Specific determination of bromobenzene metabolites by high-performance liquid chromatography

A chromatographic method for the quantitative and simultaneous determination of phenolic and mercapturic acid type metabolites of bromobenzene is presented. Acid hydrolysis (1.5 N HCl, 100 degrees C, 10 min) yielded unconjugated bromophenols. After extraction (ethyl acetate, pH 2), evaporation to dryness and dissolution in methanol, samples were analysed by high-performance liquid chromatography (HPLC) with a c18 reverse-phase column and a phosphate buffer (0.1 M,pH 9 + 5 mM tetrabutylammonium dihydrogen phosphate) - acetonitrile gradient with ultraviolet detection (225 nm). Benzylmercapturic acid and o-chlorophenol were used as internal standards. Using this method, it was possible to determine simultaneously o, m- and p-bromophenols together with o-, m- and p-bromophenylmercapturic acids. Additional validation data were obtained by analysis of urine samples from rats treated with bromobenzene. This new method was compared to existing procedures.