Burild Anders, Frandsen Henrik L, Jakobsen Jette
Division of Food Chemistry, National Food Institute, Technical University of Denmark , Søborg , Denmark.
Scand J Clin Lab Invest. 2014 Aug;74(5):418-23. doi: 10.3109/00365513.2014.900694. Epub 2014 Apr 22.
Serum 25-hydroxy-vitamin D is the established biomarker of vitamin D status although serum concentrations of vitamin D and 24,25-dihydroxyvitamin D may also be of interest to understand the in vivo kinetics of serum 25-hydroxyvitamin D.
An LC-MS/MS method was developed and validated to quantify vitamin D₃, 25-hydroxyvitamin D₃ and 24,25-dihydroxyvitamin D₃ in serum. After protein precipitation of the serum it was loaded on a HybridSPE column to separate vitamin D metabolites from phospholipids. Vitamin D₃, 25-hydroxyvitamin D₃ and 24,25-dihydroxyvitamin D₃ in the eluate were derivatized by 4-phenyl-1,2,4-triazoline-3,5-dione to improve sensitivity in the following LC-MS/MS analysis.
Using only 100 μL serum the limit of quantification was < 0.2 ng/mL for vitamin D₃, 25-hydroxyvitamin D₃ and 24,25-dihydroxyvitamin D₃. The method was validated up to 100 ng/mL (260 nmol/L) for vitamin D₃, up to 100 ng/mL (240 nmol/L) for 24,25-dihydroxyvitamin D₃ and up to 200 ng/mL (499 nmol/L) for 25-hydroxyvitamin D₃. Precision was < 6.5% for vitamin D₃ and 25-hydroxyvitamin D₃ and < 10.2% for 24,25-dihydroxyvitamin D₃.
We demonstrate that a method including not only serum 25-hydroxyvitamin D₃ but also vitamin D₃ and 24,25-dihydroxyvitamin D₃ could easily be implemented in most modern biochemical laboratories. The method could be used to study the metabolism of endogenous synthesized vitamin D₃ as well as vitamin D₃ in intervention studies.
血清25-羟基维生素D是已确立的维生素D状态生物标志物，不过维生素D和24,25-二羟基维生素D的血清浓度对于理解血清25-羟基维生素D的体内动力学也可能具有重要意义。
开发并验证了一种液相色谱-串联质谱法（LC-MS/MS），用于定量血清中的维生素D₃、25-羟基维生素D₃和24,25-二羟基维生素D₃。血清经蛋白沉淀后，加载到HybridSPE柱上，以将维生素D代谢物与磷脂分离。洗脱液中的维生素D₃、25-羟基维生素D₃和24,25-二羟基维生素D₃用4-苯基-1,2,4-三唑啉-3,5-二酮进行衍生化，以提高后续LC-MS/MS分析的灵敏度。
仅使用100μL血清，维生素D₃、25-羟基维生素D₃和24,25-二羟基维生素D₃的定量限均<0.2ng/mL。该方法对维生素D₃的验证上限为100ng/mL（260nmol/L），对24,25-二羟基维生素D₃为100ng/mL（240nmol/L），对25-羟基维生素D₃为200ng/mL（499nmol/L）。维生素D₃和25-羟基维生素D₃的精密度<6.5%，24,25-二羟基维生素D₃的精密度<10.2%。
我们证明，一种不仅包括血清25-羟基维生素D₃，还包括维生素D₃和24,25-二羟基维生素D₃ 的方法可以在大多数现代生化实验室中轻松实施。该方法可用于研究内源性合成维生素D₃以及干预研究中维生素D₃的代谢情况。
