Abstract
Organic acids play a key role in central metabolic functions of organisms, are crucial for understanding regulatory processes and are ubiquitous inside the cell. Therefore, quantification of these compounds provides a valuable approach for studying dynamics of metabolic processes, in particular when the organism faces changing environmental conditions. However, the extraction and analysis of organic acids can be challenging and validated methods available in this field are limited. In this study, we developed a method for the extraction and quantification of organic acids from microbial samples based on solid-phase extraction on a strong anionic exchange cartridge and gas chromatographic-mass spectrometric analysis. Full method validation was conducted to determine quality parameters of the new method. Recoveries for 12 of the 15 aromatic and aliphatic acids were between 100 and 111% and detection limits between 3 and 272 ng/mL. The ranges for the regression coefficients and process standard deviations for these compound classes were 0.9874–0.9994 and 0.04–0.69 μg/mL, respectively. Limitations were encountered when targeting aliphatic acids with hydroxy, oxo or enol ester functions. Finally, we demonstrated the applicability of the method on cell extracts of the bacterium Escherichia coli and the dinoflagellate Prorocentrum minimum.Graphical abstract
Highlights
Metabolomics denotes the investigation of the metabolite composition in cells, tissues or organisms and belongs to the “-omics” disciplines—genomics, proteomics and metabolomics— converging in systems biology [1]
We developed a method for the extraction of organic acids (OAs) from cell extracts with an anionic exchange solid-phase extraction cartridge and used gas chromatography-mass spectrometry for the determination of the target compounds
We tried to apply liquid–liquid extraction (LLE), as this is a method routinely used in our lab for metabolite extraction
Summary
Metabolomics denotes the investigation of the metabolite composition in cells, tissues or organisms and belongs to the “-omics” disciplines—genomics, proteomics and metabolomics— converging in systems biology [1]. Before applying the method to biological samples, several performance indicators needed to be evaluated For this purpose, experiments to access the calibration range, limits of detection and quantification, stability as well as recovery of the acids were performed before applying the method to cell extracts of E. coli and P. minimum. To test the stability of the underivatized solutions under different conditions, the stock solutions of the analytes were diluted to a high (4 and 17.5 μg/mL for high and low response OAs, respectively) and a low (0.2 and 2 μg/mL for high and low response OAs, respectively) concentration relative to the chosen calibration range Five of these samples each were measured directly after preparing the solutions and on the same day the stock solutions were made. Confidence intervals for the concentrations provided in this way were defined according to DIN 38402 part 51 [39]
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