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

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

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(Gln)、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6.25～400μmol/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。

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.