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    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

    • 摘要: 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30 mg·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6,12,24,48,72 h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1 g∶7.5 mL的比例在每份脑组织中加入组织匀浆液并匀浆30 s,在4℃高速离心2次,每次15 min,取其上清液供色谱分析。在Hypeisil GOLD aQ色谱柱(4.6 mm×250 mm,5 μm)上,用不同比例的甲醇和30 mmol·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1 000 μg·L-1以内,其检出限(3S/N)在0.34~5.26 μg·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3 d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。

       

      Abstract: 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 h after 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 h separately 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 s with 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, 5 μm) was used as the stationary phase, and mixtures in various ratios of methanol and 30 mmol·L-1 KH2PO4-H3PO4 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 1 000 μg·L-1, with their detection limits (3S/N) in the range of 0.34-5.26 μg·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.

       

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