Analysis of adenine nucleotides and nicotinamide coenzymes by CZE in combination with field enhanced stacking
| Authors | |
|---|---|
| Year of publication | 2007 |
| Type | Article in Proceedings |
| Conference | Book of Abstracts "CECE2007 4th International Interdisciplinary Meeting on Bioanalysis" |
| MU Faculty or unit | |
| Citation | |
| Field | Biochemistry |
| Keywords | Metabolomics; Capillary Zone Electrophoresis; field enhanced stacking; |
| Description | Being the intermediates of biochemical reactions, metabolites play a very important role in connecting the many different pathways that operate within living cell. Among them adenine nucleotides and nicotinamide coenzymes are central energetic metabolites in this complex metabolic network. Determination of these metabolites is thus important for metabolomic studies, because their pool analysis characterize the energetic state of the cell under a variety of physiological conditions during cell growth. A method for determination of adenine nucleotides (ATP, ADP, AMP) and nicotinamide coenzymes (NAD+, NADH, NADP+, NADPH) by CE was developed. As low concentrations of these metabolites are presented in the cellular extracts the on-line preconcentration technique field enhanced stacking was combined with capillary zone electrophoresis (CZE) to enhance the concentration sensitivities. The determination was performed in a 75 mm fused silica capillary using separation voltage 18 kV (positive polarity), temperature of capillary 20 oC and direct detection at 260 and 340 nm. 100 mM ammonium carbonate buffer (pH 9,6) was used as the background electrolyte, but addition of beta-cyclodextrin up to 5 mM concentration was required for better resolution of given analytes. Metabolites samples were dissolved in deionised water and injected into the capillary hydrodynamically with a pressure of 35 mbar for 20 s. Under these conditions, the detection limits were in the range of 300 to 400 nM at a signal-to-noise ratio (S/N) of 3. The optimized methodology was applied on the cell extract of Paracoccus denitrificans. The concentrations of given metabolites in the bacterial cells under different growth conditions were determined. |
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