The annotation of metabolites detected in LC-MS-based untargeted metabolomics studies routinely applies accurate m/z of the intact metabolite (MS1) as well as chromatographic retention time and MS/MS data. Electrospray ionization and transfer of ions through the mass spectrometer can result in the generation of multiple "features" derived from the same metabolite with different m/z values but the same retention time. The complexity of the different charged and neutral adducts, in-source fragments, and charge states has not been previously and deeply characterized. In this paper, we report the first large-scale characterization using publicly available data sets derived from different research groups, instrument manufacturers, LC assays, sample types, and ion modes. 271 m/z differences relating to different metabolite feature pairs were reported, and 209 were annotated. The results show a wide range of different features being observed with only a core 32 m/z differences reported in >50% of the data sets investigated. There were no patterns reporting specific m/z differences that were observed in relation to ion mode, instrument manufacturer, LC assay type, and mammalian sample type, although some m/z differences were related to study group (mammal, microbe, plant) and mobile phase composition. The results provide the metabolomics community with recommendations of adducts, in-source fragments, and charge states to apply in metabolite annotation workflows.

Centre for Metabolomics Research Department of Biochemistry Cell and Systems Biology Institute of Systems Molecular and Integrative Biology University of Liverpool Liverpool L69 7ZB United Kingdom

Institute of Molecular and Translational Medicine Palacký University Olomouc Olomouc 779 00 Czech Republic

School of Biosciences University of Birmingham Birmingham West Midlands B15 2TT United Kingdom

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