柱前衍生高效液相色谱测定糖化血红蛋白  

Determination of glycated hemoglobin by pre-column derivatization and HPLC separation

作　　者：王冬环[1] 周伟燕[1] 张天娇[1] 汪静[1] 马嵘[1] 张江涛[1] 

机构地区：[1]北京医院卫生部临床检验中心,北京100730

出　　处：《中国实验诊断学》2015年第12期1991-1995,共5页Chinese Journal of Laboratory Diagnosis

基　　金：国家自然科学基金(项目号:81171665;81201337;81301488)

摘　　要：目的建立一种柱前衍生高效液相色谱(high performance liquid chromatography,HPLC)测定糖化血红蛋白(HbA1c)的方法。方法蛋白内切酶Glu-C将血红蛋白从β链N末端水解得到糖基化和非糖基化的六肽(HbA1c六肽和HbA0六肽),HbA1c六肽和HbA0六肽与对氨基苯磺酸进行衍生反应,生成糖基化和非糖基化偶氮六肽衍生物,采用高效液相色谱对衍生物进行分离、测定,根据HbA1c衍生六肽和HbA0衍生六肽的峰面积比计算样本中HbA1c的量。结果对高、中、低不同浓度血样进行反复多次分析,总变异系数CV分别为1.61%、1.74%和0.90%;HbA1c浓度在0.3%-15.6%范围内,线性回归方程为Y=1005 X+7.26,R=0.995 9,线性关系良好;对低、中、高3个浓度的HbA1c国家有证标准物质(GBW09181、GBW09182和GBW09183)进行测定,测定值与认证值的偏差在-3.81%-3.53%之间。采用本研究方法和常规方法分别对20份人血样本进行测定,相关回归方程为Y=0.928 X+0.407,相关系数为R=0.996 5,二者有良好的相关性。结论建立了柱前衍生HPLC测定HbA1c的方法,本法具有很好的稳定性、精密度和准确性。Objective A method for separation and quantification of glycated hemoglobin by pre-column derivatization and HPLC was developed.Methods Haemoglobin is cleaved into peptides by the enzyme endoproteinase Glu C,the glycated and non-glycated N-terminal hexapeptides of theβchain（HbA1chexapeptides and HbA0 hexapeptides）were obtained,separated and quantified of HbA1 chexapeptides and HbA0 hexapeptides by derivation with p-aminobenzenesulfonic acid and HPLC.The percentage of HbA1 cin the sample can be calculated from the peak area ratios for the HbA1 cderivative hexapeptides and HbA0 derivative hexapeptides.Results The investigation obtain the total coefficients of variation（CV）for sample analysls were 1.61%,1.74% and 0.90%.A standard curve regression equation Y=1005 X＋7.26,R=0.995 9,The linearity range was 0.3%-15.6% HbA1 c.The results on certified reference materials showed bias of-3.81%-3.53%%.Conclusion A highly precise and accurate method for measuring HbA1 chas been developed by pre-column derivatization and HPLC.

关 键 词：血红蛋白A 糖基化 肽 柱前衍生 色谱法 液相 

分 类 号：R446.1[医药卫生—诊断学]