Simultaneous determination of ezetimibe and simvastatin in rat plasma by stable-isotope dilution LC-ESI–MS/MS and its application to a pharmacokinetic study

A simple, sensitive and specific liquid chromatography–tandem mass spectrometry method was developed for simultaneous quantification of ezetimibe and simvastatin in rat plasma. The deuterium isotopes: ezetimibe d(4) and simvastatin d(6) were used as internal standards for ezetimibe and simvastatin, respectively. MS/MS detection involved a switch of electron spray ionization mode from negative to positive at retention time 3.01 min. Samples were extracted from plasma by liquid–liquid extraction using tertiary butyl methyl ether. Chromatographic separation was achieved with Agilent Eclipse XBD-C(18) column using mobile phase that consisted of a mixture of ammonium acetate (pH4.5; 10 mM)–acetonitrile (25:75 v/v). The method was linear and validated over the concentration range of 0.2–40.0 ng/mL for simvastatin and 0.05–15.0 ng/mL for ezetimibe. The transitions selected were m/z 408.3→271.1 and m/z 412.0→275.10 for ezetimibe and ezetimibe d(4), and m/z 419.30→285.20 and m/z 425.40→199.20 for simvastatin and simvastatin d(6). Intra- and inter-batch precisions for ezetimibe were 1.6–14.8% and 2.1–13.4%; and for simvastatin 0.94–9.56% and 0.79–12%, respectively. The proposed method was sensitive, selective, precise and accurate for the quantification of ezetimibe and simvastatin simultaneously in rat plasma. The method was successfully applied to a pharmacokinetic study by oral co-administration of ezetimibe and simvastatin in SD rats.