A protocol for untargeted metabolomics using anion-exchange chromatography and high-resolution mass spectrometry.

Polar metabolites are challenging to analyze using common reverse-phase chromatography. However, these molecules are most essential for deciphering biological phenotypes. Here, we present a protocol for polar metabolite analysis based on anion-exchange chromatography coupled with high-resolution mass spectrometry (AEC-HRMS). We outline preparation techniques for common biological matrices, instrumental setup for untargeted metabolomics, including sample analysis and essential data treatment.