Methodological investigations on the determination of n-hexane metabolites in urine

Male Wistar rats were exposed to 1000 ppm n-hexane, and the excreted urinary metabolites were analyzed by capillary gas chromatography-mass spectrometry (GC-MS). 1-Hexanol, 2-hexanol, 3-hexanol, 2-hexanone, 2,5-hexanedione, 2,5-dimethyltetrahydrofuran, 2,5-dimethyl-2,3-dihydrofuran and gamma-valerolactone were identified by their retention times and their mass spectra. Quantitative gas chromatographic analyses were performed using an FID. Experiments on the hydrolysis of conjugated n-hexane metabolites revealed that enzymatic hydrolysis (in addition to acid hydrolysis) was not required, as treatment with HCl hydrolyzed conjugates sensitive to acid as well as conjugates sensitive to beta-glucuronidase. By incorporating acid hydrolysis only and by using C18-cartridges for sample extraction, a method was developed that allowed the determination of n-hexane metabolites with a sample preparation time of only 45 min. Assay precision was assessed by repeated analyses of the same urine sample. Coefficients of variation for the individual metabolites ranged from between 1.8 and 3.3.