Development of Stability‐Indicating Spectrofluorimetric Method for Favipiravir Determination Using 4‐Fluoro‐7‐Nitrobenzo‐2‐Oxa‐1,3‐Diazole Derivatization: Application to Quality Control and Therapeutic Drug Monitoring

Abstract

ABSTRACT 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, 1 H 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.