Article

Method for the Analysis of Cannabinoids and Terpenes in Cannabis

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Abstract

The requirements for an acceptable cannabis assay have changed dramatically over the years resulting in a large number of laboratories using a diverse array of analytical methodologies that have not been properly validated. Due to the lack of sufficiently validated methods, we conducted a single- laboratory validation study for the determination of cannabinoids and terpenes in a variety of commonly occurring cultivars. The procedure involves high- throughput homogenization to prepare sample extract, which is then profiled for cannabinoids and terpenes by HPLC-diode array detector and GC-flame ionization detector, respectively. Spike recovery studies for terpenes in the range of 0.03–1.5% were carried out with analytical standards, while recovery studies for Δ⁹ -tetrahydrocannabinolic acid, cannabidiolic acid, Δ⁹ -tetrahydrocannabivarinic acid, and cannabigerolic acid and their neutral counterparts in the range of 0.3–35% were carried out using cannabis extracts. In general, accuracy at all levels was within 5%, and RSDs were less than 3%. The interday and intraday repeatabilities of the procedure were evaluated with five different cultivars of varying chemotype, again resulting in acceptable RSDs. As an example of the application of this assay, it was used to illustrate the variability seen in cannabis coming from very advanced indoor cultivation operations.

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... On the other hand, GC is inappropriate for determination of both forms of cannabinoids if not previously derivatized, since due to heat of the injector, decarboxylation of cannabinoids occurs leading just to their relative neutral forms [3]. Thus, cannabinoids are generally determined by GC/MS [3], HPLC or UHPLC coupled to diode array detector (DAD) [13][14][15][16], low-resolution MS [6,16,17], or high-resolution MS [6,18,19]. On the other hand, due to the volatile character of terpenes, GC is widely used for their determination. ...
... On the other hand, due to the volatile character of terpenes, GC is widely used for their determination. Terpenes have been mostly determined by headspace-solid phase microextraction (HS-SPME) coupled to GC/MS [11,18], GC-flame ionization detection (GC-FID) [15], or direct injection of hemp oil extract into GC/MS [19]. As reported above, the main problem is that terpenes and CBD are separately determined using different techniques and methods, which results in time-consuming protocols and requiring the use of expensive instruments (such as mass spectrometers). ...
... Pavlovic et al. [19] determined terpenes in CBD oil by HS-SPME-GC/MS in 35 min and CBD by HPLC coupled to high-resolution MS. Giese et al. [15] validated a method for the analysis of cannabinoids and terpenes (17 terpenes separated in 35 min) in cannabis by using HPLC-DAD and GC-FID, respectively. Thus, the reduction of analysis time (29 terpenes and CBD in <16 min) leads to a significant improvement in the number of samples processed per day, which is advantageous for the detection of less stable terpenes. ...
... It should be pointed out that cannabinoids content may considerably vary within hemp varieties, since several chemotypes have been identified [4]. In particular, CBDA has been reported to be the main cannabinoid in hemp inflorescences, representing from 1 to 240 mg/g dried material [40][41][42][43][44][45][54][55][56][57][58][59][60][61][62][63][64][65][66][67][68][69][70][71][72][73], while CBD content has been reported to be in the range 0.4−183 mg/g [40][41][42][43][44][45][54][55][56][57][58][59][60][61][62][63][64][65][66][67][68][69][70][71][72][73]. Other minor non-psychoactive cannabinoids are generally below 10 mg/g of dry inflorescences [40][41][42][43][44][45]. ...
... It should be pointed out that cannabinoids content may considerably vary within hemp varieties, since several chemotypes have been identified [4]. In particular, CBDA has been reported to be the main cannabinoid in hemp inflorescences, representing from 1 to 240 mg/g dried material [40][41][42][43][44][45][54][55][56][57][58][59][60][61][62][63][64][65][66][67][68][69][70][71][72][73], while CBD content has been reported to be in the range 0.4−183 mg/g [40][41][42][43][44][45][54][55][56][57][58][59][60][61][62][63][64][65][66][67][68][69][70][71][72][73]. Other minor non-psychoactive cannabinoids are generally below 10 mg/g of dry inflorescences [40][41][42][43][44][45]. ...
... In general, the most widely employed method of extraction is solidliquid extraction (SLE), which involves the use of an appropriate solvent with great affinity for cannabinoids [18]. The most suitable solvent for cannabinoids extraction is ethanol (EtOH), since it has a high extracting efficiency for these compounds [67,72]. Methanol (MeOH), ethyl acetate (EtOAc) and hexane have also been employed, alone or in combination with other solvents, such as MeOH-chloroform (CHCl 3 ) 9:1 (v/v) for both chemotype distinction [73] and quality control purposes [81]. ...
Article
Cannabidiol (CBD) is a bioactive terpenophenolic compound isolated from Cannabis sativa L. It is known to possess several properties of pharmaceutical interest, such as antioxidant, anti-inflammatory, anti-microbial, neuroprotective and anti-convulsant, being it active as a multi-target compound. From a therapeutic point of view, CBD is most commonly used for seizure disorder in children. CBD is present in both medical and fiber-type C. sativa plants, but, unlike Δ⁹-tetrahydrocannabinol (THC), it is a non-psychoactive compound. Non-psychoactive or fiber-type C. sativa (also known as hemp) differs from the medical one, since it contains only low levels of THC and high levels of CBD and related non-psychoactive cannabinoids. In addition to medical Cannabis, which is used for many different therapeutic purposes, a great expansion of the market of hemp plant material and related products has been observed in recent years, due to its usage in many fields, including food, cosmetics and electronic cigarettes liquids (commonly known as e-liquids). In this view, this work is focused on recent advances on sample preparation strategies and analytical methods for the chemical analysis of CBD and related compounds in both C. sativa plant material, its derived products and biological samples. Since sample preparation is considered to be a crucial step in the development of reliable analytical methods for the determination of natural compounds in complex matrices, different extraction methods are discussed. As regards the analysis of CBD and related compounds, the application of both separation and non-separation methods is discussed in detail. The advantages, disadvantages and applicability of the different methodologies currently available are evaluated. The scientific interest in the development of portable devices for the reliable analysis of CBD in vegetable and biological samples is also highlighted.
... Various HPLC and UPLC methods that have been reported for the analysis of cannabinoids in C. sativa L. plant samples [22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] and Cannabis consumer products, e.g. hashish, marijuana and cannabis oils, [39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55] since the year 2010, are summarised in Tables 1 and 2, and appraised in the following subsections. ...
... Both HPLC and UPLC methods have been applied to separate, identify and quantify various cannabinoids in C. sativa L. samples, including whole plants, roots, inflorescences and biomass containing Cannabis plant parts [22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] (Table 1). However, the use of simple HPLC-based methods has been observed more often than UPLC-based methods for the analysis of C. sativa plant samples in the last decade. ...
... 34 Several HPLC and UPLC-based methods for the analysis of various extracts of C. sativa whole plants have been reported. [23][24][25][26][27][28][29][30][31][32][33]35,36 While the UV or UV-PDA-based detection is quite common, ESI-MS/MS detection has been increasingly becoming the method of choice for cannabinoids analysis from C. sativa plant crude extracts by HPLC or UPLC. In an HPLC-UV based method, a 50 mm long monolithic column of C 18 packing with an internal diameter of 4.6 mm, and particle size of 5 μm has recently been used with a linear ACN-water gradient (flow rate: 2 mL/min) to determine THC (12) (at 210 nm) in the plant extract obtained (yield: >26%) by the supercritical extraction method at different pressures (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33), temperature (40-80 C) and ethanol (EtOH) as a co-solvent (0-5%) 23 . ...
