Analysis of urinary vitamin D3 metabolites by liquid chromatography/tandem mass spectrometry with ESI-enhancing and stable isotope-coded derivatization |
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Authors: | Shoujiro Ogawa Satoshi Ooki Kenta Shinoda Tatsuya Higashi |
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Affiliation: | 1. Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan
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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, 2H4-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. Graphical Abstract Scheme of procedure for urinary vitamin D3 metabolite analysis based on LC/MS/MS with ESI-enhancing and isotope-coded derivatization. |
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