Innovative analytical strategies to identify new psychoactive substances and their metabolites

Over the past decades, the number and availability of new psychoactive substances (NPS) have surged, challenging conventional approaches to drug monitoring, surveillance, regulation, and public health responses. Understanding the NPS landscape requires significant effort from analytical and toxicological fields. This thesis aims to answer questions related to the metabolism of NPS, specifically synthetic cathinones (SCs), by investigating their Phase I metabolic pathways and providing innovative analytical approaches to tackle NPS-related challenges in bioanalytical chemistry, such as discriminating NPS positional isomers and addressing the lack of commercial reference standards for NPS and their metabolites. The first part of this thesis (Chapters 2 and 3) explores the in vitro Phase I metabolic pathways of SCs using pooled liver microsomal incubations. SCs are the second largest category of NPS according to the United Nations Office on Drugs and Crime (UNODC). Despite their prevalence, their metabolism is largely unknown. Chapter 2 elucidates the tentative structure of Phase I metabolites of SCs and proposes their metabolic pathways, examining the influence of rat liver microsome sex on these pathways. It was found that sex-specific metabolites were detected only in pooled male rat liver microsomal incubations, though human liver microsomal incubations showed neither sex-specific metabolites nor significant differences in relative abundance. Chapter 3 introduces electron activated dissociation (EAD) for metabolite identification (Met-ID) of SC positional isomers. EAD, a new ion activation method, provides complementary fragmentation patterns to collision-induced dissociation (CID), enhancing confidence in structural elucidation of SCs isomers. EAD allowed distinguishing SC positional isomers based on their product ion spectra, offering a competitive approach in Met-ID workflows. As many SCs on the market are positional isomers with different legal statuses, high-confidence identification is crucial. Chapter 4 builds on Chapter 3 by proposing an EAD MS and chemometrics workflow for isomer-specific identification of ring-substituted SCs in street samples. Using various kinetic energies in the EAD cell and machine learning methods, a 97% classification accuracy for isomer identification was achieved. This workflow, applied to authentic street samples, resulted in 100% identification accuracy at 15 eV kinetic energy, demonstrating that combining advanced approaches in data acquisition and analysis enhances the confidence in identifying NPS positional isomers. The lack of commercial analytical standards for NPS and metabolites limits forensic intelligence associated with these drugs. Chapter 5 introduces a "Chemical Toolbox" to mimic enzymatic oxidation reactions of hepatic enzymes for generating Phase I metabolites. Using liver microsomal incubations, metabolites of model antidepressants were compared with oxidation products from chemical reactions. This toolbox shows potential to chemically mimic microsomal enzyme activity to produce metabolite-like products (MLPs). Once optimized for extraction and purification, this toolbox could generate pure substances for use as analytical standards in qualitative and quantitative methods. This thesis advances analytical strategies for identifying and characterizing NPS and their metabolites, enhancing understanding of NPS metabolism. The developed strategies are relevant to bioanalytical, forensic, clinical, and environmental laboratories and can be applied to various xenobiotics, including conventional drugs of abuse, pharmaceuticals, and drug candidates, broadening their scientific and societal impact.