Separation and Quantitative Estimation of Pharmacopeial Organic Impurities, Process-Related Impurities, and Degradation Products of the Anti-Schizophrenia Drug Clozapine in Pharmaceutical Formulations by Using Stability-Indicating HPLC Method

Abstract

Clozapine is an antipsychotic medication primarily used to treat severe schizophrenia symptoms. This research aimed to develop and validate a reliable, rapid, and sensitive reverse-phase high-performance liquid chromatography (RP-HPLC) method for measuring organic impurities in clozapine drug substances and products. The chromatographic separation was achieved using an Agilent Poroshell 120 EC-C₁₈ column with gradient elution, using mobile phases consisting of acetonitrile, methanol, pH 2.4 phosphate buffer, and ethanol. The method operated at a flow rate of 1.0 mL/min, with a 10-μL injection volume, a column temperature of 35°C, and detection at 257 nm. Validation was performed according to ICH Q2(R2) guidelines and USP <1225> general chapter. The method demonstrated optimal resolution between adjacent peaks, with values greater than 1.5, and provided accurate estimations free from interference. Recovery and regression values ranged from 80.0% to 120.0%, with R² values exceeding 0.9995. The quantification range spanned from the limit of quantification to 150% of the specification level. The stability-indicating capability of the RP-HPLC method was confirmed through forced degradation studies. This method has been successfully implemented in a quality control laboratory for real-time analysis of clozapine drug substances and products.