Development of quantitative sample preparation protocol for targeted metabolome analysis of adenine nucleotides and coenzymes by means of field enhanced sample stacking-capillary zone electrophoresis combination
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| Year of publication | 2008 |
| Type | Conference abstract |
| MU Faculty or unit | |
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| Description | Investigations in microbial metabolomics require reliable and reproducible analyses of the metabolites in the cell across a broad dynamic range of concentrations and from a range of different chemical functionalities. The development of robust a consistent experimental protocol for all steps in the procedure ranging from biomass cultivation, metabolism quenching, metabolites extraction to the quantitative analysis is required. Recently the selective and sensitive method for targeted metabolome analysis of adenine nucleotides and coenzymes based on field enhanced sample stacking-capillary zone electrophoresis combination has been developed. However such analytical method needs also appropriate methodology to isolate and to concentrate the target compounds from the biological matrix with minimal losses and this was the main aim of this work. Aqueous solutions containing organic solvents, such as methanol, ethanol, acetonitril and others, are widely used for extraction of intracellular metabolites from bacterial cells, because destabilize the cell wall and cell membrane proteins and lipids forming pores on the cell envelopes. In this consequence 20 - 100% ethanol, methanol, acetonitril, mixtures acetonitril/methanol/water (40:40:20), mixtures of acidic (0.1 M formic acid-containing) organic solvents and mixtures of basic (0.1 M NH4OH-containing) organic solvents were tested as an extraction media for adenine nucleotides (ATP, ADP, AMP) and coenzymes (NAD+, NADH, NADP+ and NADPH) from bacterium Paracoccus denitrificans and quantitative yields of metabolites were measured by capillary zone electrophoresis with UV detection in combination with field enhanced sample stacking and compared each other. |
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