Cannabis Plant Material Research Articles

Total Synthesis of the Dimeric Phytocannabinoid Cannabitwinol (CBD Dimer) and Its Analogues.

A new synthetic route to access the natural product phytocannabinoid cannabitwinol (1) starting from commercially available raw materials is disclosed. We have also demonstrated the synthesis of other related dimers, including their enantiomers, using the present sequence. This route avoids the legal constraints concerning the acquisition of cannabis plant material or CBD.

Identification of Marijuana Using Silver-Phosphine Ion Complexation and a Semi-Quantitative 1% Decision-Point Assay

Due to the strict federal regulations concerning ∆9-tetrahydrocannabinol (∆9-THC) content for the differentiation of hemp and marijuana outlined in the 2018 Farm Bill, the ability to quickly and reliably differentiate cannabis as marijuana or hemp is crucial within both the seized drug community and hemp industry. This study provides a novel direct mass spectrometry approach for the identification of marijuana using Ag-phosphine ion complexation and a semi-quantitative 1% decision-point assay. The main constituents of hemp and marijuana, cannabidiol (CBD) and ∆9-THC, are isomeric and cannot be differentiated using soft ionization mass spectrometry techniques alone. However, the incorporation of [Ag(PPh3)(OTf)]2 enables the formation of unique MS/MS product ions at m/z 421/423, m/z 353/355, and m/z 231 for CBD due to differences in binding affinity, allowing CBD to be differentiated from ∆9-THC. Likewise, the isomeric cannabinoid precursors ∆9-tetrahydrocannabinolic acid (∆9-THCA) and cannabidiolic acid (CBDA) can be differentiated due to the formation of unique MS/MS product ions at m/z 465/467 and m/z 379/381, which are specific to CBDA. Eight additional cannabinoids were also characterized utilizing the proposed Ag-phosphine ion complexation approach. To reduce the potential for false positives, a more conservative 1% decision-point assay was developed by fortifying 1% weight-by-volume ∆9-THC-d9 into methanolic extracts of authentic cannabis plant material, followed by assessing if the total ∆9-THC content (containing ∆9-THC and ∆9-THCA) was greater than or less than the intensity of the spiked internal standard. When the developed approach was applied to 20 methanolic extracts of authentic cannabis samples with known cannabinoid compositions, 90% of the samples were correctly identified as marijuana or not marijuana based on the 1% administrative threshold for the total ∆9-THC content. The two lone misidentifications were due to the presence of elevated ∆8-THC, which highlights the necessity to explore more selective ligands in the future. This study provides the first application of Ag-ligand ion complexation for the identification of marijuana based on a semi-quantitative 1% decision-point assay, which shows great promise as an alternative method for the rapid identification of marijuana.

Young adult retail purchases of cannabis, product category preferences and sales trends in California 2018-21: Differences compared with older adults.

The aim of this study is to identify cannabis products according to their appeal among young adults and measure product sales trends. This was a retrospective comparative study using point-of-sale data from licensed recreational cannabis retailers that include buyer age with birth year entered by retailers, set in California, USA. Cannabis purchases by young adults (aged 21-24, GenZ) were compared with older adults (age 25+) over 4years (2018-21). Sales for six cannabis product categories were analyzed using a commercial data set with imputations and a raw data set. Age-appeal metrics were dollar and unit sales to young adults, and dollar and unit share ratios (young adults/older adults), where a share ratio of 100 denotes age-appeal comparability. A product category was considered more young-adult appealing than others if its mean on a metric was at least one standard deviation above the grand mean across all product categories. Flower (cannabis plant material) and vapor pen appealed to young adults based on absolute dollar sales, dominating young-adult spending compared with other cannabis products (37.24 and 31.83%, respectively). Vapor pen and concentrate appealed to young adults based on dollar share ratios of 152, meaning these products comprised a 52% greater share of young-adult cannabis spending relative to older-adult spending (31.83/20.97% and 10.47/6.88%, respectively). Less appealing to young adults were pre-roll, edible/beverage and absorbable products (tincture/sublingual, capsule and topical). Flower showed the largest dollar sales growth (B = +$3.50million/month), next to vapor pen (B = +$1.55million/month). Vapor pen tied for highest growth in the percent of product dollars from the largest package size (B = +0.85%/month) and showed the steepest price decline (B = -0.53 price per gram/month). In California, USA, from 2018 to 2021, relative to older adults, young adults spent a greater share of their cannabis dollars on vapor pen and concentrate (products with high potency of delta-9-tetrahydrocannabinol).

