School of Pharmaceutical Sciences and Global COE Program, University of Shizuoka, Shizuoka, Japan.
J Sep Sci. 2011 Apr;34(7):725-32. doi: 10.1002/jssc.201000911. Epub 2011 Feb 17.
Vitamin D deficiency in an infant is associated with a wide range of adverse health outcomes in later life. A method for the quantification of 25-hydroxyvitamin D(3) [25(OH)D(3), the best-established indicator of vitamin D status] in neonatal dried blood spots (DBSs) using LC/ESI-MS/MS has been developed and validated. The method employed two steps of derivatization, a Diels-Alder reaction with 4-phenyl-1,2,4-triazoline-3,5-dione followed by acetylation, to enhance the detectability of 25(OH)D(3) in ESI-MS/MS and to separate 25(OH)D(3) from 3-epi-25-hydroxyvitamin D(3) [3-epi-25(OH)D(3)], a potent interfering metabolite. 25(OH)D(3) was extracted from two DBS punches (3 mm in diameter, equivalent to 5.3 μL of whole blood), purified using an Oasis HLB(®) cartridge, and subjected to derivatization prior to analysis with LC/ESI-MS/MS. 25-Hydroxyvitamin D(4) was used as the internal standard. This method was reproducible (intra- and inter-assay RSDs, <6.9%) and accurate (analytical recovery, 95.2-102.7%), and the LOQ was 3.0 ng/mL. The developed method enabled specific quantification of 25(OH)D(3) in neonatal DBSs and detection of vitamin D deficiency without interference from 3-epi-25(OH)D(3).
婴儿维生素 D 缺乏与日后生活中广泛的不良健康后果有关。本研究开发并验证了一种利用 LC/ESI-MS/MS 定量检测新生儿干血斑（DBS）中 25-羟维生素 D（3）[25（OH）D（3]的方法，这是评估维生素 D 状态的最佳指标。该方法采用两步衍生化反应，先用 4-苯-1,2,4-三唑啉-3,5-二酮进行 Diels-Alder 反应，然后进行乙酰化，以提高 25（OH）D（3）在 ESI-MS/MS 中的检测能力，并将 25（OH）D（3）与 3-差向-25-羟维生素 D（3）[3-差向-25（OH）D（3]分离，后者是一种具有很强干扰作用的代谢物。从两个 DBS 血斑（直径 3 毫米，相当于 5.3 μL 全血）中提取 25（OH）D（3），用 Oasis HLB（®）小柱纯化，衍生化后用 LC/ESI-MS/MS 进行分析。25-羟维生素 D（4）作为内标。该方法重复性好（批内和批间 RSD 均<6.9%），准确度高（分析回收率为 95.2-102.7%），定量下限为 3.0ng/mL。该方法可特异性定量检测新生儿 DBS 中的 25（OH）D（3），并可在无 3-差向-25（OH）D（3）干扰的情况下检测维生素 D 缺乏。
