[Determination of low levels of urinary trans,trans-muconic acid]

A method is described which allows determination of urinary t,t-muconic acid (MA) by means of HPLC with UV detection even at micrograms/l concentrations. The clean-up procedure of samples involves the use of strong anionic-exchange cartridges (SAX) for solid phase extraction (SPE). In order to improve the reproducibility of the retention time of MA (CV < 1%) and to obtain an adequate separation of MA from interferents, a high performance reverse-phase column (250 x 4.6 [I.D.] mm, 3 microns) is used and a careful control of the temperature (25 degrees C) is made. Also, a column-switching technique is applied to the chromatographic system in order to eliminate the highly retained peaks from the analytical column. The isocratic run is performed at a constant flow rate of 0.7 ml/min; the mobile phase consists of water: methanol: acetic acid (93.5:5.5: 1, v/v) and the UV detector is set at 259 nm. Under these conditions, MA elutes at 21.5 min and a single analysis takes 25 min; the detection limit (at a signal-to-noise ratio of 3) is 3 micrograms/l in urine, when a 200 microliters aliquot of the extract is injected in the analytical apparatus. The recovery of the clean-up procedure is > 90%; both the intra-assay and the inter-assay coefficients of variation are < 4%. The method has been applied to smokers and nonsmokers, subjects occupationally unexposed to benzene; the results showed a statistically significant difference between the two groups. Also, a close correlation was found between urinary excretion of MA measured with this method and environmental concentration of benzene in a population of workers occupationally exposed to low levels of this solvent.