Article

A Qualitative and Quantitative HPTLC Densitometry Method for the Analysis of Cannabinoids in Cannabis sativa L.

Authors:
  • Hazekamp Herbal Consulting
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Abstract

Cannabis and cannabinoid based medicines are currently under serious investigation for legitimate development as medicinal agents, necessitating new low-cost, high-throughput analytical methods for quality control. The goal of this study was to develop and validate, according to ICH guidelines, a simple rapid HPTLC method for the quantification of Delta(9)-tetrahydrocannabinol (Delta(9)-THC) and qualitative analysis of other main neutral cannabinoids found in cannabis. The method was developed and validated with the use of pure cannabinoid reference standards and two medicinal cannabis cultivars. Accuracy was determined by comparing results obtained from the HTPLC method with those obtained from a validated HPLC method. Delta(9)-THC gives linear calibration curves in the range of 50-500 ng at 206 nm with a linear regression of y = 11.858x + 125.99 and r(2) = 0.9968. Results have shown that the HPTLC method is reproducible and accurate for the quantification of Delta(9)-THC in cannabis. The method is also useful for the qualitative screening of the main neutral cannabinoids found in cannabis cultivars.

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... All instruments were controlled by Wincats software version 1.4.3. Finally, the plate was sprayed with 0.1% aqueous fast blue B salt solution for the CBD visualization [32]. ...
... Data was collected using LC solution software. Identification of standard peak was achieved by comparison of the retention time (Rt) of the standard CBD [32,33]. ...
... This observation may correlate with an Rf value of 0.8 reported earlier [50]. This result, however, differs from that reported earlier [32,26], where Rf values of 0.52 and 0.55, respectively, have been reported. The plausible explanation for this discrepancy could be that these were obtained under different chromatographic conditions. ...
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Background There are anecdotal claims on the use of Cannabis sativa L. in the treatment of Alzheimer’s disease, but there is lack of scientific data to support the efficacy and safety of Cannabis sativa L. for Alzheimer’s disease. Aim The aim of the study was to evaluate the effect of aerial parts of Cannabis sativa L. on the cholinesterases and β-secretase enzyme activity as one of the possible mechanisms of Alzheimer’s disease. Methods The phytochemical and heavy metal contents were analysed. The extracts were screened for acetylcholinesterase, butyrylcholinesterase and β-secretase activity. Cytotoxicity of extracts was performed in normal vero and pre-adipocytes cell lines. The extracts were characterized using high performance thin layer chromatography and high-performance liquid chromatography for their chemical fingerprints. Alkaloids, flavonoids and glycosides were present amongst the tested phytochemicals. Cannabidiol concentrations were comparatively high in the hexane and dichloromethane than in dichloromethane: methanol (1:1) and methanol extracts. Results Hexane and dichloromethane extracts showed a better inhibitory potential towards cholinesterase activity, while water, hexane, dichloromethane: methanol (1:1) and methanol showed an inhibitory potential towards β-secretase enzyme activity. All extracts showed no cytotoxic effect on pre-adipocytes and vero cells after 24- and 48-hours of exposure. Conclusion Therefore, this may explain the mechanism through which AD symptoms may be treated and managed by Cannabis sativa L. extracts.
... Specificity has consequences for identification and differentiation between compounds having close homology, as well as for purity testing and for quantitative determination [49]. Not all analytical procedures across the manufacturing pipeline require a high level of specificity (complete discrimination) [32,54], providing they can be compensated by one or more supporting analytical procedure(s) [49]. For example, thin layer chromatography is suitable for quantitative and semi-quantitative assessment of cannabinoids within botanical raw material [32,54]. ...
... Not all analytical procedures across the manufacturing pipeline require a high level of specificity (complete discrimination) [32,54], providing they can be compensated by one or more supporting analytical procedure(s) [49]. For example, thin layer chromatography is suitable for quantitative and semi-quantitative assessment of cannabinoids within botanical raw material [32,54]. However, more discriminatory procedures, which include targeted analysis on a larger portfolio of cannabinoids as well as interfering materials, are typically used during purification of drug forms in the manufacturing pipeline [31] (Fig. 2). ...
... Indeed, both the FDA and EMA guidelines for method validation require assessment and reduction for suppression of ionisation in MS-based analytical procedures [75,76]. Structural analogues can be used to mitigate ionisation efficiency as well as matrixinduced ion suppression [53] or enhancement [54]. However, they may lack sufficient structural similarity to coelute and provide full compensation. ...
Article
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The plant genus Cannabis is a prolific producer of unique pharmaceutically relevant metabolites, commonly referred to as cannabinoids. Robust and standardised methods for the quantification of cannabinoids within botanical and drug forms is a critical step forward for an emerging Cannabis-based pharmaceutical industry, which is poised for rapid expansion. Despite a growing body of analytical methods for the quantification of cannabinoids, few have been validated using internationally accredited guidelines. Moreover, standardised methods have yet to be developed for application at various stages of manufacture as well as for different levels of processing and refinement. Validation parameters for establishing robust standardised methods for cannabinoid quantification within Cannabis-based drug forms are critically discussed. Determining an appropriate level of specificity (discrimination) among heterogeneous botanical matrices as well as evaluating accuracy (recovery) and inter-laboratory precision (reproducibility) within strict and volatile regulatory environments are potential obstacles to the establishment of robust analytical procedures. We argue that while some of these challenges remain unique to Cannabis, others are common to botanical-based drug development and manufacture. In order to address potential barriers to analytical method standardisation, a collaborative research initiative inclusive of academic and commercial stakeholders is proposed.
... In that spirit, normal-phase HPTLC with an automated spotter is shown to achieve better separation than TLC for the main neutral phytocannabinoids. The method is comparable within a small degree of error (±0.5%) to a validated HPLC method [110]. ...
... Reverse phase (RP)-TLC is performed using RP-18 HPTLC plates [129] and RP-C 18 bonded silica gel F plates [18]. The more recent TLC/HPTLC methods most commonly use HPTLC silica gel 60 F 254 plates for successful separation of 11 phytocannabinoids (∆ 9 -THC, CBD, CBN, CBC, THCV, ∆ 8 -THC, CBDV, CBG, CBGA, CBDA, ∆ 9 -THCA) [127] or of ∆ 9 -THC, CBD, CBN and CBG [172], or for separation of ∆ 9 -THC, CBD and CBN only [125], silica gel 60 [110], silica gel 60F [135] or silica gel plates [5,126]. For some methods, for instance, the type of TLC plate was not clearly defined [130,147]. ...
... Most suitable mobile phases include xylene/hexane/diethylamine (25:10:1, v/v/v) [127], CHCl 3 , with plate prewashing with MeOH [110], hexane/diethyl ether (80:20, v/v), which allowed clear separation between ∆ 8 -THC, ∆ 9 -THC, CBD and CBN [125], cyclohexane/ toluene/diethylether (75:15:10, v/v/v) [126], benzene/n-hexane/diethylamine (25:10:1, v/v/v) [130], benzene/chloroform (50:50, v/v) [131], diethylether/petroleum ether (1:4, v/v) [120], benzene/n-hexane/diethtylamine ( [127]. Another study showed that, when using alkanes as eluents (isooctane, heptane, hexane and pentane/diethylether (90:10, v/v), the capability to separate ∆ 9 -THC, CBD and CBN decreased as the length of the carbon-bearing chain increases [125]. ...
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Cannabis is gaining increasing attention due to the high pharmacological potential and updated legislation authorizing multiple uses. The development of time- and cost-efficient analytical methods is of crucial importance for phytocannabinoid profiling. This review aims to capture the versatility of analytical methods for phytocannabinoid profiling of cannabis and cannabis-based products in the past four decades (1980–2021). The thorough overview of more than 220 scientific papers reporting different analytical techniques for phytocannabinoid profiling points out their respective advantages and drawbacks in terms of their complexity, duration, selectivity, sensitivity and robustness for their specific application, along with the most widely used sample preparation strategies. In particular, chromatographic and spectroscopic methods, are presented and discussed. Acquired knowledge of phytocannabinoid profile became extremely relevant and further enhanced chemotaxonomic classification, cultivation set-ups examination, association of medical and adverse health effects with potency and/or interplay of certain phytocannabinoids and other active constituents, quality control (QC), and stability studies, as well as development and harmonization of global quality standards. Further improvement in phytocannabinoid profiling should be focused on untargeted analysis using orthogonal analytical methods, which, joined with cheminformatics approaches for compound identification and MSLs, would lead to the identification of a multitude of new phytocannabinoids.
... The development of HPTLC has given TLC the potential to be a more attractive and powerful technique for identification of drugs for crime laboratories [51]. A HPTLC densitometric method for the analysis of cannabinoids in Cannabis sativa L. has been previously reported [52]. Accuracy for this method was determined by comparing results to a validated HPLC method. ...
... Accuracy for this method was determined by comparing results to a validated HPLC method. Chloroform was used as the mobile phase and gave adequate separation of Δ9-THC from the other major neutral cannabinoids, cannabigerol (CBG), cannabichromene (CBC), CBD and tetrahydrocannabivarin (THCV) [52]. The methods reported in the studies cited above differ in conditions of solvent(s) for mobile phase, stationary phase, prewashing and preconditioning of plates and visualization of spots. ...
... Since we did not analyze a sample below 25 ng the exact LOD was not determined. These values are slightly higher than the LOD of 10 ng for Δ9-THC and CBN reported by Fischedick [52]. The reports and HPTLC plates for the LOD analysis the xylene:n-hexane:diethylamine system are provided in the Supplemental material (S5). ...
