Planta Med 2023; 89(06): 683-696
DOI: 10.1055/a-1962-8165
Natural Product Chemistry & Analytical Studies
Original Papers

Development and Validation of a GC-FID Method for the Quantitation of 20 Different Acidic and Neutral Cannabinoids

Waseem Gul
1   ElSohly Laboratories, Inc., 5 Industrial Park Drive, Oxford, MS, USA
,
Elsayed A. Ibrahim
2   National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, University, MS, USA
3   Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt
,
Sharjeel W. Gul
1   ElSohly Laboratories, Inc., 5 Industrial Park Drive, Oxford, MS, USA
4   Sally McDonnell Barksdale Honors College, University of Mississippi, USA
5   School of Pharmacy, University of Mississippi, USA
6   Frontier Medical College, Abbottabad, KPK, Pakistan
,
Iram Shahzadi
1   ElSohly Laboratories, Inc., 5 Industrial Park Drive, Oxford, MS, USA
,
Mohamed M. Radwan
2   National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, University, MS, USA
,
Suman Chandra
2   National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, University, MS, USA
,
Hemant Lata
2   National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, University, MS, USA
,
Mahmoud A. ElSohly
1   ElSohly Laboratories, Inc., 5 Industrial Park Drive, Oxford, MS, USA
2   National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, University, MS, USA
5   School of Pharmacy, University of Mississippi, USA
7   Department of Pharmaceutics and Drug Delivery, School of Pharmacy, The University of Mississippi, University, USA
› Author Affiliations
Supported by: National Institute on Drug Abuse N01DA‑15-7793

Abstract

For decades, Cannabis sativa had been illegal to sell or consume around the world, including in the United States. However, in light of the recent 2018 Farm Bill and the legalization of hemp across the US, various cannabis preparations have flooded the market, making it essential to be able to quantitate the levels of the different acidic and neutral cannabinoids in C. sativa and to have a complete cannabinoid profile of the different chemovars of the cannabis plant. A GC-FID method was developed and validated for the analysis of 20 acidic and neutral cannabinoids as trimethylsilyl (TMS) derivatives. The analyzed cannabinoids include cannabidivarinic acid (CBDVA), cannabidiolic acid (CBDA), cannabinolic acid (CBNA), cannabielsoic acid (CBEA), cannabicyclolic acid (CBLA), cannabichromenic acid (CBCA), trans9-tetrahydrocannabivarinic acid (Δ9-THCVA), trans9-tetrahydrocannabinolic acid A (Δ9-THCAA), cannabigerolic acid (CBGA), cannabidiol (CBD), cannabicyclol (CBL), cannabidivarin (CBDV), trans9-tetrahydrocannabivarin (THCV), cannabichromene (CBC), trans8-tetrahydrocannabinol (Δ8-THC), trans9-tetrahydrocannabinol (Δ9-THC), cannabigerol (CBG), cannabinol (CBN), cannabicitran (CBT), and cannabielsoin (CBE). The method limit of detection (LOD) was as low as 0.1 µg/mL, while the limit of quantitation ranged from 0.25 µg/mL to 0.5 µg/mL. The precision (%RSD) was < 10%, while trueness ranged from 90 – 107%. The developed method is simple, accurate, and sensitive for the quantitation of all 20 acidic and neutral cannabinoids. Finally, the proposed method was successfully applied to the quantitation of the cannabinoids in different cannabis chemovars grown at the University of Mississippi.

