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  • Title: Simultaneous quantification of vitamin D₃, 25-hydroxyvitamin D₃ and 24,25-dihydroxyvitamin D₃ in human serum by LC-MS/MS.
    Author: Burild A, Frandsen HL, Jakobsen J.
    Journal: Scand J Clin Lab Invest; 2014 Aug; 74(5):418-23. PubMed ID: 24749986.
    Abstract:
    INTRODUCTION: 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. METHOD: 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. RESULTS: 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. CONCLUSION: 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.
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