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Thermal stability of cannabinoids in dried cannabis: a kinetic study
Abstract
This study was undertaken to quantitatively explore the effect of temperature on the degradation of cannabinoids in dried cannabis flower. A total of 14 cannabinoids were monitored using liquid chromatography and tandem mass spectrometry in temperature environments from − 20 to + 40 °C lasting up to 1 year. We find that a network of first-order degradation reactions is well-suited to model the observed changes for all cannabinoids. While most studies focus on high-temperature effects on the cannabinoids, this study provides high-precision quantitative assessment of room temperature kinetics with applications to shelf-life predictions and age estimates of cannabis products.
... Bioactive compounds are usually degraded during processing at elevated temperatures. It is known that acidic cannabinoids are decarboxylated by heating (even at ≥30°C) [Meija et al., 2022;Wang et al., 2016] or by the action of enzymes (e.g., CBDA synthase) in the plant to produce CBDA from CBGA [Kanabus et al., 2021]. However, there is little information on changes in the cannabinoid profile under the influence of the drying process. ...
... However, there is little information on changes in the cannabinoid profile under the influence of the drying process. Determining the thermal stability of cannabinoids is important for handling plants and selecting the method of drying or processing of C. sativa plant parts for food production [Meija et al., 2022]. Fresh fiber hemp is usually harvested at a high initial moisture content (MC) (usually 80 g/100 g) and should be dried to a safe MC value (10 g/100 g) to prevent the development of harmful microflora [Kwaśnica et al., 2020]. ...
... The enzymatic inter-conversion of CBGA to CBDA is well established in the biosynthesis pathway of cannabinoids during plant growth [Kim et al., 2022;Taura et al., 2007]. In turn, Meija et al. [2022] reported that decarboxylation of CBDA to CBD was likely to occur even during storage of dried inflorescences at ambient temperature. ...
... The plant produces a myriad of structurally and functionally diverse metabolites that play various roles in plant growth and development, and they are effective toxic compounds against herbivores. The production of cannabinoids has been modified for engineering in an in vitro cell-free system [94]. However, cannabis and cannabinoid products have evolved in response to the knowledge of profits from the phytocannabinoid system. ...
... Phytocannabinoids are being investigated for their effect based on therapeutics in brain pathology. Several studies reported in previous databases on human tissues and animal models have highlighted the promising therapeutic potential of cannabinoids in different neurological disorders, including Parkinson's and Alzheimer's disease, and the mechanisms of action behind the reported improvement in the clinical outcome and disease progression are associated with their anti-inflammatory, immunosuppressive, antioxidant, and neuroprotective properties, due to the modulation of the endocannabinoid system [94]. Another important molecular feature found in neurodegenerative diseases is the failure of protein homeostasis mechanisms, resulting in undesirable aggregation of misfolded proteins [33]. ...
... Another important molecular feature found in neurodegenerative diseases is the failure of protein homeostasis mechanisms, resulting in undesirable aggregation of misfolded proteins [33]. In this context, CBD has exerted its protective effect over several signaling cascades involved with proteostasis, consequently reducing oxidative stress in cells [94]. The treatments are based on the pharmacophore of existing cannabinoids and treating the spasticity caused by multiple sclerosis in the USA, Canada, and Australia. ...
Cannabis belongs to the family Cannabaceae, and phytocannabinoids are produced by the Cannabis sativa L. plant. A long-standing debate regarding the plant is whether it contains one or more species. Phytocannabinoids are bioactive natural products found in flowers, seeds, and fruits. They can be beneficial for treating human diseases (such as multiple sclerosis, neurodegenerative diseases, epilepsy, and pain), the cellular metabolic process, and regulating biological function systems. In addition, several phytocannabinoids are used in various therapeutic and pharmaceutical applications. This study provides an overview of the different sources of phytocannabinoids; further, the biosynthesis of bioactive compounds involving various pathways is elucidated. The structural classification of phytocannabinoids is based on their decorated resorcinol core and the bioactivities of naturally occurring cannabinoids. Furthermore, phytocannabinoids have been studied in terms of their role in animal models and antimicrobial activity against bacteria and fungi; further, they show potential for therapeutic applications and are used in treating various human diseases. Overall, this review can help deepen the current understanding of the role of biotechnological approaches and the importance of phytocannabinoids in different industrial applications.
... CBN and CBNA are the two main oxidation degradation products of THC and THCA and are normally encountered in the levels of minor cannabinoids in fresh plant material [10,35]. This observation suggests that the raw extract was probably stored at RT for a large period of time prior to its reception [36]. Anyhow, the concentration of cannabinoids present in the raw extract was enough to perform the microencapsulation. ...
... The profile can be easily changed by using extracts obtained from other chemotypes, and the amount can be raised by using more purified extracts. Together with this, the amount of neutral cannabinoids (i.e., THC, CBN, CBD, CBC and CBG) can be raised by the decarboxylation of acidic cannabinoids (i.e., THCA, CBNA, CBDA, CBCA and CBGA) [36]. In this regard, even though heating plant raw extracts is a common practice to produce products rich in neutral cannabinoids [37,40], acidic cannabinoids with known therapeutic or synergistic properties are lost [8,10] along with other synergistic constituents of cannabis, such as terpenes or flavonoids [40]. ...
... The t 1/2 was between 2 and 5 weeks in all cases, with the exceptions of CBD (60 days), THCA (90 days), CBN (170 days) and CBNA (−394 days). The oxidative degradation of cannabinoids could be the main reason for the concentration decay [57,58], but the decarboxylation of acidic cannabinoids can also occur under these conditions [36]. Based on the results, all cannabinoids, except CBNA, show similar degradation trends in microcapsules stored at light and room temperature conditions (see Figure 3A,B). ...
Cannabinoids present in Cannabis sativa are increasingly used in medicine due to their therapeutic potential. Moreover, the synergistic interaction between different cannabinoids and other plant constituents has led to the development of full-spectrum formulations for therapeutic treatments. In this work, the microencapsulation of a full-spectrum extract via vibration microencapsulation nozzle technique using chitosan-coated alginate is proposed to obtain an edible pharmaceutical-grade product. The suitability of microcapsules was assessed by their physicochemical characterization, long-term stability in three different storage conditions and in vitro gastrointestinal release. The synthetized microcapsules contained mainly ∆9-tetrahydrocannabinol (THC)-type and cannabinol (CBN)-type cannabinoids and had a mean size of 460 ± 260 µm and a mean sphericity of 0.5 ± 0.3. The stability assays revealed that capsules should be stored only at 4 °C in darkness to maintain their cannabinoid profile. In addition, based on the in vitro experiments, a fast intestinal release of cannabinoids ensures a medium–high bioaccessibility (57–77%) of therapeutically relevant compounds. The full characterization of microcapsules indicates that they could be used for the design of further full-spectrum cannabis oral formulations.
... At the same time, the phenomenon of changing the profile of cannabinoids (mainly decarboxylation of acidic forms) during thermal processing and storage of cannabis is described. It has been described that drying of plant material at a relatively low temperature of 50 • C causes a loss of acidic forms of cannabinoids [10,11]. It is known that the therapeutic value of cannabis extracts from the cannabis plant depends not only on the content of various cannabinoids, but also on the ratio of acidic and neutral forms [12]. ...
... Other authors, regarding cannabis inflorescences, noted a decrease in acidic forms during the storage of the material. Meija et al. [10] found that after 8 weeks of storage of C. sativa herbs in 40 • C THCA, they degraded from 125 to 20 mg g −1 , whereas CBDA went from 24 to 8 mg g −1 . At the same time, the amount of THC increased from 60 to 115 mg g −1 . ...
Hemp is used as a source of fiber, oil and bioactive substances including volatile and cannabinoid-containing substances. This paper presents, for the first time, results on the evaluation of drying methods (convective, vacuum–microwave and combined convective pre-drying and vacuum–microwave finishing drying) of hemp leaves on the qualitative and quantitative changes in secondary metabolites, including essential oils, cannabinoids and sterols. A ranking and descriptive test of hemp leaves was also performed. Drying kinetics was presented using three models, including logarithmic, Midilli and modified Page. The SPME-Arrow technique was used to determine 41 volatile compounds, of which caryophyllene, β-myrcene and α-humulene were dominant in dried and fresh leaves. Regarding the essential oils obtained, 64 were identified, with caryophyllene, humulene epoxide II and limonene being the dominant ones. For preserving the highest amount of oils, the best method was the convective pre-drying followed by vacuum–microwave finishing drying (CD60-VMD) combined method, where the retention of volatile compounds was 36.08%, whereas the CD70 and 240-VMD methods resulted in the highest loss of 83%. The predominant cannabinoids in fresh hemp leaves were CBDA 6.05 and CBD 2.19 mg g⁻¹. Drying caused no change in the cannabinoid profile of the plant material. β-Sitosterol, campesterol and lupeol were dominant in the phytosterol and triterpene fractions. No changes in either quality or quantity were observed in any of the variants found.
... Other recent mechanism studies have also reported comparable results and corroborated that acidic cannabinoid decarboxylations demonstrated first-order kinetics. [93][94][95][96][97] Perrotin-Brunel, citing an earlier related precedent of salicylic acid decarboxylations, 98,99 employed molecular modeling calculations to support a keto-enol mechanism as a likely lower-energy THCA-A decarboxylation pathway. 97 Khan at the University of Mississippi determined that the THCA-A decarboxylation rate constant was higher than that of both CBDA and CBGA. ...
