Submitted to: Analytical and Bioanalytical Chemistry
Publication Type: Peer reviewed journal
Publication Acceptance Date: 3/20/2008
Publication Date: 4/25/2008
Citation: Aronov, P.A., Hall, L.M., Dettmer, K., Stephensen, C.B., Hammock, B.D. 2008. Metabolic Profiling of Major Vitamin D Metabolites Using Diels-Alder Derivatization and Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry. Analytical and Bioanalytical Chemistry. Analytical and Bioanalytical Chemistyr, Springer, DOI 10.1007/s00216-008-2095-8. Interpretive Summary: Current methods for measuring vitamin D status are limited because multiple assays are required to measure all the metabolites of vitamin D that are of interest for assessing status. The method described in this paper measure most relavent metabolites simultaneously and the results appear to be less variable than the standard method, to which it was compared. This method should greatly increase the amount of information available to researchers evaluating vitamin D in human studies.
Technical Abstract: Biologically active forms of vitamin D are important analytical targets both in research and in clinical practice. Typically, each of the vitamin D metabolites is best analyzed by individual assay. However, current LC-MS technologies allow simultaneous metabolic profiling of entire biochemical pathways. The impediment for metabolic profiling of vitamin D metabolites is the low level of 1a, 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 1a, 25-dihydroxyvitamin D3, 24R, 25-dihydroxyvitamin D3, 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in 0.4 mL human serum at 25 pg/mL Lower Limit of Quantification. Precision ranged from 1.6 - 4.8 % and 5 - 16 % for 25-hydroxyvitamin D3 and 1a, 25-dihydroxyvitamin D3, respectively, using solid phase extraction.