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.