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同位素稀释液相色谱串联质谱测定血清糖化白蛋白

Establishment of a candidate reference method for serum glycated albumin based on liquid chromatography tandem mass spectrometry

摘要:

目的:建立一种基于同位素稀释液相色谱串联质谱法(ID-LC/MS/MS)检测血清糖化白蛋白(GA)的准确定量方法。方法:本研究在日本临床化学学会(JSCC)推荐的ID-LC/MS方法上进行优化,采用阴离子交换色谱柱提纯白蛋白(Alb),使用重量法准确称量提纯标本、赖氨酸(Lys)和糖化赖氨酸(DOF-Lys)的标准溶液及两种的混合内标,将Alb酸水解为氨基酸,检测Lys和DOF-Lys从而定值GA。根据C62-A文件对建立的方法进行方法学评价并与常规方法进行比对研究。结果:蛋白电泳鉴定标本Alb纯度结果为100%。Lys和DOF-Lys线性标准曲线 R 2范围分别为0.997 8~0.999 9、0.999 4~1.000 0。血清GA测定的批内、批间和总的变异系数( CV)分别为0.69%~1.97%、1.13%~2.33%、1.37%~2.54%。正确度评价中三个浓度参考物质测定结果与认定值偏差分别为-2.56%、-1.87%、-2.94%。Lys和DOF-Lys的基质效应分别为89.92%、109.98%,携带污染率分别为-1.13%、-3.27%。Lys的检测限和定量限分别为0.075、0.248 nmol/g,DOF-Lys的检测限和定量限分别为0.007、0.024 nmol/g。方法 的相对扩展不确定度为1.60%~2.03%。与常规方法的拟合回归曲线 R 2为0.970 7。 结论:建立了一种基于ID-LC/MS/MS法检测血清GA的方法,方法学评价准确可靠,有望作为血清GA检测的候选参考方法。

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abstracts:

Objective:To establish an accurate quantitative method for the determination of serum glycated albumin (GA) based on isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS).Methods:This study optimized the ID-LC/MS method recommended by the Japan Society of Clinical Chemistry (JSCC). Albumin (Alb) was purified by anion exchange chromatography. The standard solution of purified sample, lysine (Lys) and deoxyfructosyl lysine (DOF-Lys) and their mixed internal standard were weighed accurately by weight method. The Alb was hydrolyzed to amino acid, and Lys and DOF-Lys were detected to determine GA. The method was evaluated and compared with the routine method.Results:The purity of Alb was 100%. The R 2 ranges of Lys and DOF-Lys linear standard curves were 0.997 8-0.999 9 and 0.999 4-1.000 0. The results of three-day precision evaluation of low, medium and high concentrations of serum were 0.69%-1.97%, 1.13%-2.33% and 1.37%-2.54% in intra, inter and total Coefficient of variation ( CV). In the accuracy evaluation, the deviations between the results of three concentration standards and the certified value were -2.56%, -1.87% and-2.94%. The matrix effects of Lys and DOF-Lys were 89.92% and 109.98%, and the carryover rates were -1.13% and -3.27% respectively. The detection limit and quantitative limit of Lys were 0.075 nmol/g and 0.248 nmol/g, and the DOF-Lys were 0.007 nmol/g and 0.024 nmol/g. The relative expanded uncertainty was 1.60%-2.03%. The fitting regression curve R 2 with the routine method was 0.970 7. Conclusions:A method was established for the detection of serum GA. The method evaluation was accurate and reliable. It was expected to be a candidate reference method for the detection of serum GA.

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