In-Source CID Ramping and Covariant Ion Analysis of Hydrophilic Interaction Chromatography 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 predefined adduct lists that are generated empirically from reversed-phase LC-MS studies. In addition, hydrophilic interaction chromatography (HILIC) is gaining popularity in metabolomics studies due to its enhanced performance over other methods for polar compounds. HILIC methods typically use high concentrations of buffer salts to improve chromatographic performance. Therefore, it is necessary to analyze adduct formation in HILIC metabolomics. To this end, we developed covariant ion analysis (COVINA) to investigate metabolite adduct formation. Using this tool, we completely annotated 201 adduct and fragment ions from 10 metabolites. Many of the metabolite adduct ions were found to contain cluster ions corresponding to mobile phase additives. We further utilized COVINA to find the major ionized forms of metabolites. Our results show that for some metabolites, the adduct ion signals can be >200-fold higher than the signals from the deprotonated form, offering better sensitivity for targeted metabolomics analysis. Finally, we developed an in-source CID ramping (InCIDR) method to analyze the intensity changes of the adduct and fragment ions from metabolites. Our analysis demonstrates a promising method to distinguish the protonated and deprotonated ions of metabolites from the adduct and fragment ions.