Analytical Sciences

Investigation of C-3 Epimerization Mechanism of24,25-Dihydroxyvitamin D₃ in Rat Using LiquidChromatography/Mass Spectrometry

Tatsuya HIGASHI, Aki OGASAWARA, and Kazutake SHIMADA

Faculty of Pharmaceutical Sciences, Kanazawa University, Takara-machi, Kanazawa 920-0934, Japan

Studies on C-3 epimerization of 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃] were performed. 3-Epi-24,25(OH)₂D₃ wasfound to circulate in the blood of rats administered 24,25(OH)₂D₃, which was confirmed in comparison with a standardsample based on its chromatographic behavior during inclusion high-performance liquid chromatography using r` -cyclodextrinas a mobile phase additive and data obtained from liquid chromatography (LC)/mass spectrometry (MS) andgas chromatography/MS. The C-3 epimerization mechanism was investigated in in vitro experiments using [3 r^ - ²H]-24,25(OH)₂D₃. The disappearance of deuterium was monitored by LC/MS. As a result, it was clarified that the 3-epimerwas formed via the 3-oxo-form as an intermediate. This epimerization was also found to be controlled by the enzymeexisting in the liver cytosol fraction and to require NAD and NADPH as coenzymes.

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