Article
Introduction: Organic molecules that bind to cannabinoid receptors are called cannabinoids, and they have similar pharmacological properties like the plant, Cannabis sativa L. Hyphenated liquid chromatography (LC), incorporating high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC, also known as ultrahigh-performance liquid chromatography, UHPLC), usually coupled to an ultraviolet (UV), UV-photodiode array (PDA) or mass spectrometry (MS) detector, has become a popular analytical tool for the analysis of naturally occurring cannabinoids in various matrices. Objective: To review literature on the use of various LC-based analytical methods for the analysis of naturally occurring cannabinoids published since 2010. Methodology: A comprehensive literature search was performed utilising several databases, like Web of Knowledge, PubMed and Google Scholar, and other relevant published materials including published books. The keywords used, in various combinations, with cannabinoids being present in all combinations, in the search were Cannabis, hemp, cannabinoids, Cannabis sativa, marijuana, analysis, HPLC, UHPLC, UPLC, quantitative, qualitative and quality control. Results: Since 2010, several LC methods for the analysis of naturally occurring cannabinoids have been reported. While simple HPLC-UV or HPLC-UV-PDA-based methods were common in cannabinoids analysis, HPLC-MS, HPLC-MS/MS, UPLC (or UHPLC)-UV-PDA, UPLC (or UHPLC)-MS and UPLC (or UHPLC)-MS/MS, were also used frequently. Applications of mathematical and computational models for optimisation of different protocols were observed, and pre-analyses included various environmentally friendly extraction protocols. Conclusions: LC-based analysis of naturally occurring cannabinoids has dominated the cannabinoids analysis during the last 10 years, and UPLC and UHPLC methods have been shown to be superior to conventional HPLC methods.
... At the same time, a need for an efficient routine analytical method for monitoring the cannabinoid content in plant material has arisen. A number of methods for the analysis of cannabinoids in cannabis have indeed already been developed; among various approaches, the predominant is chromatographic analysis, in particular using gas chromatography (GC) [11][12][13][14][15][16][17][18] or high performance liquid chromatography (HPLC) [16,[19][20][21][22][23][24][25][26][27]. ...
... However, this represents an additional step that is often not desirable, because it increases probability for experimental error and prolongs analysis time, which may be a considerable drawback in terms of method suitability for routine use. With HPLC, all of these problems have been successfully avoided, as some relatively rapid, simple, and effective methods for the determination of both acidic and decarboxylated cannabinoids in cannabis samples have already been developed [16,[19][20][21][22][23][24][25][26][27]33]. ...
... Thus, two major approaches to chromatographic analysis of cannabinoids in hemp most often appear in the literature; direct analysis of a suitably diluted sample extract by liquid chromatography [16,[19][20][21][22][23][24][25][26][27]33], or preliminary derivatisation of the extract and subsequent analysis by gas chromatography [17,[30][31][32]. Despite its mentioned drawbacks, the latter approach is still quite in use, somewhat for traditional reasons, but also for entirely practical reasons, since GC instrumentation is simpler and less expensive than HPLC and sometimes, consequently, more economical for use or maybe even the only one available. ...
Article
Full-text available
An original gas chromatographic method has been developed for simultaneous determination of major terpenes and cannabinoids in plant samples and their extracts. The main issues to be addressed were the large differences in polarity and volatility between both groups of analytes, but also the need for an exhaustive decarboxylation of cannabinoid acidic forms. Sample preparation was minimised, also by avoiding any analyte derivatisation. Acetone was found to be the most appropriate extraction solvent. Successful chromatographic separation was achieved by using a medium polarity column. Limits of detection ranged from 120 to 260 ng/mL for terpenes and from 660 to 860 ng/mL for cannabinoids. Parallel testing proved the results for cannabinoids are comparable to those obtained from established HPLC methods. Despite very large differences in concentrations between both analyte groups, a linear range between 1 and 100 µg/mL for terpenes and between 10 and 1500 µg/mL for cannabinoids was determined.
... However, as more cannabinoids were identified and their pharmacological potential was examined, the quantification of cannabinoids other than ∆9-THC gained importance. Additional cannabinoids of interest now include ∆9-tetrahydrocannabinolic acid (THCA), cannabidiol (CBD), cannabidiolic acid (CBDA), cannabigerol (CBG), cannabigerolic acid (CBGA), ∆9-tetrahydrocannabivarin (THCV), ∆9tetrahydrocannabivarinic acid (THCVA), cannabidivarin (CBDV), and cannabidivarinic acid (CBDVA) [2,3]. A recent study on virus neutralizing capabilities of naturally occurring cannabinoids (in acid form) has found that CBDA and CBGA are capable of binding to spike protein of SARS-CoV-2 therefore possible candidates for the treatment as well as prevention of COVID-19 [4]. ...
... Until recently, the detection of column-separated cannabinoids was predominantly carried out using UV spectrophotometry, which provides low specificity and makes base-line separation of all cannabinoids imperative. However, complete separation of structurally similar cannabinoids, especially isomeric compounds such as ∆8-THC and ∆9-THC, is challenging [3]. Due to the lack of specificity of UV detection, any unknown compound/s co-eluting at the same retention time as the target cannabinoid can cause overestimation of its concentration. ...
... According to a recent review of cannabinoid analysis, variabilities in extraction (solvent/s, method, time, and temperature) significantly contribute to differential analytical results [6]. Most of the recent analytical methods have used ethanol for extraction of both acidic and neutral cannabinoids [3,9], although the exact method used for extraction varied in terms of sample to solvent ratio, the technique of extraction, and the duration of extraction. Therefore, a study of the effects of these variables on the amounts of cannabinoids extracted is warranted. ...
Article
Full-text available
With an increasing appreciation for the unique pharmacological properties associated with distinct, individual cannabinoids of Cannabis sativa, there is demand for accurate and reliable quantification for a growing number of them. Although recent methods are based on highly selective chromatography-mass spectrometry technology, most are limited to a few cannabinoids, while relying on unnecessarily sophisticated and expensive ultra-high performance liquid chromatography and tandem mass spectrometry. Here we report an optimised, simple extraction method followed by a reliable and simple high performance liquid chromatography method for separation. The detection is performed using a time-of-flight mass spectrometer that is available in most natural products research laboratories. Due to the simplicity of instrumentation, and the robustness resulting from a high resolution in the chromatography of isobaric cannabinoids, the method is well suited for routine phytocannabinoid analysis for a range of applications. The method was validated in terms of detection and quantification limits, repeatability, and recoveries for a total of 17 cannabinoids: detection limits were in the range 11–520 pg when using a 1 µL sample injection volume, and the recovery percentages ranged from 85% to 108%. The validated method was subsequently applied to determine cannabinoid composition in the inflorescences of several medicinal Cannabis sativa varieties.
... SPE, that has great advantages in terms of reproducibility and automation, is frequently used for the treatment of body fluids and human samples and is based on the employment of small columns, mostly containing C 8 and C 18 reversed-phase materials. SLE is commonly used to extract cannabinoids from plant samples given their abundancy, since their concentrations are reported to range from 0.1 to 40% of inflorescence dry weight [40]. Sonication, or more advanced techniques such as high throughput homogenization (HTH), have also been observed to improve the extraction yield from dried and powdered material [40]. ...
... SLE is commonly used to extract cannabinoids from plant samples given their abundancy, since their concentrations are reported to range from 0.1 to 40% of inflorescence dry weight [40]. Sonication, or more advanced techniques such as high throughput homogenization (HTH), have also been observed to improve the extraction yield from dried and powdered material [40]. Polar solvents such as methanol and ethanol, as well as less polar solvents (hexane, petroleum ether, benzene) can be used to treat the sample, and the n-hexane:ethyl acetate 9:1 ratio provides optimal recovery values [38,41]. ...
... On the other hand, HPLC has been described as the most suitable method to analyze the "native composition" of the plant, possibly without derivatization and avoiding decarboxylation [40]. Thus, this technique is suitable to be applied for phenotype determination and detection of undesired or restricted compounds. ...
Article
Cannabidiol (CBD) is a non-psychotropic phytocannabinoid which represents one of the constituents of the “phytocomplex” of Cannabis sativa. This natural compound is attracting growing interest since when CBD-based remedies and commercial products were marketed. This review aims at exhaustively addressing the extractive and analytical approaches that have been developed for the isolation and quantification of CBD. Recent updates on cutting-edge technologies were critically examined in terms of yield, sensitivity, flexibility and performances in general, and are reviewed alongside original representative results. As an add-on to currently available contributions in the literature, the evolution of novel, efficient synthetic approaches for the preparation of CBD, a procedure which is appealing for the pharmaceutical industry, is also discussed. Moreover, given the increasing interest on the therapeutic potential of CBD and the limited understanding of the undergoing biochemical pathways, the reader will be updated about recent in silico studies on the molecular interactions of CBD towards several different targets attempting to fill this gap. Computational data retrieved from the literature have been integrated with novel in silico experiments, critically discussed to provide a comprehensive and updated overview on the undebatable potential of CBD and its therapeutic profile.
