Cannabis sativa L. is an herbaceous plant belonging to the family of Cannabaceae. It is classified into three different chemotypes based on the different cannabinoids profile. In particular, fiber-type cannabis (hemp) is rich in cannabidiol (CBD) content. In the present work, a rapid nano liquid chromatographic method (nano-LC) was proposed for the determination of the main cannabinoids in Cannabis sativa L. (hemp) inflorescences belonging to different varieties. The nano-LC experiments were carried out in a 100 µm internal diameter capillary column packed with a C18 stationary phase for 15 cm with a mobile phase composed of ACN/H2O/formic acid, 80/19/1% (v/v/v). The reverse-phase nano-LC method allowed the complete separation of four standard cannabinoids in less than 12 min under isocratic elution mode. The nano-LC method coupled to ultraviolet (UV) detection was validated and applied to the quantification of the target analytes in cannabis extracts. The nano-LC system was also coupled to an electrospray ionization-mass spectrometry (ESI-MS) detector to confirm the identity of the cannabinoids present in hemp samples. For the extraction of the cannabinoids, three different approaches, including dynamic maceration (DM), ultrasound-assisted extraction (UAE), and an extraction procedure adapted from the French Pharmacopeia's protocol on medicinal plants, were carried out, and the results achieved were compared.

Department of Analytical Chemistry Faculty of Science Palacký University 17 listopadu 12 771 46 Olomouc Czech Republic

Istituto per i Sistemi Biologici Consiglio Nazionale delle Ricerche Area della Ricerca di Roma 1 Via Salaria Km 29 300 Monterotondo 00015 Rome Italy

See more in PubMed

Hartsel J.A., Eades J., Hickory B., Makriyannis A. Cannabis sativa and Hemp. In: Gupta R.C., editor. Nutraceutical: Efficacy, Safety and Toxicity. Elsevier Inc.; London, UK: 2016. pp. 735–754.

Brenneisen R. Chemistry and analysis of phytocannabinoids and other constituents. In: Elsohly M.A., editor. Forensic Science and Medicine: Marijuana and the Cannabinoid. Humana Press Inc.; Totowa, NJ, USA: 2007. pp. 17–49.

Thomas B.F., ElSohly M.A. The Analytical Chemistry of Cannabis: Quality Assessment, Assurance and Regulation of Medicinal Marijuana and Cannabinoid Preparations. 1st ed. Elsevier Inc.; Amsterdam, The Netherlands: 2015.

Ohlsson A., Lindgren J.E., Wahlen A., Agurell S., Hollister L.E., Gillespie H.K. Plasma delta-9-tetrahydrocannabinol concentrations and clinical effects after oral and intravenous administration and smoking. Clin. Pharmacol. Ther. 1980;28:409–416. doi: 10.1038/clpt.1980.181. PubMed DOI

Morrison P.D., Zois V., McKeown D.A., Lee T.D., Holt D.W., Powell J.F., Kapur S., Murray R.M. The acute effects of synthetic intravenous Delta9-tetrahydrocannabinol on psychosis, mood and cognitive functioning. Psychol. Med. 2009;39:1607–1616. doi: 10.1017/S0033291709005522. PubMed DOI

Borgelt L.M., Franson K.L., Nussbaum A.M., Wang G.S. The pharmacologic and clinical effects of medical cannabis. Pharmacotherapy. 2013;33:195–209. doi: 10.1002/phar.1187. PubMed DOI

Appendino G., Chianese G., Taglialatela-Scafati O. Cannabinoids: Occurrence and Medicinal Chemistry. Curr. Med. Chem. 2011;18:1085–1099. doi: 10.2174/092986711794940888. PubMed DOI

Pellati F., Borgonetti V., Brighenti V., Biagi M., Benvenuti S., Corsi L. Cannabis sativa L. and Nonpsychoactive Cannabinoids: Their Chemistry and Role against Oxidative Stress, Inflammation, and Cancer. Biomed Res. Int. 2018;2018:1691428. doi: 10.1155/2018/1691428. PubMed DOI PMC

