Two-Phase Extraction for Comprehensive Analysis of the Plant Metabolome by NMR

Metabolomics is the area of research, which strives to obtain complete metabolic fingerprints, to detect differences between them and to provide hypothesis to explain those differences (Schripsema J, Dagnino D, Handbook of chemical and biological plant analytical methods. Wiley, New York, 2015). However, obtaining complete metabolic fingerprints is not an easy task. Metabolite extraction is a key step during this process, and much research has been devoted to finding the best solvent mixture to extract as much metabolites as possible.Here a procedure is described for analysis of both polar and apolar metabolites using a two-phase extraction system. D₂O and CDCl₃ are the solvents of choice, and their major advantage is that, for the identification of the compounds, standard databases can be used because D₂O and CDCl₃ are the solvents most commonly used for pure compound NMR spectra. The procedure enables the absolute quantification of components due to the addition of suitable internal standards. The extracts are also suitable for further analysis with other systems like LC-MS or GC-MS.