A mechanistic study of fragmentation of deprotonated N,2-diphenyl-acetamides in electrospray ionization tandem mass spectrometry.

RATIONALE

Exploring the fragmentation mechanism of amide ions in mass spectrometry has attracted great interest because of the desire to analyze the amino acid sequences of peptides and proteins. However, the collision-induced dissociation (CID) mechanism of deprotonated small amides has been rarely studied in electrospray ionization mass spectrometry (ESI-MS). The fragmentation of deprotonated N,2-diphenylacetamides exhibited some characteristic fragment ions, which are not derived from the conventional cleavage route. Therefore, clarification of their fragmentation mechanism is very important and useful for structural analysis of related amides and peptides.

METHODS

All CID experiments were carried out using an electrospray ionization ion trap mass spectrometer in negative ion mode. In addition, the accurate masses of fragments were measured on an ESI quadrupole time-of-flight (Q-TOF) mass spectrometer in negative ion mode. Deuterium-labeled 2-phenyl-N-(4-trifluoromethylphenyl)acetamide was synthesized and its ESI fragmentation spectrum had been obtained. Theoretical calculations were carried out by the density functional theory (DFT) method at the B3LYP level of theory with the 6-31G++(d,p) basis set.

RESULTS

Deprotonated N,2-diphenylacetamides mainly generate four kinds of ions in CID: benzyl anion, aniline anion, phenyl-ethenone anion and isocyanato-benzene anion bearing respective substituent groups. The benzyl anion and the aniline anion can be generated by direct decomposition. The phenyl-ethenone anion and the isocyanato-benzene anion were proposed to be yielded from proton transfer within an ion-neutral complex, and the intensities of two competitive product ions are well correlated with the substituent constants. The mechanism was also supported by theoretical calculations.

CONCLUSIONS

The characteristic fragment ions of deprotonated N,2-diphenylacetamides were proposed to be produced via an ion-neutral complex mechanism, which was proved by deuterium-labeling experiments, theoretical calculations and substituent effects.