Tentative Identification of Etazene (Etodesnitazene) Metabolites in Rat Serum and Urine by Gas Chromatography–Mass Spectrometry and Accurate Mass Liquid Chromatography–Mass Spectrometry

Grigoryev, Andrej; Kavanagh, Pierce V.; Dowling, Geraldine; Rodin, I. A.

doi:10.1093/jat/bkac001

Cited by 7 publications

(16 citation statements)

References 15 publications

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“…In a recent report on N ‐piperidinyl etonitazene, it was already suggested that some metabolites would undergo glucuronide conjugation, but no actual conjugates were found 14 . The present findings are comparable with results obtained in a recent etazene metabolism study performed in zebrafish 6 and rats 21 . As metabolism of drugs may differ for zebrafish/rats and humans due to species differences in, for example, expression of metabolic enzymes, it is important to confirm this in human samples.…”

Section: Resultssupporting

confidence: 89%

“…However, two less abundant N-oxide metabolites were not detected in the human urine and conversely no phase II metabolites were found in the rat samples. 21 In the zebrafish, only the N-dealkylated metabolite M2 was detected, 6 which in the human urine sample was only present in a small amount. Next to the species difference, time between drug intake and sampling and differences in used analytical techniques should also be considered to explain these (small) discrepancies.…”

Section: Resultsmentioning

confidence: 99%

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Identification of etazene (etodesnitazene) metabolites in human urine by LC‐HRMS

Verougstraete

Verhaeghe²,

Germonpré³

et al. 2022

Drug Testing and Analysis

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Etazene (or etodesnitazene) is a novel and highly active synthetic opioid belonging to the rapidly evolving and emerging group of "nitazenes." Etazene metabolites were identified through analysis of a human urine sample. The sample was obtained from a 25-year-old man who attempted suicide by taking a new psychoactive substances (NPS) cocktail purchased online and was analyzed by ultrahigh performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). Etazene metabolites were predicted with BioTransformer 3.0, and the exact masses were added to the inclusion list. Eight possible metabolites were identified in the urine sample. N-and O-deethylation were identified as the predominant metabolism routes, resulting in M1 (O-deethylated etazene; most abundant metabolite based on the peak area), M2 (N-deethylated etazene), and M3 (N,O-dideethylated etazene) metabolites.Less abundant hydroxylated products of these deethylated metabolites and etazene were also found. Additionally, in the analysis without β-glucuronidase treatment, M1-and M3-glucuronide phase II metabolites were found. As N-and O-deethylated products seem to be the predominant urinary metabolites, the detection of these metabolites in urine can be useful to demonstrate etazene exposure.

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Section: Resultssupporting

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Identification of etazene (etodesnitazene) metabolites in human urine by LC‐HRMS

Verougstraete

Verhaeghe²,

Germonpré³

et al. 2022

Drug Testing and Analysis

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“…Grigoryev et al [ 18 ] reported eight tentative metabolites (M1–M3) and (M5–M9) of etazene ( Figure 1 ) in rat serum and urine by GC–MS and high-resolution accurate mass (HRAM) LC–MS. They tentatively identified these metabolites as products of N-deethylation, O-deethylation, hydroxylation, or N-oxidation of the benzimidazole moiety or a combination of these reactions.…”

Section: Resultsmentioning

confidence: 99%

“…To quantify the downstream metabolic products of SOs, high-performance liquid chromatography (HPLC) with mass spectrometry can be employed due to the technique’s inherent specificity, increased sensitivity, and higher throughput [ 17 ] compared to other methods of chromatography. The first report on etazene metabolites in urine was published in 2022 by Grigoryev et al [ 18 ]. Using gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–mass spectrometry (LC–MS) techniques, eight etazene metabolites were found in rat urine and serum by Grigoryev et al [ 18 ].…”

Section: Introductionmentioning

confidence: 99%

“…The first report on etazene metabolites in urine was published in 2022 by Grigoryev et al [ 18 ]. Using gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–mass spectrometry (LC–MS) techniques, eight etazene metabolites were found in rat urine and serum by Grigoryev et al [ 18 ]. These were tentatively identified as products of N-deethylation, O-deethylation, hydroxylation, or N-oxidation of the benzimidazole moiety and combinations of these processes.…”

