Development and Validation of a Simple, Fast, and Accessible HPLC-UV
Method for Cannabinoids Determination in Cannabis sativa L. Extracts and
Medicinal Oils

Fresia   Melina   Silva Sofrás; Rosario      Alonso; Daiana   Sabrina   Retta; Paola      Di Leo Lira; Martin   Federico   Desimone; Catalina   María   van Baren

doi:10.2174/1381612829666230809094304

Abstract

Introduction: Cannabis sativa L. is a well-recognized medicinal plant. Cannabis regulations in Argentina are insufficient to solve the problem of patient access to full-spectrum cannabis-based products. So, the market of artisanal products with unknown quality and dosage of cannabinoids is increasing, and so is the local demand and need for analyzing these products. However, much of the latest validated methodologies for cannabinoid quantification include expensive instrumentation that is not always available in laboratories of health institutions in Argentina.

Methods: The aim of this work was to develop and validate a simple and rapid HPLC-UV method for the identification and quantification of principal cannabinoids in cannabis resins, inflorescences, and medicinal oils using standard HPLC equipment. The cannabinoids selected for validation were cannabidiol acid (CBDA), cannabigerol (CBG), cannabidiol (CBD), cannabinol (CBN), delta-9-tetrahydrocannabinol (Δ9-THC), cannabichromene (CBC), and tetrahydrocannabinol acid (THCA). A method for the simultaneous identification and quantification of these 7 main cannabinoids was developed and then validated. Some data parameters were comparable to other reports with more sophisticated analytical instruments for the analysis of cannabis. The assessed limits of detection and the limits of quantitation ranged from 0.9 to 3.66 μg/mL and 2.78 to 11.09 μg/mL, respectively. The concentration-response relationship of the method indicated a linear relationship between the concentration and peak area with R² values of > 0.99 for all 7 cannabinoids.

Results: The relative standard deviation (RSD%) varied from 2.34 to 4.82 for intraday repeatability and from 1.16 to 3.15 for interday repeatability. The percentage of recovery values was between 94 to 115% (resins) and 80 to 103% (inflorescence extract). The cannabis industry is growing rapidly, and there is a need for reliable testing methods to ensure the safety and efficacy of cannabis products. In addition, current methods for cannabinoid analysis are often time-consuming and expensive, while the HPLC-UV method herein reported is a simple, rapid, accurate, and cost-effective alternative for the analysis of cannabinoids in cannabis resins, inflorescences, and medicinal oils.

Conclusion: This method will be proposed to be included in the Cannabis sativa L. monograph of the Argentine Pharmacopoeia.

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[1]
Kalant H. Medicinal use of cannabis: history and current status. Pain Res Manag  2001; 6(2): 80-91.
 [http://dx.doi.org/10.1155/2001/469629] [PMID: 11854770]

[2]
Zuardi AW. History of cannabis as a medicine: A review. Rev Bras Psiquiatr  2006; 28(2): 153-7.
 [http://dx.doi.org/10.1590/S1516-44462006000200015] [PMID: 16810401]

[3]
Charitos IA, Gagliano-Candela R, Santacroce L, Bottalico L. The cannabis spread throughout the continents and its therapeutic use in history. Endocr Metab Immune Disord Drug Targets  2021; 21(3): 407-17.
 [http://dx.doi.org/10.2174/22123873MTA2DNzki3] [PMID: 32433013]

[4]
Crocq MA. History of cannabis and the endocannabinoid system.  Dialogues Clin Neurosci  2020; 22(3): 223-8.
 [http://dx.doi.org/10.31887/DCNS.2020.22.3/mcrocq] [PMID: 33162765]

[5]
Mechoulam R. The Pharmacohistory of Cannabis sativa. 1st ed.  Chapman and Hall/CRC 2019; p. 20.
 [http://dx.doi.org/10.1201/9780429260667-1]

[6]
Pisanti S, Bifulco M. Medical cannabis: A plurimillennial history of an evergreen. J Cell Physiol  2019; 234(6): 8342-51.
 [http://dx.doi.org/10.1002/jcp.27725] [PMID: 30417354]

[7]
Chandra S, Lata H, ElSohly MA. Cannabis sativa L. - Botany and Biotechnology. Springer Cham 2017.

