Characterization of urinary metabolites of vitamin D(3) 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 beta-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 D(3) 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.