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

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.