Chen Xiaohong, Zhou Jian, Jin Micong
Key Laboratory of Health Risk Appraisal for Trace Toxic Chemicals of Zhejiang Province, Ningbo Municipal Center for Disease Control and Prevention, Ningbo 315010, China.
Wei Sheng Yan Jiu. 2019 Nov;48(6):981-987.
To establish a rapid and accurate method for determination of 25-hydroxyl vitamin D2, 25-hydroxyl vitamin D3 and 3-epi-25-hydroxyl vitamin D3 in serum by isotope dilution ultra-fast liquid chromatography-tandem mass spectrometry(UFLC-MS/MS).
The serum sample was extracted by nhexane after methanol/acetonitrile precipitation protein, and then the extract was concentrated by nitrogen and volumed with the primary mobile phase. The chromatographic separation was carried out on a Phenomenex Kinetex F5 column(2. 1 mm × 150 mm, 1. 7 μm) by using 0. 1%(V/V) formic acid and 0. 1%(V/V) formic acid/methanol solution as the mobile phase with the gradient elution. Detection was performed in positive multi-reaction monitoring(MRM) mode with the isotope internal labeling method for quantification.
The baseline separation was obtained within 6 min for the epimer 25-hydroxyl vitamin D3 and 3-epi-25-hydroxyl vitamin D3, and the accurate qualification was obtained for the simultaneous determination of 25-hydroxyl vitamin D2, 25-hydroxyl vitamin D3 and 3-epi-25-hydroxyl vitamin D3. The three analytes showed good linear relationship within the range of 0. 5-50. 0 μg/L with a correlation coefficient r >0. 9995. The limits of detection(LODs) and the limits of quantitation(LOQs) of the method were 0. 15 μg/L and 0. 5 μg/L, respectively. The recoveries of the method were84. 3%-109. 0%(n = 11) at the three spiked levels of 1. 0, 10. 0 and 30. 0 μg/L, and the relative standard deviations(RSDs) were between 0. 8%-6. 8%. At the same time, the certified standard reference materials(SRM) of the America National Institute of Standards and Technology(NIST) Level 1, Level 2, Level 3 and Level 4(SRM 972 a)were used as the quality control samples for verification, the relative deviations of the measurement result were less than 5% compared with the reference values.
The developed method has the characteristics of simplicity, rapidity, sensitivity and accuracy, and is suitable for the simultaneous rapid determination of 25-hydroxyl vitamin D2, 25-hydroxyl vitamin D3 and 3-epi-25-hydroxyl vitamin D3 in serum.
建立一种同位素稀释超快速液相色谱-串联质谱法（UFLC-MS/MS）快速、准确测定血清中25-羟基维生素D2、25-羟基维生素D3和3-表-25-羟基维生素D3的方法。
血清样品经甲醇/乙腈沉淀蛋白后用正己烷萃取，萃取液经氮气浓缩并用初始流动相定容。采用Phenomenex Kinetex F5柱（2.1 mm×150 mm，1.7μm），以0.1%（V/V）甲酸和0.1%（V/V）甲酸/甲醇溶液为流动相进行梯度洗脱，在正离子多反应监测（MRM）模式下采用同位素内标法定量检测。
25-羟基维生素D3差向异构体和3-表-25-羟基维生素D3在6分钟内实现基线分离，可同时准确测定25-羟基维生素D2、25-羟基维生素D3和3-表-25-羟基维生素D3。三种分析物在0.5 - 50.0μg/L范围内线性关系良好，相关系数r＞0.9995。该方法的检测限（LOD）和定量限（LOQ）分别为0.15μg/L和0.5μg/L。在1.0、10.0和30.0μg/L三个加标水平下，方法回收率为84.3% - 109.0%（n = 11），相对标准偏差（RSD）在0.8% - 6.8%之间。同时，采用美国国家标准与技术研究院（NIST）1级、2级、3级和4级（SRM 972 a）的标准参考物质（SRM）作为质量控制样品进行验证，测定结果与参考值相比相对偏差小于5%。
所建立的方法具有简便、快速、灵敏、准确的特点，适用于血清中25-羟基维生素D2、25-羟基维生素D3和3-表-25-羟基维生素D3的同时快速测定。
