Improved method for the determination of serum 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 by ultra-performance liquid chromatography tandem mass spectrometry

doi:10.3969/j.issn.1673-8640.2015.10.014

Abstract

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

CLC Number:

R446.1

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.

Figures/Tables 5

References 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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