Development of novel gradient RP-HPLC method for separation of dapagliflozin and its process-related impurities: insight into stability profile and degradation pathway, identification of degradants using LCMS

Abstract

Abstract Background In the dapagliflozin (DPF) synthesis, 5-Bromo-2-chlorobenzoic acid (5-BC impurity) and 4-Bromo-1-chloro-2-(4-ethoxybenzyl) benzene (4-BC impurity) are used as starting and reagent sources, respectively. The presence of 5-BC and 4-BC impurities in DPF could potentially affect the effectiveness of the final DPF product. The purpose of this investigation was to develop a stability indicating HPLC methodology for the separation of DPF, its process-related impurities and degradants. The method of analysis was developed on Xbridge Phenyl C18 column of dimensions, 250 × 4.6 mm, 5 μm with gradient elution using mobile phase made up of 0.05% aqueous trifluoroacetic acid and acetonitrile (AcN). Results The method proposed indicates a good linearity (R 2 = 0.9996 and 0.9993), good system precision (RSD ≤ 2%), good method precision (RSD ≤ 2%), accuracy (50–150%), LOD (0.000053 ppm and 0.0000165 ppm) and LOQ (0.00016 ppm and 0.00005 ppm) for 4-BC impurity and 5-BC impurity, respectively. LC–MS was used to detect and characterise degradants that were obtained in acid and base condition were identified and characterised. A comparison of the fragmentation pattern of the [M + H] + ions of DPF and its degradation products revealed the most likely processes for the generation of degradation products. Conclusion DPF sample quality can be evaluated using the suggested method for the presence of 4-BC impurity, 5-BC impurity and 2-(3-(4-ethoxy benzyl)-4-chloro phenyl)-tetrahydro-6-(hydroxy methyl)-2H-pyran-3,4,5-triol.

Keywords

• Process impurities
• Dapagliflozin
• HPLC
• Xbridge Phenyl
• Gradient elution