Determination of Cannabinoids in Cannabis sativa Oil and Infused Ice Cream by LC-DAD Method.

BACKGROUND: Cannabis sativa is known to produce a class of terpenophenolic compounds named cannabinoids. The two main ones are cannabidiol (CBD) and tetrahydrocannabinol (THC), which have therapeutic properties. In the development of cannabis-based preparations, it is important to have suitable analytical methods for the analysis of the principal cannabinoids. OBJECTIVE: This study aimed to develop and validate a simple and rapid HPLC method with photodiode array detection for determination of CBD and THC in Cannabis sativa oil extract and infused ice cream, including a stability study. METHOD: Chromatographic separation of CBD and THC was performed with a C18 column, with a mobile phase consisting of acetonitrile and water with formic acid (80 + 20 v/v) in isocratic elution mode, with detection at 208 nm for CBD and 280 nm for THC and 1.0 mL/min flow rate. RESULTS: The method was linear over a range of 1-5 µg/mL for CBD, and 20-100 µg/mL for THC; the relative standard deviation was <3.6%, the recovery ranged between 98.8 and 102.5% for oil and between 84 and 94% for ice cream, QL was 0.33 µg/mL for CBD and 2.30 µg/mL for THC, and the assay demonstrated adequate selectivity. CBD and THC were stable for at least 28 days under light protection at 22°C, 4°C, and -20°C in the oil and for at least 60 days at -20°C in the ice cream. CONCLUSIONS: The results showed that the method was suitable for quantitative determination of CBD and THC in Cannabis sativa oil extract and infused ice cream, and it is useful for quality control purposes. HIGHLIGHTS: The method is simple and fast, and it is useful for the quality control of a new product corresponding to an ice cream based on a Cannabis sativa oil extract.

Rapid electrochemical lateral flow device for the detection of Δ9-tetrahydrocannabinol.

Cannabis is a plant that is harmful and beneficial because it contains more than 400 bioactive compounds, and the main compounds are Δ9 tetrahydrocannabinol (THC) and cannabidiol (CBD). Currently, cannabis extracts are used in medicine, but the amount of THC as a main psychoactive component is strictly regulated. Therefore, the ability to rapidly and accurately detect THC is important. Herein, we developed a sensitive electrochemical method combining a rapid lateral flow assay (LFA) to detect THC rapidly. An electrochemical LFA device was constructed by attaching a screen-printed electrode inside a lateral-flow device to exploit the remarkable binding of THC to the cannabinoid type 2 (CB2) receptor in the test zone. The ferrocene carboxylic acid attached to the monoclonal THC antibody acts as an electroactive species when it binds to the THC in the sample before it flows continuously to the CB2 receptor region on the electrode. Under optimal conditions, the detection time is within 6 min and the devise shows excellent performance with a detection limit of 1.30 ng/mL. Additionally, the device could be applied to detect THC in hemp extract samples. The results obtained from this sensor are similar to the standard method (HPLC) for detecting THC. Therefore, this proposed device is useful as an alternative device for the on-site determination of THC because it is inexpensive, portable, and exhibits high sensitivity.

The Anti-Inflammatory Effects of Cannabis sativa Extracts on LPS-Induced Cytokines Release in Human Macrophages.

Inflammation is the response of the innate immune system to any type of injury. Although acute inflammation is critical for survival, dysregulation of the innate immune response leads to chronic inflammation. Many synthetic anti-inflammatory drugs have side effects, and thus, natural anti-inflammatory compounds are still needed. Cannabis sativa L. may provide a good source of anti-inflammatory molecules. Here, we tested the anti-inflammatory properties of cannabis extracts and pure cannabinoids in lipopolysaccharide (LPS)-induced inflammation in human THP-1 macrophages. We found that pre-treatment with cannabidiol (CBD), delta-9-tetrahydrocannabinol (THC), or extracts containing high levels of CBD or THC reduced the level of induction of various cytokines. The CBD was more efficient than THC, and the extracts were more efficient than pure cannabinoids. Finally, IL-6, IL-10, and MCP-1 cytokines were most sensitive to pre-treatments with CBD and THC, while IL-1ß, IL-8, and TNF-α were less responsive. Thus, our work demonstrates the potential of the use of cannabinoids or/and cannabis extracts for the reduction of inflammation and establishes IL-6 and MCP-1 as the sensitive markers for the analysis of the effect of cannabinoids on inflammation in macrophages.

