Identification\nof Specific Hemoglobin Adduct Patterns\nin Users of Different Tobacco/nicotine Products by Nontargeted GC–MS/MS\nAnalysis

Tobacco smoke contains several electrophilic constituents\nwhich\nare capable of forming adducts with nucleophilic sites in DNA and\nproteins like hemoglobin (Hb) and albumin. New nicotine and tobacco\nproducts are discussed as less harmful forms of tobacco use compared\nto smoking combustible cigarettes (CC) due to reduced exposure to\nharmful constituents. Hence, the adduct profile in users of various\ntobacco/nicotine products is expected to differ characteristically.\nIn this article, we present a novel nontargeted screening strategy\nusing GC–MS/MS for Hb adducts based on the analysis of the\nrespective derivatized N-terminal valine adducts after modified Edman\ndegradation. We analyzed blood samples from a clinical study with\nhabitual users of CCs, electronic cigarettes, heated tobacco products\n(HTPs), oral tobacco, nicotine replacement therapy products and nonusers\nof any tobacco/nicotine products. Our nontargeted approach revealed\nsignificant differences in the Hb adduct profiles of the investigated\ntobacco/nicotine product user groups. Adduct identification was performed\nby means of an internal database, retention time estimations based\non the theoretical boiling points, as well as in-house synthesized\nreference compounds. Several chemicals that form adducts with Hb could\nbe identified: methylating and ethylating agents, ethylene oxide,\nacrylonitrile, acrylamide, glycidamide and 4-hydroxybenzaldehyde.\nLevels were elevated in smokers compared to all other groups for Hb\nadducts from methylating agents, ethylene oxide, acrylonitrile, acrylamide\nand glycidamide. Our approach revealed higher concentrations of Hb\nadducts formed by ethylation, acrylamide and glycidamide in users\nof HTPs compared to nonusers. However, concentrations for the latter\ntwo were still lower than in smokers. Due to their long half-lives,\nHb adducts related to acrylonitrile, acrylamide (glycidamide), and\nethylene oxide exposure may be useful for the biochemical verification\nof subjects̀ compliance in longitudinal and cross-sectional\nstudies with respect to smoking and HTP use/abstinence.