Development and validation of a stability-indicating spectrofluorimetric method for the determination of H1N1 antiviral drug (oseltamivir phosphate) in human plasma through the Hantzsch reaction

Abstract

A simple and sensitive spectrofluorimetric method has been described and validated for the determination of oseltamivir phosphate (OSP) in its pure form and pharmaceutical dosage forms. The method is based on the reaction of acetylacetone and formaldehyde with the primary amino group of OSP through the Hantzsch reaction, forming a yellow fluorescent dihydropyridine derivative measured spectrofluorimetrically at 475 nm (excitation at 408 nm). At the optimum reaction conditions, the fluorescence intensity concentration plot is rectilinear over the range of 0.4–2.8 μg mL⁻¹. The lower limits of detection and quantitation were 0.08 and 0.24 μg mL⁻¹, respectively. The developed method was successfully applied for determination of OSP in its commercial capsules and suspension with an average percentage recovery of 99.86 ± 1.20 and 98.36 ± 2.42, respectively (n = 5) without interference from common excipients. The proposed method was utilized for in vitro determination of the cited drug in spiked human plasma, with a percent mean recovery (n = 3) of 98.22 ± 1.27%. Furthermore, the proposed method was extended to study the stability of OSP under different stress conditions; such as hydrolysis (acidic and alkaline), oxidation (30% H₂O₂ v/v), photolysis, (as per ICH guideline Q1B option 1) and a thermal degradation study. Also the developed method was used to investigate the kinetics of the alkaline and acidic degradations of the cited drug.