Chiral separation of ketoconazole and itraconazole anti-fungal drugs using experimental and computational approaches

Abstract

Drugs with multiple chiral centers were observed as very effective for treating various diseases. However, the enantiomeric resolution of multiple chiral center racemates is not much developed compared to racemates having a single asymmetric center. This work aimed to develop a chiral separation method for antifungal drugs using electrokinetic chromatography (EKC) and to elucidate mechanism of enantioseparation using a computer-aided molecular modelling study. Two azole antifungal drugs were selected namely ketoconazole and itraconazole, which consists of two and three chiral centers, respectively. The separation for ketoconazole was achieved using heptakis (2,3,6-tri-O-methyl)-ß-cyclodextrin (TM-ß-CD), a commonly used chiral selector, as it is relatively inexpensive and has a low UV absorbance in addition to an anionic surfactant, sodium dodecyl sulfate. The optimum conditions for chiral separation of ketoconazole was achieved using 10 mM phosphate buffer at pH 2.50 containing 20 mM TM-ß-CD, 5 mM SDS, and 1.0% (v/v) methanol with an applied voltage of 25 kV at 25°C with a 5-s hydrodynamic injection time at 50 mbar. The four ketoconazole stereoisomers were successfully resolved within 17 min (total analysis time was 28 min including capillary conditioning). The migration time precision of this method was examined to give a repeatability and reproducibility with RSDs = 5.80% (n = 3) and RSDs = 8.88% (n = 9), respectively. A computational study, using quantum mechanics calculations with AutoDock and semi-empirical PM3 calculations, were used to predict the enantiodiscrimination of ketoconazole enantiomers. A Density Functional Theory (DFT) single-point calculation at the level of B3LYP/6-311G (d,p) was performed for the PM3-optimized complexes to obtain more accurate binding energy and also electronic structures of the complexes. Molecular docking and DFT were simulated to predict the enantioresolution of itraconazole with two types of cyclodextrins (CDs), TM-ß-CD and (2-hydroxylpropyl)-?-cyclodextrin (HP-?-CD). The difference in energies of the inclusion complexes between the enantiomers and CD is a measure of chiral discrimination, which results in the separation of the enantiomers in the experimental studies. The dual-CD and triple-CD methods were developed for chiral separation of itraconazole using EKC. Highly sulfated ß-cyclodextrin (S-ß-CD), (2-hydroxylpropyl)-ß-cyclodextrin (HP-ß-CD), TM-ß-CD and HP-?-CD were screened as possible chiral selectors for enantioseparation of itraconazole. The enantioseparation of itraconazole was achieved using 10 mM phosphate buffer solution at pH 3.62 containing a mixture of 10 mM of each HP-ß-CD, TM-ß-CD and HP-?-CD and an applied voltage of 25 kV at 25°C. Both computational and experimental investigations complement each other prior to chiral recognition mechanism. Combination of molecular modelling and capillary electrophoresis appears as a new emerging method for chiral analysis of pharmaceutical drugs.