RP-LC Method Development and Validation for Dasatinib Forced Degradation Study: Isolation and Structural Characterization by NMR and HRMS.

Abstract

A reverse phase high-performance liquid chromatography (HPLC) method has been developed for the quantification of a typical drug Dasatinib (DST) and its related impurities in pharmaceuticals. Kinetex C18 (4.6 × 150mm, 5μm) column was used in the chromatographic separations, using buffer (1.36g of KH2PO4 in 1000mL of water, pH = 7.8; adjusted with diluted KOH solution) with solvent as acetonitrile and mode of elution as the gradient. The flow rate is 0.9mL/min, column oven temperature as 45°C and the overall gradient run time as 65min. The developed method was found to produce symmetric and good separation between the process-related and degradation impurities. Method optimization is achieved with photodiode array at 305nm over the concentration range of 0.5mg/mL and degradation studies were carried out under acidic, alkaline, oxidative, photolytic and thermal conditions to demonstrate the stability indicating capability of the method. Two major impurities were found in forced degradation studies in the HPLC analysis, the unknown, acid degradants were enriched and isolated by preparative HPLC, then characterized through high-resolution mass spectrometry, nuclear magnetic resonance spectroscopy and Fourier transform infrared spectroscopy. The unknown acid degradation impurity was showing Exact Mass of 521.11, molecular formula C22H25Cl2N7O2S and its chemical name as 2-(5-chloro-6-(4-(2-hydroxyethyl) piperazin-1-yl)-2-methylpyrimidin-4-ylamino)-N-(2-chloro-6-methylphenyl) thiazole-5-carboxamide. Another impurity (oxidative degradant) found as known DST N-oxide Impurity-L and its chemical name as 4-(6-((5-((2-chloro-6-methylphenyl) carbamoyl) thiazol-2-yl) amino)-2-methylpyrimidin-4-yl)-1-(2-hydroxyethyl) piperazine 1-oxide. The analytical HPLC method was further validated as per ICH guidelines.