A sensitive liquid chromatographic method for the spectrophotometric determination of urinary trans,trans-muconic acid

Benzene is a human carcinogen and its metabolite, urinary trans,trans-muconic acid (ttMA), is a biomarker for risk assessment. However, most of the existing methods were not sensitive enough for monitoring of low level exposure. This paper describes a HPLC-UV method for ttMA determination with enhanced selectivity and sensitivity. A 30 mg OasisMAX cartridge was used to clean-up 50 microl of urine sample and gradient elution was performed on a Zorbax SB-C(18) column (30 degrees C). ttMA was detected at wavelength 263 nm using a UV diode array detector (DAD). The two mobile phases used were (A) 150 mM ortho-phosphoric acid containing of 9% (v/v) methanol; and (B) 125 mM ortho-phosphoric acid containing 30% (v/v) acetonitrile. The method was validated with 61 urine samples collected from non-occupationally benzene exposed individuals and 14 quality control specimens from an international quality assessment scheme. The urinary ttMA concentrations (mean+/-S.D.microg/g creatinine) were 90+/-34 for smokers (n=26), 49+/-39 for non-smokers (n=21) and 23+/-18 for non-smoking hospital staff (n=14). A correlation coefficient, r=0.99 was found with 14 external quality specimens for ttMA ranged from 0.4 to 6.8 mg/l. The recovery and reproducibility were generally over 90% and the detection limit was 5 microg/l.