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 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.
Aronov
Pavel A
PA
Department of Entomology, University of California, Davis, CA, 95616, USA.
Hall
Laura M
LM
Dettmer
Katja
K
Stephensen
Charles B
CB
Hammock
Bruce D
BD
D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D007536:Isomerism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013997:Time Factors; D014807:Vitamin D / Q000031:analogs & derivatives / Q000097:blood*
D014807:Vitamin D