Activity-based detection of synthetic cannabinoid receptor agonists in plant materials

BackgroundSince late 2019, fortification of ‘regular’ cannabis plant material with synthetic cannabinoid receptor agonists (SCRAs) has become a notable phenomenon on the drug market. As many SCRAs pose a higher health risk than genuine cannabis, recognizing SCRA-adulterated cannabis is important from a harm reduction perspective. However, this is not always an easy task as adulterated cannabis may only be distinguished from genuine cannabis by dedicated, often expensive and time-consuming analytical techniques. In addition, the dynamic nature of the SCRA market renders identification of fortified samples a challenging task. Therefore, we established and applied an in vitro cannabinoid receptor 1 (CB1) activity-based procedure to screen plant material for the presence of SCRAs.MethodsThe assay principle relies on the functional complementation of a split-nanoluciferase following recruitment of β-arrestin 2 to activated CB1. A straightforward sample preparation, encompassing methanolic extraction and dilution, was optimized for plant matrices, including cannabis, spiked with 5 µg/mg of the SCRA CP55,940.ResultsThe bioassay successfully detected all samples of a set (n = 24) of analytically confirmed authentic Spice products, additionally providing relevant information on the ‘strength’ of a preparation and whether different samples may have originated from separate batches or possibly the same production batch. Finally, the methodology was applied to assess the occurrence of SCRA adulteration in a large set (n = 252) of herbal materials collected at an international dance festival. This did not reveal any positives, i.e. there were no samples that yielded a relevant CB1 activation.ConclusionIn summary, we established SCRA screening of herbal materials as a new application for the activity-based CB1 bioassay. The simplicity of the sample preparation, the rapid results and the universal character of the bioassay render it an effective and future-proof tool for evaluating herbal materials for the presence of SCRAs, which is relevant in the context of harm reduction.

Photodegradation of cannabidiol (CBD) and Δ9-THC in cannabis plant material

Δ9-THC, the psychotropic cannabinoid in Cannabis sativa L., for many years has been the focus of all the pharmacological attention as the main promising principle of the plant. Recently, however, cannabidiol (CBD) has brought a sudden change in the scenario, exponentially increasing the interest in pharmacology as the main non-psychotropic cannabinoid with potential therapeutic, cosmetical and clinical applications. Although the reactivity of CBD and Δ9-THC has been considered, little attention has been paid to the possible photodegradation of these cannabinoids in the vegetal matrix and the data available in the literature are, in some cases, contradictory. The aim of the present work is to provide a characterization of the photochemical behaviour of CBD and Δ9-THC in three cannabis chemotypes, namely I (Δ9-THC 2.50%w/w), II (CBD:Δ9-THC 5.82%w/w:3.19%w/w) and III (CBD 3.02%w/w).Graphical abstract

Effect of Gamma Irradiation on Cannabinoid, Terpene, and Moisture Content of Cannabis Biomass

