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.

Abstract

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 0.05ng/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 10μL of 10% CHAPS as the surfactant to prevent non-specific binding issue. Chromatographic separation was achieved on a Genesis C8 column (2.1×50mm, 4μm) 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 50ng/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.

• Authors: Hamza Kandoussi, Hao Jiang, Jianing Zeng, Naiyu Zheng, Pathanjali Kadiyala, Timothy Eley, Bing He, Tushar Garimella, Roger Demers, Laura Cojocaru, Anne-Françoise Aubry, Mark E Arnold
• Journal: Journal of pharmaceutical and biomedical analysis (J Pharm Biomed Anal) Vol. 119 Pg. 145-51 (Feb 05 2016) ISSN: 1873-264X [Electronic] England
• PMID: 26683987 (Publication Type: Journal Article)
• Copyright: Copyright © 2015 Elsevier B.V. All rights reserved.

Chemical References

• Antiviral Agents
• Isoquinolines
• NS3 protein, hepatitis C virus
• Sulfonamides
• Viral Nonstructural Proteins
• asunaprevir

Topics

• Antiviral Agents (blood, chemistry, pharmacokinetics)
• Calibration
• Chromatography, High Pressure Liquid (methods)
• Hepacivirus (enzymology)
• Humans
• Isoquinolines (blood, chemistry, pharmacokinetics)
• Limit of Detection
• Liquid-Liquid Extraction
• Molecular Structure
• Reference Standards
• Reproducibility of Results
• Sulfonamides (blood, chemistry, pharmacokinetics)
• Tandem Mass Spectrometry (methods)
• Viral Nonstructural Proteins (antagonists & inhibitors)