Determination of benzene metabolites in urine of mice by solid-phase extraction and high-performance liquid...

A method was developed for quantitative measurement of trans,trans-muconic acid, catechol, hydroquinone and phenol in urine. Hydrolysis of esterified and glucuronized phenolic compounds was effected by specific enzymes. The hydrolysed mixture was purified and separated by solid-phase extraction with an anion exchanger, followed by extraction with diethyl ether. By using a clean-up procedure the natural background from mouse urine could be reduced, so that the detection limit of the metabolites was in the range 3-60 mg/l. Optimization of the chromatographic conditions resulted in a short high-performance liquid chromatography analysis time. Phenol had the longest retention time of about 10 min. The clean-up procedure could also be used for phenylmercapturic acid, an additional benzene metabolite, but for sensitive high-performance liquid chromatographic detection of phenylmercapturic acid other conditions are necessary.