Section: Introductionmentioning

confidence: 99%

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Identification of the Putative Binding Site of a Benzimidazole Opioid (Etazene) and Its Metabolites at µ-Opioid Receptor: A Human Liver Microsomal Assay and Systematic Computational Study

et al. 2023

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The synthetic benzimidazole opioid etazene (which has a 70-times higher analgesic activity than morphine), a recreational drug, has gained popularity as a novel psychoactive substance (NPS) on the illegal/darknet market; however, no experimental information is available at the molecular level on the binding mechanism and putative binding site of etazene and its metabolites at the µ-opioid receptor (MOR). In the present study, we investigated the metabolism of etazene in human liver microsomes using ultra-high-performance liquid chromatography–mass spectrometry (UHPLC–MS). We also explored the possibilities of MOR activation by etazene and its metabolites by studying their binding mechanisms and interaction profiles at an active-state MOR model via molecular docking, binding free energy calculations, and all-atom molecular dynamics (MD) simulations. The putative metabolites of etazene were also predicted using the ADMET Predictor 10.1. The molecular docking studies and free energy calculations showed that etazene and its metabolites (M1, M2, and M5–M7) exhibited strong predicted binding affinity at MOR and showed overlapped binding orientation with MOR-bound agonist BU72, which was co-crystallized in the MOR X-ray crystal structure (PDB ID: 5C1M). MD also confirmed the stability of the MOR–etazene and MOR–M6 complexes. These results suggest that etazene and its metabolites may act as strong MOR agonists, highlighting the necessity of experimental validation. The insights from this study, such as key interactions between etazene and its metabolites and the MOR, will allow authorities to predict potential analogs and clarify the target–protein interactions associated with this illicit substance, granting advanced or rapid reactions to confiscating or banning potential emerging drugs.

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Rapid screening of 2‐benzylbenzimidazole nitazene analogs in suspect counterfeit tablets using Raman, SERS, DART‐TD‐MS, and FT‐IR

Kimani

Kern

Lanzarotta

et al. 2023

Drug Testing and Analysis

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Developing methods to rapidly screen for novel synthetic 2-benzylbenzimidazole opioids, also known as nitazenes, has become increasingly important due to their high potency. These compounds have potency comparable or exceeding that of fentanyl by up to 10 times and have been implicated in approximately 5% of all drug overdose deaths in the United States in 2021. This paper details the authenticity determination of suspect tablets and the identification of three nitazene analogs (N-pyrrolidino etonitazene, isotonitazene, and etodesnitazene) in suspect tablets seized at a mail facility using Raman and surface-enhanced Raman scattering (SERS) with handheld devices, portable Fourier transform infrared spectrometer (FT-IR), and a direct analysis in realtime ambient ionization coupled to a thermal desorption unit and a mass spectrometer (DART-TD-MS). These methods are rapid and excellent for screening opioids in suspect tablets but could not fully determine the exact structure of some of the nitazene analogs present due to spectral similarities or similar fragmentation patterns.Liquid chromatography-mass spectrometry (LC-MS) confirmed the presence of these nitazene compounds in addition to other opioids/drugs that were in trace quantities. The quantitative high-performance liquid chromatography coupled with ultraviolet (HPLC-UV) detection experiments determined that the suspect tablets contained an average of 0.817 mg of N-pyrrolidino etonitazene per tablet. The results obtained reveal that the simultaneous deployment of these complementary and orthogonal portable analytical techniques as part of a workflow allows suspect tablets to be screened and nitazene-type drugs to be identified in suspect counterfeit tablets at remote sampling sites.

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Tentative Identification of Etazene (Etodesnitazene) Metabolites in Rat Serum and Urine by Gas Chromatography–Mass Spectrometry and Accurate Mass Liquid Chromatography–Mass Spectrometry

Cited by 7 publications

References 15 publications

Identification of etazene (etodesnitazene) metabolites in human urine by LC‐HRMS

Identification of etazene (etodesnitazene) metabolites in human urine by LC‐HRMS

Identification of the Putative Binding Site of a Benzimidazole Opioid (Etazene) and Its Metabolites at µ-Opioid Receptor: A Human Liver Microsomal Assay and Systematic Computational Study

Rapid screening of 2‐benzylbenzimidazole nitazene analogs in suspect counterfeit tablets using Raman, SERS, DART‐TD‐MS, and FT‐IR

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