... Flame ionization detector (FID) and mass spectrometry (MS) are generally interfaced with GC as a detection tool. FID provides a more accurate quantitative response and greater sensitivity although MS presents highest specificity [23,24]. ...
... The recent legalization of marijuana in some countries, and the licensing of CBD for medicinal use in others [25][26][27][28][29][30] has shed new light on THC quantification in different products. Despite of the popularity of GC-MS in forensic laboratories, recently HPLC-DAD became an interesting option for THC quantification [23,[31][32][33]. This is due to the fact that some cannabinoids are thermolabile and sample vaporization on the GC/MS injector causes partial degradation of delta-9-THC leading to an underestimation of the true amount of this compound present in the sample [18,19]. ...
... GC/FID or GC/MS are the most widely adopted techniques for cannabinoids analysis based on their accuracy in providing qualitative and quantitative results [20][21][22][23][24]. More recently, HPLC-DAD has proven to be an extremely powerful tool for cannabinoids quantification [23,[31][32][33]. ...
Article
HIGHLIGHTS: (1) Backlog is a reality in many different types of Forensic Laboratories; (2) THC content decreases with storage and can be used to understand backlog impact; (3) Changes in bench procedures were able to decrease waiting time considerably; (4) Long term storage of marijuana samples sharply increases inconclusive results; (5) The escalation of inconclusive results increases the run cost of a laboratory. ABSTRACT: Forensic laboratories worldwide are struggling to keep up with the increasing number of cases submitted for analysis, regardless of the reasons, backlog of controlled substances cases is a reality in many countries. In this paper we analyse the number of petitioned examinations (from 2016 to 2020) and the data from 11,655 marijuana TLC results from the Forensic Laboratory in the Federal District Civil Police in Brazil. Data demonstrates that backlog increases inconclusive results, with storage and light playing a crucial role in the process. Additionally we explored the repercussions of delayed forensic results for controlled substances and propose an approach to overcome waiting time in this context.
... On the other hand, GC is inappropriate for determination of both forms of cannabinoids if not previously derivatized, since due to heat of the injector, decarboxylation of cannabinoids occurs leading just to their relative neutral forms [3]. Thus, cannabinoids are generally determined by GC/MS [3], HPLC or UHPLC coupled to diode array detector (DAD) [13][14][15][16], low-resolution MS [6,16,17], or high-resolution MS [6,18,19]. On the other hand, due to the volatile character of terpenes, GC is widely used for their determination. ...
... On the other hand, due to the volatile character of terpenes, GC is widely used for their determination. Terpenes have been mostly determined by headspace-solid phase microextraction (HS-SPME) coupled to GC/MS [11,18], GC-flame ionization detection (GC-FID) [15], or direct injection of hemp oil extract into GC/MS [19]. As reported above, the main problem is that terpenes and CBD are separately determined using different techniques and methods, which results in time-consuming protocols and requiring the use of expensive instruments (such as mass spectrometers). ...
... Pavlovic et al. [19] determined terpenes in CBD oil by HS-SPME-GC/MS in 35 min and CBD by HPLC coupled to high-resolution MS. Giese et al. [15] validated a method for the analysis of cannabinoids and terpenes (17 terpenes separated in 35 min) in cannabis by using HPLC-DAD and GC-FID, respectively. Thus, the reduction of analysis time (29 terpenes and CBD in <16 min) leads to a significant improvement in the number of samples processed per day, which is advantageous for the detection of less stable terpenes. ...
Article
Hemp (Cannabis sativa L.) has become widely used in several sectors due to the presence of various bioactive compounds such as terpenes and cannabidiol. In general, terpenes and cannabidiol content is determined separately which is time‐consuming. Thus, a fast Gas Chromatography with Flame Ionization Detection method was validated for simultaneous determination of both terpenes and cannabidiol in hemp. The method enabled a rapid detection of 29 different terpenes and cannabidiol within a total analysis time of 16 min, with satisfactory sensitivity (LOD = 0.03 – 0.27 μg/mL, LOQ = 0.10 – 0.89 μg/mL). The interday and intraday precision (RSD) was <7.82 % and <3.59 %, respectively. Recoveries at two spiked concentration levels (low, 3.15 μg/mL; high, 20.0 μg/mL) were determined on both apical leaves (78.55 – 101.52 %) and inflorescences (77.52 ‐ 107.10 %). The reproducibility (RSD) was <5.94 % and <5.51 % in apical leaves and inflorescences, respectively. The proposed and validated method is highly sensitive, robust, fast, and accurate for determination of the main terpenes and cannabidiol in hemp and could be routinely used for quality control. This article is protected by copyright. All rights reserved
... This makes a complete characterisation difficult and limited by the availability of analytical standards and methodological factors. Existing chromatographic methods for the analysis of cannabinoids have several limitations; this includes applicability to a small number of cannabinoids [5][6][7] (only six to eight), complex chromatographic gradient separations [5,8] that can be difficult to replicate, expensive instrumentation costs [9] with mass spectrometry, and long run times [6] up to 25 mins, which would limit sample throughput. We aimed to develop a comprehensive method for the quantification of 17 phytocannabinoids using an isocratic separation with basic HPLC equipment that could be adopted by any analytical laboratory. ...
... CBG as CBD). This method provides chromatographic separation of 17 cannabinoids, which is more than previous methods that include only six to eight cannabinoids, [5][6][7] does not require gradient separation, [5][6][7][8][9] does not require the capital expense of mass spectrometry, [9] and only takes 11 min. Overall, this is a significant improvement on existing cannabinoid methods. ...
... CBG as CBD). This method provides chromatographic separation of 17 cannabinoids, which is more than previous methods that include only six to eight cannabinoids, [5][6][7] does not require gradient separation, [5][6][7][8][9] does not require the capital expense of mass spectrometry, [9] and only takes 11 min. Overall, this is a significant improvement on existing cannabinoid methods. ...
Article
Full-text available
Although cannabis has been used for several thousand years, the exact composition of the cannabinoids patients are administered for different symptoms has remained largely unknown. While this absence of catalogued information may be accepted in some cultures, the use of cannabis as a human product in the registered medicines setting requires knowing its composition so that doses can be standardised between patients. This is particularly so in clinical trials that are currently under way to determine the efficacy of a product. Although the major cannabinoids of interest to prescribers are well known – tetrahydrocannabinol and cannabidiol and the corresponding acids tetrahydrocannabinolic acid and cannabidiolic acid, the cannabis plant contains many more phytocannabinoids. We have developed and validated a robust and fast (11min) isocratic HPLC method for the analysis of 17 phytocannabinoids. The method had an analytical range of 1–150μg mL−1 for tetrahydrocannabinolic acid and cannabidiolic acid, 0.5–75μg mL−1 for tetrahydrocannabinol and cannabidiol, and 0.5–20μg mL−1 for the remaining 13 cannabinoids. The method had excellent repeatability with a relative standard deviation of between 5 and 14% and a bias of between –8.6 and 6% for the 17 cannabinoids. The method was applied to the analysis of medicinal cannabis products, including both flos and oils with results matching the supplier’s certificate of analysis. This simple fast isocratic method with basic HPLC equipment can be easily transferred to any analytical laboratory interested in the identification and quantitation of cannabinoids.
... [5][6][7] Sampling and homogenization of plant materials is also a critical factor, especially for heterogenous materials like cannabis flower and concentrates. While sonication is the most common and supported technique, [8] a thorough understanding of sampling procedures and homogeneity should underpin all cannabis compliance testing programs. [9] As more matrices containing cannabinoids enter the marketplace (e.g., pet food, shampoo, bath bombs, cereal, etc.), there may be an evolving need for modified extraction procedures. ...
