Measurement of 25-OH-vitamin D in human serum using liquid chromatography tandem-mass spectrometry with comparison to radioimmunoassay and automated immunoassay
Measurement of 25-OH-vitamin D in human serum using liquid chromatography tandem-mass spectrometry with comparison to radioimmunoassay and automated immunoassay
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DOI：
10.1016/j.jchromb.2010.03.035
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发表时间：
2010-05-01
影响因子：
3
通讯作者：
van Daal, Henny
中科院分区：
文献类型：
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作者：
van den Ouweland, Johannes M. W.;Beijers, Antonius M.;van Daal, Henny
血浆25-OH维生素D浓度是维生素D状态的可靠生物标志物，但测定的可变性使得难以充分监测维生素D状态。我们采用同位素稀释液相色谱（LC）串联质谱（MS/MS）测定人血清中的25-OH维生素D（3）和25-OH维生素D（2）。将六氘标记的25-OH维生素D（3）内标物（IS）加入校准品（在含60 g/L白蛋白的磷酸盐缓冲盐水中制备）、对照品或患者血清中，并在乙腈/甲醇（9：1，v/v）沉淀蛋白质之前，通过加入氢氧化钠从维生素D结合蛋白中释放25-OH维生素D代谢物。随后的离线固相萃取之后，使用水/甲醇/乙酸铵梯度在C-18柱上进行色谱分离。通过大气压电喷雾电离（AP-EI）进行检测，然后进行选择的反应监测。我们比较了LC-MS/MS测定法与DiaSorin放射免疫测定法（RIA）和Roche Diagnostics最近重新标准化的自动电化学发光免疫测定法（ECLIA）。我们还分析了来自国际维生素D外部质量评估计划（DEQAS）的外部质量控制样品，并使用LC-MS与其他参与实验室进行比较。该方法在5至至少550 nmol/L范围内具有线性，具有日内和日间CV
The plasma 25-OH vitamin D concentration is a reliable biomarker for vitamin D status but assay's variability makes adequate monitoring of vitamin D status difficult. We employed isotope-dilution liquid chromatography (LC) tandem-mass spectrometry (MS/MS) for the measurement of both 25-OH vitamin D(3) and 25-OH vitamin D(2) in human serum. Hexadeuterium labelled 25-OH vitamin D(3) internal standard (IS) was added to calibrators (prepared in phosphate-buffered saline with 60 g/L albumin), controls or patient sera and 25-OH vitamin D metabolites were released from vitamin D binding protein by adding sodium hydroxide prior to protein precipitation by acetonitrile/methanol (9:1, v/v). Subsequent off-line solid-phase extraction was followed by chromatographic separation on a C-18 column using a water/methanol/ammonium acetate gradient. Detection was by Atmospheric Pressure Electrospray Ionisation (AP-EI) followed by selected reaction monitoring. We compared the LC-MS/MS assay to the DiaSorin radioimmunoassay (RIA) and a recently re-standardised version of an automated electrochemi-luminescent immunoassay (ECLIA) from Roche Diagnostics. We also analysed external quality control samples from the International Vitamin D External Quality Assessment Scheme (DEQAS) for comparison with other participating laboratories using LC-MS. The method was linear from 5 to at least 550 nmol/L with intra- and interday CV's