Simultaneous quantification of vitamin D3, 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 in human serum by LC-MS/MS

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.