Abstract
<p>This protocol describes a sequential liquid–liquid partitioning procedure for the extraction and GC–MS profiling of metabolites from plant leaf tissues. Frozen leaf material is ground under liquid nitrogen and extracted with methanol:water, 80:20, v/v. Following centrifugation, methanol is partially removed under vacuum while preserving an aqueous residue. The concentrated extract is sequentially partitioned, first with chloroform and subsequently with ethyl acetate. The lower chloroform phase and the upper ethyl acetate phase are collected separately and completely dried under vacuum. Each fraction is independently derivatized by methoximation with methoxyamine hydrochloride in anhydrous pyridine, followed by trimethylsilylation with N-methyl-N-(trimethylsilyl)trifluoroacetamide, MSTFA. The procedure was successfully applied to plant leaf samples. Both organic fractions generated suitable chromatographic profiles following analysis using a Thermo Scientific‱ Orbitrap‱ Exploris‱ GC 240 Mass Spectrometer equipped with an Agilent J&W DB-35ms capillary GC column. Although the original experimental application was conducted using a high-resolution Orbitrap GC–MS platform, the extraction, partitioning, drying, and derivatization procedures may be combined with other GC–MS or GC–HRMS systems. Instrumental conditions should be optimized according to the available instrument, analytical column, plant matrix, and objectives of each study.</p>