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Synthetic cannabinoids (SCs) have emerged as one of the most severely abused categories of new psychoactive substances (NPS), exacerbating the global drug problem and posing significant threats to public health. Presently, a class of new amide-type SCs featuring an indazole or indole core has been identified in numerous cases of illegal drug use, but there is still a lack of comprehensive analysis methods of SC detection. Herein, monoclonal antibodies (mAbs) 2E4 and AE6 targeting 36 indole-type and indazole-type SCs and their metabolites with IC 50 ranging from 0.14 to 85.28 ng/mL were prepared and the molecular mechanism of antibody recognition was elaborated. We established the indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and gold immunochromatography assay (GICA) based on mAbs 2E4 and AE6 to detect indazole-type and indole-type SCs in urine and hair samples. Under optimal conditions, the proposed method detected ADB-BUTINACA (an indazole-type SC) with limits of detection (LODs) of 0.11 ng/mL for urine and 0.024 ng/mg for hair by ic-ELISA, and 1.02 ng/mL for urine and 0.046 ng/mg for hair by GICA; the LODs of 4F-MDMB-BUTICA (an indole-type SC) detection was 0.036 ng/mL for urine and 0.012 ng/mg for hair by ic-ELISA, and 0.54 ng/mL for urine and 0.03 ng/mg for hair by GICA. Collectively, our study provides a comprehensive foundation for the rapid screening and quantitation of SC derivatives in biological samples.

Background: The use of new psychoactive substances (NPS) has emerged as a significant public health concern globally, due to their unknown and unpredictable effects on both physical and mental health. Among them, synthetic cannabinoids receptor agonists (SCRAs) currently stand as the most widely consumed NPS family in Europe. Since the detection of JWH-018 in 2008, the structures of these compounds have evolved to circumvent legislation and/or enhance their effects, consequently increasing the number of reported SCRAs to be monitored. Therefore, new strategies are needed to identify these compounds, whether in seized products or in biological samples., Results: This study presents the development of an open method for detecting SCRAs employing a "pseudo-target" screening approach, a strategy previously developed and used in our laboratory for synthetic cathinones identification. The methodology involves monitoring the main product ions and neutral losses derived from 179 SCRAs of the third and fourth generations, based on their fragmentation pathways. This approach allows for the tentative identification of the SCRAs, supported also by the created database. The versatility of the developed methodology is highlighted, extending its utility beyond seizure products or 'legal highs', to biological samples. In this sense, it has been successfully applied not only to the detection of SCRAs in research chemicals but also in authentic urine from an anonymous SCRAs consumer, through the identification of a metabolite., Significance: This strategy will be particularly useful for the rapid detection of SCRAs in forensic and toxicological laboratories equipped with low-resolution MS/MS instrumentation. This is a valuable tool for the identification and monitoring of SCRAs across various contexts, significantly contributing to public health and forensic security efforts. It is especially beneficial for healthcare providers, enabling them to make informed treatment decisions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Drug-impaired driving is a significant public health and safety concern in the USA. To help assess current patterns of drug use in drivers, we evaluated 4 years of drug positivity in a large cohort of suspected impaired drivers. Samples collected between January 2017 and December 2020 were tested via a method compliant with the National Safety Council's Alcohol, Drugs, and Impairment Division's Tier I scope of recommended drugs. In 2017, NMS Labs received 17 346 driving under the influence of drugs cases, 17 471 in 2018, 19 050 in 2019, and 16 539 in 2020. The most common drug class detected was cannabinoids in ∼50% of the cases each year. The most common drugs detected over the 4 years were delta-9 tetrahydrocannabinol (delta-9 THC), ethanol, amphetamine/methamphetamine, fentanyl, and alprazolam. Delta-9 THC increased in positivity over the study, having been identified in 45% of cases in 2017, 46% in 2018, 46% in 2019, and 49% in 2020. Ethanol was found in 59% of cases in 2017, 59% in 2018, 61% in 2019, and 53% in 2020. Delta-9 THC and ethanol were the most common drug combination, found together in ∼19% of the cases every year of the study. Statistically significant increases in the average concentration of the following drugs were observed: fentanyl (5.7 ng/mL in 2017 to 9.6 ng/mL in 2020), methamphetamine (301 ng/mL in 2017 to 381 ng/mL in 2020), and delta-9-THC (6.4 ng/mL in 2017 to 7.3 ng/mL in 2020). Other findings included increases in the maximum reported concentrations between 2017 and 2020 for amphetamine (1400 to 2700 ng/mL), methamphetamine (5550 to 13 000 ng/mL), and fentanyl (56 to 310 ng/mL). Statistically significant concentration decreases were noted for several central nervous system depressants, notably prescription benzodiazepines, and several prescription narcotic analgesics., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site–for further information please contact journals.permissions@oup.com.)

