Developmet of Electrophoretically Mediated Microanalysis for Drug Metalobite Generation and Separation
| Authors | |
|---|---|
| Year of publication | 2009 |
| Type | Article in Proceedings |
| Conference | Book of Abstracts 15th American symposium on Biotechnology, Biomedical and Pharmaceutical and Industrial Application of CE and Microchip Technology |
| MU Faculty or unit | |
| Citation | |
| Field | Biochemistry |
| Keywords | method development- EMMA- drug metabolites - dextromethorphan - microsomes |
| Description | Electrophoretically mediated microanalysis (EMMA) is a technique derived from capillary electrophoresis (CE). In this method the capillary is not just a separation tool, it serves as a reaction vessel as well. Thus EMMA represents a suitable format for on-line enzymatic microreactions. Different mobility of an enzyme and its substrate are utilized to mix the zones together, accomplish the biotransformation and finally separate substrate and its metabolites. Such configuration is suitable for automation and control of the time of contact between the enzyme and its substrate. In this consequence EMMA has great potential to be a useful tool in high throughput screening of drug metabolites. The drug metabolism studies are an essential part of pharmacology related research. Human liver microsomes represent the generally accepted in vitro system for metabolism studies. They credibly mimic liver functions. The most abundant proteins in microsomes are cytochrome P450 enzymes which are responsible for phase I biotransformation of most xenobiotics. Dextromethorphan (DEX) chosen as a drug probe is under the action of microsomes transformed into three metabolites. The most involved cytochrome P450 (CYP) isoforms in DEX biotransformation are CYP 3A4 and CYP 2D6 at formation of 3-methoxymorphinan, 3-hydroxymorphinan and dextrorphan. In this work we focused on development of a method and its application to DEX biotransformation. The aim of our work was a simultaneous determination of the parent drug, DEX and its metabolites generated by in-capillary approach at direct injection of microsomes or recombinant CYP 2D6 isoenzyme. The optimal separation was reached in tetraborate buffer (80 mM, pH 9.8) with addition of 2-propanol (8 %, v/v) at 37oC. The UV detection of analytes was performed at 200 nm. The partial filling method enabled to combine separation and incubation buffers within one capillary and thus to accomplish the at-capillary incubation of whole enzymatic system presented in phosphate buffer (20 mM, pH 7.4). Main parts of optimization, operation settings and injection parameters, will be discussed. The final parameters will be applied to in-capillary incubations with microsomes and recombinant CYP2D6 isoenzyme. |
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