Determination of phytocannabinoids in cannabis samples by ultrasound-assisted solid-liquid extraction and high-performance liquid chromatography with diode array detector analysis.

The characterisation of cannabis plants, especially the determination of specific phytocannabinoids, has gained enormous importance in the last decade, mainly due to the recent changes in cannabis control in several countries or states. This is particularly relevant for the forensic, medical or recreative industry to have a rapid, inexpensive, and reliable methodology to identify and quantify phytocannabinoids. Furthermore, spiking cannabis products with Δ8-tetrahydrocannabinol (THC) is a contemporary trend that demands improving or replacing current methods to include this cannabinoid. The current study presents an ultrasound-assisted solid-liquid extraction followed by high-performance liquid chromatography with diode array detection (HPLC-DAD) methodology to identify and quantify Δ9-THC, Δ8-THC, cannabidiol, cannabinol, Δ9-tetrahydrocannabinolic acid and cannabidiolic acid in cannabis products. The herbal samples were extracted with ethanol:acetonitrile (50:50, v:v) by ultrasonication using only 50 mg of sample. The plant oils were diluted in ethanol. The optimised procedure allowed ≈100% extraction efficiency of the target cannabinoids. The validation assays showed that the method is linear (R2 > 0.997), selective, sensitive, precise and accurate, with suitable limits of detection (0.125-0.250 µg mL-1) and quantification (0.500 µg mL-1). The method was successfully applied to cannabis samples, demonstrating its suitability for routine analyses. This contribution follows the current demand for fast and straightforward analysis services of this plant and its derivatives, using small amounts of sample. The present study compares very favourably against other works, particularly in regards to the extraction efficiency, speed of the overall procedure, method sensitivity, and ability to monitor Δ8-THC spiked samples using a novel solvent mixture.

Effective isolation of cannabidiol and cannabidiolic acid free of psychotropic phytocannabinoids from hemp extract by fast centrifugal partition chromatography.

Cannabidiol (CBD), together with its precursor cannabidiolic acid (CBDA), is the major phytocannabinoid occurring in most hemp cultivars. To ensure the safe use of these compounds, their effective isolation from hemp extract is required, with special emphasis on the elimination of ∆9-tetrahydrocannabinol (∆9-THC) and ∆9-tetrahydrocannabinolic acid (∆9-THCA-A). In this study, we demonstrate the applicability of fast centrifugal partition chromatography (FCPC) as a challenging format of counter-current preparative chromatography for the isolation of CBD and CBDA free of psychotropic compounds that may occur in Cannabis sativa L. plant extracts. Thirty-eight solvent mixtures were tested to identify a suitable two-phase system for this purpose. Based on the measured partition coefficients (KD) and separation factors (α), the two-phase system consisting of n-heptane:ethyl acetate:ethanol:water (1.5:0.5:1.5:0.5; v:v:v:v) was selected as an optimal solvent mixture. Employing UHPLC-HRMS/MS for target analysis of collected fractions, the elution profiles of 17 most common phytocannabinoids were determined. Under experimental conditions, the purity of isolated CBD and CBDA was 98.9 and 95.1% (w/w), respectively. Neither of ∆9-THC nor of ∆9-THCA-A were present; only trace amounts of other biologically active compounds contained in hemp extract were detected by screening against in-house spectral library using UHPLC-HRMS.

Enantioseparation of chiral phytocannabinoids in medicinal cannabis.

