Simultaneous separation and detection of common chiral and achiral metabolites in the urine of human exposed to benzene series by LC-MS/MS

•Developed novel chiral stationary phase EACDP, achieving high resolution for PMA, BMA, and MHBMA-2 enantiomers within 50 min.•Detection limits as low as 0.109 μg/L, recoveries 78–116 %, ensuring accurate quantification in complex matrices.•Applied to 60 urine samples from an auto plant, revealing strict benzene limits but widespread toluene and xylene use.•Simultaneous detection of PMA, BMA, etc., provides precise assessment of occupational benzene exposure in non-smokers. Benzene, toluene, and xylene (BTX) are priority pollutants known for their hematotoxicity and carcinogenic properties. Benzene is further metabolized to phenyl mercapturic acid (PMA), toluene and xylene also generate benzyl mercapturic acid (BMA) in human urine. To confirm whether the exposure to benzene series comes from the workplace or from the external environment such as smoking is a very meaningful work, so accurate measurement of their biomarkers in biological samples is crucial. This study developed a novel chiral stationary phase using 6-ethylenediamine mono-derivatized-β-cyclodextrin for the simultaneous separation and detection of four chiral and achiral biomarkers PMA, BMA, N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine (DHBMA), and N-acetyl-S-(1-hydroxymethyl-2-propenyl)-L-cysteine (MHBMA) in human urine. The method demonstrated high sensitivity with detection limits below 0.211 μg/L for PMA, 0.467 μg/L for BMA, 0.246 μg/L for DHBMA, and 0.109 μg/L for MHBMA, excellent recoveries ranging from 78 to 116 % as well as the high resolutions of PMA, BMA and MHBMA-2 enantiomers being up to 1.62, 2.23 and 1.79, respectively, within 50 min under reversed-phase chromatography. Application to 60 urine samples from an automobile manufacturing plant revealed that benzene as a paint solvent has been strictly limited, while toluene and xylene are widely used, potentially posing health risks to occupational groups. The simultaneous detection of PMA, BMA, MHBMA, and DHBMA provides a more accurate assessment of non-smoking populations, enhancing the evaluation of occupational exposure to benzene series compounds.