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==Abstract==
==Abstract==
[[wikipedia:Cannabis sativa|Cannabis]] has regained much attention as a result of updated legislation authorizing many different uses, and it can be classified on the basis of the content of [[wikipedia:Tetrahydrocannabinol|Δ9-tetrahydrocannabinol]] (Δ9-THC), a psychotropic substance for which there are legal limitations in many countries. For this purpose, accurate qualitative and quantitative determination is essential. The relationship between THC and [[wikipedia:Cannabidiol|cannabidiol]] (CBD) is also significant, as the latter substance is endowed with many specific and non-psychoactive proprieties. For these reasons, it becomes increasingly important and urgent to utilize fast, easy, validated, and harmonized procedures for determination of [[wikipedia:Cannabinoid|cannabinoids]]. The procedure described herein allows rapid determination of 10 cannabinoids from the [[wikipedia:Inflorescence|inflorescences]] of ''Cannabis sativa'' L. by extraction with organic solvents. Separation and subsequent detection are by [[wikipedia:Reversed-phase chromatography|reversed-phase]] [[high-performance liquid chromatography]] with ultraviolet detector (RP-HPLC-UV). Quantification is performed by an external standard method through the construction of calibration curves using pure standard [[Chromatography|chromatographic]] reference compounds. The main cannabinoids dosed (g/100 g) in actual samples were cannabidiolic acid (CBDA), CBD, and Δ9-THC (Sample L11 CBDA 0.88 ± 0.04, CBD 0.48 ± 0.02, Δ9-THC 0.06 ± 0.00; Sample L5 CBDA 0.93 ± 0.06, CBD 0.45 ± 0.03, Δ9-THC 0.06 ± 0.00). The present validated RP-HPLC-UV method allows determination of the main cannabinoids in ''Cannabis sativa'' L. inflorescences and appropriate legal classification of it as either [[wikipedia:Hemp|hemp]] or a [[wikipedia:Cannabis (drug)|drug-type]].
[[wikipedia:Cannabis|Cannabis]] has regained much attention as a result of updated legislation authorizing many different uses, and it can be classified on the basis of the content of [[wikipedia:Tetrahydrocannabinol|Δ9-tetrahydrocannabinol]] (Δ9-THC), a psychotropic substance for which there are legal limitations in many countries. For this purpose, accurate qualitative and quantitative determination is essential. The relationship between THC and [[wikipedia:Cannabidiol|cannabidiol]] (CBD) is also significant, as the latter substance is endowed with many specific and non-psychoactive proprieties. For these reasons, it becomes increasingly important and urgent to utilize fast, easy, validated, and harmonized procedures for determination of [[wikipedia:Cannabinoid|cannabinoids]]. The procedure described herein allows rapid determination of 10 cannabinoids from the [[wikipedia:Inflorescence|inflorescences]] of ''Cannabis sativa'' L. by extraction with organic solvents. Separation and subsequent detection are by [[wikipedia:Reversed-phase chromatography|reversed-phase]] [[high-performance liquid chromatography]] with ultraviolet detector (RP-HPLC-UV). Quantification is performed by an external standard method through the construction of calibration curves using pure standard [[Chromatography|chromatographic]] reference compounds. The main cannabinoids dosed (g/100 g) in actual samples were cannabidiolic acid (CBDA), CBD, and Δ9-THC (Sample L11 CBDA 0.88 ± 0.04, CBD 0.48 ± 0.02, Δ9-THC 0.06 ± 0.00; Sample L5 CBDA 0.93 ± 0.06, CBD 0.45 ± 0.03, Δ9-THC 0.06 ± 0.00). The present validated RP-HPLC-UV method allows determination of the main cannabinoids in ''Cannabis sativa'' L. inflorescences and appropriate legal classification of it as either [[wikipedia:Hemp|hemp]] or a [[wikipedia:Cannabis (drug)|drug-type]].