Lafaye G., Karila L., Blecha L., Benyamina A. Cannabis, cannabinoids, and health. Dialogues Clin. Neurosci. 2017;19:309–316. PubMed PMC

Khan B.A., Warner P., Wang H. Antibacterial Properties of Hemp and Other Natural Fibre Plants: A Review. BioResources. 2014;9:3642–3659. doi: 10.15376/biores.9.2.3642-3659. DOI

Chandra S., Lata H., ElSohly M.A., Walker L.A., Potter D. Cannabis cultivation: Methodological issues for obtaining medical-grade product. Epilepsy Behav. 2017;70:302–312. doi: 10.1016/j.yebeh.2016.11.029. PubMed DOI

European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) Cannabis Legislation in Europe—An overview. [(accessed on 17 October 2018)];2017 Available online: http://www.emcdda.europa.eu/system/files/publications/4135/TD0217210ENN.pdf.

Fischedick J.T., Hazekamp A., Erkelens T., Choi Y.H., Verpoorte R. Metabolic fingerprinting of Cannabis sativa L., cannabinoids and terpenoids for chemotaxonomic and drug standardization purposes. Phytochemistry. 2010;71:2058–2073. doi: 10.1016/j.phytochem.2010.10.001. PubMed DOI

Citti C., Braghiroli D., Vandelli M.A., Cannazza G. Pharmaceutical and biomedical analysis of Cannabinoids: A critical review. J. Pharm. Biomed. Anal. 2018;147:565–579. doi: 10.1016/j.jpba.2017.06.003. PubMed DOI

Nahar L., Onder A., Sarker S.D. A review on the recent advances in HPLC, UHPLC and UPLC analyses of naturally occurring cannabinoids. Phytochem. Anal. 2020;31:413–457. doi: 10.1002/pca.2906. PubMed DOI

Mandrioli M., Tura M., Scotti S., Toschi T.G. Fast detection of 10 cannabinoids by RP-HPLC-UV method in Cannabis sativa L. Molecules. 2019;24:2113. doi: 10.3390/molecules24112113. PubMed DOI PMC

De Backer B., Debrus B., Lebrun P., Theunis L., Dubois N., Decock L., Verstraete A., Hubert P., Charlier C. 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. 2009;877:4115–4124. doi: 10.1016/j.jchromb.2009.11.004. PubMed DOI

Patel B., Wene D., Fan Z.H. Qualitative and quantitative measurement of cannabinoids in cannabis using modified HPLC/DAD method. J. Pharm. Biomed. Anal. 2017;146:15–23. doi: 10.1016/j.jpba.2017.07.021. PubMed DOI

Brighenti V., Pellati F., Steinbach M., Maran D. 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. doi: 10.1016/j.jpba.2017.05.049. PubMed DOI

Hädener M., König S., Weinmann W. Quantitative determination of CBD and THC and their acid precursors in confiscated cannabis samples by HPLC-DAD. Forensic Sci. Int. 2019;299:142–150. doi: 10.1016/j.forsciint.2019.03.046. PubMed DOI

Citti C., Battisti U.M., Braghiroli D., Ciccarella G., Schmid M., Vandelli M.A., Cannazza G. A metabolomic approach applied to a liquid chromatography coupled to high-resolution tandem mass spectrometry method (HPLC-ESI-HRMS/MS): Towards the comprehensive evaluation of the chemical composition of cannabis medicinal extracts. Phytochem. Anal. 2018;29:144–155. doi: 10.1002/pca.2722. PubMed DOI

Tiscione N.B., Miller R., Shan X., Sprague J., Yeatman D.T. An efficient, robust method for the determination of cannabinoids in whole blood by LC-MS-MS. J. Anal. Toxicol. 2016;40:639–648. doi: 10.1093/jat/bkw063. PubMed DOI

Azipurua-Olaizola O., Omar J., Navarro P., Olivares M., Etxenarria N., Usobiaga A. Identification and quantification of cannabinoids in Cannabis sativa L. plants by high performance liquid chromatography mass spectrometry. Anal. Bional. Chem. 2014;406:7549–7560. doi: 10.1007/s00216-014-8177-x. PubMed DOI

Protti M., Brighenti V., Battaglia M.R., 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;10:539–544. doi: 10.1021/acsmedchemlett.8b00571. PubMed DOI PMC

Asensio-Ramos M., Fanali C., D’Orazio G., Fanali S. Nano-liquid chromatography. In: Fanali S., Haddad P., Poole C., Riekkola M.L., editors. Liquid Chromatography, Fundamentals and Instrumentation. Volume I. Elsevier Inc.; Amsterdam, The Netherlands: 2017. pp. 638–687.