Article
Ten different TLC mobile phase systems were evaluated in triplicate to determine the most effective mobile phase for the analysis of cannabinoids in Cannabis sativa L. products using HPTLC. Retardation factors (RF) were recorded and the resolution was calculated for three major cannabinoids, Δ9-THC, CBD and CBN on all ten systems. HPTLC Silica gel 60 F 254 20 × 10 cm plates were used for nine systems and a RP-18 WF 254 10 × 10 cm plate was used for one additional system. Two systems, xylene-hexane-diethylamine (25:10:1) and 6% diethylamine in toluene, gave the best results in separating between the three major cannabinoids and from other phytocannabinoids. Both systems were validated according to SWGDRUG validation requirements and showed excellent precision. The results of the analysis of various cannabis products from casework are presented. Using the proper mobile phase system with HPTLC is a superior method when compared to traditional TLC systems for qualitative identification of the common cannabinoids in cannabis products. This method has the potential to provide better resolution and to generate reports for more convenient documentation for peer review of casework in crime labs.
... A high number of analytical methods has been developed for the detection of the main class of active compounds in C. sativa [16,17,21,[29][30][31] . For example, with regard to a fast quality control of medical Cannabis , a validated HPTLC method for the analysis of seven cannabinoids has been described [32] . ...
... Finding the right MP system is the next important step in HPTLC method development, in order to reach the best separation conditions necessary for the analysis of such complex samples and to solve the analytical task. A previous HPTLC method [32] was optimized for the separation of neutral cannabinoids only, making sample preparation more time-consuming and complicated, since a decarboxylation step of cannabinoic acids is necessary. However, sample pretreatment should be minimalistic to avoid compound discrimination. ...
... A non-targeted bioprofiling of any active compound present in a multicomponent hemp extract can also reveal bioactive cannabinoic acids, pesticide residues or contaminants. Halogenated solvents like chloroform [32] were tried to be avoided as well as solvents of high viscosity to achieve a fast separation. ...
Article
The scientific interest on the plant Cannabis sativa L., and in particular on its non-psychoactive or fibre-type variety (hemp), has been highly increasing in recent years, due to the pharmaceutical and nutraceutical potential of its bioactive compounds. It is characterized by a very rich chemical composition, which encompasses different classes of constituents, such as cannabinoids and terpenes. In this context, the bioanalytical testing of hemp extracts can be difficult and time-consuming. Effect-directed analysis (EDA) by the combination of high-performance thin-layer chromatography (HPTLC) with biological and enzymatic assays represents one of the latest tools available for the rapid bioprofiling of complex matrices, such as plant extracts. In this ambit, the aim of this project was the non-targeted screening of inflorescence extracts from ten different hemp varieties for components exhibiting radical scavenging, antibacterial, enzyme inhibiting and estrogen-like effects. Indeed through HPTLC-EDA, the hemp samples exhibited strong antibacterial activities against both Gram-positive Bacillus subtilis and especially Gram-negative Aliivibrio fischeri bacteria, and also estrogen-like activity. They also inhibited α- and β-glucosidase, tyrosinase and acetylcholinesterase. The characterization of two prominently multipotent bioactive compound zones was finally achieved by HPTLC-HRMS and preliminary assigned as cannabidiolic acid and cannabidivarinic acid.
... The method of extraction followed that described by Turner and Mahlberg [12] with modification. In brief, 10 g of each sample of dried cannabis (marijuana or hashish) was divided into small pieces, wellgrounded, either heated in a glass baker in boiling water at 100 ℃ for two hours [13] or used without heating. Then, unheated and heated samples were extracted with chloroform overnight to yield 2 g of dry extract and were protected from light and heat (stored at 4 ℃, and protected from light by placing in an aluminum-covered container). ...
... Decarboxylation was found to be best at 100 ℃ for two hours [13] which was implicated in the present study. Chloroform is also known to be a suitable solvent for the separation of THC from the other major neutral cannabinoids, such as CBC and CBD [13] . ...
... Decarboxylation was found to be best at 100 ℃ for two hours [13] which was implicated in the present study. Chloroform is also known to be a suitable solvent for the separation of THC from the other major neutral cannabinoids, such as CBC and CBD [13] . ...
Article
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Objective: To determine the delta-9-tetrahydrocannabinol (THC) content of cannabis seizures in Egypt. Methods: Unheated and heated extracts of cannabis seizures were prepared from the dried flowering tops and leaves (marijuana) or from the resin (hashish) and subjected to analysis using high performance liquid chromatography (HPLC). Results: The heated resin extract had the peak of THC in a relative ratio of 31.34%, while extracting the resin directly without heating contained only 18.34% of THC. On the other hand, marijuana showed minimum percentage of THC at 11.188% on heating and 9.55% without heating. Conclusions: These results indicate the high potency of the abused cannabis plant in the illicit Egyptian market.
... These data indicate that significant decarboxylation of the major cannabinoid acids occurs only by exposure to higher temperatures for extended time (oven at 145°C for 30 min), which is in agreement with previous studies [18,22]. However, under these conditions all major terpenes present were affected by significant evaporation. ...
... However, under these conditions all major terpenes present were affected by significant evaporation. Although milder decarboxylation using a boiling water bath may be efficient when applied for longer time [22], the terpene profile already changes significantly after only 5 min of treatment. For this reason, all further experiments were carried out without application of a preheating step. ...
... Some methods have been widely used for cannabinoid analysis, such as gas chromatography (GC) [10], liquid chromatography (LC) [11][12][13], and high-performance thin-layer chromatography (HPTLC) [14]. GC and LC analysis are the most used techniques; however, they have a high cost. ...
... FBBS, Fast Blue B salt (Azoic Diazo No. 48); FBRR: Fast Blue RR (Azoic Diazo No. 24); KOH, potassium hydroxide; NaOH, sodium hydroxide; NH 4 OH, ammonium hydroxide; TLC, thin-compounds, in addition to not eluting the fluorescent spots in the application (Figure S2). Similar results were found by Fischedick et al. (2009), in which they still reported that the use of ethanol caused substances to overlap in the plates[14].The TLC analysis with hexane:ethyl ether (8:2 v:v) and FBRR acid ethanolic solution is shown inFigure 2. These results revealed the presence of two different groups of cannabinoids: a first group, the least polar, composed of CBD, ∆ 9 -THC, CBG, and CBN (upper hR F values) and a second group, more polar, which consisted of cannabinoids in acid forms with lesser hR F . In this study, the less polar group, composed of CBD, ∆ 9 -THC, CBG, and CBN, travelled a greater distance. ...
Article
Cannabis sativa is the drug of abuse most cultivated, trafficked, and consumed worldwide. One of several techniques used to detect cannabinoids is based on the thin‐layer chromatography (TLC). However, the designation of the colors observed can be inaccurate and not reproducible. The designation of colors goes beyond physical and physiological aspects, because what is conventionally called color is a socio‐cultural construction. Thus, the objective of this paper was to evaluate the different TLC methods to detection of cannabinoids, and apply standardization method in naming of colors. TLC analysis performed using silica gel 60 F254 as a stationary phase. Three mobile phase compositions [hexane:chloroform (8:2 v:v), hexane:ethyl ether (8:2 v:v), and chloroform:hexane (8:2 v:v)], as well as, two different solutions of Fast Blue B salt (FBBS, Azoic Diazo No. 48) and Fast Blue RR (FBRR, Azoic Diazo No. 24) were evaluated. Determination of colors names was realized through the Sci‐Chromus® software. The best resolution was obtained using hexane:ethyl ether (8:2 v:v) as a mobile phase. It was observed that although the cannabidiol (CBD), delta‐9‐tetrahydrocannabinol (Δ⁹‐THC), cannabinol (CBN), and cannabigerol (CBG) were detect using both the FBBS‐ and FBRR‐acidified solutions, the best visualization was achieved using the latter reagent. To the best of our knowledge, this is the first study that applied and demonstrated a method for standardization and denomination of colors in the TLC analysis of cannabinoids. This method was able to reduce the subjectivity in naming the colors observed and presented several application possibilities.
... 69 The cannabis plant is a remarkably complex plant, with several phenotypes, each containing over 400 distinct chemical moieties. [70][71][72][73] Approximately 70 of these are chemically unique and classified as cannabinoids. [70][71][72][73] Delta-9 tetrahydrocannabinol (THC) and delta-8 THC appear to produce the majority of the psychoactive effects of cannabis. ...
... [70][71][72][73] Approximately 70 of these are chemically unique and classified as cannabinoids. [70][71][72][73] Delta-9 tetrahydrocannabinol (THC) and delta-8 THC appear to produce the majority of the psychoactive effects of cannabis. 74,75 Delta-9 THC, the active ingredient in dronabinol (Marinol), is the most abundant ...
Article
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Significant advances have increased our understanding of the molecular mechanisms of amyotrophic lateral sclerosis (ALS), yet this has not translated into any greatly effective therapies. It appears that a number of abnormal physiological processes occur simultaneously in this devastating disease. Ideally, a multidrug regimen, including glutamate antagonists, antioxidants, a centrally acting anti-inflammatory agent, microglial cell modulators (including tumor necrosis factor alpha [TNF-alpha] inhibitors), an antiapoptotic agent, 1 or more neurotrophic growth factors, and a mitochondrial function-enhancing agent would be required to comprehensively address the known pathophysiology of ALS. Remarkably, cannabis appears to have activity in all of those areas. Preclinical data indicate that cannabis has powerful antioxidative, anti-inflammatory, and neuroprotective effects. In the G93A-SOD1 ALS mouse, this has translated to prolonged neuronal cell survival, delayed onset, and slower progression of the disease. Cannabis also has properties applicable to symptom management of ALS, including analgesia, muscle relaxation, bronchodilation, saliva reduction, appetite stimulation, and sleep induction. With respect to the treatment of ALS, from both a disease modifying and symptom management viewpoint, clinical trials with cannabis are the next logical step. Based on the currently available scientific data, it is reasonable to think that cannabis might significantly slow the progression of ALS, potentially extending life expectancy and substantially reducing the overall burden of the disease.
