Xu Yang, Musson Donald G
Merck Research Laboratories, Department of Drug Metabolism and Pharmacokinetics, WP75A-303, West Point, PA 19486, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2008 Oct 1;873(2):195-202. doi: 10.1016/j.jchromb.2008.08.019. Epub 2008 Sep 3.
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.
MK-0974（1a），N-[(3R,6S)-6-(2,3-二氟苯基)-2-氧代-1-(2,2,2-三氟乙基)氮杂环庚烷-3-基]-4-(2-氧代-2,3-二氢-1H-咪唑并[4,5-B]吡啶-1-基)哌啶-1-甲酰胺，是一种具有两个手性中心的新型降钙素基因相关肽（CGRP）受体拮抗剂。在反相条件下，使用纤维素手性固定相Chiralcel OJ-RH柱（150 mm×4.6 mm），通过在Oasis muElution HLB 96孔固相萃取（SPE）板上萃取0.2 mL血浆，实现了其四种立体异构体（1a-d）的直接分离。在Sciex API3000上，采用涡轮离子喷雾（TIS）接口，在正离子模式下通过多反应监测进行串联质谱检测。向低有机流动相中添加三氟乙酸铵可使检测灵敏度提高30多倍。在0.5 - 5000 nM（对于1a）和0.5 - 500 nM（对于其三种异构体1b - d）范围内，验证了人血浆中四种立体异构体的同时定量。对五批人对照血浆进行的日内验证结果显示，所有四种立体异构体的精密度（变异系数%，CV）<12.4%，准确度为96.3 - 105.4%。进一步评估表明该测定具有特异性，样品在三个冻融循环后稳定，回收率合理（高于65%），并且所有四种异构体均未观察到基质效应。对1a手性完整性的研究表明，在给药后的临床样品中观察到的唯一异构体是氮杂环庚烷-3位碳发生构型翻转的非对映异构体1c，其在循环系统中占MK-0974暴露量的2.4 - 5.2%。排除了采血、血浆储存和样品制备过程中发生构型翻转的可能性，而在临床制剂中观察到构型翻转，在100 mg胶囊中1a的含量约为0.12%。
