Retention behaviour 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 tie 17β-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 flu orescence intensity of 3-(1-anthroyl)-cardiac steroid was enhanced by the addition of cyclodextrin to the mobile phase.