Development and validation of an HPLC-DAD method for the quantification of cannabigerol, cannabidiol, cannabinol and cannabichromene in human plasma and mouse matrices.

Cannabigerol, cannabidiol, cannabinol and cannabichromene are non-psychoactive phytocannabinoids, highly present in Cannabis sativa, for which numerous therapeutical applications have been described. However, additional pre-clinical and clinical data, including toxicopharmacokinetic and pharmacodynamic studies, remain required to support their use in clinical practice and new therapeutic applications. To support these studies, a new high performance liquid chromatography technique (HPLC) with diode-array detection (DAD) was developed and validated to quantify these cannabinoids in human plasma and mouse matrices. Sample extraction was accomplished by protein precipitation and double liquid-liquid extraction. Simvastatin and perampanel were used as internal standards in human and mouse matrices, respectively. Chromatographic separation was achieved in 16 min on an InfinityLab Poroshell® 120 C18 column (4.6 mm × 100 mm, 2.7 µm) at 40 °C. A mobile phase composed of water/acetonitrile was pumped with a gradient elution program at 1.0 mL min-1. The technique revealed linearity in the defined concentration ranges with a determination coefficient of over 0.99. Intra and inter-day accuracy and precision values ranged from -14.83 to 13.97% and 1.08 to 13.74%, respectively. Sample stability was assessed to ensure that handling and storage conditions did not compromise analyte concentrations in different matrices. Carry-over was absent and recoveries were over 77.31%. This technique was successfully applied for the therapeutic monitoring of cannabidiol and preliminary pre-clinical studies with cannabigerol and cannabidiol. All samples were within calibration ranges, with the exception of cannabigerol after intraperitoneal administration. This is the first HPLC-DAD technique that simultaneously quantifies cannabinoids in these biological matrices, supporting future pre-clinical and clinical investigations.

Sustainable cannabinoids purification through twin-column recycling chromatography and green solvents.

In the present study, twin-column recycling chromatography has been employed for the purification of a Cannabis extract by using a green solvent, ethanol, as the mobile phase. In particular, the complete removal of the psychoactive tetrahydrocannabinol (THC) from a Cannabis extract rich in cannabidiol (CBD) was achieved under continuous conditions. The performance of the method, in terms of compound purity, recovery, productivity and solvent consumption, was compared to that of traditional batch operations showing the potential of the twin-column recycling approach. The employment of a theoretical model to predict the band profiles of the two compounds during the recycling process has facilitated method development, thus further contributing to process sustainability by avoiding trial and error attempts or at least decreasing the number of steps significantly.

Differentiation of Δ9-THC and CBD Using Silver-Ligand Ion Complexation and Electrospray Ionization Tandem Mass Spectrometry (ESI-MS/MS).

The 2018 Farm Bill defines marijuana as Cannabis sativa L. or any derivative thereof that contains greater than 0.3% Δ9-tetrahydrocannabinol (Δ9-THC) on a dry weight basis. The main cannabinoids present in Cannabis sativa L., Δ9-THC and cannabidiol (CBD), are structural isomers that cannot be differentiated using direct mass spectrometry with soft ionization techniques alone. Due to the classification of marijuana as a Schedule I controlled substance, the differentiation of Δ9-THC and CBD is crucial within the seized drug community. This study explores the use of Ag-ligand ion complexation and electrospray ionization tandem mass spectrometry (ESI-MS/MS) for the differentiation of Δ9-THC and CBD using six different Ag complexes. Differences between the binding affinities of Δ9-THC and CBD for [Ag(PPh3)(OTf)]2 lead to the formation of unique product ions at m/z 421/423, m/z 353/355, and m/z 231 for CBD, enabling the differentiation of CBD from Δ9-THC. When applied to the analysis of known Δ9-THC:CBD mixture ratios, the developed [Ag(PPh3)(OTf)]2 ion complexation method was able to differentiate Δ9-THC-rich and CBD-rich samples based on the average abundance of the product ions at m/z 421/423. The developed approach was then applied to methanolic extracts of 20 authentic cannabis samples with known Δ9-THC and CBD compositions, resulting in a 95% correct classification rate. Even though the developed Ag-ligand ion complexation method was only demonstrated for the qualitative differentiation of Δ9-THC-rich and CBD-rich cannabis, this study establishes a foundation for the use of Ag-ligand ion complexation that is essential for future quantitative approaches.

