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Metabolism of the benzodiazepines norflurazepam, flurazepam, fludiazepam and cinolazepam by human hepatocytes using high-resolution mass spectrometry and distinguishing their intake in authentic urine samples

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Abstract

Purpose

Norflurazepam, also a metabolite of other prescription benzodiazepines, appeared on the new psychoactive substances (NPS) drug market recently, complicating the interpretation of NPS findings. The aims of the study were to tentatively identify potential metabolites of norflurazepam and structural analogues (flurazepam, fludiazepam and cinolazepam) produced by hepatocytes and in authentic human samples and to discuss the possibility to differentiate drug consumption.

Methods

Each drug (5 µmol/L) was incubated with pooled human hepatocytes, and metabolites were identified using liquid chromatography–high-resolution mass spectrometry (LC–HRMS). Similarly, urine with/without hydrolysis and blood/serum from three flurazepam and seven presumptive norflurazepam cases were analysed by LC–HRMS.

Results

No metabolites were detected for norflurazepam in hepatocytes, but six metabolites for flurazepam, two for fludiazepam and three for cinolazepam were found. In human specimens collected after flurazepam ingestion, a total of eight metabolites, in good agreement with hepatocyte metabolites, were detected. In specimens of presumptive norflurazepam intake, norflurazepam and its metabolites (four hydroxy metabolites and one glucuronide of a hydroxy metabolite) were found.

Conclusions

Based on the results, hydroxy metabolites for norflurazepam, N-(hydroxyethyl), desethyl and didesethyl for flurazepam, hydroxy for fludiazepam and glucuronides and N-(hydroxyethyl) for cinolazepam are recommended for monitoring. While flurazepam, fludiazepam and cinolazepam were metabolised by hepatocytes at side chain, norflurazepam was not, which seems to indicate that hepatocytes have difficulty in modifying the benzene/diazepine rings of some 1,4-benzodiazepines. As for confirming the intake of norflurazepam, the urine ratio of 3-hydroxy-norflurazepam/norflurazepam might be the key; a high ratio might be correlated to norflurazepam intake, thereby enabling the differentiation.

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Acknowledgements

This work was supported by the Vinnova (the EUROSTARS Psychomics project, Grant Number 10628) and Strategic Research Area in Forensic Sciences (Strategiområdet forensiska vetenskaper, grant number 2016:7) at Linköping University.

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Authors and Affiliations

  1. Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Artillerigatan 12, 587 58, Linköping, Sweden

    Shimpei Watanabe, Svante Vikingsson, Henrik Gréen & Robert Kronstrand

  2. Division of Drug Research, Department of Medical and Health Sciences, Linköping University, Linköping, Sweden

    Anna Åstrand, Henrik Gréen & Robert Kronstrand

  3. Institute of Forensic Medicine, Forensic Toxicology, Medical Center, University of Freiburg, Freiburg, Germany

    Volker Auwärter

  4. Faculty of Medicine, University of Freiburg, Freiburg, Germany

    Volker Auwärter

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  1. Shimpei Watanabe
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Correspondence to Shimpei Watanabe.

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All procedures performed in studies involving human participants were in accordance with the ethical standards of the regional ethics committee in Linköping (Approval Number 2018-186/31) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

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Watanabe, S., Vikingsson, S., Åstrand, A. et al. Metabolism of the benzodiazepines norflurazepam, flurazepam, fludiazepam and cinolazepam by human hepatocytes using high-resolution mass spectrometry and distinguishing their intake in authentic urine samples. Forensic Toxicol 38, 79–94 (2020). https://doi.org/10.1007/s11419-019-00488-9

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