Alternative matrices, especially exhaled breath (EB), have gained increasing attention for a few years. To interpret toxicological findings, knowledge on the toxicokinetic (TK) properties of a substance in EB is indispensable. While such data are already accessible for various drugs (e.g. Δ9-tetrahydrocannabinol), they are still not available for new psychoactive substances, particularly synthetic cannabinoids (SCs). As SCs raise a high public health concern, the aim of this study was to assess these data in future TK studies in pigs. For this purpose, an in vitro sampling technique of EB was initially developed, which is prospectively applied to anesthetized and ventilated pigs for the detection of SCs in a controlled and reproducible manner as exemplified by cumyl-5F-P7AICA. Furthermore, a method for the qualitative and quantitative detection of cumyl-5F-P7AICA in EB using glass fiber filters (GFFs) was established and fully validated. Therefore, cumyl-5F-P7AICA (0.5 mg/mL in ethanol absolute) was initially nebulized using a ventilation machine and a breathing tube, as they are also used in surgeries. The aerosol was delivered into a simulated pig lung. To collect EB, a pump was connected to that part of the breathing tube, which contains EB (expiratory limb), and sampling was performed repeatedly (n = 6) for 15 min (2 l EB/min) each using GFF. For extraction of the substance, the GFFs were macerated with acetone and the remaining experimental components were rinsed with ethanol. After sample preparation, the extracts were analyzed by liquid chromatography tandem mass spectrometry. In the complete experimental setup, about 40% of the initially nebulized cumyl-5F-P7AICA dose was found, with 3.6 ± 1.3% being detected in the GFF. Regarding the comparably high loss of substance, the open ventilation system and a conceivable adsorption of the SC in the ventilator have to be considered. However, the herein introduced approach is promising to determine the TK properties of cumyl-5F-P7AICA in EB., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site–for further information please contact journals.permissions@oup.com.)

Characterizing the metabolic profiles of synthetic cannabinoids (SCs), a type of new psychoactive substances, is of particular importance for forensic detection and analysis. Although the metabolism of individual SCs derived from 1-amino-3,3-dimethyl-1-oxobutan-2-yl (ADB-SCs) has been reported, their metabolites also undergo a continuous change and combination of their tail and core regions. Therefore, elucidating the metabolic characteristics and effects of these structures is essential to enhance our understanding. In this study, the human liver microsome was used as the model for studying the in vitro phase I metabolism of 12 ADB-SCs, and the metabolites obtained were analyzed using ultra-high performance liquid chromatography-tandem four-level rod-electrostatic field orbital ion trap mass spectrometry to determine type, structure, and relative contents. The results indicated that hydroxylation and N-dealkylation were the major metabolic pathways in 12 ADB-SCs. The effects of the core and tail on the metabolism of these ADB-SCs were studied using theoretical calculations. For N-dealkylation metabolism, the strong electron-withdrawing conjugative effect of the -N= moiety in the pyrazole ring, steric hindrance of the tail, and electronic effect of substituents on the tail significantly affected metabolism. Further, it changed the relative contents of N-dealkylation metabolites. For hydroxylation, the reaction types were inconsistent at different parts. For instance, the phenyl group of the core is electrophilic, and its electron cloud density determines whether the phenyl group can be hydroxylated at the specific metabolic sites. Meanwhile, hydroxylation of the neopentyl moiety of the linked group involves the oxidation of aliphatic C-H bonds, whereas amide-hydroxylamine tautomerism affects hydroxylation metabolism. When the alkyl chain in the tail contains functional groups (such as -F and >CC<), oxidative defluorination or dihydrodiol metabolites are produced. Taken together, we systematically determined d the effect of functional groups in the core and tail of ADB-SCs on their metabolism, validating confirmed the feasibility of ADB-SC metabolism prediction based on their structural characteristics., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Xuan Luo reports equipment, drugs, or supplies and travel were provided by Science and Technology Department of Guangxi Zhuang Autonomous Region. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 Elsevier B.V. All rights reserved.)

Semi-synthetic cannabinoids (SSCs) are derivatives of phytocannabinoids with slight chemical modifications. SSCs have appeared as legal alternatives to tetrahydrocannabinol (Δ 9 -THC) in recent years. This study investigates the prevalence of SSCs in seized drug samples from Danish police and custom authorities seized in Eastern Denmark in the period 2018-2023. Screening data obtained by gas chromatography-mass spectrometry (GC-MS) were reprocessed to enable detection of SSCs. Seized drug samples were categorized into six types of formulations. Δ 8 -THC was the first SSC observed and appeared in 2019 followed by hexahydrocannabinol (HHC), tetrahydrocannabidiol (H4-CBD), hexahydrocannabinol acetate (HHC-O-Acetate), hexahydrocannabiphorol (HHCP) and tetrahydrocannabiphorol (Δ 9 -THCP). Only one sample positive for SSCs was observed before the third quarter of 2021, with positive samples increasing from third quarter of 2022. Over the study period, a total of 15% (n = 216) of seized cannabis products were positive for SSCs. HHC was the most frequently identified SSC and found in 10% (n = 137) of samples, followed by H4-CBD at 4% (n = 53), Δ 8 -THC at 3% (n = 44), and HHC-O-Acetate, HHCP, and THCP each at 1% (n = 10-20). SSCs appeared in 56% of E-cigarette products, 20% of hashish, 17% of concentrates, 10% of edibles, and 10% of plant materials. In conclusion, SSCs represent a new type of cannabinoids with a rapidly growing popularity and with specific compounds dominating at different periods. Some of the observed trends were likely influenced by the scheduling of HHC in May of 2023 in Denmark., (© 2024 The Author(s). Journal of Forensic Sciences published by Wiley Periodicals LLC on behalf of American Academy of Forensic Sciences.)

In recent years, synthetic cannabinoids (SCs) have become a major public health issue. For this reason, there is a need for innovative analytical methods that allow its monitoring in biological matrices. In this work, we propose a novel methodology to screen eight SCs (AM-694, cumyl-5F-PINACA, MAM-2201, 5F-UR-144, JWH-018, JWH-122, UR-144 and APINACA) in oral fluids. A bar adsorptive microextraction method followed by microliquid desorption combined with high-performance liquid chromatography with diode array detection (BAµE-µLD/HPLC-DAD) was developed to monitor the target SCs. The main factors affecting the BAµE technology were fully optimized for oral fluid analysis. Under optimized experimental conditions, the proposed methodology showed good linear dynamic ranges from 20.0 to 2000.0 µg L -1 (r 2 > 0.99, relative residuals < 15%), limits of detection between 2.0 and 5.0 µg L -1 and suitable average recovery yields (87.9-100.5%) for the eight studied SCs. The intra- and interday accuracies (bias ≤ ± 14.7%) and precisions (RSD ≤ 14.9%) were also evaluated at three spiking levels. The validated methodology was then assayed to oral fluid samples collected from several volunteers. The proposed analytical approach showed remarkable performance and could be an effective alternative for routine monitoring of the target compounds in oral fluid., (© 2024. The Author(s).)

