Direct injection systems for high throughput in-vitro drug metabolism screening studies using capillary electrophoresis and liquid chromatography-tandem mass spectrometry.

摘要

Capillary electrophoresis (CE) and liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) have been used to set up direct injection systems for high-throughput in-vitro drug metabolism screening studies. The probe drug chosen for direct injection CE analysis was quinidine because it is metabolized by the CYP 3A4 isozyme to form (3S)-3-hydroxy quinidine (3-OHQ) and is compatible with laser induced fluorescence detection (LIF). Initially a CE method was developed in the absorbance mode for the determination 3-0HQ by direct injection of the incubation mixtures. This was validated as an analytical and a metabolism inhibition model. The limit of quantitation (LOQ) for 3-OHQ in the incubation matrix was 500 ng/ml and its formation complied with Michaelis-Menten kinetics showing Km and V max values of 94.4 ± 11.8 μM and 321 ± 4 ng/mg/hr respectively. The method was capable of screening for strong and medium inhibitors of the CYP 3A4 isozyme but lacked the sensitivity for weak inhibitors. A CE-laser induced fluorescence system was therefore constructed to decrease concentration sensitivity and this method demonstrated a LOQ of 52 ng/ml and was capable of screening for weak inhibitors of 3-OHQ. The lack of sample preparation coupled with sensitive detection and low sample volume requirements makes this an attractive approach for metabolism screening.; A direct injection LC/MS/MS method was also developed for high-throughput metabolism screening of the CYP 2D6 and the CYP 3A4 isoenzymes using dextromethorphan and midazolam as probe drugs. Direct injection was made possible by using a short LC column with large particle size packing. Matrix effects were evaluated to establish the feasibility of direct injection. Incubations were carried out in an autosampler and estimation of the half lives for the disappearance of the probe drugs validated this approach. High-throughput screening was performed using known inhibitors of the CYP 2D6 isozyme and the system was evaluated for its ability to differentiate between the potencies of these inhibitors. This technique involved no sample preparation and demonstrated analysis times of 2 minutes per injections and is therefore useful for setting up high throughput screens.