Screening of leaf extraction and storage conditions for eco-metabolomics studies

Mass spectrometry-based plant metabolomics is frequently used to identify novel natural products or study the effect of specific treatments on a plant’s metabolism. Reliable sample handling is required to avoid artifacts, which is why most protocols mandate shock freezing of plant tissue in liquid nitrogen and an uninterrupted cooling chain. However, the logistical challenges of this approach make it infeasible for many ecological studies. Especially for research in the tropics, permanent cooling poses a challenge, which is why many of those studies use dried leaf tissue instead. We screened a total of ten extraction and storage approaches for plant metabolites extracted from maize leaf tissue across two cropping seasons to develop a methodology for agroecological studies in logistically challenging tropical locations. All methods were evaluated based on changes in the metabolite profile across a 2-month storage period at different temperatures with the goal of reproducing the metabolite profile of the living plant as closely as possible. We show that our newly developed on-site liquid-liquid extraction protocol provides a good compromise between sample replicability, extraction efficiency, material logistics, and metabolite profile stability. We further discuss alternative methods which showed promising results and feasibility of on-site sample handling for field studies. Highlight We developed an on-site metabolite extraction method for leaf tissue samples from field studies in challenging logistical circumstances. We highlight extract stability and reproducibility compared to frozen or dried tissue. ### Competing Interest Statement The authors have declared no competing interest. * CE : Crude Extract (homogenisation at room temperature) CSA : Camphorsulphonic acid DCM : Dichloromethane FE : Frozen Extract (homogenisation using liquid nitrogen to freeze the leaf) LLE : Liquid-liquid extraction MeCN : Acetonitrile MeOH : Methanol OA : Oasis HLB branded SPE cartridges PCA : Principal component analysis SPE : Solid-phase extraction