毛细管电泳电化学法（CE-ECD）检测人全血中的同型半胱氨酸（Hcy）、半胱氨酸（Cys）和还原型谷胱甘肽（GSH）

 目的 建立毛细管电泳电化学技术(capillary electrophoresis with electrochemical detection,CE-ECD)检测人全血中同型半胱氨酸(homocysteine,Hcy)、半胱氨酸(cysteine,Cys)和还原型谷胱甘肽(reduced glutathione,GSH)的方法。考察缓冲液的浓度、酸碱度、分离电压、进样时间和检测电压等参数对分离和检测的影响,确定最佳的实验条件。方法 以直径为500 μm的铂圆盘电极作为检测电极,用长度为50 cm的熔融石英毛细管对一系列待检物标准溶液和人全血样本进行毛细管电泳电化学检测。结果 在最优条件下,当电极电位为+1.05 V(相对饱和甘汞电极)、分离电压为18 kV时,Hcy、Cys和GSH于100 mmol/L的磷酸盐缓冲液(pH 7.8)中在10 min内获得理想分离。检测下限(S/N=3)在0.29～0.80 μmol/L范围内,且在3倍数量级浓度范围内,3种组分的浓度与峰电流呈良好线性关系。对0.5 mmol/L的混合标准溶液连续检测7次,Hcy、Cys和GSH峰高的相对标准偏差（relative standard deviation,RSD)分别为3.7%、3.1%和2.9%。结论 CE-ECD方法可对Hcy、Cys和GSH等3种生物活性巯基化合物进行高效分离及检测,具有分析速度快、成本低、灵敏度高、试剂及样品用量小等优点,因此在生物医药领域具有广泛的应用前景。本实验采用的铂圆盘电极具有污染少、重复性好的特点。

Determination of homocysteine(Hcy),cysteine(Cys) and reduced glutathione(GSH) in human whole blood by capillary electrophoresis with electrochemical detection(CE-ECD)

Objective To establish a capillary electrophoresis with electrochemical detection(CE-ECD)method for the separation and determination of homocysteine(Hcy),cysteine(Cys) and reduced glutathione(GSH) in human whole blood simultaneously.Effects of several factors such as the acidity and concentration of the running buffer,separation voltage,injection time,and detection potential were investigated to determine optimal conditions.Methods With a 500 μm-diameter platinum disc electrode as the detection electrode,mixture standard solutions of Hcy,Cys,GSH and whole blood samples were determined using CE-ECD method in a 50 cm-long fused silica capillary.Results Under optimal conditions,Hcy,Cys and GSH were well separated within 10 minutes at the working potential of +1.05 V(vs.saturated calomel electrode) and the separation voltage of 18 kV in 100 mmol/L phosphate buffer(pH 7.8).Good linear relationship was observed between peak current and analytes concentration over about 3 orders of magnitude.The detection limits(S/N=3) ranged from 0.29 to 0.80 μmol/L.Relative standard deviations(RSD) of Hcy,Cys and GSH peaks were 3.7%,3.1% and 2.9%,respectively,based on 7 repeated assays for the standard mixture solutions containing 0.5 mmol/L Hcy,Cys and GSH.Conclusions The CE-ECD method was successfully applied to determine Hcy,Cys and GSH in human whole blood and was proved to be convenient,cheap,reliable and sensitive.It would have great potential in a wide range of bioanalytical applications.The platinum disc detection electrode has a good repeatability.