HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC SEPARATION OF DIHYDROXYLATED VITAMIN D₃ METABOLITES USING MOBILE PHASE CONTAINING CYCLODEXTRIN

Abstract

The separation of side chain dihydroxylated vitamin D₃ metabolites by inclusion high performance liquid chromatography using cyclodextrin (CD) as the mobile phase additive was examined. 24,25-Dihydroxyvitamin D₃ and its 3-epimer were clearly separated by the addition of γ-CD to the mobile phase using acetonitrile as the organic modifier. The separation of the 25-epimers of 25,26-dihydroxyvitamin D₃ has been done, to a certain extent, with the mobile phase using methanol and dimethylated β-CD as the organic modifier and the additive, respectively. These separation methods were applied to the detection of the metabolites in the plasma of the rat administered 25-hydroxyvitamin D₃. These metabolites were also identified by liquid chromatography/tandem mass spectrometry after derivatization.

ACKNOWLEDGMENTS

Part of this work was supported by grants from the Ministry of Education, Science, Sport, and Culture of Japan. We thank Kao Co. and Nihon Shokuhin Kako Co. for supplying the CDs.