Untargeted Metabolic Profiling of 4-Fluoro-Furanylfentanyl and Isobutyrylfentanyl in Mouse Hepatocytes and Urine by Means of LC-HRMS

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 us 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 4-FFUF, 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 4-FFUF 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.

Simultaneous Quantification of 25 Fentanyl Derivatives and Metabolites in Oral Fluid by Means of Microextraction on Packed Sorbent and LC–HRMS/MS Analysis

“…With regard to liquid chromatographic separation, it was decided to work with a mixed sorbent column on the basis of previous experiences with this family of compounds [ 20 ]. The mobile phases were selected thanks to the expertise gained in the field of NPS analysis and opioids; in particular, phase A, constituted by 10 mM ammonium formate acidified with 0.1% of HCOOH, was evaluated to perform a better ionization of all target analytes in the H-ESI source, as previously described by Montesano et al [ 20 ], while phase B was chosen since in the literature it is widely demonstrated that mixing different percentages of MeOH and MeCN can improve the chromatographic separation of several types of analytes if compared with the separation provided by the use of single solvents [ 21 ]. Different ratios of the two solvents were tested and the necessary peak resolution was obtained by using MeCN–MeOH with a ratio 50:50 ( v / v ).…”

Untargeted metabolomics approaches to improve casework in clinical and forensic toxicology—“Where are we standing and where are we heading?”

“…Manier et al, for example, elucidated the in vitro metabolism of the two synthetic cathinones 4-MPD and 4-MEAP (identification of 5 and 6 metabolites, respectively) by untargeted screening (Manier, Schwermer, et al, 2020). Montesano et al (2021) investigated the metabolic profile of 4F-furanylfentanyl and isobutyrylfentanyl by in vitro and in vivo experiments, detecting and putatively annotating 20 biotransformation products of 4F-furanylfentanyl and 22 metabolites of isobutyrylfentanyl.…”

Phase I In Vitro Metabolic Profiling of the Synthetic Cannabinoid Receptor Agonists CUMYL-THPINACA and ADAMANTYL-THPINACA

“…High-resolution mass spectrometry (HR-MS) data analysis software is gaining importance, as in silico-assisted workflows enable higher throughput and are able to markedly facilitate metabolite identification [ 38 , 39 ]. In this study, data analysis was assisted by the Compound Discoverer (Thermo Fisher Scientific, Reinach, Switzerland) software, which has already been proven helpful for metabolite identification and structure elucidation in previously published studies [ 38 , 40 , 41 , 42 , 43 ].…”