高效液相色谱-电化学检测法测定生物样品中单胺类神经递质及其代谢产物*

Determination of monoamine neurotransmitter and its metabolites by high performance liquid chromatography and electrochemical detection

关键词:  高效液相色谱-电化学检测法  单胺类神经递质  代谢物

Key words:  HPLC-ECD  Monoamine Neurotransmitter  Metabolites

摘要:

[目的]建立一种简单、快速、灵敏的测定生物样品中单胺类神经递质及其代谢产物的高效液相色谱方法。[方法]色谱柱采用WatersC₁₈反相色谱柱(3.9mm×150mm,5μm),流动相为水相(每500mL中含辛烷磺酸钠1.25g,磷酸二氢钾6.8g,乙二胺四乙酸18mg,pH值3.3):甲醇:乙腈=78:19:3,流速为0.8mL/min,柱温35℃;电化学检测器工作电极为玻璃碳电极,参比电极为银/氯化银(Ag/AgCl)电极,工作电压为0.7V。[结果]5种物质保留时间分别为:去甲肾上腺素3.6min,5-羟吲哚乙酸4.5min,高香草酸5.2min,多巴胺6.2min,5-羟色胺11.9min;线性范围均为20～1000μg/L,最低检测限为4～8μg/L。[结论]此方法测定生物样品中的单胺类神经递质及其代谢产物,简便,快速且灵敏度高。

Abstract:

[Objective] A simple, rapid and sensitive HPLC-ECD method was established to detect the monoamine neurotransmitter and its metabolites. [Methods] The LC was performed on a Waters @ ODS C₁₈ column (3.9×500 mm, 5μm). The mobile phase, consisted of Methanol-Acetonitrile-Water phase (containing B 8 1.25 g, KH₂PO₄ 6.8 g, EDTA 18 mg in 500 mLsolution, pH=3.3)=19:3:78. The mobile phase was at a flow-rate of 0.8 mL/min. The electrochemical detector was equipped with a glass carbon working electrode and a Ag/AgCl reference electrode. The working volt was 0.7 V. The column temperature was kept at 35℃. [Results] Their retention time were 3.6 min for NE, 4.5 min for 5-HIAA, 5.2 min for HVA, 6.1 min for DA and 11.9 min for 5-HT respectively. These five components were all found tobe linear over a concentration range of 20～1 000 μg/L. The lowlimit of quantitation was 4～8 μg/L. [Conclusion] The HPLC-ECD method was rapid, sensitive and specific for quantitative deter-mination ofmonoamine neurotransmitter and its metabolites.