Metabolomics using anion-exchange chromatography mass spectrometry for the analysis of cells, tissues and biofluids

Journal article

The direct coupling of ion-exchange chromatography with mass spectrometry using electrochemical ion suppression creates a hyphenated technique with selectivity and specificity for the analysis of highly polar and ionic compounds. The technique has enabled new applications in environmental chemistry, food chemistry, forensics, cell biology and, more recently, metabolomics. Robust, reproducible and quantitative methods for the analysis of highly polar and ionic metabolites help meet a longstanding analytical need in metabolomics. Here, we provide step-by-step instructions for both untargeted and semi-targeted metabolite analysis from cell, tissue or biofluid samples by using anion-exchange chromatography–high-resolution tandem mass spectrometry (AEC-MS/MS). The method requires minimal sample preparation and is robust, sensitive and selective. It provides comprehensive coverage of hundreds of metabolites found in primary and secondary metabolic pathways, including glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, purine and pyrimidine metabolism, amino acid degradation and redox metabolism. An inline electrolytic ion suppressor is used to quantitatively neutralize OH− ions in the eluent stream, after chromatographic separation, enabling AEC to be directly coupled with MS. Counter ions are also removed during this process, creating a neutral pH, aqueous eluent with a simplified matrix optimal for negative ion MS analysis. Sample preparation through to data analysis and interpretation is described in the protocol, including a guide to which metabolites and metabolic pathways are suitable for analysis by using AEC-MS/MS.

Authors: Williams, R; Walsby-Tickle, J; Hvinden, IC; Legge, I; Kacerova, T

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Springer Nature
• Journal: Nature Protocols
• Volume: 21
• Issue: 3
• Pages: 924-958
• Publication date: 2025-08-22
• Acceptance date: 2025-05-29
• DOI: 10.1038/s41596-025-01222-z
• EISSN: 1750-2799
• ISSN: 1754-2189
• Pmid: 40847241
• Language: English
• Keywords: mass spectrometry; metabolomics; molecular medicine