A green stability-indicating RP-HPLC-UV method using factorial design for determination of ribavirin, sofosbuvir and ledipasvir: Application to average content, acid degradation kinetics and in vitro drug interactions study

Abstract

A novel optimization approach based on full factorial design was generated for developing a green, rapid and simple RP- HPLC-UV gradient method for simultaneous assay of recent antiviral drugs of hepatitis C (sofosbuvir and ledipasvir) with ribavirin as coadministered drug in 0.1 N HCl within 10 min. Gradient elution at a flow rate of 1 mL min−1 and injection volume of 10 μL was employed on Promosil CN (250 mm × 4.6 mm i.d., 5 µm particle size, 100 Ao pore size) at 25 °C. The detector (UV) was set at 220 nm and changed to 261 nm at 4.5 min then to 324 nm at 9 min. The mobile phase consisted of methanol and 0.005 M Heptane-1-sulphonic acid sodium salt adjusted to pH 2.5 by phosphoric acid. The retention times for ribavirin, sofosbuvir and ledipasvir were 3.700 ± 0.005, 8.526 ± 0.039 and 9.360 ± 0.027 min, respectively. The calibration curves of ribavirin, sofosbuvir and ledipasvir were linear over concentration ranges of 0.2–500, 5–500 and 1–112.5 μg mL−1, respectively with coefficient of determination (R2) > 0.9999 and recovery > 99.7% for each analyte. The linearity ranges of ribavirin, sofosbuvir and ledipasvir could be from 0.2 to 25, 5–50 and 1–6 μg mL−1, respectively according to their small limits of quantitation (0.161, 2.933 and 0.521 μg mL−1, respectively), but these linearity ranges were not recommended to avoid predilution procedures for the obtained samples from average content and dissolution tests. This makes our proposed method simpler in sample preparation; besides decreasing the resulted errors. The limits of detection of ribavirin, sofosbuvir and ledipasvir were 0.055, 0.880 and 0.156, respectively. The proposed method was highly precise as indicated by low percentage RSD values of less than 1.7% for each analyte. The method was green according to Eco-Scale and the Green Analytical Procedure Index (GAPI) guidelines. The average content for ribavirin, sofosbuvir/ledipasvir and sofosbuvir dosage forms was estimated. This method is stability-indicating and is applicable for simultaneous measuring of ribavirin, sofosbuvir and ledipasvir and their related acid degradation impurities after being exposed to forced and mild acidic degradation. The reaction orders, rate constants, half-life times and activation energies of the acid degradation process were calculated for all drugs. It was applied to study the degree of the in vitro interactions between ribavirin and sofosbuvir/ledipasvir dosage forms in 0.1 N HCl to simulate gastric fluid. There was statistically significant difference in the effect of sofosbuvir/ledipasvir dosage form on the dissolution profile of ribavirin, and vice versa. The co-administration of ribavirin with sofosbuvir/ledipasvir could alter their bioavailability.