LC-MS Screening Assay for Abused Drug Exposure Based on Covalent Peptide/Protein Modification

Authors

Ludmyla Santos TavaresFollow

Document Type

Dissertation

Degree

Doctor of Philosophy (PhD)

Major/Program

Chemistry

First Advisor's Name

Anthony De Caprio

First Advisor's Committee Title

Committee Chair

Second Advisor's Name

Jose Almirall

Second Advisor's Committee Title

Committee Member

Third Advisor's Name

David Becker

Third Advisor's Committee Title

Committee Member

Fourth Advisor's Name

De Etta Kay Mills

Fourth Advisor's Committee Title

Committee Member

Fifth Advisor's Name

Kevin O'Shea

Fifth Advisor's Committee Title

Committee Member

Keywords

analytical chemistry, chemistry, toxicology

Date of Defense

10-27-2022

Abstract

In vitro metabolic assays are commonly used in forensic toxicology to assess the extent and rate of drug metabolism and to identify specific metabolites formed. Available in vitro systems include synthetic metalloporphyrins, HLM assays, and electrochemical assays. These assays also have utility in identifying reactive metabolites when a nucleophilic trapping molecule is included. While enzyme-based assays are widely employed for drug metabolite generation in forensic toxicology, alternative in vitro systems have not been extensively tested for identification of stable (SM) and reactive (RM) metabolites of drugs of abuse. The capability of reactive drug metabolites to form adducts with glutathione and a specific β-Hb tryptic peptide containing a highly reactive nucleophilic thiol moiety (i.e., GTFATLSELH⁹³CDK; β⁹³Cys peptide) was also investigated.

Covalent binding of drugs to proteins and/or peptides is an alternative to hair analysis for long-term or retrospective assessment of drug abuse or exposure. Identification of such peptide or protein modifications (i.e., adducts) can offer valuable information about exposure history. Protein adducts have the potential to survive throughout the life of the protein (e.g., 120 days in the case of human hemoglobin).

In this project, the comparison of the three in vitro assays systems revealed that the metabolites obtained exhibited a few common derivatives but also compounds unique to each system. In addition, major reported in vivo metabolites for each drug were also found with all three in vitro systems, i.e., NAPQI, MDA, amphetamine, and 11-COOH-THC from acetaminophen, methamphetamine, MDMA, and D⁹-THC, respectively. The electrochemical oxidation and synthetic metalloporphyrin systems appeared to generate a wider variety of metabolites than encountered with human liver microsomes, including stable and potentially reactive derivatives such as HFA, HPA, an imine derivative, 11-CHO-THC, and methylidene-THC. These results indicate that use of all three in vitro systems may provide a more complete profile of potential Phase I oxidative SM and RM for a variety of drugs of abuse that may be targeted for analysis in forensic toxicological studies and that may reveal possible adduct forming species.

Results of the in vitro trapping assay studies with GSH and the β⁹³Cys peptide demonstrated that the EC assay can be employed in the generation and trapping of RM by peptides containing reactive thiol moieties. 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 monitored as an alternative to clinical and forensic hair analysis and could be usefully applied in areas of drug testing and forensic toxicological analysis.

Identifier

FIDC010939

ORCID

https://orcid.org/0000-0002-3690-8622

Recommended Citation

Santos Tavares, Ludmyla, "LC-MS Screening Assay for Abused Drug Exposure Based on Covalent Peptide/Protein Modification" (2022). FIU Electronic Theses and Dissertations. 5177.
https://digitalcommons.fiu.edu/etd/5177