... The specificity of the analysis was investigated by comparison of the UV spectra of CBD, THC, and CBN to spectra reported in previous work; our spectra were found to be similar to previous reports (Fig. 1). The UV spectrum of CBD was similar to that of Hazekamp et al. (2005), the THC spectrum was similar to Ameur et al. (2013), Fischedick et al. (2009), andHazekamp et al. (2005), and the CBN spectrum was similar to Fischedick et al. (2009) and Hazekamp et al. (2005). Therefore, the analysis method was confirmed with respect to specificity. ...
... The specificity of the analysis was investigated by comparison of the UV spectra of CBD, THC, and CBN to spectra reported in previous work; our spectra were found to be similar to previous reports (Fig. 1). The UV spectrum of CBD was similar to that of Hazekamp et al. (2005), the THC spectrum was similar to Ameur et al. (2013), Fischedick et al. (2009), andHazekamp et al. (2005), and the CBN spectrum was similar to Fischedick et al. (2009) and Hazekamp et al. (2005). Therefore, the analysis method was confirmed with respect to specificity. ...
Article
The aim of this work was to validate a high-performance liquid chromatography (HPLC) method for determination of the stability of cannabidiol, ∆9-tetrahydrocannabinol, and cannabinol. Furthermore, degradation kinetics were also investigated. Five stress conditions—acid degradation, alkaline degradation, oxidation, thermal degradation, and photodegradation—were evaluated. The results showed that the HPLC method had a linear response (R2 ≥ 0.9999) in the test range of 1–200 μg/mL. The method was specific, precise, and accurate. The limits of both detection and quantitation are also reported. According to the stress test, the three cannabinoids (cannabidiol, ∆9-tetrahydrocannabinol, cannabinol) were stable during exposure to a range of thermal conditions for 24 h. They were unstable when being subjected to acid conditions; cannabidiol under alkaline conditions was extremely unstable. Degradation kinetic analysis demonstrated that the compounds remained at a level of approximately 8% after 5 h, and approached a first-order reaction (R2 = 0.9930) with a rate constant of − 0.5057 h− 1. In summary, the obtained data can be used as a guide for the formulation development of cannabis products in order to maintain their active compounds as well as their activities.
... The main goal of this study was the development and validation of a simple and rapid HPLC method for quantification of this molecule from possible pharmaceutical dosage forms derived from nanoparticle systems. Several other HPLC methods were also developed for the determination of cannabinoids15161718. All of these methods, however, are not employed for the determination of CB13 in a possible pharmaceutical dosage form and usually employed to quantify illicit substances in biological fluids. ...
... All of these methods, however, are not employed for the determination of CB13 in a possible pharmaceutical dosage form and usually employed to quantify illicit substances in biological fluids. Fischedick et al. [18] developed a HPLC method for cannabinoids quantification extracted from plant material. Mercolini et al. [16] and Abbara et al. [17] developed HPLC methods for the analysis of cannabinoids in urine and plasma after solidphase extraction. ...
Article
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A simple, fast, and reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed and validated for determining of a cannabinoid derivate, which displays potent antihyperalgesic activity, 1-naphthalenyl[4-(pentyloxy)-1-naphthalenyl]methanone (CB13) into PLGA nanoparticles. Separation was achieved in a C18 column using a mobile phase consisting of two solvents: solvent A, consisting of acetonitrile : water : acetic acid (75 : 23.7 : 1.3 v/v), and solvent B, consisting of acetonitrile. An isocratic method (70 : 30 v/v), with a flow rate of 1.000 mL/min, and a diode array detector were used. The developed method was precise, accurate, and linear over the concentration range of analysis with a limit of detection and a limit of quantification of 0.5 and 1.25 μg/mL, respectively. The developed method was applied to the analysis of CB13 in nanoparticles samples obtained by three different procedures (SEV, FF, and NPP) in terms of encapsulation efficiency and drug release. Nanoparticles size and size distribution were also evaluated founding that NPP method presented the most lowest particle sizes with narrow-size distribution (≈320 nm) and slightly negative zeta potential (≈-25 mV) which presumes a suitable procedure for the synthesis of PLGA-CB13 nanoparticles for oral administration.
... The range of quantification was determined to be 50-500 ng, using UV light at 206 nm for detection. The results of this method showed it to be comparable to validated HPLC methods, making it potentially useful for forensic analysis and the quality control of hemp and medicinal C. sativa [113]. ...
Chapter
Cannabis (Cannabis sativa, or hemp) and its constituents—in particular the cannabinoids—have been the focus of extensive chemical and biological research for almost half a century since the discovery of the chemical structure of its major active constituent, Δ9-tetrahydrocannabinol (Δ9-THC). The plant’s behavioral and psychotropic effects are attributed to its content of this class of compounds, the cannabinoids, primarily Δ9-THC, which is produced mainly in the leaves and flower buds of the plant. Besides Δ9-THC, there are also non-psychoactive cannabinoids with several medicinal functions, such as cannabidiol (CBD), cannabichromene (CBC), and cannabigerol (CBG), along with other non-cannabinoid constituents belonging to diverse classes of natural products. Today, more than 560 constituents have been identified in cannabis. The recent discoveries of the medicinal properties of cannabis and the cannabinoids in addition to their potential applications in the treatment of a number of serious illnesses, such as glaucoma, depression, neuralgia, multiple sclerosis, Alzheimer’s, and alleviation of symptoms of HIV/AIDS and cancer, have given momentum to the quest for further understanding the chemistry, biology, and medicinal properties of this plant. This contribution presents an overview of the botany, cultivation aspects, and the phytochemistry of cannabis and its chemical constituents. Particular emphasis is placed on the newly-identified/isolated compounds. In addition, techniques for isolation of cannabis constituents and analytical methods used for qualitative and quantitative analysis of cannabis and its products are also reviewed.
... The Cannabis plant, also known as marijuana, contains over 500 natural compounds and about 70 of these are classified as cannabinoids (Fischedick et al., 2009). The discovery of Δ9 -tetrahydrocannabinol (THC) as the major psychoactive principle in Cannabis, as well as the identification of numerous non-psychoactive cannabinoids such as cannabidiol (CBD), cannabigerol (CBG), cannabinol (CBN), cannabichromene (CBC), Δ9 -tetrahydrocannabivarin ( Δ9 -THCV) and cannabidivarin (CBDV), has led to a significant growth in research aimed at understanding the therapeutic effects of these compounds. ...
Article
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Amyotrophic lateral sclerosis (ALS) is the most common degenerative disease of the motor neuron system. Over the last years, a growing interest was aimed to discovery new innovative and safer therapeutic ap-proaches in the ALS treatment. In this context, the bioactive compounds of Cannabis sativa have shown antioxidant, anti-inflammatory and neuroprotective effects in preclinical models of central nervous system disease. However, most of the studies proving the ability of cannabinoids in delay disease progression and prolong survival in ALS were performed in animal model, whereas the few clinical trials that investigated cannabinoids-based medicines were focused only on the alleviation of ALS-related symptoms, not on the control of disease progression. The aim of this report was to provide a short but important overview of evidences that are useful to better characterize the efficacy as well as the molecular pathways modulated by cannabinoids.
... The analysis involves an immunoassay, GC-MS and LC-MS for the confirmation and quantification of the drugs. [9][10][11][12][13][14][15][16][17][18][19][20][21][22] The immunochemical method is generally used as a preliminary analysis because of its evident advantage in rapidity. However, the main drawback is the low selectivity of the target compound. ...
Article
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A reliable method using supercritical fluid chromatography with mass spectrometry (SFC-MS) was developed for cannabinoids using compressed carbon dioxide (CO2) and methanol as the mobile-phase. The cannabinoids, i.e., cannabicyclohexanol (CCH: cis-isomer), trans-CCH, 5-(1,1-dimethylheptyl)-2-[(1R,3S)-3-hydroxycyclohexyl]-phenol (CP-47497), 5-(1,1-dimethylheptyl)-2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)-cyclohexyl]-phenol (CP-55940), 3-(1,1'-dimethylheptyl)-6aR,7,10,10aR-tetrahydro-1-hydroxy-6,6-dimethyl-6Hdibenzo[b,d]pyran-9-methanol (HU-210), 2-[1R-3-methyl-6R-(1-methylethenyl)-2-cyclohexen-1-yl]-5-pentyl-1,3-benzenediol (CBD), (1-pentyl-1H-indol-3-yl)-1-naphthalenyl-methanone (JWH-018), (1-butyl-1H-indol-3-yl)-1-naphthalenyl-methanone (JWH-073) and 1-(1-pentyl-1H-indol-3-yl)-2-(2-methoxyphenyl)-ethanone (JWH-250), were determined within 12 min using a conventional column (2-EP) for SFC. Furthermore, two optical isomers of CCH and trans-CCH were completely and rapidly separated by a chiral stationary phase column (AMY1). A highly sensitive detection (0.002-3.75 ppb) was also obtained by these methods using 2-EP and AMY1 columns. These methods were applied to the qualitative and quantitative determination of cannabinoids in dried plant products. Although the concentration and species were different in the products, JWH-018, JWH-073 and CCH, including the cis-isomer, trans-isomer and the optical isomers, were detected in the products. Therefore, the proposed SFC-MS method seems to be useful as an alternative method to GC-MS and LC-MS for illegal drugs, such as cannabinoids.
