Urinary trans,trans-muconic acid determined by liquid chromatography: application in biological monitoring of benzene exposure.

We describe a sensitive and specific high-performance liquid-chromatographic method for determining the benzene metabolite, trans,trans-muconic acid (ttMA) in urine by measuring ultraviolet absorbance at 265 nm. We mix 1 mL of urine sample with 2 mL of Tris buffer containing vanillic acid as internal standard (IS) and percolate this through a preconditioned ion-exchange column. After rinsing the column with phosphoric acid solution, acetate buffer, and deionized water, we elute the analytes with 2 mL of an equivolume solution of 1.5 mol/L sodium chloride and methanol. Of this, 5 microL is injected into the HPLC column. The mobile phase used consists of, per liter, 10 mL of acetic acid, 100 mL of methanol, and the rest 5 mmol/L sodium acetate. The flow rate was started at 1 mL/min and increased to 1.5 mL/min after 6 min. ttMA and IS were detected at 5.2 and 10.2 min, respectively. The lowest detection limit is 125 pg. Analytical recovery and reproducibility generally exceeded 90%. We validated the method with urine samples collected from normal persons and from refinery workers exposed to benzene concentrations < 1 microL/L. The results show that urinary ttMA is a promising biological marker for risk assessment of low-concentration benzene exposure.