改良超高效液相色谱串联质谱法测定25-羟基维生素D2和25-羟基维生素D3

doi:10.3969/j.issn.1673-8640.2015.10.014

摘要/Abstract

摘要： 目的

建立可同时区分3-epi 25-羟基维生素D3[3-epi 25(OH)D3]、25-羟基维生素D3[25(OH)D3]和25-羟基维生素D2[25(OH)D2]的改良超高效液相色谱串联质谱法(UPLC-MS/MS)。

方法

采用甲醇、硫酸锌沉淀蛋白,以稳定同位素为内标,正己烷提取,色谱采用甲醇和水以一定比例进行梯度洗脱,质谱采用正离子条件下多离子反应监测模式,3-epi 25(OH)D3和25(OH)D3的定量离子通道质荷比(mass to charge, m/z)均为413.3→395.3,25(OH)D2的m/z为401.4→383.4;同位素内标25(OH)D2-d3和25(OH)D3-d3的m/z分别为416.3→398.3和404.4→386.4。25(OH)D3和25(OH)D2分别以各自的标准品和同位素内标制作标准曲线;3-epi 25(OH)D3采用25(OH)D3的标准曲线和内标。评价方法的线性、精密度和准确度等,同时用本法和常规UPLC-MS/MS测定307例临床样本,考察方法的一致性,判别3-epi 25(OH)D3对常规UPLC-MS/MS测定结果的影响。

结果

本法可在13 min内将3-epi 25(OH)D3、25(OH)D3和25(OH)D2完全区分开,3种物质互不干扰,线性相关系数(r)>0.999, 25(OH)D3总变异参数(CV)的均值(范围)为2.82%(2.45%~3.21%),批内CV为1.82%(1.76%~1.91%);25(OH)D2总CV的均值(范围)为4.34%(2.88%~7.01%),批内CV为2.62%(1.91%~3.66%)。25(OH)D2、25(OH)D3和3-epi 25(OH)D3的准确度分别104.5%~106.8%、98.9%~106.9%、108.0%~109.9%。随机测定307例临床样本,结果显示样本3-epi 25(OH)D3浓度均在3.0 ng/mL以下,本法和常规UPLC-MS/MS分析25-羟基维生素D[25(OH)D]的结果分别为15.53±8.58和(15.98±9.08)ng/mL,偏差为-0.46 ng/mL,百分偏差为-2.44%,2种方法的结果差异无统计学意义(t=0.631,P=0.53),R²=0.978。用本法测定得到的维生素D不足、缺乏和充足率分别为76.9%、14.3%、8.8%,常规法的结果分别为75.6%、15.3%、9.1%,2种方法对临床判定的一致率达94.1%,3-epi 25(OH)D3对临床常规UPLC-MS/MS基本无影响。

结论

成功建立了可同时区分3-epi 25(OH)D3、25(OH)D3和25(OH)D2的改良UPLC-MS/MS,该法准确、特异,为评价临床常规UPLC-MS/MS及自动化免疫分析25(OH)D的方法提供了方法学基础。

关键词: 25-羟基维生素D, 3-epi 25-羟基维生素D3, 液相色谱串联质谱法

Abstract: Objective

To establish a method for the determinations of serum 3-epi 25-hydroxyvitamin D3 [3-epi 25(OH)D3], 25-hydroxyvitamin D3 [25(OH)D3] and 25-hydroxyvitamin D2 [25(OH)D2] by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS).

Methods

Serum samples were mixed with internal standard [25(OH)D2-d3 and 25(OH)D3-d3] and treated with methanol and ZnSO4 solution to precipitate protein, and then extracted with hexane. Gradient elution with methanol and water was used in chromatography, and chromatography system was in the positive electro-spray ionization mode and multiple reaction monitor mode, and the transitions used for each analyte were: 3-epi 25(OH)D3 to 25(OH)D3 mass to charge (m/z) 413.3→395.3,3-epi 25(OH)D3 to 25(OH)D2 m/z 401.4→383.4, 25 (OH)D2-d3 m/z 416.3→398.3 and 25(OH)D3- d3 m/z 404.4→386.4. 25(OH)D3 and 25(OH)D2 were quantified by internal standard and standard curves. The quantification of 3-epi 25(OH)D3 used the same internal standard and standard curves as 25(OH)D3. The linearity, precision and accuracy were evaluated. A total of 307 samples were determined by this method and routine UPLC-MS/MS established early to evaluate consistency and the influence of 3-epi 25(OH)D3 on routine UPLC-MS/MS.

