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 (150 mm x 4.6 mm), under reversed-phase 
condition, following the extraction of 0.2 mL plasma on Oasis muElution 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 API3000. 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 
1a 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 1a 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 approximately 0.12% of 1a in a 100-mg capsule.