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 analy sis in human urine of the ring opened benzene metabolite, trans.trans-mucon ic acid (t,t-MA) is described. The method was tested using urine samples co llected from five refinery workers exposed to low concentrations of airborn e benzene (0.2-0.5 ppm), and from non-exposed volunteers. The analytical co lumns used were of 50X4.6 mm I.D. packed with 3 mum p.s. Microspher C-18 ma terial as the first column (C-1), and a 100X4.6 mm I.D. column packed with 3 mum 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 (10:9 0, 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. Calibr ation plots of standard additions of t,t-MA to pooled urine taken from five non-exposed subjects were linear (r >0.9991 over a wide concentration rang e (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 b e processed automatically or with minimal human labour, at low cost, and wi th a convenient turn-around time. (C) 2001 Elsevier Science B.V. All rights reserved.