Introduction: Consensus in sample preparation for untargeted human fecal metabolomics is lacking.
Objectives: To obtain sample preparation with broad metabolite coverage for high-throughput LC-MS.
Methods: Extraction solvent, solvent ratio and fresh frozen-vs-lyophilized samples were evaluated by metabolite feature quality.
Results: Methanol at 5 mL per g wet feces provided a wide metabolite coverage with optimal balance between signal intensity and saturation for both fresh frozen and lyophilized samples. Lyophilization did not affect SCFA and is recommended because of convenience in normalizing to dry matter.
Conclusion: The suggested sample preparation is simple, efficient and suitable for large-scale human fecal metabolomics.
Keywords: Fecal sample; Freeze-drying; Method optimization; Short-chain fatty acids; Untargeted fecal metabolomics; XCMS.