Stereoselective interactions and liquid chromatographic enantioseparation of thalidomide on cyclodextrin-bonded stationary phases

The racemic mixture of thalidomide (THA) has been separated by liquid chromatography using cyclodextrin (CD) bonded stationary phases. Three different chiral columns (β-, hydroxypropyl-β- and carboxymethyl-β-CD) were screened, investigating their enantiodiscriminating capacities. β-CD stationary phase was identified as the most promising one and was selected for further method optimization. In order to improve resolution (R s ), an orthogonal experimental design was employed, altering the concentration of organic modifier, column temperature and flow rate, in a multivariate manner. The optimized method (mobile phase: 0.01 % acetic acid in water/acetonitrile 95/5 (v/v), column temperature: 5 °C, flow rate: 0.6 ml/min) was successful for baseline separation of THA enantiomers (R s = 1.68 ± 0.02) and was validated in terms of linearity, precision, accuracy, limits of detection and quantification. Inclusion complexation of THA with the CDs used as bonded chiral selectors was characterized in terms of stability constants, stoichiometries, spatial geometry and thermodynamic aspects. Atomic level intermolecular host–guest connections are proposed on the basis of NMR investigations in conjunction with a molecular modeling. It was found that the inclusion complex is of 1:1 composition, the aromatic ring can be accommodated in the CD cavity and the inclusion process is enthalpically driven. Molecular modeling revealed that S-THA formed a more stable inclusion complex with β-CD, than its antipode, which is in good agreement with chromatographic results, where R-THA eluted faster than S-THA. Our method offers an effective way for determination of THA enantiomers, which might gain further significance if chiral switching of the molecule is attained.