... Although most decarboxylation studies have reported results at higher temperatures, a very recent publication (ahead of print) examined the rate of acidic cannabinoid decarboxylation at near ambient temperature for long periods of time. 93 Studies such as this will be valuable to establish Cannabis product shelf life. Interestingly, very recent results of Canadian researchers also suggested that a specific decarboxylation mechanism may be possible for acidic cannabinoids in a more lipophilic environment. ...
Introduction: Cannabis is a valuable plant, cultivated by humans for millennia. However, it has only been in the past several decades that biologists have begun to clarify the interesting Cannabis biosynthesis details, especially the production of its fascinating natural products termed acidic cannabinoids. Discussion: Acidic cannabinoids can experience a common organic chemistry reaction known as decarboxylation, transforming them into structural analogues referred to as neutral cannabinoids with far different pharmacology. This review addresses acidic and neutral cannabinoid structural pairs, when and where acidic cannabinoid decarboxylation occurs, the kinetics and mechanism of the decarboxylation reaction as well as possible future directions for this topic. Conclusions: Acidic cannabinoid decarboxylation is a unique transformation that has been increasingly investigated over the past several decades. Understanding how acidic cannabinoid decarboxylation occurs naturally as well as how it can be promoted or prevented during harvesting or storage is important for the various stakeholders in Cannabis cultivation.
... Similarly, low content of CBN (the main degradation product of THC's oxidation) was observed in all capsules, which is a fact of great interest in the case of CI and CII capsules (both containing high amounts of THC). Based on this result, we can conclude that the employed cannabis flowers were fresh (the higher CBN content is, the older a cannabis product is) and that THC was not oxidized neither in the preparation of the cannabis extracts nor in the encapsulation process [64,65]. This could be achieved by avoiding high temperatures (after decarboxylation), prolonged exposure to light and exposure to oxidant species. ...
Cannabinoids, such as ∆ ⁹ -tetrahydrocannabinol (THC) and cannabidiol (CBD), are effective bioactive compounds that improve the quality of life of patients with certain chronic conditions. The copolymer poly(lactic-co-glycolic acid) (PLGA) has been used to encapsulate such compounds separately, providing pharmaceutical grade edible products with unique features. In this work, a variety of PLGA based nanoformulations that maintain the natural cannabinoid profile found in the plant (known as full-spectrum) are proposed and evaluated. Three different cannabis sources were used, representing the three most relevant cannabis chemotypes. PLGA nanocapsules loaded with different amounts of cannabinoids were prepared by nanoemulsion, and were then functionalized with three of the most common coating polymers: pectin, alginate and chitosan. In order to evaluate the suitability of the proposed formulations, all the synthesized nanocapsules were characterized, and their cannabinoid content, size, zeta-potential, morphology and in vitro bioaccessibility was determined. Regardless of the employed cannabis source, its load and the functionalization, high cannabinoid content PLGA nanocapsules with suitable particle size and zeta-potential were obtained. Study of nanocapsules’ morphology and in vitro release assays in gastro-intestinal media suggested that high cannabis source load may compromise the structure of nanocapsules and their release properties, and hence, the use of lower content of cannabis source is recommended.
Graphical Abstract
... Furthermore, inaccurate labeling practices and differences in extraction efficiency and sample preparation techniques can also contribute to the variation in reported cannabinoid concentrations [172][173][174][175]. It is also important to consider the degradation of cannabinoids, once formulated, in products and the limited shelf life resulting from this process [176][177][178]. ...
The resurgence of cannabis (Cannabis sativa L.) has been propelled by changes in the legal framework governing its cultivation and use, increased demand for hemp-derived products, and studies recognizing the industrial and health benefits of hemp. This has led to the creation of novel high-cannabidiol, low-Δ⁹-tetrahydrocannabinol varieties, enabling hemp crop expansion worldwide. This review elucidates the recent implications for hemp cultivation in Europe, with a focus on the legislative impacts on the cultivation practices, prospective breeding efforts, and dynamic scientific landscape surrounding this crop. We also review the current cultivars’ cannabinoid composition of the European hemp market and its major differences with that of the United States.
... Remarkably, trichomes maintain their physical integrity even after the drying process owing to the protective cuticle layer covering their heads. This preservation of trichome heads suggests that no spatial limitations hinder the catalytic activity of THCAS and CBDAS during the drying period (Taura et al., 2007;Meija et al., 2022). This study provides limited evidence to support the preserved functional capacity of cannabinoid synthesis ( Figure 5). ...
Cannabis sativa L. is an industrially valuable plant known for its cannabinoids, such as cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC), renowned for its therapeutic and psychoactive properties. Despite its significance, the cannabis industry has encountered difficulties in guaranteeing consistent product quality throughout the drying process. Hyperspectral imaging (HSI), combined with advanced machine learning technology, has been used to predict phytochemicals that presents a promising solution for maintaining cannabis quality control. We examined the dynamic changes in cannabinoid compositions under diverse drying conditions and developed a non-destructive method to appraise the quality of cannabis flowers using HSI and machine learning. Even when the relative weight and water content remained constant throughout the drying process, drying conditions significantly influenced the levels of CBD, THC, and their precursors. These results emphasize the importance of determining the exact drying endpoint. To develop HSI-based models for predicting cannabis quality indicators, including dryness, precursor conversion of CBD and THC, and CBD : THC ratio, we employed various spectral preprocessing methods and machine learning algorithms, including logistic regression (LR), support vector machine (SVM), k-nearest neighbor (KNN), random forest (RF), and Gaussian naïve Bayes (GNB). The LR model demonstrated the highest accuracy at 94.7–99.7% when used in conjunction with spectral pre-processing techniques such as multiplicative scatter correction (MSC) or Savitzky–Golay filter. We propose that the HSI-based model holds the potential to serve as a valuable tool for monitoring cannabinoid composition and determining optimal drying endpoint. This tool offers the means to achieve uniform cannabis quality and optimize the drying process in the industry.
... Storage studies performed under controlled conditions, in air-tight bags in darkness showed preservation of secondary metabolites [48]. However, it has been recently observed that cannabinoids show different degradation kinetics and thermal stability depending on their molecular structure [49,50]. This indicates that each cannabis chemovar may exhibit unique degradation paths based on the content and type of cannabinoids. ...
The increasing interest in hemp and cannabis poses new questions about the influence of drying and storage conditions on the overall aroma and cannabinoids profile of these products. Cannabis inflorescences are subjected to drying shortly after harvest and then to storage in different containers. These steps may cause a process of rapid deterioration with consequent changes in precious secondary metabolite content, negatively impacting on the product quality and potency. In this context, in this work, the investigation of the effects of freeze vs tray drying and three storage conditions on the preservation of cannabis compounds has been performed. A multi-trait approach, combining both solid-phase microextraction (SPME) two-dimensional gas chromatography coupled to mass spectrometry (SPME-GC × GC–MS) and high-performance liquid chromatography (HPLC), is presented for the first time. This approach has permitted to obtain the detailed characterisation of the whole cannabis matrix in terms of volatile compounds and cannabinoids. Moreover, multivariate statistical analyses were performed on the obtained data, helping to show that freeze drying conditions is useful to preserve cannabinoid content, preventing decarboxylation of acid cannabinoids, but leads to a loss of volatile compounds which are responsible for the cannabis aroma. Furthermore, among storage conditions, storage in glass bottle seems more beneficial for the retention of the initial VOC profile compared to open to air dry tray and closed high-density polyethylene box. However, the glass bottle storage condition causes formation of neutral cannabinoids at the expenses of the highly priced acid forms. This work will contribute to help define optimal storage conditions useful to produce highly valuable and high-quality products.
Graphical Abstract
... Similar findings were obtained for drying of cannabis using IR (Chen et al., 2021;Oduola et al., 2023), MW (Uziel et al., 2022) and high temperature (Challa, 2020) as they also found these thermal treatments increased the production of active cannabinoids by decreasing the acidic cannabinoids. Postharvest thermal treatments or storage of cannabis inflorescence or extracts are likely to accelerate the decarboxylation when the temperature is >25ºC, even in dark conditions (Meija et al., 2022;Milay et al., 2020). ...
Conventional cannabis drying is time-consuming and energy intensive. A quick and dependable drying of cannabis is essential to ensure high-quality products to meet increasing demands. This study explored a combined microwave and infrared (MI) drying on cannabis comparing with control environmental (CE) drying. MI was very efficient with a short drying time of 16-200 min, high moisture diffusivity of 7.95×10 − 09-8.70×10 − 08 m 2 /s, and low energy consumption of 390.49-1611.42 kJ. The Page and Modified Page drying models fitted well to describe and predict the MI drying characteristics of cannabis. Microstructural images identified shrinkage in glandular trichomes of cannabis, whereas colorimetric assessment resulted in alteration of color attributes due to MI drying. It also facilitated the conversion of acidic cannabinoids to their neutral forms by decreasing tetra-hydrocannabinolic acid (THCA) in g/g of dry matter from 20.15% to 7.57% and increasing tetrahydrocannabinol (THC) from 6.31% to 16.65% that insignificantly (p≥0.05) affected the total THC level. MI drying resulted in a total terpenes concentration (%g/g of dry matters) of 0.541-0.730, insignificantly lower than CE drying (0.768). Overall, the study highlights MI as a rapid and energy-efficient drying for obtaining high quality cannabis, particularly for medicinal applications.
