Analytical and Bioanalytical Chemistry

, Volume 406, Issue 29, pp 7549–7560 | Cite as

Identification and quantification of cannabinoids in Cannabis sativa L. plants by high performance liquid chromatography-mass spectrometry

  • Oier Aizpurua-OlaizolaEmail author
  • Jone Omar
  • Patricia Navarro
  • Maitane Olivares
  • Nestor Etxebarria
  • Aresatz Usobiaga
Paper in Forefront


High performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) has been successfully applied to cannabis plant extracts in order to identify cannabinoid compounds after their quantitative isolation by means of supercritical fluid extraction (SFE). MS conditions were optimized by means of a central composite design (CCD) approach, and the analysis method was fully validated. Six major cannabinoids [tetrahydrocannabinolic acid (THCA), tetrahydrocannabinol (THC), cannabidiol (CBD), tetrahydrocannabivarin (THCV), cannabigerol (CBG), and cannabinol (CBN)] were quantified (RSD < 10%), and seven more cannabinoids were identified and verified by means of a liquid chromatograph coupled to a quadrupole-time-of-flight (Q-ToF) detector. Finally, based on the distribution of the analyzed cannabinoids in 30 Cannabis sativa L. plant varieties and the principal component analysis (PCA) of the resulting data, a clear difference was observed between outdoor and indoor grown plants, which was attributed to a higher concentration of THC, CBN, and CBD in outdoor grown plants.

Graphical Abstract

Representative figure of the identification and quantification process of cannabinoids


Cannabinoid analysis Liquid chromatography Mass spectrometry Fingerprinting 



The authors are grateful to GANJAZZ ART CLUB for supplying the plant material used in this study, to IDOKI SCF S.L. for their support in the SFE extraction, and to the Central Service of Analysis of the University of the Basque Country for the UPLC-QToF analysis. O.A. and J.O. are grateful to the Basque Government and the University of the Basque Country for their PhD fellowship.