'''Keywords''': cannabinoids, ''Cannabis sativa'' L., HPLC, validation
'''Keywords''': cannabinoids, ''Cannabis sativa'' L., HPLC, validation


==Introduction==
''[[wikipedia:Cannabis|Cannabis]]'' is classified into the family of Cannabaceae and initially encompassed three main species: ''[[wikipedia:Cannabis sativa|Cannabis sativa]]'', ''[[wikipedia:Cannabis indica|Cannabis indica]]'', and ''[[wikipedia:Cannabis ruderalis|Cannabis ruderalis]]''.<ref name="MontserratHemp14">{{cite journal |title=Hemp (''Cannabis sativa'' L.) seed oil: Analytical and phytochemical characterization of the unsaponifiable fraction |journal=Journal of Agricultural and Food Chemistry |author=Montserrat-de la Paz, S.; Marín-Aguilar, F.; García-Giménez, M.D. et al. |volume=62 |issue=5 |pages=1105–10 |year=2014 |doi=10.1021/jf404278q |pmid=24422510}}</ref> Nowadays, ''Cannabis'' has only one species due to continuous crossbreeding of the three species to generate hybrids. In fact, all plants are categorized as belonging to ''Cannabis sativa'' and classified into chemotypes based on the concentration of the main [[wikipedia:Cannabinoid|cannabinoids]]. Depending on the ratio of [[wikipedia:Tetrahydrocannabinolic acid||tetrahydrocannabinolic acid]] (THCA) to cannabidiolic acid (CBDA) (the THCA/CBDA ratio), some chemotypes have been distinguished. In particular, chemotype I or “drug plants” have a TCHA/CBDA ratio >1.0, chemotype II plants exhibit an intermediate ratio, chemotype III or “fiber plants” have a THCA/CBDA ratio <1.0, chemotype IV plants contain cannabigerolic acid (CBGA) as the main cannabinoid, and chemotype V plants contain almost no cannabinoids.<ref name="AppendinoCannab11">{{cite journal |title=Cannabinoids: Occurrence and medicinal chemistry |journal=Current Medicinal Chemistry |author=Appendino, G.; Chianese, G.; Taglialatela-Scafati, O. |volume=18 |issue=7 |pages=1085–99 |year=2011 |doi=10.2174/092986711794940888 |pmid=21254969}}</ref><ref name="AndreCannabis16">{{cite journal |title=''Cannabis sativa'': The Plant of the Thousand and One Molecules |journal=Frontiers in Plant Science |author=Andre, C.M.; Hausman, J.F.; Guerriero, G. |volume=7 |pages=19 |year=2016 |doi=10.3389/fpls.2016.00019 |pmid=26870049 |pmc=PMC4740396}}</ref><ref name="Aizpurua-OlaizolaEvo16">{{cite journal |title=Evolution of the Cannabinoid and Terpene Content during the Growth of Cannabis sativa Plants from Different Chemotypes |journal=Journal of Natural Products |author=Aizpurua-Olaizola, O.; Soydaner, U;. Öztürk, E. et al. |volume=79 |issue=2 |pages=324–31 |year=2016 |doi=10.1021/acs.jnatprod.5b00949 |pmid=26836472}}</ref><ref name="BrighentiDevelop17">{{cite journal |title=Development of a new extraction technique and HPLC method for the analysis of non-psychoactive cannabinoids in fibre-type Cannabis sativa L. (hemp) |journal=Journal of Pharmaceutical and Biomedical Analysis |author=Brighenti, V.; Pellati, F.; Steinbach, M. et al. |volume=143 |pages=228–36 |year=2017 |doi=10.1016/j.jpba.2017.05.049 |pmid=28609672}}</ref>


