Liquid chromatography with mass spectrometry enantioseparation of pomalidomide on cyclodextrin-bonded chiral stationary phases and the elucidation of the chiral recognition mechanisms by NMR spectroscopy and molecular modeling

A sensitive and validated liquid chromatography with mass spectrometry method was developed for the enantioseparation of the racemic mixture of pomalidomide, a novel, second-generation immunomodulatory drug, using beta-cyclodextrin-bonded stationary phases. Four cyclodextrin columns (beta-, hydroxypropyl-beta-, carboxymethyl-beta-, and sulfobutyl-beta-cyclodextrin) were screened and the effects of eluent composition, flow rate, temperature, and organic modifier on enantioseparation were studied. Optimized parameters, offering baseline separation (resolution = 2.70 +/- 0.02) were the following: beta-cyclodextrin stationary phase, thermostatted at 15 degrees C, and mobile phase consisting of methanol/0.1% acetic acid 10: 90 v/v, delivered with 0.8 mL/min flow rate. For the optimized parameter at multiple reaction monitoring mode 274.1-201.0 transition with 20 eV collision energy and 100 V fragmentor voltage the limit of detection and limit of quantitation were 0.75 and 2.00 ng/mL, respectively. Since enantiopure standards were not available, elution order was determined upon comparison of the circular dichroism signals of the separated pomalidomide enantiomers with that of enantiopure thalidomide. The mechanisms underlying the chiral discrimination between the enantiomers were also investigated. Pomalidomide-beta-cyclodextrin inclusion complex was characterized using nuclear magnetic resonance spectroscopy and molecular modeling. The thermodynamic aspects of chiral separation were also studied.