  1. 1.
    Clarke RC, Watson DP (2007) Cannabis and natural cannabis medicines. In: ElSohly MA (ed) Marijuana and the cannabinoids. Humana Press, Totowa, pp 1–15Google Scholar
  2. 2.
    Hazekamp A (2007) Cannabis; extracting the medicine, PhD thesis, Universiteit Leiden, The NetherlandsGoogle Scholar
  3. 3.
    ElSohly MA, Slade D (2005) Chemical constituents of marijuana: the complex mixture of natural cannabinoids. Life Sci 78:539–548CrossRefGoogle Scholar
  4. 4.
    Ahmed SA, Ross SA, Slade D, Radwan MM, Zulfiqar F, ElSohly MA (2008) Cannabinoid ester constituents from high-potency Cannabis sativa. J Nat Prod 71:536–542CrossRefGoogle Scholar
  5. 5.
    Radwan MM, ElSohly MA, Slade D, Ahmed SA, Khan IA, Ross SA (2009) Biologically active cannabinoids from high-potency Cannabis sativa. J Nat Prod 72:906–911CrossRefGoogle Scholar
  6. 6.
    Russo EB (2007) History of cannabis and its preparations in saga, science, and sobriquet. Chem Biodivers 4:1614–1648CrossRefGoogle Scholar
  7. 7.
    Grotenhermen F (2002) Effects of cannabis and the cannabinoids. In: Grotenhermen F, Russo E (eds) Cannabis and cannabinoids-pharmacology, toxicology, and therapeutic potential. Haworth Press, New York, pp 55–65Google Scholar
  8. 8.
    Fishbein M, Gov S, Assaf F, Gafni M, Keren O, Sarne Y (2012) Long-term behavioral and biochemical effects of an ultra-low dose of Δ9-tetrahydrocannabinol (THC): neuroprotection and ERK signaling. Exp Brain Res 221:437–448CrossRefGoogle Scholar
  9. 9.
    Schweinsburg AD, Brown SA, Tapert SF (2008) The influence of marijuana use on neurocognitive functioning in adolescents. Curr Drug Abus Rev 1:99–111CrossRefGoogle Scholar
  10. 10.
    Lisdahl KM, Wright NE, Medina-Kirchner C, Maple KE, Shollenbarger S (2014) Considering the cannabis: the effects of regular cannabis use on neurocognition in adolescents and young adults. Curr Addict Rep 1:144–156CrossRefGoogle Scholar
  11. 11.
    Consroe P (1998) Brain cannabinoid systems as targets for the therapy of neurological disorders. Neurobiol Dis 5:534–551CrossRefGoogle Scholar
  12. 12.
    McPartland JM, Russo EB (2001) Cannabis and cannabis extracts: greater than the sum of their parts? J Cannabis Ther 1:103–132CrossRefGoogle Scholar
  13. 13.
    Mechoulam R, Parker LA, Gallity R (2002) Cannabidiol: an overview of some pharmacological aspects. J Clin Pharmacol 42:11S–19SCrossRefGoogle Scholar
  14. 14.
    Izzo AA, Borrelli F, Capasso R, Di Marzo V, Mechoulam R (2009) Non-psychotropic plant cannabinoids: new therapeutic opportunities from an ancient herb. Trends Pharmacol Sci 30:515–527CrossRefGoogle Scholar
  15. 15.
    Bergamaschi MM, Queiroz RH, Chagas MH, De Oliveira DC, De Martinis BS, Kapczinski F, Quevedo J, Roesler R, Schröder N, Nardi AE, Martín-Santos R, Hallak JE, Zuardi AW, Crippa JA (2011) Cannabidiol reduces the anxiety induced by simulated public speaking in treatment-naïve social phobia patients. Neuropsychopharmacology 36:1219–1226CrossRefGoogle Scholar
  16. 16.
    Morgan CJ, Das RK, Joye A, Vurran HV, Kamboj SK (2011) Cannabidiol reduces cigarette consumption in tobacco smokers: preliminary findings. Addict Behav 38:2433–2436CrossRefGoogle Scholar
  17. 17.
    Ryan D, Drysdale AJ, Pertwee RG, Platt B (2006) Differential effects of cannabis extracts and pure plant cannabinoids on hippocampal neurons and glia. Neurosci Lett 408:236–341CrossRefGoogle Scholar
  18. 18.
    Farrimond JA, Whalley BJ, Williams CM (2012) Non-Δ 9tetrahydrocannabinol phytocannabinoids stimulate feeding in rats. Behav Pharmacol 23:113–117CrossRefGoogle Scholar
  19. 19.
    Roser P, Gallinat J, Weinberg G, Juckel G, Gorynia I, Stadelmann AM (2009) Psychomotor performance in relation to acute oral administration of Δ9-tetrahydrocannabinol and standardized cannabis extract in healthy human subjects. Eur Arch Psychiatry Clin Neurosci 259:284–292CrossRefGoogle Scholar
  20. 20.
    Whalley BJ, Wilkinson JD, Williamson EM, Constanti A (2004) A novel component of cannabis extracts potentiates excitatory synaptic transmission in rat olfactory cortex in vitro. Neurosci Lett 365:58–63CrossRefGoogle Scholar
  21. 21.
    Wilkinson JD, Whalley BJ, Baker D, Pryce G, Constanti A, Gibbons S, Williamson EM (2003) Medicinal Cannabis: is Δ9-tetrahydrocannabinol necessary for all its effects? J Pharm Pharmacol 55:1687–1694CrossRefGoogle Scholar