... To date more than 200 terpenes have been identified in cannabis, but the 58 monoterpenes and 38 sequiterpenes confirmed represent the compound classes found in the greatest frequency and abundance. [8] Parsing down these exhaustive lists further, cannabis chemotypes are generally dominated by a subset of 10-20 highly prevalent and highly abundant terpenes, with the most studied and frequently observed being b-myrcene, d-limonene, a-terpineol, c-terpinolene, a/b-pinene, linalool, b-caryophyllene (the most abundant sesquiterpene), a-humulene and caryophyllene oxide (the chemical that narcotics control dogs smell when searching for cannabis [8] ). [9,22] Beyond their natural functions in plants (pest defense, etc.), terpenes represent the dominant organoleptic contributors to cannabis-it is these compounds that drive consumer preference for certain types of cannabis due to their distinct aroma and flavor profiles. ...
... To date more than 200 terpenes have been identified in cannabis, but the 58 monoterpenes and 38 sequiterpenes confirmed represent the compound classes found in the greatest frequency and abundance. [8] Parsing down these exhaustive lists further, cannabis chemotypes are generally dominated by a subset of 10-20 highly prevalent and highly abundant terpenes, with the most studied and frequently observed being b-myrcene, d-limonene, a-terpineol, c-terpinolene, a/b-pinene, linalool, b-caryophyllene (the most abundant sesquiterpene), a-humulene and caryophyllene oxide (the chemical that narcotics control dogs smell when searching for cannabis [8] ). [9,22] Beyond their natural functions in plants (pest defense, etc.), terpenes represent the dominant organoleptic contributors to cannabis-it is these compounds that drive consumer preference for certain types of cannabis due to their distinct aroma and flavor profiles. ...
Article
Owing to the lack of federal oversight of recreational and medical cannabis in the United States, a patchwork of regulatory guidelines exists for compliance testing. Adding to this complexity is the fact that Canadian cannabis regulations differ from those in any of the state mandated regulatory jurisdictions and, at the time of writing, cannabis was only recently legalized in Mexico. Therefore, from a North American perspective, cannabis testing represents a significant regulatory landscape to navigate. This not only makes things confusing for those involved in cannabis production and processing, it also creates challenges for those in the analytical testing world when they have to understand and develop methods to be compliant with these various regulatory jurisdictions. In this review article, the current state of analytical chemistry knowledge for cannabis compliance testing is summarized, with an emphasis on suitable techniques and some common problems to avoid. This includes summaries of analytical methods for potency, terpenes, pesticides, mycotoxins, residual solvents, heavy metals and microbiology.
... Cannabinoids can be analyzed by employing both GCand LC-based analyzers (Giese et al., 2015;Leghissa et al., 2018b). However, issues with their conversion under the high temperatures of the injection port of the former, make their absolute quantification tricky, and their analyses preferable by using LC-based analyzers. ...
... On the other hand, although terpenoids can be recorded by EI detectors, their structural similarities make their absolute identification challenging. Thus, GC/FID platforms are suitable for the analyses of terpenoid profiles (Giese et al., 2015;Leghissa et al., 2018b). Additionally, the linear range of the detector facilitates the recording of the wide range of terpene concentrations in Cannabis extracts. ...
... For QC purposes, the implementation of different analyzers is required for the monitoring of metabolites across the various groups of Cannabis metabolites, which exhibit highly diverse physicochemical properties, making their detection and quantification challenging tasks. The employment of LC-diode array detector (LC-DAD) and GC/FID platforms have enabled the repeatable detection of cannabinoids and terpenes with low relative standard deviations (RSDs), using EtOH for extraction (Giese et al., 2015). ...
Article
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Cannabis (Cannabis sativa L.) is a complex, polymorphic plant species, which produces a vast array of bioactive metabolites, the two major chemical groups being cannabinoids and terpenoids. Nonetheless, the psychoactive cannabinoid tetrahydrocannabinol (Δ 9 -THC) and the non-psychoactive cannabidiol (CBD), are the two major cannabinoids that have monopolized the research interest. Currently, more than 600 Cannabis varieties are commercially available, providing access to a multitude of potent extracts with complex compositions, whose genetics are largely inconclusive. Recently introduced legislation on Cannabis cultivation in many countries represents a great opportunity, but at the same time, a great challenge for Cannabis research and development (R&D) toward applications in the pharmaceutical, food, cosmetics, and agrochemical industries. Based on its versatility and unique capabilities in the deconvolution of the metabolite composition of complex matrices, metabolomics represents an ideal bioanalytical tool that could greatly assist and accelerate Cannabis R&D. Among others, Cannabis metabolomics or cannabinomics can be applied in the taxonomy of Cannabis varieties in chemovars, the research on the discovery and assessment of new Cannabis-based sources of bioactivity in medicine, the development of new food products, and the optimization of its cultivation, aiming for improvements in yield and potency. Although Cannabis research is still in its infancy, it is highly foreseen that the employment of advanced metabolomics will provide insights that could assist the sector to face the aforementioned challenges. Within this context, here, the current state-of-the-art and conceptual aspects of cannabinomics are presented.
... As Leghissa and colleagues note [23], conclusively identifying terpenes in cannabis is challenging due to the large variety of possible candidates and a lack of commercial standards for a large number of them. With the increased interest in medicinal cannabis and the contribution of terpenes to the entourage effect, methods to quantify terpenes in medicinal cannabis biomass and products are becoming more available in the literature [24][25][26][27]. Often, these methods require large quantities of biomass (1-5 g), use large quantities of organic solvents (up to 100 mL/sample), and include separations with gradient runtimes exceeding 60 min. ...
... A 5 °C/min gradient was chosen as a trade-off between shortest possible run time and best possible resolution. The runtime achieved is shorter than that reported by Giese and colleagues using a 3.5 °C gradient [24], while also resolving five additional, later eluting sesquiterpenes, including the validation compounds guaiol and bisabolol. Furthermore, by employing column backflushing after elution of bisabolol, we can prevent cannabinoids, column bleed, and other high-boiling compounds from entering the MS, reducing contamination of the source and therefore the need for more frequent source cleaning. ...
Article
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Cannabis and its secondary metabolite content have recently seen a surge in research interest. Cannabis terpenes and terpenoids in particular are increasingly the focus of research efforts due to the possibility of their contribution to the overall therapeutic effect of medicinal cannabis. Current methodology to quantify terpenes in cannabis biomass mostly relies on large quantities of biomass, long extraction protocols, and long GC gradient times, often exceeding 60 min. They are therefore not easily applicable in the high-throughput environment of a cannabis breeding program. The method presented here, however, is based on a simple hexane extract from 40 mg of biomass, with 50 μg/mL dodecane as internal standard, and a gradient of less than 30 min. The method can detect 48 individual terpenes and terpenoids and was validated for selectivity, linearity, LOD/LOQ, precision, intermediate precision, and accuracy (recovery) for 22 terpenes and terpenoids. The validation parameters are comparable to previously published studies that employ significantly longer runtimes and/or more complex extraction protocols. It is currently being applied to medicinal cannabis precision breeding programs.
... Additionally, oxygenated terpenoids can also be found (Leghissa et al., 2018). In forthcoming years, terpenoids have received great attention because of their sensorial properties, with peculiar chemical fingerprinting for various Cannabis cultivars, and investigations concerning their synergism with phytocannabinoids (Giese et al., 2015). Many studies have proposed the application of extracts, so-called phytocomplexes, containing a mixture of phytocannabinoids and terpenoids, rather than pure synthetic molecules, thus suggesting the existence of complementary or synergistic interactions, often called entourage effects (De Vita et al., 2019;Elzinga et al., 2015). ...
... Terpenoid yields usually vary from 0.01 to 1.5 % of the inflorescence dry weight (Giese et al., 2015). The hemp volatile fraction, as mentioned, consists of monoterpenes, such as α-pinene, myrcene and terpinolene, and bitter-tasting sesquiterpenes, such as E-caryophyllene, α-humulene, and caryophyllene oxide (Fiorini et al., 2019). ...
