Dextromethorphan metabolite profiles generated by in-capillary microreaction and subsequent electrophoretic separation
| Title in English | and subsequent electrophoretic separation |
|---|---|
| Authors | |
| Year of publication | 2010 |
| Type | Article in Proceedings |
| Conference | Book of Abtracts 16th Latin-American Symposium LACE 2010 |
| MU Faculty or unit | |
| Citation | |
| Keywords | EMMA; Cytochromes P450; Drug Metabolism; Dextromethorphan |
| Description | The determination of metabolite profile of potential pharmaceuticals has a substantial role in the drug development phase. Different human-derived in vitro systems like liver slices, microsomes or recombinant enzyme systems are utilized for metabolite identification. Human liver microsomes represent the generally accepted in vitro system for these studies. They credibly mimic liver functions as they contain many drug-metabolizing enzymes, mainly cytochromes P450 (CYPs). Alternatively, recombinant cytochrome P450 enzymes (rCYP) are suitable for "frontline" predictive human metabolism studies. rCYP are a favorite in vitro system for their availability and ease employment in high throughput assays. They are a valuable tool in the CYP phenotyping, i.e. searching for which CYP enzyme is involved in the biotransformation of new agents. The metabolism pathway of dextromethorphan (DEX) was chosen as a model system. DEX is an antitusive component of many medications which is chemically a non-narcotic synthetic analog of codeine. DEX is a probe drug of CYP2D6 enzyme. CYP 2D6 is one of the most important enzymes catalyzing biotransformation of xenobiotics. Further, CYP 3A4, the most abundant CYP enzyme also contributes to DEX metabolism. Thus DEX metabolite profile should be completely contained when microsomes are applied. Nowadays, most of drug metabolism studies are performed by liquid chromatography-mass spectrometry methods characterized by off-line setup. However, capillary electrophoresis (CE) represents an innovative approach to perform automated enzyme assays. Different methods and procedures of enzymatic activity studies have been reported. The aim of our work was a simultaneous determination of the parent drug, DEX and its metabolites generated by in-capillary microreaction at direct injection of microsomes or rCYP enzymes. The analytes were separated in 50-um fused silica capillary (48 cm total length) at 14 kV. The background electrolyte consisted of tetraborate buffer (80 mM, pH 9.7) with addition of 2-propanol (8 %, v/v). The capillary temperature was kept at 37 oC and the data were recorded at 200 nm. The partial filling method enabled to combine separation and incubation buffers within one capillary thus maintaining the optimal incubation conditions. Different injection strategies will be shown and compared in the term of reaction efficacy and versatility. |
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