反相高效液相色谱法测定大鼠脑组织中4种氨基酸类神经递质

摘要

Objective:To develop a high performance liquid chromatography (HPLC) method with fluorescence detection for simultaneous separation and rapid determination of four amino acid neurotransmitters in rat brain tissues,aspartate (Asp),glu-tamate (Glu),taurine (Tau) and y-aminobutyric acid (y-GABA). Methods:The HPLC column,Dikma Inertsil ODS (250 mm × 4. 6 mm,3. 5 μ/m) was protected with a security guard cartridge (Dikma,C18,4. 0 mm× 3. 0 mm). The precolumn derivat-ization reagent was o-phthalaldehyde (OPA),and homoserine was used as the internal standard. The mobile phase was a mixture of potassium dihydrogen phosphate buffer (0. 1 mol/L,pH 6. 0) : methanol : acetonitrile (6 : 3 : 1) with a flow rate of 1 mL/min. Results: Four amino acids were separated within 15 min. A good linear correlation was obtained in the range of 0. 1-2 μmol /L amino acid concentration with good extracted recoveries. Conclusions: The established HPLC method is rapid,accurate and sensitive. It is suitable for the detection of neurotransmitter levels in rat brain tissues.%目的:建立一种高效液相色谱(high performance liquid chromatography,HPLC)荧光分析技术,用于测定大鼠脑组织中天门冬氨酸(aspartate,Asp)、谷氨酸(glutamate,Glu)、牛磺酸(taurine,Tau)和γ氨基丁酸(γ aminobutyric acid,γ-GA-BA).方法:色谱柱为Dikma Inertsil ODS (250 mm×4.6 mm,3.5 μm),预柱为Dikma C18柱,4.0 mm×3.0 mm;流动相为磷酸二氢钾缓冲液(0.1 mol/L,pH 6.0):甲醇:乙腈为6∶3∶1；流速为1 mL/min.结果:4种氨基酸在15 min内完全分离,其在0.1～2 μmol /L浓度范围内与峰面积有良好的线性关系,提取回收良好.结论:此种HPLC荧光分析法快速、准确、灵敏度高,适用于大鼠脑组织中氨基酸类神经递质含量的测定.