... Factor effect analyses and Student's t pairwise comparisons showed that Δ9-THC concentrations were not affected by pre-freezing and drying temperature and they were not significantly (p < 0.05) different compared to the reference samples for all accessions. Although significant decarboxylation of THCA to Δ9-THC was not observed in this study, likely due to the low freeze drying temperature used, storage studies have shown that postharvest processing of cannabis inflorescence or extracts at 25 • C or temperatures higher even in a dark room can cause cannabinoid decarboxylation (Meija et al., 2021;Milay et al., 2020). Major terpene content was similarly determined and compared for inflorescence from the three cannabis accessions subjected to the same postharvest drying conditions (Figs. 12 and 13). ...
... La cantidad de THC en muestras de cannabis puede disminuir por interconversión a cannabinol (CBN), un cannabinoide que no está presente en la planta, con muy bajo poder psicoactivo y que se generada por calentamiento u oxidación del material vegetal. Altas concentraciones de CBN en las muestras de cannabis pueden indicar un almacenamiento prolongado o mala manipulación de la muestra (10) . ...
El cannabis o marihuana es una de las sustancias psicoactivas más consumida en todo el mundo, por lo que conocer la composición y el tipo de cannabis que se comercializa en los entornos urbanos es un insumo necesario para el diseño de políticas en salud pública sustentadas en la evidencia científica. Este estudio caracterizó los principales fitocannabinoides de muestras de marihuana (cigarrillos o cogollos) obtenidas en áreas urbanas y rurales de la ciudad Medellín, en octubre de 2021. Se realizó un muestreo no probabilístico a conveniencia en el que se recolectaron 87 muestras de marihuana donadas por consumidores en diferentes puntos de recolección en toda la ciudad, aplicando las técnicas de cromatografía de gases masas e ionización de llama para la caracterización de los fitocanabinoides. Se encontró el tetrahidrocannabinol como el constituyente principal de la marihuana circulante en Medellín, donde el 67,8% de las muestras presentaba un rango toxicológico alto o superior para THC; lo anterior en un contexto donde el mercado desregulado limita la posibilidad que tienen los consumidores en la práctica de calibrar o decidir la concentración de cannabinoides en sus dosis.
... Their results show an overall decrease in concentrations of THC and CBD, with an increase in the concentration of CBN when heated at 40°C for a period of one year. 19 Based on these reports, the stability of CBD depends on a variety of conditions including light exposure, storage temperature, and the physical state of CBD. In all studies, higher temperatures resulted in an increased CBD degradation. ...
Cannabidiol (CBD) containing products have flooded consumer markets in the past few years as the United States, Canada, and several European countries have legalized the cultivation of hemp. Due to complex legal histories of marijuana and cannabinoids, the stability and safety of CBD is still largely uncertain as research has been restricted globally. Conversion of CBD to its psychoactive isomers, delta-9-Tetrahydrocannabinol (Δ9-THC) and delta-8-Tetrahydrocannabinol (Δ8-THC), presents a significant safety issue for consumers and producers of CBD products. For some users, unintentional conversion, leading to intoxication, could significantly impair routine activities while others may seek to intentionally convert CBD for recreational use. Previous studies investigating the stability of CBD have focused mainly on replicating conditions experienced during long-term storage at room temperature or lower. Here, we report the thermal stability of CBD at 175°C. After 30 minutes of heating, both Δ8-THC and Δ9-THC were produced in significant amounts (~4.5% and 2.0%) in aerobic and anaerobic conditions without an acid catalyst. Dynamic 1 H-NMR experiments and computational electronic structure calculations were used to characterize possible reaction paths from CBD to THC. Our findings support an energetically feasible reaction route that is favorable due to both an increase in phenol acidity at high temperatures and intramolecular OH-hydrogen bonding. Introduction:
... Their results showed an overall decrease in concentrations of THC and CBD, with an increase in the concentration of CBN when heated at 40°C for a period of 1 year. 19 Based on these reports, the stability of CBD depends on a variety of conditions, including light exposure, storage temperature, and the physical state of CBD. In each study, higher temperatures resulted in an increased CBD degradation. ...
Introduction: In the last decade, the market for Cannabidiol (CBD) has grown to become a near $2 billion dollar industry in the United States alone. This growth can be attributed to a growing social acceptance of marijuana, a more detailed understanding of many health benefits attributed to cannabinoids, and the low cost and wide availibility of hemp-derived cannabinoids. Due to the complex legal histories of marijuana and cannabinoids, the stability and safety of CBD is still an area of interest as research has been restricted globally. Conversion of CBD to its psychoactive isomers, most notably delta-9-Tetrahydrocannabinol (Δ9-THC), presents a significant safety issue for consumers and producers of CBD products. Methods: Previous studies investigating the stability of CBD have focused mainly on replicating conditions experienced during long-term storage at room temperature or lower. Here, we report the thermal stability of CBD at 175°C. Dynamic 1H-NMR experiments and computational electronic structure calculations were used to characterize possible reaction paths from CBD to THC. Results: After 30 minutes of heating, Δ9-THC was produced in detectable amounts in aerobic and anaerobic conditions without an acid catalyst. Conclusions: Our findings support an energetically feasible reaction route that is favorable due to both an increase in phenol acidity at high temperatures and the presence of intramolecular OH-π hydrogen bonding.
... Cannabinoid analysis method developed and described previously by the National Research Council of Canada was modified and used for this study [46,47]. Extracted crude cannabis oil samples were centrifuged at 5000 rpm for 5 min. ...
Limited studies have explored different extraction techniques that improve cannabis extraction with scale-up potential. Ultrasound-assisted and microwave-assisted extraction were evaluated to maximize the yield and concentration of cannabinoids and terpenes. A central composite rotatable design was used to optimize independent factors (sample-to-solvent ratio, extraction time, extraction temperature, and duty cycle). The optimal conditions for ultrasound- and microwave-assisted extraction were the sample-to-solvent ratios of 1:15 and 1:14.4, respectively, for 30 min at 60 °C. Ultrasound-assisted extraction yielded 14.4% and 14.2% more oil and terpenes, respectively, compared with microwave-assisted extracts. Ultrasound-assisted extraction increased cannabinoid concentration from 13.2–39.2%. Considering reference ground samples, tetrahydrocannabinolic acid increased from 17.9 (g 100 g dry matter⁻¹) to 28.5 and 20 with extraction efficiencies of 159.2% and 111.4% for ultrasound-assisted and microwave-assisted extraction, respectively. Principal component analyses indicate that the first two principal components accounted for 96.6% of the total variance (PC1 = 93.2% and PC2 = 3.4%) for ultrasound-assisted extraction and 92.4% of the total variance (PC1 = 85.4% and PC2 = 7%) for microwave-assisted extraction. Sample-to-solvent ratios significantly (p < 0.05) influenced the secondary metabolite profiles and yields for ultrasound-assisted extracts, but not microwave-assisted extracts.
... A cannabinoid analysis method developed and described previously by the National Research Council of Canada was modified and used for this study [39,40]. Extracted crude cannabis oil samples were centrifuged at 489 relative centrifugal force for 5 min. ...
Efficient cannabis biomass extraction can increase yield while reducing costs and minimizing waste. Cold ethanol extraction was evaluated to maximize yield and concentrations of cannabinoids and terpenes at different temperatures. Central composite rotatable design was used to optimize two independent factors: sample-to-solvent ratio (1:2.9 to 1:17.1) and extraction time (5.7 min–34.1 min). With response surface methodology, predicted optimal conditions at different extraction temperatures were a cannabis-to-ethanol ratio of 1:15 and a 10 min extraction time. With these conditions, yields (g 100 g dry matter⁻¹) were 18.2, 19.7, and 18.5 for −20 °C, −40 °C and room temperature, respectively. Compared to the reference ground sample, tetrahydrocannabinolic acid changed from 17.9 (g 100 g dry matter⁻¹) to 15, 17.5, and 18.3 with an extraction efficiency of 83.6%, 97.7%, 102.1% for −20 °C, −40 °C, and room temperature, respectively. Terpene content decreased by 54.1% and 32.2% for extraction at −20 °C and room temperature, respectively, compared to extraction at −40 °C. Principal component analysis showed that principal component 1 and principal component 2 account for 88% and 7.31% of total variance, respectively, although no significant differences in cold ethanol extraction at different temperatures were observed.
... The latter is evidenced with acidic cannabinoids, which experience a decarboxylation process due to thermal treatments. Additionally, some other researchers have previously mentioned the conversion of Δ 9 -THC into CBN due to the aging of the plant (Dujourdy and Besacier, 2017;Meija et al., 2021). In this context, our study explores the effect of temperature in the GC injector port. ...
