New synthetic opioids: development of analytical methods for their characterization and determination by means of (U)HPLC- HRMS/MS

In recent years, the synthesis and introduction into the illicit market of New Psychoactive Substances (NPS) has reached alarming levels. More than 790 compounds have been identified by the European Monitoring Center for Drugs and Drug Addiction. Among the newest NPS, synthetic opioids deserve special attention, in particular fentanyls, which covered more than 70% of the opioid’s world demand. In this context it is essential to have the right tools to identify these recent NPS and verify their consumption. The present project involved the characterization of new fentanyls, the identification of their main metabolites and the development of innovative, fast and simple analytical methods, for the determination of these compounds in different biological matrices. Results presented in the experimental part illustrates the multi-pronged strategy that was studied in this 3-years project. On the one hand miniaturized preparation techniques, such as micro extraction by packed sorbent (MEPS), parallel artificial liquid membrane extraction (PALME), were optimized for the analyses of fentanyl, fentalogs and their metabolites, conducted in HPLC- (HR)MS/MS. All methods involved the use of a small amount of biological sample (100 μL) On the other hand, advanced data mining tools for untargeted analysis involving molecular networking were investigated and allowed to connect unknown compounds to “standard networks,” simplifying the annotation of new drugs. Different complementary analytical techniques, such as IR, Raman, GC-MS, LC-HRMS/MS and NMR were then applied to characterize the unknown substances and for the first time isobutyrylfentanyl (iBF) and 4-flouro- furanylfentanyl (4F-FuF) were characterized. Metabolic profiles of iBF and 4F-FuF were investigated by means of in silico, in vitro and in vivo studies, and their major metabolites were identified for the first time. For the first compound the N-dealkylated metabolite was identified as the main biomarker, while for 4F-FUF, the main metabolite was the dihydrodiol derivative, which was further N-dealkylated. And then, thanks to the pharmaco-toxicological studies, it was possible to study the distribution of the two drugs in the different tissues. The project was carried out in collaboration with the Scientific Investigations Department of the Arma dei Carabinieri of Rome. Unpayable support was also provided by Professor Marti, head of the toxicology unit of the University of Ferrara. Thanks to this work, the knowledge of this particular class of NPS and the associated metabolic processes has been expanded, providing a fundamental contribution to forensic science as well as pharmaco-toxicology. Furthermore, it was possible to make an effective contribution to the estimate of the real propagation of NPS in the European illegal market.