TY - JOUR
T1 - Improved coupled column liquid chromatographic method for high-speed direct analysis of urinary trans,trans-muconic acid, as a biomarker of exposure to benzene
AU - Marrubini, Giorgio
AU - Hogendoorn, Elbert A.
AU - Coccini, Teresa
AU - Manzo, Luigi
PY - 2001/2/25
Y1 - 2001/2/25
N2 - A coupled column liquid chromatographic (LC-LC) method for high-speed analysis 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 μm C18 column (50×4.6 mm I.D.) as the first column (C-1) and a 5 μm C18 semi-permeable surface (SPS) column (150×4.6 mm I.D.) as the second column (C-2). The mobile phases applied 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-1 in between analysis. Under these conditions t,t-MA eluted 11 min after injection. Using relatively non-specific UV detection at 264 nm, the selectivity of the assay was enhanced remarkably by the use of LC-LC allowing detection of t,t-MA at urinary levels as low as 50 ng/ml (S/N>9). The study indicated that t,t-MA analysis can be performed by this 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) showed excellent linearity (r>0.999). The method was validated using urine samples 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%.
AB - A coupled column liquid chromatographic (LC-LC) method for high-speed analysis 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 μm C18 column (50×4.6 mm I.D.) as the first column (C-1) and a 5 μm C18 semi-permeable surface (SPS) column (150×4.6 mm I.D.) as the second column (C-2). The mobile phases applied 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-1 in between analysis. Under these conditions t,t-MA eluted 11 min after injection. Using relatively non-specific UV detection at 264 nm, the selectivity of the assay was enhanced remarkably by the use of LC-LC allowing detection of t,t-MA at urinary levels as low as 50 ng/ml (S/N>9). The study indicated that t,t-MA analysis can be performed by this 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) showed excellent linearity (r>0.999). The method was validated using urine samples 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%.
KW - Benzene
KW - Coupled column
KW - Exposure biomarker
KW - Large volume injection
KW - Muconic acid
KW - Urine analysis
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U2 - 10.1016/S0378-4347(00)00497-7
DO - 10.1016/S0378-4347(00)00497-7
M3 - Article
C2 - 11236089
AN - SCOPUS:0035946018
VL - 751
SP - 331
EP - 339
JO - Journal of Chromatography B: Biomedical Sciences and Applications
JF - Journal of Chromatography B: Biomedical Sciences and Applications
SN - 1387-2273
IS - 2
ER -