高效液相色谱法测定血清叶黄素和玉米黄素

Determination of lutein and zeaxanthin in serum by high performance liquid chromatography

目的:建立C30-高效液相色谱(high performance liquid chromatography,HPLC)内标法测定血清叶黄素、玉米黄素和β-胡萝卜素。方法:应用Develosil C30-UG色谱柱(250 mm×4.6 mm,5μm),流动相A为乙腈∶甲醇(体积比60∶40),流动相B为甲基叔丁基醚,梯度洗脱。流速1 mL/min,检测波长450 nm,进样量20μL,柱温25℃。内标物为全反式-β-阿扑-8'-胡萝卜醛。结果:血清叶黄素、玉米黄素和β-胡萝卜素的分离效果良好,保留时间分别为9.9min、10.3 min和21.2 min,日内相对标准差分别为3.22%、3.81%和1.60%。血清叶黄素、β-胡萝卜素在0.012 5～12.5 mg/L(r=0.999 5、r=0.999 7),玉米黄素在0.005～5.0 mg/L(r=1)范围内呈良好线性关系。58名正常老年人(年龄>50岁)血清叶黄素、玉米黄素和β-胡萝卜素的平均浓度分别为0.410μmol/L、0.054μmol/L和0.128μmol/L。结论:该C30-HPLC内标法是定量分析血清叶黄素、玉米黄素和β-胡萝卜素的可靠方法之一。

Objective：To establish a simultaneous determination method for measuring lutein, zeaxan- thin and β-carotene in serum by internal standard on C30-HPLC. Methods： Experimental data were as follows ： stationary phase, Develosil carotenoid column C30 （ 250 mmx 4.6 ram, 5 μm） ; mobile phase A, acetonitrile ： methanol （3 ： 2, v/v） ; mobile phase B, MTBE ; grads elution ; flow rate, I mL/min ; monitoring wavelength, 450 nm ; injection volume, 20 wL; column temperature, 25 ℃. Results： Lutein, zeaxanthin and β-carotene were thoroughly separated with the average retention time of 9.9 rain, 10.3 min and 21.2 rain, respectively. The intra-day relative standard deviation （RSD） values were 3.22% , 3.81% and 1.60%. The linear ranges of serum concentrations of lutein and β-carotene were both 0.012 5 -12.5 mg/L （r =0.999 5, r =0.999 7）, and that of zeaxanthin was 0.005 -5.0 mg/L （r = 1 ）. The mean serum concentrations of lutein, zeaxanthin and β-carotene for 58 healthy elder inhabitants （ 〉50 years） were 0. 410 μmol/L, 0. 054 μmo]/L and 0. 128 μmol/L, respectively. Con- clusion： This established method can be used for determination of lutein, zeaxanthin and B-carotene in serum.