Journal of Chromatography B, 921–922 (2013) 81–86 Contents lists available at SciVerse ScienceDirect Journal of Chromatography B j ourna l ho me page: www.elsevier.com/locate/chromb A rugged and accurate liquid chromatography–tandem mass spectrometry method for the determination of asunaprevir, an NS3 protease inhibitor, in plasma Long Yuan a , Hao Jiang a , Zheng Ouyang b , Yuan-Qing Xia b,1 , Jianing Zeng a,∗ , Qianping Peng c , Robert W. Lange d , Yuzhong Deng a,2 , Mark E. Arnold a , Anne-Franc ¸ oise Aubry a a Analytical and Bioanalytical Development, Research and Development, Bristol-Myers Squibb, Princeton, NJ 08543, United States b Bioanalytical and Discovery Analytical Sciences, Research and Development, Bristol-Myers Squibb, Princeton, NJ 08543, United States c Drug Safety Evaluation, Research and Development, Bristol-Myers Squibb, New Brunswick, NJ 08903, United States d Drug Safety Evaluation, Research and Development, Bristol-Myers Squibb, Mt. Vernon, IN 47620, United States a r t i c l e i n f o Article history: Received 21 November 2012 Accepted 15 January 2013 Available online 4 February 2013 Keywords: Asunaprevir BMS-650032 Hepatitis C virus (HCV) Quantitative LC–MS/MS a b s t r a c t Asunaprevir (BMS-650032) is a potent hepatitis C virus (HCV) non-structural protein protease inhibitor currently in Phase III clinical trials for the treatment of HCV infection. A rugged and accurate LC–MS/MS method was developed and validated for the quantitation of asunaprevir in rat, dog, monkey, rabbit and mouse plasma. A systematic method screening and optimization strategy was applied to achieve optimized mass spectrometry, chromatography, and sample extraction conditions. The validated method utilized stable-isotope labeled D 9 -asunaprevir as the internal standard. The samples were extracted by liquid–liquid extraction using 10% ethyl acetate in hexane. Chromatographic separation was achieved with gradient elution on a Waters Atlantis dC18 analytical column. Analyte and its internal standard were detected by positive ion electrospray tandem mass spectrometry. The standard curve, which ranged from 5.00 to 2000 ng/mL for asunaprevir, was fitted to a 1/x 2 weighted linear regression model. The intra-assay precision was within ±3.6% CV, inter-assay precision was within ±4.0% CV, and the assay accuracy was within ±8.1% of the nominal values in all the species. The method was successfully applied to support multiple pre-clinical toxicokinetic studies in different species. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Hepatitis C virus (HCV) infects an estimated 170 million people worldwide, and is a major cause of chronic liver disease [1]. Most infections progress to chronic hepatitis, which can lead to cirrho- sis, liver failure, and hepatocellular carcinoma. In the United States, HCV infections cause more than 10,000 deaths annually [1] and are the leading indication for liver transplantation [2]. The viral non- structural protein 3 (NS3) protease, a serine protease located in the N-terminal region of NS3, interacts with its activating cofactor NS4A to form an active proteolytic complex required for subse- quent viral replication [3]. The inhibition of the NS3 serine protease ∗ Corresponding author. E-mail address: jianing.zeng@bms.com (J. Zeng). 1 Current address: AB Sciex, 500 Old Connecticut Path, Framingham, MA 01701, USA. 2 Current address: Drug Metabolism and Pharmacokinetics, Genentech, South San Francisco, CA 94080, USA. activity could effectively block viral replication, which makes the protease inhibitor an attractive target for new anti-HCV drugs [4]. Two protease inhibitors, telaprevir and boceprevir, were approved in the United States and Europe in 2011 for treating chronic HCV, and multiple protease inhibitors are in clinical development [4]. Asunaprevir (BMS-650032; Fig. 1), a potent HCV NS3 protease inhibitor, is currently in Phase III clinical trials for the treatment of HCV infection. Asunaprevir demonstrated robust antiviral activity in patients with HCV genotype 1 infection in single-ascending-dose and multiple-ascending-dose clinical studies [5]. Asunaprevir was previously used as the model compound to develop a convenient strategy for quantitative bioanalytical assay in tissue samples [6]. In this manuscript, we report the method development and validation of an LC–MS/MS method for the quan- tification of asunaprevir in rat, dog, monkey, rabbit and mouse plasma. A systematic method screening and optimization strategy [7,8] was applied during method development to achieve opti- mized mass spectrometry, chromatography, and sample extraction conditions. Incurred samples were used for method development and optimization, which ensured the quality of the method and 1570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.01.029