微透析-HPLC-ECD联用在线测定烟碱暴露大鼠脑内单胺神经递质的动态变化

In vivo determination of monoamine neurotransmitters in brain striatum of rat using online microdialysis coupled with high performance liquid chromatography-electrochemical detector

为探讨烟碱依赖的形成机理,实时评价烟碱引起的单胺神经递质变化效应,采用在线微透析和高效液相色谱-电化学检测器(HPLC-ECD)联用方法测定了大鼠脑纹状体中多巴胺(DA)、3,4-二羟基苯乙酸(DOPAC)、高香草酸(HVA)、5-羟色胺(5-HT)和5-羟吲哚乙酸(5-HIAA)的含量。以2.0μL/min灌流,每15 min收集1次透析液并自动注入五氟苯基柱,在梯度洗脱条件下分离检测。结果表明:方法的线性范围为0.2~500.0 ng/m L,日内和日间精密度均≤9.7%,加标回收率为86.2%~105.9%,体外透析探针回收率为25.3%~28.3%。以2.0 mg/kg烟碱进行腹腔注射后,大鼠纹状体中DA和5-HT的释放增加但未见持续增强,而HVA和5-HIAA的量明显升高。该方法简便快速,灵敏度高,选择性好,适合于烟碱作用下活体动物脑内单胺类神经递质变化的动态分析。

To investigate potential mechanism of nicotine dependence and evaluate the monoamine neurotransmitter metabolism caused by nicotine as a function of time, online microdialysis sampling coupled with high performance liquid chromatography and electrochemical detector was developed and applied to determine the contents of dopamine （DA）, 3,4-dihydroxyphenyl acetic acid （DOPAC）, homovanillic acid （HVA）, serotonin （5-HT） and 5-hydroxylindole acetic acid （5-HIAA） in the brain striatum of live rats after nicotine injection. Striatum dialysates were collected every 15 rain at a flow rate of 2.0 μL/min and separated immediately on a HS F5 column under the gradient elution condition. The results showed that the linear range of the method were from 0.2 to 500.0 ng/mL, the recoveries ranged from 86.2% to 105.9%, while the intra-day and inter-day precisions were no more than 9.7%. ht vitro microdialysate recoveries of DA, DOPAC, HVA,5-HT and 5-HIAA ranged from 25.3% to 28.3%. After intraperitoneal injection of nicotine at a rate of 2.0 mg/ kg, the release of DA and 5-HT in brain striatum of rat did not increase continuously, while the release of HVA and 5-HIAA rose significantly. The method is simple, fast, sensitive and suitable for the dynamic analysis of monoamine neurotransmitters in brain of live animal administrated by nicotine via digestion.