Original Paper

Analytical and Bioanalytical Chemistry

, Volume 391, Issue 5, pp 1917-1930

First online:

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

  • Pavel A. AronovAffiliated withDepartment of Entomology, University of California
  • , Laura M. HallAffiliated withDepartment of Nutrition and USDA Western Human Nutrition Research Center, University of California
  • , Katja DettmerAffiliated withInstitute of Functional Genomics, University of Regensburg
  • , Charles B. StephensenAffiliated withDepartment of Nutrition and USDA Western Human Nutrition Research Center, University of California
  • , Bruce D. HammockAffiliated withDepartment of Entomology and U.C. Davis Cancer Research Center, University of California Email author 

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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 D3 25-Hydroxyvitamin D3 24R,25-Dihydroxyvitamin D3 UPLC LC-MS Metabolic profiling Derivatization