Supplementary Materials.docx

Benzene, toluene and xylene have strong blood toxicities and carcinogenic effects. Benzene is further metabolized to phenyl mercapturic acid (PMA), toluene and xylene also generate benzyl mercapturic acid (BMA) in human urine. While, cigarette smoke is rich in carcinogens such as benzene and toluene, therefore, if the smokers in the workplace may interfere with the correct assessment of the source of benzene and benzene series. Some metabolites, such as PMA, BMA, N-acetyl-S-(3,4-dihydroxybutyl-L-cysteine (DHBMA), N-acetyl-S-(1-hydroxymethyl-2-propenyl)-L-cysteine (MHBMA-1) and (R,S)-N-acetyl-S-(2-hydroxy-3-butenyl)-L-cysteine (MHBMA-2) were simultaneously determined so as to confirm whether the exposure to benzene series comes from the workplace or from the external environment such as smoking. A new β-cyclodextrin-based chiral stationary phase (EACDP) was prepared. After the separation conditions were optimized, a new LC-MS/MS method on EACDP column for the determination of common chiral and achiral metabolites in urine of human exposed to benzene series was established. The excellent separations of the above metabolites were achieved, in which the high resolutions of PMA, BMA and MHBMA-2 enantiomers were up to 1.62, 2.23 and 1.79, respectively, within 50 min under reversed-phase chromatography. Moreover, sensitive and selective multiple reaction monitoring (MRM) was also employed. The detection limits (LODs) of enantiomers were all less than 0.366，0.467, 0.246 and 0.153 μg/L for PMA, BMA, DHBMA, MHBMA, separately. The averaged recoveries of enantiomers were in the range of 78~93% for PMA, 80~116% for BMA, 86~102% for DHBMA and 80~92% for MHBMA. Also, the method had good reproducibilities. By simultaneous measuring the contents of four biomarkers PMA, BMA, DHBMA and MHBMA in human urine metabolites, the non-smoking population could be more accurately estimated, and toluene exposure also has a certain degree of harm to the body, so as to make a more reasonable evaluation of the people exposed to benzene series in the workplace.