HPLC-FLD测定不同脑区3种单胺神经递质及其代谢产物含量

Content Determination of Three Kinds of Monoamine Neurotrasmitters and Their Metabolites in Rat Different Brain Tissue by HPLC with Fluorescence Detection

关键词:  大鼠脑组织  单胺类神经递质  高效液相色谱-荧光检测

Key words:  rat brain tissue  monoamine transmitters  HPLC-FLD

摘要:

目的 建立并优化测定脑组织中单胺类神经递质及其代谢产物含量的高效液相色谱荧光检测方法。方法 采用Inertsustain C₁₈色谱柱（250 mm×4.6 mm，5 μm），流动相为甲醇和水，其中水相中每升含柠檬酸30 mmol、乙酸钠40 mmol、EDTA-2Na 0.2 mmol、辛烷磺酸钠0.4 mmol（pH=3.8），梯度洗脱，流速为1.0 mL·min^-1，荧光检测波长λex=280 nm，λem=330 nm。结果 重酒石酸去甲肾上腺素、盐酸多巴胺、盐酸5-羟色胺、3-甲氧基-4-羟基苯乙胺、3，4-二羟基苯乙酸、高香草酸、5-羟吲哚乙酸7种成分在45 min内洗脱完全，分离效果良好；浓度0.9~400.0 μg·L^-1内线性关系良好，且相关系数>0.99，平均回收率>80%。结论 本方法简便、准确、分离度高、重复性好，适用于一般实验室脑组织中单胺类神经递质及其代谢产物的测定。

Abstract:

OBJECTIVE To establish a method for determination of the monoamine neurotrasmitters and their metabolites in brain tissues of the rats by HPLC with fluorescence detection. METHODS Inertsustain C₁₈ column(250 mm×4.6 mm, 5 μm) was used, the mobile phase composed of methanol and water, included 30 mmol citric acid, 40 mmol NaAc, 0.2 mmol EDTA-2Na and 0.4 mmol 1-octanesulfonic acid sodium salt in 1 L water(pH=3.8), gradient elution, the flow rate was 1.0 mL·min^-1, fluorometric detector wave length setting of excitation at 280 nm and emission at 330 nm. RESULTS Norepinephrine, dopamine, serotonin hydrochloride, 2-(4-hydroxy-3-methoxyphenyl)ethylamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, 5-hydroxyindole-3-acetic acid were completely eluted and well separated in 45 min. Good linear response was obtained for the seven components within the range of 0.9-400.0 μg·L^-1 with correlation coefficients >0.99. The average recovery rate of these components was >80%. CONCLUSION The method is simple, accurate, high separation and good repeatability. It is suitable for measuring the monoamine neurotransmitters and their metabolites in cerebral tissue in common laboratory.