Economical chiral and achiral separation of seven impurities in brivaracetam by supercritical fluid chromatography with polysaccharide-based chiral stationary phases.

Due to the presence of multiple chiral centers in its structure, the analysis of brivaracetam and its enantiomers, diastereomers, as well as non-isomeric impurities imposes high requirements on the separation capability of chromatographic methods. In this study, a rapid, accurate, and environmentally friendly supercritical fluid chromatography (SFC) method was developed for the simultaneous separation and quantification of seven isomeric and non-isomeric impurities in brivaracetam. The effects of chromatographic parameters-including stationary phase, mobile phase composition, column temperature, and back pressure-on the selective separation and quantification of the target compounds were systematically investigated. The results indicated that optimal separation was achieved using an amylose tris(3,5-dimethylphenylcarbamate)-based stationary phase, with a mobile phase consisting of carbon dioxide and methanol (containing 0.2 % diethylamine, v/v) under gradient elution conditions. Van't Hoff analysis revealed that the separation process was primarily enthalpy-driven. Molecular docking studies further confirmed that hydrogen bonding and π-σ interactions were the main binding forces between the analytes and the stationary phase. These thermodynamic and molecular simulation results complemented each other and provided a rational explanation for the elution order of the impurities. The method demonstrated good accuracy (95.5 % - 105.5 %) and intermediate precision (RSD < 3 %) over the validated concentration range, with limits of detection (LOD) ranging from 1.743 to 4.207 ng, indicating high reliability and sensitivity. Moreover, the developed SFC method outperformed the reference methods from the European Pharmacopoeia in terms of analysis throughput and environmental friendliness. This study serves as a reference for the development of efficient and environmentally sustainable impurity analysis methodologies for drugs with multiple chiral centers.