title: Simultaneous quantification of vitamin D₃, 25-hydroxyvitamin D₃ and 24,25-dihydroxyvitamin D₃ in human serum by LC-MS/MS.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Division of Food Chemistry, National Food Institute, Technical University of Denmark , Søborg , Denmark.
authors.forename: Anders
authors.initials: A
authors.lastname: Burild
authors.forename: Henrik L
authors.initials: HL
authors.lastname: Frandsen
authors.forename: Jette
authors.initials: J
authors.lastname: Jakobsen
mesh_terms: D001774:Blood Chemical Analysis; D002762:Cholecalciferol / Q000097:blood*; D002850:Chromatography, Gel / Q000592:standards; D002851:Chromatography, High Pressure Liquid / Q000592:standards; D006801:Humans; D057230:Limit of Detection; D012015:Reference Standards; D052616:Solid Phase Extraction; D053719:Tandem Mass Spectrometry / Q000592:standards*
chemical_list: D002762:Cholecalciferol
keywords: Liquid chromatography; mass spectroscopy; vitamin D