Chiral separation of a basic drug with two chiral centers by electrokinetic chromatography for its pharmaceutical development

•CE method to separate four stereoisomers of a drug development as single enantiomer.•Two in-capillary preconcentration techniques allow sensitivity gains of 3000-fold.•CE method enables the control of stereoisomeric impurities of a new drug substance.•CE method enables to investigate the chiral stability of a new drug product.•CE method enables to investigate the in vivo metabolism of a single enantiomer drug.

A chiral method using capillary electrophoresis was developed for the separation of the four stereoisomers of a new chiral substance currently undergoing drug development as single enantiomer. After the selection of highly sulfated β-CD as chiral selector, an exhaustive study on the influence of several experimental variables on the resolution was performed, being the substitution degree of the CD a very decisive factor. Run time and resolutions were about 20 min and higher than 2.0, respectively. The method was validated in terms of selectivity, linearity, accuracy, precision, and limits of detection and quantitation according to the requirements of the International Conference on Harmonisation for the determination of the chiral purity of a drug substance. The usefulness of the method was demonstrated in the control of stereoisomeric impurities in raw material as well as in the determination of the chiral stability of the drug in the solid state and in dosage forms used in safety assessment. Finally, the chiral method was used to investigate the possible in vivo inversion in biological samples.