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

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.