Analysis of homemade cannabis edibles by UHPLC-HRMS after standard addition method.

With recent evolution of cannabis legalization around the world, cannabis edibles are booming, and determining their concentration in Δ9-tetrahydrocannabinol (Δ9-THC), the regulated psychoactive substance, remains a challenge for toxicology laboratories, which must prove whether the product has legal status or not. Cannabinoids are a large family of structurally similar and lipophilic molecules, requiring dedicated pre-analytical methods, as well as efficient chromatographic separation to differentiate cannabinoid isomers which are distinguished by their psychoactive properties and their legal status. Here, we present two independent cases of cannabis edibles, for which we performed analysis of homemade cannabis chocolate cakes and of the resins and herbs used for cooking. Quantitation was carried out with a new developed standard addition method, to avoid matrix effects and matrix-dependent calibration. Extraction by QuEChERs method, followed by targeted and non-targeted analysis by ultra-high performance liquid chromatography hyphenated to high resolution mass spectrometry (UHPLC-HRMS) allowed the identification of several phytocannabinoids, mainly Δ9-tetrahydrocannabinol (Δ9-THC), cannabidiol (CBD) and their acid precursors Δ9-THC acid (THCA) and CBD acid (CBDA). Δ9-THC was identified in significant concentrations (mg/g) in both edibles, even though one was prepared with CBD herb. This work highlights the need to analyze cannabis edibles, as well as the resins and herbs used in their preparation if it is homemade, and it proposes a reliable analytical method for toxicology laboratories.

Complexity of Translating Analytics to Recent Cannabis Use and Impairment.

While current analytical methodologies can readily identify cannabis use, definitively establishing recent use within the impairment window has proven to be far more complex, requiring a new approach. Recent studies have shown no direct relationship between impairment and Δ9-tetra-hydrocannabinol (Δ9-THC) concentrations in blood or saliva, making legal "per se" Δ9-THC limits scientifically unjustified. Current methods that focus on Δ9-THC and/or metabolite concentrations in blood, saliva, urine, or exhaled breath can lead to false-positive results for recent use due to the persistence of Δ9-THC well outside of the typical 3-4 h window of potential impairment following cannabis inhalation. There is also the issue of impairment due to other intoxicating substances-just because a subject exhibits signs of impairment and cannabis use is detected does not rule out the involvement of other drugs. Compounding the matter is the increasing popularity of hemp-derived cannabidiol (CBD) products following passage of the 2018 Farm Bill, which legalized industrial hemp in the United States. Many of these products contain varying levels of Δ9-THC, which can lead to false-positive tests for cannabis use. Furthermore, hemp-derived CBD is used to synthesize Δ8-THC, which possesses psychoactive properties similar to Δ9-THC and is surrounded by legal controversy. For accuracy, analytical methods must be able to distinguish the various THC isomers, which have identical masses and exhibit immunological cross-reactivity. A new testing approach has been developed based on exhaled breath and blood sampling that incorporates kinetic changes and the presence of key cannabinoids to detect recent cannabis use within the impairment window without the false-positive results seen with other methods. The complexity of determining recent cannabis use that may lead to impairment demands such a comprehensive method so that irresponsible users can be accurately detected without falsely accusing responsible users who may unjustly suffer harsh, life-changing consequences.

Identification and quantification of both isomers of hexahydrocannabinol, (9R)-hexahydrocannabinol and (9S)-hexahydrocannabinol, in three different matrices by mass spectrometry.