In recent years, cannabis has been proposed and promoted not only as a medicine for the treatment of a variety of illnesses, but also as an industrial crop for different purposes. Being an agricultural product, cannabis inflorescences may be contaminated by environmental pathogens at high concentrations, which might cause health problems if not controlled. Therefore, limits have to be placed on the levels of aerobic bacteria as well as yeast and mold. To ensure the safety of cannabis plant material and related products, a remediation process has to be put in place. Gamma irradiation is a sterilization process mainly used for pharmaceuticals, foods, cosmetics, agricultural, and herbal products including cannabis plant material. This study was designed to determine the effect of irradiation on the microbial count as well as on the chemical and physical profiles of the cannabis biomass, particularly cannabinoids, terpenes, and moisture content. The full cannabinoid profile was measured by GC/FID and HPLC analysis, while terpene profile and moisture content were determined using GC/MS and Loss on Drying (LoD) methods, respectively. Analyses were conducted on the samples before and after gamma irradiation. The results showed that the minimum and maximum doses were 15 and 20.8 KiloGray (KGY), respectively. Total Aerobic Microbial Count (TAMC) and Total Yeast and Mold Count (TYMC) were determined. The study showed that irradiation has no effect on the cannabinoids and little effect on terpenes and moisture content, but it did result in the virtual sterilization of the plant material, as evidenced by the low levels of bacterial and fungal colony-forming units (CFUs) < 10 after gamma irradiation.

Chemical identification and optimization of the 4-aminophenol colorimetric test for the differentiation between hemp-type and marijuana-type cannabis plant samples.

The 4- Aminophenol (4-AP) colorimetric test is a fast, easy-to-use, and cost-effective presumptive assay of cannabis plant material producing different chromophores with THC-rich cannabis (blue color) and with CBD-rich cannabis (pink color). The main drawback of the 4-AP test is a brief observation window where the color rapidly changes to black, limiting the utility of the test. We now report for the first time, the identification of the product chromophores between 4-AP and CBD/THC as well as propose an explanation and a solution for the color degradation of the chromophores. The identification of the chromophores is provided by spectroscopic (UV-Vis), chromatography, and mass spectrometry (TLC and LC-QToF-MS). Oxidation of excess 4-AP (Reagent A) in the presence of NaOH (Reagent B) produces the black color observed for the previously reported 4-AP tests and reported in the literature. The adjustment of reactants concentrations and volumes of 4-AP:THC/CBD to a 1:1 ratio significantly reduces the black oxidation by-product and increases the observation window up to 2 h instead of the previously reported 5-10 min. For the first time, mass spectrometry and chromatography confirmed that the reaction of THC and CBD with 4-AP produced chromophores with m/z (M + H) = 420, consistent with proposed indophenol structures. The TLC method developed confirmed the separation between CBD and THC chromophores. The specificity of the test is also reported, showing false positive results for the presence of THC (blue color) for samples of thyme and oregano. LDA and SIMCA models showed that the optimized 4-AP procedure performs better than the previously reported 4-AP color test.

Evaluating cannabis exposure in survey items: Insights, strategies, and remaining challenges identified from cognitive interviewing

BackgroundThe diversity in characteristics of cannabis products and behavior patterns make evaluation of cannabis exposure in population-based, self-report surveys challenging. Accurate identification of cannabis exposure and related outcomes necessitates a thorough understanding of participants’ interpretations of survey questions assessing cannabis consumption behaviors. ObjectivesThe current study utilized cognitive interviewing to gain insight on participants’ interpretation of items in a self-reported survey instrument used to estimate the quantity of THC consumed in population samples. MethodsCognitive interviewing was used to evaluate survey items assessing cannabis use frequency, routes of administration, quantity, potency, and perceived “typical patterns” of use. Ten participants ≥18 years (n = 4 cisgender-men; n = 3 cisgender-women; n = 3 non-binary/transgender) who had used cannabis plant material or concentrates in the past week were recruited to take a self-administered questionnaire and subsequently answer a series of scripted probes regarding survey items. ResultsWhile most items presented no issues with comprehension, participants identified several areas of ambiguity in question or response item wording or in visual cues included in the survey. Generally, participants with irregular use patterns (i.e., non-daily use) reported more difficulty recalling the time or quantity of cannabis use. Findings resulted in several changes to the updated survey, including updated reference images and new quantity/frequency of use items specific to the route of administration. ConclusionIncorporating cognitive interviewing into cannabis measurement development among a sample of knowledgeable cannabis consumers led to improvements in assessing cannabis exposure in population surveys, which may otherwise have been missed.