The substantial increase in legalization and subsequent regulation of cannabis has intensified the control and analytical monitoring of cannabis products to assure sample quality and control the cannabinoid content of the crop. In this sense, the restriction on cultivating legal cannabis plants has been limited to 0.2–0.3% of Δ⁹-THC content, depending on the host country’s laws. Thereby, cannabis flowers containing more than this limit are considered illicit drug-type cultivations and require the obtention of specific permits to work with them. The official method established by the European Commission set the gas chromatography/flame ionization detector (GC-FID) as the proper instrument to analyze the delta-9 tetrahydrocannabinol (Δ⁹-THC) content. In the present work, the potential drawbacks associated with the utilization of the official method for the evaluation of the Δ9-THC content have been described. Thus, the effect of the GC injector port temperature in the degradation of cannabinoids was evaluated, observing the degradation of CBD by 20%, generating Δ⁹-THC and CBN as by-products. Likewise, 17.2% of Δ⁹-THC was degraded, producing CBN as a by-product. Therefore, despite the brief residence of cannabinoids in the GC inlet, the effect of temperature is noteworthy and must be considered. Derivatization of cannabinoids should be a mandatory step to prevent the thermal degradation of cannabinoids, assuring the accuracy of the results. Furthermore, the evaluation of cannabinoid degradation thermally treated for longer periods of time was carried out. The kinetic degradation of CBD was evaluated in this way, observing a degradation of 0.22 μg/L per second. At the same time, the kinetics of the appearance of Δ⁹-THC demonstrates the intermediate nature of this cannabinoid, being degraded at 0.03 s⁻¹ μM⁻¹. The degradation of CBD also produced CBN and CBE as by-products.
... Samples were stored frozen to reduce heat-induced evaporation and oxidation of the volatile components that are the basis of Cannabis aroma. Benefits of low temperature storage have been demonstrated for moisture content and cannabinoid concentrations and likely apply to terpenes and terpenoids as well (Das et al., 2022;Meija et al., 2022). The stimuli were exchanged for fresh samples midway through the study. ...
Cannabis sativa L. is grown and marketed under a large number of named strains. Strains are often associated with phenotypic traits of interest to consumers, such as aroma and cannabinoid content. Yet genetic inconsistencies have been noted within named strains. We asked whether genetically inconsistent samples of a commercial strain also display inconsistent aroma profiles. We genotyped 32 samples using variable microsatellite regions to determine a consensus strain genotype and identify genetic outliers (if any) for four strains. Results were used to select 15 samples for olfactory testing. A genetic outlier sample was available for all but one strain. Aroma profiles were obtained by 55 sniff panelists using quantitative sensory evaluation of 40 odor descriptors. Within a strain, aroma descriptor frequencies for the genetic outlier were frequently at odds with those of the consensus samples. It appears that within-strain genetic differences are associated with differences in aroma profile. Because these differences were perceptible to untrained panelists, they may also be noticed by retail consumers. Our results could help the cannabis industry achieve better control of product consistency.
... Cannabis is notoriously difficult to homogenize, because of the uneven distribution of cannabinoids in the trichomes of the flower. 17 To mitigate this, a large sample was milled and mixed thoroughly before separating the samples into the respective conditions. Furthermore, three samples were stored in each condition and sampled individually for the triplicate analysis. ...
... indica to determine the effect of irradiation on THC levels and it was reported that THC significantly increased due to irradiation treatment [110]. Storage analysis revealed that the higher the storage temperature, the higher was the degradation of phytocannabinoids and it was suggested that a 4 • C temperature could be the optimum for storage of cannabis along with olive oil [136,137]. Considering these few pieces of literature, it can be summarized that decarboxylation of cannabinoids to their neutral forms depends on thermal treatments applied during post-harvest processing. However, more investigations have yet to be conducted to determine the optimum drying methods and conditions, pre-treatments, extraction methods and time, storage, and many others. ...
In recent years, cannabis (Cannabis sativa L.) has been legalized by many countries for production, processing, and use considering its tremendous medical and industrial applications. Cannabis contains more than a hundred biomolecules (cannabinoids) which have the potentiality to cure different chronic diseases. After harvesting, cannabis undergoes different postharvest operations including drying, curing, storage, etc. Presently, the cannabis industry relies on different traditional postharvest operations, which may result in an inconsistent quality of products. In this review, we aimed to describe the biosynthesis process of major cannabinoids, postharvest operations used by the cannabis industry, and the consequences of postharvest operations on the cannabinoid profile. As drying is the most important post-harvest operation of cannabis, the attributes associated with drying (water activity, equilibrium moisture content, sorption isotherms, etc.) and the significance of novel pre-treatments (microwave heating, cold plasma, ultrasound, pulse electric, irradiation, etc.) for improvement of the process are thoroughly discussed. Additionally, other operations, such as trimming, curing, packaging and storage, are discussed, and the effect of the different postharvest operations on the cannabinoid yield is summarized. A critical investigation of the factors involved in each postharvest operation is indeed key for obtaining quality products and for the sustainable development of the cannabis industry.
... Although these phenotypes have been suggested as a visual heuristic for harvest timing, little scientific work describes them during flower development and trichome maturation. Previous work supports the idea that browning of trichome heads is associated with quality degradation in dried cannabis [3], but the shelf life of dried cannabis [4] and progressive trichome browning in storage makes extrapolation of results to fresh cannabis tissue unpredictable. In order to understand the role of trichome phenotypes during trichome maturation, it is necessary to obtain measurements in situ during flower development. ...
... On the other hand, LC-MS, where decarboxylation is avoided, has been used for the quantitative detection of THCA and CBDA along with other cannabinoids from dried cannabis [7], hemp seed batches, food and feed products [8,9], seized street cannabis samples and medicines [10], as well as hemp consumer products such as oils, plant material, creams, and cosmetics [11]. An interesting kinetic study on the thermal degradation of 14 phytocannabinoids was performed by low resolution mass spectrometry (LRMS) targeted analysis from dried plant material [12]. The aforementioned examples demonstrate the usefulness of LC-MS-based techniques for the quantification of cannabinoids, but this approach requires multistep sample preparation (drying, grinding, homogenization, extraction, etc.) [13] leading to a loss in the spatial information. ...
In recent years, cannabis and hemp-based products have become increasingly popular for recreational use, edibles, beverages, health care products, and medicines. The rapid detection and differentiation of phytocannabinoids is, therefore, essential to assess the potency and the therapeutic and nutritional values of cannabis cultivars. Here, we implemented SpiderMass technology for in vivo detection of cannabidiolic acid (CBDA) and ∆⁹-tetrahydrocannabinolicacid (∆⁹-THCA), and other endogenous organic plant compounds, to access distribution gradients within the plants and differentiate between cultivars. The SpiderMass system is composed of an IR-laser handheld microsampling probe connected to a mass spectrometer through a transfer tube. The analysis was performed on different plant organs from freshly cultivated cannabis plants in only a few seconds. SpiderMass analysis easily discriminated the two acid phytocannabinoid isomers via MS/MS, and the built statistical models differentiated between four cannabis cultivars. Different abundancies of the two acid phytocannabinoids were found along the plant as well as between different cultivars. Overall, these results introduce direct analysis by SpiderMass as a compelling analytical alternative for rapid hemp analysis.
... When the oil was stored at 20 and 25 • C, the half-lives were 49 and 20 days, respectively. Meija et al. [145] conducted stability testing on seven cannabinoid pairs (CBC, ∆9-THC, CBN, CBG, CBD, ∆9-THCV, and CBDV, as well as their acidic forms) in dried hemp material stored in the dark at temperatures ranging from −20 to 40 • C). The average monthly degradation of ∆9-THCA + ∆9-THC was 2% at 20 • C. It was observed that the storage of these compounds at 4 • C did not ensure long-term (more than 12 months) cannabinoid stability. ...
Scientific demonstrations of the beneficial effects of non-psychoactive cannabinoids on the human body have increased the interest in foods containing hemp components. This review systematizes the latest discoveries relating to the characteristics of cannabinoids from Cannabis sativa L. var. sativa, it also presents a characterization of the mentioned plant. In this review, we present data on the opportunities and limitations of cannabinoids in food production. This article systematizes the data on the legal aspects, mainly the limits of Δ9-THC in food, the most popular analytical techniques (LC-MS and GC-MS) applied to assay cannabinoids in finished products, and the available data on the stability of cannabinoids during heating, storage, and access to light and oxygen. This may constitute a major challenge to their common use in food processing, as well as the potential formation of undesirable degradation products. Hemp-containing foods have great potential to become commercially popular among functional foods, provided that our understanding of cannabinoid stability in different food matrices and cannabinoid interactions with particular food ingredients are expanded. There remains a need for more data on the effects of technological processes and storage on cannabinoid degradation.
Background
In Colorado, regulations for recreational and medical cannabis sales require Tetrahydrocannabinol (THC) concentration is printed on all products. Labeled THC concentrations can vary by +/-15% of what is in the product. Studies show THC concentrations recorded on product labels are not always reflective of the THC concentration in the cannabis product and there is evidence consumers make purchasing decisions based on label claims.
Aims
Explore the accuracy of cannabis product labels and differences between THC label accuracy and product type.
Design
Data for this analysis come from a larger observational study of cannabis impairment. N=74 flower, concentrate, and edible product samples from licensed Colorado dispensaries were collected and independently tested for THC concentration.
Setting
This study was conducted in Colorado, in the Denver Metro Area.
