High performance liquid chromatographic separation of dihydroxylated vitamin D3 metabolites using mobile phase containing cyclodextrin

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