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目的 建立快速、准确检测血清中25-羟基维生素D3[25-hydroxyvitamin D3,25(OH)D3]、25-羟基维生素D2[25-hydroxyvitamin D2,25(OH)D2]含量的柱前衍生-稳定同位素标记-超高效液相色谱-四极杆静电场轨道阱高分辨质谱的方法，并用此方法诊断维生素D缺乏症，评估人群维生素D营养状况。方法 加入同位素内标的血清样品采用乙酸乙酯-正己烷溶液(2∶1,V/V)提取，离心后氮气吹干，以4-苯基-1,2,4-三唑啉-3,5-二酮(4-phenyl-1,2,4-triazoline-3,5-dione, PTAD)作为衍生化试剂与目标化合物进行衍生化反应。反应产物采用BEH C18色谱柱(2.1 mm×50 mm, 1.7μm)进行分离，以0.1%甲酸水溶液-乙腈梯度洗脱。质谱采用电喷雾正离子模式和平行反应监测对目标物进行检测，内标法定量。结果 25(OH)D3和25(OH)D2的检出限为0.01 ng/mL,定量限为0.03 ng/mL。25(OH)D2浓度的线性范围为0.5～40.0 ng/mL,25(OH)D3浓度的线性范围为2.5～200.0 ng/mL,二者在浓度范围内线性良好，相关系数(r2)大于0.995。25(OH)D3和25(OH)D2的回收率分别为95.7%～101.3%和98.7%～108.6%,相对标准偏差分别为0.5%～4.9%和2.2%～3.4%(n=5)。测定参考物质NIST SRM 972a的Level 2血清中25(OH)D3和25(OH)D2的准确度分别为104.8%和94.9%。利用该方法检测北京市116名孕妇孕早期血清中25(OH)D3和25(OH)D2水平[M(P25,P75)]分别为25.7(20.8,32.6)ng/mL和0.8(0.4,1.1)ng/mL。结论 本方法灵敏度高、定性准确，适用于人群维生素D营养状况的评价和监测。
Abstract：OBJECTIVE To establish a rapid and accurate method for the determination of 25-hydroxyvitamin D3(25(OH)D3) and 25-hydroxyvitamin D2(25(OH)D2)in serum by pre-column derivatization with stable isotope labeling and ultra performance liquid chromatography-quadrupole electrostatic field orbitrap high-resolution mass spectrometry, which could be used to diagnose vitamin deficiency and to assess the nutritional status of vitamin D in the population.METHODS The serum samples with isotopic internal standard were extracted by mixed extraction solvent(ethyl acetate∶hexane = 2∶1, V/V), centrifuged and dried by the nitrogen blowing, and derivatized with 4-phenyl-1,2,4-triazoline-3,5-dione(PTAD). The reaction products were separated on a BEH C18 column(2.1 mm×50 mm, 1.7 μm), and eluted with a 0.1% formic acid/water solution-acetonitrile gradient. The mass spectrometry was performed in positive electrospray ionization(ESI+) and parallel reaction monitoring(PRM) for the detection of the targets, and quantified by isotope internal standard.RESULTS The limits of detection both for 25(OH)D3 and 25(OH)D2 were 0.01 ng/mL and the limits of quantification were 0.03 ng/mL. The concentration series of 25(OH)D2 ranged from 0.5 to 40.0 ng/mL and the concentration series of 25(OH)D3 ranged from 2.5 to 200.0 ng/mL. The recoveries(n=5) were 95.7%-101.3% for 25(OH)D3, and 98.7%-108.6% for 25(OH)D2, with the relative standard deviations(RSDs) of 0.5%-4.9% and 2.2%-3.4%, respectively. The accuracy of the determination of 25(OH)D3 and 25(OH)D2 in Level 2 serum of the 25(OH)D standard reference material NIST SRM 972a was 104.8% and 94.9%, respectively. The M(P25,P75)serum levels of 25(OH)D3 and 25(OH)D2 for 116 pregnant women at the first trimester from Beijing were 25.7(20.8, 32.6) ng/mL and 0.8(0.4, 1.1) ng/mL, respectively.CONCLUSION This method is highly sensitive, qualitatively accurate and suitable for evaluation and monitoring the nutritional status of vitamin D in the population.
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基本信息:
DOI：10.19813/j.cnki.weishengyanjiu.2023.01.022
中图分类号:O657.63;R446.11
引用信息:
[1]韩枫,王雨昕,李敬光,等.柱前衍生-稳定同位素标记-超高效液相色谱-四极杆静电场轨道阱高分辨质谱法测定血清中25-羟基维生素D[J].卫生研究,2023,52(01):129-135.DOI:10.19813/j.cnki.weishengyanjiu.2023.01.022.
基金信息:
科技基础资源调查专项(No.2019FY101002)
2022-07-18
2022
2022-10-20
2022-10-21
2022
1
2023-01-20
2023-01-20