Preparation of a new benzylureido-β-cyclodextrin-based column and its application for the determination of phenylmercapturic acid and benzylmercapturic acid enantiomers in human urine by LC/MS/MS

Abstract

A benzylureido-β-cyclodextrin was synthesized by the reaction of 6-amino-β-cyclodextrin with an active benzyl isocyanate. Then, it was bonded to silica gel by a thiol-ene addition reaction, obtaining a new benzylureido-β-cyclodextrin-based chiral stationary phase (BzCDP). Its chemical structure was characterized by infrared spectroscopy, elemental analysis, and solid-state nuclear magnetic resonance spectroscopy. The BzCDP was successfully used to separate phenylmercapturic acid (PMA) and benzylmercapturic acid (BMA) enantiomers, which were confirmed as biomarkers of exposure to benzene and toluene in human urine. The enantiomeric separations were also optimized through the investigation of related factors. The resolutions of PMA and BMA enantiomers could be up to 2.25 and 2.14, respectively, within 30 min under reversed-phase chromatography. Based on the optimal chromatographic and mass spectrometry conditions, a new LC-MS/MS quantitative method for the PMA and BMA enantiomers was established by negative ion multiple reaction monitoring (MRM) and an isotope-labeled PMA (d2-PMA) as an internal standard. The limits of detection (LODs) of enantiomers were less than 0.17 μg/L for PMA and 0.14 μg/L for BMA, and the averaged recoveries of enantiomers were in the range of 86~100% for PMA and 86~113% for BMA. The method had good reproducibility levels with the RSDs (3.5~11.3% for intra-day and 3.9~13.1% for inter-day). The method was successfully applied to urine testing of 60 painting and printing workers. The results showed that only L-PMA was detected in the urine of the Printers, while a high content of L-PMA (27.5~106 μg/L) and D-PMA (19.9~82.8 μg/L) can be detected simultaneously in the urine of the Painters, indicating that benzene pollution was more serious in this group. The positive rate of BMA was rather higher, indicating that toluene pollution was more common than benzene. BMA also existed in the form of two enantiomers (L-BMA and D-BMA), but the difference between the two types of occupational groups was small. It is a meaningful work to deeply study the existence and content of chiral markers in human urine, which will help to better understand and evaluate the harmful effects of benzene series on human beings. [Figure not available: see fulltext.].