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 electrospra y (ion spray) mass spectrometry was evaluated for the characterization of low-molecular-mass compounds such as a variety of substituted benz oic acids and their glycine conjugates. Each substituted benzoic acid and its glycine conjugate formed with rat liver mitochondria were sepa rated 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 wer e dominated by [M + H - H2O](+) ions, with the exception of [M + H](+) ions for acids having an amino, an acetylamino or a dimethylamino gro up. 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 functi onal group. Further, the alkoxyl group gave a characteristic fragmenta tion representing loss of the alkyl moiety. The abundances of the frag ment ions due to the functional groups gave information on the positio n of the ring substituents in the negative-ion mass spectra. The posit ive- and negative-ion mass spectra of the glycine conjugates revealed the presence of glycine.