This paper describes the investigation of synthetic cannabinoid news psychoactive substances in drug rehabilitation patients in the UAE. This represents the latest data for such drugs in the Gulf region., (© 2024 John Wiley & Sons Ltd.)

The US Food and Drug Administration's (FDA) regulatory oversight over electronic cigarettes (e-cigs) includes access restriction for persons <21 years of age and flavor restrictions for "cartridge-based" products. Despite the restrictions, consumption by US youth perseveres. Studies on youth e-cig use are limited by the reliability and accuracy of self-reports. As an alternative to self-reports, the current study examined nicotine, cannabinoid, and unlabeled e-cigs and other vaping products confiscated from Virginia public schools to characterize trends among students. Findings highlight a shift from JUUL and pod-based products to single use disposable e-cigs following the FDA flavor restrictions on cartridge-based e-cigs. Chemical analysis of e-liquids by gas chromatography-mass spectrometry identified a wide variety of flavorants and an increase in the prevalence of synthetic coolants. Most confiscated products were nicotine salt formulations, but the prevalence of cannabinoid-based vaping products increased. The popularity of flavored disposable e-cigs highlights the need for further restrictions to reduce youth consumption. The increasing use of synthetic coolants instead of menthol may suggest that manufacturers are employing tactics to bypass regulations. Continued youth access to e-cigs and the abundance of cannabinoid-based products is problematic from health and safety perspectives. Continued research incorporating confiscated product analysis can be used to understand youth access to vaping products and evolutions in manufacturing practices., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site–for further information please contact journals.permissions@oup.com.)

Synthetic cannabinoids emerged in the early 21st century and have continued to evolve and flourish to present day. Like other novel psychoactive substances (NPS), synthetic cannabinoids have been sold under the guise of legitimate products. Some examples include "potpourri," "incense," and herbal material. Between May 2020 and December 2023, the United States Army Criminal Investigation Laboratory, Drug Chemistry Division (USACIL) received 29 seized drug cases mentioning "blue lotus" or "valerian root." In 90% of these cases, at least one exhibit contained one or more synthetic cannabinoids. During the same timeframe, the Armed Forces Medical Examiner System, Division of Forensic Toxicology received 65 toxicology cases that contained synthetic cannabinoids and/or their corresponding metabolites where case history mentioned "blue lotus." The most frequently observed synthetic cannabinoids between laboratories were 5F-MDMB-PICA, ADB-BUTINACA, and MDMB-4en-PINACA. Innocuous branding and marketing may deceive law enforcement, investigators, and healthcare providers into believing that the adverse effects of erratic behavior, sedation, slurred speech, and hallucinations are a result of toxicity from botanical extracts (e.g. apomorphine and nuciferine in blue lotus). Due to the dangerous nature of these NPS, synthetic cannabinoid screening is recommended for all cases where there is suspected use of vaping products suggested to contain "blue lotus" or "valerian root" as vendors continue to conceal the presence of these compounds., (Published by Oxford University Press 2024. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Yoghurts are the most popular fermented dairy products. Consumer attention is directed towards products containing inputs that enrich the product with bioactive substances. The growing interest in the Cannabis sativa L. plant has resulted in the development of a market for hemp products. The main aim of this research work was to determine the effect of the lactic fermentation process on the stability of cannabinoids in fermented milk beverages containing hemp inputs (hemp oil, dried hemp, ethanolic hemp extract) at 0.5, 1 and 2% ( w / v ). The effect of the type of hemp input on the technological process (i.e., pH value, viability of LAB during 28 days of storage) was also checked and the sensory quality of the prepared samples was evaluated. It was shown that the choice of type/form and amount of hemp addition influenced the final cannabinoid content of the product after fermentation and post-fermentation, but no effect on the survival of yoghurt bacteria or pH changes during storage was confirmed. Fermented milk drinks containing hemp oil had the highest cannabinoid content. The QDA results of the sensory evaluation of the yoghurts show that the most acceptable product in terms of overall quality is the yoghurt containing 0.5% hemp extract and 2% hemp oil.

In this study, we developed and validated a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of eight phytocannabinoids in various cannabidiol (CBD) products from Japanese market. This method was combined with electrospray ionization in positive mode and sample preparation with QuEChERS. Three types of commercial products such as honey, chocolate, and gummies were used to perform accurate quantification with unified protocol of LC-MS/MS and QuEChERS. The limit of detection and quantification were 5-20 µg g -1 and 10-40 µg g -1 , respectively. Reproducibility was ensured using matrices free of target foods, resulting in an accuracy within ±10 % and a precision with a relative standard deviation of less than 5 % for all targets. Finally, this analytical method was applied to 8 series of commercial samples from the Japanese market. This unified protocol will serve as a reference as an official method in Japan., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: The prevalence of cannabis as the most commonly used illicit substance in the United States and around the globe is well-documented. Studies have highlighted a noticeable uptrend in the potency of cannabis in the United states. This report examines the concentration of cannabinoids in illicit cannabis samples seized by the United States Drug Enforcement Administration (DEA) over the last 10 years (2013-2022)., Methods: Samples received during the course of study (2013-2022) were categorized based on the geographical region where collected, as Western Region, Midwest Region, Northeast Region, South East Region, Southern Region as well as Alaska and Hawaii. These samples were processed for analysis using a validated gas chromatography with flame ionization detector method., Results: The data showed that the cannabinoids profile of all high Δ 9 -THC cannabis samples, regardless of the state or region from which the samples are seized or the state from which the sample is produced under a state medical marijuana program, is basically the same with the major cannabinoid being Δ 9 -THC (>10% for most samples) and all other cannabinoids with less than 0.5%, with the exception of CBG (<1%) and CBN (<1%)., Conclusion: Overall, it appears the cannabinoids profile is controlled by the genetics of the plant and is not affected much by the geographical location in which the plants are cultivated., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 ElSohly, Majumdar, Chandra and Radwan.)

