LC-ESI-MS/MS method for quantification of ambrisentan in plasma and application to rat pharmacokinetic study Ramakrishna Nirogi*, Vishwottam Kandikere, Prashanth Komarneni, Raghupathi Aleti, NagaSuryaPrakash Padala and Ilayaraja Kalaikadhiban ABSTRACT: A sensitive high-performance liquid chromatography–positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of ambrisentan in plasma. The analyte and the internal standard (armodafinil) were extracted from plasma by acetonitrile precipitation and they were separated on a reversed-phase C 18 col- umn with a gradient program. The MS acquisition was performed with multiple reaction monitoring mode using the respective [M + H] + ions, m/z 379–347 for ambrisentan and m/z 274–167 for the IS. The assay exhibited a linear dynamic range of 1–2000 ng/mL for ambrisentan in plasma. Acceptable precision (<10%) and accuracy (100  8%) were obtained for concentrations over the standard curve range. The method was successfully applied to quantify ambrisentan concentrations in a rodent pharmacokinetic study after a single oral administration of ambrisentan at 2.5 mg/kg to rats. Following oral administration the maximum mean concentration in plasma (C max ; 1197  179 ng/mL) was achieved at 1.0  0.9 h (T max ), and the area under the curve (AUC) was 6013  997 ng h/mL. Therefore, development of such a simple and sensitive method in rat plasma should translate into a method for ambrisentan in human plasma for clinical trials. Copyright © 2012 John Wiley & Sons, Ltd. Keywords: ambrisentan; liquid chromatography–tandem mass spectrometry; plasma Introduction Pulmonary arterial hypertension (PAH) is a rare, life-threatening disease characterized by a progressive increase in pulmonary vascular resistance. Disease progression is associated with signif- icant increases in pulmonary vascular resistance and sustained elevations in pulmonary artery pressure, which ultimately lead to right ventricular failure and death (Galiè et al., 1998, 2008). PAH can occur with no known underlying cause, or it can occur in association with other conditions such as connective tissue disease, congenital heart defects, cirrhosis of the liver and HIV infection (Farber and Loscalzo, 2004). Therapies approved for PAH target include one of the three main pathways involved in the pathogenesis of PAH – endothelin (ET-1), nitric oxide (NO) and the prostacyclin pathways (Humbert et al., 2004). Endothelin receptor antagonists (ERAs) have emerged as a promising class of therapeutic agents for PAH pathways (Humbert et al., 2004). Sulfonamide-based ERAs have proven effective in the treatment of PAH (Rubin et al., 2002; Barst et al., 2004); however, these have been shown to alter warfarin metabolism by affecting the activ- ity of CYP3A4 and CYP2C9. Ambrisentan is a nonsulfonamide class ET A -selective ERA that is approved in the USA, Canada, EU and other countries world- wide for the treatment of PAH. The S-enantiomer of ambrisentan is most active and the in vivo inversion of S-ambrisentan to R-ambrisentan is negligible. Ambrisentan has been shown to improve exercise capacity, delay time to clinical progression, re- duce disease symptoms and improve cardiopulmonary hemody- namics in patients with PAH (Galiè et al., 2005; Rubin et al., 2005; Gilead Sciences, 2007). Ambrisentan is rapidly absorbed into the systemic circulation (time to maximum mean concentration in plasma, T max , ~2 h) and is highly bound to plasma protein. Concentrations decline over time in a multi-exponential manner. Both maximum mean concentration in plasma (C max ) and area under the curve (AUC) increase proportionally over the thera- peutic range of the drug (Rubin et al., 2005), and pharmacokinet- ics were similar in fed and fasted states. After repeated adminis- tration of ambrisentan 5 or 10 mg/day, mean C max values were 539 and 1147 ng/mL. Mean AUC from time 0 to 24 h values were 4804 and 12591 ng h/mL at steady state (Croxtall and Keam, 2008). In contrast to the sulfonamide-based ERAs, ambrisentan is primarily metabolized via hepatic glucuronidation (Galiè et al., 2005; Rubin et al., 2005; Gilead Sciences, 2007), and does not undergo significant phase 1 metabolism by CYP pathways. The hydroxylated ambrisentan is the major metabolite observed in human plasma and was also seen in mouse, rat and rabbit plasma. The AUC for the hydroxylated ambrisentan was approx- imately 4% relative to the parent ambrisentan AUC (Spence et al., 2009; Gilead Sciences, 2007). Pharmacokinetic data show that systemic exposure is greater in patients with PAH than in * Correspondence to: Ramakrishna Nirogi, Discovery Research, Suven Life Sciences Ltd, Serene Chambers, Road 5, Avenue 7, Banjara Hills, Hyderabad 500034, India. E-mail: ramakrishna_nirogi@yahoo.co.in Discovery Research, Suven Life Sciences Ltd, Serene Chambers, Road 5, Avenue 7, Banjara Hills, Hyderabad 500034, India Abbreviation used: ERA, endothelin receptor antagonists; PAH, pulmonary arterial hypertension. Biomed. Chromatogr. 2012 Copyright © 2012 John Wiley & Sons, Ltd. Research article Received: 31 October 2011, Accepted: 9 November 2011 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/bmc.2670