Validated UPLC/MS/MS assay for quantitative bioanalysis of elbasvir in rat plasma and application to pharmacokinetic study.

Rapid, sensitive, selective and accurate ultra performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the quantification of elbasvir (ELB) in rat plasma with deuterated elbasvir (ELB-D6) as internal standard (IS).Sample preparation was done by protein precipitation using acetonitrile containing 50 ng/mL IS. Chromatographic separation was achieved by an UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) column with a gradient mobile phase consisting of acetonitrile-water (containing 5.0mM ammonium acetate with 0.01% acetic acid, pH 4.5) as mobile phase at a flow rate of 0.3 mL/min for 3 min. ELB was monitored using positive electrospray triple quadrupole mass spectrometer (Waters Xevo TQ-S) via multiple reaction monitoring (MRM) mode. The monitored transitions were set at m/z 882.51→656.42 and m/z 888.49→662.43 for ELB and ELB-D6, respectively. The achieved lower limit of quantification was 1.0 ng/mL. The validated method had an excellent linearity in the range of 1.0-2000 ng/mL (r(2)>0.996). Recovery efficiency at three levels QC concentrations of 2.0 (low), 160 (medium) and 1600 (high) ng/mLwas in the range of 98.29-106.40% for ELB. Matrix effect was found to be minimal. The intra- and inter-day precisions were less than 7.01%. The intra- and inter-day accuracies were determined to be within ±6.23% for all accuracy measurements. The validated simple and rapid UPLC-MS/MS method was successfully used to the pharmacokinetics study of ELB in rats, providing its applicability in relevant preclinical studies.