In Italy, the interest in ''Cannabis sativa'' L. has increased recently, mainly due to December 2016 legislation (Legge 2 Dicembre 2016, n. 242).<ref name="ItalyLegge16">{{cite web |url=https://www.gazzettaufficiale.it/eli/id/2016/12/30/16G00258/sg |title=Legge 2 Dicembre 2016, n. 242 - Disposizioni per la promozione della coltivazione e della filiera agroindustriale della canapa |work=Gazzetta Ufficiale della Repubblica Italiana |date=30 December 2016 |accessdate=01 June 2019}}</ref> As a result, requests have been made to develop cost-effective and easy-to-use quantitative and qualitative methods for analysis of cannabinoids.
The Italian regulatory framework has classified two types of ''Cannabis sativa'' L. depending on the content of [[wikipedia:Tetrahydrocannabinol|Δ9-tetrahydrocannabinol]] (Δ9-THC). In particular, fiber-type plants of ''Cannabis sativa'' L., also called “hemp,” are characterized by a low content of Δ9-THC (<0.2% w/w). If the content of Δ9-THC is >0.6% w/w, it is considered as drug-type, also called “therapeutic” or “marijuana.”
Industrial hemp is used in several sectors, such as in the pharmaceutical, cosmetic, food, and textile industries, as well as in energy production and building. In general, fiber-type plants are less used in the pharmaceutical field, where drug-type plants are more often employed.<ref name="BrighentiDevelop17" /> However, there is also an increased interest in hemp varieties containing non-psychoactive compounds. In fact, the European Union has approved 69 varieties of ''Cannabis sativa'' L. for commercial use.<ref name="EUPlant19">{{cite web |url=http://ec.europa.eu/food/plant/plant_propagation_material/plant_variety_catalogues_databases/search//public/index.cfm |title=EU Plant variety database |publisher=European Commission |date=2019 |accessdate=01 June 2019}}</ref>
Hemp has a complex chemical composition that includes [[wikipedia:Terpeniod|terpenoids]], sugars, alkaloids, stilbenoids, quinones, and the characteristic compounds of this plant, namely cannabinoids. ''Cannabis sativa'' L. has several chemotypes, each of which is characterized by a different qualitative and quantitative chemical profile.<ref name="BrighentiDevelop17" /> The cannabinoids, [[wikipedia:Terpene|terpenes]], and phenolic compounds in hemp are formed through secondary metabolism.<ref name="AndreCannabis16" /><ref name="PisantiCannabidiol17">{{cite journal |title=Cannabidiol: State of the art and new challenges for therapeutic applications |journal=Pharmacology and Therapeutics |author=Pisanti, S.; Malfitano, A.M.; Ciaglia, E. et al. |volume=175 |pages=133-150 |year=2017 |doi=10.1016/j.pharmthera.2017.02.041 |pmid=28232276}}</ref> The term “cannabinoid” indicates terpenophenols derived from ''Cannabis''. More than 90 cannabinoids are known, and some are derived from breakdown reactions.<ref name="PisantiCannabidiol17" /> Mechoulam and Gaoni<ref name="MechoulamRecent67">{{cite journal |title=Recent Advances in the Chemistry of Hashish |journal=Progress in the Chemistry of Organic Natural Products |author=Mechoulam, R.; Gaoni, Y. |volume=25 |pages=175–213 |year=1967 |doi=10.1007/978-3-7091-8164-5_6}}</ref> were the first to define cannabinoids “as a group of C21 compounds typical of and present in ''Cannabis sativa'', their carboxylic acids, analogs, and transformation products.” Currently, cannabinoids have been classified according to their chemical structure, with the primary cannabinoids constituting seven types of [[wikipedia:Cannabigerol|cannabigerol]] (CBG); five types of [[wikipedia:Cannabichromene|cannabichromene]] (CBC); seven types of [[wikipedia:Cannabidiol|cannabidiol]] (CBD); the main psychoactive cannabinoid Δ9-tetrahydrocannabinol (Δ9-THC) in nine different forms, including its acid precursor (Δ9-tetrahydrocannabinolic acid, Δ9-THCA); Δ8-tetrahydrocannabinol (Δ8-THC), which is a more stable isomer of Δ9-THC but 20% less active; three types of [[wikipedia:Cannabicyclol|cannabicyclol]] (CBL); five different forms of cannabielsoin (CBE); seven types of [[wikipedia:Cannabinol|cannabinol]] (CBN), which is the oxidation artifact of Δ9-THC; cannabitriol (CBT); [[wikipedia:Cannabivarin|cannabivarin]] (CBDV); and [[wikipedia:Tetrahydrocannabivarin|tetrahydrocannabivarin]] (THCV).