异硫氰酸苯酯柱前衍生RP-HPLC测定大鼠脑组织中9种氨基酸类神经递质含量

Content Determination of Nine Kinds of Amino Acids Neurotrasmitters in Rat Brain Tissue by HPLC with Pre-column PITC Derivatization

关键词:  高效液相色谱法  柱前衍生  异硫氰酸苯酯  氨基酸类神经递质

Key words:  HPLC  precolumn derivatization  phenylisothiocyanate  amino acid neurotransmitter

摘要:

目的 建立一种脑组织中氨基酸类神经递质的测定方法。方法 醋酸钠-乙腈-水为流动相，反相高效液相色谱洗脱，异硫氰酸苯酯(PITC)柱前衍生和紫外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9种氨基酸类神经递质(谷氨酸、门冬氨酸、甘氨酸、牛磺酸、γ-氨基丁酸、谷氨酰胺、酪氨酸、色氨酸、丝氨酸)的含量。结果 9种氨基酸在50 min内洗脱完全，分离效果良好；浓度在5~400 μmol·L^-1内线性关系良好，且相关系数>0.99，平均回收率≥80%。结论 本方法简便、准确、灵敏度高、重复性好，适用于一般实验室脑组织中氨基酸类神经递质的测定。

Abstract:

OBJECTIVE Establish the method of measuring the amino acid neurotransmitters contents in rat brain tissue. METHODS Sodium acetate-acetonitrile-water was used as the mobile phase. The reversed phase HPLC system with pre-column derivatization by Phenyl isothiocyanate(PITC) and with UV detection was used to measure the contents of nine kinds of amino acid neurotransmitters including glutamate(Glu), aspartate(Asp), glycine(Gly), taurine(Tau), γ-aminobutyric acid(GABA), glutamine(Gln), tyrosine(Tyr), tryptophan(Trp) and serine(Ser) in the cerebral cortex, hippocampus, striatum, mesencephalon, cerebellum and hypothalamus of rats. RESULTS The nine kinds of amino acids were well separated using elution within 50 minutes. Good linear response was obtained for the nine kinds of amino acids within the range of 5 to 400 μmol·L^-1 with correlation coefficients not less than 0.99. The average recovery rate of these amino acids was not less than 80%. CONCLUSION The method is simple, accurate, high sensible and better repeatability. It was saitable for measuring the amino acid neurotransmitters in brain tissue in common laboratory.