Analysis of the isomeric composition of the proline peptide bond in an angiotensin-converting enzyme inhibitor using capillary electrophoresis

Abstract

The cis and trans isomeric composition of a proline peptide bond can be determined by routine free-solution capillary electrophoresis measurements provided that one isomeric form is preferentially stabilized by a dissociable ionic group. This capability is illustrated using the angiotensin converting enzyme (ACE) inhibitor (S)-1- N-[1-(ethoxycarbonyl)-3-phenylpropyl]- l-ala- l-pro, which has the trade name enalapril. Electropherograms indicate that the two isomeric forms of enalapril can be separated with baseline resolution at 15 °C using capillary buffers having pH values in the dissociation ranges of the enalapril carboxyl group, p K cis and p K trans of 2.6 and 3.1, and of the enalapril amine group, p K cis and p K trans of 5.9 and 5.6. Such baseline resolution indicates that the isomeric composition does not change during analysis, facilitating measurement of the isomer composition of a sample prior to its injection into the capillary. Thus the effect of pH, ionic strength, or an aprotic solvent on the isomeric composition of enalapril can be measured under uniform analytical conditions. The trans isomer composition changes from 68% in the cationic form, pH <2, to 50% in the isoelectric form, pH ∼4.5, to 60% in the anionic form, pH >7. Addition of salt to the isoelectric form or addition of an aprotic solvent to any form prior to analysis increases the trans isomer composition. Similar analyses can be made using the alternative ACE inhibitors captopril and enalaprilat.