CONTEXT: Hexahydrocannabinol (HHC), a compound derived from synthetic production using cannabidiol (CBD) or delta-9-tetrahydrocannabinol (Δ9 -THC), has gained recent attention due to its presence in seized materials across Europe. Sold legally in various forms, HHC poses potential health risks, particularly as a legal alternative to THC in some countries. Despite its historical description in the 1940s, limited toxicology data, pharmacological understanding, and analytical methods for HHC exist. METHOD: This study proposes analytical techniques using mass spectrometry to detect, identify, and quantify (9R)-HHC and (9S)-HHC, concurrently with THC and CBD in various matrices, including oral fluid, whole blood, and seized material. Three distinct methods were employed for different matrices: GC/MS for seized material, GC/MS/MS for whole blood, and UHPLC/MS/MS for oral fluid. Methods were validated qualitatively for oral fluid with a FLOQSwab® device and quantitatively in whole blood and seized material according to Peters et al's recommendations and ICH guidelines. RESULTS: Validated methods were considered reliable in detecting and quantifying HHC isomers in terms of repeatability, reproducibility, and linearity with r2 systematically >0.992. These methods were applied to authentic cases, including seized materials and biological samples from traffic control (whole blood and oral fluid). In seized materials, (9R)-HHC levels ranged from 2.09% to 8.85% and (9R)-HHC/(9S)-HHC ratios varied from 1.36 to 2.68. In whole blood sample, (9R)-HHC and (9S)-HHC concentrations were, respectively, 2.38 and 1.39 ng/mL. For all analyzed samples, cannabinoids such as THC and CBD were also detected. CONCLUSION: This research contributes analytical insights into differentiating and simultaneously analyzing (9R)-HHC and (9S)-HHC, using widely applicable mass spectrometric methods. The study emphasizes the need for vigilance among toxicologists, as new semisynthetic cannabinoids continue to emerge in Europe, with potential health implications. The findings underscore the importance of reliable analytical methods for monitoring these compounds in forensic and clinical settings.

Sequential purification of cannabidiol by two-dimensional liquid chromatography combined with modeling and simulation of elution profiles.

Cannabidiol (CBD) has garnered significant attention for its neuroprotective properties, and research on its therapeutic effects has increased dramatically in recent years. However, the systematic purification of CBD through scalable processes has remained bottleneck due to the structural similarities of the cannabinoids. Although preparative chromatography is considered as a potential solution, it is usually time-consuming and expensive. Therefore, the development of scalable strategy via fast and accurate optimization approach is crucial. The present study aimed to develop a sequential process for the scalable purification of CBD through an eco-friendly ethanolic extraction using ultrasonic assisted extraction, decarboxylation of cannabidiolic acid optimized by response surface methodology, followed by the development of off-line two-dimensional semi-preparative chromatography, boosted with stacked injection overloading. In the first dimension, a column packed with macroporous resin allows to enrich the target substance and then, the behavior of resin column for scale-up procedure were predicted and optimized by developed mathematical model. A C18 column was used in the second dimension. The CBD purity and recovery obtained were 94.3 and 82.1 %, respectively. A robust and reliable method was employed for CBD enrichment/purification, which can be generalized to other bioactive compounds in complex matrices.

Potency trends of cannabis in Jamaica during the period of 2014 to 2020.

Reports suggest that cannabis potency has dramatically increased over the last decade in the USA and Europe. Cannabinoids are the terpeno-phenolic compounds found in the cannabis plant and are responsible for its pharmacological activity. The two most prominent cannabinoids are delta-9-tetrahydrocannabinol (Δ9 THC) and cannabidiol (CBD). Cannabis potency is measured not only by the Δ9 THC levels but also by the ratio of Δ9 THC to other non-psychoactive cannabinoids, namely, CBD. Cannabis use was decriminalized in Jamaica in 2015, which opened the gates for the creation of a regulated medical cannabis industry in the country. To date, there is no information available on the potency of cannabis in Jamaica. In this study, the cannabinoid content of Jamaican-grown cannabis was examined over the period 2014-2020. Two hundred ninety-nine herbal cannabis samples were received from 12 parishes across the island, and the levels of the major cannabinoids were determined using gas chromatography-mass spectrometry. There was a significant increase (p < 0.05) in the median total THC levels of cannabis samples tested between 2014 (1.1%) and 2020 (10.2%). The highest median THC was detected in the central parish of Manchester (21.1%). During the period, THC/CBD ratios increased from 2.1 (2014) to 194.1 (2020), and there was a corresponding increase in the percent freshness of samples (CBN/THC ratios <0.013). The data show that a significant increase in the potency of locally grown cannabis has occurred in Jamaica during the last decade.

