同位素稀释液相色谱串联质谱定量检测血清载脂蛋白E及其表型分型的方法学建立  

Quantification of serum apolipoprotein E and patient phenotyping using isotope dilution liquid chromatography-tandem mass spectrometry

作　　者：李卿 居漪 孙贺伟 范霄宇 张素洁 金中淦 孙立山[2] Li Qing;Ju Yi;Sun Hewei;Fan Xiaoyu;Zhang Sujie;Jin Zhonggan;Sun Lishan(Department of Reference Measurement Laboratory,Shanghai Center for Clinical Laboratory,Shanghai 200126,China;Department of Laboratory Medicine,Shanghai East Hospital,Shanghai 200120,China)

机构地区：[1]上海市临床检验中心参考测量实验室,上海200126 [2]上海市东方医院医学检验科,上海200120

出　　处：《中华检验医学杂志》2019年第8期629-633,共5页Chinese Journal of Laboratory Medicine

基　　金：上海市卫生和计划生育委员会面上项目(201640180).

摘　　要：目的采用同位素稀释液相色谱串联质谱技术(ID-LC-MS),建立一种对血清载脂蛋白E定量检测并分型的方法.方法方法学建立.以同位素标记精氨酸的肽段为内标,对标本进行变性、烷基化和胰蛋白酶酶解处理,液相色谱部分采用SB-C18色谱柱分离,质谱部分采用正离子模式和多反应监测定量和分型.评价该方法的精密度、准确度和线性范围等.收集上海市东方医院2018年10至12月40份血清标本对ID-LC-MS和免疫法进行方法学比较.Deming回归和Bland-Altman分析用于方法学比较.SPSS24软件用于线性评价中的多项式回归分析.结果ApoE的所选酶解肽段在4h内即达释放峰值,SE肽段3h时达到释放峰值.对ApoE分型发现E3/E3、E2/E3和E3/E4型.实验室内不精密度均值为5.2%.低浓度和高浓度准确性评价标本的测定值与靶值的相对偏差分别为7.6%和3.6%.与免疫法比对,Deming回归的截距的95%置信区间为6.44~11.44(P<0.05),斜率的95%置信区间为0.77~0.89(P<0.05),两者的相关性r=0.97.两者差值的均值是-2.95mg/L,95%置信区间为-4.26~1.65mg/L.本方法在16.9~58.5mg/L内呈线性关系.结论本研究建立的ID-LC-MS方法可用于定量血清ApoE,并对ApoE进行蛋白分型.Objective To establish an isotope dilution liquid chromatography tandem mass spectrometry method (ID-LC-MS) for quantification of serum apolipoprotein E and phenotyping. Methods Method establishment. Samples underwent denaturing, alkylation and trypsin digestion with addition of internal standards as isotope labelling arginine. SB-C18 column was used for the liquid chromatographic separation and mass spectrometry positive ion mode and multiple reaction monitoring were employed for quantification and phenotyping. Precision, accuracy and linearity were investigated for method evaluation. 40 serum samples from Shanghai Dongfang Hospital during Oct. to Dec., 2018 were used for method comparison between ID-LC-MS and immunoassay. Deming regression and Bland-Altman were used for method comparison analysis and SPSS 24 for linearity. Results Target peptides reached their releasing maximum within 4 hours and SE did at 3 hours. 3 phenotyping of ApoE were observed, such as E3/E3, E2/E3 and E3/E4. The imprecision of IQC was 5.2%. The relative bias for low and high levels of accuracy-based samples was 7.6% and 3.6%, respectively. Deming regression showed the intercept with 95% confidence interval (CI) was 6.44-11.44 (P<0.05 and the 95% confidence interval for the slopewas 0.77-0.89 (P<0.05). The coefficient was r=0.97. The mean difference was -2.95 mg/L with 95% CI -4.26--1.65 mg/L. The linearity covered from 16.9 to 58.5 mg/L. Conclusion ID-LC-MS can be used to quantify serum apolipoprotein E and simultaneously detect its phenotyping.

关 键 词：载脂蛋白E类 表型 色谱法 液相 串联质谱法 

分 类 号：O657.63[理学—分析化学] R446.11[理学—化学]