| Summary: | HCV-genotype-4 (HCV-GT4) is the cause of approximately 20% of the 170 million cases of chronic hepatitis C virus (HCV) in the world. Around 95% of patients with chronic HCV infection can be cure by utilizing direct-acting antiviral treatment. Two anti-HCV genotype 4 co-administered ravidasvir (RAV) and sofosbuvir (SOF) were simultaneously quantified in rat plasma by a validated and sensitive LC-MS/MS method using aciclovir as an internal standard. Chromatographic resolution for all analytes was performed with an Eclipse plus C18 column (50 mm × 2.1 mm, 1.8 µm) with isocratic mobile phase consisted of 10 mM ammonium formate: acetonitrile (61:39, v/v, pH 4.0) at a flow rate of 0.25 mL/min. Sample pre-treatment involved protein precipitation in plasma and stable internal standard resulted in a sensitive and robust method. Positive multiple reaction monitoring (MRM) mode was chosen to identify RAV, SOF, and IS. The developed assay was validated for accuracy, precision, linearity, selectivity, extraction recovery, matrix effect, carry-over, dilution integrity, and stability in accordance with US-FDA bioanalytical method validation guidelines. The method was linear over the ranges of 0.5–600 and 1–3000 ng/mL of RAV and SOF, respectively (r2 ≥ 0.997). After injection of the HLOQ sample, carry-over in the blank sample was less than 20% of the LLOQ of RAV, SOF and less than 5% of the IS. The mean relative standard deviation (RSD) of the results of accuracy and precision were ≤9.74%, and the overall recoveries of RAV and SOF from rat plasma were in the range 92.53–107.25%. The current methodology is the first LC-MS/MS for the quantification of RAV and SOF in rat plasma and to applied the pharmacokinetics of these agents in rats.
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