The use of capillary electrophoresis (CE) modified with cyclodextrin (CD) for the separation of stereoisomers of peptides is well established. To increase the solubility of β‐CD, urea is often added to the buffer which may influence the complexation of a CD with a guest molecule. The aim of the present study was to investigate the influence of urea on the complexation between dipeptides and β‐CD using Ala‐Phe and Ala‐Tyr as model compounds. For this purpose three different analytical methods were employed: capillary electrophoresis (CE), 1H‐NMR spectroscopy and molecular dynamics simulations (MD). Electropherograms of the peptide enantiomers were different in the presence and absence of urea. For example, at pH 2.5 in the absence of urea the enantiomers of Ala‐Tyr are not separated in contrast to the use of buffers containing urea. Applying “complexation‐induced chemical shift (CICS)” in NMR spectroscopy and rotating frame Overhauser enhancement spectroscopy (ROESY) revealed differences in the complexation of the peptide enantiomers by β‐CD in the absence and presence of urea suggesting the stabilization of the complex through the phenolic hydroxyl group of tyrosine. MD simulations for different complexes were carried out with consideration of both water and urea molecules in solution. Simulations were performed for 1 ns. In conclusion, NMR spectroscopy and MD methods help to understand the structure of peptide‐CD complexes and the separation and migration behavior in CE. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

An enantioselective CE assay for the simultaneous determination of the enantiomeric purity and of related substances of etomidate has been developed and validated using a binary chiral selector system employing 30 mg/mL beta-CD and 4.6 mg/mL sulfated-beta-CD in a 150 mM potassium phosphate buffer, pH 2.1. The method was validated with respect to specificity, range, linearity, LOQ and LOD, precision and accuracy. The assay allowed the detection and determination of related substances including (S)-etomidate at the 0.05% w/w level, the reporting threshold as defined by the International Conference on Harmonisation guidelines as well as the European Pharmacopoeia. Robustness testing was carried out by an "Augmented Plackett-Burman" design. Quantitation of the compounds was performed by calibration graphs with respect to lidocaine hydrochloride as internal standard and by peak area normalization, the procedure usually applied by pharmacopoeias. Although data obtained from the calibration graphs constructed with the aid of the internal standard were more accurate based on compound recovery, peak area normalization may also be used without significant loss of accuracy and precision.

The cover picture shows the route that was used to confirm the formation of the possible complex between ?‐cyclodextrin and Ala‐Tyr in the presence of urea. Capillary electrophoresis shows obvious differences in the enantioseparation of the dipeptide both in the presence and absence of urea. Experimental (NMR spectroscopy) and theoretical (molecular dynamics) approaches were used to investigate these differences. Both methods confirm that a complex is formed in which urea is involved in the binding through hydrogen bonds. Details are discussed in the article by U. Holzgrabe et al. on p. 2921 ff.