Analysis of urinary vitamin D₃ metabolites by liquid chromatography/tandem mass spectrometry with ESI-enhancing and stable isotope-coded derivatization.

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.