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.SPSS 24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4 h内即达释放峰值,SE肽段3 h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-2.95 mg/L,95％置信区间为-4.26～1.65 mg/L.本方法在16.9～58.5 mg/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.

abstractsObjective 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&nbsp;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.