HPLC法测定tremor大鼠脑组织中谷氨酸和GABA含量

Determination of glutamate and GABA in tremor rat by HPLC

目的建立测定tremor大鼠(tm)脑内海马组织中GLU及GABA浓度的高效液相色谱(HPLC)方法。方法冰上剥离13wk的tm/Wistar大鼠海马组织,低温匀浆处理,利用邻苯二甲醛(OPA)柱前衍生HPLC法结合荧光检测器测定GLU和GABA的含量。0.1mol·L-1醋酸钾和甲醇进行二元梯度洗脱,峰面积定量。结果GLU的线性方程为Y^=1E+06X(r2=0.9937),线性范围为0.15～5.88mg·L-1,具有良好的线性关系,在0.74、1.47、3.68mg.L-13个浓度下样本回收率分别为97.7%、98.9%、100.5%;样品中GLU日内变异系数为3.8%,隔日测定后,日间变异系数为5.9%;GABA的线性方程为Y^=4E+06X(r2=0.998),线性范围为0.10～2.15mg·L-1,具有良好的线性关系,在0.2、0.5、1.0mg·L-13个浓度下样本回收率分别为98.3%、101.0%、102.7%;样品GABA日内变异系数为2.3%,隔日测定后,日间变异系数为3.9%。结论该方法较简便、快速且干扰相对较小,满足测定要求,可用于氨基酸的含量测定研究。

Aim To set up a reversed-phase HPLC method for determining the concentration of glutamate（GLU）and GABA in tremor rats.Methods The brain of 13 W tremor rat and Wistar rat was removed and the hippocampus was dissected out on an ice-cold plate,weighted with OPA derivatives by means of high performance liquid chromatography method.The mobile phase consisted of 0.1 mol·min-1 potassium acetate and methanol by gradient elution and measured through the peak areas.Result A good linearity was obtained from 0.15~5.88 mg·L^-1 of GLU with r2=0.9937,The standard curve of GLU was Y^=1E＋06X（r2=0.9937）.The recoveries of three concentration （0.74,1.47,3.68 mg·L^-1） were 97.7%,98.9%,100.5%respectively.The RSD of intraday variation of GLU was 3.8% and that of interday variation was 5.9%.A good linearity was obtained from 0.10~2.15 mg·L^-1 of GABA with r2=0.998.The standard curve of GLU was Y^=4E＋06X（r2=0.998）.The recoveries of three concentration （0.2,0.5,1.0 mg·L^-1） were 98.3%,101.0%,102.7% respectively.The RSD of intraday variation of GLU was 2.3% and that of interday variation was 3.9%.Conclusion The method is simple,accurate and has low interference,and can be used to study other amnio acids.