血清总同型半胱氨酸候选参考测量程序（液相色谱串联质谱法）的建立及性能评估

doi:10.3969/j.issn.1673-8640.2018.011.011

摘要/Abstract

摘要：

目的建立一种基于液相色谱串联质谱（LC-MS/MS）技术的血清总同型半胱氨酸（Hcy）候选参考测量程序并对其性能进行评价。方法采用一种简单的蛋白沉淀方法对血清样本进行前处理,然后采用LC-MS/MS定量检测总Hcy,参照美国临床实验室标准化协会（CLSI） C62-A文件和C50-A文件对建立的候选参考方法进行线性、检测限与定量限、基质效应、精密度、正确度等基本分析性能验证。结果 LC-MS/MS检测总Hcy的线性范围为0.5~200.0 μmol/L。定量限和检测限分别为0.31 nmol/g、0.06 nmol/g。3种不同比例（1∶1、80∶20、20∶80）的血清与溶液混合物的相对基质效应分别为1.94%、1.91%、1.78%。批内、批间变异系数（CV）分别为<2%和<1%。3种浓度（30.58、49.21、65.42 nmol/g）的加标样本平均加标回收率分别为99.8%、100.2%、100.8%。测定NIST SRM 1950标准物质的结果偏移<1%。样本处理后分别在室温[（23±2 ）℃]和自动进样器（温度为10 ℃）中放置24 h,检测结果均非常稳定。结论成功建立了基于LC-MS/MS技术的血清总Hcy候选参考测量程序。该参考测量程序准确度高、精密度好,能够用于常规临床检验方法的量值溯源,保证测定结果的准确性。

关键词: 总同型半胱氨酸, 同位素稀释液相色谱串联质谱, 参考测量程序, 性能评估

Abstract:

Objective To establish a candidate reference measurement procedure for serum total homocysteine （Hcy） by liquid chromatography-tandem mass spectrometry （LC-MS/MS）,and to evaluate its analytical performance. Methods Samples were prepared by a simple protein precipitation method,and LC-MS/MS was used to determine serum total Hcy quantitatively. According to the Clinical and Laboratory Standards Institute（CLSI） C62-A and C50-A documents,the performance,including linearity,limit of detection,limit of quantitation,relative matrix effect,precision and trueness,was evaluated. Results The linear range of total Hcy by LC-MS/MS was 0.5-200.0 μmol/L. The limit of quantitation and limit of detection were 0.31 nmol/g and 0.06 nmol/g,respectively. The relative matrix effects were 1.94%,1.91% and 1.78% for sera and solution mixtures in 3 different ratios（1∶1,80∶20 and 20∶80）. The within-run and between-run coefficients of variation（CV）were <2% and <1%. The average recovery rates of 3 levels （30.58,49.21 and 65.42 nmol/g） were 99.8%,100.2%,100.8%, respectively. The bias of NIST standard reference material （SRM） 1950 was <1%. After the samples were processed,they were placed at room temperature [（23±2）℃] and autosampler（10℃） for 24 h, and the results were stable. Conclusions The candidate reference measurement procedure for serum total Hcy by LC-MS/MS has been established,and it has good accuracy and precision,which can be used for measurement traceability.

Key words: Total homocysteine, Isotope-dilution liquid chromatography-tandem mass spectrometry, Reference measurement procedure, Performance evaluation

中图分类号:

R446.1

沈敏, 杨晓东, 王琳, 邹继华, 张曼, 邹炳德. 血清总同型半胱氨酸候选参考测量程序（液相色谱串联质谱法）的建立及性能评估[J]. 检验医学, 2018, 33(11): 1018-1025.

SHEN Min, YANG Xiaodong, WANG Lin, ZOU Jihua, ZHANG Man, ZOU Bingde. Establishment and performance evaluation of candidate reference measurement procedure for serum total homocysteine by liquid chromatography-tandem mass spectrometry[J]. Laboratory Medicine, 2018, 33(11): 1018-1025.

图/表 9

参考文献 23

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名称	定量/ 定性	母离子（m/z）	子离子（m/z）	去簇电压（V）	碰撞能量（V）
Hcy	定量	136.1	90.0	44	15
	定性	136.1	118.0	44	10
Hcy-d4	定量	140.1	94.0	40	15
	定性	140.1	122.1	40	11

名称	定量/ 定性	母离子（m/z）	子离子（m/z）	去簇电压（V）	碰撞能量（V）
Hcy	定量	136.1	90.0	44	15
	定性	136.1	118.0	44	10
Hcy-d4	定量	140.1	94.0	40	15
	定性	140.1	122.1	40	11

样本名称	理论浓度（nmol/g）	样本数	测定均值（nmol/g）	平均S/N	CV（%）	偏移（%）
定量限样本1	0.31	10	0.33	16	9.72	6.45
定量限样本2	0.51	10	0.53	24	7.81	3.92
定量限样本3	0.76	10	0.78	38	5.57	2.63

样本名称	理论浓度（nmol/g）	样本数	测定均值（nmol/g）	平均S/N	CV（%）	偏移（%）
定量限样本1	0.31	10	0.33	16	9.72	6.45
定量限样本2	0.51	10	0.53	24	7.81	3.92
定量限样本3	0.76	10	0.78	38	5.57	2.63

样本	批次	平均值（μmol/L）	批内CV（%）	总体平均值（μmol/L）	批间CV（%）
混合血清1	1	11.33	1.54	11.24	0.76
	2	11.18	1.22
	3	11.19	1.72
混合血清2	1	48.72	1.29	48.83	0.22
	2	48.84	1.29
	3	48.93	0.98
混合血清3	1	81.41	1.00	81.68	0.28
	2	81.81	1.18
	3	81.80	1.51