金纳米粒子富集-高效液相色谱同时测定人血浆中三种氨基硫醇

建立了金纳米粒子富集-高效液相色谱-紫外检测(HPLC-UVD)同时测定人血浆中3种氨基硫醇(半胱氨酸(Cys)、高半胱氨酸(Hcys)、谷胱甘肽(GSH))的新方法。以Tween 20修饰的金纳米粒子作为选择探针萃取富集氨基硫醇。经二硫苏糖醇脱附后,采用SpursilTM C18柱(250 mm×4.6 mm, 5 μm)分离氨基硫醇,以60 mmol/L磷酸盐缓冲溶液(pH 2.0)等度洗脱,检测波长为200 nm。3种氨基硫醇的浓度分别在0.025～350 μmol/L、0.02～60 μmol/L、0.01～50 μmol/L内与峰面积具有良好的线性关系,相关系数均高于0.99。方法检出限(信噪比为3)分别为5.0、6.0和2.5 nmol/L,回收率为92.8%～106.0%。该方法能显著降低血浆样品中内源性物质的干扰,提高HPLC-UVD的选择性和灵敏度。将该方法应用于心血管病人血浆中上述氨基硫醇的分离测定,结果显示: 与对照组相比,疾病组血浆中的Hcys和GSH水平存在显著性差异,Cys不存在显著性差异。

Simultaneous determination of three aminothiols in human plasma by high performance liquid chromatography with gold nanoparticle enrichment

A high performance liquid chromatography (HPLC) method with ultraviolet detection (UVD) for the simultaneous determination of cysteine (Cys), homocysteine (Hcys) and glutathione (GSH) in human plasma was established. Tween 20-capped gold nanoparticles (Tween 20-AuNPs) were used as selective probes for the extraction and enrichment of aminothiols. The aminothiols removed from the Tween 20-AuNPs by dithiothreitol were separated on a SpursilTMC18 column (250 mm×4.6 mm, 5 μm) with a mobile phase of 60 mmol/L phosphate buffer solution (pH 2.0). The column eluent was monitored using UVD at the wavelength of 200 nm. The linear ranges were found between peak area and concentration in the ranges of 0.025~350 μmol/L for Cys, 0.02~60 μmol/L for Hcys and 0.01~50 μmol/L for GSH, with linear regression coefficients all above 0.99. The limits of detection (LOD, signal to noise ratio of 3) for the aminothiols were between 2.5 nmol/L and 6.0 nmol/L and the recoveries were between 92.8% and 106.0%. Due to the significant interference reduction of endogenous substances in the plasma and the subsequent improvement of the selectivity and sensitivity of HPLC-UVD, the present method was applied for the simultaneous analysis of the forementioned aminothiols in the plasma samples of cardiovascular disease patients. The results showed that the levels of Hcys and GSH had significant differences between the patients and the controls, while that of Cys had no significant difference.