The Sampling and Sample Preparation Problem in Microbial Metabolomics

The reproducibility and accuracy of analysis methods has expanded rapidly over the past years. Hyphenated methods, for example, GC-MS and LC-MS/MS (liquid chromatography-mass spectrometry), are now almost routinely used for (semi-) quantitative analysis of tens to hundreds of metabolites in biological samples. It should be realized, however, that the quality of the applied analytical techniques alone is not sufficient to obtainmeaningful results. Other important aspects, which are often overlooked, are the methods that are applied to withdraw the samples from a microbial cultivation and the subsequent sample processing procedures. Immediately after withdrawal of the samples, the metabolism should be arrested to preserve the metabolic snapshot represented by the sample by preventing further metabolic conversions. Subsequently, an appropriate extraction method should be applied,which guarantees complete andunbiased release of allmetabolites from the cells without significant degradation and inactivation of enzymatic activity. Finally, an accurate and high throughput analytical platform is required for metabolite quantification. In this chapter, we focus on the essential requirements of the sampling and sample processing procedures as well as the methods to validate if these requirements are met in practice.