Supporting Information



Publication History

Received: 02 June 2022

Accepted after revision: 18 October 2022

Accepted Manuscript online:
18 October 2022

Article published online:
24 January 2023

© 2023. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

 
  • References

  • 1 Pathak S, Deruiter J, Ramesh S, Govindarajulu M, Almaghrabi M, Nadar R, Moore T, Agrawal DC, Dhanasekaran M. The Legality of Use and Consumption of Cannabis (Marijuana) in the United States of America. Singapore: Springer Link; 2022: 113
  • 2 Pollastro F, Minassi A, Fresu LG. Cannabis phenolics and their bioactivities. Curr Med Chem 2018; 25: 1160-1185
  • 3 Radwan MM, Chandra S, Gul S, ElSohly MA. Cannabinoids, phenolics, terpenes and alkaloids of cannabis. Molecules 2021; 26: 2774
  • 4 Mazurkiewicz-Bełdzińska M, Zawadzka M. Use of cannabidiol in the treatment of epilepsy. Neurol Neurochir Pol 2022; 56: 14-20
  • 5 Sharma A, Szaflarski J. Cannabidiol decreases seizure severity and brain temperature in treatment-resistant epilepsy. (P1–8.004). Neurology 2022; 98: 3625
  • 6 Sarma ND, Waye A, ElSohly MA, Brown PN, Elzinga S, Johnson HE, Marles RJ, Melanson JE, Russo E, Deyton L. Cannabis inflorescence for medical purposes: USP considerations for quality attributes. J Nat Prod 2020; 83: 1334-1351
  • 7 Johnson MS, Wallace JG. Genomic and chemical diversity of commercially available high-CBD industrial hemp accessions. Front Genet 2021; 12: 1160
  • 8 Crini G, Lichtfouse E, Chanet G, Morin-Crini N. Applications of hemp in textiles, paper industry, insulation and building materials, horticulture, animal nutrition, food and beverages, nutraceuticals, cosmetics and hygiene, medicine, agrochemistry, energy production and environment: A review. Environ Chem Lett 2020; 18: 1451-1476
  • 9 Salami SA, Martinelli F, Giovino A, Bachari A, Arad N, Mantri N. It is our turn to get cannabis high: Put cannabinoids in food and health baskets. Molecules 2020; 25: 4036
  • 10 Oh DA, Parikh N, Khurana V, Smith CC, Vetticaden S. Effect of food on the pharmacokinetics of dronabinol oral solution versus dronabinol capsules in healthy volunteers. Clin Pharmacol 2017; 9: 9
  • 11 Nielsen S, Germanos R, Weier M, Pollard J, Degenhardt L, Hall W, Buckley N, Farrell M. The use of cannabis and cannabinoids in treating symptoms of multiple sclerosis: a systematic review of reviews. Curr Neurol Neurosci Rep 2018; 18: 1-12
  • 12 Food and Drug Administration. Highlights of prescribing information: Epidiolex (cannabidiol) oral solution. Accessed August 22, 2022 at: https://www.epidiolex.com/sites/default/files/pdfs/EPIDIOLEX_Full_Prescribing_Information_04_16_2020.pdf
  • 13 Food and Drug Administration. FDA approves first drug comprised of an active ingredient derived from marijuana to treat rare, severe forms of epilepsy. Accessed August 22, 2022 at: https://www.fda.gov/news-events/press-announcements/fda-approves-first-drug-comprised-active-ingredient-derived-marijuana-treat-rare-severe-forms
  • 14 European Medicines Agency. Epidyolex. Accessed August 22, 2022 at: https://www.ema.europa.eu/en/medicines/human/EPAR/epidyolex
  • 15 Lah TT, Novak M, Pena Almidon MA, Marinelli O, Žvar Baškovič B, Majc B, Mlinar M, Bošnjak R, Breznik B, Zomer R. Cannabigerol is a potential therapeutic agent in a novel combined therapy for glioblastoma. Cells 2021; 10: 340
