Analysis of Cyclosporin A and Main Degradation Impurities by Cyclodextrin–Modified Micellar Electrokinetic Chromatography

Abstract

Separation of the immunosuppressive drug cyclosporin A (CyA) from the closely related degradation impurities cyclosporin H (CyH, a CyA diastereomer) and isocyclosporin A (IsoCyA) was accomplished by means of cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC). Heptakis (2,3,6-tri-O-methyl)-β-cyclodextrin (TM-βCD) showed to be an effective modifiers of the sodium dodecyl sulfate (SDS) micellar system, allowing for the CD-MEKC separation of CyA from CyH and IsoCyA. By means of electric current measurements carried out using the capillary electrophoretic apparatus, the critical micelle concentration of SDS in the presence of different neutral cyclodextrins was estimated. Interestingly, it was found that TM-βCD strongly inhibited the micellization of SDS compared to β-, and hydroxypropyl-β-cyclodextrin. This unusual behavior was considered to be a key factor of the specific ability of the methylated cyclodextrin in providing CD-MEKC separation of hydrophobic cyclosporins. The optimized method consisted of: 50 mM sodium dodecyl sulfate (SDS) in 50 mM tetraborate buffer (pH 9.2) supplemented with 15 mM TM-βCD. The electrophoretic runs were performed at 30°C under the application of 22 kV in a fused-silica capillary (50 µm id, 64.5 cm total length, 56 cm length to the detector). The method was validated for linearity of CyA (within 0.25–5.00 mg/mL) and CyH and IsoCyA (within 0.25–5.0%, w/w with respect to CyA), sensitivity (LOQ 5.0 µg/mL), accuracy, and precision. The applicability of the method was proved by analysis of a commercially available pharmaceutical (gelatin capsules).