Enantioseparation of Mirabegron Using Cyclodextrin-based Chiral Columns: High-performance Liquid Chromatography and Molecular Modeling Study

Abstract

A novel high-performance liquid chromatography method for the enantioseparation of mirabegron (R-mirabegron), a selective β-3 adrenergic receptor agonist, using cyclodextrin (CD)-based chiral stationary phases (CSPs) was developed. Seven different CSPs containing β-, γ-, hydroxypropyl-β-, sulfobutyl-β-, carboxymethyl-β-, permethyl-β-, and phenylcarbamate-β-cyclodextrin were evaluated under both polar organic and reversed-phase conditions. Only the phenylcarbamate-β-cyclodextrin containing the Chiral CD-Ph column displayed enantiorecognition. Optimization of conditions using a full factorial design led to the determination of the most suitable conditions: a mobile phase composition of 90:10:0.1 methanol:water:diethylamine, a flow rate of 0.8 mL/min, and a column temperature of 40°C with enantiomeric elution order, where the impurity S-mirabegron elutes first. Using the optimized conditions, enantioseparation with R_s = 1.9 was achieved within 10 min. The developed method was validated according to current guidelines and successfully applied for the determination of S-mirabegron, as a chiral impurity in pharmaceutical formulations. The enantiorecognition mechanism was investigated by molecular docking and thermodynamic analysis. Using molecular modeling, the interactions between CDs and the analyte were analyzed at the molecular level, revealing that mirabegron interacts primarily with the phenylcarbamate groups on the outer surface of the structure. Enthalpy-controlled enantioseparation was consistently observed across all eluent compositions, regardless of the conditions. The developed and validated method is highly suitable for routine determination of the enantiomeric purity of mirabegron, offering a reliable tool for regulatory compliance.