Retention behavior of cardiac steroids using cyclodextrin in the mobile phase in high-performance liquid chromatography.

The retention behaviour of twenty cardiac steroids and four fluorescent derivatives was examined by the addition of cyclodextrin to the mobile phase in reversed-phase high-performance liquid chromatography. The addition of a suitable cyclodextrin improved the separation of isomeric cardiac steroids. The steroid A/B ring junction is the most important factor in the choice of the optimum cyclodextrin to be added; the C/D ring junction is less important. The hydroxyl group at the 3- or 12-position of the steroid enhanced the changes in retention times of these compounds. The effect of an unsaturated lactone ring at the 17 beta-position on the retention in the presence of cyclodextrin was also determined with cardenolide (five-membered ring) and bufadienolide (six-membered ring) but little difference was observed. Isomeric cardiac steroids, whose separation has not been done by the conventional method, were clearly separated by this method. The fluorescence intensity of 3-(1-anthroyl)-cardiac steroid was enhanced by the addition of cyclodextrin to the mobile phase.