Daily associations with cannabis use and sleep quality in anxious cannabis users.

INTRODUCTION: Cannabis is increasingly used to self-treat anxiety and related sleep problems, without clear evidence of either supporting or refuting its anxiolytic or sleep aid effects. In addition, different forms of cannabis and primary cannabinoids ∆9-tetrahydrocannabinol (THC) and cannabidiol (CBD) have differing pharmacological effects. METHODS: Thirty days of daily data on sleep quality and cannabis use were collected in individuals who use cannabis for mild-to-moderate anxiety (n = 347; 36% male, 64% female; mean age = 33 years). Participants self-reported both the form (flower or edible) and the ratio of THC to CBD in the cannabis used during the observation period. RESULTS: Individuals who reported cannabis use on a particular day also reported better sleep quality the following night. Moderation analyses showed that better perceived sleep after cannabis use days was stronger for respondents with higher baseline affective symptoms. Further, respondents who used cannabis edibles with high CBD concentration reported the highest perceived quality of sleep. CONCLUSIONS: Among individuals with affective symptoms, naturalistic use of cannabis was associated with better sleep quality, particularly for those using edible and CBD dominant products.

Fast and sensitive LC-MS/MS method for quantification of cannabinoids and their metabolites in plasma of cattle fed hemp.

Hemp-based materials have gained interest as alternative feed ingredients for livestock. However, safety concerns arise regarding the transfer of cannabinoids from the plant to the animals. Addressing these concerns requires the use of methods capable of detecting and quantifying cannabinoids in livestock. In this study, a fast and sensitive method was developed for quantification of cannabinoids and cannabinoid metabolites in cattle plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The extraction of cannabinoids from the plasma matrix was achieved by combining the Captiva Enhanced Matrix Removal-Lipid clean-up and salting-out assisted liquid-liquid extraction procedure. The developed method underwent validation using various analytical parameters, and the results demonstrated good accuracy, precision, specificity, and high sensitivity. The method was applied to real plasma samples obtained from cattle fed hemp for 2 weeks, and successfully detected various cannabinoids, including delta-9-tetrahydrocannabinol. Furthermore, the study revealed that 7-carboxy cannabidiol, a metabolite of cannabidiol, was the predominant cannabinoid present in the cattle plasma throughout the feeding period, which could remain detectable for weeks after the hemp feeding had ended.

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.

Miniaturized paper-based analytical device for the portable analysis of phyto-cannabinoids in plant and oral fluids.

In this work, a low-cost and eco-friendly paper-based analytical device (PAD) method is described for the determination of phyto-cannabinoids in cannabis and oral fluids based on a simple colorimetric reaction. The PAD was able to distinguish tetrahydrocannabinol (THC)- and cannabidiol (CBD)-rich plant samples by using 4-aminophenol (4-AP) and later on to quantify total phyto-cannabinoid content (THC + CBD + CBN) in plant and oral fluids by using the Fast Corinth V reagent. The chemical and physical properties regarding paper type and reagent concentration in the PAD were optimized to achieve the best analytical performance. After that, analytical features were obtained, including a linear range of 0.01-0.1 mg mL-1, a limit of detection (LOD) of 0.003 mg mL-1, and a suitable precision, expressed as relative standard deviation (RSD) lower than 10%. Furthermore, no significant interferences were observed in colorimetric reactions when tea, herbs, and drug samples were analyzed. Additionally, the PAD proved color stability up to 1 month after the sampling at 25 °C. The developed PAD was suitable for determining total phyto-cannabinoid content in plants and oral fluids, obtaining good results compared to GC-MS. Overall, this method showed good reliability resulting in an operational on-site device for drug monitoring.

