HPLC-ECD法测定大鼠脑组织中单胺类神经递质的含量

目的:建立测定大鼠脑组织中的单胺类神经递质含量的方法。方法:采用高效液相色谱-电化学法,以2,5-二羟基苯甲酸为内标,测定大鼠大脑皮层、小脑、海马组织、下丘脑、嗅球中以去甲肾上腺素(NE)和5-羟色胺(5-HT)为代表的单胺类神经递质的含量。色谱柱为DIKMAC18,流动相为缓冲盐溶液(醋酸钠、庚烷磺酸钠、乙二胺四乙酸二钠和柠檬酸)-甲醇(梯度洗脱),流速为1.0 ml/min,工作电压为+0.7 V。结果:NE、5-HT的检测质量浓度线性范围分别为0.084⁴.2μg/ml(r=0.999 9)和0.0054.2μg/ml(r=0.999 9)和0.005⁰.25μg/ml(r=0.999 1),平均加样回收率分别为98.85%(RSD=2.89%,n=3)和101.5%(RSD=2.41%,n=3),日内(n=5)和日间(n=3)RSD均不大于3%;NE在小脑中的含量最低(0.343±0.14)mg/g],在下丘脑中含量最高(3.062±1.51)mg/g];5-HT在小脑中的含量最低(0.059±0.04)mg/g],在大脑皮层中含量最高(0.383±0.21)mg/g]。结论:本方法灵敏、简便、快速,可用于大鼠脑组织中NE、5-HT含量的测定。

Content Determination of Monoamines Neurotransmitter in Cerebral Tissues of Rats by HPLC-ECD

OBJECTIVE： To establish a method for the content determination of monoamines neurotransmitter in cerebral tissues of rats. METHODS： Using 2,5-dihydroxybenzoic acid as intemal standard, HPLC-ECD was adopted to determine the contents of monoamines neurotransmitter as norepinephrine （NE） and 5-hydroxytryptamine （5-HT） in pallium, cerebellum, hippocampus, hypothalamus and apophysis mamillaris of rats. The determination was performed on DIKMA C18 column with mobile phase consisted of buffer salt （sodium acetate, sodium heptanesulfonate, ETAs, citric acid）-methanol （gradient elution） at the flow rate of 1.0 ml/min. The voltage of electrode was ＋0.7 V. RESULTS： The linear range of NE and 5-HT were 0.084-4.2μg/ml （r=0.999 9） and 0.005-0.25 μg/ml （r=0.999 1）, respectively. The average recoveries were 98.85% （RSD=2.89%, n=3） and 101.5% （RSD= 2.41%, n=3）, respectively. RSD of intra-day （n=5） and inter-day （n=3） were no more than 3%. Content of NE in cerebellum was the lowest （0.343 ±0.14） mg/g], and content of it in hypothalamus was the highest （3.062 ±1.51） mg/g]. Content of 5-HT in cerebellum was the lowest （0.059 ±0.04） mg/g], and content of it in pallium was the highest （0.383 ±0.21） mg/g]. CONCLUSIONS： The method is sensitive, simple and rapid, and can be used for the content determination of NE and 5-HT in cerebral tissue of rats.