Maciel E.V.S., de Toffoli A.L., Sobieski E., Domingues Nazario C.E., Lanças F.M. Miniaturized liquid chromatography focusing on analytical columns and mass spectrometry: A review. Anal. Chim. Acta. 2020;1103:11–31. doi: 10.1016/j.aca.2019.12.064. PubMed DOI

Welch C.J., Wu N., Biba M., Hartman R., Brkovic T., Gong X., Helmy R., Schafer W., Cuff J., Pirzada Z., et al. Greening analytical chromatography. Trends Anal. Chem. 2010;29:667–680. doi: 10.1016/j.trac.2010.03.008. DOI

Fanali C., Dugo L., Dugo P., Mondello L. Capillary-liquid chromatography (CLC) and nano-LC in food analysis. Trends Anal. Chem. 2013;52:226–238. doi: 10.1016/j.trac.2013.05.021. DOI

Aturki Z., Rocco A., Rocchi S., Fanali S. Current applications of miniaturized chromatographic and electrophoretic techniques in drug analysis. J. Pharm. Biomed. Anal. 2014;101:194–220. doi: 10.1016/j.jpba.2014.03.041. PubMed DOI

Merola G., Aturki Z., D’Orazio G., Gottardo R., Macchia T., Tagliaro F., Fanali S. Analysis of synthetic cannabinoids in herbal blends by means of nano-liquid chromatography. J. Pharm. Biomed. Anal. 2012;71:45–53. doi: 10.1016/j.jpba.2012.08.008. PubMed DOI

Jornet-Martínez N., Ortega-Sierra A., Verdú-Andrés J., Herráez-Hernández R., Campíns-Falcó P. Analysis of contact traces of cannabis by in-tube solid-phase microextraction coupled to nano-liquid chromatography. Molecules. 2018;23:2359. doi: 10.3390/molecules23092359. PubMed DOI PMC

Wang Y.H., Avula B., El-Sohly M.A., Radwan M.M., Wang M., Wanas A.S., Mehmedic Z., Khan I.A. Quantitative determination of Δ9-THC, CBG, CBD, their acid precursors and five other neutral cannabinoids by UHPLC-UV-MS. Planta Med. 2018;84:260–266. doi: 10.1055/s-0043-124873. PubMed DOI

Peschel W., Politi M. 1H NMR and HPLC/DAD for Cannabis sativa L. chemotype distinction, extract profiling and specification. Talanta. 2015;140:150–165. doi: 10.1016/j.talanta.2015.02.040. PubMed DOI

Mateos-Vivas M., Fanali S., Rodríguez-Gonzalo E., Carabias-Martínez R., Aturki Z. Rapid determination of nucleotides in infant formula by means of nano-liquid chromatography. Electrophoresis. 2016;37:1873–1880. doi: 10.1002/elps.201500526. PubMed DOI

D’Orazio G., Fanali C., Gentili A., Tagliaro F., Fanali S. Nano-liquid chromatography for enantiomers separation of baclofen by using vancomycin silica stationary phase. J. Chromatogr. A. 2019;1605:360358. doi: 10.1016/j.chroma.2019.07.012. PubMed DOI

Fanali S., Aturki Z., D’Orazio G., Rocco A. Separation of basic compounds of pharmaceutical interest by using nanoliquid chromatography coupled with mass spectrometry. J. Chromatogr. A. 2007;1150:252–258. doi: 10.1016/j.chroma.2006.10.021. PubMed DOI

Zöllner T., Schwarz M. Herbal Reference Standards: Applications, definitions and regulatory requirements. Braz. J. Pharmacogn. 2013;23:1–21. doi: 10.1590/S0102-695X2012005000144. DOI