[8]
Andre CM, Hausman JF, Guerriero G. Cannabis sativa: The plant of the thousand and one molecules. Front Plant Sci  2016; 7: 19.
 [http://dx.doi.org/10.3389/fpls.2016.00019] [PMID: 26870049]

[9]
Flores-Sanchez IJ, Verpoorte R. Secondary metabolism in cannabis. Phytochem Rev  2008; 7(3): 615-39.
 [http://dx.doi.org/10.1007/s11101-008-9094-4]

[10]
Liu Y, Liu HY, Li SH, et al. Cannabis sativa bioactive compounds and their extraction, separation, purification, and identification technologies: An updated review. Trends Analyt Chem  2022; 149: 116554.
 [http://dx.doi.org/10.1016/j.trac.2022.116554]

[11]
Mechoulam R, Parker LA. The endocannabinoid system and the brain. Annu Rev Psychol  2013; 64(1): 21-47.
 [http://dx.doi.org/10.1146/annurev-psych-113011-143739] [PMID: 22804774]

[12]
Di Marzo V, Piscitelli F. The endocannabinoid system and its modulation by phytocannabinoids. Neurotherapeutics  2015; 12(4): 692-8.
 [http://dx.doi.org/10.1007/s13311-015-0374-6] [PMID: 26271952]

[13]
Piomelli D, Mabou Tagne A. Endocannabinoid-based therapies. Annu Rev Pharmacol Toxicol  2022; 62(1): 483-507.
 [http://dx.doi.org/10.1146/annurev-pharmtox-052220-021800] [PMID: 34516291]

[14]
Lu HC, Mackie K. Review of the endocannabinoid system. Biol Psychiatry Cogn Neurosci Neuroimaging  2021; 6(6): 607-15.
 [PMID: 32980261]

[15]
Ben Amar M. Cannabinoids in medicine: A review of their therapeutic potential. J Ethnopharmacol  2006; 105(1-2): 1-25.
 [http://dx.doi.org/10.1016/j.jep.2006.02.001] [PMID: 16540272]

[16]
Kowal MA, Hazekamp A, Grotenhermen F. Review on clinical studies with cannabis and cannabinoids 2010-2014. Mult Scler J  2016; (6): 1515.

[17]
Hazekamp A, Grotenhermen F. Review on clinical studies with cannabis and cannabinoids 2005-2009. Cannabinoids  2010; 5: 1-21.

[18]
Whiting PF, Wolff RF, Deshpande S, et al. Cannabinoids for medical use: A systematic review and meta-analysis. JAMA  2015; 313(24): 2456-73.
 [http://dx.doi.org/10.1001/jama.2015.6358] [PMID: 26103030]

[19]
MacCallum CA, Russo EB. Practical considerations in medical cannabis administration and dosing. Eur J Intern Med  2018; 49(49): 12-9.
 [http://dx.doi.org/10.1016/j.ejim.2018.01.004] [PMID: 29307505]

[20]
Roy A, Kaur S, Sharma N. Role of cannabinoids in various diseases: A review. Curr Pharm Biotechnol  2022; 23(11): 1346-58.
 [http://dx.doi.org/10.2174/1389201023666211223164656] [PMID: 34951355]

[21]
Bilbao A, Spanagel R. Medical cannabinoids: A pharmacology-based systematic review and meta-analysis for all relevant medical indications. BMC Med  2022; 20(1): 259.
 [http://dx.doi.org/10.1186/s12916-022-02459-1] [PMID: 35982439]

[22]
MacCallum CA, Lo LA, Boivin M. “Is medical cannabis safe for my patients?” A practical review of cannabis safety considerations. Eur J Intern Med  2021; 89: 10-8.
 [http://dx.doi.org/10.1016/j.ejim.2021.05.002] [PMID: 34083092]