... HPTLC method was validated in terms of accuracy, precision, repeatability and linearity (Reich & Schibli 2007;Fischedick et al. 2009;Thomas et al. 2010;Renjith et al. 2013). ...
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Camptothecin (CPT), a modified monoterpene indole alkaloid, is a potential anticancer drug, and due to high demand, search for its new plant-based sources is a priority. Genus Ophiorrhiza is a candidate group in the search for new resources of CPT. Here, CPT contents in 38 Ophiorrhiza accessions, belonging to 11 species and 3 varieties, collected from the southern Western Ghats region in India were quantified by HPTLC-densitometry. Ophiorrhiza mungos (396.54 µg/g, dr. wt.) and O. mungos var. angustifolia (373.19 µg/g, dr. wt.) were the two best CPT sources among the screened species/varieties. O. rugosa var. decumbens (18.55 µg/g, dr. wt.) and O. hirsutula (17.14 µg/g, dr. wt.) showed moderate contents of CPT. This is the first systematic CPT screening of O. hirsutula, O. barnesii, O. incarnata, O. radicans and O. villosa. This study shows the significance of choosing high CPT-yielding ecotypes/chemotypes of Ophiorrhiza species or varieties for commercial purposes.
... The separation of phytocannabinoids is mainly achieved by using silica gel as stationary phase, reversed phase for the non-polar system and normal phase for the polar system. Two different reagents for the visualisation of cannabinoids, fast blue and vanillin-sulphuric acid, can be used [11,30,31]. Figure 3 shows high performance thin layer chromatography (HPTLC) chromatogram of cannabis ethanolic extracts, representing THC and CBD predominant types, respectively. ...
... During the flowering stage, a pool of inflorescences (flowers and leaves) were collected from each pot. The 9tetrahydrocannabinol (THC) concentration was measured in each genotype by the HPTLC densitometry method according to Fischedick et al. (2009). ...
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The seed of Cannabis sativa L. is an expanding source of proteins and oil for both humans and animals. In this study, the proximate composition of a collection of hemp cultivars and accessions of different geographical origins grown under the same conditions for 1 year was analyzed in order to identify potential accessions to improve hemp cultivars. Fatty acids, tocopherols, and antinutritional components, as well as concentrations of crude protein and oil were quantified. The seed oil concentrations varied between 285 and 360 g kg(-1) dry seed (DS), while crude protein ranged between 316 and 356 g kg(-1) dry matter (DM). The seed oil was mainly composed of unsaturated fatty acids and, as expected, the dominant fatty acids were linoleic and a-linolenic acid. A high variability among the cultivars and accessions was also detected for polyphenolic content which ranged from 5.88 to 10.63 g kg(-1) DM, cv. Felina was the richest, whereas cv. Finola had the lowest polyphenolic content. Regarding antinutritional compounds in seed, a high variability was detected among all genotypes analyzed and phytic acid was particularly abundant (ranging between 43 and 75 g kg(-1) DM). In conclusion, our results reveal noticeable differences among hemp seed genotypes for antinutritional components, oil and protein content. Collectively, this study suggests that the hemp seed is an interesting product in terms of protein, oil and antioxidant molecules but a reduction of phytic acid would be desirable for both humans and monogastric animals. The high variability detected among the different genotypes indicates that an improvement of hemp seed might be possible by conventional and/or molecular breeding.
... Several methods have been reported in the literature for the analysis of cannabinoids in cannabis biomass, extracts, and preparations. These include high-performance thin-layer chromatography and TLC [17,18]. Different separation techniques (GC, HPLC, and UHPLC) have been mainly used in most studies in the field of cannabis analysis [19][20][21][22][23][24][25]. ...
Article
Cannabis (Cannabis sativa L.) is an annual herbaceous plant that belongs to the family Cannabaceae. Trans-Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) are the two major phytocannabinoids accounting for over 40% of the cannabis plant extracts, depending on the variety. At the University of Mississippi, different strains of C. sativa, with different concentration ratios of CBD and Δ9-THC, have been tissue cultured via micropropagation and cultivated. A GC-FID method has been developed and validated for the qualitative and quantitative analysis of acid and neutral cannabinoids in C. sativa extracts. The method involves trimethyl silyl derivatization of the extracts. These cannabinoids include tetrahydrocannabivarian, CBD, cannabichromene, trans-Δ8-tetrahydrocannabinol, Δ9-THC, cannabigerol, cannabinol, cannabidiolic acid, cannabigerolic acid, and Δ9-tetrahydrocannabinolic acid-A. The concentration-response relationship of the method indicated a linear relationship between the concentration and peak area ratio with R2 > 0.999 for all 10 cannabinoids. The precision and accuracy of the method were found to be ≤ 15% and ± 5%, respectively. The limit of detection range was 0.11 – 0.19 µg/mL, and the limit of quantitation was 0.34 – 0.56 µg/mL for all 10 cannabinoids. The developed method is simple, sensitive, reproducible, and suitable for the detection and quantitation of acidic and neutral cannabinoids in different extracts of cannabis varieties. The method was applied to the analysis of these cannabinoids in different parts of the micropropagated cannabis plants (buds, leaves, roots, and stems).
... The chloroform extract was prepared with the modification previously described by Turner and Mahlberg (1984) at the laboratory of Toxicology and Narcotics Department (NRC, Cairo, Egypt). The dry extract was suspended in ethanol-saline (2%), and HPLC (Fischedick et al. 2009) quantification revealed that the mixture contained 10% THC. ...
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Nicotine (Nic) and cannabis are considered to be the most abused drugs worldwide that are progressively taken concomitantly. The present study aimed to investigate the modulatory effect of Nic on cannabis extract–induced neuro-inflammation, oxidative status, and the associated behavioral/biochemical alterations. Nic (0.25 mg/kg) and/or cannabis extract expressed as ∆⁹-tetrahydrocannabinol (THC10/20; 10 and 20 mg/kg) were given intraperitoneally for 30 days to Wistar rats. Nic shortened the floating time in forced swimming test, increased locomotion in the open field test, and decreased escape latency in the Morris water maze when co-administered with THC. These effects were associated with the inhibition of THC-mediated elevations in brain interleukin-1 beta, lipid peroxidation, superoxide dismutase, and ascorbic acid. Additionally, Nic increased serum butyrylcholinesterase (BChE) when combined with THC without affecting the serum acetylcholinesterase enzyme. The combinations spiked the brain glucose content above normal. In conclusion, the co-administration of Nic reduced THC-induced depressive-like behavior and memory impairment as well as hypo-locomotion associated with THC20. Such effects could be linked to Nic-mediated inhibition of brain oxidative stress, inflammation, and decreased serum BChE deactivity.
... 5). In the presence of the examined sediment matrices, exclusively CBN reacts under the described conditions with FBS resulting in red bands (FBS is known as a selective detection reagent for cannabinoids, see Fischedick et al.[21][22][23]). Thus, a postchromatographic detection with FBS reagent was found to be appropriate as a pretest to confirm the presence of low CBN concentrations in sediment samples.R F values of CBN obtained by developing TLC or HPTLC plates in n-heptane/diethyl ether/formic acid (75:25:0.3 v/v/v), n-heptane/diethyl ether (90:10 v/v), and n-hexane/acetone/ triethylamine (40:20:2 v/v/v) as well as observed colors of the bands are summarized in ...
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Cannabis products have been used in various fields of everyday life for many centuries, and applications in folk medicine and textile production have been well-known for many centuries. For traditional textile production, hemp fibers were extracted from the stems by water retting in stagnant or slow-moving waters. During this procedure, parts of the plant material‚ among them phytocannabinoids‚ are released into the water. Cannabinol (CBN) is an important degradation product of the predominant phytocannabinoids found in Cannabis species. Thus, it is an excellent indicator for present as well as ancient hemp water retting. In this study, we developed and validated a simple and fast method for the determination of CBN in sediment samples using high-performance thin-layer chromatography (HPTLC) combined with electrospray ionization mass spectrometry (ESI-MS), thereby testing different extraction and cleanup procedures‚ as well as various sorbents and solvents for planar chromatography. This method shows a satisfactory overall analytical performance with an average recovery rate of 73%. Our protocol enabled qualitative and quantitative analyses of CBN in samples of a bottom sediment core‚ having been obtained from a small lake in Northern India, where intense local retting of hemp was suggested in the past. The analyses showed a maximum CBN content in pollen zone 4 covering a depth range of 262–209 cm, dating from approximately 480 BCE to 1050 CE. These findings correlate with existing records of Cannabis-type pollen. Thus, the method we propose is a helpful tool to track ancient hemp retting activities. Graphical Abstract
... Merck 20 Â 10 cm silica gel 60 aluminum plates, chloroform mobile phase in CAMAG twin trough chamber, and a CAMAG ATS 4 applicator and TLC Scanner 3 controlled by winCATS software version 1.4.3 were employed. [42] OPLC separation of D 9 -THC, CBD, CBN, CBG, and CBC was achieved on silica bonded amino (NH 2) F plates using dichloromethane single component mobile phase. Employing bidirectional development (from both ends to the plate to the center), 30 samples were analyzed on a 10 x 20 cm plate within 4 min. ...
