Liquid chromatographic enantioseparation of thalidomide and its derivatives on cyclodextrin-bonded stationary phases

Abstract

Abstract The chiral separation of three racemic immunomodulatory drugs, thalidomide, pomalidomide and lenalidomide was studied, using three cyclodextrin bonded stationary phases (β-, hydroxypropyl-β- and carboxymethyl-β-CD) in reversed-phase and polar organic mode. In polar organic mode, using acetonitrile and methanol, no chiral separation was observed. In reversed-phase mode pomalidomide showed chiral interactions with all selectors, while lenalidomide showed no chiral interactions with any of the cyclodextrins employed. Thalidomide showed chiral interactions with β-and carboxymethyl-β-CD, only. Based on these observations it can be concluded that the oxo group at position two is necessary for chiral recognition, while the aromatic primary amine group enhances it. Orthogonal experimental design was used to investigate the effect of the eluent composition, flow rate, and the column temperature on chiral separation. Concentration of the organic modifier was the most important factor among the investigated three variables showing high impact on the chiral separations. In the case of thalidomide optimized parameters (β-cyclodextrin-based stationary phase, 0.1% acetic acid/acetonitrile 95/5 (v/v), 5 °C column temperature, 0.6 ml/min flow rate) resulted in a resolution of 1.68 ± 0.02 between enantiomers. For pomalidomide, this value was 2.70 ± 0.02, under the circumstances as follows: β-cyclodextrin-based stationary phase, 0.1% acetic acid/acetonitrile 90/10 (v/v), 15 °C column temperature and 0.8 mL/min flow rate. Utilizing the experimental conditions employed on an LC-MS/MS system, concentrations as low as 2 ng/mL could be determined from mouse plasma for both substances. Elution sequences were determined with enantiopure standards and in both cases the R-enantiomers eluted first. The methods developed are suitable for the chiral separation of the abovementioned compounds and are sound starting points for bioanalytical method development.