[23]
Johal H, Devji T, Chang Y, Simone J, Vannabouathong C, Bhandari M. Cannabinoids in chronic non-cancer pain: A systematic review and meta-analysis. Clin Med Insights Arthritis Musculoskelet Disord  2020; 13: 1179544120906461.
 [http://dx.doi.org/10.1177/1179544120906461] [PMID: 32127750]

[24]
Spanagel R, Bilbao A. Approved cannabinoids for medical purposes – Comparative systematic review and meta-analysis for sleep and appetite. Neuropharmacology  2021; 196: 108680.
 [http://dx.doi.org/10.1016/j.neuropharm.2021.108680] [PMID: 34181977]

[25]
Amin MR, Ali DW. Pharmacology of medical cannabis. Adv Exp Med Biol  2019; 1162: 151-65.
 [http://dx.doi.org/10.1007/978-3-030-21737-2_8] [PMID: 31332738]

[26]
Argentinian republic. Law. 2017. Available from:  http://www.msal.gob.ar/dlsn/categorias/salud-publica/cannabis-uso-medicinal/ley-27350

[27]
Argentinian republic. Resolution. 2022. Available from: https://www.boletinoficial.gob.ar/detalleAviso/primera/260768/20220411

[28]
Argentinian republic. Joint Resolution No. 5/2021. 2021. Available from: https://www.argentina.gob.ar/noticias/inase-habilita- la-inscripcion-de- variedades-de-cannabis-sativa-l

[29]
Taschwer M, Schmid MG. Determination of the relative percentage distribution of THCA and Δ9-THC in herbal cannabis seized in Austria – Impact of different storage temperatures on stability. Forensic Sci Int  2015; 254(254): 167-71.
 [http://dx.doi.org/10.1016/j.forsciint.2015.07.019] [PMID: 26247127]

[30]
Gambaro V, Dell’Acqua L, Farè F, Froldi R, Saligari E, Tassoni G. Determination of primary active constituents in cannabis preparations by high-resolution gas chromatography/flame ionization detection and high-performance liquid chromatography/UV detection. Anal Chim Acta  2002; 468(2): 245-54.
 [http://dx.doi.org/10.1016/S0003-2670(02)00660-8]

[31]
Burnier C, Esseiva P, Roussel C. Quantification of THC in cannabis plants by fast-HPLC-DAD: A promising method for routine analyses. Talanta  2019; 192(192): 135-41.
 [http://dx.doi.org/10.1016/j.talanta.2018.09.012] [PMID: 30348368]

[32]
Andonova L, Ivanov E, Pencheva I, Konstantinov S. Elaboration and validation of HPLC/DAD method for quality control of products containing cannabidiol. Curr Pharm Anal  2021; 17(9): 1218-23.
 [http://dx.doi.org/10.2174/1573412916999200805163154]

[33]
Ambach L, Penitschka F, Broillet A, König S, Weinmann W, Bernhard W. Simultaneous quantification of delta-9-THC, THC-acid A, CBN and CBD in seized drugs using HPLC-DAD. Forensic Sci Int  2014; 243(243): 107-11.
 [http://dx.doi.org/10.1016/j.forsciint.2014.06.008] [PMID: 25005819]

[34]
Madej K, Chmiołek A, Szlachta K, Piekoszewski W. Hplc-dad analysis of hemp oil supplements for determination of four cannabinoids: Cannabidiol, cannabidiolic acid, cannabinol and delta 9-tetrahydrocannabinol. Separations  2021; 8(12): 227.
 [http://dx.doi.org/10.3390/separations8120227]

[35]
Reuter WM. Development of an Improved Method for Cannabis Potency Analysis Utilising HPLC/PDA in Preference to UHPLC. Chromatography Today 2018. Available from: https://www.chromatographytoday.com/article/hplc-uhplc/31/perkin-elmer/pdevelopment-of-an-improved-method-for-cannabis-potency-analysis-utilising-hplcpdanbspin-preference-to-uhplcp/2380

[36]
Madej K, Kózka G, Winiarski M, Piekoszewski W. A simple, fast, and green oil sample preparation method for determination of cannabidioloic acid and cannabidiol by hplc-dad. Separations  2020; 7(4): 60.
 [http://dx.doi.org/10.3390/separations7040060]

[37]
Carvalho VM, Aguiar AFL, Baratto LC, Souza FLC, Rocha ED. Cannabinoids quantification in medicinal cannabis extracts by high performance liquid chromatography. Quim Nova  2020; 43(1): 90-7.

