Measurement of covalent binding of xenobiotics to blood proteins, such as hemoglobin (Hb), to yield adducted species has been successfully applied in environmental and occupational exposure assessment. Such adducts have the potential to survive throughout the life of the protein (~120 days for human Hb) if they do not trigger increased protein turnover. Nevertheless, the potential use of this technology for clinical or forensic analysis of abused drug exposure has not been extensively explored.
In this study, the capability of reactive drug metabolites to form adducts with the -Hb tryptic peptide containing a highly reactive thiol moiety (i.e., GTFATLSELH93CDK; -93Cys peptide) was investigated. Analysis of adducted peptide formed with acetaminophen (APAP), 3,4-methylenedioxymethamphetamine (MDMA), cocaine (COC), and Δ9-tetrahydrocannabinol (THC) was performed using HRMS on an Agilent 1290/6530 LC-QTOF-MS system. For the generation and trapping of reactive drug metabolites by synthetic -93Cys peptide, two methods were applied: 1) an in vitro metabolic trapping assay with human liver microsomes and 2) electrochemical oxidation using glassy carbon as working electrode, calomel standard as reference electrode, and platinum rod as counter electrode. Predicted covalent thiol modifications by drugs and metabolites were entered as target ions for screening using BioConfirm software for data analysis. For subsequent targeted MS/MS studies, the spectra were collected and compared to a theoretical peak list supplied by Protein Prospector software.
Adducted Hb peptides were identified and structure confirmed for APAP and metabolites (i.e., NAPQI, 1,4-benzoquinone), MDMA and metabolites (i.e., HHA and an aminochrome intermediate), COC metabolite (i.e., ecgonidine), and THC metabolite, (i.e., carbaldehyde-THC). The ability of abused drugs and/or metabolites to covalently modify the β-Hb peptide containing the reactive 93Cys suggests that such modifications could be monitor