Investigation of microorganisms in cannabis after heating in a commercial vaporizer.

There are concerns about microorganisms present on cannabis materials used in clinical settings by individuals whose health status is already compromised and are likely more susceptible to opportunistic infections from microbial populations present on the materials. Most concerning is administration by inhalation where cannabis plant material is heated in a vaporizer, aerosolized, and inhaled to receive the bioactive ingredients. Heating to high temperatures is known to kill microorganisms including bacteria and fungi; however, microbial death is dependent upon exposure time and temperature. It is unknown whether the heating of cannabis at temperatures and times designated by a commercial vaporizer utilized in clinical settings will significantly decrease the microbial loads in cannabis plant material. To assess this question, bulk cannabis plant material supplied by National Institute on Drug Abuse (NIDA) was used to assess the impact of heating by a commercial vaporizer. Initial method development studies using a cannabis placebo spiked with Escherichia coli were performed to optimize culture and recovery parameters. Subsequent studies were carried out using the cannabis placebo, low delta-9 tetrahydrocannabinol (THC) potency and high THC potency cannabis materials exposed to either no heat or heating for 30 or 70 seconds at 190°C. Phosphate-buffered saline was added to the samples and the samples agitated to suspend the microorganism. Microbial growth after no heat or heating was evaluated by plating on growth media and determining the total aerobic microbial counts and total yeast and mold counts. Overall, while there were trends of reductions in microbial counts with heating, these reductions were not statistically significant, indicating that heating using standard vaporization parameters of 70 seconds at 190°C may not eliminate the existing microbial bioburden, including any opportunistic pathogens. When cultured organisms were identified by DNA sequence analyses, several fungal and bacterial taxa were detected in the different products that have been associated with opportunistic infections or allergic reactions including Enterobacteriaceae, Staphylococcus, Pseudomonas, and Aspergillus.

Comprehensive comparison of industrial cannabinoid extraction techniques: Evaluation of the most relevant patents and studies at pilot scale

Cannabinoids are gaining attention in many sectors, including the pharmaceutical, nutraceutical, and cosmetic sectors. To date, several conventional and alternative techniques have been applied for Cannabis sativa L (C. sativa L.) extraction at the industrial scale. The conventional methods are liquid solvent extraction, including polar and non-polar solvents such as ethanol, hexane, petroleum ether, and other solvent extraction. Pressurized gas extraction is another conventional method and comprises gaseous hydrocarbon extraction such as n-butane and n-propane, supercritical or subcritical carbon dioxide extraction, and extraction using a refrigerant gas such as 1,1,1,2-tetrafluoroethane (HFC 134a). Alternative extraction methods include microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE), hydrodynamic cavitation, and pulsed electric fields (PEF). This review thoroughly analyzes and compares the main extraction techniques and technologies at the industrial scale found in the patent literature. In addition, several aspects of the pretreatment of cannabis plant material and its influence on extraction are also discussed.

NIDA sets 5 mg of THC as standard cannabis unit for research

According to the National Institute on Drug Abuse (NIDA), one of the barriers to research on cannabis is the lack of a standard unit. “Existing experimental data are often hard to interpret due to the wide variability in potency of cannabis plant material and extracts, the lack of standard measures of use, and the wide variety of ways people consume cannabis,” according to a May 10 blog post by NIDA Director Nora Volkow, M.D. To help rectify this, NIDA, along with the National Cancer Institute, the National Heart, Lung, and Blood Institute, and the National Institute of Mental Health, published a notice in the NIH Guide (https://grants.nih.gov/grants/guide/notice‐files/NOT‐DA‐21‐049.html) directing researchers funded by these institutes to measure and report their findings from clinical research on cannabis using a standard unit of delta‐9‐tetrahydrocannabinol (THC) of 5 milligrams.