The comprehensive detection of new psychoactive substances, including synthetic cannabinoids along with their associated metabolites in biological samples, remains an analytical challenge. To detect these chemicals, untargeted approaches using appropriate bioinformatic tools such as molecular networks are useful, albeit it necessitates as a prerequisite the identification of a node of interest within the cluster. To illustrate it, we reported in this study the identification of synthetic cannabinoids and some of their metabolites in seized e-liquid, urine, and hair collected from an 18-year-old poisoned patient hospitalized for neuropsychiatric disorders. A comprehensive analysis of the seized e-liquid was performed using gas chromatography coupled with electron ionization mass spectrometry, 1 H NMR, and liquid chromatography coupled with high resolution tandem mass spectrometry combined with data processing based on molecular network strategy. It allowed researchers to detect in the e-liquid known synthetic cannabinoids including MDMB-4en-PINACA, EDMB-4en-PINACA, MMB-4en-PINACA, and MDMB-5F-PICA. Compounds corresponding to transesterification of MDMB-4en-PINACA with pentenol, glycerol, and propylene glycol were also identified. Regarding the urine sample of the patient, metabolites of MDMB-4en-PINACA were detected, including MDMB-4en-PINACA butanoic acid, dihydroxylated MDMB-4en-PINACA butanoic acid, and glucurono-conjugated MDMB-4en-PINACA butanoic acid. Hair analysis of the patient allowed the detection of MDMB-4en-PINACA and MDMB-5F-PICA in the two investigated hair segments. This untargeted analysis of seized materials and biological samples demonstrates the utility of the molecular network strategy in identifying closely related compounds and metabolites of synthetic cannabinoids. It also emphasizes the need for developing strategies to anchor molecular networks, especially for new psychoactive substances.

Synthetic cannabinoids (SCs), which are among the most widely abused new psychoactive substances, are much more potent and have greater efficacy than natural cannabis. SCs can be disguised in various ways and are commonly sold in the form of electronic cigarette oil. SCs belong to a large family with structures consisting of a core with substituents, linker, ring with substituents, and tail. New SCs can be developed by adding substituents, such as halogen, alkyl, and alkoxy groups, to the aromatic ring system or by changing the alkyl chain length. Since the emergence of so-called first-generation SCs, subsequent developments have led to eighth-generation indole/indazole amide-based SCs. As of July 1, 2021, the entire category of SCs was added to the list of controlled substances, but implementation requires urgent improvements in detection technologies. Typically, each method is limited to a few SCs. Owing to the vast number of chemically diverse SCs and their fast update speed, the determination and identification of various types of SCs using a single method is challenging. Therefore, rapid, sensitive, and accurate quantitative methods that includes various types of SCs must be developed to meet the demand for the qualitative and quantitative analysis of new SCs in seized electronic cigarette oil. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of 102 SCs in electronic cigarette oil. The mass spectrometry and liquid-phase conditions influencing SC separation and determination were optimized. Using the external standard method, 102 SCs were successfully identified in electronic cigarette oil. The samples were extracted using methanol. Target analytes were separated on a Shimadzu Shim-pack GIST-HP C18 AQ column (100 mm×2.1 mm, 1.9 μm) at a column temperature of 40 ℃. The mobile phases consisted of (A) 0.1% formic acid aqueous solution and (B) methanol-acetonitrile (1∶1, v/v). The gradient elution conditions were as follows: 0-8 min, 55%A-15%A; 8-15 min, 15%A; 15-16 min, 15%A-55%A; 16-18 min, 55%A. The flow rate was 0.4 mL/min and the injection volume was 1 μL. Operating in the multiple reaction monitoring mode, the 102 SCs were identified within 18 min. Each SC exhibited a good linear relationship in the range of 1-100.0 μg/L with a correlation coefficient ( r )≥0.9915. The limits of detection were 0.01-0.30 μg/L and the limits of quantification were 0.04-0.99 μg/L, which meet the requirements for analyzing SCs in actual samples. Precision was determined using standard solutions with 2, 10, and 50 μg/L of the SCs. The precisions ( n =6) were 0.3%-6.0%. The recoveries of the 102 SCs, as evaluated by spiking electronic cigarette oil at low (2 μg/mL), medium (10 μg/mL), and high (50 μg/mL) levels, were 80.1%-119.8%. Good performance was observed for the analysis of real samples. The developed method is accurate, rapid, sensitive, and effective for the determination of the 102 SCs in electronic cigarette oil, satisfying the requirements for practical qualitative and quantitative analysis.