Participants
Participants in the study voluntarily enrolled and provided one-gram samples of the cannabis they consumed during the study to be independently tested. The cannabis tested for this analysis was donated on a voluntary basis, not all participants chose to donate.
Measurement
The main outcomes of interest for this analysis are accuracy of cannabis product labels compared to observed THC content, accuracy in the context of legally allowable variation, and difference between accuracy by product.
Findings
Overall, label values were higher than observed values in flower and edible products (p < 0.001) but was not significant for concentrates (p = 0.85). Flower products were observed to be significantly lower on labels versus the 15% legally allowable range (p = 0.04). Concentrate and edible products were not significantly different (p = 0.9 and p = 0.5, respectively).
Conclusions
There is tension between legally allowable THC concentration claims on cannabis product labels and how consumers purchase cannabis. As cannabis policy evolves, standards and regulations that ensure accurate THC concentrations are reported on product labels are urgently needed.
Hang-drying of cannabis at room conditions is a slow process and leads to the risk of microbial growth. This method can sometimes prevent cannabis from reaching the equilibrium moisture content (EMC) below the safe storage threshold. On the other hand, high-temperature drying techniques are faster but negatively impact the secondary metabolites. Cold plasma (CP) is a novel technique explored in this study to treat cannabis at various operational conditions of plasma jet (power: 300, 350, and 400 W, time: 20, 30, and 40 s) prior to drying at environmental conditions of 25°C and 50 % RH. The findings revealed that untreated cannabis samples reached an equilibrium moisture content (EMC) of approximately 16 % in 1260 min. In contrast, CP-pretreated samples achieved lower EMCs of 10–14 % within 690–840 min. CP pretreatment also resulted in high moisture diffusivity, lower energy consumption, and higher energy efficiency. Increasing CP power and residence time accelerated the decarboxylation of cannabinoids, leading to the formation of more tetrahydrocannabinol (THC) and less tetrahydrocannabinolic acid (THCA), without significantly affecting the total THC (27.45 % untreated vs. 25.82 % - 28.36 % g/ g of dry matter in CP pretreated samples). Compared with untreated dried inflorescences, the 400 W and 30 s CP treated inflorescences resulted in the retention of 96 % of terpenes, whereas all 300 W CP treated samples retained > 90 % of terpenes. Overall, the study highlights that CP pretreatment is a promising technology for the cannabis industry in shortening the drying time and preserving the product quality, especially terpenes.
Cannabis‐infused foods are currently on the rise in markets all around the world. Meanwhile, there are concerns over the health implications for consumers. Studies have explored the therapeutic potential and nutritional and economic benefits of cannabis usage. Yet, the phytonutrients, processing methods, and health implications of cannabis‐infused foods have not been well explored. This review evaluates existing evidence on the nutritional, processing, safety, and phytonutrient composition of cannabis‐infused food products and their medicinal and functional prospects. Cannabis seeds contain the highest amount of dietary nutrients, while flowers contain the highest amount of bioactive constituents. Oils, butter, seeds, flowers, and leaf extracts are the plant forms currently incorporated into food products such as beverages, baked products, cooking ingredients, functional foods, nutraceuticals, and nootropics. Cannabis‐infused foods have been found to offer therapeutic benefits for pain management, brain function, gut health, and certain cancers. Findings also show significant constraints associated with cannabis‐infused foods regarding dosage guidelines, limited research, efficacy, and long‐term health effects on consumers. This is further worsened by the lack of policies that regulate the industry. To realize the full potential of cannabis use in the food and health industries and in research, regulatory guidelines are needed to control dosages and improve its efficient use in these industries. This will go a long way to ensure the safety of cannabis users and enhance responsible production, marketing, and distribution.
THC (Δ⁹-tetrahydrocannabinol) is the major psychoactive constituent of cannabis, accountable for instigating euphoric effects in individuals. With the imperative need for a roadside testing device for the detection of THC, this study aims to overcome the challenges associated with the stability of a biomolecule-free sensor capable of detecting ultra-low concentrations of THC. Since THC is highly susceptible to oxidation, this study explores different avenues to create optimal storage conditions to minimize the oxidation of THC prior to detection. Hence, all experiments were conducted by controlling the principal factors contributing to the oxidation of THC: temperature, humidity, airflow and light, to extend the shelf-life of the manufactured electrodes and engender stable electrochemical signals. It is concluded that frozen storage conditions and a second acidic pH modification were ideal for improving the stability of the modified electrodes from day one up to a period of six months.
To investigate cannabinoid content and profiles, 16 cannabinoids were quantified in 30 commercial hemp seed edible oils. In addition, one hemp seed oil was subjected to thermal processing up to 200 °C for up to 60 min. UHPLC-MS/MS was used for analysis. The content of cannabinoids in the samples ranged from 9 to 279 mg kg−1 (sum) and for Δ9-tetrahydrocannabinol (Δ9-THC) from 0.2 to 6.7 mg kg−1. Three samples exceeded the EU Δ9-THC equivalent maximum levels of 7.5 mg kg−1 for hemp seed oils. Cannabinoid profiles can provide indications of different product characteristics (e.g. degree of processing, variety of plant material). Furthermore, intense thermal processing (200 °C, 60 min) led to 38% decrease in sum cannabinoid content (sum of all analysed cannabinoids in this study), 99% decrease in cannabinoid acids, and 22% increase in Δ9-THC.
This study focused on the investigation of cannabinoid profiles and contents of 23 different hemp teas and on the individual transfer of 16 cannabinoids from hemp teas into their tea infusions. The total cannabinoid content in the dry products averaged 14,960 mg kg-1, with CBD&CBDA (sum of cannabidiol (CBD) and cannabidiolic acid (CBDA)) being the major component, accounting for 87% of the total cannabinoid content. The Δ9-tetrahydrocannabinol (Δ9-THC) content ranged from 16 mg kg-1 to 935 mg kg-1 and was on average 221 mg kg-1. For each hemp tea, an infusion was prepared according to a standardized protocol issued by the German Standardisation body DIN and transfer rates per cannabinoid were estimated by comparing the contents in the dry material with the concentrations in the aqueous infusion. The limited water solubility of cannabinoids results in limited extraction efficiency for cannabinoids using boiling water to prepare a tea infusion and the average transfer rate of the psychoactive Δ9-THC was only 0.5%.
Cannabis (Cannabis sativa L.) is cultivated by licensed producers in Canada for medicinal and recreational uses. The recent legalization of this plant in 2018 has resulted in rapid expansion of the industry, with greenhouse production representing the most common method of cultivation. Female cannabis plants produce inflorescences that contain bracts densely covered by glandular trichomes, which synthesize a range of commercially important cannabinoids (e.g., THC, CBD) as well as terpenes. Cannabinoid content and quality varies over the 8-week flowering period to such an extent that the time of harvest can significantly impact product quality. Cannabis flower maturation is accompanied by a transition in the color of trichome heads that progresses from clear to milky to brown (amber) and can be seen visually using low magnification. However, the importance of this transition as it impacts quality and describes maturity has never been investigated. To establish a relationship between trichome maturation and trichome head color changes (phenotype), we developed a novel automatic trichome gland analysis pipeline using deep learning. We first collected a macro-photography dataset based on 4 commercially grown cannabis strains, namely 'Afghan Kush', 'Green Death Bubba', 'Pink Kush', and 'White Rhino'. Images were obtained in two modalities: conventional macroscopic light photography and macroscopic UV induced fluorescence. We then implemented a pipeline where the clear-milky-brown heuristic was injected into the algorithm to quantify trichome phenotype progression during the 8-week flowering period. A series of clear, milky, and brown phenotype curves were recorded for each strain over the flowering period that were validated as indicators of trichome maturation and corresponded to previously described parameters of trichome development, such as trichome gland head diameter and stalk elongation. We also derived morphological metrics describing trichome gland geometry from deep learning segmentation predictions that profiled trichome maturation over the flowering period. We observed that mature and senescing trichomes displayed fluorescent properties that were reflected in the clear, milky, and brown phenotypes. Our method was validated by two experiments where factors affecting trichome quality and flower development were imposed and the effects were then quantified using the deep learning pipeline. Our results indicate the feasibility of automated trichome analysis as a method to evaluate the maturation of female flowers cultivated in a highly variable environment, regardless of strain. These findings have broad applicability in a growing industry in which cannabis flower quality is receiving increased circumspection for medicinal and recreational uses.
Drug stability is an important concern of forensic toxicological testing, particularly postmortem samples that may be stored for an extensive period of time before analysis. In postmortem toxicology, the complex assessment of analyte stability in biological matrices can profoundly impact the interpretation of toxicological results and the outcome of forensic casework. The aim of this work is to assess the stability of Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), 11-hydroxy-THC (11-OH-THC), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THCCOOH), and 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid glucuronide (THCCOOH-glucuronide) in brain stored at three different temperatures (4°C, -20°C and -70°C) up to over 12 months (390 days) in order to establish the best storage condition for preventing potential drug degradation during the storage period. Brain is suitable and useful for xenobiotic concentrations and a valuable specimen in the interpretation of postmortem toxicological results. In our study, pooled brain specimens were spiked at low and high concentrations to evaluate the change in concentration over time. Stable compounds were quantified within ±20% of the target concentration (the mean concentration resulting from the initial analysis). According to stability criteria, our preliminary findings revealed that all the cannabinoids studied are stable in frozen brain samples (-20°C and -70°C) for over 12 months: all the analytes' concentrations remained unaffected during storage over time, the analytical variation staying within ±20%. On the contrary, under refrigeration conditions (4°C), 11-OH-THC, THCCOOH and THCCOOH-glucuronide were instable. Authentic brain samples, collected from eight cases during the autopsy, were analyzed and the stability was evaluated. This study provided new data on cannabinoid stability in brain. The stability of the brain samples, both in spiked samples and authentical caseworks, highlights the importance of the brain as a valid testing matrix when retesting is required after a long period of time or when laboratories are faced with backlog.
