ABSTRACT:A flow rate of 1 ml/min along with 220

ABSTRACT:A simple, accurate, economical, rapid, selective, reverse phase high performance liquid chromatography (RP-HPLC) was developed for simultaneous estimation of sofosbuvir, velpatasvir and voxilaprevir in its tablet dosage form. The separation was carried out using a mobile phase of Buffer and acetonitrile in the ratio of 50:50 pumped at a flow rate of 1 ml/min along with 220 nm as a UV detection wavelength. The stationary phase used was column Waters C18 250 x 4.6 mm, 5m.(. Sofosbuvir, velpatasvir and voxilaprevir were eluted at a retention times were 2.091 for sofosbuvir, 2.640 for velpatasvir and 3.349 min for voxilaprevir. Analytical method of the proposed RP-HPLC procedure was statistically validated with as per ICH guidelines by considering the parameters such as, system suitability, linearity, ranges, precision, accuracy, specificity, robustness, limit of detection and quantification limits. The linearity ranges were 100–600, 25–150 and 25–150 ppm for sofosbuvir, velpatasvir and voxilaprevir respectively with correlation coefficients >0.9993. The three samples were subjected to stress conditions of acidic and alkaline hydrolysis, oxidation, photolysis and thermal degradation. The proposed method proved to be stability-indicating by resolution of the analytes from their forced-degradation products. The developed RP-HPLC method can be used for routine analysis of sofosbuvir, velpatasvir and voxilaprevir in combinational dosage form. The proposed method made use of PDA as a tool for peak identification and purity confirmation. Key words: sofosbuvir, velpatasvir, voxilaprevir RP-HPLC method development, Validation, 1. IntroductionVoseviTM drug (sofosbuvir, velpatasvir and voxilaprevir) is a combination product that Contains Sofosbuvir (SOF) is a once-daily pangenotypic HCV NS5B nucleotide polymerase inhibitor approved in the United States and abroad for the treatment of chronic HCV infection Lawitz et al., 2013. It is an oral pro drug that undergoes hepatic metabolism into an active nucleotide analog that competitively inhibits the NS5B polymerase, thereby preventing viral replication. The structure of SOF showed in (1A) and chemically (S)-Isopropyl2-((S)-(((2R, 3R, 4R, 5R)-5-(2, 4-dioxo-3, 4dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl) methoxy (phenoxy) phosphorylamino) propanoate (Keating G.M., 2014). Velpatasvir (VEL) is a once-daily pangenotypic HCV NS5A protein inhibitor, including equivalent potency in vitro against genotype 3 compared with other genotypes, approved in the United States and abroad for the treatment of HCV infection in combination with SOF (Feld JJ, Jacobson IM, Hezode T, e al., 2015 and Ravikumar et al., 2016 ). Inhibition of the NS5A protein disrupts HCV replication, assembly and possibly egress Ross-T. The structure of VEL showed in (1B) and chemically it is Methyl{ (1R)-2-(2S,4S)-2-(5-{2(2S,5S)-1- {(2S)-2- (methoxycarbonyl) amino-3-methylbutanoyl} -5- methylpyrrolidin-2-yl 1,11dihydro2benzopyrano4′,3′:6,7 naphtha 1,2-d imidazol-9-yl} -1H-imidazol-2-yl) -4(methoxymethyl) pyrrolidin-1-yl-2-oxo-1phenylethyl}carbamate (Ross-Thriepland D, Harris M.2015). Voxilaprevir (VOX) is a novel macro cyclic NS3/4A protease inhibitor that has recently completed phase III clinical development in combination with SOF/VEL. It has excellent activity across all HCV genotypes and against most of the RASs associated with first-generation protease inhibitors. The structure of VOX showed in (1C) and chemically it is (1aR,5S,8S,9S,10R,22aR) -5-tert -butyl N- {(1R,2R)-2-(difluoromethyl)-1-(1-methylcyclopropanesulfonyl)carbamoylcyclopropyl}-9-ethyl-18,18-difluoro-14-methoxy-3,6-dioxo-1,1a,3,4,5,6,9,10,18,19,20,21,22,22a-tetradecahydro-8H-7,10-methanocyclopropa 18,191,10,3,6 dioxadiazacyclononadecino11,12-b quinoxaline-8-carboxamide7. Now, sofosbuvir, velpatasvir and voxilaprevir are official in US pharmacopeia 8. (Wjpps1-4). Literature survey reveals that a few analytical methods have been reported for the quantitative estimation of SOF, VEL some of them were being: Development and validation for estimation sofosbuvir in pure and tablet dosage form Asian J. Pharm. Tech. 2017; 7(3):153-156, Development and validation of RP-HPLC method for quantitative analysis of sofosbuvir in pure and pharmaceutical formulation Shaikh et al. World Journal of Pharmacy and Pharmaceutical Sciences volume 6, issue 8, 2017, Development and validation of new RP-HPLC method for the determination of sofosbuvir in pure form Vikas et al, World Journal of Pharmacy and Pharmaceutical Sciences volume 5, issue 5, 2016, a stability indicating RP-HPLC method for simultaneous estimation of velpatasvir and sofosbuvir in combined tablet dosage forms Nalla et al, World Journal of Pharmacy and Pharmaceutical Sciences volume 6, and issue 9, 2017, A new RP-HPLC method development and validation for simultaneous estimation of sofosbuvir and velpatasvir in pharmaceutical dosage form.J.Sandya Rani IJETSR volume 4 issue 11 2017, and a new RP-HPLC Method for the simultaneous assay of sofosbuvir and ledipasvir in Combined Dosage Form S.V.M.Vardhan et al/International Journal of ChemTech Research, 2017, 10(7): 761-768 also Evolution of sofosbuvir, velpatasvir plus voxilaprevir as fixed dose co formulation for treating hepatitis C in Soriano et al. Expert Opin Drug Metab Toxicol 2017, besides that several methods reported for the determination of SOF, VEL and VOX like Quantitative detection methods applying mass spectrometric detection are limited to four UPLC-MSRezk et al; shi et al 2015; pan et al 2016; Ariaudo et al and two LC–MS/MS methodsPadala et al 2017; Bahrami et al To the best of our knowledge, there is no official method for this combination.  As per literature survey several methods have been reported for estimation of sofosbuvir, velpatasvir and voxilaprevir individually or with the combination of some other drugs. With this current proposed method sofosbuvir, velpatasvir and voxilaprevir estimates simple and economical formulations.The purpose of this research was to establish and validate, in accordance with International Conference on Harmonization (ICH) guidelines, an accurate, economical, and reproducible procedure for quantitative analysis of sofosbuvir, velpatasvir and voxilaprevir as the bulk drug and in tablet dosage forms. It was thought worthwhile to develop precise, accurate, simple RP-HPLC method for simultaneous estimation of sofosbuvir, velpatasvir and voxilaprevir in tablets.