Cannabis is the most widely consumed illicit drug worldwide. As consumption rates increase, partially due to the decriminalization of its use for medicinal and recreational purposes, analytical methods for monitoring different cannabinoids in several biological matrices have been developed. Herein, a simple and fast extraction procedure to extract natural cannabinoids from oral fluid (OF) samples was developed and fully validated according to the ANSI/ASB 2019 Standard Practices for Method Validation in Forensic Toxicology. Using only 0.2 mL of neat OF, the analytes [Δ 9 -tetrahidrocannabinol (THC), 11-hydroxy-Δ 9 -tetrahydrocannabinol (THC-OH), 11-nor-9-carboxy-Δ 9 -tetrahydrocannabinol (THC-COOH), cannabinol (CBN) and cannabidiol (CBD)] were extracted by protein precipitation with a mixture of methanol:acetonitrile (80:20, v/v); the extracts were centrifuged, evaporated to dryness and reconstituted in 100 µL of methanol. Analysis was performed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The developed methodology produced linear results for all compounds, with working ranges of 0.1-50 ng/mL for THC, 0.5-50 ng/mL for THC-OH, CBN and CBD, and 0.05-1 ng/mL for THC-COOH. Ion suppression was observed for THC, CBN and CBD, which did not impair sensitivity considering the low limits of quantification (LOQs) and limits of detection (LODs) obtained (which varied between 0.05 and 0.5 ng/mL). The extraction procedure produced great recoveries, and the compounds were stable. No interferences were found, and the method proved to be extremely fast, selective, precise, and accurate for use in routine analysis. The method was successfully applied to authentic samples., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

As cannabidiol (CBD) is not considered to be a drug and because of its potential health claims, it is an interesting compound that is often found in cosmetics. However, the safety of CBD, as well as the presence of trace amounts of other phytocannabinoids, including the psychoactive substance ∆ 9 -tetrahydrocannabinol (THC), is still being debated. A robust analytical technique capable of analysing cosmetic products and determining their phytocannabinoid content will be crucial in assessing the safety of these products. This systematic review aims to highlight the current analytical tools that could be used to analyse phytocannabinoids in cosmetics. The ideal method would be able to analyse high levels of CBD in combination with trace levels of THC and their acids. The method should provide good recoveries and accuracies in a variety of matrices while providing information on up-coming phytocannabinoids such as cannabichromene (CBC), cannabigerol (CBG) and cannabinol (CBN). The systematic review approach was based on the Preferred Reporting Items for Systematic review and Meta-Analyses method. The research focused on studies published from January 2010 to December 2022 in PubMed and Scopus. A total of 15 datasets met the inclusion and exclusion criteria and were tabulated to allow easy comparison. Although some of the reviewed methods can handle multiple matrices and provide satisfactory recoveries, this review process did not identify an ideal method. The most suitable methods either could not quantify phytocannabinoid acids or were not sensitive enough to quantify trace levels of psychoactive phytocannabinoids., (© 2024 John Wiley & Sons Ltd.)

Detection and characterization of newly synthesized cannabinoids (NSCs) is challenging due to the lack of availability of reference standards and chemical data. In this study, a binary classification system was developed and validated using partial least square discriminant analysis (PLS-DA) by utilizing readily available mass spectral data of known drugs to assist in the identification of previously unknown NCSs. First, a binary classification model was developed to discriminate cannabinoids and cannabinoid-related compounds from other drug classes. Then, a classification model was developed to discriminate classical (THC-related) from synthetic cannabinoids. Additional models were developed based on the most abundant functional groups including core groups such as indole, indazole, azaindole, and naphthoylpyrrole, as well as head and tail groups including 4-fluorobenzyl (FUB) and 5-Fluoropentyl (5-F). The predictive ability of these models was tested via both cross-validation and external validation. The results show that all models developed are highly accurate. Additionally, latent variables (LVs) of each model provide useful mass to charge ( m / z ) for discrimination between classes, which further facilitates the identification of different functional groups of previously unknown drug molecules.

Besides many other uses, dried Cannabis may be used for "tea" preparation. This study focused on a comprehensive characterization of an aqueous infusion prepared according to a common practice from three fairly different Cannabis cultivars. The transfer of 42 phytocannabinoids and 12 major bioactive compounds (flavonoids) into the infusion was investigated using UHPLC-HRMS/MS. Phytocannabinoid acids were transferred generally in a higher extent compared to their counterparts; in the case of Δ 9 -THC, it was only in the range of 0.4-1.9% of content in the Cannabis used. A dramatic increase of phytocannabinoids, mainly of the neutral species, occurred when cream was added during steeping, and the transfer of Δ 9 -THC into "tea" achieved a range of 53-64%. Under such conditions, drinking a 250 mL cup of such tea by a 70 kg person might lead to multiple exceedance of the Acute Reference Dose (ARfD), 1 μg/kg b.w., even in the case when using hemp with a Δ 9 -THC content below 1% in dry weight for preparation.

