Chromatographic separation of 24(R),25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3-26,23-lactone using a cyano-bonded phase packing.

A high-performance liquid chromatographic system is described for the baseline resolution of 25-hydroxyvitamin D3, 24(R),25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3-26,23-lactone, the three principal circulating metabolites of vitamin D3 in the vitamin D-replete animal. The system is based upon a cyano-bonded phase packing and the solvent hexane--isopropanol--methanol (94:5:1). Of particular interest is the strong retention of carbonyl-containing vitamin D metabolites. The new system can be used for unequivocal analysis of vitamin D metabolites in plasma samples from clinical and animal studies and in the separation and identification of renal metabolites generated in vitro.