高效液相荧光法测定大鼠脑内氨基酸类神经递质方法的改良

目的 改良测定大鼠脑组织氨基酸类神经递质的反相高效液相色谱荧光法.方法 改良使用磷酸盐-甲醇-乙腈作为流动相,反相高效液相色谱洗脱,高丝氨酸作为内标,邻苯二甲醛柱前衍生和荧光检测器,检测大鼠大脑皮质、海马、纹状体、中脑、小脑和下丘脑6个脑区中天冬氨酸(Asp)、谷氨酸(Glu)、谷氨酰胺(G1n)、甘氨酸(Gly)、γ-氨基丁酸(GABA)和牛磺酸(Tau)6种氨基酸类神经递质含量.结果 6种氨基酸在20 min内洗脱完全,分离效果良好；在6.25～ 400 μmol/L浓度范围有较好的线性关系,其相关系数不低于0.99;6种氨基酸日内试验精密度范围为1.38％ ～7.59％；日间试验精密度为2.7％～8.68％；6种氨基酸回收率不低于80％.结论 改良后的反相高效液相色谱荧光法灵敏度较高、重复性好,能有效分离检测大鼠脑组织分区中氨基酸类神经递质含量.

Improvement of high-performance liquid chromatography with fluorescence detection to measure amino acid neurotransmitters in the rat brain

Objective To improve the method of reversed phase high-performance liquid chromatography with fluorescence detection（HPLC-FL） to measure amino acid neurotransmitters in rat brain tissues.Methods Sodium phosphate-methanol-acetonitrile was used as the mobile phase.The reversed phase HPLC system with pre-column derivatization by o-phthalaldehyde and with fluorescence detection was used to measure the amino acid neurotransmitters including aspartate（Asp）,glutamate（Glu）,glutamine（Gln）,glycine（Gly）,γ-aminobutyric acid（GABA） and taurine（Tau） in the cerebral cortex,hippocampus,striatum,mesencephalon,cerebellum and hypothalamus of rats.Results The six kinds of amino acids were well separated using elution within 20 minutes.Good linear response was obtained for the 6 standards within the range of 6.25 to 400 μmol/L with correlation coefficients not less than 0.99.For these amino acids,good precisions was obtained by 1.38% to 7.59% for the intra-day precision（RSD,%） and 2.7% to 8.68% for the inter-day precision.The recovery of added amino acids was not less than 80%.The feasibility and advantages of this improved method were validated by determination of the 6 kinds of amino acid in 6 regions of brain samples from 5 rats.Conclusions The improved method of reversed phase HPLC with fluorescence detection is more sensitive,has better reproducibility,and can effectively separate and measure the amino acid neurotransmitters in rat brain.