Abstract
The benzene metabolite trans,trans-muconic acid (tt-MA) is widely used as a biological indicator of exposure to this xenobiotic. An analytical method was developed and validated for the determination of urinary tt-MA using solid phase extraction onto a Strata SAX cartridge. The analysis was performed by HPLC with an Aminex HPX-87H ion exclusion column and a UV diode array detector. The sample preparation conditions were optimized using a 24−1 factorial design. Comparison of the slopes of standard analytical curves prepared in aqueous solution and urine showed that the biological matrix suppressed the tt-MA signal by around 50%, so the analytical curves were prepared with tt-MA standard at low concentrations in pooled urine. The analytical curves in the range of 5–500 μg·L−1 showed determination coefficients values (R2) > 0.99 for tt-MA standard in water and R2 > 0.98 for tt-MA standards in pooled urine. The coefficients of variation obtained using seven replicates were lower than 3.6%, and recoveries of tt-MA from solutions containing 5, 25, 50 and 100 μg·L−1 of the analyte were in the range 85%–90%, demonstrating the satisfactory precision and accuracy of the method. The limits of detection and quantification were 0.11 and 0.36 µg ·L−1, respectively. The benefits of this new method developed is the possibility of complete chromatographic peak separation for the determination of tt-MA at baseline, without matrix components’ interference as normally found in the C18 column. This is the first time that this chromatographic column has been used for the analysis of tt-MA in urine.
Highlights
Despite substantial progress over the last 20 years in understanding the risks posed by benzene, there is a continuing need for the development of sensitive techniques able to determine the compound at low levels, in order to assist in the implementation of control strategies
The precision of the technique was measured using the coefficient of variation (CV) value obtained for repetitions of the entire procedure, from sample preparation up to injection onto the chromatographic column
It is recommended that a pretreatment might be required in order to obtain a satisfactory chromatography performance
Summary
Despite substantial progress over the last 20 years in understanding the risks posed by benzene, there is a continuing need for the development of sensitive techniques able to determine the compound at low levels, in order to assist in the implementation of control strategies. An alternative to the evaluation of exposure to benzene by directly measuring its concentration in air is to determine the concentrations of its metabolites in urine or blood that involve the use of biological exposure indicators (BEIs). Separations 2016, 3, 14 be used as a BEI are trans,trans-muconic acid (tt-MA) and S-phenyl mercapturic acid (SPMA), which are biomarkers that are more sensitive and specific, compared to urinary phenol [11] This exposure biomarker has been adopted in Brazilian legislation for the monitoring of occupational exposure to benzene [12]. The objective of this work was to develop a new technique for the measurement of tt-MA in urine, using SPE (SAX cartridges) and HPLC with an Aminex HPX-87H column and UV detection
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