Impurity profiling by capillary electrophoresis: definition of the design space within quality by design approach and investigation of separation mechanism and selectivity

A Quality by Design (QbD) approach has been applied for the set-up of a capillary electrophoresis (CE) method for the simultaneous determination of the enantiomeric purity and of related substances of the chiral drug ambrisentan, an orphan drug used in the treatment of pulmonary arterial hypertension. QbD scouting made it possible to select cyclodextrin modified-micellar electrokinetic chromatography as operative mode, using sodium dodecyl sulphate (SDS) as surfactant for obtaining the micellar pseudostationary phase and γ-cyclodextrin (γ-CD) as chiral selector. This system involved complex separation mechanisms, including electrophoretic mobility, partitioning into the micelles and inclusion complexation into the cyclodextrin, and was essential for obtaining the separation of ambrisentan enantiomers and the other impurities. A first screening phase was followed by response surface methodology, which allowed the design space to be defined in combination with Monte-Carlo simulations. The built mathematical models represented a step forward in the comprehension of the electrophoretic behaviour of the analytes confirming the successful binomium of multivariate strategies and CE. Molecular Dynamics (MD) and NMR studies, essential tools for exploring the mechanism of molecular recognition, contributed to the understanding of the involved intermolecular interactions and to evaluate the mechanism of separation. MD simulations and NMR experiments underlined the ability of γ-CD of including SDS monomer forming inclusion complexes and the pivotal role of this surfactant in modulating the different affinities of the analytes for the chiral selector.