Cannabigerol (CBG) signal enhancement in its analysis by gas chromatography coupled with tandem mass spectrometry.

PURPOSE: According to recent reports, cannabigerol (CBG) concentration level in blood and body fluids may have forensic utility as a highly specific albeit insensitive biomarker of recent cannabis smoking. While the analytical sensitivity of cannabidiol (CBD), Δ9-tetrahydrocannabinol (Δ9-THC), cannabichromene (CBC) or cannabinol (CBN) estimation by gas chromatography-mass spectrometry (GC-MS) is similar and sufficiently high, it is exceptionally low in the case of CBG (ca. 25 times lower than for the other mentioned cannabinoids). The purpose of this study is to explain the reasons for the extremely low analytical sensitivity of GC-MS in estimating CBG and to present possible ways of its improvement. METHODS: Nuclear magnetic resonance (NMR) data and GC-MS responses to CBG and its various derivatization and transformation products were studied. RESULTS: The validation data of individual derivatives of CBG and its transformation products were established. CBG silylation/acylation or hydration allows to decrease LOD about 3 times, whereas the formation of pyranic CBG derivative leads to 10-times decrease of LOD. The paper enriches the literature of the subject by providing MS and NMR spectra, not published so far, for derivatives of CBG and its transformation products. The most likely cause of low GC-MS response to CBG is also presented. CONCLUSIONS: The presented results shows that although the signal increase of CBG can be obtained through its derivatization by silylation and/or acylation, the greatest increase is observed in the case of its cyclization to the pyranic CBG form during the sample preparation process. The CBG cyclization procedure is very simple and workable in estimating this cannabinoid in blood/plasma samples.

Ultrasound-assisted dispersive solid-liquid microextraction with eutectic solvents for the determination of cannabinoids in different hemp products.

The wide range of applications of hemp products, together with the environmental benefits that come from hemp cultivation are driving up the market demand for Cannabis sativa L. plant. One of the main restrictions for hemp cultivation and marketing concerns the content of delta-9-tetrahydrocannabidiol (Δ9-THC), which is known to have psychotomimetic effect. If the recent growing of hemp market is beneficial by an economic and environmental point of view, it is necessary to develop reliable analytical methods for the chemical characterization of hemp products, to guarantee the safety of use for the customers. This study aimed to develop a simple ultrasound-assisted dispersive solid-liquid microextraction (UA-DSLME) method for the extraction of cannabinoids in hemp products, using eutectic solvents (ESs) as extraction material. Two types of ESs were compared: one prepared with a [Ch+][Br-]-modified salts as hydrogen bond acceptor and one based on natural terpenoids. The ultrasound-assisted dispersive solid-liquid microextraction method was optimized to be applied for the analysis of aerial parts of hemp collected before flowering, hemp inflorescences and a commercial sample called CBD oil, and proved to be robust and versatile. Under optimal conditions, only 100 µL of ES and 2 mL of water as co-solvent were used in the US-assisted extraction, before the analysis in the UHPLC-PDA system. The developed approach allowed to obtain the same chemical profile of conventional methods, while improving the greenness of the method and the enrichment of the marker analytes. To overcome the strong matrix effect for cannabinoids, a matrix-matched calibration was used. Blank matrices of the samples under study were easily obtained by performing an exhaustive extraction of the marker analytes in the hemp samples. These matrices were successfully used for validation, achieving accuracy values between 82% and 118%.

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.

Confirmation of cannabinoids in forensic toxicology casework by isomer-selective UPLC-MS-MS analysis in urine.

