In-Source CID Ramping (InCIDR) and Co-Variant Ion Analysis of Hydrophilic Interaction Chromatography (HILIC) Metabolomics

A large proportion of the complexity and redundancy of LC-MS metabolomics data comes from adduct formation. To reduce such redundancy, many tools have been developed to recognize and annotate adduct ions. These tools rely on pre-defined adduct lists which are learned empirically from reverse phase LC-MS studies. Meanwhile, hydrophilic interaction chromatography (HILIC) is gaining popularity in metabolomics studies due to better performance on polar compounds. HILIC methods typically use high concentration of buffer salts for improved chromatography performance. It is therefore necessary to analyze the adduct formation in HILIC metabolomics. To this end, we developed co-variant ion analysis (COVINA) to investigate the metabolite adduct formation. Using this tool, we completely annotated 201 adduct and fragment ions of 10 metabolites. Many of the metabolite adduct ions are found to contain cluster ions of mobile phase additives. We further utilized COVINA to find the major ionization forms of metabolites. Our results show that for some metabolites the adduct ion signals can be >200-fold higher than the deprotonated form, offering better sensitivity for targeted metabolomics analysis. Finally, we developed the in-source CID ramping (InCIDR) method to analyze the intensity changes of the adduct and fragment ions of the metabolites. Our analysis demonstrates a promising method to distinguish the protonated/deprotonated ions of the metabolites from the adduct and fragment ions.