A 25-year-old man visited his general practitioner with generalized muscle pain and severe abdominal pain including nausea, cramping and diarrhea, asking help for detoxification. The patient stated that his current complaints were the result of the use of high doses of N-piperidinyl etonitazene. After repeated intranasal administrations, he declared to have acquired a high tolerance for the substance. N-piperidinyl etonitazene (‘etonitazepipne’) represents a recent addition to the rapidly expanding class of 2-benzylbenzimidazole nitazene opioids. Here, we report on the full chemical and pharmacological characterization of N-piperidinyl etonitazene.

A serum and a urine sample from the patient, collected at the same time, and the online-sourced N-piperidinyl etonitazene powder were available for analysis. The powder and patient samples were analyzed with a liquid chromatography - high resolution mass spectrometry (LC-HRMS) screening method using data-dependent acquisition. Additionally, an accurate HRMS method using targeted selected ion monitoring was developed for the quantification of N-piperidinyl etonitazene. A cell-based bioassay was used to determine the extent of MOR activation in both the patient samples and in the powder. Furthermore, MOR binding affinity via radioligand binding assays, in vitro pharmacological characterization and in vivo effects (in rats) were examined.

Analysis of the powder via different analytical techniques (LC-HRMS, GC-MS, UHPLC-DAD, FT-IR) led to the unequivocal identification of N-piperidinyl etonitazene. The obtained concentrations in serum and urine from the patient were 1.21 and 0.51ng/mL, respectively. Via molecular networking of the obtained HRMS data, various urinary metabolites were identified, with O-dealkylation as the primary metabolism route. Further characterization revealed that N-piperidinyl etonitazene had a high MOR binding affinity in vitro. Moreover, a high potency (EC50=2.49nM; 53 and 6.7 times more potent than morphine and fentanyl, respectively) and relative efficacy (Emax=183% versus hydromorphone; reaching 80% and 36% higher relative efficacies than morphine and fentanyl, respectively) in activating human MOR in vitro was obtained. Despite its equal efficacy, N-piperidinyl etonitazene was still less potent than etonitazene, the most potent nitazene identified to date. The compound showed characteristic opioid-mediated behavior in rats, as evidenced by catalepsy, increased hot plate latency and hypothermia. Using the bioassay, we could derive that the activity exerted by the patient's serum concentration is equivalent to that exerted by 2.5–10ng/mL fentanyl or 10–25ng/mL hydromorphone, which is compatible with the declared tolerance.

N-piperidinyl etonitazene was identified for the first time in an online-sourced powder and in the serum and urine of a patient. Taken together, our findings indicate that etonitazepipne is a highly potent opioid with the potential to cause harm in users.