Hydrogen Radical Removal Causes Complex Overlapping Isotope Patterns of Aromatic Carboxylic Acids in Negative-ion Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

We studied the ionization process of aromatic carboxylic acids, including ones with or without hydroxy groups in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), because many natural products, metabolites, and drags contain those structural units. In the actual experimental data, benzoic acid was ionized as only deprotonated molecule [M−H]−. In contrast, both of negative molecular ion M− and deprotonated molecule [M−H]− were generated from 2-naphthoic acid and 2-anthracenecarboxylic acid, and the ratio of negative molecular ion to deprotonated molecule M−/[M−H]− was increased in 2-anthracenecarboxylic acid. In addition, the ratio of 2-anthracenecarboxylic acid was much higher than those of 1- and 9-anthracenecarboxylic acids among the three isomers. Therefore, 2-substitution induced the generation of the negative molecular ion M−, which can made us prediction of the substituted positions from their overlapping peak isotope patterns. 2,5-Dihydroxybenzoic acid (2,5-DHBA) showed two deprotonated molecules, [M−H]− and [M−H*−H]−, which was generated from a neutral hydrogen radical (H*) removal from a phenolic hydroxy group. The deprotonated molecule [M−H*−H]− of 2,5-DHBA was the most abundant among six DHBAs and three hydroxybenzoic acids (hBAs). This observation raises the possibility that such a property of 2,5-DHBA could be a clue to explain its highest efficiency as a MALDI matrix. The order of the hydrogen radical removal from the phenolic hydroxy groups was the 3-<4-≪5-positions in the DHBAs, and the 3-<4-positions in hBAs. We can distinguish among six DHBA isomers and three hBA isomers from their spectral pattern around the deprotonated molecules [M−H*−H]− and [M−H]−. The intra-molecular hydrogen bonding between 1-carboxy and 2-hydroxy groups was an important factor in hydrogen radical removal in the hydroxylbenzoic acids and dihydroxybenzoic acids.