Development and application of a HILIC UHPLC-MS method for polar fecal metabolome profiling

The fecal metabolome is a complex mixture of endogenous, microbial metabolites, and food derived compounds. Hydrophilic interaction liquid chromatography (HILIC) enables the analysis of polar compounds, which is a valuable alternative to reversed-phase liquid chromatography in the field of metabolomics due to its ability to retain a greater portion of the polar metabolome. The objective of the study was to find the optimal chromatographic solution to perform non-targeted metabolomics of feces by means of HILIC ultra-high-pressure liquid chromatography mass spectrometry (UHPLC-Q-TOF-MS). The performance was systematically investigated analyzing a pooled fecal sample, and mixtures of 150 metabolites from different families, including for example amino acids, amines, indole derivatives, fatty acids and carbohydrates. Three different stationary phases (zwitterionic, amide and unbonded silica) were operated at three pH values (4.6, 6.8 and 9.0), and three salt gradient conditions (5-5, 5-10 and 5-25 mM ammonium acetate). Amide and zwitterionic stationary phases performed similarly at low pH, with highest number of detected standards, which increased by increasing the salt gradient. The amide column showed slightly better performance in terms of separation of isomers and peak widths and remarkably good performance at basic pH, with highest number of metabolite features in the non-targeted analysis. The zwitterionic column operated best in terms of number of detected standards, retention time distribution of standards and metabolite feature across whole chromatographic run. Thus, the zwitterionic column was proven to suit for non-targeted analysis of fecal samples, resulting in good coverage of especially amino acids and carbohydrates.