Article
Cannabis has been used as a medicinal plant for thousands of years. There are now over 700 varieties of cannabis that contain hundreds of compounds, including fatty cannabinoids that are the main biologically active constituents and volatile terpenes that have distinct odors. This is a selective review that includes important examples of the analysis and study of cannabis and its components and synthetic cannabinoids by thin layer chromatography (TLC) related to its medical and recreational uses. The TLC methods described in this review complement the more expensive and difficult to perform and sustain high performance liquid chromatography (HPLC), HPLC/mass spectrometry (HPLC/MS), gas chromatography (GC), and GC/MS methods. These TLC methods are especially valuable and often sufficient for use in resource-limited countries. Since this is apparently the first review devoted only to the TLC of cannabis in the literature, even earlier TLC references have been included for completeness.
... However, the results are not as accurate as those obtained by GC and LC and the sensitivity is not high enough when very low limits of psychotropic cannabinoids have to be detected. Nonetheless, a reliable quantitative high-performance TLC method has been published by Fischedick et al. [97]. This technique, regardless of the improvements made [98], is still far from being recognized and included among the official methods for cannabinoid analysis. ...
Article
The chemical analysis of cannabis potency involves the qualitative and quantitative determination of the main phytocannabinoids: Δ9-tetrahydrocannabinol (Δ9-THC), cannabidiol (CBD), cannabigerol (CBG), cannabichromene (CBC), etc. Although it might appear as a trivial analysis, it is rather a tricky task. Phytocannabinoids are present mostly as carboxylated species at the aromatic ring of the resorcinyl moiety. Their decarboxylation caused by heat leads to a greater analytical variability due to both reaction kinetics and possible decomposition. Moreover, the instability of cannabinoids and the variability in the sample preparation, extraction, and analysis, as well as the presence of isomeric forms of cannabinoids, complicates the scenario. A critical evaluation of the different analytical methods proposed in the literature points out that each of them has inherent limitations. The present review outlines all the possible pitfalls that can be encountered during the analysis of these compounds and aims to be a valuable help for the analytical chemist. Graphical abstract
... In order to differ between hemp preparations, the content of ∆ 9 -THC and CBD can be determined by various chromatographic methods. In literature, several methods for analysis of cannabinoids in Cannabis sativa have been published, including high-performance thin-layer chromatography (HPTLC) [5,6], supercritical fluid chromatography (SFC) [7][8][9], gas chromatography (GC) [9,10] and high-performance liquid chromatography (HPLC) [11][12][13][14][15]. However, due to the decarboxylation of cannabidiolic acid (CBDA) and THCA at high temperature, it is not possible to detect these corresponding acids by GC ( Figure 1). ...
Article
Cannabis sativa is known to be the most abused illegal drug worldwide. To date it is not only used as a medicine but has been established as a lifestyle product. The most relevant phytocannabinoids represent the ingredients delta-9-tetrahydrocannabinol (9-THC) and cannabidiol (CBD), whereby only 9-THC shows a psychoactive effect. Since 2017, the so-called CBD-hemp containing CBD as main ingredient is distributed in many countries as a legal alternative. In these products, 9-THC must not exceed a certain percentage. It is hardly possible to differentiate between THC-hemp and CBD-hemp presenting a major challenge for authorities. Therefore, there is the need to develop fast and efficient analysis methods to distinguish between fibre-type, drug-type and intermediate-type cannabis products. The aim of this study was to compare two simple and inexpensive HPLC-UV and GC-FID methods for their ability to quantify phytocannabinoids in dried cannabis plant material. For this purpose, a set of 37 fresh and dried cannabis samples randomly chosen from seizures of Austrian police was subject to complementary quantification of 9-THC and CBD. After having taken into account decomposition of certain phytocannabinoids, the result of this quantitative study showed good correlation between HPLC-UV and GC-FID regardless of quantifying cannabis leaves or buds.
... Recently, the medicinal use of cannabis has been legalized in several countries [2]. Some of the medical purposes include, but are not limited to, multiple sclerosis, chronic pain, glaucoma, appetite stimulant, asthma and cardiovascular conditions, and as an antiemetic [3]. The active cannabinoids are present in the cannabis flower of the female species. ...
Article
The solubilities of two different non-psychoactive cannabinoids i.e., cannabigerol (CBG) and cannabidiol (CBD), in supercritical carbon dioxide (CO2) have been determined at 315, 326 and 334K and in the pressure range from 11.3 to 20.6MPa. These solubility data have been compared to the previously determined solubilities of two psychoactive cannabinoids i.e., (−)-Δ9-tetrahydrocannabinol (Δ9-THC) and cannabinol (CBN), in supercritical CO2. An analytical method with a quasi-flow apparatus was used for the experimental determination. Within the investigated temperature and pressure range, the molar solubility of CBG ranged from 1.17 to 1.91×10−4 and the molar solubility of CBD ranged from 0.88 to 2.69×10−4. The solubility of the different cannabinoids in supercritical CO2 increases at 326K in the following order: Δ9-THC
... Dozens of methods are used to quantify phytocannabinoids, based on many techniques: LC-HRMS [61], LC-DAD [62], HPTLC [63], GC-FID [31,64], GC-MS [65], Nuclear Magnetic Resonance (NMR) [66], and Triple quadrupole Multiple Reaction Monitoring (QqQ-MRM) [67], among others. Techniques and methods used in Cannabis quality control monographs differ between countries. ...
Article
Cannabis sativa has a long history of domestication both for its bioactive compounds and its fibers. This has produced hundreds of varieties, usually characterized in the literature by chemotypes, with Δ9-THC and CBD content as the main markers. However, chemotyping could also be done based on minor compounds (phytocannabinoids and others). In this work, a workflow, which we propose to name cannabinomics, combines mass spectrometry of the whole metabolome and statistical analysis to help differentiate C. sativa varieties and deciphering their characteristic markers. By applying this cannabinomics approach to the data obtained from 20 varieties of C. sativa (classically classified as chemotype I, II, or III), we compared the results with those obtained by a targeted quantification of 11 phytocannabinoids. Cannabinomics can be considered as a complementary tool for phenotyping and genotyping, allowing the identification of minor compounds playing a key role as markers of differentiation.
... During the flowering stage, a pool of inflorescences (flowers and leaves) were collected from each pot. The 9tetrahydrocannabinol (THC) concentration was measured in each genotype by the HPTLC densitometry method according to Fischedick et al. (2009). ...
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Hempseed could be a new source of proteins and oil for both humans and animals. In this study, the proximate composition of a collection of hempseed cultivars and accessions of different geographical origins grown under the same conditions was analyzed. Fatty acids, tocopherols and antinutritional components, as well as concentrations of crude protein and oil were quantified. Hempseed oil concentrations varied between 285 and 360 g kg-1 dry seed (DS), while crude protein ranged between 316 and 356 g kg-1 dry matter (DM). The hempseed oil was mainly composed of unsaturated fatty acids and, as expected, the dominant fatty acids were linoleic and α-linolenic acid. A high variability among the cultivars and accessions was also detected for polyphenolic content which ranged from 5.88 to 10.63 g kg-1 DM, cv. Felina was the richest, whereas cv. Finola had the lowest polyphenolic content. Regarding antinutritional compounds in seed, a high variability was detected among all genotypes analyzed and phytic acid was particularly abundant (ranging between 43 and 75 g kg-1 DM). In conclusion, our results reveal noticeable differences among hempseed genotypes for antinutritional components, oil and protein content. Collectively, this study suggests that the hempseed is an interesting product in terms of protein, oil and antioxidant molecules but a reduction of phytic acid would be desirable for both humans and monogastric animals. The high variability detected among the different genotypes indicates that an improvement of hempseed might be possible by conventional and/or molecular breeding.
Conference Paper
Purpose: The aim of this work was to develop an HPLC method for the analysis of 11 cannabinoids and its application of the method for profiling different cannabis extracts. Methods: Eleven cannabinoids (cannabidiolic acid, CBDA ; cannabigerolic acid, CBGA ; cannabigerol, CBG ; cannabidiol, CBD ; tetrahydrocannabivarian, THCV ; cannabinol, CBN ; delta-9-trans-tetrahydrocannabinol, Δ9-THC ; delta-8-transtetrahydrocannabinol, Δ8-THC ; cannabicyclol, CBL ; cannabichromene, CBC ; and tetrahydrocannabinol acid A, THCAA ) were prepared at 9 different levels with internal standard. The peaks for the cannabinoids were observed at 220 nm. Results: All the 11 cannabinoids were successfully analyzed by HPLC-UV method and the method was validated. Thirteen different cannabis extracts were analyzed by this method and all the above cannabinoids except CBL (which was detected only in one extract) were successfully quantitated in these extracts. Conclusion: The method is reproducible and can be used successfully to analyze cannabis extracts. Acknowledgements The authors are thankful to Candice Tolbert and Shahbaz Gul for their technical assistance and data collection and assembly. Abstract Link: http://abstracts.aaps.org/Verify/AAPS2014/PosterSubmissions/W4215.pdf
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The flavonoid-rich hydro-acetone extract of red onion (Allium cepa peel, ACPE) was studied for its spasmolytic and bronchodilator activities using ex-vitro and in-vivo assays. In isolated rabbit jejunum preparations, ACPE produced a concentration-dependent (0.03-1 mg L-1) relaxation of spontaneous and high K+(80 mM)-induced contractions equipotently, nearly similar to that caused by papaverine, whereas, verapamil was relatively more potent against K+-induced contractions. ACPE also caused the right ward shift in the Ca++ concentration-response curves (CRCs), similar to that of verapamil and papaverine. In normotensive anesthetized rats, ACPE dose-dependently (3-30 mg kg(-1)) suppressed the carbachol (CCh, 1 mg kg(-1)) induced bronchoconstriction similar to the effect observed with aminophylline. In guinea-pig tracheal preparation, ACPEexhibited concentration-dependent relaxation of both CCh (1 mu M) and high K+-induced contraction at similar concentrations (0.3-3 mg mL(-1)) and also shifted the isoprenaline-induced inhibitory CRCs to the left, similar to that caused by papaverine. Theresults of this study indicated that the spasmolytic and bronchodilatory activities of ACPE are mediated through the dual inhibition of Ca++ channels and phosphodiesterase enzyme like-mechanisms, which might add an evidence-based medicinal value to the red onion peel in the treatment of gastrointestinal and respiratory disorders, e.g. diarrhea and asthma, respectively.
