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

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 profilin...

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Bibliographic Details
Published in:Analytical and bioanalytical chemistry 2008-07, Vol.391 (5), p.1917-1930
Main Authors: Aronov, Pavel A., Hall, Laura M., Dettmer, Katja, Stephensen, Charles B., Hammock, Bruce D.
Format: Article
Language:English
Subjects:
Analytical Chemistry
Biochemistry
Calibration
Characterization and Evaluation of Materials
Chemistry
Chemistry and Materials Science
Chromatography, Liquid - methods
Food Science
Humans
Isomerism
Laboratory Medicine
Monitoring/Environmental Analysis
Original Paper
Reproducibility of Results
Sensitivity and Specificity
Solid Phase Extraction - methods
Tandem Mass Spectrometry - methods
Time Factors
Vitamin D - analogs & derivatives
Vitamin D - blood
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Summary: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 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 1α,25-dihydroxyvitamin D 3 , 1α,25-dihydroxyvitamin D 2 , 24 R ,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 1α,25-dihydroxyvitamin D 3 , respectively, using solid-phase extraction.
ISSN:1618-2642
1618-2650
DOI:10.1007/s00216-008-2095-8