Abstract
A sensitive and sustainable electroanalytical approach for the simultaneous determination of 5-fluorouracil (5-FU) and empagliflozin (EMPA) is desirable for pharmaceutical and bioanalytical investigations, particularly in preliminary studies exploring their concomitant use and potential protective effects. Herein, a square-wave voltammetric (SWV) method was developed using a sequentially fabricated poly-XO@poly-EUC/PGE sensing interface, prepared by successive electropolymerization of euchrysine (EUC) and xylenol orange (XO) on the surface of pencil graphite electrode (PGE). Electrochemical and morphological characterization confirmed successful electrode modification and improved interfacial electron-transfer properties. The sequential dual-polymer architecture produced a more pronounced analytical response than the corresponding individual polymer-modified electrodes, supporting a synergistic contribution of the two polymeric layers. Under optimized conditions, the proposed SWV method exhibited good linearity, precision (RSD ≤ 1.51%), and accuracy (recoveries of 96.21-103.59%), with detection limits of 0.33 and 0.02 µM for 5-FU and EMPA, respectively. The method also provided satisfactory recoveries in spiked human plasma, demonstrating its potential applicability to biological samples. An Environmental, Performance, and Practicality Index (EPPI) score of 78.0 highlighted the favorable sustainability and practical performance of the method. Density functional theory (DFT) calculations were performed using isolated EUC and XO molecular models and suggested favorable analyte-modifier interactions and provided qualitative molecular-level support for the proposed sensing mechanism, while the estimated oxidation pathway indicated a one-electron irreversible process. Overall, the poly-XO@poly-EUC/PGE platform provides a sensitive, selective, practical, and sustainable approach for the simultaneous electrochemical determination of 5-FU and EMPA, with potential utility in pharmaceutical quality control and bioanalytical studies.