High performance liquid chromatography for the quantitative determination of the urinary methabolities of toluene, xylene, and styrene.

A new high-pressure liquid chromatography (HPLC) method for simultaneous quantitative determination of the urinary metabolites of toluene, m-xylene, and styrene (hippuric acid, m-methylhippuric acid, phenylglyoxylic acid, mandelic acid) is described. The extraction procedure was performed on acidified urines, after addition of 4-hydroxybenzoic acid as internal standard, using a butylchloride/isopropanol mixture and drying 0.5 ml of the organic layer under nitrogen flow. The residue obtained was dissolved in 0.1 ml water/acetonitrile and 5 microliters were injected into an HPLC apparatus equipped with a 0.26 X 25 cm HC ODS SIL X column. Absorbance measures were performed at 225 nm throughout the investigation. All metabolites were clearly separated in a short time (12 min) and the amounts of other urinary compounds affecting the analysis were so small that the measurement of low concentrations of the urinary metabolites could be easily performed. Linear calibration curves were obtained from 0.1 to 3 mg/ml and a correlation coefficient greater than 0.99 was found between concentrations of the standards and areas of the peaks. Statistical analysis confirms that this method, which has a high reproducibility, is simple, reliable, and useful for the biologic monitoring of industrial exposure to aromatic compounds