A highly sensitive and selective LC–MS/MS method to quantify asunaprevir, an HCV NS3 protease inhibitor, in human plasma in support of pharmacokinetic studies

•Sensitive LC–MS/MS assay was developed to quantify asunaprevir in human plasma.•CHAPS was added during LLE to prevent non-specific binding issue.•Assay was fully validated and achieved accurate, precise and reproducible results.•Assay equivalency was demonstrated by cross validation after method transfers.•It has been applied to pharmacokinetic evaluation in many clinical studies.

Asunaprevir (BMS-650032) is a selective hepatitis C virus (HCV) NS3 protease inhibitor with potent activity against HCV genotypes 1, 4, 5 and 6. It has been developed in conjunction with direct-acting antiviral agents, in interferon- and ribavirin-free regimen, to improve existing therapies for HCV infection. To support the pharmacokinetic analyses in asunaprevir clinical studies, we have developed and validated a highly sensitive and robust LC–MS/MS method to quantify asunaprevir in human EDTA plasma with an LLOQ of 0.05 ng/mL, which was a 20-fold sensitivity improvement over a previously reported assay for asunaprevir. A deuterated labeled [D9]-asunaprevir was used as the internal standard (IS). The analyte and the IS were extracted using a semi-automated liquid–liquid extraction (LLE) at pH 7 with methyl-t-butyl ether (MTBE) in a 96-well plate containing 10 μL of 10% CHAPS as the surfactant to prevent non-specific binding issue. Chromatographic separation was achieved on a Genesis C8 column (2.1 × 50 mm, 4 μm) with a gradient elution using 0.1% formic acid in water as mobile phase A and a mixture of methanol: acetone: formic acid (95:5:0.1; v/v/v) as the mobile phase B. Positive electrospray ionization was performed using multiple reaction monitoring (MRM) with transitions of m/z 748 → 648 for asunaprevir and m/z 757 → 649 for [D9]-asunaprevir,and a collision energy of 30 electron Volts (eV). The assay was validated over a standard curve range from 0.05 to 50 ng/mL for asunaprevir in human plasma. The intra- and inter assay precisions were within 7.1% CV, and the % deviation was within 5.5% of their nominal concentrations. This assay has been successfully applied to multiple clinical studies with excellent assay ruggedness and reproducibility.

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