The popularity of vaping cannabis products has increased sharply in recent years. In 2019, a sudden onset of electronic cigarette/vaping-associated lung injury (EVALI) was reported, leading to thousands of cases of lung illness and dozens of deaths due to the vaping of tetrahydrocannabinol (THC)-containing e-liquids that were obtained on the black market. A potential cause of EVALI has been hypothesized due to the illicit use of vitamin E acetate (VEA) in cannabis vape cartridges. However, the chemistry that modifies VEA and THC oil, to potentially produce toxic byproducts, is not well understood under different scenarios of use. In this work, we quantified carbonyls, organic acids, cannabinoids, and terpenes in the vaping aerosol of pure VEA, purified THC oil, and an equal volume mixture of VEA and THC oil at various coil temperatures (100-300 °C). It was found under the conditions of our study that degradation of VEA and cannabinoids, including Δ9-THC and cannabigerol (CBG), occurred via radical oxidation and direct thermal decomposition pathways. Evidence of terpene degradation was also observed. The bond cleavage of aliphatic side chains in both VEA and cannabinoids formed a variety of smaller carbonyls. Oxidation at the ring positions of cannabinoids formed various functionalized products. We show that THC oil has a stronger tendency to aerosolize and degrade compared to VEA at a given temperature. The addition of VEA to the e-liquid nonlinearly suppressed the formation of vape aerosol compared to THC oil. At the same time, toxic carbonyls including formaldehyde, 4-methylpentanal, glyoxal, or diacetyl and its isomers were highly enhanced in VEA e-liquid when normalized to particle mass.
Although several extraction methodologies have been reported to date, the development of novel, more powerful, and alternative extraction processes using hemp as the vegetable matrix is still a challenging field of research. In this manuscript the performance of the subcritical butane promoted extraction of a panel of 5 non psychoactive cannabinoids from hemp inflorescences from 2 hemp varieties, under a counter-current mode using a handmade extraction apparatus,was studied. All secondary metabolites under investigation have been finally quantified by gas chromatographic analyses coupled to mass spectrometry. Phyto canabinoids have been extracted in yields from 3.3-fold to 55-fold higher with respect to traditional methods that were adopted herein as a comparison. The use of subcritical butane as the extraction solvent for cannabinoids from hemp inflorescences is reported herein for the first time and could be easily adopted for other matrices (e.g. seeds) deriving from the same vegetable source.
This review is a collection of recent applications of mass spectrometry studies for the characterization of phytocannabinoids in cannabis and hemp plant material and related products. The focus is mostly on recent applications using mass spectrometry as detector, in hyphenation to typical separation techniques (i.e., liquid chromatography or gas chromatography), but also with less common couplings or by simple direct analysis. The papers are described starting from the most common approach for targeted quantitative analysis, with applications using low-resolution mass spectrometry equipment, but also with the introduction of high-resolution mass analysers as the detectors. This reflects a common trend in this field, and introduces the most recent applications using high-resolution mass spectrometry for untargeted analysis. The different approaches used for untargeted analysis are then described, from simple retrospective analysis of compounds without pure standards, through untargeted metabolomics strategies, and suspect screening methods, which are the ones currently allowing to achieve the most detailed qualitative characterization of the entire phytocannabinoid composition, including minor compounds which are usually overlooked in targeted studies and in potency evaluation. These approaches also represent powerful strategies to answer questions on biological and pharmacological activity of cannabis, and provide a sound technology for improved classification of cannabis varieties. Finally, open challenges are discussed for future directions in the detailed study of complex phytocannabinoid mixtures.
This book delineates a forward looking vision for the pervasive ways that measurement and the rigorous evaluation of measurement uncertainty plays in this 21st century while honoring the scientific traditions that we hold dear.
The increase in production of cannabis for medical and recreational purposes in recent years has led to a corresponding increase in laboratories performing cannabinoid analysis of cannabis and hemp. We have developed and validated a quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method that is simple, reliable, specific, and accurate for the analysis of 17 cannabinoids in cannabis and hemp. Liquid-solid sample extraction coupled with dilution into a calibration ranges from 10 to 10,000 ng/mL and LC-MS/MS analysis provides quantification of samples ranging from 0.002 to 200 mg/g (0.0002 to 20.0%) in matrix. Linearity of calibration curves in methanol was demonstrated with regression r2 ≥ 0.99. Within-batch precision (0.5 to 6.5%) and accuracy (91.4 to 108.0%) and between-batch precision (0.9 to 5.1%) and accuracy (91.5 to 107.5%) were demonstrated for quality control (QC) samples in methanol. Within-batch precision (0.2 to 3.6%) and accuracy (1.4 to 6.1%) and between-batch precision (1.4 to 6.1 %) and accuracy (90.2 to 110.3%) were also evaluated with a candidate cannabis certified reference material (CRM). Repeatability (1.5 to 12.4% RSD) and intermediate precision (2.2 to 12.8% RSD) were demonstrated via analysis of seven cannabis samples with HorRat values ranging from 0.3 to 3.1. The method provides enhanced detection limits coupled with a large quantitative range for 17 cannabinoids in plant material. It is suitable for a wide range of applications including routine analysis for delta-9-tetrahydrocannabinol (Δ9-THC), delta-9-tetrahydrocannabinolic acid (Δ9-THCA), cannabidiol (CBD), cannabidiolic acid (CBDA), and cannabinol (CBN) as well as more advanced interrogation of samples for both major and minor cannabinoids.
There is an active and growing interest in cannabis female inflorescence (Cannabis sativa) for medical purposes. Therefore, a definition of its quality attributes can help mitigate public health risks associated with contaminated, substandard, or adulterated products and support sound and reproducible basic and clinical research. As cannabis is a heterogeneous matrix that can contain a complex secondary metabolome with an uneven distribution of constituents, ensuring its quality requires appropriate sampling procedures and a suite of tests, analytical procedures, and acceptance criteria to define the identity, content of constituents (e.g., cannabinoids), and limits on contaminants. As an independent science-based public health organization, United States Pharmacopeia (USP) has formed a Cannabis Expert Panel, which has evaluated specifications necessary to define key cannabis quality attributes. The consensus within the expert panel was that these specifications should differentiate between cannabis chemotypes. Based on the secondary metabolite profiles, the expert panel has suggested adoption of three broad categories of cannabis. These three main chemotypes have been identified as useful for labeling based on the following cannabinoid constituents: (1) tetrahydrocannabinol (THC)-dominant chemotype; (2) intermediate chemotype with both THC and cannabidiol (CBD); and (3) CBD-dominant chemotype. Cannabis plants in each of these chemotypes may be further subcategorized based on the content of other cannabinoids and/or mono- and sesquiterpene profiles. Morphological and chromatographic tests are presented for the identification and quantitative determination of critical constituents. Limits for contaminants including pesticide residues, microbial levels, mycotoxins, and elemental contaminants are presented based on toxicological considerations and aligned with the existing USP procedures for general tests and assays. The principles outlined in this review should be able to be used as the basis of public quality specifications for cannabis inflorescence, which are needed for public health protection and to facilitate scientific research on cannabis safety and therapeutic potential.
Simple Summary
The use of a two-component error model for the evaluation of chemical degradation data is reported for the first time in this contribution.
Abstract
In the kinetic evaluation of chemical degradation data, degradation models are fitted to the data by varying degradation model parameters to obtain the best possible fit. Today, constant variance of the deviations of the observed data from the model is frequently assumed (error model “constant variance”). Allowing for a different variance for each observed variable (“variance by variable”) has been shown to be a useful refinement. On the other hand, experience gained in analytical chemistry shows that the absolute magnitude of the analytical error often increases with the magnitude of the observed value, which can be explained by an error component which is proportional to the true value. Therefore, kinetic evaluations of chemical degradation data using a two-component error model with a constant component (absolute error) and a component increasing with the observed values (relative error) are newly proposed here as a third possibility. In order to check which of the three error models is most adequate, they have been used in the evaluation of datasets obtained from pesticide evaluation dossiers published by the European Food Safety Authority (EFSA). For quantitative comparisons of the fits, the Akaike information criterion (AIC) was used, as the commonly used error level defined by the FOrum for the Coordination of pesticide fate models and their USe(FOCUS) is based on the assumption of constant variance. A set of fitting routines was developed within the mkin software package that allow for robust fitting of all three error models. Comparisons using parent only degradation datasets, as well as datasets with the formation and decline of transformation products showed that in many cases, the two-component error model proposed here provides the most adequate description of the error structure. While it was confirmed that the variance by variable error model often provides an improved representation of the error structure in kinetic fits with metabolites, it could be shown that in many cases, the two-component error model leads to a further improvement. In addition, it can be applied to parent only fits, potentially improving the accuracy of the fit towards the end of the decline curve, where concentration levels are lower.
