Optimization and modeling of a green dual detected RP-HPLC method by UV and fluorescence detectors using two level full factorial design for simultaneous determination of sofosbuvir and ledipasvir: Application to average content and uniformity of dosage unit testing

Abstract

A simple, rapid, selective, linear, precise, accurate and eco-friendly RP-HPLC-UV-Fluorescence method was developed by 23 full factorial design and validated for simultaneous assay of sofosbuvir and ledipasvir mixture in tablet dosage form within 7 min without interference of tablet excipients. Isocratic elution at a flow rate of 1.2 mL min−1 was employed on C18 (250 mm × 4.6 mm, 5 μm in particle size) at ambient temperature. The column equipped with UV detector then fluorescence detector. The fluorescence detector is an optional extra tool for a more sensitive analysis of LED. UV detector can be used for both drugs. UV detector was set at 261 nm and switched online at 5 min to 333 nm. On the other hand, the fluorescence detector was set at an emission wavelength of 404 nm with excitation at 333 nm. The mobile phase consisted of acetonitrile:methanol:0.01% triethylamine adjusted to pH 3 by glacial acetic acid [35: 35:30] (v/v/v). After injection of 30μL sample, the retention time for sofosbuvir by UV detector was 3.353 ± 0.017 min. While for ledipasvir, it was 6.102 ± 0.006 min by UV detector and 6.255 ± 0.004 min by florescence detector. Using the proposed method, calibration graphs were found linear for sofosbuvir over the concentration range of 1–40 μg mL−1 with coefficient of determination (R2) > 0.9999 and mean percentage recovery of 100.33 ± 1.11 by UV detector. While for ledipasvir, calibration graphs were linear over the concentration range of 0.4–20 μg mL−1 with coefficient of determination (R2) > 0.9999 and mean percentage recovery of 100.06 ± 0.59 by UV detector and over the concentration range of 0.1–10 μg mL−1 with coefficient of determination (R2) > 0.9999 and mean percentage recovery of 100.55 ± 1.22 by fluorescence detector. Florescence detection enhances method selectivity and opens the door for biological and toxicological application of the proposed method.The proposed method was highly precise as indicated by low percentage RSD values of <1.4% for sofosbuvir and <1.9% for ledipasvir. The method was found to be customarily green as being evaluated according to analytical Eco-Scale and the Green Analytical Procedure Index (GAPI) tools guidelines. The proposed methods were validated according to international conference of harmonization (ICH) requirements and statistically compared to published reference methods. The ANOVA test confirmed that there is no significant differences between UV and florescence detectors for ledipasvir determination. The average content and uniformity of dosage unit for sofosbuvir/ledipasvir combined dosage form and sofosbuvir single dosage form were estimated by the developed method according to British pharmacopeia (BP) requirements.