EVALUATION OF LIQUID-CHROMATOGRAPHY ION-SPRAY MASS-SPECTROMETRY FOR THE DETERMINATION OF SUBSTITUTED BENZOIC-ACIDS AND THEIR GLYCINE CONJUGATES

Liquid chromatography combined with pneumatically assisted electrospray (ion spray) mass spectrometry was evaluated for the characterization of low-molecular-mass compounds such as a variety of substituted benzoic acids and their glycine conjugates. Each substituted benzoic acid and its glycine conjugate formed with rat liver mitochondria were separated with a conventional C-8 packed column or a semimicro C-8 column using three mobile phases. The positive- and negative-ion mass spectra of the glycine conjugates gave abundant [M + H]+ and [M - H](-) ions, respectively. However, the positive-ion mass spectra of the acids were dominated by [M + H - H2O](+) ions, with the exception of [M + H](+) ions for acids having an amino, an acetylamino or a dimethylamino group. All of the negative-ion mass spectra of acids gave dominant [M - H](-) ions. The negative-ion mass spectra of acids with halogen, nitro and cyano groups showed the ions equivalent to the mass of each functional group. Further, the alkoxyl group gave a characteristic fragmentation representing loss of the alkyl moiety. The abundances of the fragment ions due to the functional groups gave information on the position of the ring substituents in the negative-ion mass spectra. The positive- and negative-ion mass spectra of the glycine conjugates revealed the presence of glycine.