Facultad de Medicina

A rapid and simple capillary zone electrophoresis (CZE) method for the determination of terbinafine and eight of its main metabolites after incubation with rat hepatic S9 fraction was developed. The effect of the concentration and type of the running electrolyte as well as of the addition of an organic solvent were studied with emphasis on selectivity and sensitivity. All nine components obtained (unmetabolized terbinafine and up to eight of its metabolites) are baseline resolved in less than 4 minutes using a 0.05 M phosphate buffer (pH 2.2) without additives, after a solid-phase clean-up procedure of these in-vitro samples. In addition, under the conditions described, no endogenous components of the sample interfere at the detection wavelength chosen. After optimization of the separation conditions, some analytical characteristics of the developed CZE method were investigated. A limit of detection of only 0.08 lg/mL was obtained for terbinafine using a standard solution. Finally, the use of on-line CZE/ diode array detection enabled to identify tentatively the presence of unmetabolized parent terbinafine and its metabolites in the mixture of nine components separated.

A simple and robust solid-phase extraction (SPE) procedure for the cleanup and sample preconcentration of antifungals (ketoconazole, clotrimazole, itraconazole, fluconazole, and voriconazole) and their metabolites after incubation with human liver microsomes, as well as a simplified capillary zone electrophoresis (CZE) method for their rapid analysis, have been developed to determine the stability of these compounds in in vitro samples. Three different sample pretreatment procedures using SPE with reversed-phase sorbents (100 mg C8, 100 mg C18, and 30 mg Oasis-HLB) were studied. The highest and most reproducible recoveries were obtained using a 30mg Oasis- HLB sorbent and methanol containing2% acetic acid as eluent. Enrichment by a factor of about four times was achieved by reconstituting the final SPE eluates to a small volume. For the CZE separation, good separations without interfering peaks due to the in vitro matrix were obtained with a simple running electrolyte using a fused-silica capillary. The best separation for all components originated by each tested drug after incubation with human liver microsomes (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 sensitivity. Performance levels in terms of precision, linearity, limits of detection, and robustness were determined.