Analytical and cheminformatic framework for studying drugs and their metabolites in human plasma using high resolution mass spectrometry

Abstract Pharmacological interventions are required to manage a wide array of chronic diseases. Drug metabolism is heavily influenced by individual-level genomic and environmental factors, and there is substantial variability in drug efficacy for many medications. Novel strategies are needed to evaluate drug performance and improve therapeutic effectiveness. Recent technological advancements in high-resolution mass spectrometry (HRMS) have enabled broad exposomic analyses that can evaluate how an individual metabolizes drugs. Here, we present a new analytical framework to annotate drug metabolites in human plasma samples. We performed non-targeted analyses using liquid-chromatography (LC)-HRMS and developed an analytical workflow that leverages tandem mass spectra for structural annotation of unknown molecular features. In addition to database matching, we implemented molecular networking to link parent pharmaceutical compounds to potential metabolite features using shared fragmentation patterns followed by in silico chemical formula and structure prediction. In doing so, we successfully annotated downstream metabolites of an array of pharmaceutical agents and created a new investigational pipeline towards measuring drug metabolites in human samples for which reference standards or spectra are rarely available. The analytical framework presented in this study can be used to develop new pharmacokinetic models where both the parent drugs and the metabolites are simultaneously measured and monitored. Similarly, this workflow can be applied for annotating xenobiotic chemicals in various matrices. Our results contribute to efforts in developing a personalized approach to measuring drug metabolites and may provide new opportunities to maximize therapeutic response and improve drug efficacy in humans.