Combined chiral–achiral supercritical fluid chromatography method for the impurity analysis of dapoxetine reveals insights in entropy-driven retention and acid-modulated selectivity

Chiral analysis is an essential part of the development and quality control of enantiopure pharmaceuticals. Supercritical fluid chromatography (SFC) emerged as a powerful tool for the chiral separation of drugs, however, its implementation in the pharmacopoeia remains limited. In this study, we developed a rapid SFC-UV method for the impurity control of S-dapoxetine, enabling simultaneous chiral and achiral analysis in less than 20min. The method was validated according to ICH Q2(R2) guidelines and demonstrated sufficient sensitivity, precision and accuracy down to 2.5µgmL^-1. A binary acid-base additive mixture was used to modify chemo- and stereoselectivity, achieving baseline separation of all relevant analytes. During method development, an unusual retention behaviour of basic analytes was observed, leading to a thermodynamic investigations using the Van 't Hoff analysis. This revealed a rare case of entropy-driven retention, with strong acidic additives like trifluoroacetic acid leveraging the effect for basic analytes. Further investigations involving 15 diverse chiral amines, as well as molecular dynamic simulations, showed that isopropanol stabilizes the helical amylose backbone by reducing excessive flexibility, thereby increasing the enantioselective separation efficiency. Additionally, strong acids were found to form transient ion-pairs with basic analytes, which are favoured in the supercritical environment. The neutral complexes impact retention depending on the amine substitution pattern and may suppress polar and enhance hydrophobic interactions. These results underscore the potential application of combined additive systems to enhance SFC applications in the field of ionizable analytes.