Abstract
The diffusion of new psychoactive substances (NPS) is highly dynamic and the available substances change over time, resulting in forensic laboratories becoming highly engaged in NPS control. In order to manage NPS diffusion, efficient and innovative legal responses have been provided by several nations. Metabolic profiling is also part of the analytical fight against NPS, since it allows to identify the biomarkers of drug intake which are needed for the development of suitable analytical methods in biological samples. We have recently reported the characterization of two new analogs of fentanyl, i.e., 4-fluoro-furanylfentanyl (4F-FUF) and isobutyrylfentanyl (iBF), which were found for the first time in Italy in 2019; 4F-FUF was identified for the first time in Europe and was notified to the European Early Warning System. The goal of this study was the characterization of the main metabolites of both drugs by in vitro and in vivo experiments. To this end, incubation with mouse hepatocytes and intraperitoneal administration to mice were carried out. Samples were analyzed by means of liquid chromatography-high resolution mass spectrometry (LC–HRMS), followed by untargeted data evaluation using Compound Discoverer software with a specific workflow, designed for the identification of the whole metabolic pattern, including unexpected metabolites. Twenty metabolites were putatively annotated for 4F-FUF, with the dihydrodiol derivative appearing as the most abundant, whereas 22 metabolites were found for iBF, which was mainly excreted as nor-isobutyrylfentanyl. N-dealkylation of 4F-FUF dihydrodiol and oxidation to carbonyl metabolites for iBF were also major biotransformations. Despite some differences, in general there was a good agreement between in vitro and in vivo samples.
Highlights
The use of new psychoactive substances (NPSs), introduced as legal alternatives for controlled drugs [1], has become a worldwide phenomenon since the early 90s and it has been exacerbated by the increase in online selling points and sales [2]
Materials and Methods, diclofenac and testosterone were used as positive controls respectively for phase I and phase II metabolism to ensure that proper incubation conditions were maintained: hydroxydiclofenac was observed in the diclofenac positive control, confirming phase I hepatocyte metabolic activity, whereas testosterone glucuronide and sulphate were detected in the testosterone control, showing that phase II metabolic activity occurred
The metabolic profiles of iBF and 4F-FUF were investigated in this study
Summary
The use of new psychoactive substances (NPSs), introduced as legal alternatives for controlled drugs [1], has become a worldwide phenomenon since the early 90s and it has been exacerbated by the increase in online selling points and sales [2]. The NPS market is highly dynamic and the available substances change over time, resulting in forensic laboratories becoming highly engaged in the fight against NPSs. From an analytical point of view, NPS detection in both seizures and biological samples is a challenge due to the lack of analytical standards, library spectra and pharmacokinetic information. Characterization of drug metabolites is usually performed using in vitro and/or in vivo studies, which can be assisted by in-silico prediction tools to make LC-HRMS data analysis easier [7]. Controlled human studies would be best suited but are not practicable for ethical reasons and the lack of preclinical safety data
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