Bioanalytical Method Development and Validation for The Estimation of Metformin and Empagliflozin in Blood Plasma by using RP-HPLC
Abstract
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 0.25g/ml L for MET and 0.75g/ml for EPG. Stability studies under various conditions confirmed that both…
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