The evaluation of the chiral composition of phytocannabinoids in the cannabis plant is particularly important as the pharmacological effects of the (+) and (-) enantiomers of these compounds are completely different. Chromatographic attempts to assess the presence of the minor (+) enantiomers of the main phytocannabinoids, cannabidiolic acid (CBDA) and trans-Δ9-tetrahydrocannabinolic acid (trans-Δ9-THCA), were carried out on heated plant extracts for the determination of the corresponding decarboxylated species, cannabidiol (CBD) and trans-Δ9-tetrahydrocannabinol (trans-Δ9-THC), respectively. This process produces an altered phytocannabinoid composition with several new and unknown decomposition products. The present work reports for the first time the stereoselective synthesis of the pure (+) enantiomers of the main phytocannabinoids, trans-CBDA, trans-Δ9-THCA, trans-CBD and trans-Δ9-THC, and the development and optimization of an achiral-chiral liquid chromatography method coupled to UV and high-resolution mass spectrometry detection in reversed phase conditions (RP-HPLC-UV-HRMS) for the isolation of the single compounds and evaluation of their actual enantiomeric composition in plant. The isolation of the peaks with the achiral stationary phase ensured the absence of interferences that could potentially co-elute with the analytes of interest in the chiral analysis. The method applied to the Italian medicinal cannabis variety FM2 revealed no trace of the (+) enantiomers for all phytocannabinoids under investigation before and after decarboxylation, thus suggesting that the extraction procedure does not lead to an inversion of configuration.

Characterization of the Antitumor Potential of Extracts of Cannabis sativa Strains with High CBD Content in Human Neuroblastoma.

Cannabis has been used for decades as a palliative therapy in the treatment of cancer. This is because of its beneficial effects on the pain and nausea that patients can experience as a result of chemo/radiotherapy. Tetrahydrocannabinol and cannabidiol are the main compounds present in Cannabis sativa, and both exert their actions through a receptor-mediated mechanism and through a non-receptor-mediated mechanism, which modulates the formation of reactive oxygen species. These oxidative stress conditions might trigger lipidic changes, which would compromise cell membrane stability and viability. In this sense, numerous pieces of evidence describe a potential antitumor effect of cannabinoid compounds in different types of cancer, although controversial results limit their implementation. In order to further investigate the possible mechanism involved in the antitumoral effects of cannabinoids, three extracts isolated from Cannabis sativa strains with high cannabidiol content were analyzed. Cell mortality, cytochrome c oxidase activity and the lipid composition of SH-SY5Y cells were determined in the absence and presence of specific cannabinoid ligands, with and without antioxidant pre-treatment. The cell mortality induced by the extracts in this study appeared to be related to the inhibition of the cytochrome c oxidase activity and to the THC concentration. This effect on cell viability was similar to that observed with the cannabinoid agonist WIN55,212-2. The effect was partially blocked by the selective CB1 antagonist AM281, and the antioxidant α-tocopherol. Moreover, certain membrane lipids were affected by the extracts, which demonstrated the importance of oxidative stress in the potential antitumoral effects of cannabinoids.

Phytocannabinoids - An Overview of the Analytical Methodologies for Detection and Quantification of Therapeutically and Recreationally Relevant Cannabis Compounds.

The legalization of the cultivation of low Δ9-tetrahydrocannabinol (Δ9-THC) and high cannabidiol (CBD) Cannabis Sativa plants is gaining momentum around the world due to increasing demand for CBD-containing products. In many countries where CBD oils, extracts and CBD-infused foods and beverages are being sold in health shops and supermarkets, appropriate testing of these products is a legal requirement. Normally this involves determining the total Δ9-THC and CBD and their precursor tetrahydrocannabinolic acids (THCA) and cannabidiolic acid (CBDA). As our knowledge of the other relevant cannabinoids expands, it is likely so too will the demand for them as additives in many consumer products ensuring a necessity for quantification methods and protocols for their identification. This paper discusses therapeutically relevant cannabinoids found in Cannabis plant, the applicability and efficiency of existing extraction and analytical techniques as well as the legal requirements for these analyses.

Cannabinoid Content in Cannabis Flowers and Homemade Cannabis-Based Products Used for Therapeutic Purposes in Argentina.

