Separation of vitamin D metabolites and their analogues by high-pressure liquid chromatography.

The separation of vitamin D3 metabolites and their analogues by high-pressure liquid chromatography was investigated using a Zorbax SIL column. Each metabolite can be separated effectively with a solvent consisting of 2% methanol-methylene chloride or gradient elution from 0.02% to 6% methanol-methylene chloride. The C-24 epimers of 1alpha, 24-dihydroxy- and 1alpha,24,25-trihydroxy-vitamin D3 are resolved as their free forms, while the epimers of 24-hydroxy- and 24,25-dihydroxy-vitamin D3 can be separated as their trimethylsilyl derivatives.