Difference between revisions of "Journal:Fast detection of 10 cannabinoids by RP-HPLC-UV method in Cannabis sativa L."
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[[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
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.|
|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|
|Volume and issue||24(11)|
|Distribution license||Creative Commons Attribution 4.0 International|
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
Cannabis is classified into the family of Cannabaceae and initially encompassed three main species: Cannabis sativa, Cannabis indica, and Cannabis ruderalis. 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.
In Italy, the interest in Cannabis sativa L. has increased recently, mainly due to December 2016 legislation (Legge 2 Dicembre 2016, n. 242). 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. 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.
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. The cannabinoids, terpenes, and phenolic compounds in hemp are formed through secondary metabolism. The term “cannabinoid” indicates terpenophenols derived from Cannabis. More than 90 cannabinoids are known, and some are derived from breakdown reactions. Mechoulam and Gaoni 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). 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).
There are numerous methods to quantify cannabinoids, some of which require expensive mass spectrometry detectors. 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. 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.
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
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
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.
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.
This research received no external funding; this trial received financial support from Enecta Srl.
Conflicts of interest
The authors declare no conflict of interest.
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