Bioanalytical Method Development and Validation for The Estimation of Metformin and Empagliflozin in Blood Plasma by using RP-HPLC

The development and validation of a bioanalytical method for the simultaneous estimation of metformin (MET) and empagliflozin (EPG) in blood plasma using reversed-phase high-performance liquid chromatography (RP-HPLC) are presented. The method development involved optimizing chromatographic conditions, including the mobile phase composition, flow rate, and detection wavelength, to achieve well-resolved peaks for both analytes. The validation process adhered to regulatory guidelines, assessing precision, accuracy, specificity, sensitivity, linearity, and stability. The RP-HPLC method utilized a C18 column with a mobile phase consisting of phosphate buffer and acetonitrile (pH 3) in a 20:80 (v/v) ratio, a flow rate of 1.0mL/min, and UV detection at 254nm. The retention times for MET and EPG were 5.1 and 7.1 minutes, respectively. The method demonstrated excellent linearity over the concentration ranges of 10-50ppm for MET and 2-10ppm for EPG, with correlation coefficients (r²) exceeding 0.999. The intra-day and inter-day precision, expressed as relative standard deviation (RSD), were below 2% for both analytes, indicating high reproducibility. Accuracy, evaluated through recovery studies, ranged from 98.5% to 101.2%, confirming the method's reliability. Specificity tests showed no interference from endogenous plasma components, and the limits of detection (LOD) were 0.25g/ml L for MET and 0.75g/ml for EPG. Stability studies under various conditions confirmed that both analytes remained stable with negligible degradation. The validated method was successfully applied to pharmacokinetic studies, demonstrating its practical utility in clinical and research settings.