Comprehensive analysis of pig feces metabolome by chromatographic techniques coupled to mass spectrometry in high resolution mode: Influence of sample preparation on the identification coverage

Abstract

Pig feces is an interesting biological sample to be implemented in metabolomics experiments by virtue of the information that can be deduced from the interaction between host and microbiome. However, pig fecal samples have received scant attention, especially in untargeted metabolomic studies. In this research, an analytical strategy was planned to maximize the identification coverage of metabolites found in pig fecal samples. For this purpose, two complementary platforms such as LC–QTOF MS/MS and GC–TOF/MS were used. Concerning sample preparation six extractant solvents with different polarity grade were tested to evaluate the extraction performance and, in the particular case of GC–MS, two derivatization protocols were compared. A total number of 303 compounds by combination of all the extractants and analytical platforms were tentatively identified. The main identified families were amino acids, fatty acids and derivatives, carbohydrates and carboxylic acids. For GC–TOF/MS analysis, the recommended extractant is methanol, while methoxymation was required in the derivatization protocol since this step allows detecting the α-keto acids, which are direct markers of the microbiome status. Concerning LC–QTOF MS/MS analysis, a dual extraction approach with methanol (MeOH) or MeOH/water and ethyl acetate is proposed to enhance the detection of polar and non-polar metabolites.