Method development for the simultaneous analysis of trans,trans-muconic acid, 1,2-dihydroxybenzene, S-phenylmercapturic acid and S-benzylmercapturic acid in human urine by liquid chromatography/tandem mass spectrometry
This study reports a method to simultaneously determine trans,trans-muconic acid (t,t-MA), 1,2-dihydroxybenzene (or catechol, abbreviated as 1,2-DB), S-phenylmercapturic acid (S-PMA) and S-benzylmercapturic acid (S-BMA) in human urine. Samples were prepared through a solid phase extraction and analyzed by ultra-high performance liquid chromatography/tandem mass spectrometer in negative electrospray ionization mode. The method was completely validated through the studies of precision, accuracy, matrix effects, detection limit, linear range, stability and real urine sample tests. Calibration curves of all the target analytes showed a favorable linearity within the wide concentration range of 0.2–4000 μg L−1. The detection limits in 10 times diluted pooled urine ranged from 0.08 to 7.8 μg L−1. The method showed satisfactory accuracies and precisions. Except for the low spiked quality control (QC) level of 1,2-DB (73.1% recovery), the recoveries were in the range of 100% ± 15% with a variation coefficient of less than 15%. The target analytes were stable in stock solutions and spiked urine samples under storage and test conditions. Thirty three anonymous real urine samples from non-occupational donors were analyzed for all the target analytes. The preliminary results showed that S-BMA was a sensitive and specific biomarker of toluene exposure and had a significant correlation with t,t-MA (r = 0.631, p < 0.01). Moreover, S-BMA and S-PMA together could reflect the overall exposure to BT at low levels.