Proposal for field sampling of plants and processing in the lab for environmental metabolic fingerprinting

BACKGROUND: Samples for plant metabolic fingerprinting are prepared generally by metabolism quenching, grinding of plant material and extraction of metabolites in solvents. Further concentration and derivatisation steps follow in dependence of the sample nature and the available analytical platform. For plant material sampled in the field, several methods are not applicable, such as, e.g., collection in liquid nitrogen. Therefore, a protocol was established for sample pre-treatment, grinding, extraction and storage, which can be used for analysis of field-collected plant material, which is further processed in the laboratory. Ribwort plantain (Plantago lanceolata L., Plantaginaceae) was used as model plant. The quality criteria for method suitability were high reproducibility, extraction efficiency and handling comfort of each subsequent processing step. RESULTS: Highest reproducibility of results was achieved by sampling fresh plant material in a solvent mixture of methanol:dichloromethane (2:1), crushing the tissue with a hand-held disperser and storing the material until further processing. In the laboratory the material was extracted threefold at different pH. The gained extracts were separated with water (2:1:1 methanol:dichloromethane:water) and the aqueous phases used for analysis by LC-MS, because the polar metabolites were in focus. Chromatograms were compared by calculating a value Ξ for similarities. Advantages and disadvantages of different sample pre-treatment methods, use of solvents and solvent mixtures, influence of pH, extraction frequency and duration, and storing temperature are discussed with regard to the quality criteria. CONCLUSIONS: The proposed extraction protocol leads to highly reproducible metabolic fingerprints and allows optimal handling of field-collected plant material and further processing in the laboratory, which is demonstrated for an exemplary field data-set. Calculation of Ξ values is a useful tool to judge similarities between chromatograms.