<ref name="RadwanNatural17">{{cite book |chapter=Natural Cannabinoids of Cannabis and Methods of Analysis |title=''Cannabis sativa'' L. - Botany and Biotechnology |author=Radwan, M.M.; Wanas, A.S.; Chandra, S. et al. |editor=Chandra, S.; Lata, H.; ElSohly, M.A. |publisher=Springer |year=2017 |isbn=9783319545646 |doi=10.1007/978-3-319-54564-6_7}}</ref><ref name="LeghissaARev18">{{cite journal |title=A review of methods for the chemical characterization of cannabis natural products |journal=Journal of Separation Science |author=Leghissa, A.; Hildenbrand, Z.L.; Schug, K.A. et al. |volume=41 |issue=1 |pages=398-415 |year=2018 |doi=10.1002/jssc.201701003 |pmid=28986974}}</ref> THC, CBD, CBG, CBN, and CBC are not biosynthesized in ''Cannabis sativa'', and the plant produces the carboxylic acid forms of these cannabinoids (THCA, CBDA, CBGA, CBNA, and CBCA). Cannabinoid acids undergo a chemical [[Wikipedia:Decarboxylation|decarboxylation]] reaction triggered by different factors, mainly temperature. This decarboxylation reaction leads to the formation of the respective neutral cannabinoids (THC, CBD, CBG, CBN, and CBC).<ref name="CittiAnalysis18">{{cite journal |title=Analysis of cannabinoids in commercial hemp seed oil and decarboxylation kinetics studies of cannabidiolic acid (CBDA) |journal=Journal of Pharmaceutical and Biomedical Analysis |author=Citti, C;. Pacchetti, B;. Vandelli, M.A. et al. |volume=149 |pages=532–40 |year=2018 |doi=10.1016/j.jpba.2017.11.044 |pmid=29182999}}</ref><ref name="CittiPharma18">{{cite journal |title=Pharmaceutical and biomedical analysis of cannabinoids: A critical review |journal=Journal of Pharmaceutical and Biomedical Analysis |author=Citti, C.; Braghiroli, D.; Vandelli, M.A. et al. |volume=147 |pages=565-579 |year=2018 |doi=10.1016/j.jpba.2017.06.003 |pmid=28641906}}</ref>
There are numerous methods to quantify cannabinoids<ref name="RodriguesDeterm18">{{cite journal |title=Determination of cannabinoids in hair of CBD rich extracts consumers using gas chromatography with tandem mass spectrometry (GC/MS-MS) |journal=Forensic Science International |author=Rodrigues, A.; Yegles, M.; Van Elsué, N. et al. |volume=292 |pages=163–66 |year=2018 |doi=10.1016/j.forsciint.2018.09.015 |pmid=30317054}}</ref><ref name="CardeniaDevel18">{{cite journal |title=Development and validation of a fast gas chromatography–mass spectrometry method for the determination of cannabinoids in ''Cannabis sativa'' L |journal=Journal of Food and Drug Analysis |author=Cardenia, V.; Toschi, T.G.; Scappini, S.; Rubino, R.C.; Rodriguez-Estrada, M.T. |volume=26 |issue=4 |pages=1283–92 |year=2018 |doi=10.1016/j.jfda.2018.06.001 |pmid=30249327}}</ref><ref name="LeghissaDeterm18">{{cite journal |title=Determination of cannabinoids from a surrogate hops matrix using multiple reaction monitoring gas chromatography with triple quadrupole mass spectrometry |journal=Journal of Separation Science |author=Leghissa, A.; Hildenbrand, Z.L.; Foss, F.W. et al. |volume=41 |issue=2 |pages=459–68 |year=2018 |doi=10.1002/jssc.201700946 |pmid=29094798}}</ref><ref name="PatelQual17">{{cite journal |title=Qualitative and quantitative measurement of cannabinoids in cannabis using modified HPLC/DAD method |journal=Journal of Pharmaceutical and Biomedical Analysis |author=Patel, B.; Wene, D.; Fan, Z.T. |volume=146 |pages=15–23 |year=2017 |doi=10.1016/j.jpba.2017.07.021 |pmid=28841427}}</ref><ref name="BurnierQuant19">{{cite journal |title=Quantification of THC in Cannabis plants by fast-HPLC-DAD: A promising method for routine analyses |journal=Talanta |author=Burnier, C.; Esseiva, P.; Roussel, C. |volume=192 |pages=135-141 |year=2019 |doi=10.1016/j.talanta.2018.09.012 |pmid=30348368}}</ref><ref name="PellatiNew18">{{cite journal |title=New Methods for the Comprehensive Analysis of Bioactive Compounds in ''Cannabis sativa'' L. (hemp) |journal=Molecules |author=Pellati, F.; Brighenti, V.; Sperlea, J. et al. |volume=23 |issue=10 |page=E2639 |year=2018 |doi=10.3390/molecules23102639 |pmid=30322208 |pmc=PMC6222702}}</ref><ref name="CiolinoComm18">{{cite journal |title=Commercial cannabis consumer products part 2: HPLC-DAD quantitative analysis of cannabis cannabinoids |journal=Forensic Science International |author=Ciolino, L.A.; Ranieri, T.L.; Taylor, A.M. |volume=289 |page=438–47 |year=2018 |doi=10.1016/j.forsciint.2018.05.033 |pmid=30025568}}</ref><ref name="FeketeImplem18">{{cite journal |title=Implementation of a generic liquid chromatographic method development workflow: Application to the analysis of phytocannabinoids and Cannabis sativa extracts |journal=Journal of Pharmaceutical and Biomedical Analysis |author=Fekete, S.; Sadat-Noorbakhsh, V; Scheilling, C. et al. |volume=155 |page=116-124 |year=2018 |doi=10.1016/j.jpba.2018.03.059 |pmid=29627728}}</ref>, some of which require expensive mass spectrometry detectors.<ref name="PurschkeDevelop16">{{cite journal |title=Development and validation of an automated liquid-liquid extraction GC/MS method for the determination of THC, 11-OH-THC, and free THC-carboxylic acid (THC-COOH) from blood serum |journal=Analytical and Bioanalytical Chemistry |author=Purschke, K.; Heinl, S.; Lerch, O. et al. |volume=408 |issue=16 |pages=4379-88 |year=2016 |doi=10.1007/s00216-016-9537-5 |pmid=27116418 |pmc=PMC4875941}}</ref><ref name="PacificiEval17">{{cite journal |title=Evaluation of cannabinoids concentration and stability in standardized preparations of cannabis tea and cannabis oil by ultra-high performance liquid chromatography tandem mass spectrometry |journal=Clinical Chemistry and Laboratory Medicine |author=Pacifici, R.; Marchei, E.; Salvatore, F. et al. |volume=55 |issue=10 |pages=1555–1563 |year=2017 |doi=10.1515/cclm-2016-1060 |pmid=28207408}}</ref><ref name="CasiraghiExtract18">{{cite journal |title=Extraction Method and Analysis of Cannabinoids in Cannabis Olive Oil Preparations |journal=Planta Medica |author=Casiraghi, A.; Roda, G.; Casagni, E. et al. |volume=84 |issue=4 |pages=242–49 |year=2018 |doi=10.1055/s-0043-123074 |pmid=29202510}}</ref><ref name="LinUrine18">{{cite journal |title=Urine specimen validity test for drug abuse testing in workplace and court settings |journal=Journal of Food and Drug Analysis |author=Lin, S.Y.; Lee, H.H.; Lee, J.F. et al. |volume=26 |issue=1 |pages=380–84 |year=2018 |doi=10.1016/j.jfda.2017.01.001 |pmid=29389577}}</ref> Furthermore, there is a great deal of uncertainty around the use of [[gas chromatography]] (GC) for the titration of cannabinoids due to the high temperature of the injector and detector, which can lead to the decarboxylation of cannabinoid acids if not derivatized correctly.<ref name="MudgeLeaner17">{{cite journal |title=Leaner and greener analysis of cannabinoids |journal=Analytical and Bioanalytical Chemistry |author=Mudge, E.M.; Murch, S.J.; Brown, P.N. |volume=409 |issue=12 |pages=3153–63 |year=2017 |doi=10.1007/s00216-017-0256-3 |pmid=28233028 |pmc=PMC5395585}}</ref> Moreover, recent studies have reported that cannabinoid acid decarboxylation is only partial, and as a result the actual value is underestimated. A [[high-performance liquid chromatography]] (HPLC) system allows for determination of the actual cannabinoid composition of both neutral and acid forms without the necessity of the derivatization step.<ref name="CittiPharma18" />
It is necessary, in addition to honed methods, to develop new procedures with a view to discriminate different ''Cannabis'' varieties in order to identify and titrate cannabinoids in a simple way. These methods should ideally be fast, easy, robust, and cost-efficient, as they can be used not only by research laboratories but also by small companies with a view on quality control.
This study focuses on the development, validation, and step-by-step explanation of a rapid, simple, and reproducible HPLC ultraviolet detector-based (HPLC-UV) method for identification and quantification of the main cannabinoids in hemp inflorescences. The method described is focused on the quantification of CBD but can also be applied to check the levels of THC.
==Results and discussion==
===Method development===





