Simultaneous quantification of vitamin D₃, 25-hydroxyvitamin D-3 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