Analytical and Bioanalytical Chemistry

, 391:1917

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


  • Pavel A. Aronov
    • Department of EntomologyUniversity of California
  • Laura M. Hall
    • Department of Nutrition and USDA Western Human Nutrition Research CenterUniversity of California
  • Katja Dettmer
    • Institute of Functional GenomicsUniversity of Regensburg
  • Charles B. Stephensen
    • Department of Nutrition and USDA Western Human Nutrition Research CenterUniversity of California
    • Department of Entomology and U.C. Davis Cancer Research CenterUniversity of California
Original Paper

DOI: 10.1007/s00216-008-2095-8

Cite this article as:
Aronov, P.A., Hall, L.M., Dettmer, K. et al. Anal Bioanal Chem (2008) 391: 1917. doi:10.1007/s00216-008-2095-8


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 1α,25-dihydroxyvitamin D3 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 1α,25-dihydroxyvitamin D3, 1α,25-dihydroxyvitamin D2, 24R,25-dihydroxyvitamin D3, 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 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 D3 and 1α,25-dihydroxyvitamin D3, respectively, using solid-phase extraction.


1α,25-Dihydroxyvitamin D325-Hydroxyvitamin D324R,25-Dihydroxyvitamin D3UPLCLC-MSMetabolic profilingDerivatization

Supplementary material

216_2008_2095_MOESM1_ESM.pdf (186 kb)
ESM 1(PDF 185 kb)

Copyright information

© Springer-Verlag 2008