Metabolomics is the comparative analysis of metabolites found in sets of similar biological samples. Since metabolites play vital roles in biological systems, metabolomics can be useful for finding and identifying biomarkers, or for obtaining a better understanding of the effects of drugs or diseases on both known and unexpected biological pathways. Successful metabolomic research requires effective metabolite extraction. For non-targeted metabolomics, extraction methods need to capture a broad range of cellular and biofluid metabolites, while excluding components such as proteins that are not intended for analysis. Extraction is made more challenging by the physico-chemical diversity of metabolites and by metabolite abundances that can vary by many orders of magnitude. Biphasic, liquid-liquid extraction is often used to extract metabolites. The nature of the organic and aqueous solvents, their volumes, solvent ratios, and aqueous solvent pH, however, must be considered carefully. They can significantly affect the total number of metabolites extracted and experimental reproducibility. This application note describes a method for liquid-liquid extraction of metabolites from erythrocytes. It demonstrates the importance of adjusting the aqueous/organic ratio to favor biphasic extraction.
Metabolomics research requires effective metabolite extraction, often using biphasic liquid-liquid extraction with organic and aqueous solvents. This document details the challenges posed by the physico-chemical diversity of metabolites and the importance of adjusting solvent ratios and aqueous-phase pH to capture a broad range of cellular components.
The method uses a mixture of methanol, water, and chloroform to form a two-phase system.
Varying the pH allows for the extraction of different classes of metabolites, such as organic acids or bases, that might otherwise remain in one phase.
The erythrocyte samples should be centrifuged at 1000 g for 2 minutes.