Simultaneous Estimation of Empagliflozin and Metformin by High-Performance Thin-Layer Chromatography Using Quality-by-Design Approach

The present research work highlights the benefits of analytical quality-by-design (QbD) approach to optimize high-performance thin-layer chromatography (HPTLC) method for the simultaneous quantification of empagliflozin and metformin in fixed dose combination. With a QbD model, the present study endeavors to establish the method operable design region (MODR) for optimization of the HPTLC method assay by means of design of experiments (DOE) and response surface methodology, in order to achieve a good separation and quantification of all analyzed compounds along with an acceptable analysis time. A deep understanding of the quality target product profile (QTPP) and of the analytical target profile (ATP), followed by a risk assessment for variables that affect the efficiency of the method, led to the development of a precise, accurate, and cost-effective method. The method was established on pre-coated silica gel 60 F-254 aluminum plates using ammonium acetate (2%)-methanol-acetonitrile-ethyl acetate (3:1:4.5:1.5) as the mobile phase at a detection wavelength of 228 nm. Numerical optimization was performed using the Derringer's desirability approach and ANOVA to develop optimized chromatography. The R-F values were 0.49 and 0.81 for metformin and empagliflozin, respectively. The limits of detection for empagliflozin and metformin were found to be 3.49 ng per band and 109.7 ng per band, respectively, while the limits of quantification for empagliflozin and metformin were found to be 10.58 ng per band and 330.54 ng per band, respectively. The intra-and inter-day precisions were less than 2%, with accuracies between 98 and 102% of the true values. The method was successfully applied to quantify empagliflozin and metformin in fixed dose combinations.