A stability indicating method development and validation for separation of process related impurities and characterization of unknown impurities of tyrosine kinase inhibitor Nilotinib by RP-HPLC, NMR spectroscopy and ESI-MS

Abstract

A selective RP-HPLC method for separation and determination of potential related impurities (process related, and degradants) of Nilotinib (NTB) drug substance has been developed and validated. The separation was accomplished on YMC Triart C-18 (150 × 4.6 mm, 3.0 µm) column connected to a photodiode array (PDA) detector using 10 mM NH 4 H 2 PO 4 (pH: 3.5 ± 0.05 adjusted with diluted Ortho phosphoric acid) as a buffer and acetonitrile in a ratio of 85:15 (%v/v) respectively as mobile phase-A, and 10 mM NH 4 H 2 PO 4 buffer, acetonitrile and methanol in a ratio of 20:27.5:52.5 (%v/v/v) respectively as mobile phase B, under gradient elution. The flow rate and column oven temperature were 1.0 mL/min and 50 °C respectively. Detection was carried out at 230 nm. NTB was stressed to degrade under various stress conditions of photolysis, thermal, hydrolysis and oxidation stress as per ICH Q1A (R2). The drug was highly susceptible to acidic and alkaline hydrolytic stress conditions. The drug degraded to two degradation products (DPs), while the remains stable under thermal, photolytic, neutral hydrolytic and oxidative stress conditions. Characterization of DPs was performed by 1 D and 2 D NMR, FT-IR and LC/MS/MS. Optimized method was validated in agreement with ICH guidelines for specificity, limit of detection, limit of quantification, linearity, accuracy, precision, robustness and ruggedness. This optimized method can be used for quality control of both drug substance and drug product of NTB.