Method development and validation for the quantification of organic acids in microbial samples using anionic exchange so
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RESEARCH PAPER
Method development and validation for the quantification of organic acids in microbial samples using anionic exchange solid-phase extraction and gas chromatography-mass spectrometry Simone Heyen 1
&
Barbara M. Scholz-Böttcher 1
&
Ralf Rabus 1 & Heinz Wilkes 1
Received: 10 June 2020 / Revised: 29 July 2020 / Accepted: 13 August 2020 # The Author(s) 2020
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. Keywords Calibration . Recovery . Stability . Limit of detection . Limit of quantification . Statistics
Introduction 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]. In the last years, growing interest has shed light on this emerging field especially in biotechnology and clinical research [2, 3]. With an increasing number of publications relating to metabolomics, more analytical approaches have advanced, including gas or liquid chromatography coupled to mass spectrometry (GC-MS, LC-MS) and Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00216-020-02883-3) contains supplementary material, which is available to authorized users. * Heinz Wilkes [email protected] 1
Institute for Chemistry and Biology of the Marine Environment (ICBM), Carl von Ossietzky University of Oldenburg, P.O. Box 2503, 26111 Oldenburg, Germany
nuclear magnetic resonance (NMR) spectroscopy [4, 5]. Besides an overall examination of the metabolome [6], two separate fields have developed—the analysis of the metabolic composition outside of the cell, also called the exometabolome or metabolic footprinting [7] and the anal
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