Green Spectrofluorometric Method for Labetalol Determination Based on Electron Transfer Inhibition With Density Functional Theory Validation and Statistical Design Optimization

ABSTRACT

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 ( R ²  = 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.