Endocannabinoid system, including endocannabinoid neurotransmitters (eCBs), has gained much attention over the last years due to its involvement with the pathophysiology of diseases and the potential use of Cannabis sativa (marijuana). The identification of eCBs and phytocannabinoids in biological samples for forensic, clinical, or therapeutic drug monitoring purposes constitutes a still significant challenge. In this scoping review, the recent advantages, and limitations of the eCBs and phytocannabinoids quantification in biological samples are described. Published studies from 2018-2023 were searched in 8 databases, and after screening and exclusions, the selected 38 articles had their data tabulated, summarized, and analyzed. The main characteristics of the eCBs and phytocannabinoids analyzed and the potential use of each biological sample were described, indicating gaps in the literature that still need to be explored. Well-established and innovative sample preparation protocols, and chromatographic separations, such as GC, HPLC, and UHPLC, are reviewed highlighting their respective advantages, drawbacks, and challenges. Lastly, future approaches, challenges, and tendencies in the quantification analysis of cannabinoids are discussed., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Carolina Henkes Inamassu reports financial support was provided by National Council for Scientific and Technological Development (CNPq). Luisa Raspini e Silva reports financial support was provided by Coordination for the Improvement of Higher Education Personnel (CAPES). If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The cannabis plant is being increasingly researched due to its numerous therapeutic properties leading to the need for analytical techniques to assess substances present in extracts of the cannabis plant in carrier oils, such as medium chain triglycerides (MCT) oil. Awareness of the environmental impact of activities related to analysis led to the development of greenness assessment metrics. This study aimed to assess the environmental impact of analytical techniques applied in the analysis of cannabinoids in oil using Green Analytical Chemistry metrics. The first phase of the study consisted of a systematic literature review to identify high performance liquid chromatography and ultra high performance liquid chromatographic methods of analysis for cannabinoids in oil. In the second phase, the identified methods were assessed using the National Environmental Method Index (NEMI), Analytical Eco-scale, Analytical Greenness Calculator (AGREE) and Green Analytical Procedure Index (GAPI). Out of 124 identified studies, 8 were considered for the comparative analysis. The identified analytical methods consisted of high performance liquid chromatography (HPLC) using high resolution MS ( n = 1), DAD ( n = 2), UV ( n = 1), UV and MS ( n = 2) and MS/MS ( n = 2) as detectors. When the analytical methods were assessed using the Analytical Eco-Scale, 7 out of 8 methods achieved a score ranging between 50 and 73, categorising them as acceptable green methods of analysis. One method achieved a total score of 80, categorising the method as an excellent green analysis. The application of Green Analytical Chemistry and respective metrics during the development of analytical methods contributes towards a reduction in the environmental footprint which results from related activities.

Cannabis is one of the most consumed illicit drugs and the potency of cannabis products is of note due to health-related concerns. Hand-rubbed hashish is the ancient technique of extracting psychoactive resin from cannabis plants and is practiced in the Indian Himalayas. This study establishes the cannabinoid profile and potency of hand-rubbed hashish collected from 20 regions of the northwest Himalayas. Fifty-eight hashish samples were analyzed using a validated high-performance liquid chromatography-diode array detection (HPLC-DAD) method. Ten cannabinoids were quantified including acidic (THCA & CBDA), and neutral compounds (CBDV, THCV, CBD, CBG, CBN, Δ 9 -THC, Δ 8 -THC, and CBC). The mean concentration (w/w%) of Δ 9 -THC is 26%; THCA is 15% and THC Total is 40% is observed in the studied hashish samples. The majority (70%) of the hashish samples were categorized in chemotype I with the THC:CBD:CBN ratio of 91:3:4, and the remaining 30% were categorized under chemotype II with the ratio of 76:15:8. Diverse qualities of hashish are produced in the studied regions as per the seed, plant selection, and skills of manual rubbing, which results in potency variations. The average difference between the least and highest potent hand-rubbed hashish of a region is 27 w/w% (THC Total ). The other studied non-psychoactive cannabinoids have a mean w/w% of <5%, followed by 6% of CBDA. It is concluded that the cultivated and wild cannabis fields in the northwest Himalayas belong to the drug-type cannabis subspecies. Hand-rubbed hashish holds traditional significance and impacts the current policies of legislation., (© 2024 American Academy of Forensic Sciences.)

Rationale: With an increasing appreciation for the unique pharmacological properties associated with distinct, individual cannabinoids of Cannabis sativa, there is demand for accurate and reliable quantification for a growing number of them. In this study, we developed rapid, sensitive, selective, accurate, and validated liquid chromatography-tandem mass spectrometry for the quantification of cannabinoids., Methods: Crushed industrial hemp flower and leaf sample was extracted by 95% methanol aqueous, sonicated for 30 min. UPLC-MS/MS analysis using Waters Acquity BEH-C18 column and electrospray ionization(ESI) mass spectrometry detector., Results: The method was validated to demonstrate its reproducibility and precision, linearity, recovery investigation, and investigation of matrix effect. The concentration-response relationship for all analyzed cannabinoids were linear with R2 values >0.99, with intra- and inter-day precision and relative errors below 12%. The recovery and matrix effect were measured as 66.1%-104.1% and 70.42%-110.75%., Conclusions: This study established a UHPLC-MS/MS method for the simultaneous and rapid quantitative determination of twelve cannabinoids in industrial hemp flowers and leaves in 11 min. The method was used to analyze 43 industrial hemp flower and leaf samples, with the data being statistically analyzed. Based on the statistical analysis of the cannabinoids, hemp from different regions and different varieties were well distinguished by the PLS-DA model, with the main contributing substances being cannabidiol, Δ9-tetrahydrocannabinol, and Δ8-tetrahydrocannabinol., (© 2024 John Wiley & Sons Ltd.)

Tea is a recommended way of administration of prescribed cannabis plant products in Denmark. We aimed to investigate the cannabinoid and terpene doses contained in different teas. We analysed tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THCA), cannabidiol (CBD), cannabidiolic acid (CBDA), and terpene concentrations in three repeated preparations of each type of tea, and in plant material. In standard tea, concentrations of THC were [median (min-max)] 9.5 (2.3-15), 19 (13-34), and 36 (26-57) μg/mL for products with a labelled content of 6.3%, 14%, and 22% total THC (THC + THCA), respectively. The CBD concentration in tea from a product labelled with 8% total CBD (CBD + CBDA) was 7.5 (1.9-10) μg/mL. Based on this, the recommended starting amount of 0.2 L of the different teas would contain between 0.46 and 11.3 mg THC, and 0.38 to 2.0 mg CBD. Adding creamer before, but not after boiling, increased the THC and CBD concentration 2.3-4.4 and 2.1-fold, respectively. Terpenes were detected in plant material, but not in tea. The study elucidates THC and CBD doses in different teas, which may assist the clinician's choice of cannabis product. Moreover, it underscores the need for caution as administration as tea can result in exposure to different doses, even when the same cannabis product is used., (© 2024 The Author(s). Basic & Clinical Pharmacology & Toxicology published by John Wiley & Sons Ltd on behalf of Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

