Application of capillary zone electrophoresis with off-line solid-phase extraction to in vitro metabolism studies of antifungals

A simple and robust solid-phase extraction (SPE) procedure for the cleanup and sample preconcentration of antifungals (ketoconazole, clotrimazole, itr aconazole, fluconazole, and voriconazole) and their metabolites after incub ation with human liver microsomes, as well as a simplified capillary zone e lectrophoresis (CZE) method for their rapid analysis, have been developed t o determine the stability of these compounds in in vitro samples. Three dif ferent sample pretreatment procedures using SIDE with reversed-phase sorben ts (100 mg C8, 100 mg C18, and 30 mg Oasis-HLB) were studied. The highest a nd most reproducible recoveries were obtained using a 30 mg Oasis-HLB sorbe nt and methanol containing 2% acetic acid as eluent. Enrichment by a factor of about four times was achieved by reconstituting the final SPE eluates t o a small volume. For the CZE separation, good separations without interfer ing peaks due to the in vitro matrix were obtained with a simple running el ectrolyte using a fused-silica capillary. The best separation for all compo nents originated by each tested drug after incubation with human liver micr osomes (unmetabolized parent drug and its metabolites) was obtained using a 0.05 M phosphate running buffer (pH 2.2) without additives. The effect of the injection volume was also investigated in order to obtain the best sens itivity. Performance levels in terms of precision, linearity, limits of det ection, and robustness were determined.