Revision as of 23:04, 2 December 2019

Full article title Fast detection of 10 cannabinoids by RP-HPLC-UV method in Cannabis sativa L.
Journal Molecules
Author(s) Mandrioli, Mara; Tura, Matilde; Scotti, Stefano; Toschi, Tullia Gallina
Author affiliation(s) University of Bologna, Shimadzu Italia
Primary contact Email: tullia dot gallinatoschi at unibo dot it
Editors Locatelli, Marcello
Year published 2019
Volume and issue 24(11)
Page(s) 2113
DOI 10.3390/molecules24112113
ISSN 1420-3049
Distribution license Creative Commons Attribution 4.0 International
Website https://www.mdpi.com/1420-3049/24/11/2113/htm
Download https://www.mdpi.com/1420-3049/24/11/2113/pdf (PDF)

Abstract

Cannabis has regained much attention as a result of updated legislation authorizing many different uses, and it can be classified on the basis of the content of Δ9-tetrahydrocannabinol (Δ9-THC), a psychotropic substance for which there are legal limitations in many countries. For this purpose, accurate qualitative and quantitative determination is essential. The relationship between THC and cannabidiol (CBD) is also significant, as the latter substance is endowed with many specific and non-psychoactive proprieties. For these reasons, it becomes increasingly important and urgent to utilize fast, easy, validated, and harmonized procedures for determination of cannabinoids. The procedure described herein allows rapid determination of 10 cannabinoids from the inflorescences of Cannabis sativa L. by extraction with organic solvents. Separation and subsequent detection are by reversed-phase high-performance liquid chromatography with ultraviolet detector (RP-HPLC-UV). Quantification is performed by an external standard method through the construction of calibration curves using pure standard chromatographic reference compounds. The main cannabinoids dosed (g/100 g) in actual samples were cannabidiolic acid (CBDA), CBD, and Δ9-THC (Sample L11 CBDA 0.88 ± 0.04, CBD 0.48 ± 0.02, Δ9-THC 0.06 ± 0.00; Sample L5 CBDA 0.93 ± 0.06, CBD 0.45 ± 0.03, Δ9-THC 0.06 ± 0.00). The present validated RP-HPLC-UV method allows determination of the main cannabinoids in Cannabis sativa L. inflorescences and appropriate legal classification of it as either hemp or a drug-type.

