LC-QqQ-MS screening assay for abused drug exposure based on covalent peptide/protein modification

Long-term assessment of drug use or exposure is limited to analysis of hair nowadays, for which numerous methods and a large literature database exists. Although widely used for this purpose, hair analysis presents many challenges. An alternative to hair analysis is the study of covalent binding of drugs to proteins and/or peptides, such as hemoglobin (Hb), human serum albumin (HSA), and glutathione (GSH) to form drug-protein or drug-peptide “adducts.” Such protein modifications remain in the body for the lifetime of the protein and can provide a much longer window of detection of exposure than is generally possible by direct measurement of a parent compound or a metabolite in blood. Such data may be critically important in forensic toxicology, such as evidence in drug-facilitated crimes, measurement of drug compliance or abstinence in pain drug management, addiction rehabilitation programs, and probation/parole criminal justice situations. While widely used in human exposure assessment for environmental and occupational chemicals, applications of protein adducts as markers of illicit drug exposure are virtually nonexistent.
The first goal of this research is to utilize three different methods to generate reactive metabolites of drugs of abuse: in vitro metabolic assays, chemical oxidation, and electrochemical oxidation to produce reactive metabolites of drugs of abuse. After the metabolites have been produced, the capability of these reactive species to form adducts with reactive biological thiols present in proteins and peptides will be assessed. By comparing all these systems, the following can be determined: 1) the similarities in the metabolites generated, 2) which system can yield a higher quantity of the desired metabolites, and 3) what is the most efficient method for the formation of the drug-protein adducts. Analysis of potential adducts will be performed through liquid chromatography (LC) mass spectrometry (MS). The end goal is to develop and validate a LC-MS method for routine detection of Hb and HAS modifications by drugs of abuse. For each adduct found, two to four precursor/product ion pairs will be selected as targeted multiple reaction monitoring (MRM) transitions for confirmation of a specific drug adduct. Finally, the developed method will be employed to analyze authentic blood specimens, in order to analyze the adducts formed in vivo. This goal will be facilitated by collaborations with local physicians who manage drug rehabilitation programs with in-house laboratory facilities.
Note: This project contains a research and/or development component, as defined in applicable law, and complies with Part 200 Uniform Requirements - 2 CFR 200.210(a)(14). CA/NCF