9-芴甲基氯甲酸酯柱前衍生反相高效液相色谱法测定大鼠脑内海马组织中谷氨酸与γ-氨基丁酸含量  

The Changes of GLutamic Acid and Gamma Aminobutyric Acid in Brain Hippocampus of Rats by Reversed-Phase High Performance Liquid Chromatography with Precolumn 9-fluorenylmethyl Chloroformate Derivatization

作　　者：曾春梅 雷丹青[2] 董乐 廖宇晗 黄琪[1] 吴原[1] ZENG Chunmei;LEI Danqing;DONG Le(Department of Neurology,the First Affiliated Hospital of Guangxi Medical University,Nanning (530021),Guangxi,China)

机构地区：[1]广西医科大学第一附属医院神经内科,广西南宁530021 [2]广西医科大学

出　　处：《癫痫与神经电生理学杂志》2019年第1期1-7,共7页Journal of Epileptology and Electroneurophysiology(China)

基　　金：国家自然科学基金资助项目(NO:81760242;名称:Parvalbumin介导的GLu-GABA代谢途径在癫痫发作终止中的作用及分子机制).

摘　　要：目的:建立并改良柱前衍生反相高效液相色谱法(RP-HPLC),测定大鼠脑内海马组织中氨基酸类神经递质,研究戊四唑(PTZ)致病大鼠脑内海马组织中谷氨酸(GLu)及γ-氨基丁酸(GABA)的含量变化。方法:通过PTZ皮下注射SD大鼠进行癫痫动物造模,将大鼠随机分为正常对照组(皮下注射生理盐水)、癫痫组(皮下注射PTZ),样品经甲醇及乙腈处理后,采用RedClassical AQ-C18色谱柱(5μm,250 mm*4.6 mm),以9-芴甲基氯甲酸酯(FMOC-CL)为柱前衍生剂,流动相A:50 mmol/L乙酸钠溶液(PH4.8)410 mL+去离子水85 mL+四氢呋喃5 mL;流动相B:乙腈500 mL。流速:1.0mL/min,在柱温30~32℃下采用梯度洗脱程序进行洗脱,检测波长265nm。结果:GLu和GABA在30min内分离完全,在质量浓度为20~200μg/mL范围内有良好的线性关系,线性相关系数均大于0.9995;两种氨基酸加标回收率均大于100%,加标回收率的RSD均小于2%。结论:改良后的FMOC-CL柱前衍生反相高效液相色谱法稳定、简单、反应快速、灵敏度高、特异性及重复性好,能有效分离并检测大鼠脑内海马组织中的GLu与GABA。Objective:To establish and improve reversed-phase high performance liquid chromatography (RP-HPLC)of precolumn derivatization for the determination of amino acid neurotransmitters in brain hippocampus of rats and study the changes of GLutamic acid (GLu)and gamma aminobutyric acid (GABA)in brain hippocampus of PTZ-induced epileptic rats.Methods..A model of temporal lobe epilepsy was induced via PTZ kindling in SD male rats by hypodermic injection.The rats were divided into the control group and the epileptic group.When amino acids in samples were pretreated with methanol and acetonitrile,the chromatographic separation was performed on a RedClassical AQ-18 column (250*4.6ram,5μm)using 9-fluorenylmethyl chloroformate (FMOC-CL)as pre-column derivatization and A [50mmoi/L sodium acetate (PH4.8)410 mL+water 85 mL+diethylene oxide5mL]and B racetonitrile500mL]in the mobile phase at a current velocity being 1.0 mL/min. This method used gradient elution and the chromatogram was detected at a wavelength of 265 nm with the column temperature being from 30℃ to 32℃.Results:GLu and GABA were completely separated within 30 min.The linear relation was well in the range.from 20μg/mL to 200μg/mL,their concentrations had a high linear relationship with its peak area (r>0.999 5).The sample adding standard recoveries of this two amino acid were more than 100%,and the relative standard deviation of sample adding standard recoveries were less than 2%.Conclutions:The method has a series of advantages, such as good stability,simpleness,high sensitivity,promptness,good specificity and repeatabiltity. Improved the precolumn derivatization reverse phase high performance liquid chromatography with a reaction with FMOC-CL can effectively separate and measure GLu and GABA in brain hippocampus of rat.

关 键 词：反相高效液相色谱法(RP-HPLC) 9-芴甲基氯甲酸酯(FMOC-CL) 谷氨酸(GLu) γ-氨基丁酸(GABA) 海马 

分 类 号：O657.72[理学—分析化学]