Center of Diagnostics and Therapeutics, Georgia State University, 50 Decatur Street, Atlanta, GA 30303, USA.
J Chromatogr A. 2013 Jan 4;1271(1):207-16. doi: 10.1016/j.chroma.2012.11.046. Epub 2012 Nov 26.
The enantioseparation of warfarin (WAR) along with the five positional and optical isomers is challenging because of the difficulty to simultaneously separate and quantitate these chiral compounds. Currently, no effective chiral CE-MS methods exist for the simultaneous enantioseparation of WAR and all its hydroxylated metabolites in a single run. Polymeric surfactants (aka. molecular micelles) are particularly compatible with micellar electrokinetic chromatography-mass spectrometry (MEKC-MS) because they have a wider elution window for enantioseparation and do not interfere with the MS detection of chiral drugs. Using polysodium N-undecenoyl-L,L-leucylvalinate (poly-L,L-SULV) as a chiral pseudophase in MEKC-MS baseline separation of WAR, its five metabolites along with the internal standard was obtained in 45 min. This is in comparison to 100 min required for separation of the same mixture with packed column CEC-MS using a vancomycin chiral stationary phase. Serum samples were extracted with mixed-mode anion-exchange (MAX) cartridge with recoveries of greater than 85.2% for all WAR and hydroxywarfarin (OH-WAR) metabolites. Utilizing the tandem MS and multiple reaction monitoring mode, the MEKC-MS/MS method was used to simultaneously generate calibration curves over a concentration range from 2 to 5000 ng/mL for R- and S-warfarin, 5 to 1000 ng/mL for R- and S-6-, 7-, 8- and 10-OH-WAR and 10 to 1000 ng/mL for R and S-4'-OH-WAR. For the first time, the limits of detection and quantitation for most WAR metabolites by MEKC-MS/MS were found to be at levels of 2 and 5 ng/mL, respectively. The method was successfully applied for the first time to analyze WAR and its metabolites in plasma samples of 55 patients undergoing WAR therapy, demonstrating the potential of chiral MEKC-MS/MS method to accurately quantitate with high sensitivity.
华法林（WAR）及其五个位置和光学异构体的对映体分离具有挑战性，因为同时分离和定量这些手性化合物具有一定难度。目前，尚无有效的手性毛细管电泳-质谱（CE-MS）方法可在单次运行中同时分离和定量 WAR 及其所有羟基化代谢物。聚合物表面活性剂（又称分子胶束）与胶束电动色谱-质谱（MEKC-MS）特别兼容，因为它们具有更宽的手性药物洗脱窗口，并且不会干扰 MS 检测。在 MEKC-MS 中，使用聚 N-十一烯酰基-L,L-亮氨酰缬氨酸（poly-L,L-SULV）作为手性伪相，在 45 分钟内实现了 WAR、其五个代谢物和内标物的基线分离。相比之下，使用万古霉素手性固定相的填充柱 CEC-MS 分离相同混合物则需要 100 分钟。血清样品用混合模式阴离子交换（MAX）小柱提取，WAR 和羟基华法林（OH-WAR）代谢物的回收率均大于 85.2%。利用串联质谱和多反应监测模式，MEKC-MS/MS 方法可同时在 2 至 5000 ng/mL 范围内为 R-和 S-华法林、5 至 1000 ng/mL 范围内为 R-和 S-6-、7-、8-和 10-OH-WAR 以及 10 至 1000 ng/mL 范围内为 R 和 S-4'-OH-WAR 生成校准曲线。首次发现，MEKC-MS/MS 对华法林大多数代谢物的检测限和定量限分别为 2 和 5 ng/mL。该方法首次成功应用于 55 名接受 WAR 治疗的患者的血浆样品中 WAR 和其代谢物的分析，证明了手性 MEKC-MS/MS 方法具有高灵敏度的准确定量潜力。
