title: Analysis of urinary vitamin D₃ metabolites by liquid chromatography/tandem mass spectrometry with ESI-enhancing and stable isotope-coded derivatization.
abstract: The determination of the urinary vitamin D₃ metabolites might prove helpful in the assessment of the vitamin D status. We developed a method for the determination of trace vitamin D₃ metabolites, 25-hydroxyvitamin D₃ [25(OH)D₃] and 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃], in urine using liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) combined with derivatization using an ESI-enhancing reagent, 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD), and its isotope-coded analogue, (2)H4-DAPTAD (d-DAPTAD). The urine samples were treated with β-glucuronidase, purified with an Oasis hydrophilic-lipophilic balanced (HLB) cartridge, and then subjected to the derivatization. The DAPTAD derivatization enabled the highly sensitive detection (detection limit, 0.25 fmol on the column), and the use of d-DAPTAD significantly improved the assay precision [the intra- (n = 5) and inter-assay (n = 3) relative standard deviations did not exceed 9.5%]. The method was successfully applied to urine sample analyses and detected the increases of the urinary 25(OH)D₃ and 24,25(OH)₂D₃ levels due to vitamin D₃ administration.
authors.affiliation: Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan.
authors.forename: Shoujiro
authors.initials: S
authors.lastname: Ogawa
authors.forename: Satoshi
authors.initials: S
authors.lastname: Ooki
authors.forename: Kenta
authors.initials: K
authors.lastname: Shinoda
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Higashi
mesh_terms: D002112:Calcifediol / Q000652:urine*; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D007554:Isotopes / Q000032:analysis; D057230:Limit of Detection; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000652:urine
chemical_list: D007554:Isotopes; C118756:dihydroxy-vitamin D3; D014807:Vitamin D; D002112:Calcifediol