[38]
Hanuš LO, Meyer SM, Muñoz E, Taglialatela-Scafati O, Appendino G. Phytocannabinoids: A unified critical inventory. Nat Prod Rep  2016; 33(12): 1357-92.
 [http://dx.doi.org/10.1039/C6NP00074F] [PMID: 27722705]

[39]
Citti C, Braghiroli D, Vandelli MA, Cannazza G. Pharmaceutical and biomedical analysis of cannabinoids: A critical review. J Pharm Biomed Anal  2018; 147(147): 565-79.
 [http://dx.doi.org/10.1016/j.jpba.2017.06.003] [PMID: 28641906]

[40]
Leghissa A, Hildenbrand ZL, Schug KA. The imperatives and challenges of analyzing cannabis edibles. Curr Opin Food Sci  2019; 28(28): 18-24.
 [http://dx.doi.org/10.1016/j.cofs.2019.02.010]

[41]
Leghissa A, Hildenbrand ZL, Schug KA. A review of methods for the chemical characterization of cannabis natural products. J Sep Sci  2018; 41(1): 398-415.
 [http://dx.doi.org/10.1002/jssc.201701003] [PMID: 28986974]

[42]
Leghissa A. New methods for discovery, fingerprinting, and analysis of Cannabis sativa natural products. Theses and Dissertations University of Texas Arlington 2018.

[43]
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  2022; 1209: 339184.
 [http://dx.doi.org/10.1016/j.aca.2021.339184] [PMID: 35569861]

[44]
Borille BT, González M, Steffens L, Ortiz RS, Limberger RP. Cannabis sativa: A systematic review of plant analysis. Drug Anal Res  2017; 1(1): 1-23.
 [http://dx.doi.org/10.22456/2527-2616.73676]

[45]
Micalizzi G, Vento F, Alibrando F, Donnarumma D, Dugo P, Mondello L. Cannabis sativa L.: A comprehensive review on the analytical methodologies for cannabinoids and terpenes characterization. J Chromatogr A  2021; 1637: 461864.
 [http://dx.doi.org/10.1016/j.chroma.2020.461864] [PMID: 33422797]

[46]
Pellati F, Brighenti V, Sperlea J, Marchetti L, Bertelli D, Benvenuti S. New methods for the comprehensive analysis of bioactive compounds in Cannabis sativa L. (hemp). Molecules  2018; 23(10): 2639.
 [http://dx.doi.org/10.3390/molecules23102639] [PMID: 30322208]

[47]
Brighenti V, Marchetti L, Anceschi L, et al. Separation and non-separation methods for the analysis of cannabinoids in Cannabis sativa L. J Pharm Biomed Anal  2021; 206: 114346.
 [http://dx.doi.org/10.1016/j.jpba.2021.114346] [PMID: 34537622]

[48]
Mercolini L, Mandrioli R, Protti M. New Trends in the Analysis of Cannabis-Based Products.  Boca Raton: CRC Press 2021; pp. 111-30.
 [http://dx.doi.org/10.1201/9780429274893-5]

[49]
Nahar L, Onder A, Sarker SD. A review on the recent advances in HPLC, UHPLC and UPLC analyses of naturally occurring cannabinoids (2010–2019). Phytochem Anal  2020; 31(4): 413-57.
 [http://dx.doi.org/10.1002/pca.2906] [PMID: 31849137]

[50]
Stefkov G, Cvetkovikj Karanfilova I, Stoilkovska Gjorgievska V, et al. Analytical techniques for phytocannabinoid profiling of cannabis and cannabis-based products-a comprehensive review. Molecules  2022; 27(3): 975.
 [http://dx.doi.org/10.3390/molecules27030975] [PMID: 35164240]

