Development and Validation of a Trace-Level LC–MS/MS Method for the Determination of N-Nitroso Clozapine in Clozapine Drug Substance
Abstract:
The global pharmaceutical industry continues to face significant regulatory scrutiny due to the presence of nitrosamine impurities and nitrosamine drug substance related impurities (NDSRIs), which are recognized as potent mutagenic and carcinogenic compounds. A key analytical gap remains in the availability of a validated, robust, and highly sensitive LC–MS/MS method for the determination of N -nitroso clozapine in clozapine drug substance. In the present work, selective, and sensitive LC–MS/MS method was developed using multiple reaction monitoring (MRM) in positive electrospray ionization mode and subsequently validated in accordance with International Council for Harmonisation (ICH) guidelines. Chromatographic separation was achieved on a Shim-pack Scepter C18-120 column (150 × 4.6 mm, 3.0 µm) using gradient elution with 10 mM ammonium formate containing 0.1% formic acid in water as mobile phase A and 0.1% formic acid in methanol as mobile phase B, at a flow rate of 0.5 mL/min and a column temperature of 40°C. The MRM transitions m/z 356.0 → 326.1 (quantifier) and m/z 356.0 → 243.1 (qualifier) were employed for N -nitroso clozapine. The method demonstrated excellent linearity (r 2 ≥ 0.999) over the validated range from the LOQ to 200%, with acceptable precision (%RSD < 5%), accuracy (97.52%–115.30%), and robustness. The limits of detection and quantification were 0.0539 µg/mL and 0.165 µg/mL, respectively. The validated method is suitable for routine quality control, stability assessment, and regulatory compliance for monitoring trace level N -nitroso clozapine in clozapine drug substance.