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.
Stan is a probabilistic programming language for specifying statistical models. A Stan program imperatively defines a log probability function over parameters conditioned on specified data and constants. As of version 2.14.0, Stan provides full Bayesian inference for continuous-variable models through Markov chain Monte Carlo methods such as the No-U-Turn sampler, an adaptive form of Hamiltonian Monte Carlo sampling. Penalized maximum likelihood estimates are calculated using optimization methods such as the limited memory Broyden-Fletcher-Goldfarb-Shanno algorithm. Stan is also a platform for computing log densities and their gradients and Hessians, which can be used in alternative algorithms such as variational Bayes, expectation propagation, and marginal inference using approximate integration. To this end, Stan is set up so that the densities, gradients, and Hessians, along with intermediate quantities of the algorithm such as acceptance probabilities, are easily accessible. Stan can be called from the command line using the cmdstan package, through R using the rstan package, and through Python using the pystan package. All three interfaces support sampling and optimization-based inference with diagnostics and posterior analysis. rstan and pystan also provide access to log probabilities, gradients, Hessians, parameter transforms, and specialized plotting.
Introduction: Decarboxylation is an important step for efficient production of the major active components in cannabis, for example, Δ⁹-tetrahydrocannabinol (Δ⁹-THC), cannabidiol (CBD), and cannabigerol (CBG). These cannabinoids do not occur in significant concentrations in cannabis but can be formed by decarboxylation of their corresponding acids, the predominant cannabinoids in the plant. Study of the kinetics of decarboxylation is of importance for phytocannabinoid isolation and dosage formulation for medical use. Efficient analytical methods are essential for simultaneous detection of both neutral and acidic cannabinoids.
Methods:C. sativa extracts were used for the studies. Decarboxylation conditions were examined at 80°C, 95°C, 110°C, 130°C, and 145°C for different times up to 60 min in a vacuum oven. An ultra-high performance supercritical fluid chromatography/photodiode array-mass spectrometry (UHPSFC/PDA-MS) method was used for the analysis of acidic and neutral cannabinoids before and after decarboxylation.
Results: Decarboxylation at different temperatures displayed an exponential relationship between concentration and time indicating a first-order or pseudo-first-order reaction. The rate constants for Δ⁹-tetrahydrocannabinolic acid-A (THCA-A) were twice those of the cannabidiolic acid (CBDA) and cannabigerolic acid (CBGA). Decarboxylation of THCA-A was forthright with no side reactions or by-products. Decarboxylation of CBDA and CBGA was not as straightforward due to the unexplained loss of reactants or products.
Conclusion: The reported UHPSFC/PDA-MS method provided consistent and sensitive analysis of phytocannabinoids and their decarboxylation products and degradants. The rate of change of acidic cannabinoid concentrations over time allowed for determination of rate constants. Variations of rate constants with temperature yielded values for reaction energy.
Traditional tinctures of Cannabis sativa L. became obsolete before elucidation of the main cannabinoids and routine quality testing for medicines. In view of increasing medicinal use of cannabinoids and associated safety concerns, tinctures from a ∆9-tetrahydrocannabinol (THC)-type chemovar were studied. High-performance liquid chromatography with diode-array detection (HPLC/DAD) was used to determine THC, ∆9-tetrahydrocannabinolic acid A (THCA), cannabinol (CBN), cannabidiol (CBD), cannabidiolic acid (CBDA), cannabigerol (CBG), cannabigerolic acid (CBGA), cannflavin A/B, and total phenolics. Derived group and ratio markers describe absolute and relative profiles when varying plant part (flos, folium), extraction solvent (EtOH percentage), storage conditions (‘shelf’ or ‘fridge’ up to 15 months), and pasteurization (2 h 70 °C, 20 min 80 °C). Tinctures from female flowering tops contained ten-fold more cannabinoids than tinctures from leaves; tinctures (80%–90% EtOH) contained ten-fold more cannabinoids than tinctures (40% EtOH). The analysis of CBGA + CBG, the main co-cannabinoids aside from THCA + THC, appears more relevant than CBDA + CBD. The decarboxylation of THCA to THC—the main change during storage of freshly prepared tinctures—is after 15 months in the ‘fridge’ comparable to 3 months on the ‘shelf’. Minimally increased CBN totals did not correlate to diminished totals of THCA and THC (up to 15% after 3 months ‘shelf’, 45% after 15 months ‘fridge’). Instead, total cannabinoids or acidic/neutral cannabinoid ratios are better stability markers. Moderate changes after pasteurization and partial losses below 10% for total cannabinoids after 9 months ‘fridge’ indicate possibilities for a reasonable shelf life. Yet storage and use of non-stabilized tinctures remain critical without authorized specification and stability data because a consistent cannabinoid content is not guaranteed.
Accelerated stability coupled with modeling to predict the stability of compounds, blends, and products at long-term storage conditions provides significant benefits in science-based decision-making throughout drug substance and drug product development. The study can often be completed, including data analysis in the space of three working weeks, and the information gathered and learning made in this time period can rival years of traditional analysis. The speed of the studies allows an earlier assessment of risk to quality enabling appropriate risk mitigation strategies to be implemented in a timely manner. The scientific foundation is based upon Arrhenius kinetic equations that can be linear or nonlinear in time, and can be based upon water vapor pressure or liquid water activity (relative humidity). A variety of kinetic models are evaluated, and the best model is chosen based upon both Bayesian information criteria and an automated assessment of kinetic model parameters fitting within acceptable ranges. Confidence intervals are estimated based upon a bootstrapping approach. Moisture vapor transmission rate models are applied on top of the resulting kinetic models in order to simulate different packaging types and the use of desiccant. The kinetic models are integrated with the prediction of packaging humidity over time to create a long-term prediction of impurities and other phenomena. The resulting models have been shown to be useful for not only the prediction of drug product impurities in long-term storage but other physical phenomena as well such as hydrate development and solvate loss.
Kinetic models are essential to quantitatively understand and predict the behaviour of metabolic networks. Detailed and thermodynamically feasible kinetic models of metabolism are inherently difficult to formulate and fit. They have a large number of heterogeneous parameters, are non-linear and have complex interactions. Many powerful fitting strategies are ruled out by the intractability of the likelihood function. Here, we have developed a computational framework capable of fitting feasible and accurate kinetic models using Approximate Bayesian Computation. This framework readily supports advanced modelling features such as model selection and model-based experimental design. We illustrate this approach on the tightly-regulated mammalian methionine cycle. Sampling from the posterior distribution, the proposed framework generated thermodynamically feasible parameter samples that converged on the true values, and displayed remarkable prediction accuracy in several validation tests. Furthermore, a posteriori analysis of the parameter distributions enabled appraisal of the systems properties of the network (e.g., control structure) and key metabolic regulations. Finally, the framework was used to predict missing allosteric interactions.
The evolution of major cannabinoids and terpenes during the growth of Cannabis sativa plants was studied. In this work, seven different plants were selected: three each from chemotypes I and III and one from chemotype II. Fifty clones of each mother plant were grown indoors under controlled conditions. Every week, three plants from each variety were cut and dried, and the leaves and flowers were analyzed separately. Eight major cannabinoids were analyzed via HPLC-DAD, and 28 terpenes were quantified using GC-FID and verified via GC-MS. The chemotypes of the plants, as defined by the tetrahydrocannabinolic acid/cannabidiolic acid (THCA/CBDA) ratio, were clear from the beginning and stable during growth. The concentrations of the major cannabinoids and terpenes were determined, and different patterns were found among the chemotypes. In particular, the plants from chemotypes II and III needed more time to reach peak production of THCA, CBDA, and monoterpenes. Differences in the cannabigerolic acid development among the different chemotypes and between monoterpene and sesquiterpene evolution patterns were also observed. Plants of different chemotypes were clearly differentiated by their terpene content, and characteristic terpenes of each chemotype were identified.
When the Guide to the Expression of Uncertainty in Measurement (GUM) and methods from its supplements are not applicable, the Bayesian approach may be a valid and welcome alternative. Evaluating the posterior distribution, estimates or uncertainties involved in Bayesian inferences often requires numerical methods to avoid high-dimensional integrations. Markov chain Monte Carlo (MCMC) sampling is such a method—powerful, flexible and widely applied. Here, a concise introduction is given, illustrated by a simple, typical example from metrology.
The Metropolis–Hastings algorithm is the most basic and yet flexible MCMC method. Its underlying concepts are explained and the algorithm is given step by step. The few lines of software code required for its implementation invite interested readers to get started. Diagnostics to evaluate the performance and common algorithmic choices are illustrated to calibrate the Metropolis–Hastings algorithm for efficiency. Routine application of MCMC algorithms may be hindered currently by the difficulty to assess the convergence of MCMC output and thus to assure the validity of results. An example points to the importance of convergence and initiates discussion about advantages as well as areas of research. Available software tools are mentioned throughout.
