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 and simvastatin d 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 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 / 408.3→271.1 and / 412.0→275.10 for ezetimibe and ezetimibe d, and / 419.30→285.20 and / 425.40→199.20 for simvastatin and simvastatin d. 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.