Direct analysis of urinary trans,trans-muconic acid by coupled column liquid chromatography and spectrophotometric ultraviolet detection: method applicability to human urine

A coupled column liquid chromatographic (LC–LC) method for the direct analysis in human urine of the ring opened benzene metabolite, trans,trans-muconic acid (t,t-MA) is described. The method was tested using urine samples collected from five refinery workers exposed to low concentrations of airborne benzene (0.2–0.5 ppm), and from non-exposed volunteers. The analytical columns used were of 50×4.6 mm I.D. packed with 3 μm p.s. Microspher C₁₈ material as the first column (C-1), and a 100×4.6 mm I.D. column packed with 3 μm p.s. Hypersil ODS material as the second one (C-2). The mobile phases applied consisted, respectively, of methanol–0.074% trifluoroacetic acid (TFA) in water (4:96, v/v) on C-1, and of methanol–0.074% TFA in water (10:90, v/v) on C-2. Under these conditions t,t-MA eluted 15 min after injection. The present method, coupling the LC–LC technique with UV detection at 264 nm, permits the quantitation of t,t-MA directly in urine at levels as low as 0.05 mg/l. The determination is performed with a sample throughput of 2 h⁻¹ requiring only pH adjustment and centrifugation of the sample. Calibration plots of standard additions of t,t-MA to pooled urine taken from five non-exposed subjects were linear (r>0.999) over a wide concentration range (0.05, 0.1, 0.5, 1.0, 2.0 mg/l). The precision of the method (RSD) was in the range of 0.5 to 3.8%, and the within-session repeatability on workers urine samples (levels 0.06, 0.1, 0.2, 1.0 mg/l) was in the range of 3 to 8%. The present method improves the applicability of routine t,t-MA analysis, where it is most desirable that a large number of biological samples can be processed automatically or with minimal human labour, at low cost, and with a convenient turn-around time.