Abstract
Objective: This study points to build up and validate a simple methodology to quantify the most used drug sofosbuvir for the treatment of hepatitis C virus (HCV) infection, in human plasma by using atazanavir as an Internal Standard (IS) for preclinical studies and validate as per USFDA guidelines.Methods: Sofosbuvir was isolated from plasma samples by liquid-liquid extraction method using acetonitrile; good chromatographic separation was achieved on Kromasil Column (250 mm ×4.6 mm, 5 µm). The mobile phase consisted of 0.1 % orthophosphoric acid (OPA) buffer pH 2 and acetonitrile in the ratio of (68:32, v/v), respectively. The analysis time was 7 min at a flow rate 1 ml/min. The photodiode array detector (PDA) detection was carried out at 228 nm. The suggested method was validated by performing linearity, system suitability, specificity and sensitivity, accuracy and precision, recovery, ruggedness, stability studies. The method was validated as per USFDA guidelines.Results: The developed method resulted in retention times of sofosbuvir and IS were found out to be 4.7 and 4.2 min respectively. The calibration curves are linear (r2 = 0.999) over the concentration range of 0.050-2.0 µg/ml of plasma analytes concentration. LOQ value was found to be 0.050 µg/ml with precision and accuracy. Within-batch % mean accuracy of the method ranged between 96.00% and 109.09%, and within-batch and total precision, expressed as the coefficient of variation, was 1.40–10.33%. Overall percentage mean recovery of sofosbuvir from spiked plasma was 84.14%. All the validated parameters were found to be within the limit.Conclusion: A simple, accurate, precise, linear, rugged and rapid RP-HPLC method was developed for quantitative estimation of sofosbuvir in human plasma and should be suitable for conducting pharmacokinetics studies and therapeutic drug monitoring.
Highlights
Sofosbuvir is a prodrug nucleotide analogue used as a portion of combination therapy to treat hepatitis C virus (HCV) infection or to handle co-infection of HIV and HCV its function has proven successful in the treatment of HCV in patients co-infected with HIV [1]
The primary goal of this work was to produce a simple, sensitive, specific and economic bioanalytical reverse phase high-performance liquid chromatography (RP-HPLC)-photodiode array detector (PDA) detection method for the determination of sofosbuvir from human plasma that is applicable in further pharmacokinetics studies
The mobile phase consisting of 0.1 % orthophosphoric acid, acetonitrile (68: 32) was selected because it was found to give good separation for the peaks of sofosbuvir (Rt-4.78 min) and Internal Standard (IS) (Rt-4.2 min) respectively
Summary
Sofosbuvir is a prodrug nucleotide analogue used as a portion of combination therapy to treat hepatitis C virus (HCV) infection or to handle co-infection of HIV and HCV its function has proven successful in the treatment of HCV in patients co-infected with HIV [1].In 2013, the FDA approved sofosbuvir in combination with ribavirin (RBV) for dual oral therapy of HCV genotypes 2 and 3, and for triple therapy with injected pegylated interferon (peg IFN) and RBV for treatment-naive patients with HCV genotypes 1 and 4. Sofosbuvir is a prodrug nucleotide analogue used as a portion of combination therapy to treat hepatitis C virus (HCV) infection or to handle co-infection of HIV and HCV its function has proven successful in the treatment of HCV in patients co-infected with HIV [1]. In 2014, a combination of sofosbuvir with the viral NS5A inhibitor ledipasvir was approved. A new treatment, which is a combination of two direct-acting antiviral agents, ledipasvir 90 mg (anti-NS5A) and sofosbuvir 400 mg (anti-NS5B), has been approved in the US and the European Union for the treatment of chronic hepatitis C viral infection [2]. The primary goal of this work was to produce a simple, sensitive, specific and economic bioanalytical RP-HPLC-PDA detection method for the determination of sofosbuvir from human plasma that is applicable in further pharmacokinetics studies. The developed method was validated according to USFDA guidelines [10]
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