Determinations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in serum by liquid chromatography tandem-mass spectrometry

doi:10.3969/j.issn.1673-8640.2015.08.012

Abstract

Abstract:

Objective To establish a method for the determinations of 25-hydroxyvitamin D2[25(OH)D2]and 25- hydroxyvitamin D3[25(OH)D3] contents in serum by liquid chromatography tandem-mass spectrometry (LC-MS/MS). Methods After proteins in serum were precipitated by ethanol, liquid-liquid extraction by n-Hexane, then the residuals were re-dissolved in acetonitrile and analyzed by LC-MS/MS in the positive electrospray ionization(ESI+) mode and multiple reaction monitor(MRM) mode. The quantitative analysis for 25(OH)D2 and 25(OH)D3 contents was carried out by deuterium isotope as internal standard, and the methodology validation was performed. Results The within-run precision of 25(OH)D2 and 25(OH)D3 was 0.88%-7.69%, and between-run precision was 1.56%-9.90%. The good correlation coefficients were 0.999 1 and 0.999 9 at the concentration of 0.5-10.0 ng/mL 25(OH)D2 and 5-100 ng/mL 25(OH)D3, respectively. The accuracy of quality control materials of 25(OH)D2 and 25(OH)D3 was 95.5%-101.2%. 25(OH)D2 content did not have statistical significance(P>0.05), and 25(OH)D3 content had statistical significance(P<0.05)between males and females of 281 volunteers. Conclusions This method is sensitive, accurate and stable, satisfying the quantification requirements of 25(OH)D2 and 25(OH)D3 in serum, and it can be used in clinical analysis.

Key words: 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, Liquid chromatography tandem-mass spectrometry, Internal standard method, Serum

CLC Number:

R446.1

XU Fengxian, YU Jiaping.. Determinations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in serum by liquid chromatography tandem-mass spectrometry[J]. Laboratory Medicine, 2015, 30(8): 821-824.

Figures/Tables 11

时间	流速(mL/min)	水(0.2%甲酸)∶ 甲醇(0.2甲酸)
0 min 1 s	0.40	70∶30
0 min 45 s	0.40	70∶30
1 min 30 s	0.50	17∶83
3 min 45 s	0.55	3∶97
4 min 30 s	0.55	3∶97
5 min 0 s	0.40	70∶30
6 min 0 s	0.40	70∶30

25(OH)D2	批内精密度			批间精密度
25(OH)D2	$x ?$ (ng/mL)	s	变异系数(%)	$x ?$ (ng/mL)	s	变异系数(%)
高值	6.03	0.27	4.47	5.65	0.39	6.89
中值	2.61	0.19	6.48	2.75	0.20	7.21
低值	1.03	0.08	7.69	1.08	0.11	9.90

25(OH)D2	批内精密度			批间精密度
25(OH)D2	$x ?$ (ng/mL)	s	变异系数(%)	$x ?$ (ng/mL)	s	变异系数(%)
高值	6.03	0.27	4.47	5.65	0.39	6.89
中值	2.61	0.19	6.48	2.75	0.20	7.21
低值	1.03	0.08	7.69	1.08	0.11	9.90

25(OH)D3	批内精密度			批间精密度
25(OH)D3	$x ?$ (ng/mL)	s	变异系数(%)	$x ?$ (ng/mL)	s	变异系数(%)
高值	58.18	0.51	0.88	58.45	0.91	1.56
中值	29.8	0.44	1.47	29.42	0.72	2.46
低值	10.42	0.16	1.54	10.23	0.42	4.13

25(OH)D3	批内精密度			批间精密度
25(OH)D3	$x ?$ (ng/mL)	s	变异系数(%)	$x ?$ (ng/mL)	s	变异系数(%)
高值	58.18	0.51	0.88	58.45	0.91	1.56
中值	29.8	0.44	1.47	29.42	0.72	2.46
低值	10.42	0.16	1.54	10.23	0.42	4.13

级别	项目	靶值 (ng/mL)	允许区间下限 (ng/mL)	允许区间上限 (ng/mL)	测定结果 (ng/mL)	正确度 (%)
L1	25(OH)D2	17.6	14.1	21.1	16.8	95.5
L1	25(OH)D3	16.3	13.1	19.6	16.5	101.2
L2	25(OH)D2	41.4	33.1	49.7	41.0	99.0
L2	25(OH)D3	38.0	30.4	45.6	37.9	99.7

参考区间	男性			女性
参考区间	25(OH)D2	25(OH)D3	25(OH)D	25(OH)D2	25(OH)D3	25(OH)D
下限	0.46	12.30	13.23	0.51	10.90	11.87
上限	1.65	29.90	30.62	1.61	26.60	27.35

