Stability-indicating HPLC-DAD determination of ribavirin in capsules and plasma

A simple, selective and stability-indicating high-pressure liquid chromatographic method was developed for the analysis of ribavirin. Chromatographic separation was achieved by using a CPS Hypersil cyano column (4.6 × 250 mm, 5 µm particle size) with isocratic elution of the mobile phase, which was composed of 50 mM phosphate buffer, adjusted at pH 4 with phosphoric acid. The mobile phase was pumped at a flow rate of 0.8 mL/min. The detector was set at 240 nm and quantification of the analyte was based on peak area measurement. The method was validated with respect to linearity, range, precision, accuracy, selectivity, robustness, limit of detection and limit of quantitation. The calibration curve was linear in the range of 5-200 µg/mL with correlation coefficient > 0.999. Ribavirin was subjected to forced degradation studies under two conditions: mild and extensive stress testing. These studies included the effects of hydrolysis (neutral, acidic and alkaline) and oxidation, photolysis and dry heat). The proposed method was proved to be stability-indicating by the resolution of the drug from its forced degradation products, making use of the diode array detector as a tool for confirmation of peak identity and purity. Moreover, the kinetics of alkaline degradation of ribavirin were investigated, an Arrhenius plot was constructed and the activation energy was calculated. The developed method was also extended to analyze ribavirin in capsules and in human plasma with good recovery values.