Structural identification of compounds containing tertiary amine side chains using ESI-MS3 combined with fragmentation pattern matching to chemical analogues – Benzimidazole derivatives as a case study

• Dissociation processes of benzimidazoles containing tertiary amine side chains were investigated utilizing QTRAP-MS3.
• Characteristic fragmentation pathways were proposed.
• Structural identification of the benzimidazole coreand side chain related ions was demonstrated, based on selectivity enhancement using MS3experiments.
• Confirmation of MS3-based ions was obtained via fragmentation pattern matching with analogues .

The identification of benzimidazoles containing tertiary amine side chains, some of which are drugs and may also be used as drugs of abuse and poisons, is an important analytical challenge. The GC–MS and LC–MS/MS spectra of these compounds exhibit fragments and product ions mostly representing the amine-containing residue, while ions representing the benzimidazole core and side chain are infrequently observed. This lack of information leads to unavoidable ambiguity in the identification process. The fragmentation behavior of benzimidazole derivatives containing tertiary amine side chains was investigated in this study by LC–ESI-MS2 utilizing a quadrupole linear ion trap (QTRAP) mass spectrometer. MS3 experiments were subsequently carried out to enhance the selectivity of the low abundance ions observed in the MS/MS spectrum, establish their common fragmentation patterns and expose product ions indicative of the core structures. A large number of heterolytic and homolytic cleavages, including ions that require two-bond cleavages for formation, were observed only with MS3. Therefore, most product ion structures proposed following ESI-MS3 could not be supported by ESI-QTOF-MS2 exact mass measurements. To overcome this difficulty, the structures and fragmentation pathways proposed based on low resolution MS3 spectra were confirmed by utilizing fragmentation pattern matching to a chemical analogue that had a similar or identical benzimidazole core but lacked the amine moiety. Detailed MS fragmentation pathways were proposed by QTRAP-MS2, MS3 and quadrupole time-of-flight mass spectrometric (QTOF/MS/MS) accurate mass measurements. The fragmentation of the original compound and its analogue were compared, enabling confirmation of the proposed molecular structure and a higher degree of certainty in analyte identification. Characteristic fragmentation pathways of the benzimidazole core included the sequential loss of two HCN groups generated via benzimidazole ring opening. This new analytical approach can be extended to compounds with core groups other than benzimidazole.

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