参考区间	25(OH)D2	25(OH)D3	25(OH)D
下限	0.51	11.3	12.39
上限	1.7	29.9	30.78

References 9

[1]	周建烈,杨宗麟. 维生素D补充与药物选择[J]. 上海医药, 2011,32(10):472-474.
[2]	王剑,朱玮琪,沈立松. 高效液相色谱法测定血清中25-羟基维生素D3[J]. 检验医学,2008,23(4):380-383.
[3]	SAENGER AK,LAHA TJ,BREMNER DE, et al.Quantification of serum 25-hydroxyvitamin D(2) and D(3) using HPLC-tandem mass spectrometry and examination of reference intervals for diagnosis of vitamin D deficiency[J]. Am J Clin Pathol,2006,125(6): 914-920.
[4]	HØJSKOV CS,HEICKENDORFF L,MØLLER HJ. High-throughput liquid-liquid extraction and LCMSMS assay for determination of circulating 25(OH) vitamin D3 and D2 in the routine clinical laboratory[J].Clin Chim Acta,2010,411(1-2): 114-116.
[5]	VAN DEN OUWELAND JM,BEIJERS AM,DEMACKER PN,et al. Measurement of 25-OH-vitamin D in human serum using liquid chromatography tandem-mass spectrometry with comparison to radioimmunoassay and automated immunoassay[J]. J Chromatogr B Analyt Technol Biomed Life Sci,2010,878(15-16): 1163-1168.
[6]	毛旭东,吴彦,盛宏光,等. 液相色谱-质谱联用法(LC-MS/MS)和酶联免疫法(ELISA)对体内25(OH) D3水平的测定[J]. 中国骨质疏松杂志,2013,19(6):584-586.
[7]	王春平,毛旭东,盛宏光. 上海市徐汇区住院患者血中维生素D水平分析[J]. 中国临床医学,2014,21(1):75-77.
[8]	熊燏,钱士匀,熊小泉,等. 海南地区成人血清25-羟基-维生素D参考区间的初步调查[J].检验医学,2014,29(5):464-467.
[9]	HOLICK MF,BINKLEY NC,BISCHOFF-FERRARI HA,et al.Evaluation,treatment,and prevention of vitamin D deficiency: an endocrine society clinical practice guideline[J]. J Clin Endocrinol Metab, 2011,96(7): 1911-1930.

[1]	HUANG Yuan, CHEN Feng, MAO Leili, ZHANG Linglin, LÜ Qian, YAN Jun, CUI Wei. Application of serum peptidomics based on MALDI-TOF MS in the differential diagnosis of benign and malignant pancreatic diseases [J]. Laboratory Medicine, 2023, 38(1): 1-7.
[2]	ZHANG Meng, LI Sha, CHANG Yanli, WANG Lu, XU Jianjun, ZHANG Qingyun. Role of vascular endothelial growth factor in monitoring treatment outcome and evaluating the prognosis of patients with advanced melanoma [J]. Laboratory Medicine, 2022, 37(9): 821-825.
[3]	Danhong , FANG Huiling, LIN Feiran, ZHU Yuqing. Comparison of testing efficiency of simulated emergency biochemical samples among different instruments GE [J]. Laboratory Medicine, 2022, 37(8): 766-771.
[4]	HAN Guang, XU Qiongfeng, DUAN Shumin, LUO Jiahuan, WANG Yunxiu, ZHANG Xiujuan, WAN Zemin, KE Peifeng, HUANG Xianzhang. Evaluation of the interference of serum index in the determination of prealbumin by immunoscattering turbidimetry according to CLSI EP07-A3 document [J]. Laboratory Medicine, 2022, 37(2): 108-111.
[5]	LU Youli, YANG Shuangshuang, ZHANG Meiwei, OU Meixian, DONG Chunxia, SHEN Weiwei, JIANG Fengli, LI Shuijun. Simultaneous determination of 5 steroid hormones in human serum by ultra-performance liquid chromatography-tandem mass spectrometry and establishment of reference intervals in healthy adults [J]. Laboratory Medicine, 2022, 37(2): 165-173.
[6]	XIONG Zhongbo, WANG Lei, YIN Beiqi, DAI Yue, WEI Xiaoqing, LU Pei. Clinical roles of SAA and CRP in pathological staging and metastasis of gastrointestinal cancer [J]. Laboratory Medicine, 2022, 37(1): 51-55.
[7]	SUN Jingfang, LIU Yun, JIANG Qingqing, SONG Shuang, LI Shibao, MA Ping, DING Shuang. Clinical role of high-fluorescent cell and serum ascites albumin gradient in the diagnosis of malignant ascites [J]. Laboratory Medicine, 2022, 37(1): 60-62.
[8]	YU Ting, SHEN Min, QU Shoufang, SUN Nan, SUN Jing, HUANG Jie. Development of the national standard materials of total bilirubin in frozen human serum [J]. Laboratory Medicine, 2021, 36(9): 957-961.
[9]	YANG Fan, DONG Danfeng, LU Yide. Application of patient-based real-time quality control using exponentially weighted moving average method on internal quality control procedures for serum ion items [J]. Laboratory Medicine, 2021, 36(9): 962-968.
[10]	LIU Jinyan, ZHAO Juntao, XIANG Mingjie. Mechanism and application of serum amyloid A in clinical diseases [J]. Laboratory Medicine, 2021, 36(7): 756-760.
[11]	LIU Fei, HU Yanwei. Influence of pre-analysis factors on the determination of serum renal function items [J]. Laboratory Medicine, 2021, 36(4): 369-373.
[12]	ZHANG Weiwei, JING Rongrong, JI Huoyan, WANG Jianxin, WANG Feng, WANG Huimin. Isotope dilution mass spectrometry applied in measurement uncertainty of serum creatinine concentration [J]. Laboratory Medicine, 2021, 36(3): 263-269.
[13]	LI Bingling, CHENG Yating, ZHAO Beibei, SHE Xuhui, DONG Heng, YU Mujun. Evaluation of surrogate matrices for serum 25-hydroxyvitamin D calibrators by LC-MS/MS [J]. Laboratory Medicine, 2021, 36(2): 213-218.
[14]	YU Ting, SHEN Min, QU Shoufang, SUN Nan, SUN Jing, HUANG Jie. Development of the national standard materials of testosterone in frozen human serum [J]. Laboratory Medicine, 2021, 36(10): 1074-1077.
[15]	ZOU Jihua, ZHANG Wei, SHEN Min, ZHANG Man, TU Minmin, WANG Huimin, ZHOU Houqing. Research progress in the separation of serum lipoprotein subfractions and lipoprotein（a） [J]. Laboratory Medicine, 2021, 36(10): 1078-1086.