INVESTIGATIONS IN THE DETERMINATION AND METABOLISM OF AROMATIC AMINES BY LIQUID CHROMATOGRAPHY AND ELECTROCHEMISTRY (ANILINE, PEROXIDASES, N-OXIDIZED COMPOUNDS)
Abstract
Liquid chromatography/electrochemistry (LCEC) is a useful analytical method for studying the metabolism of xenobiotics. LCEC offers efficient separation and low detection limits, typically in the sub-picomole range. In this laboratory LCEC has been applied to metabolism studies of the xenobiotics benzidine, acetaminophen and their metabolites. This work describes further applications of LCEC to the understanding of foreign compound metabolism, focusing on the biotransformations of primary N-arylamines. The mechanism by which aromatic amines form toxic reactive intermediates in vivo has not yet been fully elucidated. Electroanalytical studies of several primary arylamines including aniline, naphtylamines and aminobiphenyls illustrate the electrochemical oxidations these species undergo. The relationship between the electrochemical oxidation and the mechanism of arylamine metabolism is discussed. Hydroxyl radicals may be formed in a xanthine oxidase/hypoxanthine system, where the superoxide anion radical O(,2) and H(,2)O(,2) are produced. Aniline and phenol are excellent hydroxyl radical scavengers in this system. LCEC is used to determine hydroxylated products with little sample preparation. The effects of additional reagents and scavengers on the hydroxylation of aniline is presented. Methods were developed to study mouse liver microsomal metabolism of aniline, 4-nitroaniline and the azo dye Disperse Orange 3. Metabolites, including the labile hydroxylamines were directly determined. The dual-electrode detector in the parallel-adjacent configuration is used to confirm chromatographic peak identity. The series-mode of this detector permits determination of 4-nitroaniline in the presence of other metabolites without oxygen interference. Detection limits for these compounds are in the sub-picomole range. The cooxidation of aniline by the enzymes, horseradish peroxidase and prostaglandin H synthase was investigated using LCEC. Products resulting from aniline oxidation, presumably first forming a cation-radical, were determined. The initial oxidation product was found to preferentially interact with nucleophiles, rather than form coupling products such as N-phenyl-p-phenylenediamine. This may implicate the one electron oxidation product of aromatic amines as a damaging reactive intermediate.
Degree
Ph.D.
Subject Area
Analytical chemistry
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