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

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 D-3, 25-hydroxyvitamin D-3 and 24,25-dihydroxyvitamin D-3 in serum. After protein precipitation of the serum it was loaded on a HybridSPE column to separate vitamin D metabolites from phospholipids. Vitamin D-3, 25-hydroxyvitamin D-3 and 24,25-dihydroxyvitamin D-3 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 L serum the limit of quantification was <0.2 ng/mL for vitamin D-3, 25-hydroxyvitamin D-3 and 24,25-dihydroxyvitamin D-3. The method was validated up to 100 ng/mL (260 nmol/L) for vitamin D-3, up to 100 ng/mL (240 nmol/L) for 24,25-dihydroxyvitamin D-3 and up to 200 ng/mL (499 nmol/L) for 25-hydroxyvitamin D-3. Precision was <6.5% for vitamin D-3 and 25-hydroxyvitamin D-3 and <10.2% for 24,25-dihydroxyvitamin D-3. Conclusion. We demonstrate that a method including not only serum 25-hydroxyvitamin D-3 but also vitamin D-3 and 24,25-dihydroxyvitamin D-3 could easily be implemented in most modern biochemical laboratories. The method could be used to study the metabolism of endogenous synthesized vitamin D-3 as well as vitamin D-3 in intervention studies.