TY - UNPB
T1 - An optimised monophasic faecal extraction method for LC-MS analysis and its application in gastrointestinal disease
AU - Kelly, Patricia E.
AU - Ng, H Jene
AU - Farrell, Gillian
AU - McKirdy, Shona
AU - Russell, Richard K.
AU - Hansen, Richard
AU - Rattray, Zahra
AU - Gerasimidis, Konstantinos
AU - Rattray, Nicholas J.W.
N1 - The copyright holder for this preprint is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
This study was supported by Shimadzu UK and the University of Strathclyde through joint contribution to P.E.K’s studentship. The clinical studies were funded by the Glasgow Children Hospital Charity and the Nutricia Research Foundation. ZR would like to acknowledge the EPSRC Multiscale Metrology Suite (EP/V028960/1).
PY - 2022/9/21
Y1 - 2022/9/21
N2 - 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, a standardised method for extracting metabolites from faecal samples for large-scale metabolomic studies is yet to be defined. 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.
AB - 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, a standardised method for extracting metabolites from faecal samples for large-scale metabolomic studies is yet to be defined. 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.
KW - mass spectrometry
KW - metabolite extraction
KW - inflammatory bowel disease
KW - Crohn’s disease
KW - coeliac disease
U2 - 10.1101/2022.09.19.22280109
DO - 10.1101/2022.09.19.22280109
M3 - Preprint
BT - An optimised monophasic faecal extraction method for LC-MS analysis and its application in gastrointestinal disease
PB - medRxiv
CY - Cold Spring Harbor Laboratory
ER -