Isomeric Differentiation and Acidic Metabolite Identification by Piperidine-Based Tagging, LC–MS/MS, and Understanding of the Dissociation Chemistries

We demonstrate a method for facile differentiation of acidic, isomeric metabolites by attaching high proton affinity, piperidine-based chemical tags to each carboxylic acid group. These tags attach with high efficiency to the analytes, increase the signal, and result in the formation of multiply-charged cations. We illustrate the present approach with citrate and isocitrate, which are isomeric metabolites each containing three carboxylic acid groups. We observe a 20-fold increase in signal-to-noise for citrate and an 8-fold increase for isocitrate as compared to detection of the untagged analytes in negative mode. Collision-induced dissociation of the triply tagged, triply charged analytes results in distinct tandem mass spectra. The phenylene spacer groups limit proton mobility and enable access to structurally informative C–C bond cleavage reactions. Modeling of the gas-phase structures and dissociation chemistry of these triply charged analyte ions highlights the importance of hydroxyl proton mobilization in this low proton mobility environment. Tandem mass spectrometric analyses of deuterated congeners and MS 3 spectra are consistent with the proposed fragment ion structures and mechanisms of formation. Direct evidence that these chemistries are more generally applicable is provided by subsequent analyses of doubly tagged, doubly charged malate ions. Future work will focus on applying these methods to identify new metabolites and development of general rules for structural determination of tagged metabolites with multiple charges.