Method for the Determination of Benzene Metabolite t,t-Muconic Acid in Urine by HPLC-UV with an Ion Exclusion Column

Abstract

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 2 4´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 (R 2 ) > 0.99 for tt-MA standard in water and R 2 > 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.

Different Analytical Techniques, Pretreatment Methods and Adsorbent Materials for the Determination of Trans, Trans-Muconic Acid in Urine Samples

“…Mudiam et al [24] reported a MIP using methacrylic acid (MAA) as a monomer followed by simultaneous derivatization using ethyl chloroformate and pre-concentration using dispersive liquid-liquid microextraction (DLLME) for the determination of tt-MA in urine samples. Gomes et al [25] developed an analytical method for the determination of urinary tt-MA using solid phase extraction (SPE) onto a Strata SAX cartridge. Rismanchian et al [26] detected tt-MA in urine samples using partitioned dispersive liquid-liquid microextraction technique (PDLLME) coupled with high-performance liquid chromatography-UV detection.…”

Solidified floating organic droplet microextraction coupled with HPLC for rapid determination of trans, trans muconic acid in benzene biomonitoring

“…However, in low levels of benzene (lower than 1 ppm), no association was found between urinary levels of phenol and catechol compounds 11 . It has been found that there is a good correlation between urinary t,t-MA and concentration of benzene in air and blood in low levels of benzene exposure 12 , 13 . Furthermore, the American Conference of Governmental Industrial Hygienists (ACGIH) has introduced t,t-MA as a urinary biomarker of benzene exposure 14 .…”

Exposure assessment of wastewater treatment plant employees to BTEX: a biological monitoring approach

“…Urine metabolites may act as indicators of occupational or environmental exposure, especially for water-soluble chemicalssuch as benzene, which includes phenol, hydroquinone, tt-MA acid, urinary hippuric acid (uHA), and S-phenylmercuric acid 17 – 19 . Among them, urinary tt-MA is recognized as a reliable and relatively facile biomarker 20 and represents about 1% of the absorbed amount of benzene 21 . The range of metabolic conversion of benzene to tt-MA is about 2–25% and dependent on the benzene exposure level, simultaneous exposure to toluene, and probably also to genetic factors 22 .…”