Eco-friendly second-derivative synchronous fluorescence method for the determination of empagliflozin and sitagliptin in tablets and plasma samples

Abstract Empagliflozin and sitagliptin are commonly co-administered to manage type 2 diabetes mellitus due to their complementary mechanisms of action on renal glucose reabsorption and insulin secretion, respectively. This synergistic effect enhances glycemic control with a minimal risk of hypoglycemia. In this study, a sensitive, simple, rapid, and selective spectrofluorimetric method was developed for the simultaneous determination of empagliflozin in the presence of sitagliptin in pharmaceutical formulations and spiked human plasma. Both drugs exhibit native fluorescence; however, their emission spectra significantly overlap, complicating their simultaneous analysis with conventional spectrofluorimetric techniques. To overcome this limitation, second-derivative synchronous spectrofluorimetry was applied. Measurements were performed within an optimized wavelength interval (Δλ = 60 nm), enabling selective determination of sitagliptin at 393 nm and empagliflozin at 310 nm without mutual interference. Experimental parameters affecting fluorescence intensity, including solvent type, pH, and Δλ, were carefully optimized to achieve maximum sensitivity and selectivity. The proposed method demonstrated excellent analytical performance, with limits of detection of 0.031 µg/mL and 0.011 µg/mL, and limits of quantification of 0.093 µg/mL and 0.033 µg/mL for sitagliptin and empagliflozin, respectively. Good linearity was observed over concentration ranges of 0.1–4 µg/mL for sitagliptin and 0.04–1.2 µg/mL for empagliflozin. The method was successfully applied to spiked human plasma samples and validated in accordance with ICH guidelines, demonstrating satisfactory accuracy, precision, robustness, and selectivity. A statistical comparison with a reported method showed no significant difference in performance. The environmental sustainability of the proposed method was comprehensively evaluated using several green analytical chemistry assessment tools, including the Carbon Footprint Reduction Index (CaFRI), Eco-Scale, AGREEprep, CompMoGAPI, and NEMI. In addition, the Blue Applicability Grade Index (BAGI) was employed to assess the method’s blueness, reflecting its practical applicability. Furthermore, the RGB 12 algorithm was applied to evaluate the method’s overall whiteness by integrating its environmental impact (greenness), analytical performance (redness), and practical efficiency (blueness). The obtained results consistently confirmed the excellent eco-friendly profile of the proposed method, demonstrating minimal environmental impact, reduced solvent consumption, strong compliance with green analytical chemistry principles, and high overall performance.