Lemna minor studies under various storage periods using extended-polarity extraction and metabolite non-target screening analysis

Plant metabolomic studies cover a broad band of compounds, including various functional groups with different polarities and other physiochemical properties. For this reason, specific optimized methods are needed in order to enable efficient and non-destructive extraction of molecules over a large range of LogD values. This study presents a simple and efficient extraction procedure for Lemna minor samples demonstrating polarity extension of the molecular range. The Lemna samples chosen were kept under the following storage conditions: 1) fresh, 2) stored for a few days at −80 °C, and 3) stored for 6 months at −80 °C. The samples were extracted using five specifically chosen solvents: 100 % ethanol, 100 % methanol (MeOH), acidic 90 % MeOH (MeOH-water-formic acid (FAC) (90:9.5:0.5, v/v/v), MeOH-water (50:50, v/v), and 100 % water. The final extraction procedure was conducted subject to three solvent conditions, and the subsequent polarity-extended analysis was applied for Lemna minor samples using RPLC-HILIC-ESI-TOF-MS. The extraction yield is in descending order (acidic 90 % MeOH), 50 % MeOH, 100 % water and 100 % MeOH. The results displayed significant molecular differences, both in the extracts investigated and in the fresh Lemna samples, compared to stored samples, in terms of the extraction yield and reducing contents as well as the number of features. The storage of Lemna minor resulted in changes to the fingerprint of its metabolites as the reducing contents increased. The comparisons enable a direct view of molecule characterizations, in terms of their polarity, molecular mass, and signal intensity. This parametric information would appear ideal for further statistical data analysis. Consequently, the extraction procedure and the analysis/data evaluation are highly suitable for the so-called extended-polarity non-target screening procedure.