Global Profiling of Urinary Mercapturic Acids Using Integrated Library-Guided Analysis

Urinary mercapturic acids (MAs) are often used as biomarkers for monitoring human exposures to occupational and environmental xenobiotics. Untargeted mass spectrometry-based approaches have been applied for the broad characterization of MAs, but the metabolite coverage was limited due to the lack of comprehensive MA databases. In this study, we developed an integrated library-guided analysis (ILGA) workflow using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). This method includes expanded assignment criteria and a curated library of ~240 MAs and addresses the shortcomings of previous untargeted approaches. We employed this workflow to profile MAs in the urine of 70 participants - 40 nonsmokers and 30 smokers. We identified approximately 500 MA candidates in each urine sample, and 118 MAs were putatively assigned with the ILGA approach. These include 29 previously unreported MAs derived mostly from alkenals and hydroxyalkenals. In addition, we observed that the levels of 70 MAs were comparable in nonsmokers and smokers, 2 MAs were higher in nonsmokers, and the levels of 46 MAs were elevated in the smokers. These included previously unreported mercapturates of polyaromatic hydrocarbons and hydroxyalkenals and well-documented MAs derived from toxicants present in cigarette smoke (e.g., acrolein, 1,3-butadiene, isoprene, acrylamide, benzene, and toluene). Our untargeted workflow led to the effective identification and discovery of known and unreported MAs derived from endogenous and environmental sources, and the levels of several of these MAs were increased by smoking. Our method can also be expanded and applied to other exposure-wide association studies.