High performance liquid chromatography for determination of urinary metabolites of toluene, xylene and styrene and its application.

The purpose of this research was to determine mandelic, phenylglyoxylic, hippuric, o-, m- and p-methylhippuric acids, the six urinary metabolites of styrene, toluene and xylene by high performance liquid chromatography (HPLC). These metabolites were extracted in an acid medium, transferred into a basic solution and back extracted again using ethyl acetate, and the organic phase was evaporated to dryness under a compressed air flow at room temperature. The residue obtained was dissolved in 1 ml mobile phase solution of 0.01 M potassium orthophosphate in 0.3% acetic acid (adjusted to a pH of 2.5 with orthophosphoric acid):tetrahydrofuran:acetonitrile(87:5:8) and 100 microl was injected into a HPLC equipped with a 4.6 x 250 mm ODS3-C18 reversed phased column and ultraviolet (UV) detector at a wavelength of 254 nm. All metabolites were clearly separated within 21 minutes. The detection limits of the method were 1.1 ng/ml for PGA, 4.9 ng/ml for HA, 17.0 ng/ml for MA, 2.5 ng/ml for o-MHA, 1.7 ng/ml for p-MHA and 2.0 ng/ml for m-MHA. The percent recoveries of the six metabolites were 99.2-101.8% with percent coefficients of variation of less than 2%. The method was applied to the analysis of urine samples of twelve workers exposed to toluene, xylene and styrene in a paint factory. The 5-day post-shift urinary excretions of the six metabolites in these workers are presented. The metabolites were found at levels greater than the Biological Exposure Index (BEI) recommended by the American Conference of Governmental Industrial Hygienists (ACGIH).

Simultaneous determination of trans, transmuconic acid and s-phenylmercapturic acid by high pressure liquid chromatography and its application.

The simultaneous determination of urinary trans,trans-muconic acid (t,t-MA) and S-phenylmercapturic acid (S-PMA) was performed by liquid extraction with ethyl acetate and reversed-phase high performance liquid chromatography (RP-HPLC) on a Hypersil-ODS column using the gradient mobile phase of methanol and 0.0012 N perchloric acid and diode array detection at 205 and 264 nm for S-PMA and t,t-MA, respectively. The retention times for t,t-MA and S-PMA were 3.8 and 12.3 minutes, respectively. The recoveries of t,t-MA and S-PMA were > 97%; between-day precisions were all within 8% RSD (100x SD/mean). The method was applied to analyze the urinary t,t-MA and S-PMA of 59 service station attendants exposed to average benzene concentrations in the air of 0.20+/-0.18 ppm. Significant differences in pre-shift and post-shift urinary t,t-MA between smokers and non-smokers were found.