A novel factorial design implemented spectrofluorimetric determination of caffeic acid: individual and combined assays with curcumin

Abstract In this research, we developed, for the first time, sensitive, selective, and reliable spectrofluorimetric methods for the quantitative analysis of caffeic acid (CAF) alone and in combination with curcumin (CUR). Two fluorimetric methods were established for the determination of CAF alone; a native fluorescence method (Method I) was optimized using 2 3 factorial model, exploiting the compound’s intrinsic fluorescence properties. The excitation and emission wavelengths were set at λex/λem = [324/443] nm. For the simultaneous analysis of CAF and CUR, the synchronous fluorescence (SF) technique was employed (Method II). The constant wavelength difference was optimized at 100 nm, allowing the simultaneous quantification of both analytes in a single measurement without prior separation. Under optimized conditions, the native fluorescence method for CAF alone showed a linear range of 30.0–800.0 ng/mL with a lower detection limit of 9.44 ng/mL. The SF method for the mixture demonstrated linear ranges of 50.0-2000.0 ng/mL for CAF and 20.0-200.0 ng/mL for CUR, with LOD values of 15.40 and 5.94 ng/mL, respectively. Both methods were successfully validated according to ICH guidelines, demonstrating excellent accuracy, precision, and robustness. The proposed methods were applied to different matrices, including green tea (method I), synthetic mixtures, and spiked human plasma (methods I and II). The ecological impact of the proposed methods was investigated. They provide a simple, cost-effective, and green alternative to chromatographic techniques for the routine quality control of caffeic acid and curcumin.