Green Extraction Method: Microwave-Assisted Water Extraction Followed by HILIC-HRMS Analysis to Quantify Hydrophilic...

Area of Science:

• Plant Science
• Analytical Chemistry
• Biochemistry

Background:

• Plant cells contain abundant hydrophilic compounds like amino acids, organic acids, and sugars.
• These metabolites significantly influence plant product quality and plant-pathogen interactions.
• Accurate quantification of these compounds is crucial for plant research.

Purpose of the Study:

• To develop and validate a comprehensive analytical workflow for quantifying hydrophilic compounds in plant tissues.
• The workflow combines water-based extraction with rapid hydrophilic interaction liquid chromatography-high-resolution mass spectrometry (HILIC-HRMS).
• The goal is to enable absolute quantification of key polar metabolites.

Main Methods:

• Comparison of water-based microwave-assisted extraction (MAE) methods for hydrophilic compounds.
• Utilized HILIC-HRMS for fast separation and detection.
• Developed a method with a 20-second MAE and a 10-minute HILIC-HRMS analysis.
• Validated the bioanalytical method for 24 polar metabolites, assessing selectivity, LOD, LOQ, calibration range, and precision.

Main Results:

• A rapid method was established, involving 20s MAE and 10 min HILIC-HRMS analysis.
• Quantification limits ranged from 0.10 µM to 4.50 µM, depending on the metabolite.
• Between-run relative standard deviations (RSDs) were below 20% for most compounds in *Vitis vinifera* and *Arabidopsis* samples.
• The method demonstrated effectiveness in both lignified (*V. vinifera*) and non-lignified (*Arabidopsis*) plant tissues.

Conclusions:

• A water-based MAE method coupled with HILIC-HRMS was successfully developed for absolute quantification of free amino acids, organic acids, and sugars in plant tissues.
• The method is effective for diverse plant types, including woody and herbaceous species.
• This approach is suitable for medium- to high-throughput analysis of essential polar metabolites from small plant sample volumes.