title: 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.affiliation: Analytical & Bioanalytical Development, Bristol-Myers Squibb Company, Princeton, NJ 08543, USA. Electronic address: hamza.kandoussi@bms.com.
authors.forename: Hamza
authors.initials: H
authors.lastname: Kandoussi
authors.affiliation: Analytical & Bioanalytical Development, Bristol-Myers Squibb Company, Princeton, NJ 08543, USA. Electronic address: hao.jiang@bms.com.
authors.forename: Hao
authors.initials: H
authors.lastname: Jiang
authors.affiliation: Analytical & Bioanalytical Development, Bristol-Myers Squibb Company, Princeton, NJ 08543, USA.
authors.forename: Jianing
authors.initials: J
authors.lastname: Zeng
authors.affiliation: Analytical & Bioanalytical Development, Bristol-Myers Squibb Company, Princeton, NJ 08543, USA.
authors.forename: Naiyu
authors.initials: N
authors.lastname: Zheng
authors.affiliation: Analytical & Bioanalytical Development, Bristol-Myers Squibb Company, Princeton, NJ 08543, USA.
authors.forename: Pathanjali
authors.initials: P
authors.lastname: Kadiyala
authors.affiliation: Early Clinical and Translational Research, Bristol-Myers Squibb Company, Hopewell, NJ 08543, USA.
authors.forename: Timothy
authors.initials: T
authors.lastname: Eley
authors.affiliation: Early Clinical and Translational Research, Bristol-Myers Squibb Company, Hopewell, NJ 08543, USA.
authors.forename: Bing
authors.initials: B
authors.lastname: He
authors.affiliation: Early Clinical and Translational Research, Bristol-Myers Squibb Company, Hopewell, NJ 08543, USA.
authors.forename: Tushar
authors.initials: T
authors.lastname: Garimella
authors.affiliation: Tandem Laboratory, A LabCorp Division, Trenton, NJ 08628, USA.
authors.forename: Roger
authors.initials: R
authors.lastname: Demers
authors.affiliation: Tandem Laboratory, A LabCorp Division, Trenton, NJ 08628, USA.
authors.forename: Laura
authors.initials: L
authors.lastname: Cojocaru
authors.affiliation: Analytical & Bioanalytical Development, Bristol-Myers Squibb Company, Princeton, NJ 08543, USA.
authors.forename: Anne-Françoise
authors.initials: AF
authors.lastname: Aubry
authors.affiliation: Analytical & Bioanalytical Development, Bristol-Myers Squibb Company, Princeton, NJ 08543, USA.
authors.forename: Mark E
authors.initials: ME
authors.lastname: Arnold
mesh_terms: D000998:Antiviral Agents / Q000097:blood* / Q000737:chemistry / Q000493:pharmacokinetics; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D016174:Hepacivirus / Q000201:enzymology*; D006801:Humans; D007546:Isoquinolines / Q000097:blood* / Q000737:chemistry / Q000493:pharmacokinetics; D057230:Limit of Detection; D059625:Liquid-Liquid Extraction; D015394:Molecular Structure; D012015:Reference Standards; D015203:Reproducibility of Results; D013449:Sulfonamides / Q000097:blood* / Q000737:chemistry / Q000493:pharmacokinetics; D053719:Tandem Mass Spectrometry / Q000379:methods*; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000998:Antiviral Agents; D007546:Isoquinolines; D013449:Sulfonamides; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C571889:asunaprevir
keywords: Asunaprevir (BMS-650032); HCV NS3; Human plasma; LC–MS/MS; Pharmacokinetics; Quantitation