Complete separation of urinary metabolites of xylene in HPLC/DAD using β-cyclodextrin: application for biological monitoring

To determine the biomarkers of exposure to xylene, urinary 2-, 3- and 4-methyl-hippuric acids, a new HPLC/DAD analytical method has been developed, which uses β-cyclodextrin as an additive for elution; its complexing abilities are exploited to achieve complete chromatographic separation of the three isomers. The mobile phase was a 3% aqueous solution of β-cyclodextrin, pH 3, and methanol, 80:20, in isocratic conditions, with a flow rate of 1 mL/min. To optimize quantitative analysis three wavelengths were employed for detection: λ=198 nm, λ=200 nm, and λ=202 nm. SPE was applied for the extraction from urine samples of analytes. Validation parameters show recoveries always above 82%; LOD was set at 1 μg/mL with an LOQ of 3 μg/mL. The linear dynamic range (from 4 to 100 μg/mL) showed excellent correspondence. This method is rapid and inexpensive and can be applied to several samples simultaneously using a manifold for SPE extraction. The analytes were separated completely and could be fully quantified. The method was used for the analysis of urine samples from 54 workers exposed to xylene in hospital laboratories and showed a good applicability while allowing quantification even at low doses.