反相高效液相色谱法测定大鼠脑组织中4种氨基酸类神经递质

Reverse Phase High Performance Liquid Chromatography for the Determination of Four Amino Acid Neurotransmitters in Rat Brain Tissues

目的:建立一种高效液相色谱(high performance liquid chromatography,HPLC)荧光分析技术,用于测定大鼠脑组织中天门冬氨酸(aspartate,Asp)、谷氨酸(glutamate,Glu)、牛磺酸(taurine,Tau)和γ-氨基丁酸(γ-aminobutyric acid,γ-GA-BA)。方法:色谱柱为Dikma Inertsil ODS(250mm×4.6mm,3.5μm),预柱为Dikma C18柱,4.0mm×3.0mm;流动相为磷酸二氢钾缓冲液(0.1mol/L,pH 6.0):甲醇:乙腈为6∶3∶1;流速为1mL/min。结果:4种氨基酸在15min内完全分离,其在0.1～2μmol/L浓度范围内与峰面积有良好的线性关系,提取回收良好。结论:此种HPLC荧光分析法快速、准确、灵敏度高,适用于大鼠脑组织中氨基酸类神经递质含量的测定。

Objective：To develop a high performance liquid chromatography （HPLC） method with fluorescence detection for simultaneous separation and rapid determination of four amino acid neurotransmitters in rat brain tissues, aspartate （Asp）, glu- tamate （Glu）, taurine （Tau） and γ-aminobutyric acid （y-GABA）. Methods：The HPLC column, Dikma Inertsil ODS （250 mm × 4.6 mm, 3.5 μm） was protected with a security guard cartridge （Dikma, C18, 4.0 mm × 3.0 mm）. The precolumn derivat ization reagent was o-phthalaldehyde （OPA）, and homoserine was used as the internal standard. The mobile phase was a mix ture of potassium dihydrogen phosphate buffer （0.1 tool/L, pH 6.0） ： methanol ： acetonitrile （6 ： 3 ： 1） with a flow rate of 1 mL/min. Results： Four amino acids were separated within 15 rain. A good linear correlation was of tained in the range of 0.1- 2 μmol /L amino acid concentration with good extracted recoveries. Conclusions： The established HPLC method is rapid, accu rate and sensitive. It is suitable for the detection of neurotransmitter levels in rat brain tissues.