高效液相色谱法测定多巴胺-β-羟化酶活性

Determination of dopamine-β-hydroxylase in rat serum and brain tissue by high performance liquid chromatography with electrochemical detector

目的建立一种通过高效液相色谱法测定酶促反应的产物真蛸胺(Octopamine)的量来确定大鼠血清及脑组织中多巴胺-β-羟化酶活性的方法。方法采用ODS柱,流动相为缓冲液(0.02mol/L柠檬酸三钠和0.05mol/L磷酸氢二钠)∶甲醇=85∶15的溶液,流速1.0mL/min。结果待测物真蛸胺标准溶液的保留时间和峰面积的RSD分别在0.29～0.67之间和0.09～0.36之间,重现性好;回收率在85.3%～95.4%之间,检测限1.58pmol/mL;真蛸胺的线性范围在10～160pmol/mL之间,相关系数为0.9996。结论该方法为神经毒理学、药理学等研究提供了一个简单灵敏、稳定可靠的方法。

Objective To develop a rapid, reliable and simple method detecting dopamine-β- hydroxylase in rat serum and brain tissue by high performance liquid chromatography with electrochemical detector. Methods An ODS column was selected as separation column at room temperature, and the mobile phase （pH4.50） consisted of 0.02 mol/L trisodium citrate and 0.05 mol/L disodium phosphate containing 15% methanol （volume ratio） in distilled water. The mobile phase was pumped at a flow rate of 1. 0 mL/min and the oxidation potential was set at ＋ 0.75 V. Octopamine in the brain tissue （striatum） and serum was separated and determined under the above conditions. Results RSD% of the retention time and peak area of standard sample was in a range of 0.29 - 0.67 and 0.09 - 0.36, respectively. The recovery of the analyte was over 85 %. The detection limit of 1.58 pmol/mL for octopamine was achieved with standard solutions. The linearity was over a range of 10- 160 pmol/mL for octopamine, and the linearity relation coefficient was 0. 999 6. Conclusions The assay has been applied successfully to measure the cortex and serum concentrations of dopamine-β-hydroxylases in rats.