Keywords: cannabinoids, Cannabis sativa L., HPLC, validation

Introduction

Cannabis is classified into the family of Cannabaceae and initially encompassed three main species: Cannabis sativa, Cannabis indica, and Cannabis ruderalis.[1] Nowadays, Cannabis has only one species due to continuous crossbreeding of the three species to generate hybrids. In fact, all plants are categorized as belonging to Cannabis sativa and classified into chemotypes based on the concentration of the main cannabinoids. Depending on the ratio of |tetrahydrocannabinolic acid (THCA) to cannabidiolic acid (CBDA) (the THCA/CBDA ratio), some chemotypes have been distinguished. In particular, chemotype I or “drug plants” have a TCHA/CBDA ratio >1.0, chemotype II plants exhibit an intermediate ratio, chemotype III or “fiber plants” have a THCA/CBDA ratio <1.0, chemotype IV plants contain cannabigerolic acid (CBGA) as the main cannabinoid, and chemotype V plants contain almost no cannabinoids.[2][3][4][5]

In Italy, the interest in Cannabis sativa L. has increased recently, mainly due to December 2016 legislation (Legge 2 Dicembre 2016, n. 242).[6] As a result, requests have been made to develop cost-effective and easy-to-use quantitative and qualitative methods for analysis of cannabinoids.

The Italian regulatory framework has classified two types of Cannabis sativa L. depending on the content of Δ9-tetrahydrocannabinol (Δ9-THC). In particular, fiber-type plants of Cannabis sativa L., also called “hemp,” are characterized by a low content of Δ9-THC (<0.2% w/w). If the content of Δ9-THC is >0.6% w/w, it is considered as drug-type, also called “therapeutic” or “marijuana.”

Industrial hemp is used in several sectors, such as in the pharmaceutical, cosmetic, food, and textile industries, as well as in energy production and building. In general, fiber-type plants are less used in the pharmaceutical field, where drug-type plants are more often employed.[5] However, there is also an increased interest in hemp varieties containing non-psychoactive compounds. In fact, the European Union has approved 69 varieties of Cannabis sativa L. for commercial use.[7]

Hemp has a complex chemical composition that includes terpenoids, sugars, alkaloids, stilbenoids, quinones, and the characteristic compounds of this plant, namely cannabinoids. Cannabis sativa L. has several chemotypes, each of which is characterized by a different qualitative and quantitative chemical profile.[5] The cannabinoids, terpenes, and phenolic compounds in hemp are formed through secondary metabolism.[3][8] The term “cannabinoid” indicates terpenophenols derived from Cannabis. More than 90 cannabinoids are known, and some are derived from breakdown reactions.[8] Mechoulam and Gaoni[9] were the first to define cannabinoids “as a group of C21 compounds typical of and present in Cannabis sativa, their carboxylic acids, analogs, and transformation products.” Currently, cannabinoids have been classified according to their chemical structure, with the primary cannabinoids constituting seven types of cannabigerol (CBG); five types of cannabichromene (CBC); seven types of cannabidiol (CBD); the main psychoactive cannabinoid Δ9-tetrahydrocannabinol (Δ9-THC) in nine different forms, including its acid precursor (Δ9-tetrahydrocannabinolic acid, Δ9-THCA); Δ8-tetrahydrocannabinol (Δ8-THC), which is a more stable isomer of Δ9-THC but 20% less active; three types of cannabicyclol (CBL); five different forms of cannabielsoin (CBE); seven types of cannabinol (CBN), which is the oxidation artifact of Δ9-THC; cannabitriol (CBT); cannabivarin (CBDV); and tetrahydrocannabivarin (THCV).[10][11] THC, CBD, CBG, CBN, and CBC are not biosynthesized in Cannabis sativa, and the plant produces the carboxylic acid forms of these cannabinoids (THCA, CBDA, CBGA, CBNA, and CBCA). Cannabinoid acids undergo a chemical decarboxylation reaction triggered by different factors, mainly temperature. This decarboxylation reaction leads to the formation of the respective neutral cannabinoids (THC, CBD, CBG, CBN, and CBC).[12][13]

