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Novel Blood Protein Modification Assay for Retrospective Detection of Drug Exposure – Development and Validation

NCJ Number
Date Published
64 pages

This project report details the author’s efforts to achieve the main research goal of extending the capabilities of current biomonitoring methods for testing long-term drug use or exposure.


This document reports on a project that addressed two research questions: to detect and characterize thiol modifications of human hemoglobin (Hb) and serum albumin (SA) by reactive metabolites (RM) of selected drugs following in vitro incubation in a human liver microsome (HLM) based assay; and to explore a technique for enrichment of the adducted protein species by selective removal of unadducted protein for enhancement of assay sensitivity and selectivity, specifically addressing the question of whether a published method for enrichment of adducted SA could be adapted to Hb, as it had not previously been reported in the literature. The major goals and objectives of the project were to support further development of a robust blood protein adduct detection technology for retrospective monitoring of drugs of abuse as a potential alternative, or complement, to the current biomonitoring approach of hair analysis. The report provides a detailed discussion of the research design, methods, analytical and data analysis techniques. The results and findings section discusses: identification of stable and reactive drug metabolites by in vitro HLM assay; generation of reactive drug metabolites by electrochemical oxidation; generation of reactive drug metabolites by biomimetic chemical catalysts; identification of peptide-drug adducts by in vitro trapping assay; enrichment assay for adducted Hb; and identification of Hb adducts by top-down and bottom-up proteomics.

Date Published: January 1, 2022