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New approaches in analytical and preparative free fluid electrophoresis

English title New approaches in analytical and preparative free fluid electrophoresis
Applicant Thormann Wolfgang
Number 129706
Funding scheme Project funding (Div. I-III)
Research institution Institut für Klinische Pharmakologie Universität Bern
Institution of higher education University of Berne - BE
Main discipline Biophysics
Start/End 01.04.2010 - 31.08.2013
Approved amount 288'000.00
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Keywords (11)

capillary electrophoresis; isoelectric focusing; computer simulation of electrophoresis; electrophoresis in free solution; isotachophoresis; electrokinetic capillary chromatography; enantiomeric resolution; stereoselective drug metabolism; alcohol marker; computer simulation; electrokinetic chromatography

Lay Summary (English)

Lead
Lay summary
Electrophoresis is a key technology in genomics, proteomics and metabolomics (areas whose combined information will help to obtain an integrated understanding of cell biology), in clinical, forensic and pharmaceutical analysis, and in many other fields of application. Our research laboratory specializes in the exploration, development and application of novel approaches in analytical and preparative free fluid electrophoresis. Electrophoresis in free solution can be applied to small molecular mass compounds, peptides and proteins. Three projects are currently executed. Project A represents fundamental work dealing with electrokinetic transport and separations using theoretical predictions obtained by computer simulations and, whenever possible, experimental validation in capillaries. It is anticipated that our generalized, dynamic, high-resolution model for electrophoresis is employed to study fundamental aspects of isoelectric focusing, zone electrophoresis and isotachophoresis. The model is also extended to permit the study of molecular interactions, such as those involved in enantiomeric separations with chiral buffer additives and electrokinetic capillary chromatography. The underlying transport processes can thereby be studied and visualized under realistic time scales and complexity that were hitherto inaccessible. The investigated configurations are important to understand and design new separation schemes and for capillary electrophoresis-mass spectrometry (CE-MS) of proteins used in proteomics. Project B focuses on finding proper capillary electrophoretic conditions for analysis of alcohol markers in human serum and urine. Project C is concerned with fundamental aspects of enantiomeric resolution of low molecular mass pharmaceuticals and/or their metabolites under the influence of an electric field. It is planned to apply enantioselective capillary electrophoresis to the exploration of the in vivo and in vitro stereoselectivity of the metabolism of drugs and to use free solution electrophoresis approaches for the preparation of standards of drug enantiomers. The data obtained in the three projects will contribute to the better understanding of electrokinetic separations and will pave the way for improved and widespread use of free fluid electrophoretic technology in proteomics, pharmaceutical and pharmacological sciences, clinical analysis and forensic case work. The proposed work will include collaborations with scientists in the Czech Republic, Hungary, the US, Australia and Switzerland.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Associated projects

Number Title Start Funding scheme
119911 Ketamine: stereoselective pharmacokinetic modeling and CYP-dependent metabolism in vitro - a comparison between species 01.06.2008 Project funding (Div. I-III)
139231 Advancement of functional genomics research at the University of Bern by extension of LC-MS platform 01.07.2012 R'EQUIP
112116 New approaches in analytical and preparative free fluid electrophoresis 01.06.2006 Project funding (Div. I-III)
146161 New approaches in analytical and preparative free fluid electrophoresis 01.09.2013 Project funding (Div. I-III)

Abstract

Electrophoresis is a key technology in genomics, proteomics and metabolomics, in clinical, forensic and pharmaceutical analysis, in biomedical research and many other fields of application. Our research laboratory specializes in the exploration, development and application of novel approaches in analytical and preparative free fluid electrophoresis. Electrophoresis in free solution can be applied to small molecular mass compounds, ions, peptides and proteins. A continuation of the work conducted during the previous grant period is proposed as three parts. Part A comprises fundamental work dealing with electrokinetic transport and separations in capillaries and microchannels using predictions obtained by computer simulation and, whenever possible, experimental validation. It is anticipated that our dynamic PC model for electrophoresis, which was recently extended with the first algorithms dealing with chemical equilibria between solutes and a buffer additive, is employed to study (i) aspects of isoelectric focusing, namely focusing of amphoteric analytes in mixtures of good and poor carrier ampholytes, with decreasing amounts of carrier ampholytes and in presence of electroosmosis and having different sample application strategies, ii) interactions between chiral analytes and a neutral or charged cyclodextrin as buffer additive in continuous and discontinuous buffers, and iii) the temporal behavior of system and analyte peaks in CE and electrokinetic capillary chromatography (EKC). The underlying transport processes can thereby be studied and visualized under realistic time scales and complexity that were hitherto inaccessible. In part B it is anticipated that our work in finding proper capillary electrophoretic conditions for analysis of alcohol markers, including ethyl glucuronide and ethyl sulfate in human serum and urine, is continued. It is proposed that CE-MS with nanoelectrospray ionization is being applied to the analyses of the two direct ethanol metabolites. The use of micellar EKC with fluorescence detection to assess alcohol related metabolic changes, including the urinary 5-hydroxytryptophol to 5-hydroxyindoleacetic acid ratio and the amount of salsolinol in urine and serum, is another anticipated topic. Furthermore, the elucidation of a simple immunocapture and concentration protocol for serum transferrin is proposed, a procedure that would allow monitoring of carbohydrate-deficient transferrin in sera of patients with progressed liver cirrhosis that typically have a low transferrin level and interferences in the ?-region. Electrokinetic aspects on enantiomeric resolution of low molecular mass pharmaceuticals and their metabolites comprises part C. Major focus is on the use of CE to assess the stereoselectivity in drug metabolism. Proposed investigations include i) in-line enantioselective CE for assessment of drug metabolism in vitro using electrophoretically mediated microanalysis in the plug-plug mode with single CYP450 enzymes, ii) the use of CE to assess the stereoselectivity of N-oxidation of drugs in vivo and in vitro, and iii) the electrokinetic preparation of enantiomer standards of drugs and metabolites from their racemic mixtures using charged cyclodextrins and different instrumental setups. The data obtained will contribute to the better understanding of electrokinetic separations and will pave the way for improved and widespread use of free fluid electrophoretic technology in proteomics, pharmaceutical and pharmacological sciences, clinical analysis and forensic case work. The proposed work will include collaborations with scientists in the Czech Republic, USA, Australia, Hungary and Switzerland.
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