Liquid chromatography–negative ion electrospray tandem mass spectrometry method for the quantification of ezetimibe in human plasma

Abstract

A simple, reliable and sensitive liquid chromatography–tandem mass spectrometry method (LC–MS/MS) was developed and validated for quantification of free and total ezetimibe in human plasma. The analyte and internal standard ( 13C6-ezetimibe) were extracted by liquid–liquid extraction with methyl tert-butyl ether. The reversed-phase chromatographic separation was performed on a Capcell C18 column, and the plasma extract was eluted with a gradient consisting of acetonitrile and 5 mM ammonium acetate. The analyte was detected using negative ionization by multiple reaction monitoring mode. The mass transition pairs of m/ z 408.5 → 270.8 and m/ z 414.5 → 276.8 were used to detect ezetimibe and internal standard, respectively. The assay exhibited linear ranges from 0.02 to 20 ng/ml for free ezetimibe and 0.25 to 250 ng/ml for total ezetimibe in human plasma. Acceptable precision and accuracy were obtained for concentrations of the calibration standard and quality control. The validated method was successfully used to analyze human plasma samples for application in a pharmacokinetic study.