Metabolic profiling of major vitamin D metabolites using Diels-Alder derivatization and ultra-performance liquid chromatography-tandem mass spectrometry

Anal Bioanal Chem. 2008 Jul;391(5):1917-30. doi: 10.1007/s00216-008-2095-8. Epub 2008 Apr 24.


Biologically active forms of vitamin D are important analytical targets in both research and clinical practice. The current technology is such that each of the vitamin D metabolites is usually analyzed by individual assay. However, current LC-MS technologies allow the simultaneous metabolic profiling of entire biochemical pathways. The impediment to the metabolic profiling of vitamin D metabolites is the low level of 1alpha,25-dihydroxyvitamin D(3) in human serum (15-60 pg/mL). Here, we demonstrate that liquid-liquid or solid-phase extraction of vitamin D metabolites in combination with Diels-Alder derivatization with the commercially available reagent 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) followed by ultra-performance liquid chromatography (UPLC)-electrospray/tandem mass spectrometry analysis provides rapid and simultaneous quantification of 1alpha,25-dihydroxyvitamin D(3), 1alpha,25-dihydroxyvitamin D(2), 24R,25-dihydroxyvitamin D(3), 25-hydroxyvitamin D(3) and 25-hydroxyvitamin D(2) in 0.5 mL human serum at a lower limit of quantification of 25 pg/mL. Precision ranged from 1.6-4.8 % and 5-16 % for 25-hydroxyvitamin D(3) and 1alpha,25-dihydroxyvitamin D(3), respectively, using solid-phase extraction.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Calibration
  • Chromatography, Liquid / methods*
  • Humans
  • Isomerism
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Solid Phase Extraction / methods*
  • Tandem Mass Spectrometry / methods*
  • Time Factors
  • Vitamin D / analogs & derivatives
  • Vitamin D / blood*


  • Vitamin D