Development and validation of a green spectrofluorimetric method using a rhodamine 6G probe and FCCCD-optimized...

Tenuazonic acid (TeA) is a major Alternaria mycotoxin frequently contaminating dried figs, yet its routine surveillance remains constrained by reliance on costly chromatographic instrumentation. To address this analytical gap, a novel "turn-off" spectrofluorimetric method was developed for TeA determination in dried fig samples using rhodamine 6G as a cationic xanthene fluorescent probe. Given the complexity of the sugar-rich matrix, dispersive liquid-liquid microextraction was first optimized through a face-centered central composite design coupled with Derringer's desirability function. Optimal conditions comprised 350 µL of 1-octanol as extractant, 700 µL of acetonitrile as disperser, pH 3.3, and 5% (w/v) ammonium sulfate, yielding an experimental recovery of 95.23% ± 1.48%. Subsequently, spectral characterization demonstrated strong native fluorescence of rhodamine 6G at 555 nm (λ _ex 526 nm), progressively quenched in the presence of TeA. Multi-temperature Stern-Volmer analysis, Job's continuous variation method, and thermodynamic evaluation confirmed a static quenching mechanism involving 1 : 1 ground-state complex formation. Comprehensive validation demonstrated linearity across 20-4000 µg kg^-1 (R ² = 0.9981), with an LOQ of 19.2 µg kg^-1. Mean recoveries ranged from 94.9% to 99.7%, intra- and inter-day precision remained below 11%, expanded measurement uncertainty was within ±22.4%, and matrix effects were negligible (-3.4%). Application to 60 Egyptian dried fig samples revealed widespread contamination (90% incidence; mean 726 ± 483 µg kg^-1), with 26.7% exceeding the 1000 µg kg^-1 indicative level. Green chemistry evaluation using AGSA-Prep (65%), WECA (81%), and EPPI (84.2) confirmed the method's sustainability, establishing it as a practical alternative to LC-MS/MS for routine TeA surveillance in resource-limited laboratories.