Efficient tetrahydrocannabinol (Δ9-THC) production from cannabis is important for its medical application and as basis for the development of production routes of other drugs from plants. This work presents one of the steps of Δ9-THC production from cannabis plant material, the decarboxylation reaction, transforming the Δ9-THC-acid naturally present in the plant into the psychoactive Δ9-THC. Results of experiments showed pseudo-first order reaction kinetics, with an activation barrier of 85kJmol−1 and a pre-exponential factor of 3.7×108s−1.Using molecular modeling, two options were identified for an acid catalyzed β-keto acid type mechanism for the decarboxylation of Δ9-THC-acid. Each of these mechanisms might play a role, depending on the actual process conditions. Formic acid proved to be a good model for a catalyst of such a reaction. Also, the computational idea of catalysis by water to catalysis by an acid, put forward by Li and Brill, and Churchev and Belbruno was extended, and a new direct keto-enol route was found. 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. The presented approach is important for the development of a sustainable production of Δ9-THC from the plant.
The time course of cannabinoid accumulation in the leaves of individual plants of three Cannabis accessions was determined by gas-chromatographic analysis in greenhouse-grown plants. The total amounts and the concentration
ratios of CBD, THC and CBG were determined; two accessions (an experimental hybrid, (21R×15R)×NL, and plants from a seized
seed lot) were found chemotypically uniform, with all plants belonging to chemotpe II (mixed) and I (high THC) respectively.
The Carmagnola accession showed chemotypic heterogeneity, with a majority of plants belonging to chemotype III. The CBD/THC
and CBG/CBD ratios were shown to be largely constant in the leaves, since 28 and until 103days after sowing, and consistent
with the ratios determined on mature inflorescences. CBD and THC maximum amounts in the leaves showed a peak in the leaves
around 80days from sowing, and were shown to be simultaneous during the growth period, irrespective of the chemotypes. Callus
cultures were obtained from all the five different chemotypes (I, II, III, IV, V), and GC analyses were performed. Independently
of the type and amount of cannabinoids in the mother plants, it was confirmed that callus cultures of Cannabis were not able to produce detectable amounts of any cannabinoids.
Cannabinoids from three samples of cannabis obtained from the Pitt-Rivers Museum, Oxford, and dating from the turn of the century were examined by gas chromatography and mass spectometry for the presence of cannabinoids. Although the samples were from different geographical locations, the profiles of constituent cannabinoids were similar. In common with other aged material, most of the cannabinoid content was present as cannabinol (CBN), the main chemical degradation product of the major psychoactive constituent, delta-9-tetrahydrocannabinol (delta-9-THC). However, a substantial concentration of CBN acid-A was also present; this compound is unstable to heat and readily undergoes decarboxylation to CBN. Methyl and propyl homologues of CBN, together with delta-9-THC and its naturally occurring acid-A were also found at low concentrations in all samples. Intermediates in the formation of CBN from delta-9-THC, previously identified in aged solutions of the drug, were absent or present in only trace concentrations. However, oxidation products involving hydroxylation at the benzylic positions, C-11 and C-1', not seen in solution, were identified in substantial abundance. The results suggest that decomposition of cannabis samples may proceed more slowly than originally thought.
The aim of this study was to investigate the role of time and different real-life storage conditions on the composition of different varieties of cannabis products (hashish and marijuana). Six high-potency cannabis products constituted by herbal and resin materials containing different initial concentrations of delta 9-Tetrahydrocannabinol (THC) were employed for this study. Four representative samples were collected from each study material and were maintained for a prolonged time (four years) under different controlled storage conditions: (A) light (24 h) and room temperature (22 °C); (B) darkness (24 h) and room temperature; (C) darkness and refrigeration (4 °C); (D) darkness and freezing (−20 °C). The concentration of the three main cannabinoids, i.e. THC, Cannabinol (CBN, produced from the degradation of THC), and Cannabidiol (CBD), were measured by GC-FID around every 100 days along the four-year study. Significant changes in the THC (degradation) and CBN (formation) content were detected under storage conditions A and B, and almost 100% of THC was degraded after four years. A mono-exponential function was able to well fit both THC degradation and CBN formation, suggesting that these processes occur with a first order kinetics. Data treatment indicated that the storage temperature and light exposure had two different effects on the conversion of THC to CBN: temperature changed only the speed, light changed both the speed and the stoichiometry of this conversion. Models were proposed which allow to predict the storage time, if unknown, and the initial content of THC (i.e. the concentration of THC at the starting storage time), from the measurement of THC and CBN content at any time under storage condition A. Values predicted are more uncertain at larger storage times and have an accuracy of around 5-10%. These models were also tested on data reported in the literature, and can represent a starting point for further improvements. Prediction models may be helpful for forensic purposes, if the initial concentration of THC or the approximate age of a degraded material need to be estimated, or to plan the storage of delicate samples which need to be re-examined over time.
Hemp seed oil from Cannabis sativa L. is a very rich natural source of important nutrients, not only polyunsaturated fatty acids and proteins, but also terpenes and cannabinoids, which contribute to the overall beneficial effects of the oil. Hence, it is important to have an analytical method for the determination of these components in commercial samples. At the same time, it is also important to assess the safety of the product in terms of amount of any psychoactive cannabinoid present therein. This work presents the development and validation of a highly sensitive, selective and rapid HPLC-UV method for the qualitative and quantitative determination of the main cannabinoids, namely cannabidiolic acid (CBDA), tetrahydrocannabinolic acid (THCA), cannabidiol (CBD), tetrahydrocannabinol (THC), cannabinol (CBN), cannabigerol (CBG) and cannabidivarin (CBDV), present in 13 commercial hemp seed oils. Moreover, since decomposition of cannabinoid acids generally occurs with light, air and heat, decarboxylation studies of the most abundant acid (CBDA) were carried out in both open and closed reactor and the kinetics parameters were evaluated at different temperatures in order to evaluate the stability of hemp seed oil in different storage conditions.
The concentration of Δ9-tetrahydrocannabinol (THC) and cannabinol (CBN) in cannabis plant material (marijuana) of different varieties stored at room temperature (20-22°Celsius (C)) over a four-year period was determined. The percentage loss of THC was proportional to the storage time. On average, the concentration of THC in the plant material decreased by 16.6% ±7.4 of its original value after one year and 26.8% ±7.3, 34.5% ±7.6 and 41.4% ±6.5 after two, three and four years, respectively. A relationship between the concentration ratio of CBN to THC and the storage time was developed and could serve as a guide in determining the approximate age of a given marijuana sample stored at room temperature.
Decarboxylation of cannabidiolic and tetrahydrocannabinolic acids was studied in open reactors in order to investigate the accuracy and reliability of the decarboxylation sample preparation process applied prior to indirect methods, which has been widely used for the determination of cannabinoid acids. The rate of the decarboxylation reaction was followed by the high-performance liquid chromatographic determination of the neutral cannabinoids formed. The effects of different parameters (temperature, solvents, sorbent phases, salts) on decarboxylation were investigated. Reliable results could only be obtained by the mathematical correction of data obtained from experiments in an open reactor.
The Cannabis plant (Cannabis sativa L.) has a long history as a recreational drug, but also as part of traditional medicine in many cultures. Based on the number of publications, it is one of the best-studied plants in the world. The relatively recent discovery of cannabinoid receptors and the human endocannabinoid system has opened up a new and exciting field of research. But despite the pharmaceutical potential of Cannabis, its classification as a narcotic drug has prevented its successful development into modern medicine.
Fortunately, the chemistry of Cannabis has been studied in much detail. In particular the psychoactive cannabinoid tetrahydrocannabinol (THC) has received great scientific attention, and much is known about its biological effects and mechanisms of action. Besides an extensive description of the chemistry of the cannabinoids, this chapter also introduces the lesser-known terpenoids, flavonoids, and other constituents of the Cannabis plant. Comprehensive information on a variety of subjects is presented, including chromatographic analytical methods, pharmacokinetics, and structure-activity relationships. The known biological effects of Cannabis constituents are discussed in relationship to the development of modern cannabinoid-based medications. Finally, some practical aspects of working with Cannabis are discussed.
Solutions of pure cannabinoids, nine samples of herbal and two of resin cannabis (one freshly prepared) were stored in varying conditions for up to 2 years. Exposure to light (not direct sunlight) was shown to be the greatest single factos in loss of cannabinoids especially in solutions, which should therefore be protected from light during analytical and phytochemical operations. Previous claims that solutions in ethanol were stable have not been substantiated. The effect of temperature, up to 20 degrees, was insignificant but air oxidation did lead to significant losses. These could be reduced if care was taken to minimize damage to the glands which act as "well filled, well closed containers". Loss of tetrahydrocannabinol after exposure to light does not lead to an increase in cannabinol, but air oxidation in the dark does. It is concluded that carefully prepared herbal or resin cannabis or extracts are reasonably stable for 1 to 2 years if stored in the dark at room temperature.
A simple procedure based on a common silica gel column chromatography for the isolation of Delta9-tetrahydrocannabinolic acid A (Delta9-THCA-A) from hemp in a multi-milligram scale is presented. Further, the decarboxylation reaction of Delta9-THCA-A to the toxicologically active Delta9-tetrahydrocannabinol (Delta9-THC) at different analytical and under-smoking conditions is investigated. Maximal conversion in an optimised analytical equipment yields about 70% Delta9-THC. In the simulation of the smoking process, only about 30 % of the spiked substance could be recovered as Delta9-THC.
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