Article
In recent years, hemps health and nutritional properties recognition has led to an impressive growth of Cannabis research, industrial processing, and the related market. Moreover, the demand for natural Cannabis-derived compounds (i.e. terpenes, polyphenols, and cannabinoids) is constantly growing. In spite of the strict regulation of some countries, the global market needs suitable technologies for the smart recovery of bioactive Cannabis metabolites. Conventional extraction procedures can show drawbacks, in terms of environmental impact and their high energy consumption. Microwaves (MW), a mature technique for extraction-process intensification, is attracting great amounts of attention in academic-research and industrial-application fields for its technological advantages. This work aims to design a fast and cost-efficient MW-assisted cascade protocol for bioactive Cannabis compounds recovery in a pilot-scale reactor. Microwave-assisted hydrodistillation (MAHD) can provide a volatile hydrodistillate that is rich in monoterpenes, sesquiterpenes, and a small amount of phytocannabinoids. Using non-canonical protocol of hydrodistillation, the definition of "volatile fraction" is generally considered more appropriate than "essential oil". The health-promoting activity of this combination has been proposed in literature, and can constitute matter of further investigations. The optimized MAHD procedure yielded 0.35 ± 0.02 % w/w of hydrodistillate, while conventional hydrodistillation gave only 0.12 ± 0.01 %, w/w (in relation to dry inflorescence mass). The water resulting in the vessel after MAHD showed a high total polyphenolic content (5.35 ± 0.23 %, w/w). Two flavones known for their beneficial effects to health, namely luteolin-7-O-glucoside and apigenin-7-O-glucoside, were detected and quantified. An attempt to recover phytocannabinoid using the MW-assisted hydrodiffusion and gravity method (MAHG) was also carried out. Cannabinoids (CBD and THC) content was determined in fresh Cannabis and in production streams. During MAHD, phytocannabinoid decarboxylation inside the residual matrix was around 70 % (69.01 ± 0.98 % and 74.32 ± 1.02 % for THC and CBD respectively). Furthermore, the overall content of these metabolites was not affected by the hydrodistillation, preserving the processed plant material for subsequent ethanolic extraction.
... For this reason, our experiments did not use cannabis products nor included cannabinoids. Instead, we focused on concomitant substances present in VCCs, including monoterpenes (βmyrcene, d-limonene, α-pinene, β-pinene), terpene alcohols (linalool), sesquiterpenes (β-caryophyllene, α-humulene), sesquiterpenoid alcohols (cedrol, α-bisabolol), triterpenes (friedelin), lignans (secoisolariciresinol), and flavonoids (quer- 13,14,16 To evaluate the impact of these substances on indoor air quality after heating and decomposition, we studied a "full terpenoid" mixture comprising nine compounds commonly found in VCCs and two separate mixtures containing the corresponding light and heavy terpenoid fractions. In addition, a "complex" mixture was prepared by adding a small amount (∼0.5%) of high MW compounds (triterpenes, lignans, and flavonoids) to the full terpenoid mixture. ...
... Mixing ratios were based on those reported in the literature. 14,16 All mixtures were prepared separately by combining research-grade pure compounds (between 80% and 95% purity, Sigma Aldrich and TCI America), most of which were in the liquid form. ...
Article
Vaporizable cannabis concentrates (VCCs) consumed as a liquid (vaping) or a waxy solid (dabbing) are becoming increasingly popular. However, their associated emissions and impacts have not been fully described. Mixtures containing different proportions of 12 VCC terpenoids and high MW compounds were heated at 100-500 °C inside a room-sized chamber to simulate emissions. Terpenoids, thermal degradation byproducts, and ultrafine particles (UFPs) were quantified in the chamber air. Air samples contained over 50% of emitted monoterpenes and less than 40% of released sesquiterpenes and terpene alcohols. Eleven degradation byproducts were quantified, including acrolein (1.3-3.9 μg m-3) and methacrolein (2.0 μg m-3). A large amount of UFPs were released upon heating and remained airborne for at least 3 h. The mode diameter increased from 80 nm at 100 °C to 140 nm at 500 °C, and particles smaller than 250 nm contributed to 90% of PM1.0. The presence of 0.5% of lignin, flavonoid, and triterpene additives in the heated mixtures resulted in a threefold increase in the particle formation rate and PM1.0 concentration, suggesting that these high-molecular-weight compounds enhanced aerosol inception and growth. Predicted UFP emission rates in typical consumption scenarios (6 × 1011-2 × 1013 # min-1) were higher than, or comparable with, other common indoor sources such as smoking and cooking.
... Three monoterpenes, limonene, b-myrcene, and a-pinene, and two sesquiterpenes, b-caryophyllene and a-humulene, were abundant in the majority of chemovars (Mudge et al., 2019). In North American chemovars, the following eight terpenes were predominant: b-myrcene, terpinolene, ocimene, limonene, a-pinene, humulene, linalool, and b-caryophyllene (Giese et al., 2015). But the work of Allen et al. (2019) highlighted that the quantity of the common top five terpenes, including b-myrcene, a-pinene, limonene, b-caryophyllene, and terpinolene, showed large variance; they can be the single most abundant terpene in certain chemovars, or minor components, or even under the limit of detection in other chemovars. ...
... However, several papers in the literature can be found without validation of analytical process, which could result in unreliable data. To increase the reproducibility of terpene profile analysis, rigorous studies need to be performed analyzing terpenes in plant materials produced under controlled environmental conditions, harvested in certain developmental stages, and analyzed using validated and standardized analytical methods (Giese et al., 2015;Ibrahim et al., 2019). Consistent cultivation and handling conditions must be applied to all plant materials used for medicinal purposes. ...
Article
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Pain prevalence among adults in the United States has increased 25% over the past two decades, resulting in high health-care costs and impacts to patient quality of life. In the last 30 years, our understanding of pain circuits and (intra)cellular mechanisms has grown exponentially, but this understanding has not yet resulted in improved therapies. Options for pain management are limited. Many analgesics have poor efficacy and are accompanied by severe side effects such as addiction, resulting in a devastating opioid abuse and overdose epidemic. These problems have encouraged scientists to identify novel molecular targets and develop alternative pain therapeutics. Increasing preclinical and clinical evidence suggests that cannabis has several beneficial pharmacological activities, including pain relief. Cannabis sativa contains more than 500 chemical compounds, with two principle phytocannabinoids, Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD). Beyond phytocannabinoids, more than 150 terpenes have been identified in different cannabis chemovars. Although the predominant cannabinoids, Δ9-THC and CBD, are thought to be the primary medicinal compounds, terpenes including the monoterpenes β-myrcene, α-pinene, limonene, and linalool, as well as the sesquiterpenes β-caryophyllene and α-humulene may contribute to many pharmacological properties of cannabis, including anti-inflammatory and antinociceptive effects. The aim of this review is to summarize our current knowledge about terpene compounds in cannabis and to analyze the available scientific evidence for a role of cannabis-derived terpenes in modern pain management. SIGNIFICANCE STATEMENT: Decades of research have improved our knowledge of cannabis polypharmacy and contributing phytochemicals, including terpenes. Reform of the legal status for cannabis possession and increased availability (medicinal and recreational) have resulted in cannabis use to combat the increasing prevalence of pain and may help to address the opioid crisis. Better understanding of the pharmacological effects of cannabis and its active components, including terpenes, may assist in identifying new therapeutic approaches and optimizing the use of cannabis and/or terpenes as analgesic agents.
... Hemp terpenes in the EO contribute to the aroma of various cannabis genotypes, and so far, around 140 different terpenes have been reported in this plant [1,[6][7][8]. Current thinking is that terpenes have played a key role in the selection of medical/recreational and CBD type cannabis because their concentration is positively correlated to some of the cannabinoids [9]. ...
... The 10 constituents whose means were significantly different are shown in Table 2. The comparative concentrations of the EO constituents in this and previous reports cited here are summarized in Table 3. Table 2. Mean concentration (%) of isocaryophyllene (γ-caryophyllene) [1], β-caryophyllene [2], α-(E)-bergamotene [3], caryophyllene oxide [4], humulene epoxide 2 [5], selina-6-en-4-ol [6], caryophylla-4(12),8(13)-dien-5β-ol [7], β-bisabolol [8], α-bisabolol [9], and δ9-tetrahydrocannabinol (dronabinol) [10] obtained from the 9 cultivars. ...
