液相色谱串联质谱临床检测方法的开发与验证

doi:10.3969/j.issn.1673-8640.2019.03.001

摘要/Abstract

摘要：

液相色谱串联质谱（LC-MS/MS）是近年来发展极为迅速的新技术。该技术结合了液相色谱的高分离性能和质谱的高敏感性、高特异性等优势,被广泛应用于化工、生物、医药、食品、临床医学、环境等领域。在临床检验和诊断领域,LC-MS/MS作为传统诊断技术的补充,可提供更为准确、可靠的依据,使许多疾病得到准确、快速的诊断。文章结合目前已公布的LC-MS/MS方法相关验证指南、文献及实际操作经验,系统介绍了LC-MS/MS方法开发的关键流程,并以25-羟基维生素D₃[25（OH）D₃]为实例介绍方法验证的关键要素,为LC-MS/MS技术临床应用方法的建立、验证和实施提供参考。

关键词: 液相色谱串联质谱, 方法开发, 方法验证

Abstract:

Liquid chromatography-tandem mass spectrometry （LC-MS/MS） is an emerging technology which has developed rapidly in recent years. It has combined the separation properties of liquid chromatography and the high sensitivity and specificity of mass spectrometry,which is widely applied in various areas,such as chemistry,biology,pharmaceutical science,food,clinic and environmental science. Especially in the field of clinical laboratory and diagnostics,LC-MS/MS used as a complement to traditional diagnostic techniques can often provide more accurate and reliable testing results in accurate and rapid diagnosis of diseases. In this review,some internationally published LC-MS/MS method validation guidelines,related literature and practical experience were summarized,and some key processes of LC-MS/MS development were introduced. Using 25-hydroxyvitamin D₃[25（OH）D₃] as an example,the key elements of method validation were reviewed,in order to provide a reference for the establishment,verification and implementation of LC-MS/MS.

Key words: Liquid chromatography-tandem mass spectrometry, Method development, Method validation

中图分类号:

R446.1

中国医师协会检验医师分会临床质谱检验医学专业委员会. 液相色谱串联质谱临床检测方法的开发与验证[J]. 检验医学, 2019, 34(3): 189-196.

Clinical Mass Spectrometry Committee, Chinese Medical Doctor Association of Laboratory Medicine. Consensus of method development and validation of liquid chromatography-tandem mass spectrometry in clinical laboratories[J]. Laboratory Medicine, 2019, 34(3): 189-196.

图/表 8

参考文献 15

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乙醇基质			血清基质（提取后）
样本名称	组分名称	峰面积	样本名称	组分名称	峰面积
Sample1-1	D3-IS	93 240	Sample3-1	D3-IS	259 600
Sample1-1	D3-IS	97 270	Sample3-1	D3-IS	272 200
Sample1-1	D3-IS	97 910	Sample3-1	D3-IS	270 100
Sample1-2	D3-IS	111 300	Sample3-2	D3-IS	260 900
Sample1-2	D3-IS	111 000	Sample3-2	D3-IS	261 900
Sample1-2	D3-IS	112 500	Sample3-2	D3-IS	262 300
Sample1-3	D3-IS	97 200	Sample3-3	D3-IS	256 800
Sample1-3	D3-IS	99 710	Sample3-3	D3-IS	273 500
Sample1-3	D3-IS	96 880	Sample3-3	D3-IS	261 100
Sample1-4	D3-IS	105 500	Sample3-4	D3-IS	274 100
Sample1-4	D3-IS	111 500	Sample3-4	D3-IS	272 400
Sample1-4	D3-IS	111 200	Sample3-4	D3-IS	278 100
Sample1-5	D3-IS	106 800	Sample3-5	D3-IS	276 200
Sample1-5	D3-IS	106 700	Sample3-5	D3-IS	278 600
Sample1-5	D3-IS	108 900	Sample3-5	D3-IS	272 000
Sample1-6	D3-IS	114 100	Sample3-6	D3-IS	270 100
Sample1-6	D3-IS	112 900	Sample3-6	D3-IS	263 000
Sample1-6	D3-IS	113 300	Sample3-6	D3-IS	268 800
均值		105 995	均值		268 428
CV（%）		6.61	CV（%）		2.55

乙醇基质			血清基质（提取后）
样本名称	组分名称	峰面积	样本名称	组分名称	峰面积
Sample1-1	D3-IS	93 240	Sample3-1	D3-IS	259 600
Sample1-1	D3-IS	97 270	Sample3-1	D3-IS	272 200
Sample1-1	D3-IS	97 910	Sample3-1	D3-IS	270 100
Sample1-2	D3-IS	111 300	Sample3-2	D3-IS	260 900
Sample1-2	D3-IS	111 000	Sample3-2	D3-IS	261 900
Sample1-2	D3-IS	112 500	Sample3-2	D3-IS	262 300
Sample1-3	D3-IS	97 200	Sample3-3	D3-IS	256 800
Sample1-3	D3-IS	99 710	Sample3-3	D3-IS	273 500
Sample1-3	D3-IS	96 880	Sample3-3	D3-IS	261 100
Sample1-4	D3-IS	105 500	Sample3-4	D3-IS	274 100
Sample1-4	D3-IS	111 500	Sample3-4	D3-IS	272 400
Sample1-4	D3-IS	111 200	Sample3-4	D3-IS	278 100
Sample1-5	D3-IS	106 800	Sample3-5	D3-IS	276 200
Sample1-5	D3-IS	106 700	Sample3-5	D3-IS	278 600
Sample1-5	D3-IS	108 900	Sample3-5	D3-IS	272 000
Sample1-6	D3-IS	114 100	Sample3-6	D3-IS	270 100
Sample1-6	D3-IS	112 900	Sample3-6	D3-IS	263 000
Sample1-6	D3-IS	113 300	Sample3-6	D3-IS	268 800
均值		105 995	均值		268 428
CV（%）		6.61	CV（%）		2.55

样本	峰面积比值				CV（%）	偏差（%）
样本	第1次测定	第2次测定	第3次测定	均值	CV（%）	偏差（%）
乙醇基质	1.52	1.53	1.49	1.51	1.38	1.96
血清基质	0.53	0.52	0.53	0.53	1.10
1:1混合物	1.01	1.01	0.97	1.00	2.32

样本	峰面积比值				CV（%）	偏差（%）
样本	第1次测定	第2次测定	第3次测定	均值	CV（%）	偏差（%）
乙醇基质	1.52	1.53	1.49	1.51	1.38	1.96
血清基质	0.53	0.52	0.53	0.53	1.10
1:1混合物	1.01	1.01	0.97	1.00	2.32

样本	25（OH）D₃	L-L	H-L
L1	1.11
L2	1.13	1.13
L3	1.09	1.09
H1	52.80
H2	53.45
L4	1.16		1.16
H3	53.60
H4	52.56
L5	1.10		1.10
L6	1.14	1.14
L7	1.09	1.09
L8	1.05	1.05
H5	52.28
H6	53.48
L9	1.09		1.09
H7	52.14
H8	53.09
L10	1.15		1.15
H9	52.79
H10	53.02
L11	1.12		1.12
均值		1.10	1.12
携带污染率（%）		-1.82