RP-HPLC-荧光法测定大鼠海马组织中5种氨基酸类神经递质

The determination of 5 amino acid neurotransmitters in hippocampus of rats by RP-HPLC with fluorescence detector

目的采用反相高效液相色谱（RP-HPLC）-荧光检测技术建立一种用于测定脑海马组织中5种氨基酸[天门冬氨酸（Asp）、谷氨酸（Glu）、天冬酰胺（Aspa）、谷胺酰胺（Gln）、甘氨酸（Gly）]类神经递质的方法。方法采用甲醇：水=1：1提取大鼠脑海马组织中多种氨基酸类神经递质。用6一氨基喹啉基-N-羟基-琥珀酰亚胺氨基甲酸酯（AQC）柱前衍生RP-HPLC-荧光法测定海马组织中Asp、Glu、Aspa、Gin、Gly含量。结果5种氨基酸类神经递质在15min内完全分离，分离效果良好；在0．5-100．0μg/μL范围有较好的线性关系，相关系数（r）I〉0．99990；Asp、Glu、Aspa、Gin、Gly的检出限分别为0．05、0．068、0．037、0．05、0．02μg/μL。精密度[相对标准偏差（RSD）]为0．4％～2．5％，加样回收率为70．27％～128．20％，RSD均〈5％；8只正常大鼠海马组织中Asp、Glu、Aspa、Gln、Gly含量分别为38．98±9．66、126．42±34．06、216．00±0．75、90．44±33．75、（12．95±4．42）斗g／g。结论RP-HPLC一荧光法快速、准确、灵敏度高，适用于脑组织中氨基酸类神经递质含量的测定。

Objective To establish a method of reverse phase high performance liquid chromatography （RP-HPLC） with fluorescence detector to determine 5 amino acid [ aspartate （Asp）, glutamate （Glu）, asparagine （Aspa）, glutamine （Gln） and glycine （Gly） ] neurotransmitters. Methods The amino acid neurotransmitters in hippocampus of rats were collected by methanol： water = 1 ： 1. The RP-HPLC with fluorescence detector with 6-aminoquinolyl-N- hydroxysuecinimidyl carbamate （AQC） pre-column derivatization was used to measure the 5 amino acid neurotransmitters in hippocampus of rats. Results The 5 amino acid neurotransmitters were well and completely separated using elution within 15 min. There was a good linear correlation in the range of 0.5-100.0 μg/μL, and the correlation coefficient （r） was≥0. 999 90. The detection limits of Asp, Glu, Aspa, Gin and Gly were 0.05, 0. 068, 0.037, 0.05 and 0.02 μg/μL. The precisions [ relative standard deviation（RSD） ] were 0.4% -2.5%. The recoveries of added amino acids were 70.27%-128. 20% （ RSD 〈 5% ）. The contents of Asp, Glu, Aspa, Gin and Gly in hippocampus of normal rats were 38.98 ±9.66, 126.42 ± 34.06, 216.00 ± 0.75, 90.44 ± 33.75 and （ 12.95 ±4.42） Ixg/g. Conclusions RP-HPLC with fluorescence detector is quick, reliable and sensitive to measure the amino acid neurotransmitters in hippocampus of rats.