Comparison of electrophoretic methods for dextromethorphan metabolites separation obtained by in vitro incubation with microsomes

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Authors

ZEISBERGEROVÁ Marta KONEČNÝ Jiří GLATZ Zdeněk HOOGMARTENS Jos VAN SCHEPDAEL Ann

Year of publication 2008
Type Article in Proceedings
Conference BOOK OF ABSTRACTS 16th International Symposium on Capillary Electroseparation Techniques
MU Faculty or unit

Faculty of Science

Citation
Field Biochemistry
Keywords drug metabolism; microsomes; cytochrome P450; on-capillary microreaction; electrophoresis
Description In-vitro biotransformations with microsomes represent a felicitous system for drug metabolism studies. Characterization of metabolites is essential in drug discovery and development. Microsomes, a source of cytochrome P450 enzymes credibly mimic liver function like in the human body is exhibited. All metabolic pathways of Phase I biotransformation are maintained. Dextromethorphan (DEX) chosen as a probe drug is under the action of microsomes transformed into three metabolites. The most involved cytochrome P450 isoforms 3A4 and 2D6 transform DEX to 3-methoxymorphinan, 3-hydroxymorphinan and dextrorphan. To follow DEX metabolic fate, off-line or on-line incubation way can be chosen. Capillary electrophoretic (CE) methods have been utilized in many pharmaceutical and clinical applications for drug, metabolites and biomarkers determination. The capillary can serve as a separation tool or at the same time it can be employed as a microvessel for an enzymatic reaction. This work aims for the comparison of DEX metabolites separation prepared by off-line incubation to in-capillary originated metabolites. For off-line prepared sample the optimal separation was reached at tetraborate buffer (75 mM, pH 9.8) based background electrolyte (BGE) at 25C. On the other hand to meet incubation and separation requirements for on-line method, BGE composition had to be re-evaluated. The best results were obtained with tetraborate buffer with addition of linear polyacrylamide and 2-propanol exhibiting the suitable conditions to perform the separation of incubated microsomal mixture presented in phosphate buffer. The partial filling method enabled to combine two different buffers within one capillary. The final separation was performed at 37C favorable for incubation. Parameters like injection procedure, rinsing and other operation setting will be discussed.
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