  • 16 Vigli D, Cosentino L, Raggi C, Laviola G, Woolley-Roberts M, De Filippis B. Chronic treatment with the phytocannabinoid Cannabidivarin (CBDV) rescues behavioural alterations and brain atrophy in a mouse model of Rett syndrome. Neuropharmacology 2018; 140: 121-129
  • 17 Abioye A, Ayodele O, Marinkovic A, Patidar R, Akinwekomi A, Sanyaolu A. Δ9-Tetrahydrocannabivarin (THCV): A commentary on potential therapeutic benefit for the management of obesity and diabetes. J Cannabis Res 2020; 2: 1-6
  • 18 Khodadadi H, Salles ÉL, Shin E, Jarrahi A, Costigliola V, Kumar P, Yu JC, Morgan JC, Hess DC, Vaibhav K. A potential role for cannabichromene in modulating TRP channels during acute respiratory distress syndrome. J Cannabis Res 2021; 3: 1-7
  • 19 Udoh M, Santiago M, Devenish S, McGregor IS, Connor M. Cannabichromene is a cannabinoid CB2 receptor agonist. Br J Pharmacol 2019; 176: 4537-4547
  • 20 Mandrioli M, Tura M, Scotti S, Gallina Toschi T. Fast detection of 10 cannabinoids by RP-HPLC-UV method in Cannabis sativa L. Molecules 2019; 24: 2113
  • 21 Patel B, Wene D, Fan ZT. Qualitative and quantitative measurement of cannabinoids in cannabis using modified HPLC/DAD method. J Pharm Biomed Anal 2017; 146: 15-23
  • 22 Brighenti V, Pellati F, Steinbach M, Maran D, Benvenuti S. Development of a new extraction technique and HPLC method for the analysis of non-psychoactive cannabinoids in fibre-type Cannabis sativa L.(hemp). J Pharm Biomed Anal 2017; 143: 228-236
  • 23 Citti C, Pacchetti B, Vandelli MA, Forni F, Cannazza G. Analysis of cannabinoids in commercial hemp seed oil and decarboxylation kinetics studies of cannabidiolic acid (CBDA). J Pharm Biomed Anal 2018; 149: 532-540
  • 24 Križman M. A simplified approach for isocratic HPLC analysis of cannabinoids by fine tuning chromatographic selectivity. Eur Food Res Technol 2020; 246: 315-322
  • 25 Berman P, Futoran K, Lewitus GM, Mukha D, Benami M, Shlomi T, Meiri D. A new ESI-LC/MS approach for comprehensive metabolic profiling of phytocannabinoids in Cannabis. Sci Rep 2018; 8: 1-15
  • 26 Dei Cas M, Casagni E, Saccardo A, Arnoldi S, Young C, Scotti S, de Manicor EV, Gambaro V, Roda G. The Italian panorama of cannabis light preparation: Determination of cannabinoids by LC-UV. Forensic Sci Int 2020; 307: 110113
  • 27 Gul W, Gul SW, Chandra S, Lata H, Ibrahim EA, ElSohly MA. Detection and quantification of cannabinoids in extracts of cannabis sativa roots using LC-MS/MS. Planta Med 2018; 84: 267-271
  • 28 Happyana N, Agnolet S, Muntendam R, Van Dam A, Schneider B, Kayser O. Analysis of cannabinoids in laser-microdissected trichomes of medicinal Cannabis sativa using LCMS and cryogenic NMR. Phytochemistry 2013; 87: 51-59
  • 29 Merone G, Tartaglia A, Rossi S, Santavenere F, Bassotti E, DʼOvidio C, Rosato E, de Grazia U, Locatelli M, Del Boccio P. Fast LC–MS/MS screening method for the evaluation of drugs, illicit drugs, and other compounds in biological matrices. Talanta Open 2022; 5: 100105
  • 30 Merone GM, Tartaglia A, Rossi S, Santavenere F, Bassotti E, DʼOvidio C, Bonelli M, Rosato E, De Grazia U, Locatelli M. Fast quantitative LC-MS/MS determination of illicit substances in solid and liquid unknown seized samples. Anal Chem 2021; 93: 16308-16313