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An HPLC single-laboratory validation was performed for the detection and quantification of the 11 major cannabinoids in most cannabis varieties, namely, cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabigerol (CBG), cannabidiol (CBD), tetrahydrocannabivarin (THCV), cannabinol (CBN), Δ(9)-trans-tetrahydrocannabinol (Δ(9)-THC), Δ(8)- trans-tetrahydrocannabinol (Δ(8)-THC), cannabicyclol (CBL), cannabichromene (CBC), and Δ(9)-tetrahydrocannabinolic acid-A (THCAA). The analysis was carried out on the biomass and extracts of these varieties. Methanol-chloroform (9:1, v/v) was used for extraction, 4-androstene-3,17-dione was used as the internal standard, and separation was achieved in 22.2 min on a C18 column using a two- step gradient elution. The method was validated for the 11 cannabinoids. The concentration-response relationship of the method indicated a linear relationship between the concentration and peak area with r(2) values of >0.99 for all 11 cannabinoids. Method accuracy was determined through a spike study, and recovery ranged from 89.7 to 105.5% with an RSD of 0.19 to 6.32% for CBDA, CBD, THCV, CBN, Δ(9)-THC, CBL, CBC, and THCAA; recovery was 84.7, 84.2, and 67.7% for the minor constituents, CBGA, CBG, and Δ(8)-THC, respectively, with an RSD of 2.58 to 4.96%. The validated method is simple, sensitive, and reproducible and is therefore suitable for the detection and quantification of these cannabinoids in different types of cannabis plant materials.
Article
This thesis concerns the production of natural compounds from plant material for pharmaceutical and food applications. It describes the production (extraction and isolation) of cannabinoids, the active components present in cannabis. Many cannabinoids have medicinal properties but not all cannabinoids are available in the (large) quantities necessary to develop new medicines, because so far, for large scale production, there are no economically and technically viable methods to extract those cannabinoids present in low quantities in the plant. Moreover, the currently used production process for the most important cannabinoid, tetrahydrocannabinol (Δ9-THC), has many drawbacks, such as the large use of the organic solvents, which is not only a burden to the environment but also to the safety of the operators, the production costs as well as the treatment of the produced waste. In this thesis, an alternative process using supercritical carbon dioxide is presented for the production of cannabinoids, including Δ9-THC, cannabinol (CBN), cannabigerol (CBG) and cannabidiol (CBD). One of the steps of Δ9-THC production from cannabis plant material, is the decarboxylation reaction, transforming the Δ9-THC-acid naturally present in the plant into the psychoactive Δ9-THC. Experiments showed a pseudo first order reaction, with an activation barrier of 85 kJ.mol-1 and a pre-exponential factor of 3.7x108 s-1. Using molecular modeling, two options for an acid catalysed β-keto acid type mechanism were identified. Each of these mechanisms might play a role, depending on the actual process conditions. Formic acid was shown to be a good model for a catalyst of such a reaction. A direct keto-enol mechanism catalyzed by formic acid seems to be the best explanation for the observed activation barrier and the pre-exponential factor of the decarboxylation of Δ9-THC-acid. Evidence for this was found by performing an extraction experiment with Cannabis Flos. It revealed the presence of short chain carboxylic acids supporting this hypothesis. Then, in order to develop the supercritical fluid extraction process, the solubility of Δ9-THC, CBN, CBG and CBD in supercritical carbon dioxide has been determined using an analytical method with a quasi-flow apparatus. First the solubility of Δ9-THC has been determined at 315, 327, 334 and 345 K and in the pressure range from 13.2 to 25.1 MPa. The molar solubility for Δ9-THC ranged from 0.20 to 2.95x10-4. Then, the solubility of CBN, CBG and CBD in supercritical carbon dioxide has been determined at 314, 327 and 334 K and in the pressure range from 11.3 to 20.6 MPa. The molar solubility of CBN, CBG and CBD ranged from 1.26 x 10-4 to 4.16 x 10-4, from 1.17 to 1.91 x 10-4 and from 0.88 to 2.69 x 10-4, respectively. These solubility data have been compared to each other. The solubility of the different cannabinoids in supercritical CO2 increases at 326 K in the following order: Δ9-THC < CBG < CBD < CBN. The solubility data were correlated using the Peng-Robinson equation of state in combination with Van der Waals mixing rules. To continue, supercritical fluid extraction (SFE) using carbon dioxide was performed with Cannabis Sativa L. in a pilot scale set-up at 313 and 323 K in the pressure range from 18 to 23 MPa. The SFE yield of Δ9-THC is at maximum 98 %, which is comparable to classical hexane extraction. CBN and CBG can be extracted in higher amounts with SFE than with hexane extraction. Waxes are co-extracted with the cannabinoids. They can be easily removed via a winterization step. The purity of the final extract after winterization was 85 % Δ9-THC at the optimal experimental conditions found in these experiments. With a two-steps extraction, it is possible to selectively extract minor cannabinoids (i.e. CBN, CBD and CBG) in a first step at low pressure (~15 MPa), and Δ9-THC in a second step at higher pressure (~20 MPa). The last step of the process is performed using Centrifugal Partition Chromatography. It uses a two-phase liquid system, instead of a solid stationary phase, as it is the case in High Pressure Liquid Chromatography (HPLC). Separation is realized by the partitioning of compounds between the two phases. With this technique, a successful separation of Δ9- THC, CBN and CBG is presented using the two-phase system hexane / acetone / acetonitrile. A purity higher than 99% is achieved with Δ9- THC. With CBN and CBG the best purity obtained is higher than 90%. To conclude, an economical and ecological evaluation of two production routes to obtain pure Δ9-THC is presented: the current process using organic solvents is compared with the alternative process using supercritical carbon dioxide developed in this thesis. The alternative process is significantly cheaper than the current one, although the high price of the starting material cannabis dominates the ultimate cost price. From an ecological point of view, the alternative process is also more sustainable as it consumes less energy and generates less waste. Therefore, this alternative process is preferred from an economical and ecological point of view.
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Unlike hospice, long-term drug safety is an important issue in palliative medicine. Opioids may produce significant morbidity. Cannabis is a safer alternative with broad applicability for palliative care. Yet the Drug Enforcement Agency (DEA) classifies cannabis as Schedule I (dangerous, without medical uses). Dronabinol, a Schedule III prescription drug, is 100% tetrahydrocannabinol (THC), the most psychoactive ingredient in cannabis. Cannabis contains 20% THC or less but has other therapeutic cannabinoids, all working together to produce therapeutic effects. As palliative medicine grows, so does the need to reclassify cannabis. This article provides an evidence-based overview and comparison of cannabis and opioids. Using this foundation, an argument is made for reclassifying cannabis in the context of improving palliative care and reducing opioid-related morbidity.
Chapter
A wide assortment of methods are available to assess the chemical constituents of cannabis products and thereby direct product formulation for optimal efficacy and safety as medicines. This chapter emphasizes the necessity for and difficulty of quality control and identifies methods, primarily forms of mass spectrometry and nuclear magnetic resonance, for particular tasks. The very many variables in moving from herbal starting materials that are themselves liable to wide variation, through extraction of desired cannabinoids, to end products prepared by methods subject to wide variation, imposes a burden of quantitation and process control that is currently lacking. The chapter is intended to point the way forward, to the extent that the whole enterprise of making and dispensing cannabis-based medicines in the real world can be confidently directed.
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Cannabis products have recently regained much attention due to the high pharmacological potential of their cannabinoid content. In this review, the most widely used sample preparation strategies for the extraction of cannabinoids are described for the specific application to either plant materials or biological matrices. Several analytical techniques are described pointing out their respective advantages and drawbacks. In particular, chromatographic methods, such as TLC, GC and HPLC, are discussed and compared in terms of selectivity and sensitivity. Various detection methods are also presented based on the specific aim of the cannabinoids analysis. Lastly, critical considerations are mentioned with the aim to deliver useful suggestions for the selection of the optimal and most suitable method of analysis of cannabinoids in either biomedical or cannabis derived samples.
Chapter
Cannabis has gained a lot of popularity in last few years not only because of its use as illicit drug but due to its use as food, fiber and medicine. It is a complex mixture of constituents which contain a unique class of secondary metabolites called phytocannabinoids. In general, so far a total of 565 constituents including 120 phytocannabinoids have been reported in Cannabis sativa. This chapter discusses the chemistry of phytocannabinoids in the plant with particular emphasis on the Δ9-THC type of cannabinoids and different analytical methods available for cannabinoids analysis in cannabis plant and cannabis products.
