Characterization of urinary metabolites of vitamin D3 in man under physiological conditions using liquid chromatography-tandem mass spectrometry

The characterization of the urinary metabolites of vitamin D-3 in man under physiological conditions was performed using liquid chromatography-tandem mass spectrometry (LC-MS-MS). The urine specimens obtained from healthy volunteers were treated with P-glucuronidase, purified with disposal solid-phase extraction cartridges, derivatized with a Cookson-type reagent, 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione, and subjected to LC-MS-MS. The derivatization was employed to increase the ionization efficiencies of the vitamin D-3 metabolites, which enabled detection of the metabolites in the picogram range. The identification of the genin parts of the metabolites was done by comparison with authentic samples based on their LC-MS-MS data. The glucuronides of 23S,25-dihydroxyvitamin D-3 and 24R,25-dihydroxyvitamin D3 were obtained as the main metabolites from the urine in almost equal amounts. In contrast to the fact that the plasma/serum concentration of the former is much lower than that of the latter, the hydroxylation at the C-23 position was considered to be the important side-chain modification of 25(OH)D-3 to excrete the excess vitamin D-3 in man. In addition, 23S,25-dihydroxy-24-oxovitamin D-3 occurred as its glucuronide in most of the urine, which suggested that this metabolite also plays a part in the excretion of vitamin D-3 in man. (C) 2002 Elsevier Science B.V. All rights reserved.