A novel method for the accurate quantification of two isomeric mercapturic acids of 1,3-dichlorobenzene in human urine using isotope dilution online-SPE-LC-MS/MS

•1,3-dichorobenzene is an occupationally relevant noxious agent in silicone rubber industry.•First method to quantify urinary mercapturates of 1,3-dichlorobenzene using online-SPE LC-MS/MS.•Authentic mercapturate standards and labelled analogues were self-synthesized.•The method is fully automated, precise, sensitive and accurate.•Post-shift urine of exposed workers shows that mercapturates are suitable biomarkers. 1,3-dichlorobenzene (1,3-DCB) is an aromatic solvent that might be formed during thermal decomposition of bis(2,4-dichlorobenzoyl)peroxide used as initiator in silicone rubber production with many workers exposed worldwide. During metabolism of 1,3-DCB, two isomeric mercapturic acids can be formed from ring oxidation of 1,3-DCB in the liver, namely 2,4-dichlorophenylmercapturic acid (24CPhMA) and 3,5-dichlorophenylmercapturic acid (35CPhMA). These urinary mercapturic acids might serve as biomarkers of the toxicologically relevant absorbed dose of 1,3-DCB and have not been determined so far. Thus, we were aimed to develop an analytical method for quantification of these biomarkers. Authentic standards of both mercapturic acids as well as deuterium-labelled analogues were self-synthesized. A method for the quantification of both CPhMAs in human urine using online-SPE LC/MS/MS was developed and validated with an LOQ of 0.1 ng mL−1 for both CPhMAs. The analytes were extracted from urine by online-SPE on a restricted access material phase, transferred to the analytical column and quantified by tandem mass spectrometry. Interday (n = 6) and Intraday (n = 10) precision for both CPhMAs ranged from 1.7 to 4.3 % with accuracies between 99.4 and 109.9 % at concentrations of 0.6 and 3 ng mL−1. We applied the method on post-shift urine samples of 16 workers of the silicone rubber industry with occupational exposure to 1,3-DCB. Both CPhMAs were above LOQ in 15 of 16 urine samples with median levels (range) for 24CPhMA and 35CPhMA of 1.64 ng mL−1 (