Spent hemp biomass (SHB) contains trace amounts of cannabinoids, including Δ 9 -tetrahydrocannabinol (Δ 9 -THC), that may accumulate in the tissues of animals consuming SHB. We measured cannabinoid residues in the liver, adipose tissue, and muscle of finishing lambs fed either 10% or 20% SHB for 8 weeks, or 4 weeks followed by 4 weeks SHB withdrawal. We detected multiple cannabinoids in the liver at a similar proportion to the SHB. However, CBD and Δ 9 -THC were enriched >20-fold in the adipose and muscle, compared to their proportion in SHB. The highest concentration of Δ 9 -THC was detected in adipose tissue and was 7.4-times higher than in muscle. Most cannabinoids were undetectable in tissues after 4 weeks of clearance. The consumers' exposure assessment on Δ 9 -THC revealed tissue levels of total THC (THCA+Δ 9 -THC) that exceed the acute reference dose of 1 μg/kg BW across population groups. When consuming meat from the lambs fed 10% and 20% SHB, the maximum total THC exposure was 2.03 and 7.32 μg/kg BW, respectively, equal to or below the Lowest Observed Adverse Effect Level of 36 μg/kg BW, the No Observed Adverse Effect Level of 12 μg/kg BW or a tolerable dose intake of 7 μg/kg BW., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Synthetic cannabinoid receptor agonists (SCRAs) are a diverse class of new psychoactive substances (NPS) and new structural scaffolds have emerged on the recreational drug market since the enactment of Chinese SCRA analog controls in 2021. This study reports the first SCRAs to be detected with a bromide at the 5 position (5'Br) on the phenyl ring of the indazole core and without a tail moiety. ADB-5'Br-INACA (ADMB-5'Br-INACA) and MDMB-5'Br-INACA were detected in seized samples from Scottish prisons, Belgian customs, and US forensic casework. The brominated analog with a tail moiety, ADB-5'Br-BUTINACA (ADMB-5'Br-BUTINACA), was also detected in Scottish prisons and US forensic casework. The metabolites of these compounds and the predicted compound MDMB-5'Br-BUTINACA were identified through incubation with primary human hepatocytes to aid in their toxicological identification. The bromide on the indazole remains intact on metabolites, allowing these compounds to be easily distinguished in toxicological samples from their non-brominated analogs. Glucuronidation was more common for tail-less analogs than their butyl tail-containing counterparts. Forensic toxicologists are advised to update their analytical methods with the characteristic ions for these compounds, as well as their anticipated urinary markers: amide hydrolysis and monoOH at tert-butyl metabolites (after β-glucuronidase treatment) for ADB-5'Br-INACA; monoOH at tert-butyl and amide hydrolysis metabolites for ADB-5'Br-BUTINACA; and ester hydrolysis metabolites with additional metabolites for MDMB-5'Br-INACA and MDMB-5'Br-BUTINACA. Toxicologists should remain vigilant to the emergence of new SCRAs with halogenation of the indazole core and tail-less analogs, which have already started to emerge., (© 2023 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Synthetic cannabinoids are a class of novel psychoactive substances that emerged in the drug market in the early 2010s. Since then, a wide range of different synthetic cannabinoids has been detected in drug materials and in biological specimens collected from intoxication cases. In general, synthetic cannabinoids are reported first in seized materials. In this study, the identification of the novel synthetic cannabinoid, ADB-5'Br-BINACA is reported. A plant material suspected to contain a synthetic cannabinoid was extracted and analyzed. Analyses were performed using gas chromatography-mass spectrometry (GC-MS), liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and one dimensional and two-dimensional nuclear magnetic resonance (NMR) spectroscopy. An aliquot of the sample was extracted using methanol and deuterated chloroform, and analyzed via GC-MS and NMR, respectively. Further dilution of the methanolic extract was analyzed via LC-QTOF-MS. For ATR-FTIR analyses, a few drops of the extract in deuterated chloroform were analyzed. GC-MS, LC-QTOF-MS, and 1 H NMRwere successfully used to elucidate and confirm the structure of ADB-5'Br-BINACA in the drug sample. ATR-FTIR and 13 C NMR analyses of the extracts did not result in significant information for the confirmation of ADB-5'Br-BINACA in the plant material likely due to low amount of drug material and high background noise. The chemical characterization of ADB-5'Br-BINACA in an authentic sample is reported herein, and chromatographic, mass spectrometric and spectroscopic data are provided for use in future analysis of this drug in suspected samples., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cannabis sativa L. is a plant belonging to the Cannabaceae family known primarily for its recreational use due to the psychoactive properties of Δ 9 -tetrahydrocannabinol (THC). Despite this, several compounds belonging to the category of phytocannabinoids have shown in recent years a number of potentially promising therapeutic effects that have increased the interest in the pharmaceutical field towards this plant. However, the content of these compounds is very variable and influenced by different factors, such as growing conditions and time of the year. An indication of the status and age of Cannabis samples is provided by the content of CBN, a minor phytocannabinoid and degradation product of other phytocannabinoids, including THC. In this research work an innovative, solid state analytical approach has been developed to observe and evaluate the variations in the content of two phytocannabinoids (CBN and CBD) in Cannabis-derived products over time. In order to simulate the ageing of the Cannabis samples, an artificially accelerated ageing procedure has been developed and optimised by using high temperatures. The analyses were carried out using an innovative ATR-FTIR method for solid state analysis, enabling direct analysis of a solid sample without any pretreatment phase. This study has allowed the development of an innovative analytical approach for the evaluation of the age and state of conservation of Cannabis samples and may be a useful tool both in the industrial, pharmaceutical and forensic fields., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Drug addiction is associated with significant health risks, including cardiovascular complications, cancer, and mental disorders. Illicit drugs, such as cannabinoids