Development of an LC-HRMS non-targeted method for comprehensive profiling of the exposome of nicotine and tobacco product users - A showcase for cigarette smokers.

The global prevalence of electronic cigarettes, heated tobacco products, and other smokeless alternatives has grown significantly in the last ten years. These products have been suggested as combustion-free alternatives for conventional tobacco products like cigarettes, aiming to reduce the negative health impacts associated with smoking. However, the impact of those products on the health and safety of the general population are still unclear, as the absolute exposure from those products has not been thoroughly studied, yet. In this project, a non-targeted LC-HRMS method was developed comprising four different analytical modes for the investigation of the exposure profile in urine of the product users. The method is characterized by its high sensitivity and reproducibility, as shown during method validation. As a proof of concept, we first applied this method to detect significant differences in biomarkers of exposure (BoEs) between smokers and non-smokers. We observed a total of 171 BoEs significantly elevated in smokers, including several well-known biomarkers of smoke exposure like nicotine and its metabolites, mercapturic acid derivatives, and phenolic compounds. Some of the detected biomarkers are present at low ng/mL concentrations in urine, proving the high sensitivity needed for a holistic exploration of the exposome. Moreover, we were able to identify BoEs that have not been reported previously for smoking, such as 2,6-dimethoxyphenol and 7-methyl-1-naphthol glucuronide.