Extraction/derivation procedures for the determination of urinary acid metabolites of aromatic hydrocarbons by gas chromatography.

Aromatic hydrocarbons (toluene, styrene, xylenes, ethylbenzene …) are among the most widely used chemicals in industry. Some of their urinary acid metabolites are classically used to assess the occupational exposure of workers to these solvents; for example hippuric acid for toluene, methylhippuric acids for xylene and mandelic and phenylglyoxylic acids for ethylbenzene and styrene. Although minor metabolites, the mercapturic acids due to their specificity have also been proposed as candidate biomarkers for human biomonitoring studies; for example benzyl- and p-toluyl-mercapturic acids for toluene, dimethylphenylmercapturic acids for xylenes and phenylhydroxyethylmercapturic acids for styrene.
We therefore sought to develop rapid, simple, and sensitive method(s) to detect these metabolites in rat urine.
First, a convenient and reliable gas chromatographic method was developed for the simultaneous determination of six aromatic acid metabolites of styrene and styrene-oxide in rat urine; i.e., benzoic , phenylacetic, mandelic, phenylglyoxylic, hippuric and phenylaceturic acids. The method involved a one pot esterification-extraction procedure, performed directly on urine without prior treatment. Analyses were performed by gas chromatography on a RTX-1701 capillary column and the recovered isopropyl esters derivatives were detected by flame ionization detection.
This method has subsequently been adapted and improved (using microwave assisted esterification) for the determination by gas chromatography– tandem mass spectrometry (GC-MS/MS) of urinary toluene mercapturic acids derived from side chain and ring oxidation, i.e., benzylmercapturic acid and the three isomers o-, m- and p-toluylmercapturic acids.
The method was successfully applied to quantitatively analyze styrene, styrene-oxide, ethylbenzene and toluene metabolites in urine samples from rats exposed by inhalation to these compounds at levels close to the occupational threshold limit values. It could have applications as part of biological monitoring for workers exposed to styrene or related compounds. From a more fundamental point of view, the optimized method allowed the identification of metabolites never seen before.