Analysis of urinary biomarkers for exposure to alkyl benzenes by isotope dilution gas chromatography-mass spectrometry

A validated GC-MS method for the analysis of urinary metabolites of alkyl benzenes is reported. Metabolites for exposure to toluene, xylene and ethylbenzene were analyzed simultaneously using stable isotope substituted internal standards. The method entailed acidic deconjugation of urine samples followed by extractive alkylation with pentafluorobenzyl bromide as alkylating agent. The resulting pentafluorobenzyl derivatives of ortho-, meta-, para-cresol, mandelic acid (MA), hippuric acid (HA) and ortho-, meta-, para-methylhippuric acid (MHA) were then quantified by SIM. Optimized reaction conditions for the extractive alkylation step are reported. The derivatives were found to be sufficiently stable for overnight batch analysis. The LODs were below 0.1 mu mol/L for the cresols and below 1 mu mol/L for MA and the HAs. Within-batch precision for o-MHA was 7%, for m-MHA 5%, for p-MHA 5.2% and below 5% for the rest of the analytes.