Li-Bo Dai, Miao Yan, Huan-De Li, Ping-Fei Fang, Feng Wang, Yang Deng
Clinical Pharmacy and Pharmacology Research Institute, The Second Xiangya Hospital, Central South University, 139 Middle Renmin Road, Changsha, Hunan Province 410011, PR China.
Biomed Chromatogr. 2013 Sep;27(9):1143-9. doi: 10.1002/bmc.2919. Epub 2013 Apr 25.
A simple and sensitive analytical method based on ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) has been developed for determination of moclobemide in human brain cell monolayer as an in vitro model of blood-brain barrier. Brucine was employed as the internal standard. Moclobemide and internal standard were extracted from cell supernatant by ethyl acetate after alkalinizing with sodium hydroxide. The UPLC separation was performed on an Acquity UPLC(TM) BEH C18 column (50 × 2.1 mm, 1.7 µm, Waters, USA) with a mobile phase consisting of methanol-water (29.5:70.5, v/v); the water in the mobile phase contained 0.05% ammonium acetate and 0.1% formic acid. Detection of the analytes was achieved using positive ion electrospray via multiple reaction monitoring mode. The mass transitions were m/z 269.16 → 182.01 for moclobemide and m/z 395.24 → 324.15 for brucine. The extraction recovery was 83.0-83.4% and the lower limit of quantitation (LLOQ) was 1.0 ng/mL for moclobemide. The method was validated from LLOQ to 1980 ng/mL with a coefficient of determination greater than 0.999. Intra- and inter-day accuracies of the method at three concentrations ranged from 89.1 to 100.9% for moclobemide with precision of 1.1-9.6%. This validated method was successfully applied to bidirectional transport study of moclobemide blood-brain barrier permeability.
已开发出一种基于超高效液相色谱-串联质谱法（UPLC-MS/MS）的简单灵敏的分析方法，用于测定作为血脑屏障体外模型的人脑细胞单层中的吗氯贝胺。马钱子碱用作内标。用氢氧化钠碱化后，通过乙酸乙酯从细胞上清液中提取吗氯贝胺和内标。UPLC分离在Acquity UPLC(TM) BEH C18柱（50×2.1 mm，1.7 µm，美国沃特世公司）上进行，流动相由甲醇-水（29.5:70.5，v/v）组成；流动相中的水含有0.05%乙酸铵和0.1%甲酸。通过多反应监测模式，使用正离子电喷雾实现对分析物的检测。吗氯贝胺的质量转移为m/z 269.16→182.01，马钱子碱的质量转移为m/z 395.24→324.15。吗氯贝胺的提取回收率为83.0-83.4%，定量下限（LLOQ）为1.0 ng/mL。该方法在LLOQ至1980 ng/mL范围内得到验证，测定系数大于0.999。该方法在三个浓度下的日内和日间准确度对于吗氯贝胺而言在89.1%至100.9%之间变动，精密度为1.1-9.6%。该经过验证的方法已成功应用于吗氯贝胺血脑屏障通透性的双向转运研究。