Confirmation of cannabinoid use by forensic toxicology testing in urine has been traditionally focused on ∆9-tetrahydrocannabinol (∆9-THC) with analysis of its major metabolite, 11-nor-9-carboxy-∆9-THC (∆9-cTHC), in free and conjugated forms. Legalization of hemp, however, has led to the widespread production and sale of cannabidiol (CBD) derivatives with psycho-activity, including ∆8-THC and ∆10-THC isomers. The increasing availability and growing use of isomer derivatives necessitate an expanded scope of cannabinoid confirmation test protocols. We report a quantitative, isomer-selective method of cannabinoid confirmation by liquid chromatography-tandem mass spectrometry determination of parent drug isomers (∆8-THC, ∆9-THC, ∆10-THC and CBD) as well as isomeric metabolites (∆8-cTHC and ∆9-cTHC). An efficient C18 phase chromatography on 1.6-µm solid core particles was used with a step gradient for near isocratic separation of both early-eluting THC metabolite isomers and later-eluting CBD and THC isomers. A rapid method of hydrolysis, dilution and analysis was employed for the quantitative co-determination of free and conjugated analytes, using stable isotope internal standardization. Method validation is reported, along with interference assessment from a prior confirmation method. Casework experience with the isomer-selective method revealed a 14% prevalence of ∆8-cTHC positive cases with a pattern of concomitant ∆8-THC and ∆9-THC use. A comparison of ∆8-cTHC and ∆9-cTHC phase two metabolism is also reported.

Potency levels of regulated cannabis products in Michigan 2021-2022.

Evaluation of cannabinoid concentrations in products from the legal cannabis market has been fraught with uncertainty. The lack of standardized testing methodology and the susceptibility of cannabinoids to degradation under certain storage conditions complicates the efforts to assess total tetrahydrocannabinol (THC) levels across wide geographic areas. There are few peer-reviewed surveys of cannabinoid concentrations in regulated products. Those that have been done have not characterized the effects of differences in analytical methodology, sample population, and storage conditions. Viridis Laboratories, which operates two cannabis safety compliance facilities in Michigan, has analyzed over 34,000 cannabis products throughout 2021 and 2022 before the sale in the regulated market. Fifteen cannabinoids in cannabis flower, concentrates, and infused products were tested using methanolic extraction and analysis by high-performance liquid chromatography with diode-array detection. Methods were validated before use, and the flower analysis procedure was certified by the Association of Analytical Collaboration. All the samples were tested before submission for sale and therefore had not undergone prolonged storage. The results are compared with those seen in other states as well as in the illicit market. Total THC levels in cannabis flower from the regulated market are significantly higher than those seen in illicit products. The distribution of cannabinoid levels is similar in flowers intended for either the medicinal or adult-use markets, with an average potency of 18%-23% of total THC. Total THC in concentrates averages up to 82%. Other cannabinoids are observed at significant levels, mostly in products specifically formulated to contain them. These results may act as a benchmark for potency levels in the regulated market.

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.

Quality test of cannabidiol profiling in CBD oil products and evaluation of residual THC at high temperature based on liquid chromatography coupled with tandem mass spectrometry.

Cannabidiol (CBD) oil products are available in Japan as cosmetics, fragrances, food and items. Herein, quality testing of cannabinoid profiling in CBD oil products and the evaluation of possible residual tetrahydrocannabinol (THC) in these products using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was conducted. A simple, sensitive, and selective LC-MS/MS assay (electrospray positive ion mode) was employed for the simultaneous quantification of eight cannabinoids. This quantification with three different oil samples showed that accuracy rates ranged from 87.7 to 106.9% (RSD > 3.5%). Furthermore, the quantification limit of THC is 0.001 mg/g of CBD oil products for suitable levels lower than the regulatory value. Notably, this method was used to evaluate CBD oil products from the Japanese market. Additionally, we investigated the THC conversion in CBD oil products at a high temperature (70°C) which has a minor effect on CBD stability in oil products with additives. Herein, the developed LC-MS/MS assay is applied to monitor the quality of CBD, trace THC and other components in CBD oil products.

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.