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In recent years, the Cannabis plant (Cannabis sativa L.) has been rediscovered as a source of new medicines around the world. Despite the fact that a number of registered medicines have been developed on the basis of purified cannabis components, there is a rapid increasing acceptance and use of cannabis in its herbal form. Licensed producers of high quality cannabis plants now operate in various countries including The Netherlands, Canada, Israel, and Australia, and in many US states. The legal availability of cannabis flowers allows to prescribe and prepare different cannabis galenic preparations by pharmacists. It is believed that synergy between cannabis components, known as “entourage effect”, may be responsible for the superior effects of using herbal cannabis versus isolated compounds. So far, only a few cannabis components have been properly characterized for their therapeutic potential, making it unclear which of the isolated compounds should be further developed into registered medicines. Until such products become available, simple and accessible galenic preparations from the cannabis plant could play an important role. In cannabis, phytochemical and pharmacological attention has been attributed mainly to four major cannabinoids (Δ9- tetrahydrocannabinol, cannabidiol, cannabigerol and cannabichromene) and to terpene components. This means a basic knowledge of these compounds and their bioavailability in different administration forms is useful for producers as well as prescribers of galenic preparations. This work will outline the most important aspects of cannabinoids and terpenes, and their behaviors during preparation and use of various administration forms including vaporizing, cannabis oils and extracts, tea, and skin creams.
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To the best of our knowledge, this is the first report on the content of cannabinoids in seized marijuana in our country, and the first densitometric HPTLC method for the quantitative analysis of 3 main compounds in marijuana. The proposed method can be easily implemented in forensic laboratories in order to establish a chemical profile of these 3 components in seized cannabis with low cost and suitable for law enforcement purposes.
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The simple isolation method of Cannabinol compounds from cannabis plants by maceration combined with ultrasonication assisted extraction, fractionation, separation and purification was carried out to obtain Cannabinol isolates which can be used as a reference standard analysis material. Ultrasonication was proven to shorten the extraction time, where extraction for 5 h with ultrasonication assisted for 15 min produced 5.536 % yield greater than the 24 h extraction. The results of extraction were fractionated using n-hexane and then chromatographically isolated with a column containing Silica Gel 60, with 2.5 cm diameter and a 15.3 cm height, eluted by n-hexane-ethyl acetate (90:10) solution. The fraction containing Cannabinol was purified using HPTLC preparative with eluent n-hexane-ethyl acetate (80:20). The purification of the Cannabinol isolate was further characterized by Spectrophotometer UV, FTIR, DCS, GCMS and LCMSMS and compared its profile to the reference standard of Cannabinol from Lipomed. The characterization results showed that the purified isolates had UV spectra with λ max at 219.0 nm and 284.5 nm, FTIR spectra at wave numbers 1620.21 cm ⁻¹ , 1051.20 cm ⁻¹ , 1581.63 cm ⁻¹ , 1026.13 cm ⁻¹ , 1128.36 cm ⁻¹ and 1232.51 cm ⁻¹ . The DSC thermogram shows the melting point of compound is 74.36 °C with 99.35 % purity, GCMS fragmentation at m/z 295, 296, 238 and 310, LCMSMS with [M + H] ⁺ at 311.1 and MS ² at 222.95, 292.95 and 240.95 confirmed the chemical structure of the compound. The results of the characterization of pure isolates indicate that the compound produced was Cannabinol in accordance with the standard characterization profile of the reference standard of Cannabinol used.
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Among the analytical advances, hyphenated HPTLC offers great potential for solving pressing questions. It provides straightforward information about effects arising from individual compounds in complex or natural samples separated in parallel. The chromatographic separation is combined with effect-directed detection using enzymatic or biological assays. This helps to select from the thousands of compounds in a sample the important ones that need to be further characterized using high-resolution mass spectrometry. Unique benefits are discussed exemplarily arising from its super-hyphenation, minimum requirements for sample preparation, detection of multi-modulating compounds or agonistic versus antagonistic effects, and miniaturized on-surface metabolization. HPTLC stands for a versatile, creative and flexible open-format technique. As miniaturized open-source lab-to-go system, it shows the potential to be applied as Citizen Science.
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Cannabis sativa has a long history of domestication both for its bioactive compounds and its fibers. This has produced hundreds of varieties, usually characterized in the literature by chemotypes, with Δ 9-THC and CBD content as the main markers. However, chemotyping could also be done based on minor compounds (phytocannabinoids and others). In this work, a workflow, which we propose to name cannabinomics, combines mass spectrometry of the whole metabolome and statistical analysis to help differentiate C. sativa varieties and deciphering their characteristic markers. By applying this cannabinomics approach to the data obtained from 20 varieties of C. sativa (classically classified as chemotype I, II, or III), we compared the results with those obtained by a targeted quantification of 11 phytocannabinoids. Cannabinomics can be considered as a complementary tool for phenotyping and genotyping, allowing the identification of minor compounds playing a key role as markers of differentiation.
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Cannabis sativa, widely known as ‘Marijuana’ poses a dilemma for being a blend of both good and bad medicinal effects. The historical use of Cannabis for both medicinal and recreational purposes suggests it to be a friendly plant. However, whether the misuse of Cannabis and the cannabinoids derived from it can hamper normal body physiology is a focus of ongoing research. On the one hand, there is enough evidence to suggest that misuse of marijuana can cause deleterious effects on various organs like the lungs, immune system, cardiovascular system, etc. and also influence fertility and cause teratogenic effects. However, on the other hand, marijuana has been found to offer a magical cure for anorexia, chronic pain, muscle spasticity, nausea, and disturbed sleep. Indeed, most recently, the United Nations has given its verdict in favour of Cannabis declaring it as a non-dangerous narcotic. This review provides insights into the various health effects of Cannabis and its specialized metabolites and indicates how wise steps can be taken to promote good use and prevent misuse of the metabolites derived from this plant.
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Cannabis is one of the oldest cultivated plant, which has been used by humankind for thousands of years due to its biological properties and a wide range of applications. In total, hemp plants contain over 500 different substances while the characteristic components are the cannabinoids. The most important cannabinoids are (-)-Δ⁹-trans-tetrahydrocannabinol (Δ⁹-THC), cannabidiol (CBD), and cannabinol (CBN – the latter being an oxidation product resulting from Δ⁹-THC). In the course of recent years, a paradigm shift has taken place with regard to the use of products and ingredients derived from hemp, especially CBD. Thus, an ever-increasing number of products containing CBD are on the market; this ranges from classic CBD oil to CBD chewing gum and even CBD shampoo. Despite an increasing presence of these products in the market, the regulation of cannabinoids in these products is very inconsistent in different countries, except for Δ⁹-THC whose limit is 0.2% for many products and many countries. The enormous abundance of CBD-containing products calls for the development of new analytical techniques that allow a reliable and quick determination of the main cannabinoids usually found in hemp. This seems all the more necessary since previous examinations of CBD oils often revealed a difference between the declared amount and the actual content of the ingredients. Many methods usually applied to determine cannabinoids are rather time-consuming and associated with high costs. In this study, we developed and validated a sensitive, simple, reliable as well as fast method for the determination of CBN, CBD and Δ⁹-THC in commercially available CBD oils using high-performance thin-layer chromatography (HPTLC) combined with electrospray ionization mass spectrometry (ESI-MS). Thus, for this method, a recovery rate of ≥90% was determined. This procedure enables both qualitative and quantitative analyses of CBN, CBD and Δ⁹-THC in CBD oils of different matrices such as hempseed oil, olive oil or sunflower oil. Thus, this method is a helpful and fast tool to investigate a broad variety of commercially available CBD oils.
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Cannabis has garnered a great deal of new attention in the past couple of years in the United States due to the increasing instances of its legalization for recreational use and indications for medicinal benefit. Despite a growing number of laboratories focused on cannabis analysis, the separation science literature pertaining to the determination of cannabis natural products is still in its infancy despite the plant having been utilized by humans for nearly 30 000 years and it being now the most widely used drug world-wide. This is largely attributable to the restrictions associated with cannabis as it is characterized as a Schedule 1 drug in the United States. Presented here are reviewed analytical methods for the determination of cannabinoids (primarily) and terpenes (secondarily), the primary natural products of interest in cannabis plants. Focus is placed foremost on analyses from plant extracts and the various instrumentation and techniques that are used, but some coverage is also given to analysis of cannabinoid metabolites found in biological fluids. The goal of this work is to provide a collection of relevant separation science information, upon which the field of cannabis analysis can continue to grow.
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Chromatographic and spectroscopic data was determined for 16 different major cannabinoids from Cannabis sativa plant material as well as 2 human metabolites of Δ‐tetrahydrocannabinol. Spectroscopic analysis included UV absorbance, infrared‐spectral analysis, (GC‐) mass spectrometry, and spectrophotometric analysis. Also, the fluorescent properties of the cannabinoids are presented. Most of this data is available from literature but scattered over a large amount of scientific papers. In this case, analyses were carried out under standardised conditions for each tested cannabinoid so spectroscopic data can be directly compared. Different methods for the analysis of cannabis preparations were used and are discussed for their usefulness in the identification and determination of separate cannabinoids. Data on the retention of the cannabinoids in HPLC, GC, and TLC are presented.
