色谱

色谱 ›› 2019, Vol. 37 ›› Issue (4): 404-411.DOI: 10.3724/SP.J.1123.2018.11012

• 研究论文 • 上一篇    下一篇

维生素C抗氧化-同位素内标稀释-超高效液相色谱-串联质谱同时测定新生期大鼠海马体神经递质的含量

邝洪轩1, 张海彬1, 谭建华3, 雒亦凡1, 刘舒华1, 庞启华1,2, 李楚华1, 范瑞芳1   

  1. 1. 华南师范大学生命科学学院, 广东省药食资源生物加工及综合利用工程技术研究中心, 广东 广州 510631;
    2. 广东工业大学环境科学与工程系, 广州市环境催化与污染控制重点实验室, 广东 广州 510006;
    3. 广州质量监督检测研究院, 广东 广州 511447
  • 收稿日期:2018-11-08 出版日期:2019-04-08 发布日期:2015-02-14
  • 通讯作者: 范瑞芳.Tel:(020)85211372,E-mail:20001047@m.scnu.edu.cn
  • 基金资助:

    国家自然科学基金项目（21777048）；广州市环境催化与污染控制重点实验室开放基金（GKLECPC-09）.

Simultaneous determination of five neurotransmitters in neonatal rat hippocampus by adding vitamin C coupled with isotope dilution-ultra-high performance liquid chromatography-tandem mass spectrometry

KUANG Hongxuan1, ZHANG Haibin1, TAN Jianhua3, LUO Yifan1, LIU Shuhua1, PANG Qihua1,2, LI Chuhua1, FAN Ruifang1   

  1. 1. Guangdong Provincial Engineering Technology Research Center for Drug and Food Biological Resources Processing and Comprehensive Utilization, School of Life Sciences, South China Normal University, Guangzhou 510631, China;
    2. Guangzhou Key Laboratory of Environmental Catalysis and Pollution Control, School of Environmental Science and Engineering, Institute of Environmental Health and Pollution Control, Guangdong University of Technology, Guangzhou 510006, China;
    3. GuangZhou Quality Supervision and Testing Institute, Guangzhou 511447, China
  • Received:2018-11-08 Online:2019-04-08 Published:2015-02-14
  • Supported by:

    National Natural Science Foundation of China (No. 21777048); Open Fund of Guangzhou Key Laboratory of Environmental Catalysis and Pollution Control (No. GKLECPC-09).

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摘要：

脑区神经递质的测定对于研究神经系统的作用机制具有重要意义。该研究建立了同位素内标稀释-超高效液相色谱-串联质谱同时测定双酚A暴露后大鼠海马体中5种神经递质含量的方法，包括谷氨酸、γ-氨基丁酸、乙酰胆碱、多巴胺以及5-羟色胺。选用2%（体积分数）乙酸水-甲醇（9：1，v/v）溶液配制标准样品及溶解样品，以Ultimate AQ-C18（150 mm×4.6 mm，3 μ m）为分离柱，0.1%（体积分数）甲酸水和甲醇为流动相，在28℃柱温下，梯度洗脱，所有化合物在3 min内出峰。添加维生素C能显著减少多巴胺和5-羟色胺在前处理以及样品储存过程中的氧化，极大地提高神经递质的稳定性，从而准确定量。该方法标准曲线线性关系良好，相关系数R2>0.998，检出限和定量限低，日内和日间精密度为0.39%~13.6%，加标回收率为92.9%~119%，实际进样残留低，已被成功地应用于新生期双酚A暴露后大鼠海马体神经递质含量的测定。

关键词: 超高效液相色谱-串联质谱, 海马体, 神经递质, 维生素C

Abstract:

Analysis of neurotransmitters in brain tissue is useful for mechanistic studies of the central nervous system. Isotope dilution coupled with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for the simultaneous determination of glutamic acid, γ-amino butyric acid, acetylcholine, dopamine, and serotonin in the hippocampus tissue. A 2% (v/v) acetic acid in water-methanol (9:1, v/v) solution was used to prepare the standards and re-dissolute samples after nitrogen drying. An Ultimate AQ-C18 column (150 mm×4.6 mm, 3 μm) was used as the analytical column and 0.1% (v/v) formic acid in water and methanol were used as the mobile phase. Gradient elution was performed and all target compounds were eluted over 3 min at 28℃. Vitamin C spiked during the sample pretreatment and storage periods significantly retarded the oxidation of dopamine and serotonin and improved the neurotransmitter stability. The developed method showed good linearities (correlation coefficient (R2)>0.998), low detection limits (0.15-1.0 μg/L), good inter-and intra-day precisions (relative standard deviations:0.39%-13.6%), good accuracy (92.9%-119%), low carry-over, and excellent stability. Moreover, the method was successfully applied and validated in the determination of rat hippocampus exposed to bisphenol A.

Key words: hippocampus, neurotransmitters, ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), vitamin C

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