[51]
Pourseyed Lazarjani M, Torres S, Hooker T, Fowlie C, Young O, Seyfoddin A. Methods for quantification of cannabinoids: A narrative review. J Cannabis Res  2020; 2(1): 35.
 [http://dx.doi.org/10.1186/s42238-020-00040-2] [PMID: 33526084]

[52]
Madden O, Walshe J, Kishore Patnala P, Barron J, Meaney C, Murray P. Phytocannabinoids - an overview of the analytical methodologies for detection and quantification of therapeutically and recreationally relevant cannabis compounds. Crit Rev Anal Chem  2021; 53(1): 211-31.
 [PMID: 34328047]

[53]
Welling MT, Liu L, Hazekamp A, Dowell A, King GJ. Developing robust standardised analytical procedures for cannabinoid quantification: Laying the foundations for an emerging cannabis-based pharmaceutical industry. Med Cannabis Cannabinoids  2019; 2(1): 1-13.
 [http://dx.doi.org/10.1159/000496868] [PMID: 34676328]

[54]
Desimone MF, Silva SFM. Entourage effect and analytical chemistry: Chromatography as a tool in the analysis of the secondary metabolism of Cannabis sativa L. Curr Pharm Des  2023; 29(6): 394-406.
 [http://dx.doi.org/10.2174/1381612829666221103093542] [PMID: 36330630]

[55]
De Backer B, Debrus B, Lebrun P, et al. 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 Analyt Technol Biomed Life Sci  2009; 877(32): 4115-24.
 [http://dx.doi.org/10.1016/j.jchromb.2009.11.004] [PMID: 19932642]

[56]
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(11): 2113.
 [http://dx.doi.org/10.3390/molecules24112113] [PMID: 31167395]

[57]
Gul W, Gul SW, Radwan MM, et al. Determination of 11 cannabinoids in biomass and extracts of different varieties of cannabis using high-performance liquid chromatography. J AOAC Int  2015; 98(6): 1523-8.
 [http://dx.doi.org/10.5740/jaoacint.15-095] [PMID: 26651563]

[58]
Giese MW, Lewis MA, Giese L, Smith KM. Development and validation of a reliable and robust method for the analysis of cannabinoids and terpenes in cannabis. J AOAC Int  2015; 98(6): 1503-22.
 [http://dx.doi.org/10.5740/jaoacint.15-116] [PMID: 26651562]

[59]
Jin D, Jin S, Yu Y, Lee C, Chen J. Classification of cannabis cultivars marketed in canada for medical purposes by quantification of cannabinoids and terpenes using HPLC-DAD and GC-MS. J Anal Bioanal Tech  2017; 8(1)
 [http://dx.doi.org/10.4172/2155-9872.1000349]

[60]
Peschel W, Politi M. 1 H NMR and HPLC/DAD for Cannabis sativa L. chemotype distinction, extract profiling and specification. Talanta  2015; 140: 150-65.
 [http://dx.doi.org/10.1016/j.talanta.2015.02.040] [PMID: 26048837]

[61]
Križman M. A simplified approach for isocratic HPLC analysis of cannabinoids by fine tuning chromatographic selectivity. Eur Food Res Technol  2020; 246(2): 315-22.
 [http://dx.doi.org/10.1007/s00217-019-03344-7]

[62]
Ciolino LA, Ranieri TL, Taylor AM. Commercial cannabis consumer products part 2: HPLC-DAD quantitative analysis of cannabis cannabinoids. Forensic Sci Int  2018; 289: 438-47.
 [http://dx.doi.org/10.1016/j.forsciint.2018.05.033] [PMID: 30025568]

[63]
Zivovinovic S, Alder R, Allenspach MD, Steuer C. Determination of cannabinoids in Cannabis sativa L. samples for recreational, medical, and forensic purposes by reversed-phase liquid chromatography-ultraviolet detection. J Anal Sci Technol  2018; 9(1): 27.
 [http://dx.doi.org/10.1186/s40543-018-0159-8]

