Effect of Cyclodextrin Complexation on the Separation of Insulin Analogues in Capillary Electrophoresis Using...

The simultaneous separation of therapeutic insulin analogues by capillary electrophoresis (CE) remains challenging because of their high structural similarity and their tendency to adsorb onto the capillary surface. This study investigated the applicability of cyclodextrin (CD) derivatives as selectivity-modifying additives for the CE separation of human insulin and five therapeutic insulin analogues using five-layer poly(diallyldimethylammonium chloride)/poly(sodium 4-styrenesulfonate) (PDADMAC/PSS)_2.5 and four-layer (PDADMAC/PSS)₂ successive multiple ionic-polymer layer (SMIL)-coated capillaries under acidic and alkaline conditions, respectively. A broad range of CD derivatives was systematically evaluated under optimized background electrolyte conditions. Under acidic conditions, neutral and predominantly uncharged CD derivatives improved separation selectivity, with carboxyethyl-β-CD providing the most favorable discrimination within the critical insulin lispro-human insulin-insulin aspart migration group. In contrast, highly charged anionic CD derivatives disrupted the PDADMAC-terminated coating and could not be applied. The PSS-terminated coating remained stable under alkaline conditions, enabling the evaluation of both neutral and anionic CD derivatives. Under these conditions, the migration order of the insulin species was completely reversed, while several CD derivatives further modulated the relative spacing of neighboring peaks. The use of organic modifiers produced system-dependent effects on separation selectivity and peak shape. These findings demonstrate that the appropriate selection of CD derivatives and SMIL coating strategy provides an effective approach for modulating selectivity in the CE separation of structurally related therapeutic peptides.