Method development and validation for the chiral separation of peptides in the presence of cyclodextrins using capillary electrophoresis and experimental design

The present study describes the application of statistical experimental design to the optimization of enantioselective separations of peptides in capillary electrophoresis in order to obtain optimal operating conditions for routine work. Hydroxypropyl-β-cyclodextrin was used as chiral selector and Ala–PheOMe as model peptide. The experiments were performed according to a face centered cube response surface experimental design for obtaining information how the factors such as concentration of the chiral selector, pH, buffer concentration and voltage affected the two response goals, resolution and analysis time. In order to achieve the simultaneous optimization of these two major electrophoretic performance goals for efficient and fast separation, the Derringer desirability functions were tested. While in the predefined experiments the analysis time for baseline separation was 25 min the desirability functions proposed a CE method, which diminished the analysis time and permitted the complete separation of the peptide enantiomers within 9 min.