Introduction: A recent law (DCTO-2020-883-APN-PTE-Law No. 27,350. Regulation) passed in Argentina put an end to the ban imposed for the last 60 years on cannabis cultivation within the country. The law permits restricted access to cannabis derivatives for medicinal, therapeutic, and palliative use by individuals and communities, allowing self- and community-based cannabis production. This is cause for concern in view of the lack of quality controls for cannabis derivatives. The several varieties of cannabis grown in Argentina have different chemical profiles and are processed in a variety of ways-mostly by alcohol extraction or maceration at different temperatures and for different amounts of times-making the cannabinoid content of these preparations highly variable. Determining the characteristics of home- and community-grown cannabis products will facilitate the implementation of public policies conducive to their safety and improvement. Objective: The aim of this study was to determine the cannabinoid chemotypes used for therapeutic purposes in Argentina and evaluate whether the cannabinoids present in homemade derivatives are comparable to those in commercially available products. Materials and Methods: High performance liquid chromatography with ultraviolet and diode array detector (HPLC/UV-DAD) analysis of 436 samples (oils, resins, and inflorescences) was carried out to determine the identity and concentration of five cannabinoids: tetrahydrocannabinolic acid (THCA), tetrahydrocannabinol (THC), cannabidiolic acid (CBDA), cannabidiol (CBD), and cannabinol (CBN). From three different sources, the samples represent the type of medical cannabis preparations to which patients have access. Results: The results indicate that the medium-to-low cannabinoid concentration in a significant number of homemade oil samples is similar to that found in commercial products. Most of the samples have a THC/CBD ratio >1 or only contain THC. Acidic cannabinoids were detected in homemade preparations, but were not reported in package inserts of commercial products. Conclusions: Our results indicate that despite their considerable variability, homemade preparations as a whole show cannabinoid levels and profiles equivalent to the commercially available products commonly used for medicinal, therapeutic, and palliative purposes in Argentina.

Will tetrahydrocannabinol be formed from cannabidiol in gastric fluid? An in vivo experiment.

Cannabidiol (CBD) products have ascribed an uprising trend for their health-promoting effects worldwide. In contrast to Δ9-tetrahydrocannabinol (THC), CBD exhibits no state of euphoria. Since conversion of CBD into THC in an acidic environment has been reported, it has not been proved whether this degradation will also occur in human gastric fluid. A total of 9 subjects ingested 400 mg CBD as a water-soluble liquid together with lecithin as an emulsifier and ethanol as a solubilizer. Blood samples were taken up to 4 h, and urine samples were submitted up to 48 h. THC, 11-hydroxy-Δ9-THC (THC-OH), 11-nor-9-carboxy-Δ9-THC (THC-COOH), CBD, 7-hydroxy cannabidiol (7-OH-CBD), and 7-carboxy cannabidiol (7-CBD-COOH) were determined in blood and THC-COOH and 7-CBD-COOH in urine by LC-MS/MS. Neither THC, THC-OH, nor THC-COOH were detectable in any serum specimen. Only two urine samples revealed THC-COOH values slightly above the threshold of 10 ng/ml, which could also be caused by trace amounts of THC being present in the CBD liquid. It can be concluded that negative consequences for participants of a drug testing program due to a conversion of CBD into THC in human gastric fluid appear unlikely, especially considering a single intake of dosages of less than 400 mg. Nevertheless, there is a reasonable risk for consumers of CBD products being tested positive for THC or THC metabolites. However, this is probably not caused by CBD cyclization into THC in human gastric fluid but is most likely due to THC being present as an impurity of CBD products.

Cannabidiol in urine is not a proof of CBD consumption-lesson learned from urine sample analysis in routine caseworks.

PURPOSE: Cannabidiol (CBD) has been gaining popularity in recent years. Knowing that CBD products can contain more tetrahydrocannabinol (THC) than expected, interpretation of cannabinoids concentration in urine can be tricky, especially when low amounts of THC and CBD are found. Moreover, interpretation can also be difficult due to interindividual variation in pharmacokinetics. The objective of this work was to take a critical look at the data from our daily practice as a toxicology laboratory. METHODS: We have collected results obtained in a first batch of 1074 urine samples submitted to cannabinoids analysis, and results of cannabinoids content of a second batch of 719 seized materials. RESULTS: CBD was detected in 163 urine specimens (15%). Its concentration was higher than the limit of quantification of 5 ng/mL in 108 samples only (10% of the sampling population). Most of CBD-positive samples were associated with a high THC-COOH concentration (> 500 ng/mL in 63.8% of CBD-positive samples) suggesting only a few CBD consumers in our population. Cannabinoids composition of seized plant materials (drug type at first glance) revealed CBD in 110 of them (15% of the sampling population), with a concentration mostly below 1%. All of the resin samples were CBD positive, and contained more THC compared to flowers. CONCLUSIONS: We can conclude that urine samples from drug-type cannabis users contained a low amount of CBD, what was not described previously. These findings are useful for the interpretation of cannabinoids results in daily practice.

