基于液相色谱-串联质谱技术(LC-MS/MS)检测癫痫小鼠毛发中内源性大麻素(2-AG、AEA)的含量

Liquid Chromatography and Tandem Mass Spectrometry Determined the Content (2-AG,AEA) Endocenic Cannabinoids in Epileptic Mice

目的:基于液相色谱-串联质谱技术(LC-MS/MS)对癫痫小鼠毛发中内源性大麻素(2-AG、AEA)进行含量检测。方法:匹鲁卡品腹腔注射方法建立小鼠癫痫模型,采用LC-MS/MS检测毛发中2-AG、AEA的含量,对比分析不同癫痫发作分级小鼠2-AG、AEA含量差异。结果:标准品溶液中AEA、2-AG的保留时间分别为14.3 min,18.1 min,毛发样品中AEA、2-AG的保留时间分别为14.0 min,17.7 min。毛发样品中AEA的检测限、定量限、回收率为0.7 pg/mg、2.1 pg/mg和97.9%,毛发样品中2-AG的检测限、定量限、回收率为3.2 pg/mg、10.9 pg/mg和99.3%,且二者的日内、日间变异系数均低于15%。癫痫发作1级、2级、3级、4级、5级这五个分级癫痫小鼠的AEA含量和2-AG含量依次升高,不同癫痫发作分级小鼠毛发样品中AEA含量和2-AG含量比较(P均<0.05)。结论:LC-MS/MS测定癫痫小鼠毛发中2-AG、AEA的表达量,具有灵敏度更高,样品使用量更少等优点,可大规模样本研究。

Objective:The content of endocannabinoids(2-AG,AEA)was examined based on liquid chromatography and tandem mass spectrometry(LC-MS/MS)in epileptic mice.Methods:The mouse epilepsy model was established by pilocarpine peritoneal injection,methanol incubation and solid phase extraction column extraction,qualitatively tested under atmospheric chemical ionizing positive ion mode and multi-reaction monitoring,quantitatively tested 2-AG and AEA content by liquid chromatography and series mass spectrometry,compared the differences in 2-AG and AEA content of different seizure grades of epileptic mice.Result:The retention times of AEA and 2-AG in the standard solution were 14.3 min,18.1 min,and those of AEA and 2-AG in the hair samples were 14.0 min,17.7 min,respectively.Hair AEA,2-AG detection limits were 0.7 pg/mg and 3.2 pg/mg,quantitative limits of 2.1 pg/mg and 10.9 pg/mg,respectively,the recovery rates were 97.9%and 99.3%,respectively,and the day and daytime coefficient of variation was less than 15%.AEA and 2-AG were increased in grade seizures,grade 1,grade 3,grade 4,and grade 2,and AEA and 2-AG were compared in hair samples of different seizure grades(P<0.05).Conclusion:The determination of endocannabinoids(2-AG,AEA)used by LC-MS/MS has the advantages of higher sensitivity and less sample use,and is conducive to large-scale sample research.