RESUMEN
Polyphenolic compounds play an essential role in plant growth, reproduction, and defense mechanisms against pathogens and environmental stresses. Extracting these compounds is the initial step in assessing phytochemical changes, where the choice of extraction method significantly influences the extracted analytes. However, due to environmental factors, analyzing numerous samples is necessary for statistically significant results, often leading to the use of harmful organic solvents for extraction. Therefore, in this study, a novel DES-based shaking-assisted extraction procedure for the separation of polyphenolic compounds from plant samples followed by LC-ESI-QTOF-MS analysis was developed. The DES was prepared from choline chloride (ChCl) as the hydrogen bond acceptor (HBA) and fructose (Fru) as the hydrogen bond donor (HBD) at various molar ratios with the addition of 30% water to reduce viscosity. Several experimental variables affecting extraction efficiency were studied and optimized using one-variable-at-a-time (OVAT) and confirmed by response surface design (RS). Nearly the same experimental conditions were obtained using both optimization methods and were set as follows: 30 mg of sample, 300 mg of ChCl:Fru 1:2 DES containing 30% w/w of water, 500 rpm shaking speed, 30 min extraction time, 10°C extraction temperature. The results were compared with those obtained using conventional solvents, such as ethanol, methanol and water, whereby the DES-based shaking-assisted extraction method showed a higher efficiency than the classical procedures. The greenness of the developed method was compared with the greenness of existing procedures for the extraction of polyphenolic substances from solid plant samples using the complementary green analytical procedure index (ComplexGAPI) approach, while the results for the developed method were better or comparable to the existing ones. In addition, the practicability of the developed procedure was evaluated by application of the blue applicability grade index (BAGI) metric. The developed procedure was applied to the determination of spruce root samples with satisfactory results and has the potential for use in the analysis of similar plant samples.
RESUMEN
We present concise results of method validation for trehalose quantitation by LC-MS/MS in spruce ectomycorrhizal roots in order to describe spruce health status, mainly in connection to contamination by a pathogenic fungus, Gemmamyces piceae. The procedure is based on Rogatsky et al. (2005) developed for human plasma. We found out that the best extraction yield was achieved with 80% methanol/water (v/v) solution and optimal extraction temperature was set between 50-60°C. In contrast to previous papers, we minimized the activity of trehalase enzyme by putting root samples into liquid N2 immediately after root excavation, followed by freeze-drying in order to stop trehalase activity. Higher content of trehalose was recorded in healthy trees, confirming the idea that ectomycorrhiza plays a significant role in plant-pathogen interactions.