Semiquantitative Screening of THC Analogues by Silica Gel TLC with an Ag(I) Retention Zone and Chromogenic Smartphone Detection.

With the ever-evolving cannabis industry, low-cost and high-throughput analytical methods for cannabinoids are urgently needed. Normally, (potentially) psychoactive cannabinoids, typically represented by Δ9-tetrahydrocannabinol (Δ9-THC), and nonpsychoactive cannabinoids with therapeutic benefits, typically represented by cannabidiol (CBD), are the target analytes. Structurally, the former (tetrahydrocannabinolic acid (THCA), cannabinol (CBN), and THC) have one olefinic double bond and the latter (cannabidiolic acid (CBDA), cannabigerol (CBG), and CBD) have two, which results in different affinities toward Ag(I) ions. Thus, a silica gel thin-layer chromatography (TLC) plate with the lower third impregnated with Ag(I) ions enabled within minutes a digital chromatographic separation of strongly retained CBD analogues and poorly retained THC analogues. The resolution (Rs) between the closest two spots from the two groups was 4.7, which is almost 8 times higher than the resolution on unmodified TLC. After applying Fast Blue BB as a chromogenic reagent, smartphone-based color analysis enabled semiquantification of the total percentage of THC analogues (with a limit of detection (LOD) of 11 ng for THC, 54 ng for CBN, and 50 ng for THCA when the loaded volume is 1.0 µL). The method was validated by analyzing mixed cannabis extracts and cannabis extracts. The results correlated with those of high-performance liquid chromatography with ultraviolet detection (HPLC-UV) (R2 = 0.97), but the TLC approach had the advantages of multi-minute analysis time, high throughput, low solvent consumption, portability, and ease of interpretation. In a desiccator, Ag(I)-TLC plates can be stored for at least 3 months. Therefore, this method would allow rapid distinction between high and low THC varieties of cannabis, with the potential for on-site applicability.

Rapid quantification of cannabidiol from oils by direct analysis in real time mass spectrometry.

This work is the first to describe the use of Direct Analysis in Real Time Mass Spectrometry (DART-MS) for the rapid quantification of cannabidiol (CBD) in CBD oils. For this study, self-prepared samples spiked with CBD in hemp seed oil as well as commercial CBD oils from the Austrian market with different CBD contents were analyzed. CBD concentrations were between 5 and 30% (m/m) for the spiked samples as well as between 5 and 15% (m/m) for the real samples. The performance of quantification by means of DART-MS was assessed against a validated liquid chromatography-mass spectrometry (LC-MS) method. The correlation of the quantification results of both methods was high with a correlation factor greater than 0.98 and a maximum bias of 9.8%. Furthermore, the relative standard deviation values of the DART-MS measurments were below the tolerable limit of 12%. These results demonstrate that quantification of CBD by DART-MS is reliable and hence suitable as a rapid and cost-effective alternative method for quality control of CBD content in CBD oils.

Application of experimental design in HPLC method optimisation for the simultaneous determination of multiple bioactive cannabinoids.

