van den Ouweland Johannes M W, Beijers Antonius M, van Daal Henny
Department of Clinical Chemistry, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands.
Department of Clinical Chemistry, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands.
J Chromatogr B Analyt Technol Biomed Life Sci. 2014 Sep 15;967:195-202. doi: 10.1016/j.jchromb.2014.07.021. Epub 2014 Jul 31.
An LC-MS/MS method was developed for simultaneous quantification of 25-hydroxyvitamin D3 (25(OH)D3), 3-epi-25(OH)D3, and 25(OH)D2 in human serum.
Sample preparation consisted of protein precipitation followed by off-line SPE. Calibration curves for each vitamin D metabolite were constructed in phosphate-buffered saline with 60 g/L albumin including its corresponding stable isotope labelled (SIL) internal standard. A pentafluorophenyl (PFP) analytical column was used to resolve 25(OH)D3 from 25(OH)D2 and 3-epi-25(OH)D3, followed by SRM registration using positive ESI-MS/MS. Accuracy was assessed from measurement of samples with NIST reference method procedure (RMP) assigned values. The PFP LC-MS/MS method was compared to an in-house C18 column LC-MS/MS method, not resolving 25(OH)D3 from 3-epi-25(OH)D3, using adult and newborn samples.
Intra-assay and inter-assay coefficients of variation were less than 4% and 7.5%, respectively for all three vitamin D metabolites; lower limits of quantification were 1, 1 and 2 nmol/L and linearity of methods were 1-500, 1-200 and 2-500 nmol/L for 25(OH)D3, 3-epi-25(OH)D3 and 25(OH)D2, respectively. The PFP LC-MS/MS method showed minimal bias to the NIST RMP. Method comparison revealed that in the C18 LC-MS/MS method, the 3-epi-25(OH)D3 concentration is overestimated inadvertently not only from co-elution of both analytes, but also by an additional 30-40% higher ionisation efficiency of 3-epi-25(OH)D3 when compared to 25(OH)D3.
This accurate LC-MS/MS method allows the simultaneous measurement of 25(OH)D3, 3-epi-25(OH)D3, and 25(OH)D2 in human serum. Due to increased ionisation efficiency, the contribution of the 3-epi-25(OH)D3 metabolite to the total 25(OH)D3 concentration is significantly overestimated in MS methods that do not resolve 3-epi-25(OH)D3 from 25(OH)D3 and may compromise its use in infant samples known to have significant amounts of 3-epi-25(OH)D3.
建立了一种液相色谱-串联质谱法（LC-MS/MS），用于同时定量测定人血清中的25-羟基维生素D3（25(OH)D3）、3-表-25-羟基维生素D3（3-epi-25(OH)D3）和25-羟基维生素D2（25(OH)D2）。
样品制备包括蛋白质沉淀，然后进行离线固相萃取（SPE）。每种维生素D代谢物的校准曲线在含60 g/L白蛋白的磷酸盐缓冲盐水中构建，包括其相应的稳定同位素标记（SIL）内标。使用五氟苯基（PFP）分析柱分离25(OH)D3与25(OH)D2和3-表-25(OH)D3，随后使用正离子电喷雾电离串联质谱（ESI-MS/MS）进行选择反应监测（SRM）。通过使用美国国家标准与技术研究院（NIST）参考方法程序（RMP）赋值的样品测量来评估准确性。使用成人和新生儿样本，将PFP LC-MS/MS方法与内部C18柱LC-MS/MS方法进行比较，后者无法分离25(OH)D3与3-表-25(OH)D3。
所有三种维生素D代谢物的批内和批间变异系数分别小于4%和7.5%；25(OH)D3、3-表-25(OH)D3和25(OH)D2的定量下限分别为1、1和2 nmol/L，方法的线性范围分别为1-500、1-200和2-500 nmol/L。PFP LC-MS/MS方法对NIST RMP的偏差最小。方法比较显示，在C18 LC-MS/MS方法中，3-表-25(OH)D3浓度不仅因两种分析物的共洗脱而被无意高估，而且与25(OH)D3相比，3-表-25(OH)D3的电离效率还额外高出30-40%。
这种准确的LC-MS/MS方法可同时测定人血清中的25(OH)D3、3-表-25(OH)D3和25(OH)D2。由于电离效率增加，在未将3-表-25(OH)D3与25(OH)D3分离的质谱方法中，3-表-25(OH)D3代谢物对总25(OH)D3浓度的贡献被显著高估，这可能会影响其在已知含有大量3-表-25(OH)D3的婴儿样本中的应用。
