Analytical and Bioanalytical Chemistry

, Volume 408, Issue 13, pp 3571–3591 | Cite as

Identification of main human urinary metabolites of the designer nitrobenzodiazepines clonazolam, meclonazepam, and nifoxipam by nano-liquid chromatography-high-resolution mass spectrometry for drug testing purposes

  • Markus R. MeyerEmail author
  • Madeleine Pettersson Bergstrand
  • Anders Helander
  • Olof Beck
Research Paper


Among the new psychoactive substances (NPS), so-called designer benzodiazepines have become of particular importance over the last 2 years, due to their increasing availability on the internet drug market. Therapeutically used nitrobenzodiazepines such as flunitrazepam are known to be extensively metabolized via N-dealkylation to active metabolites and via nitro reduction to the 7-amino compounds. The aim of the present work was to tentatively identify phase I and II metabolites of the latest members of this class appearing on the NPS market, clonazolam, meclonazepam, and nifoxipam, in human urine samples. Nano-liquid chromatography-high-resolution mass spectrometry was used to provide data about their detectability in urine. Data revealed that clonazolam and meclonazepam were extensively metabolized and mainly excreted as their amino and acetamino metabolites. Nifoxipam was also extensively metabolized, but instead mainly excreted as the acetamino metabolite and a glucuronic acid conjugate of the parent. Based on analysis of human urine samples collected in cases of acute intoxication within the Swedish STRIDA project, and samples submitted for routine drug testing, the most abundant metabolites and good targets for urine drug testing were 7-aminoclonazolam for clonazolam, 7-acetaminomeclonazepam for meclonazepam, and 7-acetaminonifoxipam for nifoxipam.


New psychoactive substances NPS Metabolism Benzodiazepines Urine Mass spectrometry 



The STRIDA project was supported by grants from the Public Health Agency of Sweden.

Compliance with ethical standards

The study was conducted in accordance with the Helsinki Declaration. The use of de-identified leftover volumes from the routine samples pool (No. 00-230) and the STRIDA project (No. 2013/116–31/2) were approved by the regional ethical review board.

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

216_2016_9439_MOESM1_ESM.pdf (112 kb)
ESM 1 (PDF 112 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Markus R. Meyer
    • 1
    • 2
    Email author
  • Madeleine Pettersson Bergstrand
    • 1
  • Anders Helander
    • 1
    • 3
  • Olof Beck
    • 1
    • 3
  1. 1.Karolinska Institutet, Department of Laboratory MedicineStockholmSweden
  2. 2.Department of Pharmacology and PharmacoepidemiologyHeidelberg University HospitalHeidelbergGermany
  3. 3.Department of Clinical PharmacologyKarolinska University LaboratoryStockholmSweden

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