A high-performance liquid chromatography with fluorescence detection method for the simultaneous quantitation of monoamine neurotransmitters and their metabolites in subregions of rat brain  

高效液相色谱荧光检测法同时测定大鼠脑亚区中单胺类神经递质及其代谢产物（英文）

作　　者：徐鹏[1,2] 白燕平[2] 杨海松[1] 李静[1] 卢炜[1] 凌笑梅[1] 

机构地区：[1]北京大学医学部药学院,北京100191 [2]公安部禁毒情报技术中心,北京100193

出　　处：《Journal of Chinese Pharmaceutical Sciences》2015年第7期458-466,共9页中国药学（英文版）

基　　金：National Natural Science Foundation(Grant No.813 73372);the Open Foundation of State Key Laboratory of Natural and Biomimetic Drugs(Grant No.SKL2012004);Specialized Research Fund for the Doctoral Program of Higher Education of China(Grant No.20110001110021 and 20130001110059)

摘　　要：Abstract： In the presem study, we simultaneously quantified the levels of monoamine neurotransmitters （MANTs） and their metabolites （levodopa, norepinephrine, epinephrine, dopamine, 5-HT, 3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindole-3-acetic acid） in different brain subregions of rats using a newly developed simple, sensitive and selective high-performance liquid chromatography with fluorescence detection （HPLC-FLD） method. In this new HPLC-FLD method, analytes were directly extracted and separated without deriveatization step within 20 min. The FLD wavelength was set at 280 nm and 330 nm for excitation and emission, respectively. The analytes were separated on an Agilent Eclipse Plus Cls column （4.6 mm×150 mm, 5.0 μm） equipped with an Agilent XDB-C18 security guard column （4.6 mm×12.5 mm, 5.0 lam）, and the column temperature was maintained at 35 ℃. The mobile phase for elution was isocratic. The mobile phase consisted of citric acid buffer （50 mmol/L citric acid, 50 mmol/L sodium acetate, 0.5 mmol/L octane sulfonic acid sodium salt, 0.5 mmol/L Na2EDTA and 5 mmol/L triethylamine, pH 3.8） and methanol （90：10, v/v） at a flow rate of 1.0 mL/min. The detection limit （DL） was 0.9-23 nM for all the MANTs and their metabolites with a sample volume of 50 μL. The method was shown to be highly reproducible in terms of peak area （intraday, 0.08%-1.85% RSD, n = 5）. The simultaneous measurement of these MANTs and their metabolites improved our understanding of the neurochemistry in the central nervous system （CNS） in relation to different addictive drugs （methamphetamine, heroin and their mixture） in drug-addicted rat models.建立并优化了一种简单、灵敏、高选择性的高效液相色谱荧光检测法,可同时定量检测大鼠不同脑亚区中的单胺类神经递质及其代谢产物(左旋多巴,去甲肾上腺素,肾上腺素,多巴胺,5-羟色胺,二羟基苯乙酸,高香草酸和5-羟基吲哚乙酸)。在这种新方法中,所有待测物均采用未经过衍生化步骤而直接提取,并在20分钟内达到良好分离。荧光检测的激发波长280 nm,发射波长330 nm。色谱柱:Agilent Eclipse Plus C18柱(美国Agilent公司,4.6 mm×150 mm,5.0μm),保护柱:Agilent XDB-C18(美国Agilent公司,4.6 mm×12.5 mm,5.0μm),柱温:35 oC。流动相:柠檬酸–乙酸钠–辛烷磺酸钠缓冲体系(50 mmol/L柠檬酸,50 mmol/L无水乙酸钠,0.5 mmol/L辛烷磺酸钠,0.5 mmol/L Na2-EDTA,5 mmol/L三乙胺)–甲醇(90:10,v/v,p H 3.8),等度洗脱,流速:1.0 mL/min。所有待测物的检出限为0.9–23 nM,进样体积为50μL。该方法重现性良好,日内精密度在0.08%–1.85%之间(n=5)。对这些物质的同时测定可加深我们对不同成瘾性药物(甲基苯丙胺,海洛因及其二者的混合物)在大鼠中枢神经系统中神经生化作用机制的理解。

关 键 词：High performance liquid chromatography Fluorescence detection Monoamine neurotransmitters Addictive drug Brain subregions Corticolimbic system 

分 类 号：R965[医药卫生—药理学]