An Investigation into the Adduct Forming Potential of Drugs of Abuse with Peptides and Proteins

Hemoglobin and serum albumin, two prevalent proteins in human blood, contain unbound cysteine thiol moieties, creating a nucleophilic site with the potential for covalent modification by reactive chemical species. These covalent modifications, called “adducts”, are stable entities that accumulate during acute and chronic exposure and remain covalently bound for the life-span of the protein. Despite their current use as exposure markers for a variety of compounds, the use of adducts in assessing exposure to drugs of abuse has not yet been explored. The goal of this work was to examine the in vitro adduct forming capability of selected drugs of abuse with hemoglobin to provide additional proof of principle for the development of a real-world detection and monitoring analysis method. This goal was accomplished by first analyzing the binding capabilities of the drugs of interest with glutathione, a smaller tripeptide. Use of protein adducts as biomarkers of drug exposure may allow for an increased window of detection, from several days to several months, as compared to current blood analysis methods. In total, there were 16 drugs analyzed in the research, and they covered a wide range of abused drugs, including cocaine, methamphetamine, and Δ 9 -THC. Results from the glutathione trials showed that 10 of the 16 the drugs of interest were able to form covalent adducts with the free thiol moiety, with four drugs forming more than one novel adduct. The MS results for hemoglobin showed 11 adducts formed for five of the drugs under investigation. Additional MS/MS confirmatory data was obtained for two of those 11 adducts. I successfully identifyied adducts formed between drugs of abuse and glutathione and hemoglobin, which have the potential to be used as long-term biomarkers of exposure