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Identification of AB-FUBINACA metabolites in human hepatocytes and urine using high-resolution mass spectrometry

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Abstract

AB-FUBINACA, N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide, is an indazole synthetic cannabinoid identified in drug seizures around the world. Few metabolism data are available, despite the need for human urinary markers to detect AB-FUBINACA intake. Our main objective was to identify suitable analytical targets by analyzing human hepatocyte incubation samples with high-resolution mass spectrometry (HRMS) and to confirm the results in authentic urine specimens. We also determined AB-FUBINACA’s metabolic stability in human liver microsomes (HLMs) and compared hepatocyte and urine results with in silico predictions. The metabolic stability of AB-FUBINACA was determined in pooled HLMs (1 µmol/l, up to 1 h). The metabolite profile of human hepatocytes (10 µmol/l, 1 and 3 h) and urine samples from two subjects were determined by HRMS using information-dependent tandem-mass spectrometry (MS-MS) acquisition. Data were analyzed with MetabolitePilot™ software utilizing different processing algorithms, including generic peak finding, mass defect filtering, neutral loss, and product ion filtering. In silico metabolite prediction was performed with MetaSite™ software. AB-FUBINACA’s half-life in HLMs was 62.6 ± 4.0 min. AB-FUBINACA produced 11 metabolites (2 glucuronides) in human hepatocytes and 10 were identified in authentic human urine. Major metabolic pathways were terminal amide hydrolysis, acyl glucuronidation and hydroxylation at the aminooxobutane moiety. Epoxidation followed by hydrolysis, hydroxylation at the indazole moiety and dehydrogenation were minor pathways. Defluorination did not occur. Seventeen first-generation metabolites were predicted in silico, of which seven were observed in vitro and eight in vivo. We recommend AB-FUBINACA carboxylic acid, hydroxy AB-FUBINACA carboxylic acid, dihydrodiol AB-FUBINACA and dihydrodiol AB-FUBINACA carboxylic acid as suitable urinary markers.

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Acknowledgments

This research was supported by the Intramural Research Program of the National Institute on Drug Abuse, National Institutes of Health. AB-FUBINACA was generously donated by the US Drug Enforcement Administration. Molecular Discovery Ltd kindly provided the MetaSite™ software.

Conflict of interest

There are no financial or other relations that could lead to a conflict of interest.

Ethical approval

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

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Authors and Affiliations

  1. Chemistry and Drug Metabolism, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, 251 Bayview Boulevard, Suite 200 Room 05A-721, Baltimore, MD, 21224, USA

    Marisol S. Castaneto, Ariane Wohlfarth, Karl B. Scheidweiler & Marilyn A. Huestis

  2. Program in Toxicology, University of Maryland, School of Medicine, Baltimore, MD, USA

    Marisol S. Castaneto

  3. SCIEX, Redwood City, CA, USA

    Shaokun Pang

  4. Department of Biotransformation, Bristol-Myers Squibb, Research and Development, Princeton, NJ, USA

    Mingshe Zhu

  5. Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Linköping University, Linköping, Sweden

    Robert Kronstrand

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

    Robert Kronstrand

Authors
  1. Marisol S. Castaneto
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  2. Ariane Wohlfarth
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  3. Shaokun Pang
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  4. Mingshe Zhu
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  5. Karl B. Scheidweiler
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  7. Marilyn A. Huestis
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Correspondence to Marilyn A. Huestis.

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Castaneto, M.S., Wohlfarth, A., Pang, S. et al. Identification of AB-FUBINACA metabolites in human hepatocytes and urine using high-resolution mass spectrometry. Forensic Toxicol 33, 295–310 (2015). https://doi.org/10.1007/s11419-015-0275-8

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  • DOI: https://doi.org/10.1007/s11419-015-0275-8

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