Chiral Separation and Molecular Simulation of Five Quinolones on a Bonded Amylose[(S)-α-Methylbenzyl Carbamate] Column (CHIRALPAK IH) and the Elucidation of Its Recognition Mechanism.

In this work, the silica gel bonded amylose[(S)-α-methylbenzyl carbamate] (CHIRALPAK IH) was chosen as the chiral stationary phase (CSP) for the separation of five quinolone enantiomers by high-performance liquid chromatography (HPLC), namely, ofloxacin, flumequine, nadifloxacin, lomefloxacin, and clinafloxacin. The mobile phase composition, organic modifier, and acid-base additive were systematically investigated for the baseline separation of these five interested quinolones. The optimized mobile phases were n-hexane-ethanol-acetic acid-diethylamine (60:40:0.2:0.2, v/v/v/v) for ofloxacin and nadifloxacin, n-hexane-ethanol-acetic acid-diethylamine (60:40:0.2:0.2, v/v/v/v) for flumequine, n-hexane-isopropanol-acetic acid-diethylamine (60:40:0.3:0.3, v/v/v/v) for lomefloxacin, and n-hexane-ethanol-acetic acid-diethylamine (70:30:0.3:0.3, v/v/v/v) for clinafloxacin. The interaction forces between the amylose CSP and target quinolones were simulated by computerized molecular docking to study their enantiorecognition mechanisms. The results indicated that hydrogen bonding, π-π stacking, and hydrophobic interactions collectively contributed to the stereoselective binding. The differences in these interaction forces between the quinolone enantiomers, particularly the greater contribution of hydrogen bonding in one enantiomer compared to the other, led to a significant difference in binding energy. This differential binding energy ultimately governed the elution order and enabled chiral recognition of the five quinolone enantiomers.