Validated High Resolution Mass Spectrometry-Based Approach for Metabolomic Fingerprinting of the Human Gut Phenotype

Fecal samples are an obvious choice for metabolomic approaches, since they can be obtained noninvasively and allow one to study the interactions between the gut microbiota and the host. The use of ultrahigh performance liquid chromatography hyphenated to Orbitrap high-resolution mass spectrometry (UHPLC-Orbitrap HRMS) in this field is unique. Hence, this study relied on Orbitrap HRMS to develop and validate a metabolic fingerprinting workflow for human feces and in vitro digestive fluids. After chemometric sample extraction optimization, an aqueous dilution appeared necessary to comply to the dynamic range of the MS. The method was proven “fit-for-purpose” through a validation procedure that monitored endogenous metabolites in quality control samples, which displayed in both matrices an excellent linearity (R2 > 0.990), recoveries ranging from 93% to 105%, and precision with coefficients of variation (CVs) P in vivo and in vitro, offering a window to reveal disease related biomarkers and potential insight into the mechanisms behind pathologies.