The scientific interest in Cannabis sativa L. analysis has been rapidly increasing in recent years, especially for what concerns cannabinoids, plant secondary metabolites which are well known for having many biological properties. High-performance liquid chromatography (HPLC) is frequently used for both the qualitative and quantitative analysis of cannabinoids in plant extracts from C. sativa and its derived products. Many studies have been focused on the main cannabinoids, such as ∆9-tetrahydrocannabinolic acid (∆9-THCA), cannabidiolic acid (CBDA), cannabigerolic acid (CBGA) and their decarboxylated derivatives, such as ∆9-tetrahydrocannabinol (∆9-THC), cannabidiol (CBD) and cannabigerol (CBG). In addition to the abovementioned compounds, the plant produces other metabolites of the same chemical class, and some of them have shown interesting biological activities. In the light of this, it is important to have efficient analytical methods for the simultaneous separation of cannabinoids, which is quite complex since they present similar chemical-physical characteristics. The present work is focused on the use of the Design of Experiments technique (DoE) to develop and optimise an HPLC method for the simultaneous separation of 14 cannabinoids. Experimental design optimisation was applied by using a Central Composite Face-Centered design to achieve the best resolution with minimum experimental trials. Five significant variables affecting the chromatographic separation, including ammonium formate concentration, gradient elution, run time and flow rate, were studied. A multivariate strategy, based on Principal Component Analysis (PCA) and Partial Least Squared (PLS) regression, was used to define the best operative conditions. The developed method allowed for the separation of 12 out of 14 cannabinoids. Due to co-elution phenomena, HPLC coupled with a triple quadrupole mass analyser (HPLC-ESI-MS/MS) was applied, monitoring the specific transitions of each compound in the multiple reaction monitoring (MRM) mode. Finally, the optimised method was applied to C. sativa extracts having a different cannabinoid profile to demonstrate its efficiency to real samples. The methodology applied in this study can be useful for the separation of other cannabinoid mixtures, by means of appropriate optimisation of the experimental conditions.

Analysis of Anti-Cancer and Anti-Inflammatory Properties of 25 High-THC Cannabis Extracts.

Cannabis sativa is one of the oldest cultivated plants. Many of the medicinal properties of cannabis are known, although very few cannabis-based formulations became prescribed drugs. Previous research demonstrated that cannabis varieties are very different in their medicinal properties, likely due to the entourage effect-the synergistic or antagonistic effect of various cannabinoids and terpenes. In this work, we analyzed 25 cannabis extracts containing high levels of delta-9-tetrahydrocannabinol (THC). We used HCC1806 squamous cell carcinoma and demonstrated various degrees of efficiency of the tested extracts, from 66% to 92% of growth inhibition of cancer cells. Inflammation was tested by induction of inflammation with TNF-α/IFN-γ in WI38 human lung fibroblasts. The efficiency of the extracts was tested by analyzing the expression of COX2 and IL6; while some extracts aggravated inflammation by increasing the expression of COX2/IL6 by 2-fold, other extracts decreased inflammation, reducing expression of cytokines by over 5-fold. We next analyzed the level of THC, CBD, CBG and CBN and twenty major terpenes and performed clustering and association analysis between the chemical composition of the extracts and their efficiency in inhibiting cancer growth and curbing inflammation. A positive correlation was found between the presence of terpinene (pval = 0.002) and anti-cancer property; eucalyptol came second, with pval of 0.094. p-cymene and ß-myrcene positively correlated with the inhibition of IL6 expression, while camphor correlated negatively. No significant correlation was found for COX2. We then performed a correlation analysis between cannabinoids and terpenes and found a positive correlation for the following pairs: α-pinene vs. CBD, p-cymene vs. CBGA, terpenolene vs. CBGA and isopulegol vs. CBGA. Our work, thus, showed that most of high-THC extracts demonstrate anti-cancer activity, while only certain selected extracts showed anti-inflammatory activity. Presence of certain terpenes, such as terpinene, eucalyptol, cymene, myrcene and camphor, appear to have modulating effects on the activity of cannabinoids.

Rapid quantification of cannabinoids in beef tissues and bodily fluids using direct-delivery electrospray ionization mass spectrometry.

