用高效液相色谱电化学检测器测定大小鼠脑内单胺类物质的研究

Assay Procedures for the Determination of Monoamines in Rat and Mouse Brain by Means of Reversed-phase High-performance Liquid Chromatography with Electrochemical Detection

应用反相高效液相色谱法和电化学检测器对单胺类物质如去甲肾上腺素、多巴胺、5-羟色胺及酸性代谢产物3,4二羟基苯乙酸、5-羟吲哚醋酸和高香草酸的色谱条件及样品的制备进行了研究.结果表明:降低流动相的pH及甲醇含量或增加离子对B_8的含量可使样品的保留时间延长,而增加二正丁胺的含量可缩短保留时间。当pH=3.7,甲醇10％,二正丁胺1.3mmol,离子对B_81.2 mmol时,分离效果最好,能在25 min内将七种物质完全基线分离.在样品的制备方面,用高氯酸沉淀蛋白提取单胺类物质,45,000g×20 min一次离心取上清液20μl直接进样,所含杂质少且不干扰单胺类物质的测定.本实验方法简单,快速、灵敏,比较实用.

The determination of noradrenaline, dopamine, 5-hydroxytryptamine and their acidic metabolites was carried out by high-performance liquid chromatography with electrochemical detection. Results showed that it is important for the separation of samples to be adjusted to proper pH and composition of the mobile phase. The decrease of pH and methanol or increase of ion-pairing B8 all prolong the retentive time of samples, while the increase of dibutylamine might shorten it. The best separation was obtained when the mobile phase contains 10% methanol, 1.2 mmol/L ion-pairing Ba, 1.3 mmol/L dibutylamine and pH = 3.7. Seven monoamines were separated thoroughly within 25 minutes in this condition. In the procedures of preparing samples, the sample was treated with 0.1 mol/L HCIOs, then centri-fuged for 20 minutes at 45000 g at 4℃. The supernatants were injected directly into the HPLC system. Therefore, it is recommended as a simple, rapid and sensitive method for determination of monoamines in rat and mouse brain.