Identification of the new synthetic opioid N-piperidinyl etonitazene (etonitazepipne) in postmortem alternative matrices

The new synthetic opioid N-piperidinyl etonitazene, commonly referred to as etonitazepipne, belongs to the benzimidazole group, and was formally notified in Europe to the European Monitoring Centre for Drugs and Drug Addiction's (EMCDDA) Early Warning System (EWS) on NPS in February 2021 [1]. To date, there are very few published references in the literature regarding the molecule's detection in biological matrices. Furthermore, no scientific article mentions the identification of etonitazepipne in alternative biological matrices. These alternative matrices, often overlooked, play a complementary role to conventional matrices in toxicological analyses. Alternative matrices, including keratin matrices are extremely important for forensic toxicology because nails and hair have been shown to accumulate substances within them consistently over time [2-3]. The affinity for the MOR receptor of etonitazepipne is almost equivalent to that of morphine and slightly higher than that of fentanyl [4]. The molecule is more than 50 times more potent and 80% more effective than morphine in activating the receptor. As a result, etonitazepipne can be identified as a substance hazardous to human health. The fact that the molecule is highly effective makes it plausible that even a small dose of the substance is sufficient to achieve the narcotic effect. This makes the detection process difficult in biological matrices such as blood and urine. This presentation aims to comment on the finding of etonitazepipne in postmortem samples of hair, nail, and vitreous humor in a case where the cause of death was ascribed to fatal etonitazepipne intoxication. Samples taken during the judicial autopsy were stored in special containers at temperature of 4 °C until analysis. Hair and nail were washed with H2O, dried and cut into small fragments. A validated method for the sought of new synthetic opioids has been followed for the extraction [5]. Briefly, samples were fortified with methanol and incubated at 52 °C for 24 and 72 h for hair and nail, respectively. Then, the samples were centrifuged, the supernatant of each was recovered and evaporated. The extracts were resuspended with 50 µL of phase B and submitted to the analysis by liquid chromatography at high-resolution coupled with the Thermo Exactive Plus Orbitrap (UHPLC–HR-MS). Vitreous humor was subjected to a liquid/liquid extraction. The pH of the sample was adjusted at 9 and a methanol-chloroform solution (50/50 v/v) was added. After extraction, the sample was brought to dryness and resuspended with 50 µL of organic phase B for subsequent chromatographic injection. The finding of the molecule in these matrices leads to several considerations. Since its presence in the vitreous humor can be traced back to the presence of etonitazepipne in the blood, as it is known that morphine-like compounds are present in the vitreous humor in lower amounts than in the blood [6,7]. It follows that positivity for the drug in this matrix could be indicative of acute or fatal intoxication. Furthermore, the presence of the substance in keratin matrices indicates repeated exposures over time that have led to the accumulation of the molecule.