Metabolite extraction as a coverage gate in metabolomics.

Metabolomics aims to measure a chemically diverse metabolome, but the observable metabolome is first defined by extraction. Extraction creates a matrix environment in which metabolites can (1) bind residual proteins, precipitated surfaces, membranes, metals, and other sample components, or (2) serve as substrates for residual catalytic activity. These metabolite-matrix interactions are not fixed; they depend on solvent composition, water content, pH, salt, protein state, and, critically, extraction concentration. As the biomass-to-solvent ratio increases, the concentrations of metabolites, binding sites, and active residual enzymes increase together, raising the opportunity for matrix retention and post-extraction conversion. Consequently, broad metabolome coverage requires treating extraction as an analytical selection step, with solvent conditions, matrix effects, biomass-to-solvent ratio, and extraction linearity controlled, validated, and reported as core determinants of metabolite recovery.