LC-MS/MS同时测定小鼠脑组织中7种神经递质含量

Simultaneous Determination of Seven Neurotransmitters in Mice Brain Tissue by LC-MS /MS

目的:建立一种同时测定小鼠脑组织中7种神经递质的LC-MS/MS分析方法。方法:小鼠脑组织加入4倍量冰冷的0.2%甲酸-水超声匀浆,匀浆液加入0.2%甲酸-乙腈沉淀蛋白,离心,取上清液,加入内标,混匀后进LC-MS/MS分析。采用TSK Gel amide 80(4.5 mm×150 mm,5μm)色谱柱,柱温35℃,流动相乙腈-6 mmol·L-1甲酸铵水溶液(pH 5.5)(67.5∶32.5),流速0.2 mL·min-1,采用正离子多反应检测(MRM)模式进行扫描。通过动物试验验证LC-MS/MS的可靠性,并考察酸枣果肉对小鼠神经递质水平的影响。结果:多巴胺(DA)、肾上腺素(E)、去甲肾上腺素(NE)、5-羟色胺(5-HT)、乙酰胆碱(Ach)、γ-氨基丁酸(GABA)、谷氨酸(Glu)的最低检测限均<0.1μg·L-1,精密度RSD分别为0.52%,0.71%,0.98%,0.35%,0.32%,0.46%,0.94%,准确度RSD分别为0.67%,0.46%,0.53%,0.72%,0.48%,0.59%,0.82%,均符合生物样品的分析要求。动物试验显示与空白组相比,小鼠给予多奈哌齐后,海马和皮层中乙酰胆碱水平极显著升高;而东莨菪碱则作用相反,给药后会降低脑内乙酰胆碱含量,与空白组相比,具有极显著差异。小鼠口服酸枣果肉后,脑内多种神经递质明显发生改变,且变化规律与阳性药物舒乐安定相同。结论:建立的LC-MS/MS分析方法可应用于神经精神类药物的药理作用机制研究。

Objective：To establish a LC-MS/MS method for simultaneous determination of seven neurotransmitters in mice brain tissue.Method：Mice brain tissue was weighed and homogenized with 4 times the amount of ice-cold 0.2％ aqueous formic acid,then homogenates mixed with 0.2％ formic acid-acetonitrile for protein precipitation,centrifugation,the supernatant was collected and mixed with internal standard solution,then the mixture was injected into LC-MS/MS system for assay.Chromatographic separation was performed on a TSK Gel amide 80 column （4.5 mm× 150 mm,5 μm）,column temperature 35 ℃,mobile phase of acetonitrile6 mmol · L-1 ammonium formate solution （pH 5.50） （67.5 ∶ 32.5）,flow rate was 0.2 mL · min-1,taking positive ion multiple reaction monitoring （MRM） mode for scanning.Reliability of LC-MS/MS was verified by animal tests,and effect of jujube pulp on mice neurotransmitters levels was investigated.Result：The minimum detection limit of dopamine （DA）,epinephrine （E）,norepinephrine （NE）,5-hydroxytryptamine （5-HT）,acetylcholine （Ach）,γ-aminobutyric acid （GABA）,glutamate （Glu） were less than 0.1 μg ·L-1,precision RSD of them were 0.52％,0.71％,0.98％,0.35％,0.32％,0.46％,0.94％,respectively; Accuracy RSD of them were 0.67％,0.46％,0.53％,0.72％,0.48％,0.59％,0.82％,respectively.Compared with the blank group animal,after mice were given donepezil,acetylcholine levels in hippocampus and cortex were significantly increased; While scopolamine had opposite effects after administrated,the content of acetylcholine significantly decreased by comparing with the blank group.After oral administration with jujube pulp,neurotransmitters in mice brain tissue significantly changed,and variation was the same with estazolam.Conclusion：This LC-MS/MS analysis method could be applied to pharmacological mechanisms study of neuropsychiatric drugs.