Systematic optimization of reverse phase ultra‐high‐performance liquid chromatography method for quantification of nilotinib and its related substances in bulk drug and pharmaceutical formulation using quality by design approach

Abstract

A sensitive ultra‐high‐performance liquid chromatography (UHPLC) method was developed for the quantification of trace levels of nilotinib and its related substances (RSs) in the nilotinib capsule dosage form. Critical method parameters were optimized using the design of experiments, employing the ACQUITY UHPLC BEH Phenyl column (2.1 × 100 mm, 1.7 μm) at a constant flow rate of 0.6 mL/min. The isocratic mobile phase, consisting of aqueous and organic phases, achieved efficient chromatographic separation within 8 min at 261 nm. The mean retention time for nilotinib was 6.1339, and for RSs, it ranged from 2.10 to 6.91 min, with a resolution exceeding 2 for all peaks. The method demonstrated robustness, separating known impurities and degradation products. The linearity was assessed over a concentration range of 0.02–80 ppm for nilotinib and 0.015–0.12 ppm for impurities. The limit of detection and limit of quantitation (LOQ) for nilotinib were 0.01 and 0.02 μg/mL, respectively, and 0.01 and 0.015 μg/mL for individual impurities. Recovery studies at LOQ, 100%, and 150% of the specification limit yielded percent recoveries ranging from 92.27% to 102.45%. Precision results showed low relative standard deviations, below 2% for nilotinib and below 8% for impurities. This method is deemed suitable for pharmaceutical formulation quantification.