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MK-0974 (1a), N-[(3R,6S)-6-(2,3-difluorophenyl)-2-oxo-1-(2,2,2-trifuoroethyl)azepan-3-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo-[4,5-B] pyridine-1-yl)piperidine-1-carboxamide, is a novel calcitonin gene-related peptide (CGRP) receptor antagonist with two chiral centers. Direct separation of its four stereoisomers (1a-d) was achieved using a cellulose chiral stationary phase, a Chiralcel OJ-RH column (150mm x 4.6mm), under reversed-phase condition, following the extraction of 0.2 mL plasma on Oasis mu Elution HLB 96-well solid-phase-extraction (SPE) plate. The tandem mass spectrometric detection was conducted in the positive-ion mode with a turbo-ion-spray (TIS) interface using multiple-reaction- monitoring on a Sciex AP13000. Addition of ammonium trifluoroacetate to low-organic mobile phase improved detection sensitivity by more than 30-fold. The simultaneous quantification of the four stereoisomers in human plasma was validated over the ranges of 0.5-5000 nM for la and 0.5-500 nM for its three isomers (1b-d). Intraday validation, conducted with five lots of human control plasma, resulted in < 12.4% (% coefficient of variation, CV) precision and 96.3-105.4% accuracy for all four stereoisomers. Further evaluation indicated that the assay was specific, the samples were stable after three freeze/thaw cycles, the recovery was reasonable (above 65%) and no matrix effect was observed for all four isomers. Investigation on the chiral integrity of la indicated that the diastereomer 1c, inversion at azepinone-3 carbon, was the only isomer observed in the post-dose clinical samples and accounted for 2.4-5.2% of MK-0974 exposure in the circulatory system. The possibility of inversion during blood collection, plasma storage and sample preparation was ruled out, while inversion was observed in the clinical formulation accounting for similar to 0.12% of 1a in a 100-mg capsule, (C) 2008 Elsevier B.V. All rights reserved.