The AAPS Journal

, Volume 17, Issue 3, pp 660–677 | Cite as

Pentylindole/Pentylindazole Synthetic Cannabinoids and Their 5-Fluoro Analogs Produce Different Primary Metabolites: Metabolite Profiling for AB-PINACA and 5F-AB-PINACA

  • Ariane Wohlfarth
  • Marisol S. Castaneto
  • Mingshe Zhu
  • Shaokun Pang
  • Karl B. Scheidweiler
  • Robert Kronstrand
  • Marilyn A. Huestis
Research Article


Whereas non-fluoropentylindole/indazole synthetic cannabinoids appear to be metabolized preferably at the pentyl chain though without clear preference for one specific position, their 5-fluoro analogs’ major metabolites usually are 5-hydroxypentyl and pentanoic acid metabolites. We determined metabolic stability and metabolites of N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-pentyl-1H-indazole-3-carboxamide (AB-PINACA) and 5-fluoro-AB-PINACA (5F-AB-PINACA), two new synthetic cannabinoids, and investigated if results were similar. In silico prediction was performed with MetaSite (Molecular Discovery). For metabolic stability, 1 μmol/L of each compound was incubated with human liver microsomes for up to 1 h, and for metabolite profiling, 10 μmol/L was incubated with pooled human hepatocytes for up to 3 h. Also, authentic urine specimens from AB-PINACA cases were hydrolyzed and extracted. All samples were analyzed by liquid chromatography high-resolution mass spectrometry on a TripleTOF 5600+ (AB SCIEX) with gradient elution (0.1% formic acid in water and acetonitrile). High-resolution full-scan mass spectrometry (MS) and information-dependent acquisition MS/MS data were analyzed with MetabolitePilot (AB SCIEX) using different data processing algorithms. Both drugs had intermediate clearance. We identified 23 AB-PINACA metabolites, generated by carboxamide hydrolysis, hydroxylation, ketone formation, carboxylation, epoxide formation with subsequent hydrolysis, or reaction combinations. We identified 18 5F-AB-PINACA metabolites, generated by the same biotransformations and oxidative defluorination producing 5-hydroxypentyl and pentanoic acid metabolites shared with AB-PINACA. Authentic urine specimens documented presence of these metabolites. AB-PINACA and 5F-AB-PINACA produced suggested metabolite patterns. AB-PINACA was predominantly hydrolyzed to AB-PINACA carboxylic acid, carbonyl-AB-PINACA, and hydroxypentyl AB-PINACA, likely in 4-position. The most intense 5F-AB-PINACA metabolites were AB-PINACA pentanoic acid and 5-hydroxypentyl-AB-PINACA.


5-fluoro-AB-PINACA AB-PINACA in silico prediction metabolism synthetic cannabinoids 









Counts per second


Cytochrome P450


Extraction ratio


Electrospray ionization


Food and Drug Administration


Human liver microsomes


High-resolution mass spectrometry


Information-dependent acquisition


Liquid chromatography-mass spectrometry


Mass defect filter


Mass spectrometry


Molecular weight


Nicotinamide adenine dinucleotide phosphate reduced form


Novel psychoactive substances




Quadrupole/time of flight




Time of flight



This research was supported by the Intramural Research Program of the National Institute on Drug Abuse, National Institutes of Health. AB-PINACA and 5F-AB-PINACA were generously donated by the Drug Enforcement Administration. Molecular Discovery kindly provided the MetaSite software.

Conflict of Interest


Supplementary material

12248_2015_9721_Fig8_ESM.jpg (450 kb)
Supplementary Fig. A

Proposed human hepatic metabolic pathway of AB-PINACA; ambiguous assignments of functional groups are shown as Markush structures (JPEG 449 kb)

12248_2015_9721_MOESM1_ESM.eps (1.9 mb)
High resolution image (EPS 1899 kb)
12248_2015_9721_Fig9_ESM.jpg (425 kb)
Supplementary Fig. B

Proposed human hepatic metabolic pathway of 5F-AB-PINACA; ambiguous assignments of functional groups are shown as Markush structures (JPEG 424 kb)

12248_2015_9721_MOESM2_ESM.eps (1.8 mb)
High resolution image (EPS 1840 kb)
12248_2015_9721_MOESM3_ESM.docx (49 kb)
Supplementary Table 1 (DOCX 48.5 kb)
12248_2015_9721_MOESM4_ESM.docx (49 kb)
Supplementary Table 2 (DOCX 49 kb)
12248_2015_9721_MOESM5_ESM.docx (13 kb)
Supplementary Table 3 (DOCX 13 kb)


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Copyright information

© American Association of Pharmaceutical Scientists 2015

Authors and Affiliations

  • Ariane Wohlfarth
    • 1
  • Marisol S. Castaneto
    • 1
  • Mingshe Zhu
    • 2
  • Shaokun Pang
    • 3
  • Karl B. Scheidweiler
    • 1
  • Robert Kronstrand
    • 4
    • 5
  • Marilyn A. Huestis
    • 1
  1. 1.Chemistry and Drug Metabolism, Intramural Research Program, National Institute on Drug AbuseNational Institutes of HealthBaltimoreUSA
  2. 2.Department of BiotransformationBristol-Myers Squibb, Research and DevelopmentPrincetonUSA
  3. 3.AB SCIEXRedwood CityUSA
  4. 4.Department of Forensic Genetics and Forensic ToxicologyNational Board of Forensic MedicineLinköpingSweden
  5. 5.Division of Drug ResearchLinköping UniversityLinköpingSweden

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