  22. 22.
    Hazekamp A, Fischedick JT (2012) Cannabis—from cultivar to chemovar. Drug Test Anal 4:660–667CrossRefGoogle Scholar
  23. 23.
    Fischedick JT, Hazekamp A, Erkelens T, Choi YH, Verpoorte R (2010) Metabolic fingerprinting of Cannabis sativa L., cannabinoids and terpenoids for chemotaxonomic and drug standardization purposes. Phytochemistry 71:2058–2073CrossRefGoogle Scholar
  24. 24.
    Herrero M, Castro-Puyana M, Mendiola JA, Ibañez E (2013) Compressed fluid for the extraction of bioactive compounds. Trends Anal Chem 43:67–83CrossRefGoogle Scholar
  25. 25.
    Guiochon G, Tarafder A (2011) Fundamental challenges and opportunities for preparative supercritical fluid chromatography. J Chromatogr A 1218:1037–1114CrossRefGoogle Scholar
  26. 26.
    Omar J, Olivares M, Alzaga M, Etxebarria N (2013) Optimization and characterization of marihuana extracts obtained by supercritical fluid extraction and focused ultrasound extraction and retention time locking GC-MS. J Sep Sci 36:1397–1404CrossRefGoogle Scholar
  27. 27.
    Grauwiler SB, Scholer A, Drewe J (2007) Development of a LC/MS/MS method for the analysis of cannabinoids in human EDTA-plasma and urine after small doses of cannabis sativa extracts. J Chromatogr B 850:515–522CrossRefGoogle Scholar
  28. 28.
    De Backer B, Debrus B, Lebrun P, Theunis L, Dubois N, Decock L, Verstraete A, Hubert P, Charlier C (2009) Innovative development and validation of an HPLC/DAD method for the qualitative and quantitative determination of major cannabinoids in cannabis plant material. J Chromatogr B 877:4115–4124CrossRefGoogle Scholar
  29. 29.
    Gröger T, Schäffer M, Pütz M, Ahrens B, Drew K, Eschner M, Zimmermman R (2008) Application of two-dimensional gas chromatography combined with pixel-based chemometric processing for the chemical profiling of illicit drug samples. J Chromatogr A 1200:8–16CrossRefGoogle Scholar
  30. 30.
    Omar J, Olivares M, Amigo JM, Etxebarria N (2014) Resolution of co-eluting compounds of Cannabis sativa in comprehensive two-dimensional gas chromatography/mass spectrometry detection with multivariate curve resolution-alternating least squares. Talanta 121:273–280CrossRefGoogle Scholar
  31. 31.
    Bäckström B, Cole MD, Carrott MJ, Jones DC, Davidson G, Coleman K (1997) A preliminary study of the analysis of Cannabis by supercritical fluid chromatography with atmospheric pressure chemical ionisation mass spectroscopic detection. Science and Justice. J Forensic Sci Soc 37:91–97Google Scholar
  32. 32.
    Lacorte S, Fernandez-Alba AR (2006) Time of flight mass spectrometry applied to the liquid chromatographic analysis of pesticides in water and food. Mass Spectrom Rev 25:866–880CrossRefGoogle Scholar
  33. 33.
    Zhou JL, Qi LW, Li P (2009) Herbal medicine analysis by liquid chromatography/time-of-flight mass spectrometry. J Chromatogr A 1216:7582–7594CrossRefGoogle Scholar
  34. 34.
    Bobeldijk I, Viessers JPC, Kearney G, Major H, Van Leerdam JA (2001) Screening and identification of unknown contaminants in water with liquid chromatography and quadrupole-orthogonal acceleration-timeof-flight tandem mass spectrometry. J Chromatogr A 929:63–74CrossRefGoogle Scholar
  35. 35.
    Stolker AAM, Van Schoonhoven J, de Vries AJ, Bobeldijk-Pastorova I, Vaes WHJ, Van den Berg R (2004) Determination of cannabinoids in cannabis products using liquid chromatography-ion trap mass spectrometry. J Chromatogr A 1058:143–151CrossRefGoogle Scholar
  36. 36.
    Kneisel S, Auwärter V (2012) Analysis of 30 synthetic cannabinoids in serum by liquid chromatography-electrospray ionization tandem mass spectrometry after liquid–liquid extraction. J Mass Spectrom 47:825–835CrossRefGoogle Scholar
  37. 37.
    Brenneisen R (2007) Chemistry and analysis of phytocannabinoids. In: ElSohly MA (ed) Marijuana and the cannabinoids. Humana Press, Totowa, pp 17–48Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Oier Aizpurua-Olaizola
    • 1
    Email author
  • Jone Omar
    • 1
  • Patricia Navarro
    • 1
  • Maitane Olivares
    • 1
  • Nestor Etxebarria
    • 1
  • Aresatz Usobiaga
    • 1
  1. 1.Analytical Chemistry DepartmentUniversity of the Basque Country (UPV/EHU)LeioaSpain

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