RP-HPLC Method Development and Validation for Simultaneous Estimation of Metformin, Dapagliflozin and Glimepiride in fixed dose Combinations

For the concurrent assessment of Metformin (MET), Dapagliflozin (DAPA) and Glimepiride (GLIME) in fixed-dose combinations, a straightforward, exact, accurate, and unique method has been established. We utilized RP-HPLC, a sort of high-performance liquid chromatography, to accomplish this estimate. The method employs a mobile phase comprising 350 ml of buffer (pH 3, adjusted through ortho-phosphoric acid) and 650 ml of acetonitrile. A solution of 100 ml of water, 900 ml of methanol and 2 ml with triethylamine (TEA) is used for dissolution of standard compounds. A chromatographic column (250 × 4.6 mm) is used to separate the components of the mixture. The detection wavelength for MET, DAPA and GLIME is set at 248 nm. The mobile phase’s discharge rate remains unchanged at 1 ml/min, with an amount of 20 μl for injection. The chromatographic analysis period is around 15 minutes, completing MET resolution (Retention time (RT) = 4.365 min), DAPA resolution (RT = 2.187 minutes) and GLIME resolution (RT = 7.269 min). The presented approach was validated using ICH (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use) recommendations, evaluating variables like robustness, linearity, precision, limit of detection (LOD), accuracy, limit of quantification (LOQ), linearity, specificity as well as system adaptability. Under optimized chromatographic conditions, MET demonstrated linearity at concentrations of 1-120 μg ml^-1. DAPA demonstrated linearity at concentrations of 0.5-2.5 μg ml^-1. GLIME demonstrated linearity at concentrations of 0.2-1.2 μg ml^-1. The values for MET, DAPA and GLIME LOD and LOQ had been achieved at concentrations of (5.26 μg ml^-1 and 15.94 μg ml^-1), (0.06 μg ml^-1 and 0.17 μg ml^-1) and (0.02 μg ml^-1 and 0.08 μg ml^-1). The analytical method is confirmed to be appropriate for its intended purpose and to meet the criteria given in ICH guideline Q2 (R2) according to the validation results.