Investigation of the In Vitro and In Vivo Metabolism and μ‐Opioid Receptor Affinity of the Nitazene N ‐Pyrrolidino Fluetonitazene

ABSTRACT N ‐pyrrolidino fluetonitazene ( N ‐pyrrolidino‐4′‐(2‐fluoroethoxy) nitazene) also known as fluetonitazepyne, is the fluorinated analogue of etonitazepyne, belonging to the group of new synthetic opioids (NSOs), which are among the fastest‐growing classes of new psychoactive substances. In the present study, the N ‐pyrrolidino fluetonitazene metabolism was investigated in vitro using pooled human liver microJsomes (pHLM) and in authentic samples from a forensic postmortem case. Qualitative analysis was performed using liquid chromatography–high‐resolution tandem mass spectrometry (LC‐HR‐MS/MS). Confirmation and semiquantitative analysis of N ‐pyrrolidino fluetonitazene in urine and blood were carried out using liquid chromatography tandem mass spectrometry (LC–MS/MS). The μ‐opioid (MOR) receptor affinity of N ‐pyrrolidino fluetonitazene was determined using an LC–MS/MS‐based competitive binding assay. In total, eight different metabolites for N ‐pyrrolidino fluetonitazene were tentatively identified in vitro in pHLM incubations of which three ( M2 O ‐dealkylation, M6 oxidative deamination and M8 carboxylation to N ‐butanoic acid) were also found in the postmortem urine sample. In the blood samples, only one metabolite was found ( M9 formed by N ‐acetylation of 5‐aminofluetodesnitazene). This metabolite was only observed in the in vivo samples and was the most abundant metabolite in blood and urine samples. We also detected metabolite M2 (formed by 4′‐hydroxylation), which is common in the metabolism of other nitazepyne‐type substances. For the confirmation of N ‐pyrrolidino fluetonitazene, we recommend including M8 and M9 as analytical target compounds, since they are specific N ‐pyrrolidino fluetonitazene metabolites. Furthermore, we demonstrated that the LC–MS/MS‐based MOR receptor affinity assay yields valid results for N ‐pyrrolidino fluetonitazene that are consistent with those obtained using established radioligand‐based methods.