Determination of phenol in urine by high-performance liquid chromatography with on-line precolumn enzymatic hydrolysis of the conjugates

A precolumn enzyme reactor containing beta-glucosidase immobilized on LC-NH2 packed-material beads was used on-line with HPLC for determining the glucuronide/sulphate metabolites of benzene. After dilution with phosphate buffer (pH 6.8), the urine sample was injected into the HPLC system directly. Subsequently, after hydrolysis of the conjugates, phenol was produced in the enzyme reactor and was separated from other urinary components on a reversed-phase C18 column with fluorescence detection. A switching valve assembly was used to control the passage of the sample and the eluent into the reactor to prevent damage to the enzyme by the elution solvent. Factors affecting the enzymatic hydrolysis were investigated. The proposed method provides a simple and rapid procedure for urinary phenol determination. The calibration graph was linear in the range 0.25-5.0 ppm with a good correlation coefficient (r = 0.999), and in the range 0.05-1.0 ppm with r = 0.981. The detection limit was 10 ppb and the relative standard deviation was less than 2.27%. Application of the method is illustrated by the analysis of a urine sample collected from a gas station worker.