Development and validation of an environmentally friendly fluorescence quenching method for linagliptin quantification using eosin Y: optimization <i>via</i> design of experiment and comparative greenness assessment

Diabetes management has increasingly relied on dipeptidyl peptidase-4 inhibitors like linagliptin, creating a need for environmentally sustainable analytical methods to replace conventional chromatographic techniques that often involve complex sample preparation, organic solvent usage, and expensive instrumentation. A sensitive and selective "turn-off" fluorescence quenching method was developed and validated for the determination of linagliptin using eosin Y as the fluorescent probe. The spectral characteristics and sensing mechanisms were investigated using Stern-Volmer analysis, Job's method, and thermodynamic studies, revealing a static quenching process driven by the formation of a non-fluorescent 1 : 1 linagliptin-eosin Y complex with a high Stern-Volmer constant (<i>K</i> _sv = 6.46 × 10⁵ M^-1). The influencing factors, including pH, buffer volume, eosin Y concentration, and incubation time, were optimized using a Box-Behnken experimental design. A significant reduced quadratic regression model was established, and the optimal conditions were found to be pH 5.25, buffer volume of 1 mL, eosin Y volume of 1.25 mL, and an incubation time of 5 min based on desirability function analysis that maximizes the quenching efficiency. The developed method demonstrated linearity in the range of 0.1-3.0 μg mL^-1 with a correlation coefficient of 0.9999, a limit of detection of 0.03 μg mL^-1, and accuracy of 99.59 ± 1.360%, in accordance with ICH guidelines. Selectivity was confirmed by the lack of interference from common pharmaceutical excipients and endogenous plasma components. The eosin Y-based fluorescence quenching method was successfully applied for the determination of linagliptin in pharmaceutical dosage forms and spiked human plasma samples. Statistical comparison of the proposed method with the reported HPLC-UV method revealed comparable analytical performance as evident by non-significant differences in the accuracy and precision profiles as well as interval equivalence testing. Furthermore, a comprehensive assessment of the environmental impact and analytical practicality of the proposed method was conducted, confirming its "green" and "blue" analytical profile. These findings establish the eosin Y-based fluorescence quenching method as a viable and environmentally friendly alternative for the routine analysis of linagliptin in various pharmaceutical and bioanalytical applications shedding light on the potential of spectrofluorometric techniques in green analytical chemistry and bioanalysis.