Assessment of Biphasic Extraction Methods of Mouse Fecal Metabolites for Liquid Chromatography–Mass Spectrometry-Based Metabolomic Studies

With the increasing knowledge about the important roles of gut microbiota on the biological system, a systematic strategy to profile the fecal metabolome is urgently needed. Thus, an unbiased, efficient, and reproducible fecal metabolite extraction protocol needs to be established; however, the effect of biphasic extraction methods for the fecal samples remains unclear. In this study, five different methods were assessed in the extraction of polar and non-polar metabolites for the liquid chromatography–mass spectrometry (LC–MS)-based mouse fecal metabolomic study. First, the detection coverage of two extraction systems, the Bligh and Dyer extraction method (M1, chloroform/methanol/water, 2/2/1.8) and Matyash method (M2, methyl tert-butyl ether (MTBE)/methanol/water, 10/3/2.5), was compared; then, MTBE/methanol/water system with different solvent ratios (M3, 2.6/2.0/2.4; M4, 4.5/1/2.5; and M5, 3/2.5/2.5) were further evaluated. The results showed that M2 showed higher detection coverage than M1. For the MTBE/methanol/water system with different solvent ratios, M3 showed the largest detection coverage based on peak numbers and numbers of putatively annotated metabolites, while M4 presented the least overlap between two phases, higher peak intensities of metabolites, and superior reproducibility. Based on the above evidence, M4 was recommended for the biphasic extraction of fecal metabolites in the LC–MS-based mouse fecal metabolomic study.