[64]
Ternelli M, Brighenti V, Anceschi L, et al. Innovative methods for the preparation of medical cannabis oils with a high content of both cannabinoids and terpenes. J Pharm Biomed Anal  2020; 186: 113296.
 [http://dx.doi.org/10.1016/j.jpba.2020.113296] [PMID: 32334134]

[65]
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(146): 15-23.
 [http://dx.doi.org/10.1016/j.jpba.2017.07.021] [PMID: 28841427]

[66]
Protti M, Brighenti V, Battaglia MR, Anceschi L, Pellati F, Mercolini L. Cannabinoids from Cannabis sativa L.: A new tool based on HPLC-DAD-MS/MS for a rational use in medicinal chemistry. ACS Med Chem Lett  2019; 10(4): 539-44.
 [http://dx.doi.org/10.1021/acsmedchemlett.8b00571] [PMID: 30996793]

[67]
Elkins AC, Deseo MA, Rochfort S, Ezernieks V, Spangenberg G. Development of a validated method for the qualitative and quantitative analysis of cannabinoids in plant biomass and medicinal cannabis resin extracts obtained by super-critical fluid extraction. J Chromatogr B Analyt Technol Biomed Life Sci  2019; 1109(1109): 76-83.
 [http://dx.doi.org/10.1016/j.jchromb.2019.01.027] [PMID: 30738340]

[68]
Deidda R, Avohou HT, Baronti R, et al. Analytical quality by design: Development and control strategy for a LC method to evaluate the cannabinoids content in cannabis olive oil extracts. J Pharm Biomed Anal  2019; 166: 326-35.
 [http://dx.doi.org/10.1016/j.jpba.2019.01.032] [PMID: 30685656]

[69]
Mudge EM, Brown PN. Determination of cannabinoids in Cannabis sativa dried flowers and oils by LC-UV: Single-laboratory validation, first action 2018.10. J AOAC Int  2020; 103(2): 489-93.
 [http://dx.doi.org/10.5740/jaoacint.19-0197] [PMID: 31561754]

[70]
Franklin E, Wilcox M. Gradient HPLC-UV Method for Cannabinoid Profiling.  Chromatogr Today 2019; pp. 34-7.

[71]
Hall D, Sinclair J, Bhuyan D, Khoo C, Open CLT. Quality control of cannabis inflorescence and oil products: Response factors for the cost-efficient determination of ten cannabinoids by HPLC. Talanta open  2022; (5): 100112.

[72]
Büttenbender S, Carlos G, Steppe M, Ortiz RS, Limberger RP, Mendez ASL. Fast and reliable profiling of cannabinoids in seized samples using the method of HPLC–DAD followed by chemometrics. Forensic Toxicol  2022; 40(2): 407-13.
 [http://dx.doi.org/10.1007/s11419-022-00625-x] [PMID: 36454417]

[73]
Analakkattillam S, Langsi VK, Hanrahan JP, Moore E. Analytical method validation for assay determination of cannabidiol and tetrahydrocannabinol in hemp oil infused products by RP-HPLC. Sci Rep  2022; 12(1): 12453.
 [http://dx.doi.org/10.1038/s41598-022-13737-6] [PMID: 35864137]

[74]
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(143): 228-36.
 [http://dx.doi.org/10.1016/j.jpba.2017.05.049] [PMID: 28609672]

[75]
Bartkutė M, Sadaunykas A, Naujalis E. HPLC method development and validation for main cannabinoids determination in hemp biomass and oil.   Available from:  https://www.openreadings.eu/thesismanager/posters21/186Bartkut%C4%97.pdf

[76]
International Conference of Harmonization (ICH). Validation of Analytical Procedures.   Available from:  https://database.ich.org/sites/default/files/ICH_Q2R2_Document_Step2_Guideline_2022_0324.pdf

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DOI https://dx.doi.org/10.2174/1381612829666230809094304	Print ISSN 1381-6128
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Development and Validation of a Simple, Fast, and Accessible HPLC-UV Method for Cannabinoids Determination in Cannabis sativa L. Extracts and Medicinal Oils

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