Hempseed cake is a byproduct of hempseed oil extraction and is potentially a useful source of protein and fiber for use in ruminant diets. However, data are lacking on the appearance and/or clearance of cannabinoids in tissues of animals fed hempseed cake. To this end, a rapid method for quantifying cannabinol (CBN), cannabidiol (CBD), cannabinolic acid (CBNA), cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabichromenic acid (CBCA), cannabidivarin (CBDV), cannabidivarinic acid (CBDVA), tetrahydrocannabinol (THC) and tetrahydrocannabinolic acid (THCA) in cattle tissues, plasma, and urine was developed using rapid screen electrospray ionization mass spectrometry (RS-ESI-MS). Regression coefficients of matrix-matched standard curves ranged from 0.9946 to >0.9999 and analyte recoveries averaged from 90.2 ± 15.5 to 108.7 ± 18.7% across all compounds. Limits of detection and quantification ranged from 0.05 to 2.79 ng · mL-1 and 0.17 to 9.30 ng · mL-1, respectively, while the inter-day relative standard deviation ranged from 5.1 to 15.1%. Rapid screening electrospray ionization mass spectrometry (RS-ESI-MS) returned no false positives for any cannabinoid in plasma, urine, and tissue (liver, skeletal muscle) samples from 6 non-dosed control animals (n = 90 samples; of which 72 samples were plasma or urine and 18 samples were tissues). Across-animal cannabinoid concentrations measured in 32 plasma samples of cattle dosed with ground hemp were quantified by RS-ESI-MS; analytical results correlated well (r2 = 0.963) with independent LC-MS/MS analysis of the same samples.

DoE-assisted development and validation of a stability-indicating HPLC-DAD method for simultaneous determination of five cannabinoids in Cannabis sativa L. based on analytical quality by design (AQbD) concept.

INTRODUCTION: Medical uses of Cannabis sativa L. have gained interest in recent decades, which highlights the need for defining appropriate quality specifications for Cannabis-based products. However, the complexity of plant matrices and structural similarity between cannabinoids make analytical development a challenging task. Thus, the application of analytical quality by design (AQbD)-driven approaches can favour the development of fit-for-purpose methods. OBJECTIVES: To develop a high-performance liquid chromatography diode array detector (HPLC-DAD) method for simultaneous quantification of cannabidiol, Δ9 -tetrahydrocannabinol, cannabidiolic acid, tetrahydrocannabinolic acid, and cannabinol in C. sativa by applying an AQbD-driven approach. MATERIALS AND METHODS: Critical method attributes (CMA) were established following the analytical target profile. Critical method variables (CMV) were categorised based on risk assessment and literature review. Selected CMV regarding sample preparation and chromatographic conditions were optimised using response surface methodology (RSM). The working point was estimated by multiple response optimisation using Deringer's desirability function. The validity of the optimal conditions was confirmed experimentally. Method validation was performed according to ANVISA and ICH guidelines. Relative response factors (RRFs) were also determined. RESULTS AND DISCUSSION: Baseline resolution of 12 major cannabinoids was achieved in a 35 min chromatographic analysis. All experimental responses obtained during confirmatory analyses were within the prediction intervals (PI95% ). Method's selectivity, linearity (10-100 µg/mL), precision, bias, extraction recovery, and ruggedness were satisfactorily demonstrated. CONCLUSIONS: The application of an AQbD-driven approach allowed for a better understanding of the effects of the ensemble of CMV on the analyte's behaviour, enabling the definition of appropriate conditions to ensure consistent achievement of the intended method's performance.

Direct Quantitation of Phytocannabinoids by One-Dimensional 1H qNMR and Two-Dimensional 1H-1H COSY qNMR in Complex Natural Mixtures.

The widespread use of phytocannabinoids or cannabis extracts as ingredients in numerous types of products, in combination with the legal restrictions on THC content, has created a need for the development of new, rapid, and universal analytical methods for their quantitation that ideally could be applied without separation and standards. Based on previously described qNMR studies, we developed an expanded 1H qNMR method and a novel 2D-COSY qNMR method for the rapid quantitation of ten major phytocannabinoids in cannabis plant extracts and cannabis-based products. The 1H qNMR method was successfully developed for the quantitation of cannabidiol (CBD), cannabidiolic acid (CBDA), cannabinol (CBN), cannabichromene (CBC), cannabichromenic acid (CBCA), cannabigerol (CBG), cannabigerolic acid (CBGA), Δ9-tetrahydrocannabinol (Δ9-THC), Δ9-tetrahydrocannabinolic acid (Δ9-THCA), Δ8-tetrahydrocannabinol (Δ8-THC), cannabielsoin (CBE), and cannabidivarin (CBDV). Moreover, cannabidivarinic acid (CBDVA) and Δ9-tetrahydrocannabivarinic acid (Δ9-THCVA) can be distinguished from CBDA and Δ9-THCA respectively, while cannabigerovarin (CBGV) and Δ8-tetrahydrocannabivarin (Δ8-THCV) present the same 1H-spectra as CBG and Δ8-THC, respectively. The COSY qNMR method was applied for the quantitation of CBD, CBDA, CBN, CBG/CBGA, and THC/THCA. The two methods were applied for the analysis of hemp plants; cannabis extracts; edible cannabis medium-chain triglycerides (MCT); and hemp seed oils and cosmetic products with cannabinoids. The 1H-NMR method does not require the use of reference compounds, and it requires only a short time for analysis. However, complex extracts in 1H-NMR may have a lot of signals, and quantitation with this method is often hampered by peak overlap, with 2D NMR providing a solution to this obstacle. The most important advantage of the COSY NMR quantitation method was the determination of the legality of cannabis plants, extracts, and edible oils based on their THC/THCA content, particularly in the cases of some samples for which the determination of THC/THCA content by 1H qNMR was not feasible.

