Development of Stability-Indicating Spectrofluorimetric Method for Favipiravir Determination Using...

A sensitive, accurate, and stability-indicating spectrofluorimetric method was developed and validated for favipiravir determination in the presence of its acid-induced degradation product. The method employs selective chemical derivatization with 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-F) to overcome inherent analytical challenges associated with weak native fluorescence and spectral overlap between the parent compound and degradation products. Comprehensive characterization of the acid-induced degradation product was achieved through infrared spectroscopy, ¹H NMR, and mass spectrometry, confirming amide hydrolysis to the corresponding carboxylic acid derivative. The derivatization mechanism exploits functional group differences, where favipiravir's primary amide undergoes nucleophilic substitution with NBD-F forming a highly fluorescent adduct (λ_ex = 472 nm, λ_em = 530 nm), while the degradation product remains nonreactive, providing inherent selectivity. Systematic optimization of derivatization conditions established optimal parameters: pH 8.0 borate buffer, 70°C for 25 min, and 1.5 mL of 0.2% NBD-F. The method demonstrated excellent linearity over 0.01-0.25 μg/mL with remarkable sensitivity (LOD = 0.0028 μg/mL, LOQ = 0.0094 μg/mL), representing significant improvement over conventional HPLC-UV methods. The method was successfully applied to pharmaceutical formulation analysis and human plasma samples. This stability-indicating approach provides a valuable analytical tool for favipiravir quality control and therapeutic drug monitoring.