Results

By this method, serum 3-epi 25(OH)D3 , 25(OH)D3 and 25(OH)D2 were separated and quantified in 13 min. The linearity correlation coefficients(r) were > 0.999. The total and within-run coefficients of variation (CV) of 25(OH)D3 were 2.82%(2.45%-3.21%) and 1.82%(1.76%-1.91%). For 25(OH)D2, they were 4.34%(2.88%-7.01%) and 2.62%(1.91%-3.66%). The accuracies of 25(OH)D2, 25(OH)D3 and 3-epi 25(OH)D3 were 104.5%-106.8%, 98.9%-106.9% and 108.0%-109.9%, respectively. In all the 307 patients, the concentration of 3-epi 25(OH)D3 was less than 3 ng/mL. The means of this method and routine UPLC-MS/MS for 25-hydroxyvitamin D [25(OH)D] were 15.53±8.58 and (15.98 ±9.08) ng/mL, respectively, with a bias of -0.46 ng/mL, and bias% of -2.44%, and there was no statistical significance between the 2 methods(t=0.631, P=0.53). R² was 0.978. The rates of vitamin D deficiency, insufficiency and sufficiency were 76.9%, 14.3% and 8.8% by this method, respectively, and the rates for routine UPLC-MS/MS were 75.6%, 15.3% and 9.1%. The consistency of the 2 methods was 94.1%. There was no influence of 3-epi 25(OH)D3 on clinical routine UPLC-MS/MS.

Conclusions

The established UPLC-MS/MS can separate serum 3-epi 25(OH)D3, 25(OH)D2 and 25(OH)D3 . It is accurate and specific, and it provides a reference method for the evaluation of routine UPLC-MS/MS and automated immunoassays for determining 25(OH)D.

Key words: 25-hydroxyvitamin D, 3-epi 25-hydroxyvitamin D3, Liquid chromatography tandem mass spectrometry

中图分类号:

R446.1

禹松林, 方慧玲, 张瑞苹, 程歆琦, 韩建华, 苏薇, 程倩, 侯立安, 姜小梅, 邱玲. 改良超高效液相色谱串联质谱法测定25-羟基维生素D2和25-羟基维生素D3[J]. 检验医学, 2015, 30(10): 1021-1026.

YU Songlin, FANG Huiling, ZHANG Ruiping, CHENG Xinqi, HAN Jianhua, SU Wei, CHENG Qian, HOU Li'an, JIANG Xiaomei, QIU Ling. Improved method for the determination of serum 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 by ultra-performance liquid chromatography tandem mass spectrometry[J]. Laboratory Medicine, 2015, 30(10): 1021-1026.

图/表 5

参考文献 11

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质控品	25(OH)D3				25(OH)D2
质控品	均值(ng/mL)		平均批内CV(%)	总CV(%)	均值(ng/mL)	平均批内CV(%)	总CV(%)
Q1	7.38±0.21	1.79	2.81		5.12±0.36	3.66	7.01
Q2	10.91±0.27	1.91	2.45		14.65±0.46	2.30	3.14
Q3	38.16±1.23	1.76	3.21		45.34±1.31	1.91	2.88

质控品	25(OH)D3				25(OH)D2
质控品	均值(ng/mL)		平均批内CV(%)	总CV(%)	均值(ng/mL)	平均批内CV(%)	总CV(%)
Q1	7.38±0.21	1.79	2.81		5.12±0.36	3.66	7.01
Q2	10.91±0.27	1.91	2.45		14.65±0.46	2.30	3.14
Q3	38.16±1.23	1.76	3.21		45.34±1.31	1.91	2.88

SRM 972a	项目	靶值(ng/mL)	均值(ng/mL)	偏倚(%)	准确度(%)
Level 1	25(OH)D3	28.8±1.1	30.5(30.2~30.8)	5.90	105.9(104.9~106.9)
Level 2	25(OH)D3	18.1±0.4	18.0(17.9~18.1)	0.55	99.5(98.9~100.0)
Level 3	25(OH)D2	13.3±0.3	14.1(13.9~14.2)	5.64	105.6(104.5~106.8)
	25(OH)D3	19.8±0.5	19.8(19.6~20.0)	0.00	100.0(99.0~101.0)
Level 4	25(OH)D3	29.4±0.9	30.0(29.4~30.6)	2.04	102.0(100.0~104.1)
	3-epi 25(OH)D3	26.4±2.1	28.8(28.5~29.0)	8.90	108.9(108.0~109.9)

SRM 972a	项目	靶值(ng/mL)	均值(ng/mL)	偏倚(%)	准确度(%)
Level 1	25(OH)D3	28.8±1.1	30.5(30.2~30.8)	5.90	105.9(104.9~106.9)
Level 2	25(OH)D3	18.1±0.4	18.0(17.9~18.1)	0.55	99.5(98.9~100.0)
Level 3	25(OH)D2	13.3±0.3	14.1(13.9~14.2)	5.64	105.6(104.5~106.8)
	25(OH)D3	19.8±0.5	19.8(19.6~20.0)	0.00	100.0(99.0~101.0)
Level 4	25(OH)D3	29.4±0.9	30.0(29.4~30.6)	2.04	102.0(100.0~104.1)
	3-epi 25(OH)D3	26.4±2.1	28.8(28.5~29.0)	8.90	108.9(108.0~109.9)

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