[Cultivation and breeding of industrial Cannabis sativa].

Cannabis sativa,with a long history of cultivation, is a traditional industrial crop widely used for food, textiles, and me-dicine. This study discussed industrial C. sativa and medicinal C. sativa. According to the characteristics of management policies of C. sativa in different periods, we divided the development stages of C. sativa into three stages and analyzed the changes in breeding and cultivation goals under the influence of policies. Meanwhile, a comprehensive analysis was carried out based on the breeding conditions of industrial C. sativa in China. Because of the vast territory of China, the differences in agricultural planting environment, economic development, and social development in the southern and northern areas result in different used parts of C. sativa. To be speci-fic, flowers and leaves are used in Yunnan, fiber in Heilongjiang, and seeds in Shanxi. The breeding of C. sativa varieties highlights fiber, seeds, or both of them. As the value of cannabidiol is explored, medicinal C. sativa has been approved in recent years. Based on the cultivation characteristics and value of industrial C. sativa, it is proposed that industrial C. sativa has a broad application prospect as an important industrial crop, and the existing products contain almost no tetrahydrocannabinol. The cultivation of C. sativa should be rationally guided to promote the development of the C. sativa industry. Moreover, it is recommended to actively apply advanced breeding techniques such as molecular breeding to overcome the problems of the uncertainty of the existing induced breeding and the excessively long hybrid breeding cycle, and develop high value-added applications such as medicinal products of C. sativa to enhance the exploitation of the economic value of C. sativa.

LC-MS/MS Assay for the Measurement of Cannabidiol Profiling in CBD Oil from Japanese Market and Application for Convertible Tetrahydrocannabinol in Acetic Acid Condition.

Cannabidiol (CBD), a major non-psychoactive cannabinoid, has a lot of attention due to its potential relaxing properties and led the trend in commercial CBD aroma/oral hemp seed oil from the Japanese market. In this study, a routine assay for evaluating CBD oil samples was performed using LC coupled with tandem mass spectrometry (LC-MS/MS) and was used to apply the convertible tetrahydrocannabinol (THC) in acetic acid conditions. Based on the electrospray positive ion mode, the detection of cannabidiolic acid (CBDA; m/z 359 > 219), cannabigerolic acid (CBGA; m/z 361 > 343), cannabigerol (CBG; m/z 317 > 193), CBD (m/z 315 > 193), THC (m/z 315 > 193) and cannabinol (CBN; m/z 311 > 223) was performed by satisfying separation with high density of C18 column. Oil samples (50 mg) were diluted with isopropanol (5 mL), to which stable isotope internal standards were added by dilution with methanol/water (50/50), and accuracy rates ranged from 97.8 to 102.2%. This method was used to evaluate the CBD oil products (5 kinds) from the Japanese market. Our survey found obvious counterfeit (non-detectable CBD) CBD oil from Japanese market. Following that, we investigated the conversion of THC in CBD oil samples in simple conditions such as 10% acetic acid and 70 °C for 6 h and discovered that converts THC proportions are approximately 5% ((THC content/CBD content) × 100) and <1.0%. Thus, our developed LC-MS/MS assay could be applied to monitor the CBD concentration and convertible THC from CBD oil.