Development and validation of a stability-indicating LC-MS/MS method for simultaneous estimation of empagliflozin and metoprolol in pharmaceutical dosage forms with forced degradation studies and computational assessment of degradation products.

The empagliflozin and metoprolol FDC is currently in clinical evaluation (Phase III) for the management of type 2 diabetes mellitus associated with hypertension in India. The present study was carried out with the objective to develop and validate a sensitive, selective and stability indicating LC-MS/MS method for the simultaneous determination of empagliflozin and metoprolol in bulk drug substances and tablet formulations. Chromatography was performed using a Phenomenex Kinetex® C18 column (150 × 4.6 mm, 2.6 μm, 100 Å) with 5 mM ammonium formate in water with 0.1% formic acid (mobile phase A) and methanol with 5 mM ammonium formate and 0.1% formic acid (mobile phase B). The method showed good linearity for empagliflozin within the range of 100-4000 ng mL-1 and for metoprolol within the range of 250-10 000 ng mL-1 with correlation coefficients (R2) of 0.999 for both analytes. Validation in accordance with the ICH Q2(R2) guidelines demonstrated good accuracy, precision, specificity, robustness and sensitivity. Stability-indicating capability of the method was confirmed from forced degradation studies under acidic, alkaline, oxidative, thermal and photolytic conditions and tentative characterization of degradation products was achieved based on LC-MS/MS fragmentation patterns. To get a preliminary idea of their potential biological and toxicological significance, the suggested degradation products were further assessed using molecular docking and in silico toxicity prediction tools. The developed method offers a reliable analytical platform for routine quality control, stability assessment and degradation profiling of empagliflozin and metoprolol in pharmaceutical development.