Article
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Hemp (Cannabis sativa L.) is currently one of the most controversial and promising crops. This study compared nine wild hemp (C. sativa spp. spontanea V.) accessions with 13 registered cultivars, eight breeding lines, and one cannabidiol (CBD) hemp strain belonging to C. sativa L. The first three groups had similar main essential oil (EO) constituents, but in different concentrations; the CBD hemp had a different EO profile. The concentration of the four major constituents in the industrial hemp lines and wild hemp accessions varied as follows: β-caryophyllene 11-22% and 15.4-29.6%; α-humulene 4.4-7.6% and 5.3-11.9%; caryophyllene oxide 8.6-13.7% and 0.2-31.2%; and humulene epoxide 2, 2.3-5.6% and 1.2-9.5%, respectively. The concentration of CBD in the EO of wild hemp varied from 6.9 to 52.4% of the total oil while CBD in the EO of the registered cultivars varied from 7.1 to 25%; CBD in the EO of the breeding lines and in the CBD strain varied from 6.4 to 25% and 7.4 to 8.8%, respectively. The concentrations of δ9-tetrahydrocannabinol (THC) in the EO of the three groups of hemp were significantly different, with the highest concentration being 3.5%. The EO of wild hemp had greater antimicrobial activity compared with the EO of registered cultivars. This is the first report to show that significant amounts of CBD could be accumulated in the EO of wild and registered cultivars of hemp following hydro-distillation. The amount of CBD in the EO can be greater than that in the EO of the USA strain used for commercial production of CBD. Furthermore, this is among the first reports that show greater antimicrobial activity of the EO of wild hemp vs. The EO of registered cultivars. The results suggest that wild hemp may offer an excellent opportunity for future breeding and the selection of cultivars with a desirable composition of the EO and possibly CBD-rich EO production.
... To avoid the decarboxylation of acid forms, a timeconsuming derivatization before GC analysis must be performed (39), e.g. by silylation as the trimethylsilyl ethers. However, an effective derivatization yield for all components in a complex mixture is difficult to achieve (13) and may also occur the thermo-degradation of derivatized cannabinoids in injector and/or column system (40). Whereas the cannabis plant mainly contains the acid forms of cannabinoids, GC analysis presents a limited value to establish the metabolic profile of a cannabinoid sample. ...
... An accurate manner to assay the cannabis composition is to use a method that does not involve thermal stress, such as LC (40). This technique allows the simultaneous detection of both acid and neutral cannabinoids with no need of derivatization. ...
Article
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Background: Cannabis has been the most widely used illicit drug worldwide throughout many years. Reports from different countries indicate that the potency of cannabis preparation has been increasing, as well as the ratio of tetrahydrocannabinol/cannabidiol has been changing. The high consumption along with the changing chemical profile of the drug has led increasingly to the interest in researching the cannabis plant. Methods: This article reviews available literature on the analytical methods currently used for the detection and quantification of cannabinoids in cannabis plant. The papers were screened by two researchers independently and following a pre-specified protocol. Results and Discussion: The systematic review of the literature allowed to include 42 citations on cannabis plant analysis. Conclusions: The analytical methods for cannabis material published in the included articles of this systematic review showed a lack of relevant information of the development of methods on GC and LC analysis and the limits of detection and quantification of mass detectors.
... Relatively few of these compounds-about 200-are found in cannabis. According to recent publications [142,143], 50 cannabis terpenes can be found in North American chemovars, but some are more commonly found ( Figure 2). The monoterpene myrcene as well as the sesquiterpenes βcaryophyllene and α-humulene appear to be present in most cannabis cultivars. ...
Article
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In recent years, and even more since its legalization in several jurisdictions, cannabis and the endocannabinoid system have received an increasing amount of interest related to their potential exploitation in clinical settings. Cannabinoids have been suggested and shown to be effective in the treatment of various conditions. In cancer, the endocannabinoid system is altered in numerous types of tumours and can relate to cancer prognosis and disease outcome. Additionally, cannabinoids display anticancer effects in several models by suppressing the proliferation, migration and/or invasion of cancer cells, as well as tumour angiogenesis. However, the therapeutic use of cannabinoids is currently limited to the treatment of symptoms and pain associated with chemotherapy, while their potential use as cytotoxic drugs in chemotherapy still requires validation in patients. Along with cannabinoids, cannabis contains several other compounds that have also been shown to exert anti-tumorigenic actions. The potential anti-cancer effects of cannabinoids, terpenes and flavonoids, present in cannabis, are explored in this literature review.
... For analysis, the researchers used a multistep process that included analytical and olfactory testing. Several researchers 18,37 have suggested that the best way to detect cannabis odours is through multidimensional gas chromatography (GC) in tandem with human olfaction. The terpene composition of the grape, hemp, and wine samples was determined using headspace gas chromatography-mass spectrometry (MS) (HS-20 GCMS-QP2010 SE; Shimadzu Corporation, Kyoto). ...
Technical Report
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Industrial Hemp (Cannabis sativa L.) is one of the most versatile agricultural crops in the United States (US). However, until 2018, hemp had not been cultivated on a large scale in the US in over 80 years. With the recent re-authorization of hemp cultivation, the acreage under cultivation has increased tremendously while the knowledge base regarding hemp cultivation practices and interaction with other field crops has remained static. Hemp like other agricultural plants (e.g. Vitis vinefera, Eucalyptus, Lavandula, and Arabidopsis) produce copious amounts of volatile organic compounds (VOCs) such as terpenes. There are concerns about hemp VOCs tainting other agricultural crops. In this study, we examined the potential of hemp terpenes in tainting wine grapes planted in close proximity to a hemp field. Wine grape samples were collected from the vineyard over a five-week period when both the hemp plants and wine grapes were nearing harvest. Overall, the hemp plants contained high levels of terpenes. However, using a headspace GC-MS, there were no detectable levels of hemp terpenes on the wine grapes or the resultant wine made from the vineyard in this study. While the findings of this study are significant, we believe that more research is warranted to fully understand how other variables could influence hemp terpene emission and potential wine grape taint.
... HPLC-DAD differentiates between Cannabis sativa L. chemotypes based on different absorption spectra (Peschel & Polit, 2015). In order to differentiate between CBD and CBG, using HPLC-MS is preferred over HPLC-UV because UV does not provide enough resolution in this case, with certainty, in above 10% concentration of extract (Giese et al., 2015;Lazarjani et al., 2020). Some other methods have also been shown to have different advantages in the quantification of cannabinoids including LC-MS/MS and APCI (Grauwiler et al., 2007), HPLC-MS/MS (Aizpurua-Olaizola et al., 2014), and UPLC-qTOF (Lazarjani et al., 2020). ...
Article
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“Hemp” refers to non-intoxicating, low delta-9 tetrahydrocannabinol (Δ9-THC) cultivars of Cannabis sativa L. “Marijuana” refers to cultivars with high levels of Δ9-THC, the primary psychoactive cannabinoid found in the plant and a federally controlled substance used for both recreational and therapeutic purposes. Although marijuana and hemp belong to the same genus and species, they differ in terms of chemical and genetic composition, production practices, product uses, and regulatory status. Hemp seed and hemp seed oil have been shown to have valuable nutritional capacity. Cannabidiol (CBD), a non-intoxicating phytocannabinoid with a wide therapeutic index and acceptable side effect profile, has demonstrated high medicinal potential in some conditions. Several countries and states have facilitated the use of THC-dominant medical cannabis for certain conditions, while other countries continue to ban all forms of cannabis regardless of cannabinoid profile or low psychoactive potential. Today, differentiating between hemp and marijuana in the laboratory is no longer a difficult process. Certain thin layer chromatography (TLC) methods can rapidly screen for cannabinoids, and several gas and liquid chromatography techniques have been developed for precise quantification of phytocannabinoids in plant extracts and biological samples. Geographic regulations and testing guidelines for cannabis continue to evolve. As they are improved and clarified, we can better employ the appropriate applications of this uniquely versatile plant from an informed scientific perspective.
