Complexation-based spectrofluorimetric method for besifloxacin determination: Combined experimental and computational insights

Fluorescence-based analytical methods offer high sensitivity, cost-effectiveness, and simplicity for pharmaceutical analysis. Besifloxacin hydrochloride (Bfln.HCl), a fluoroquinolone antibiotic used to treat eye infections, exhibits native fluorescence that can be enhanced in acidic media. However, the mechanism of this enhancement has not been fully explored. To address this, we developed a novel spectrofluorimetric method that enhances Bfln.HCl fluorescence using acetate buffer of pH 4.0. A 10 nm redshift in the excitation wavelength and a significant increase in fluorescence intensity at 440 nm were observed upon the addition of acetate buffer. Density Functional Theory (DFT) calculations revealed that the fluorescence enhancement results from the formation of 1:2 cyclic complexes between Bfln⁺ and acetic acid (AcOH). Thermodynamic analysis indicated that the Bfln⁺/[2AcOH] complex was the most stable, exhibiting the lowest HOMO-LUMO energy gap, consistent with the observed fluorescence enhancement. The method demonstrated high sensitivity (LOD = 22.6 ng mL^-1), selectivity, and a linear range of 100-1000 ng mL^-1, and it was successfully applied to ophthalmic preparations and artificial aqueous humor samples, yielding high recoveries. This method provides a cost-effective, sensitive alternative to chromatographic and electrochemical methods for Bfln.HCl determination, eliminating the need for complex derivatization, expensive reagents, or nanomaterials fabrications. Green analytical chemistry assessments (AGREE, BAGI, and RGB models) confirmed the method's environmental sustainability. This study offers both experimental and computational insights into the fluorescence enhancement mechanism of Bfln.HCl, laying the groundwork for extending this approach to other fluoroquinolone antibiotics.