. This method has the potential to standardize therapeutic drug monitoring (TDM) for CLO, improving clinical outcomes and patient safety.