Green Spectrofluorometric Method for Labetalol Determination Based on Electron Transfer Inhibition With Density...

A novel spectrofluorimetric method for sensitive determination of labetalol hydrochloride was developed based on photoinduced electron transfer (PET) blocking mechanism through acidification-induced fluorescence enhancement. Upon acidification, the secondary amine functionality of labetalol undergoes protonation, resulting in five-fold fluorescence enhancement at 415-nm emission when excited at 298 nm. Quantum mechanical calculations revealed the electronic structure changes underlying the PET mechanism, with protonation causing 3.17-eV HOMO stabilization and decreased electron density on the secondary amine nitrogen that effectively eliminates electron transfer quenching. Subsequently, D-optimal experimental design systematically optimized the PET blocking conditions. The ANOVA analysis demonstrated statistical significance of the reduced two-factor interaction (2FI) model (F-value = 26.25, p < 0.0001) with satisfactory fit statistics (R² = 0.9459), identifying 0.6-M acetic acid, 5-min incubation time, and water-ethanol mixture as optimal conditions. The method was validated according to ICH guidelines exhibiting excellent linearity in the range of 10-300 ng/mL, detection limit of 3.23 ng/mL, and precision below 1.6% RSD. Furthermore, successful applications to pharmaceutical formulations and biological samples (plasma) demonstrated recoveries ranging from 96.36% to 104.97%. The developed PET-blocking approach provides an environmentally sustainable alternative for labetalol determination, eliminating toxic derivatization reagents while maintaining analytical reliability and sensitivity.