NIDA sets 5 mg of THC as standard cannabis unit for research

According to the National Institute on Drug Abuse (NIDA), one of the barriers to research on cannabis is the lack of a standard unit. “Existing experimental data are often hard to interpret due to the wide variability in potency of cannabis plant material and extracts, the lack of standard measures of use, and the wide variety of ways people consume cannabis,” according to a May 10 blog post by NIDA Director Nora Volkow, M.D.

NIDA sets 5 mg of THC as standard cannabis unit for research

According to the National Institute on Drug Abuse (NIDA), one of the barriers to research on cannabis is the lack of a standard unit. “Existing experimental data are often hard to interpret due to the wide variability in potency of cannabis plant material and extracts, the lack of standard measures of use, and the wide variety of ways people consume cannabis,” according to a May 10 blog post by NIDA Director Nora Volkow, M.D. To help rectify this, NIDA, along with the National Cancer Institute; the National Heart, Lung, and Blood Institute; and the National Institute of Mental Health, published a notice in the NIH Guide (https://grants.nih.gov/grants/guide/notice‐files/NOT‐DA‐21‐049.html) directing researchers funded by these institutes to measure and report their findings from clinical research on cannabis using a standard unit of delta‐9‐tetrahydrocannabinol (THC) of 5 milligrams.

Single-Laboratory Validation of an Immunoaffinity Column Cleanup LC Method for the Analysis of Aflatoxins and Ochratoxin A in Cannabis Plant Material, Resins, Vapes, Isolates, and Edible Products.

Potential fungal infection of cannabis plants during drying has raised concerns of resulting mycotoxin contamination in leaves and flowers and subsequent contamination of derived products including cannabis-containing edible products. Validated routine methods are essential to monitor cannabis and cannabis products to ensure consumer safety consistent with long-standing controls for mycotoxins such as aflatoxins and ochratoxin A in foodstuffs. To provide single-laboratory validation data to demonstrate the suitability of a method for determining aflatoxins and ochratoxin A in cannabis plant material, resins, vapes, isolates, and edible products such as chocolate. Extraction of solid and liquid matrixes with acetonitrile:water, centrifugation, and then dilution of an aliquot of supernatant with phosphate-buffered saline solution containing Tween 20 surfactant. Cleanup by passing through an immunoaffinity column containing antibodies to both aflatoxins and ochratoxin A and analyzing in a single LC chromatographic run with fluorescence detection. For within-day analysis, recoveries were in the range 77 to 99% with RSDs from 0.7 to 9.6% for aflatoxin B1. Similarly, ochratoxin A recoveries were from 64 to 94% and RSDs from 0.9 to 9.5% for mycotoxin mixtures spiked into cannabis flowers, resins, vapes, isolates, chocolate, gummies, edible oils, and beverages. A method for the determination of aflatoxins and ochratoxin A was successfully developed and single-laboratory validation data has been presented for cannabis plant material, resins, vapes, isolates, and edible products. A multi-mycotoxin immunoaffinity column cleanup with LC-fluorescence has been validated and shown to be suitable for routine control of aflatoxins and ochratoxin A in cannabis flowers and a diverse range of edible cannabis products.

PROTOCOL: The effects of cannabis liberalization laws on health, safety, and socioeconomic outcomes: An evidence and gap map.

PROTOCOL: The effects of cannabis liberalization laws on health, safety, and socioeconomic outcomes: An evidence and gap map.

Differentiation between drug-type, fibre-type and intermediate-type in cannabis samples: HPLC-UV versus GC-FID

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.

Validation of the 4-aminophenol color test for the differentiation of marijuana-type and hemp-type cannabis.

