Forensic Toxicology

, Volume 35, Issue 1, pp 20–32 | Cite as

In vitro and in vivo human metabolism of a new synthetic cannabinoid NM-2201 (CBL-2201)

  • Xingxing Diao
  • Jeremy Carlier
  • Mingshe Zhu
  • Shaokun Pang
  • Robert Kronstrand
  • Karl B. Scheidweiler
  • Marilyn A. Huestis
Original Article


In 2014, NM-2201 (CBL-2201), a novel synthetic cannabinoid (SC), was detected by scientists at Russian and US laboratories. It has been already added to the list of scheduled drugs in Japan, Sweden and Germany. Unfortunately, no human metabolism data are currently available, which makes it challenging to confirm its intake, especially given that all SCs investigated thus far have been found to be extensively metabolized. The present study aims to recommend appropriate marker metabolites by investigating NM-2201 metabolism in human hepatocytes, and to confirm the results in authentic human urine specimens. For the metabolic stability assay, 1 µM NM-2201 was incubated in human liver microsomes (HLMs) for up to 1 h; for metabolite profiling, 10 µM of NM-2201 was incubated in human hepatocytes for 3 h. Two authentic urine specimens from NM-2201-positive cases were subjected to β-glucuronidase hydrolysis prior to analysis. The identification of metabolites in hepatocyte samples and urine specimens was achieved with high-resolution mass spectrometry via information-dependent acquisition. NM-2201 was quickly metabolized in HLMs, with an 8.0-min half-life. In human hepatocyte incubation samples, a total of 13 NM-2201 metabolites were identified, generated mainly from ester hydrolysis and further hydroxylation, oxidative defluorination and subsequent glucuronidation. M13 (5-fluoro PB-22 3-carboxyindole) was found to be the major metabolite. In the urine specimens, the parent drug NM-2201 was not detected; M13 was the predominant metabolite after β-glucuronidase hydrolysis. Therefore, based on the results of our study, we recommend M13 as a suitable urinary marker metabolite for confirming NM-2201 and/or 5F-PB-22 intake.


NM-2201 CBL-2201 Synthetic cannabinoid In vitro human hepatocyte metabolism High-resolution mass spectrometry Authentic human urine specimen 



This research is supported by the Intramural Research Program of the National Institute on Drug Abuse, National Institutes of Health. NM-2201 was generously donated by the US Drug Enforcement Administration. We also appreciate help from Dr. Ariane Wohlfarth in performing the HLMs metabolic stability assays.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.


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Copyright information

© Japanese Association of Forensic Toxicology and Springer Japan (outside the USA) 2016

Authors and Affiliations

  • Xingxing Diao
    • 1
  • Jeremy Carlier
    • 1
  • Mingshe Zhu
    • 2
  • Shaokun Pang
    • 3
  • Robert Kronstrand
    • 4
    • 5
  • Karl B. Scheidweiler
    • 1
  • Marilyn A. Huestis
    • 1
    • 6
  1. 1.Chemistry and Drug Metabolism Section, Clinical Pharmacology and Therapeutics Branch, Intramural Research Program, National Institute on Drug AbuseNational Institutes of HealthBaltimoreUSA
  2. 2.Department of BiotransformationBristol-Myers Squibb, Research and DevelopmentPrincetonUSA
  3. 3.SCIEXRedwood CityUSA
  4. 4.Department of Forensic Genetics and Forensic ToxicologyNational Board of Forensic MedicineLinköpingSweden
  5. 5.Department of Drug ResearchUniversity of LinköpingLinköpingSweden
  6. 6.University of Maryland School of MedicineBaltimoreUSA

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