Publication details
In-capillary approach to drug metabolite generation with subsequent electrophoretic separation
| Authors | |
|---|---|
| Year of publication | 2009 |
| Type | Article in Proceedings |
| Conference | EURO ANALYSIS 2009 |
| MU Faculty or unit | |
| Citation | |
| Field | Biochemistry |
| Keywords | capillary electrophoresis- in-capillary microreaction-drug metabolism-microsomes-method development |
| Description | Studies of drug metabolism are essential for pharmacology related research. Human liver microsomes represent the generally accepted in vitro system for these studies. Cytochrome P450 enzymes, the most abundant proteins of microsomes, are responsible for biotransformation of xenobiotics. Dextromethorphan (DEX) chosen as a drug probe is transformed into three metabolites. The most involved cytochrome P450 (CYP) isoforms are CYP 3A4 and CYP 2D6 at formation of 3-methoxymorphinan, 3-hydroxymorphinan and dextrorphan. Electrophoretically mediated microanalysis (EMMA) is a technique derived from CE. In EMMA method the separation capillary serves as a reaction vessel and thus it represents a suitable format for on-line enzymatic microreactions. Different mobility of an enzyme and its substrate are utilized to mix the zones together and to accomplish the biotransformation and subsequent separation of the substrate and its metabolites. Such configuration is suitable for automation and control of the time of contact between the enzyme and the substrate. In this work we focused on development of a method for simultaneous determination DEX and its metabolites generated by at-capillary (i.e., capillary inlet) incubation 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 37C and allowed to perform the separation of both off-line and on-line incubated samples. The partial filling method enabled to combine separation and incubation buffers within one capillary. Main parts of optimization, operation settings and injection parameters, are discussed. The results of incubations with microsomes and recombinant CYP2D6 isoenzyme were compared. |
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