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.
