Interconversion of cyclosporin molecular form inducing peak broadening, tailing and splitting during reversed-phase liquid chromatography

Summary

Reversed-phase liquid chromatography of cyclosporin A, a peptide immunosuppressant, at various temperatures produced remarkably different chromatograms. At 60°C one sharp peak was obtained, at 23°C this became a single broad peak and between 15° and 0°C this became one high sharp peak followed by a tailing or low plateau. Remarkably different chromatograms were produced also by varying the mobile phase flow-rate. The effects of both temperature and flow-rate on the chromatogram could be accounted for by interconversion between two forms of the cyclosporin molecule. Kinetic analysis showed that one form was converted by first-order kinetics with a half-life of 2.0 min at 20°C and that the apparent activation energy for the conversion was about 18 kcal/mol. The other two immunosuppressants, cyclosporin C and D, were also shown to undergo interconversion. This kinetic analysis of the interconversion should be helpful in clarifying the relationship between molecular structure and activity of the immunosuppressants.