Protein‐Based High‐Performance Liquid Chromatography and Cyclodextrin‐Capillary Electrokinetic Chromatography for the Chiral Separation of Azoles

Liquid chromatographic (LC) and capillary electrokinetic chromatography (EKC) methods with ultraviolet/diode array detection were developed for the enantioseparation of five azoles: econazole (ECO), miconazole (MICO), imazalil (IMA), ketoconazole (KET), and penconazole (PEN). The chiral selectors investigated were human serum albumin (HSA) and eight cyclodextrins (CDs), exploited in LC and EKC, respectively. HSA LC offered partial enantioseparation of IMA and, less effectively, of PEN. Interestingly, these azoles share a high degree of molecular similarity, as confirmed by in silico calculations. This may suggest that a single enantiomer achieves preferential access to enantioselective sites of the protein. To address the limited enantioselectivity of HSA for these azoles, CDs were explored as chiral selectors where EKC was preferable to LC due to its superior separation efficiency. The chiral discrimination capabilities of eight CDs were evaluated for these azole derivatives, aiming for fast separations with minimal amounts of CDs. Effective enantioseparation was achieved for ECO, MICO, IMA, and KET, using two of the tested CDs: 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) and heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin (TM-β-CD). Satisfactory resolutions were obtained using a 40 mM phosphate buffer (pH 3.2) with 1 mM HP-β-CD for ECO and MICO and 8 mM TM-β-CD for KET. For IMA, the optimal conditions consisted of a 20 mM phosphate buffer with 3 mM HP-β-CD. None of the tested CDs were effective for the enantiomeric separation of PEN; therefore, a dual CD system was developed for the first time for this compound, employing succinyl-β-CD (Succ-β-CD) in combination with HP-β-CD in a borax buffer under basic conditions (pH 9.4), enabling complete enantioseparation of PEN. The low concentration of CDs required under optimal conditions renders the developed methods highly cost-effective.