Validation of an LC-MS/MS method for the quantitative determination of the orexin receptor antagonist almorexant and its four primary metabolites in human plasma

Abstract

A sensitive and selective LC-MS/MS method has been developed to quantify almorexant and its four primary metabolites M3, M5, M6, and M8 in human plasma samples. The method involved protein precipitation with acetonitrile in the high calibration range and liquid/liquid extraction with ethyl acetate in the low calibration range. Labeled internal standards were available for four analytes. Separation was performed with an Eclipse XDB-C18 (2.1mm×150mm, particle size 3.5μm) and a XBridge C18 column (2.1mm×50mm, particle size 3.5μm). The mobile phases were mixtures of acetonitrile, methanol, and water containing 1% formic acid; flow rate was 400μL/min. The triple stage quadrupole mass spectrometer was operated in ESI mode and the methods were linear over a range of 0.400-100ng/mL (almorexant, M5, M6), 1.00-100ng/mL (M3, M8), and 50.0-1000ng/mL (all analytes). The inter-day coefficients of variation were equal to or smaller than 10.5%. The inter-day accuracies were between 92.1% and 105.2%. The validated method was successfully applied to the pharmacokinetic assessment of almorexant and its metabolites in several phase I studies.