用液相色谱串联质谱法检测脐带血血清25-羟基维生素D2和25-羟基维生素D3浓度

doi:10.3969/j.issn.1673-8640.2015.08.013

摘要/Abstract

摘要：

目的建立一种测定脐带血25-羟基维生素D2[25(OH)D2]和25-羟基维生素D3[25(OH)D3]浓度的方法。方法脐带血血清样本前处理采用饱和硫酸锌和乙腈的蛋白沉淀法。采用液相色谱串联质谱法(LC-MS/MS)测定25(OH)D2 和25(OH)D3浓度。固定相为Poroshell^?120 EC-C18正向柱;电喷雾离子源(ESI);多反应监测模式(MRM);喷雾电压35.0 psi;喷雾温度180℃;鞘气温度350℃;鞘气流速11.0 L/min。以甲醇(80%)、超纯水(20%)为流动相进行梯度洗脱,流速为0.5 mL/min,柱温为50℃;质谱采用高温ESI,以选择反应监测模式进行定量分析。结果 25(OH)D2和25(OH)D3在3.125~100 ᠜g/L范围内呈良好的线性关系(R²=0.998 7、0.999 1)。血样冻融3次进行检测,日内、日间相对标准偏差(RSD)均<15%。对血清25-(OH)D2和25(OH)D3 质控品进行检测,结果均符合要求。结论 LC-MS/MS敏感性高,分析时间短,适用于脐带血25(OH)D2 和25(OH)D3浓度检测。

关键词: 25-羟基维生素D2, 25-羟基维生素D3, 液相色谱串联质谱, 脐带血

Abstract:

Objective To establish a method for determining the concentrations of 25-hydroxyvitamin D2[25(OH)D2] and 25-hydroxyvitamin D3[25(OH)D3] in cord blood. Methods The serum samples from cord blood were pretreated by acetonitrile and ZnSO₄.High performance liquid chromatography tandem-mass spectrometry (LC-MS/MS) was used for the determinations of 25(OH)D2 and 25(OH)D3 concentrations. The analysis was carried on a Poroshell^? 120 EC-C18 column. Electrospray ionization(ESI), multiple reaction monitor (MRM), nebulizer 35.0 psi, gas temperature 180℃, sheath gas temperature 350℃ and sheath gas flow 11.0 L/min were performed. The column temperature was 50℃ with linear gradient elution using methanol(80%)and water(20%), and the flow rate was 0.5 mL/min. The quantitative analysis was performed by selective reaction monitoring mode of heater ESI. Results A good linearity was detected in the linear range of 3.125-100 ᠜g/L for 25(OH) D2 and 25(OH) D3 (R²=0.998 7 and 0.999 1). Blood samples had frozen and thawed for 3 times. Both intra-day and inter-day relative standard deviations (RSD) were <15%. The quality control materials of serum 25(OH)D2 and 25(OH)D3 were analyzed, and the results met the requirements of quality control. Conclusions LC-MS/MS is sensitive, rapid and suitable for the determinations of serum 25(OH)D2 and 25(OH)D3 concentrations in cord blood.

Key words: 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, Liquid chromatography tandem-mass spectrometry, Cord blood

中图分类号:

R446.1

赵丽, 王伟业. 用液相色谱串联质谱法检测脐带血血清25-羟基维生素D2和25-羟基维生素D3浓度[J]. 检验医学, 2015, 30(8): 825-829.

ZHAO Li, WANG Weiye.. Determinations of serum 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 concentrations in cord blood by liquid chromatography tandem-mass spectrometry[J]. Laboratory Medicine, 2015, 30(8): 825-829.

图/表 9

参考文献 12

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反应物名称	质荷比		极性
反应物名称	母离子		子离子
25(OH)D2-d3	416.4	398.3	正极
25(OH)D2	413.3	395.3	正极
25(OH)D2	413.3	355.2	正极
25(OH)D3-d3	404.3	386.3	正极
25(OH)D3	401.3	383.2	正极
25(OH)D3	401.3	159.1	正极

反应物名称	质荷比		极性
反应物名称	母离子		子离子
25(OH)D2-d3	416.4	398.3	正极
25(OH)D2	413.3	395.3	正极
25(OH)D2	413.3	355.2	正极
25(OH)D3-d3	404.3	386.3	正极
25(OH)D3	401.3	383.2	正极
25(OH)D3	401.3	159.1	正极

项目	日内精密度(%)			日间精密度(%)
项目	3.125 ᠜g/L	25 ᠜g/L	100 ᠜g/L	3.125 ᠜g/L	25 ᠜g/L	100 ᠜g/L
25(OH)D2	0.81	1.58	1.90	8.41	4.43	1.86
25(OH)D3	1.01	1.44	1.65	2.18	2.62	1.41

项目	日内精密度(%)			日间精密度(%)
项目	3.125 ᠜g/L	25 ᠜g/L	100 ᠜g/L	3.125 ᠜g/L	25 ᠜g/L	100 ᠜g/L
25(OH)D2	0.81	1.58	1.90	8.41	4.43	1.86
25(OH)D3	1.01	1.44	1.65	2.18	2.62	1.41

保存时间 (h)	25(OH)D2
	3.125 ᠜g/L		25 ᠜g/L		100 ᠜g/L
	测定值(᠜g/L)	RSD(%)	测定值(᠜g/L)	RSD(%)	测定值(᠜g/L)	RSD(%)
0	4.71	1.52	26.09	2.14	104.22	3.71
24	3.92	0.43	25.64	1.97	105.21	2.02
48	4.12	0.89	27.81	3.95	108.80	9.59
保存时间(h)	25(OH)D3
	3.125 ᠜g/L		25 ᠜g/L		100 ᠜g/L
	测定值(᠜g/L)	RSD(%)	测定值(᠜g/L)	RSD(%)	测定值(᠜g/L)	RSD(%)
0	3.77	1.52	24.87	1.98	105.40	4.18
24	4.10	1.42	25.91	1.30	103.27	1.97
48	3.96	1.46	27.03	4.85	107.92	8.34