... Přibližně 140 terpenoidů je zodpovědných za typickou vůni konopí 11 a asi 17 nejběžnějších terpenů by mohlo být použito pro hodnocení fenotypických a/nebo biologických vlastností odrůd konopí 64 . Monoterpenoidy jsou obecně nejvíce zastoupenou skupinou terpenoidů v konopí a zahrnují převážně myrcen, α-a β-pinen, limonen, terpinolen a cis-ocimen. ...
Article
Cannabis is a plant containing a mixture of chemical substances of unique composition, and therefore it has vast array of applications. Hemp fiber and shives are used in the paper, construction and textile industries. Hemp seeds and products have high nutritional value for humans. They contain significant amounts of essential amino acids and represent a source of ω-3 and ω-6 essential fatty acids. In addition, cannabis flowers contain a broad spectrum of secondary metabolites (phytocannabinoids, terpenoids, and phenylpropanoids), which possess beneficial properties against various disorders. This review summarizes the current knowledge of cannabis chemistry, as well as its utilization for various purposes. Special attention is fo-cused on the biosynthesis of phytocannabinoids. © 2020, Czech Society of Chemical Engineering. All rights reserved.
... The higher bias (35.6%) obtained for CBD at low concentrations (specifically at 0.14% w/w) was considered acceptable because of the well-known higher CBD concentrations in cannabis inflorescences, normally in the range of 1− 15% w/w. 7,54,55 The analyzed samples were all characterized by a total CBD concentration ≥1.5% w/w. Thus, the relevance of the high-end of the calibration curve here was more important for real-world sample quantification. ...
Article
The present research reports on the development of a methodology to unravel the complex phytochemistry of cannabis. Specifically, cannabis inflorescences were considered and stir bar sorptive extraction (SBSE) was used for the preconcentration of the metabolites. Analytes were thermally desorbed into a comprehensive two-dimensional (2D) gas chromatography (GC × GC) system coupled with low- and high-resolution mass spectrometry (MS). Particular attention was devoted to the optimization of the extraction conditions, to extend the analytes' coverage, and the chromatographic separation, to obtain a robust data set for further untargeted analysis. Monoterpenes, sesquiterpenes, hydrocarbons, cannabinoids, other terpenoids, and fatty acids were considered to optimize the extraction conditions. The response of selected ions for each chemical class, delimited in specific 2D chromatographic regions, enabled an accurate and fast evaluation of the extraction variables (i.e., time, temperature, solvent, salt addition), which were then selected to have a wide analyte selection and good reproducibility. Under optimized SBSE conditions, eight different cannabis inflorescences and a quality control sample were analyzed and processed following an untargeted and unsupervised approach. Principal component analysis on all detected metabolites revealed chemical differences among the sample types which could be associated with the plant subspecies. With the same SBSE-GC × GC-MS methodology, a quantitative targeted analysis was performed on three common cannabinoids, namely, Δ9-tetrahydrocannabinol, cannabidiol, and cannabinol. The method was validated, giving correlation factors over 0.98 and <20% reproducibility (relative standard deviation). The high-resolution MS acquisition allowed for high-confidence identification and post-targeted analysis, confirming the presence of two pesticides, a plasticizer, and a cannabidiol degradation product in some of the samples.
... Up to now, most studies profiling phytocannabinoids report only the major constituents [15][16][17][18][19]. whereas it has been demonstrated that cannabis extracts from different strains showed contrasting anti-convulsant effects despite possessing equally high CBD concentrations, meaning that low-abundance cannabinoids could play a crucial role in determining the pharmaceutical properties of cannabis and its derivatives [20]. ...
Article
Phytocannabinoids are a broad class of compounds uniquely synthesized by the various strains of Cannabis sativa. Up to date, most investigation on phytocannabinoids have been addressed to the most abundant species, Δ⁹-tetrahydrocannabinol and cannabidiol, for their well-known wide range of pharmaceutical activities. However, in the recent years a large number of minor constituents have been reported, whose role in cannabis pharmacological effects is of current scientific interest. With the purpose of gaining knowledge on major and minor species and furnishing a strategy for their untargeted analysis, in this study we present an innovative approach for comprehensively identifying phytocannabinoids based on high-resolution mass spectrometry in negative ion mode, which allows discrimination of the various isomeric species. For a faster and more reliable manual validation of the tandem mass spectra of known and still unknown species, an extensive database of phytocannabinoid derivatives was compiled and implemented on Compound Discoverer software for the setup of a dedicated data analysis tool. The method was applied to extracts of the Italian FM-2 medicinal cannabis, resulting in the identification of 121 phytocannabinoids, which is the highest number ever reported in a single analysis. Among those, many known and still unknown unconventional phytocannabinoids have been tentatively identified, another piece in the puzzle of unravelling the many uncharted applications of this matrix.
... (Martyny et al., 2013;Victory et al., 2018). The harvest and trimming phases of cannabis cultivation also release high concentrations of terpenes and other VOCs, which can act as respiratory irritants (Giese et al., 2015). While the levels of PM were relatively low at this facility (<0.1 mg m −3 ), many of the tasks associated with Cannabis cultivation generate a significant amount of organic dust. ...
Article
Background: While little is known about the occupational hazards associated with Cannabis cultivation, both historical research in the hemp industry and preliminary data from modern grow houses, suggest that Cannabis workers may be at increased risk of respiratory and allergic diseases. Objectives: We sought to investigate the association between workplace exposures and health symptoms in an indoor Cannabis grow facility in Washington State, USA. Methods: We performed a cross-sectional study with all consenting employees in an indoor Cannabis grow facility in Seattle, WA using a questionnaire. The questionnaire gathered data on respiratory, ocular, nasal, and dermal symptoms. A subset of employees with work-related symptoms underwent repeated cross-shift and cross-week measurement of spirometry, fractional exhaled nitrogen oxide (FeNO), and skin prick testing for Cannabis sensitization. Exposure to Cannabis dust was classified based on self-described tasks, expert opinion, and exposure monitoring of particulate matter. Multivariable logistic regression was undertaken to examine associations between exposure to Cannabis dust (classified as low, medium, and high) and health symptoms. Linear mixed effects models examined the relationship between cross-shift and cross-week changes in spirometry and FeNO. Results: Ninety-seven percent (97%) of the employees (n = 31) surveyed were recreational cannabis users, with 81% (n = 25) smoking cannabis multiple times per day. Twenty-two (71%) employees reported one or more work-related symptoms: 65% respiratory, 39% ocular, 32% nasal, and 26% dermal symptoms. There was a trend toward increased likelihood of work-related symptoms with increasing exposure to Cannabis dust, although none of these results were statistically significant. Of the 10 employees with work-aggravated symptoms, 5 had borderline-high or high FeNO, 7 had abnormal spirometry, and 5 had evidence of Cannabis sensitization on skin prick testing. FeNO increased by 3.78 ppb (95% confidence interval 0.68-6.88 ppb) across the work-week and there was a trend toward cross-week and cross-shift reduced airflow. Conclusions: We found a high prevalence of work-related allergic- and particularly respiratory symptoms in the employees of one indoor Cannabis grow facility in Washington State. A high proportion of employees with work-aggravated symptoms had findings consistent with probable work-related asthma based on high FeNO, airflow obstruction on spirometry, and Cannabis sensitization on skin prick testing. However, due to the high incidence of recreational cannabis use among these workers, the relative influence of occupational versus recreational exposure to Cannabis dust on the respiratory health and sensitization status of these workers could not be resolved in this study.
... Přibližně 140 terpenoidů je zodpovědných za typickou vůni konopí 11 a asi 17 nejběžnějších terpenů by mohlo být použito pro hodnocení fenotypických a/nebo biologických vlastností odrůd konopí 64 . Monoterpenoidy jsou obecně nejvíce zastoupenou skupinou terpenoidů v konopí a zahrnují převážně myrcen, α-a β-pinen, limonen, terpinolen a cis-ocimen. ...
Article
Cannabis is a plant containing a mixture of chemical substances of unique composition, and therefore it has vast array of applications. Hemp fiber and shives are used in the paper, construction and textile industries. Hemp seeds and products have high nutritional value for humans. They contain significant amounts of essential amino acids and represent a source of ω-3 and ω-6 essential fatty acids. In addition, cannabis flowers contain a broad spectrum of secondary metabolites (phytocannabinoids, terpenoids, and phenylpropanoids), which possess beneficial properties against various disorders. This review summarizes the current knowledge of cannabis chemistry, as well as its utilization for various purposes. Special attention is fo-cused on the biosynthesis of phytocannabinoids.
