Abstract: A method for the determination of urinary trans, trans-muconic acid (tt-MA)（benzene metabolite） by high performance liquid chromatography （HPLC） was developed. The separation was carried out on a C18 column (Spherisorb C18, 150 mm×4.6 mm i.d., 5 μm) at 25 ℃ with glacial acetic acid-tetrahydrofuran-methanol-water (1∶2∶10∶87, v/v) as mobile phase. Urinary sample was acidified by 2 mol/L hydrochloric acid and pretreated by liquid-liquid extraction using diethyl ether. After removal of diethyl ether with a stream of nitrogen, the residue was re-dissolved in 1 mL of mobile phase for HPLC injection. Good linearity was observed within the range from 0.10 mg/L to 10.00 mg/L (r＝0.9999) and the detection limit was 0.10 mg/L. The average recoveries for tt-MA were 95.1%-100.5%. Relative standard deviations (RSD) for intra-day and inter-day determinations were 4.0%-9.0% and 6.2%-8.8%, respectively. The method was applied to 56 benzene-exposed workers and 24 controll workers. Urinary tt-MA in benzen-exposed workers were significantly higher than that in the control group. They can be correlated with benzene exposure concentrations (P<0.01). This analytical method for tt-MA is sensitive, rapid, and convenient. It is suitable for monitoring of occupational exposure to benzene and toxic kinetics studies.

Key words: benzene metabolite, trans, trans-muconic acid, urine , high performance liquid chromatography （HPLC）