Green spectrophotometric methods for simultaneous quantification of metformin, empagliflozin, and melamine

Abstract

The simultaneous determination of metformin (MET), empagliflozin (EMP), and melamine (MEL), a nephrotoxic process-related impurity of MET, presents a significant analytical challenge due to severe spectral overlap and structural similarity between MET and MEL. We report a separation-free, green spectrophotometric protocol enabling concurrent quantification of all three analytes from a single recorded zero-order spectrum through distinct mathematical transformations. Phosphate buffer (pH 3.6) was selected as the optimal medium, inducing a hyperchromic effect in MEL through protonation of its triazine ring primary amines (pKa ~ 5), thereby enhancing detectability. The method employs: (i) first-derivative spectrophotometry for MEL at 217 nm, (ii) ratio difference for MET (238–244 nm), and (iii) second derivative of ratio spectra for EMP at 284 nm. Validation per ICH Q2(R1) showed excellent linearity (0.5–14 µg/mL MEL; 1–50 µg/mL MET; 1–24 µg/mL EMP), high accuracy (98–102% recovery), precision (RSD ≤ 1.3%), and low LODs (0.141, 0.306, and 0.352 µg/mL, respectively) and LOQ (0.427, 0.926, and 1.007 µg/mL, respectively). Successfully applied to Empagliform® tablets, the method eliminates chromatographic separation, uses minimal non-hazardous reagents, and avoids organic solvents in the final measurement. Greenness was confirmed by NEMI (non-hazardous), Eco-Scale (89/100), GAPI (minimal impact), AGREE (0.71), and BAGI (85.0), collectively demonstrating a sustainable, simple, and cost-effective solution for quality control laboratories, especially in resource-limited settings.