... The monoterpenes have higher volatility and dominate in the inflorescences to repel insects, the more bitter sesquiterpenes dominate in the leaves acting as anti-herbivores for grazing animals. Moreover, different content in terpenes pattern can be associated to different cultivars of Cannabis such as Futura 75, Antal, Carmagnola, and Kompolti [15][16][17][18]. ...
Article
A piezoelectric peptide-hpDNA based gas sensor array has been used for the detection of terpenes coming from Cannabis sativa samples. The array consisted in 11 sensors, 6 having pentapeptides and 5 having hairpin DNA as binding elements. The volatile composition of 28 Cannabis sativa samples, assessed by GCMS analysis, allowed their classification into 2 groups having as monoterpenes and sesquiterpenes in different amounts. The response of the gas sensor array to the same samples demonstrated that both type of sensors are sensitive to the terpenes and contribute to classification. A satisfactory classification (79 % of correctly identified samples) was found using a PLS-DA approach. Using the same dataset and a simple ANN approach the headspace analytical profile of the two different groups was predicted with an average prediction error ≤1 %.
... The cannabinoids are usually carboxylated in plant material, and high temperature in the GC apparatus causes the degradation of the acidic forms [6,7] irreversibly. Therefore, this study aimed to take an overview of the concentration of the principal cannabinoids in cannabis light preparations by using an HPLC/UV technique which does not require any derivatization or the use of high temperature [7][8][9][10][11][12][13][14][15]. ...
Article
Cannabis light preparations are products derived or containing dried female inflorescences of Cannabis sativa belonging to Chemotype III (THC/CBD ratio <<1); the total THC (THC + THCA) content in the crop must not exceed 0.2 % in accordance with the EU regulation. In Italy the most recent law for industrial hemp (242/2016) states that only for farmers this limit is extended to 0.6 %. On the other hand, the Ministry of the Interior published a note stating that the sale or the presence in the markets of products (inflorescences, concentrates, essences and resins) or plants with concentrations higher than 0.5 % constitutes a crime. In this confusing legislation framework, it is very important to assess the legality of hemp, determining the total amount of THC. To this end a reliable LC-UV analytical method was developed and validated taking into account parameters such as precision, accuracy, linearity, repeatability of peak area and retention time, limit of detection (LOD = 0.002 % for all cannabinoids) and limit of quantification (LOQ = 0.005 % for all cannabinoids). Accuracy was expressed as the relative error (Er%), while precision was measured as the coefficient of variation (CV%). A CV% below 3 % and Er% between ± 6 % were obtained. The linearity was proven in the concentration range 0.005–1 % for THC, THCA and CBN and 0.005 %–50 % for CBD and CBDA. The analytical method was applied to more than nine hundred cannabis light samples. Based on the law 242/2016, only 18 % of the crops are to be considered legal for the market (total THC < 0.2 %). If the circular of the Ministry of the Interior should be converted as a proper law, a substantial amount of cannabis light preparations (24 %) would be considered illegal (total THC > 0.5 %). On the other hand, the most of the inflorescences (58 %) have a total THC content comprised between 0.2 % and 0.5 %, and it is not clear whether these products could be sold or not. Moreover, Cannabis light products are not authorized for human consumption, even if everybody knows that this is their primary use. In conclusion, the cannabis light panorama in Italy is quite confused and more specific and clear legislation should be proposed.
... Some studies (Casano et al., 2011;Giese et al., 2015) used GC-flame ionization detection (GC-FID) or GC-MS based on their high volatility to analyze terpenoids. Also, Fischedick (2017) identified and quantified 16 terpenoids by using GC-MS and GC-FID, respectively. ...
Article
Cannabis sativa L. is a high-value crop with a multi-billion dollar international market, yet due to the long history of prohibition, there is a significant lack of research on the plant and biotechnological techniques are in their infancy. Developing and applying modern techniques to Cannabis will help overcome some species-specific challenges to increase productivity and improve our knowledge about this plant. With regulatory environments relaxing in many parts of the world, there has been a significant increase in biotechnological research with this species. The current manuscript reviews the advances in Cannabis biotechnology, including molecular markers, microRNA, omics-based methods, and functional genes related to the terpene and cannabinoid biosynthesis as well as fiber quality. The foremost aim of this study is to a comprehensive review of the available literature to guide future cannabis studies in the field of genetic engineering and biotechnology.
... Além disso, a acetonitrila (ACN) é um organosolvente utilizado à quebra das nanopartículas, permitindo a recuperação e a detecção do ativo no sistema cromatográfico. O processo de otimização deste sistema considerou por base protocolos de terpenos e canabinoides (de Almeida Borges, Ribeiro et al. 2013, Giese, Lewis et al. 2015, com modificações para otimizar a corrida cromatográfica. Por exemplo, o principal solvente da fase móvel é a ACN, mas observou-se que ao adicionar metanol, o tempo de corrida foi reduzido em 50% (15min). ...
... The resulting TMS/TBDMS derivatives have lower polarity and increased thermal and catalytic stability and GC amenability. However, they can be thermally degraded in injector port and/or column system [162]. Versatile silylation agents are used, ordered by reactivity: hexamethyldisilazine (HMDS) [88], N-methyl-(trimethylsilyl) trifluoroacetamide (MSTFA) [153], N, O-bistrifluoroacetamide (BSTFA) [90], alone or accompanied by a catalyst, usually 1% trimethylchlorosilane (TMCS) [58,99,100,123,124,133] for TMS derivatization and N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) alone or with 1% tert-butyldimethylchlorosilane (t-BDMCS) as a catalyst for TBDMS derivatization. ...
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... Mass spectral library matching is often used, which is valid as long as a close match is found; however, the quality of the library matches is rarely reported. An examination of analytical results recently obtained by the CSIRO (data not published) as well as a selection of recently published cannabis terpene and terpenoid profiles [39][40][41][42][43][44][45][46][47][48][49][50] found that the following monoterpenes, 36-44 ( Fig. 5), sesquiterpenes, 45-49 (Fig. 6), and terpenoids, 50-57 ( Fig. 7) are most often found in cannabis varieties. Over 50 other cannabis terpenes and terpenoids have been described, but these were usually only reported in a single or small number of publications, and detected at low to very low levels. ...
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... Some essential oils like chamomile, citronella, lemongrass, ambrette seeds, neroli, musk, cyclamen, rose, tolu, tuberose, and balsam are sources of farnesol [49,50]. The research group also explored terpinolene, a cyclic structured monoterpene, as an alternative crosslinker to reduce the feedstock stock extracted from some commercially available cannabis chemovars [33,51]. In the study, terpinolene was blended with either any of the following organic crosslinkers like DIB, DCPD, and ethy- ...
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The pharmaceutical importance of cannabis continues to grow due to the interest in the natural non-psychoactive, and psychoactive cannabinoids. For medicinal and forensic purposes, effective extraction and quantification are essential to utilize the natural cannabinoids fully. The supercritical fluid extraction (SFE) process has gained increasing interest due to its selective extraction, short processing time (partly due to the efficient solvent removal process-supercritical fluid to vapor-leaving a solvent-free product that doesn't require an additional drying step), low running cost, and low impact on the environment, compared to that of most conventional organic solvent-based extraction methods. In this review, the extraction of cannabinoids through SFE methods from the literature has been summarised. The advantages of SFE of cannabinoids over conventional extraction procedures, such as microwave-assisted extraction, solid-phase microextraction, hard-cap espresso, soxhlet extraction, high-throughput homogenization, ultrasound-assisted extraction, vacuum distillation of lipid-based extract, and liquid-liquid extraction, are discussed for comparison. Although not an 'extraction method for all molecules of interest, this review examines the importance of the SFE of cannabinoids by coupling with various conventional extraction methods, separation techniques, selection of a suitable co-solvent/modifier, and appropriate sample preparation. Additionally, potential applications of SFE technology and cannabinoids are reviewed with a focus on industrial, pharmaceutical, waste by-products, and purification.
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