高效液相色谱法测定百草枯染毒后小鼠脑组织中8种单胺类神经递质的含量变化

HPLC Determination of 8 Monoamine Neurotransmitters in Brain Tissues of Mice Contaminated with Paraquat and Their Changes After Contamination

应用高效液相色谱法测定小鼠脑组织中8种单胺类神经递质(去甲肾上腺素、肾上腺素、左旋多巴、多巴胺、二羟基苯乙酸、5-羟色胺、5-羟基吲哚乙酸和高香草酸)的含量,并研究其含量随百草枯中毒时间的长短而产生的变化情况。取小鼠36只,分为6组,每组6只。其中一组为对照组,其余5组的小鼠均按30mg·kg^-1的剂量给予灌服百草枯,对照组小鼠灌服同体积的生理盐水。按给毒后达到6,12,24,48,72h时,用1%戊巴比妥钠溶液先后将其麻醉并在低温条件下取其脑组织。按1g∶7.5mL的比例在每份脑组织中加入组织匀浆液并匀浆30s,在4℃高速离心2次,每次15min,取其上清液供色谱分析。在Hypeisil GOLD aQ色谱柱(4.6 mm×250 mm,5μm)上,用不同比例的甲醇和30mmol·L^-1磷酸二氢钾-磷酸缓冲溶液(pH 4.6)的混合液为流动相进行梯度洗脱,并用荧光检测器测定各组分的峰面积,用外标法定量。上述8种单胺类神经递质的线性范围均在1000μg·L^-1以内,其检出限(3S/N)在0.34~5.26μg·L^-1之间。对样品重复测定5次,测得8组分测定值的相对标准偏差(n=5)在0.58%~4.2%之间,加标回收试验得出其回收率在90.8%~106%之间。同时测得了在百草枯染毒后3d内8种单胺类神经递质含量的变化情况,为进一步研究提供可靠依据。

HPLC was applied to the determination of 8 monoamine neurotransmitters(norepinephrine,epinephrine,levodopa,dopamine,dihydroxyphenylacetic acid,serotonin,5-hydroxyindoleacetic acid and high vanillin acid)in brain tissues of mice contaminated with paraquat and their changes in mass concentrations during time intervals within 72 hafter contamination.36 mice were separated into 6 groups with 6 mice in each group.One of the group was taken as the reference group,while the remainder 5 groups were the contaminated groups staying for 6,12,24,48,72 hseparately after contamination with paraquat by pouring down the throat a definite volume of paraquat solution giving 30 mg·kg^-1 of paraquat for each mouse.The mice of reference group were poured down with normal saline.At the end of each staying time,the mice were anesthetized with 1%sodium salt of pentabarbital and their brain tissues were taken and homogenized for 30 swith the tissue-homogenizing solution with its volume by the ratio of 1 g of brain tissue to 7.5 mL of the homogenizing solution.The mixture was centrifuged twice taking 15 min for each.The supernants were taken and used for HPLC analysis.Hypeisil GOLD aQ column(4.6 mm×250 mm,5μm)was used as the stationary phase,and mixtures in various ratios of methanol and30 mmol·L^-1 KH2 PO4-H3 PO4 buffer solution(pH 4.6)were used as mobile phases in the gradient elution.Detections of peak areas of the 8 analytes were taken by fluorescence detector.Linearity ranges of the 8 analytes were obtained all within 1000μg·L^-1,with their detection limits(3 S/N)in the range of 0.34-5.26μg·L^-1.Replicate analysis of a sample was done and values of RSDs(n=5)were found ranged from 0.58%to 4.2%.Recovery was tested by standard addition method,giving recoveries in the range of 90.8%-106%.In further,changes of mass concentrations of each of the 8 neurotransmitters in the brain tissue after contamination with paraquat and within 3 days of staying were also determined and a reliable base for further study was provided.