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

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 (2.1 mm × 150 mm, particle size 3.5 μm) and a XBridge C18 column (2.1 mm × 50 mm, particle size 3.5 μm). The mobile phases were mixtures of acetonitrile, methanol, and water containing 1% formic acid; flow rate was 400 μL/min. The triple stage quadrupole mass spectrometer was operated in ESI mode and the methods were linear over a range of 0.400-100 ng/mL (almorexant, M5, M6), 1.00-100 ng/mL (M3, M8), and 50.0-1000 ng/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.