Comparative Study on Chiral Separation of Afoxolaner on Polysaccharide‐Based Chiral Stationary Phases by Supercritical Fluid Chromatography and High‐Performance Liquid Chromatography

Supercritical fluid chromatography (SFC)-based chiral separation has been considered as one of the green, highly efficient, and precise methods for the resolution of new chiral pharmaceuticals. Herein, a comparative study of chiral separation of afoxolaner on polysaccharide-based chiral stationary phases (CSPs) by SFC and high-performance liquid chromatography (HPLC) has been conducted. First, effects of chromatographic conditions on chiral separation of afoxolaner by SFC, including CSPs, modifier types and ratios, flow rate, column temperature, and back pressure, have been discussed in detail. Cellulose tris(4-methylbenzoate)-coated CSP demonstrated the best separation performance for afoxolaner by SFC with the resolution of 2.37 among five CSPs. Afoxolaner enantiomers were eluted at 3.53 min and 4.54 min under the optimized SFC conditions, respectively, and the total analysis time was less than 6 min, much shorter than that by HPLC. Subsequently, molecular docking studies revealed that hydrogen bonds and halogen bonds formed by afoxolaner and CSPs dominated the selective interaction for the separation of afoxolaner. Additionally, hydrophobic effects and π-π stacks between afoxolaner and chiral selectors enhanced the resolution of afoxolaner. Moreover, quantitative determination results of afoxolaner by SFC showed good linearity relationships (R² > 0.999) between concentration of enantiomers and the corresponding chromatographic peak area in the range from 0.025 to 0.800 mg/mL, and the limit of quantification of enantiomers was 0.025 mg/mL. In brief, SFC separation would offer a green alternative to overcome potential technical bottlenecks in the resolution of new chiral pharmaceuticals.