The analysis of cannabis plant material submitted to seized-drug laboratories was significantly affected by the signing of the Agricultural Improvement Act of 2018, which defined hemp and removed it from the definition of marijuana in the Controlled Substances Act. As a result, field law enforcement personnel and forensic laboratories now are in need of implementing new protocols that can distinguish between marijuana-type and hemp-type cannabis. Colorimetric tests provide a cost-effective and efficient manner to presumptively identify materials prior to submission to a laboratory for analysis. This work presents the validation of the 4-aminophenol (4-AP) color test and demonstrates its utility for discriminating between marijuana-type and hemp-type cannabis (i.e., typification). Validation studies included the testing of numerous cannabinoid reference materials, household herbs, previously characterized cannabis plant samples, and real-case samples. The 4-AP test reliably produces a pink result when the level of Δ9 -tetrahydrocannabinol (THC) is approximately three times lower than the level of cannabidiol (CBD). A blue result is generated when the level of THC is approximately three times higher than that of CBD. Inconclusive results are observed when the levels of THC and CBD are within a factor of three from each other, demonstrating the limitations of the test under those scenarios.

Festival testing: A survey of suspected drugs seized from New Zealand music festivals, December 2018 to March 2019

Music festivals and other recreational events are common settings for illicit drug use with the intention of socialising, relaxing and creating a memorable experience. The use of stimulants is commonly reported at music festivals, with a general preference for MDMA, cocaine, cathinones and LSD. The results from this survey of drugs seized at New Zealand music festivals show a similar trend, with MDMA accounting for two-thirds of the 305 samples seized. The second most popular drug in this survey was cannabis plant material, which is not generally noted as being a common festival drug but reflect New Zealand drug culture. On-site drug testing services can provide valuable information and education to potential drug users. In this study, a handheld Raman spectrometer was able to correctly identify at least one of the components in 54% of the samples analysed, meaning a large proportion of samples tested were unable to be identified using this device. A collaborative testing approach between on-site testing services and laboratory-based scientists is recommended in order to improve testing accuracy and provide additional information that might assist in harm reduction from drug use at music festivals.

The Cannabis Proteome Draft Map Project.

Recently we have seen a relaxation of the historic restrictions on the use and subsequent research on the Cannabis plants, generally classified as Cannabis sativa and Cannabis indica. What research has been performed to date has centered on chemical analysis of plant flower products, namely cannabinoids and various terpenes that directly contribute to phenotypic characteristics of the female flowers. In addition, we have seen many groups recently completing genetic profiles of various plants of commercial value. To date, no comprehensive attempt has been made to profile the proteomes of these plants. We report herein our progress on constructing a comprehensive draft map of the Cannabis proteome. To date we have identified over 17,000 potential protein sequences. Unfortunately, no annotated genome of Cannabis plants currently exists. We present a method by which “next generation” DNA sequencing output and shotgun proteomics data can be combined to produce annotated FASTA files, bypassing the need for annotated genetic information altogether in traditional proteomics workflows. The resulting material represents the first comprehensive annotated protein FASTA for any Cannabis plant. Using this annotated database as reference we can refine our protein identifications, resulting in the confident identification of 13,000 proteins with putative function. Furthermore, we demonstrate that post-translational modifications play an important role in the proteomes of Cannabis flower, particularly lysine acetylation and protein glycosylation. To facilitate the evolution of analytical investigations into these plant materials, we have created a portal to host resources developed from our proteomic and metabolomic analysis of Cannabis plant material as well as our results integrating these resources.

Sample Processing and Preparation Considerations for Solid Cannabis Products.

Background: The nascent cannabis industry is often challenged by a lack of guidance, unproven methodologies and conflicting legal challenges. The actual sampling and sample preparation of cannabis can be a difficult endeavor due the economic value, material complexity and heterogeneous nature of a plant based material. Objective: Examine the challenges of cannabis material sampling and sample processing as it relates to other similar materials. Method: In this paper, the complexity of sampling and sample preparation are reviewed for use in the cannabis industry. The mechanisms for sample processing are examined and compared for the best preparation techniques for targeted analytes in cannabis analysis. Results: Cryogenic grinding is one of the best methods for sample processing for the preservation of volatile compounds. Conclusions: Proper sampling techniques and procedures, including the use of standards, ensure homogeneity and improve analysis accuracy. Highlights: Methods of sample processing and preparation for cannabis plant material were examined to ensure homogeneity, accuracy and reproducibility.