A Stability Indicating Method Development and Validation for Separation of Process Related Impurities and Characterization of Unknown Impurities of Tyrosine Kinase Inhibitor Ibrutinib Using QbD Approach by RP-HPLC, NMR Spectroscopy and ESI-MS

A selective RP-HPLC method for separation and determination of potential-related impurities (process related and degradants) of Ibrutinib drug substance has been developed and validated. The separation was accomplished on a X-Bridge C18, (150 x 4.6 mm, 3.5 μm) column connected to a photodiode array detector using 10 mM potassium dihydrogen phosphate with 0.025% of trifluoroacetic acid (pH ~ 5.5 adjusted with KOH solution) and acetonitrile in a ratio of 85:15 respectively as mobile phase A, and 10 mM potassium dihydrogen phosphate with 0.07% of trifluoroacetic acid (pH ~ 5.5 adjusted with KOH solution) and acetonitrile in a ratio of 30:70 respectively as mobile phase B, under gradient elution. The flow rate and detection wavelength were 1.0 mL/min and 220 nm, respectively. Quality by design approach using design expert software was strategically designed to optimize the critical chromatographic parameters like column temperature, flow rate and mobile phase B, pH variation in the mobile phase to achieve the separation of process impurities and thermal degradants. Two unknown impurities found in IBT thermal stability condition at more than 0.1% in HPLC analysis were enriched and isolated by preparative HPLC and structure was confirmed by 1H NMR, 13C NMR, mass spectroscopy and FT-IR spectroscopy. This method can be used for the quality control of both drug substance and drug product. The performance of the method was validated according to the International Conference on Harmonization guidelines for specificity, limit of detection, limit of quantification, linearity, accuracy, precision, ruggedness and robustness.