高效液相色谱荧光检测法测定小鼠脑组织中氨基酸类神经递质

doi:10.5246/jcps.2013.03.034

高效液相色谱荧光检测法测定小鼠脑组织中氨基酸类神经递质

王萌萌, 杜望春, 沈杰^*, 董毅, 魏文石, 宋钟娟

1. 复旦大学附属华东医院药剂科, 上海 200040
2. 上海医药高等专科学校临床医学系, 上海 201318
3. 复旦大学上海医学院脑科学研究所医学神经生物学国家重点实验室, 上海 200032
4. 复旦大学附属华东医院神经内科, 上海 200040

收稿日期:2012-11-02 修回日期:2013-03-05 出版日期:2013-05-08 发布日期:2013-05-08
通讯作者: 沈杰*

Determination of amino acid neurotransmitters in mouse brain tissue using high-performance liquid chromatography with fluorescence detection

Mengmeng Wang, Wangchun Du, Jie Shen^*, Yi Dong, Wenshi Wei, Zhongjuan Song

1. Department of Pharmacy, Huadong Hospital Affiliated to Fudan University, Shanghai 200040, China
2. Department of Medicine, Shanghai Institute of Health Sciences, Shanghai 201318, China
3. State Key Laboratory of Medical Neurobiology, Shanghai Medical College and Institutes of Brain Science, Fudan University, Shanghai 200032, China
4. Department of Neurology, Huadong Hospital Affiliated to Fudan University, Shanghai 200040, China

Received:2012-11-02 Revised:2013-03-05 Online:2013-05-08 Published:2013-05-08
Contact: Jie Shen*

摘要/Abstract

摘要：

建立并优化高效液相色谱荧光检测法, 灵敏有效地检测5种氨基酸类神经递质在小鼠脑组织中的浓度。以高丝氨酸(Hse)为内标, 经过邻苯二甲醛柱前衍生化后, 检测天门冬氨酸(Asp)、谷氨酸(Glu)、甘氨酸(Gly)、牛磺酸(Tau)和γ-氨基丁酸(GABA)。通过等度洗脱, 5种氨基酸在24分钟内被精确分离。该方法在定量范围内线性良好, 相关系数不低于0.9998, 且检测限不超过10 nmol/L。以相对标准偏差表示批内及批间重现性均小于或等于10.5%。该方法被成功应用于C57BL/6小鼠黑质和纹状体中氨基酸类神经递质浓度检测。

关键词: 高效液相色谱, 荧光检测法, 邻苯二甲醛, 氨基酸

Abstract:

Amino acid neurotransmitters facilitate the transmission of nerve messages across the synapses and play essential roles in the control and regulation of a variety of functions in the central and peripheral nervous system. In this study, we developed a sensitive and efficient method using high-performance liquid chromatography (HPLC) with fluorescence detection for the assay of five important amino acid neurotransmitters. After derivatization with ο-phthaldialdehyde (OPA), aspartate (Asp), glutamic acid (Glu), glycine (Gly), taurine (Tau) and γ-aminobutyric acid (GABA) were simultaneously detected in the presence of the internal standard homoserine (Hse). Precise separation of these five amino acids was achieved using isocratic elution within 24 min. Good linearity was found over the concentration range with correlation coefficients (r²) not less than 0.9998. The limit of detection (LOD) values were no more than 10 nmol/L. The intra- and inter-day reproducibility was adequate with the relative standard deviation (RSD) of 10.5% or below. This method has also been applied to the analysis of amino acids in the substantia nigra and striatum samples obtained from C57BL/6 mice.

Key words: HPLC, Fluorescence detection, ο-Phthaldialdehyde, Amino acids

中图分类号:

R969

Supporting:

^*Corresponding author. Tel.: 86-21-62483180

王萌萌, 杜望春, 沈杰^*, 董毅, 魏文石, 宋钟娟. 高效液相色谱荧光检测法测定小鼠脑组织中氨基酸类神经递质[J]. , DOI: 10.5246/jcps.2013.03.034.

Mengmeng Wang, Wangchun Du, Jie Shen^*, Yi Dong, Wenshi Wei, Zhongjuan Song . Determination of amino acid neurotransmitters in mouse brain tissue using high-performance liquid chromatography with fluorescence detection[J]. , DOI: 10.5246/jcps.2013.03.034.

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