An optimised monophasic faecal extraction method for LC-MS analysis and its application in gastrointestinal disease

Liquid chromatography coupled with mass spectrometry (LC-MS) metabolomic approaches are widely used to investigate underlying pathogenesis of gastrointestinal disease and mechanism of action of treatments. However, there is an unmet requirement to assess faecal metabolite extraction methods for large-scale metabolomics studies. Current methods often rely on biphasic extractions using harmful halogenated solvents, making automation and large-scale studies challenging. The present study reports an optimised monophasic faecal extraction protocol that is suitable for untargeted and targeted LC-MS analyses. The impact of several experimental parameters, including sample weight, extraction solvent, cellular disruption method, and sample-to-solvent ratio, were investigated. It is suggested that a 50 mg freeze-dried faecal sample should be used in a methanol extraction (1:20) using bead beating as the means of cell disruption. This is revealed by a significant increase in number of metabolites detected, improved signal intensity, and wide metabolic coverage given by each of the above extraction parameters. Finally, we addressed the applicability of the method on faecal samples from patients with Crohn’s disease (CD) and coeliac disease (CoD), two distinct chronic gastrointestinal diseases involving metabolic perturbations. Untargeted and targeted metabolomic analysis demonstrated the ability of the developed method to detect and stratify metabolites extracted from patient groups and healthy controls (HC), highlighting characteristic changes in the faecal metabolome according to disease. The method developed is, therefore, suitable for the analysis of patients with gastrointestinal disease and can be used to detect and distinguish differences in the metabolomes of CD, CoD, and HC.