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.