Improvement in HPLC analysis of urinary trans,trans-muconic acid, a promising substitute for phenol in the assessment of benzene exposure

Abstract

Urinary trans,trans-muconic acid (t,t-MA), a minor metabolite of benzene, is a potential candidate for biological monitoring of benzene. A clean-up procedure using SPE extraction cartridges was applied to urinary samples in order to improve the reliability of t,t-MA determinations by HPLC-UV greatly and to carry out convenient analyses on a routine scale, particularly at low levels of t,t-MA concentrations. The detection limit of the method is low enough to measure urinary t,t-MA at a concentration of 0.05-0.1 mg/l. The recovery rates and relative standard deviations from spiked urines (1 mg/l to 20 mg/l) were about 90% and 5%, respectively. t,t-MA was found to be rapidly excreted by rats and humans. In rats the background range never exceeded 0.5 mg/l with a mean concentration around 0.3 mg/l. In 49 human blank urines, t,t-MA average and median-value were respectively around 0.2 and less than 0.1 mg/l with a range of less than 0.1 to 0.5 mg/l. Experimental exposure of rats for 1 h to 10.2 ppm of benzene induced urinary excretion of 13 mg/l of t,t-MA during a 6-h post-exposure period while occupational exposures to 2.6 ppm (mean exposure level during 5 d-8 h) and 7 ppm (4 h) of benzene resulted in urinary excretion of 2.1 (mean excretion level) and 6.5 mg/l respectively at the end of the exposure. In humans, t,t-MA has a similar half-time as phenol. Analysis of urinary t,t-MA seems to be a better indicator than phenol for the assessment of exposure to low levels of benzene. Ingestion of 200 mg of sorbic acid, the only other known precursor of t,t-MA, interfered minimally with the background excretion of t,t-MA.