OPTIMIZATION OF SPE METHOD FOR TRACE LEVEL DETERMINATION OF T,T-MUCONIC ACID AS A BIOLOGICAL INDICATOR OF OCCUPATIONAL EXPOSURE TO BENZENE

In this study, parameters affecting on SPE of trance trance muconic acid (t,t-MA), including sample pH, sample concentration, sample volume, sample flow rate, washing solvent, elution solvent, and type of sorbent were evaluated. After sample preparation of muconic acid as urinary metabolite of benzene, it was determined by HPLC-UV. In chromatographic analysis, column was C18 (250 cm × 4.6 mm id, 0.5 µm), UV wave length was 259 nm, mobile phase was H2O/methanol/acetic acid (69:30:1 v/v/v) was run at flow rate of 1 ml/min. Through experimental evaluation, a strong anion exchange silica cartridge (SAX) has been found successful in simplifying sample preparation compare to C8 and C18. There were significant difference between recoverie of muconic acid when different washing solvent, sample volume, and sample flow rate were used (p< 0.001). An optimum recovery was obtained when sample pH was adjusted at seven. Other optimum conditions were: sample flow rate 1 ml/min; washing solvent acetic acid 1%, and acetic acid 10% as elution solvent. In this study, there was no significant difference when different sample concentrations were used (p>0.05). Recovery of spiked urine sample at concentrations of 0.1, 1, 10 µg/ml were more than 95%. The limit of detection of the optimized method was 0.01 µg/ml, showing 20 times less than biological exposure index (BEI). The optimized method was then validated with three different pools of samples at above mentioned concentrations and showed a good reproducibility over six consecutive days as well as six within-days experiments.