There are numerous methods to quantify cannabinoids[14][15][16][17][18][19][20][21], some of which require expensive mass spectrometry detectors.[22][23][24][25] Furthermore, there is a great deal of uncertainty around the use of gas chromatography (GC) for the titration of cannabinoids due to the high temperature of the injector and detector, which can lead to the decarboxylation of cannabinoid acids if not derivatized correctly.[26] Moreover, recent studies have reported that cannabinoid acid decarboxylation is only partial, and as a result the actual value is underestimated. A high-performance liquid chromatography (HPLC) system allows for determination of the actual cannabinoid composition of both neutral and acid forms without the necessity of the derivatization step.[13]

It is necessary, in addition to honed methods, to develop new procedures with a view to discriminate different Cannabis varieties in order to identify and titrate cannabinoids in a simple way. These methods should ideally be fast, easy, robust, and cost-efficient, as they can be used not only by research laboratories but also by small companies with a view on quality control.

This study focuses on the development, validation, and step-by-step explanation of a rapid, simple, and reproducible HPLC ultraviolet detector-based (HPLC-UV) method for identification and quantification of the main cannabinoids in hemp inflorescences. The method described is focused on the quantification of CBD but can also be applied to check the levels of THC.

Results and discussion

Method development

Supplementary material

The following are available online here (.zip):

File S1: Standard operating procedure (SOP) of the method presented in this article, Table S1: Calibration curves relating to the standard solution of 10 cannabinoids determined by RP-HPLC-UV method, Figure S1: Calibration curves relating to the standard solution of 10 cannabinoids determined by RP-HPLC-UV method, File S2: Preliminary tests carried out for development of the analytical procedure by RP-HPLC-UV

Acknowledgements

The authors gratefully acknowledge Enecta Srl for providing samples. The experimentation was conducted in the context of a PhD project entitled Harmonized procedures of analysis of medical, herbal, food and industrial cannabis: Development and validation of cannabinoids’ quality control methods, of extraction and preparation of derivatives from the plant raw material, according to the product destination and funded by Enecta Srl.

Author contributions

Conceptualization, T.G.T., M.M. and M.T.; Methodology, M.M.; Software, M.M. and S.S.; Validation, M.M.; Formal analysis, M.M.; Investigation, T.G.T. and M.M.; Resources, T.G.T. and M.M.; Data curation, M.M., T.G.T. and M.T.; Writing—original draft preparation, M.M., M.T. and T.G.T.; writing—review and editing, T.G.T. and S.S.; Visualization, T.G.T.; Supervision, T.G.T.; project administration, T.G.T.; funding acquisition, T.G.T.

Funding

This research received no external funding; this trial received financial support from Enecta Srl.

Conflicts of interest

The authors declare no conflict of interest.

References

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Notes

This presentation is faithful to the original, with only a few minor changes to presentation. Some grammar and punctuation was cleaned up to improve readability. In some cases important information was missing from the references, and that information was added.