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A simple method is presented for the preparative isolation of seven major cannabinoids from Cannabis sativa plant material. Separation was performed by centrifugal partition chromatography (CPC), a technique that permits large‐scale preparative isolations. Using only two different solvent systems, it was possible to obtain pure samples of the cannabinoids; (−)‐Δ‐(trans)‐tetrahydrocannabinol (Δ‐THC), cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), (−)‐Δ‐(trans)‐tetrahydrocannabinolic acid‐A (THCA), cannabigerolic acid (CBGA), and cannabidiolic acid (CBDA). A drug‐type and a fiber‐type cannabis cultivar were used for the isolation. All isolates were shown to be more than 90% pure by gas chromatography. This method makes acidic cannabinoids available on a large scale for biological testing. The method described in this report can also be used to isolate additional cannabinoids from cannabis plant material.
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. A central tenet underlying the use of botanical remedies is that herbs contain many active ingredients. Primary active ingredients may be enhanced by secondary compounds, which act in beneficial syn-ergy. Other herbal constituents may mitigate the side effects of dominant active ingredients. We reviewed the literature concerning medical can-nabis and its primary active ingredient, ∆ 9 -tetrahydrocannabinol (THC). Good evidence shows that secondary compounds in cannabis may enhance the beneficial effects of THC. Other cannabinoid and non-cannabinoid compounds in herbal cannabis or its extracts may reduce THC-induced anxiety, cholinergic deficits, and immunosuppression. Cannabis terpenoids and flavonoids may also increase cerebral blood flow, enhance cortical activity, kill respiratory pathogens, and provide anti-inflammatory activ-ity. [Article copies available for a fee from The Haworth Document Delivery Service: and: Cannabis Therapeutics in HIV/AIDS (ed: Ethan Russo) The Haworth Integrative Healing Press, an imprint of The Haworth Press, Inc., 2001, pp. 103-132. Single or multiple copies of this arti-cle are available for a fee from The Haworth Document Delivery Service [1-800-342-9678, 9:00 a.m. -5:00 p.m. (EST). E-mail address: getinfo@haworthpressinc.com].
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A (1)H-NMR method has been developed for the quantitative analysis of pure cannabinoids and for cannabinoids present in Cannabis sativa plant material without any chromatographic purification. The experiment was performed by the analysis of singlets in the range of delta 4.0-7.0 in the (1)H-NMR spectrum, in which distinguishable signals of each cannabinoid are shown. Quantitation was performed by calculating the relative ratio of the peak area of selected proton signals of the target compounds to the known amount of the internal standard, anthracene. For this method no reference compounds are needed. It allows rapid and simple quantitation of cannabinoids with a final analysis time of only 5 min without the need for a pre-purification step.
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The use of cannabis is illicit in numerous countries, and the increasing consumption has led to a multiplication of scientific studies. New methods of planar chromatography such as automated multiple development (AMD) and optimum performance laminar chromatography (OPLC) techniques can be used as a substitute for the traditional thin-layer chromatography for the identification and quantitation of the Indian hemp components. Each method offers its own advantage: high resolution with neither diffusion nor spot stretching for AMD and speed, efficiency, and the possibility of working in the semipreparative mode for OPLC.
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The overpressured-layer chromatographic separation of neutral cannabinaids (Δ9 -tetrahydro-cannabinol, cannabidiol, cannabinol, cannabigerol and cannabichromene) has been achieved on amino HPTLC plates with dichloromethane as mobile phase. By use of bidirectional development up to 30 samples can be analysed on a 10 cm×20 cm plate within 4 min. Evaluation was performed by slit-scanning densitometry at 200 nm. System-suitability data confirm the applicability of the method.
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Two overpressured layer chromatography (OPLC) methods have been developed for the separation of neutral and acidic cannabinoids. The first is an adaptation of Korte's well known method to the OPLC system, which improves its reproducibility. The second one is a new technique based on the phenomenon of chromatographic solvent demixing. The eluent itself is also divided into zones. In the alpha-zone the neutral cannabinoids and in the beta-zone the acidic ones are separated. As a result of the good and reproducible separation, there is a possibility to quantitate cannabinoids by densitometry. The on-line version of OPLC proved suitable for the isolation of hemp constituents.
Article
Cannabis has a potential for clinical use often obscured by unreliable and purely anecdotal reports. The most important natural cannabinoid is the psychoactive tetrahydrocannabinol (Δ9-THC); others include cannabidiol (CBD) and cannabigerol (CBG). Not all the observed effects can be ascribed to THC, and the other constituents may also modulate its action; for example CBD reduces anxiety induced by THC. A standardised extract of the herb may be therefore be more beneficial in practice and clinical trial protocols have been drawn up to assess this. The mechanism of action is still not fully understood, although cannabinoid receptors have been cloned and natural ligands identified. Cannabis is frequently used by patients with multiple sclerosis (MS) for muscle spasm and pain, and in an experimental model of MS low doses of cannabinoids alleviated tremor. Most of the controlled studies have been carried out with THC rather than cannabis herb and so do not mimic the usual clincal situation. Small clinical studies have confirmed the usefulness of THC as an analgesic; CBD and CBG also have analgesic and antiinflammatory effects, indicating that there is scope for developing drugs which do not have the psychoactive properties ofTHC. Patients taking the synthetic derivative nabilone for neurogenic pain actually preferred cannabis herb and reported that it relieved not only pain but the associated depression and anxiety. Cannabinoids are effective in chemotherapy-induced emesis and nabilone has been licensed for this use for several years. Currently, the synthetic cannabinoid HU211 is undergoing trials as a protective agent after brain trauma. Anecdotal reports of cannabis use include case studies in migraine and Tourette’s syndrome, and as a treatment for asthma and glaucoma. Apart from the smoking aspect, the safety profile of cannabis is fairly good. However, adverse reactions include panic or anxiety attacks, which are worse in the elderly and in women, and less likely in children. Although psychosis has been cited as a consequence of cannabis use, an examination of psychiatric hospital admissions found no evidence of this, however, it may exacerbate existing symptoms. The relatively slow elimination from the body of the cannabinoids has safety implications for cognitive tasks, especially driving and operating machinery; although driving impairment with cannabis is only moderate, there is a significant interaction with alcohol. Natural materials are highly variable and multiple components need to be standardised to ensure reproducible effects. Pure natural and synthetic compounds do not have these disadvantages but may not have the overall therapeutic effect of the herb.
Article
Various methods for the analysis of cannabinoids in biological materials, including plant and human body materials, are reviewed. Chromatographic methods, such as TLC, GC and HPLC, and non-chromatographic methods, mainly immunoassays, are discussed and compared. Chromatography is most commonly used in the analysis of plant material, with GC apparently offering the most advantages. Immunoassays, such as radioimmunoassay and fluorescence polarisation immunoassay, and enzyme immunoassay methods, such as enzyme multiplied immunoassay technique and enzyme-linked immunosorbent assay, can be used for human body materials; however, GC-MS is still necessary for confirmation and accurate quantification. Preferred methods are suggested for various specific purposes.
Article
The cannabis plant (Cannabis sativa L.) and products thereof (such as marijuana, hashish and hash oil) have a long history of use both as a medicinal agent and intoxicant. Over the last few years there have been an active debate regarding the medicinal aspects of cannabis. Currently cannabis products are classified as Schedule I drugs under the Drug Enforcement Administration (DEA) Controlled Substances act, which means that the drug is only available for human use as an investigational drug. In addition to the social aspects of the use of the drug and its abuse potential, the issue of approving it as a medicine is further complicated by the complexity of the chemical make up of the plant. This manuscript discusses the chemical constituents of the plant with particular emphasis on the cannabinoids as the class of compounds responsible for the drug's psychological properties.
Article
Recent studies have elucidated the biosynthetic pathway of cannabinoids and have highlighted the preference for a C-3 n-pentyl side chain in the most prominently represented cannabinoids from Cannabis sativa and their medicinally important decarboxylation products. The corresponding C-3 n-propyl side chain containing cannabinoids are also found, although in lesser quantities. Structure-activity relationship (SAR) studies performed on Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the key psychoactive ingredient of Cannabis, and its synthetic analogues have identified the C-3 side chain as the key pharmacophore for ligand affinity and selectivity for the known cannabinoid receptors and for pharmacological potency. Interestingly, the terminal n-pentyl saturated hydrocarbon side chain of endocannabinoids also plays a corresponding crucial role in conferring similar properties. This review briefly summarizes the biosynthesis of cannabinoids and endocannabinoids and focuses on their side chain SAR.
Article
Cannabis is one of the first plants used as medicine, and the notion that it has potentially valuable therapeutic properties is a matter of current debate. The isolation of its main constituent, Delta9-tetrahydrocannabinol (THC), and the discovery of the endocannabinoid system (cannabinoid receptors CB1 and CB2 and their endogenous ligands) made possible studies concerning the pharmacological activity of cannabinoids. This paper reviews some of the most-important findings in the field of THC pharmacology. Clinical trials, anecdotal reports, and experiments employing animal models strongly support the idea that THC and its derivatives exhibit a wide variety of therapeutic applications. However, the psychotropic effects observed in laboratory animals and the adverse reactions reported during human trials, as well as the risk of tolerance development and potential dependence, limit the application of THC in therapy. Nowadays, researchers focus on other therapeutic strategies by which the endocannabinoid system might be modulated to clinical advantage (inhibitor or activator of endocannabinoid biosynthesis, cellular uptake, or metabolism). However, emerging evidence highlights the beneficial effects of the whole cannabis extract over those observed with single components, indicating cannabis-based medicines as new perspective to revisit the pharmacology of this plant.
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