  • 31 Merone G, Tartaglia A, Rossi S, Santavenere F, Bassotti E, DʼOvidio C, Bonelli M, Rosato E, de Grazia U, Zanardo A. Fast liquid chromatography-tandem mass spectrometry method for the simultaneous determination of phytocannabinoids in oily based preparations. J Pharm Biomed Anal 2021; 203: 114174
  • 32 Deidda R, Dispas A, De Bleye C, Hubert P, Ziemons É. Critical review on recent trends in cannabinoid determination on cannabis herbal samples: From chromatographic to vibrational spectroscopic techniques. Anal Chim Acta 2021; 1209: 339184
  • 33 Amirav A, Neumark B, Margolin Eren KJ, Fialkov AB, Tal N. Cannabis and its cannabinoids analysis by gas chromatography–mass spectrometry with Cold EI. J Mass Spectrom 2021; 56: e4726
  • 34 Bakro F, Jedryczka M, Wielgusz K, Sgorbini B, Inchingolo R, Cardenia V. Simultaneous determination of terpenes and cannabidiol in hemp (Cannabis sativa L.) by fast gas chromatography with flame ionization detection. J Sep Sci 2020; 43: 2817-2826
  • 35 Baranauskaite J, Marksa M, Ivanauskas L, Vitkevicius K, Liaudanskas M, Skyrius V, Baranauskas A. Development of extraction technique and GC/FID method for the analysis of cannabinoids in Cannabis sativa L. spp. santicha (hemp). Phytochem Anal 2020; 31: 516-521
  • 36 Cardenia V, Toschi TG, Scappini S, Rubino RC, Rodriguez-Estrada MT. Development and validation of a Fast gas chromatography/mass spectrometry method for the determination of cannabinoids in Cannabis sativa L. J Food Drug Anal 2018; 26: 1283-1292
  • 37 Hazekamp A, Fischedick J. Cannabis‐from cultivar to chemovar. Drug Test Anal 2012; 4: 660-667
  • 38 Hazekamp A, Tejkalová K, Papadimitriou S. Cannabis: From cultivar to chemovar II–a metabolomics approach to Cannabis classification. Cannabis Cannabinoid Res 2016; 1: 202-215
  • 39 Fodor B, Molnár-Perl I. The role of derivatization techniques in the analysis of plant cannabinoids by gas chromatography mass spectrometry. Trends Analyt Chem 2017; 95: 149-158
  • 40 Fodor B, Boldizsár I, Molnár-Perl I. Alkylsilyl speciation and direct sample preparation of plant cannabinoids prior to their analysis by GC-MS. Anal Chim Acta 2018; 1021: 51-59
  • 41 Leghissa A, Hildenbrand ZL, Foss FW, Schug KA. Determination of cannabinoids from a surrogate hops matrix using multiple reaction monitoring gas chromatography with triple quadrupole mass spectrometry. J Sep Sci 2018; 41: 459-468
  • 42 Ye L, Budge SM. Sample preparation for the analysis of key metabolites from cannabinoids biosynthesis in phytoplankton using gas chromatography–mass spectrometry. J Am Oil Chem Soc 2022; 99: 115-127
  • 43 Karschner EL, Barnes AJ, Lowe RH, Scheidweiler KB, Huestis MA. Validation of a two-dimensional gas chromatography mass spectrometry method for the simultaneous quantification of cannabidiol, Δ9-tetrahydrocannabinol (THC), 11-hydroxy-THC, and 11-nor-9-carboxy-THC in plasma. Anal Bioanal Chem 2010; 397: 603-611
  • 44 Ahmed AQ, Noshad D, Li PC. Quantification of cannabinoids in cultivars of cannabis sp. by gas chromatography–mass spectrometry. Chromatographia 2021; 84: 711-717
  • 45 Ibrahim EA, Gul W, Gul SW, Stamper BJ, Hadad GM, Salam RAA, Ibrahim AK, Ahmed SA, Chandra S, Lata H. Determination of acid and neutral cannabinoids in extracts of different strains of Cannabis sativa using GC-FID. Planta Med 2018; 84: 250-259
  • 46 ICH Guidelines. Validation of analytical procedures: Text and methodology. Q2 (R1) 2005; 1: 05.