Improved coupled column liquid chromatographic method for high-speed direct analysis of urinary trans,trans-muconic acid, as a biomarker of exposure to benzene

A coupled column liquid chromatographic (LC-LC) method for high-speed analy sis of the urinary ring-opened benzene metabolite, trans,trans-muconic acid (t,t-MA) is described. Efficient on-line clean-up and concentration of t,t -MA from urine samples was obtained using a 3 mum C-18 column (50x4.6 mm I. D.) as the first column (C-1) and a 5 mum C-18 semi-permeable surface (SPS) column (150x4.6 mm I.D.) as the second column (C-2). The mobile phases app lied consisted, respectively, of methanol-0.05% trifluoroacetic acid (TFA) in water (7:93, v/v) on C-1, and of methanol-0.05% TFA in water (8:92, v/v) on C-2. A rinsing mobile phase of methanol-0.05% TFA in water (25:75, v/v) was used for cleaning C-l in between analysis. Under these conditions t,t- MA eluted ii min after injection. Using relatively non-specific UV detectio n at 264 nm, the selectivity of the assay was enhanced remarkably by the us e of LC-LC allowing detection of t,t-MA at urinary levels as low as 50 ng/m l (S/N>9). The study indicated that t,t-MA analysis can be performed by thi s procedure in less than 20 min requiring only pH adjustment and filtration of the sample as pretreatment. Calibration plots of standard additions of t,t-MA to blank urine over a wide concentration range (50-4000 ng/ml) showe d excellent linearity (r>0.999). The method was validated using urine sampl es collected from rats exposed to low concentrations of benzene vapors (0.1 ppm for 6 h) and by repeating most of the analyses of real samples in the course of measurement sequences. Both the repeatability (n=6, levels 64 and 266 ng/ml) and intra-laboratory reproducibility (n=6, levels 679 and 1486 ng/ml) were below 5%. (C) 2001 Elsevier Science B.V. All rights reserved.