and opioids, including prescription medications, are widely consumed and have profound health consequences. Understanding the health effects of the toxic elements in these substances is critical for overdose prevention and effective recovery strategies. This study aimed to determine toxic elements, including arsenic (As), cadmium (Cd), mercury (Hg), and nickel (Ni), in cannabinoid and opioid drugs and in biological samples (whole blood, scalp hair, and serum) from 311 male drug abuse patients aged 15-60 years with a history of drug abuse. The participants were categorized into three age groups. The comparative analysis involved 113 reference subjects of the same age groups. The sample preparation employed microwave-assisted acid digestion, and the toxic elements were quantified using atomic absorption spectrophotometry. Accuracy was ensured using certified reference materials for hair, whole blood, and serum samples. Drug-addicted subjects had significantly higher concentrations of toxic elements (arsenic, cadmium, mercury, and nickel) in biological samples than referent subjects (p > 0.001). Elevated levels of these toxic elements may increase susceptibility to infections, possibly due to malnutrition, drug-related effects, and additional contaminants. These findings necessitate further studies to explore the long-term health outcomes, potential treatment options, and broader socioeconomic impacts of substance abuse. This study serves as a baseline for future research in this critical public health field., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The forensic analysis of amide-based synthetic cannabinoids (SCs) in seized materials is routinely performed using gas chromatography-mass spectrometry (GC-MS); however, a major challenge associated with GC-MS is the thermolytic degradation of substances with sensitive functional groups. Herein, we report the comprehensive thermal degradation and ester transformation of amide-based SCs, such as AB-FUBINACA, AB-CHMINACA, and MAB-CHMINACA, during GC-MS analysis and their treatment with analyte protectants (APs). These SCs were found to undergo thermolytic degradation during GC-MS in the presence of non-alcohol solvents. Using methanol as an injection solvent resulted in the conversion of the amide group to an ester group, producing other SCs such as AMB-FUBINACA, MA-CHMINACA, and MDMB-CHMINACA. Degradant and ester product formation has been interpreted as the adsorption of target SCs on glass wool via hydrogen bonding interactions between the active silanol and amide groups of the SCs, followed by an addition and/or elimination process. The factors found to influence the thermal degradation and/or esterification of the amide functional group include residence time, activity of glass wool, and injection volume. This report presents the fragmentation patterns of all compounds that were produced by degradation and esterification. Using 0.5 % sorbitol (AP) in MeOH as an injection solvent resulted in complete protection and improvement of the chromatographic shape of the compounds. This method has been successfully confirmed in terms of sensitivity, linearity, accuracy, and precision for standard solutions and tablet extraction using 0.5 % sorbitol in MeOH. Using AP increased the sensitivity by ten times or more compared to the use of only MeOH. The limit of detection for all analytes was determined as 25 ng/mL, and the calibration curves were linear over the concentration range of 50-2000 ng/mL. The values of accuracy error were below 11 %, and precision was less than 13 %. The effects of phytochemicals of herbal products, tablet ingredients, and biological matrices on the degradation and/or esterification and APs performance have also been evaluated in this work., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Cannabis sativa is a psychoactive plant indigenous to Central and South Asia, traditionally used both for recreational and religious purposes, in addition to folk medicine. Cannabis is a rich source of natural compounds, the most important of which are commonly known as cannabinoids that cause a variety of effects through interaction with the endocannabinoid system. Materials and Methods: In this study, a high-performance liquid chromatography-ultraviolet/photodiode array (PDA) method was developed and validated for the analysis of 15 cannabinoids in cannabis plant materials and cannabis-based marketed products. These cannabinoids are cannabidivarinic acid, cannabidivarin, cannabidiolic acid, cannabigerolic acid, cannabigerol, cannabidiol, delta-9-tetrahydrocannabivarin, delta-9-tetrahydrocannabivarinic acid, cannabinol, delta-9-tetrahyrocannabinol, delta-8-tetrahyrocannabinol, cannabicyclol, cannabichromene, delta-9-tetrahyrocannabinolic acid A, and cannabichromenic acid. The separation was carried out using a reversed-phase Luna ® C18(2) column and a mobile phase consisting of 75% acetonitrile and 0.1% formic acid in water. A PDA detector was used, and data were extracted at λ =220 nm. Principal component analysis of cannabis four varieties was performed. Results: The method was linear over the calibration range of 5-75 μg/mL with R 2 >0.999 for all cannabinoids. This method was sensitive and gave good baseline separation of all examined cannabinoids with limits of detection ranging between 0.2 and 1.6 μg/mL and limits of quantification ranging between 0.6 and 4.8 μg/mL. The average recoveries for all cannabinoids were between 81% and 104%. The measured repeatability and intermediate precisions (% relative standard deviation) in all varieties ranged from 0.35% to 9.84% and 1.11% to 5.26%, respectively. Conclusions: The proposed method is sensitive, selective, reproducible, and accurate. It can be applied for the simultaneous determination of these cannabinoids in the C. sativa biomass and cannabis-derived marketed products.

Hemp-sprouts are emerging as a new class of attractive functional food due to their numerous health benefits when compared to other sprout species. Indeed, the high content of beneficial components including polyphenols and flavonoids makes this type of food a promising and successful market. However, the available literature on this topic is limited and often conflicting as regards to the content of phytocannabinoids. High-performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC-HRMS) was applied in an untargeted metabolomics fashion to extracts of hemp seeds, sprouts and microgreens of nine different genotypes. Both unsupervised and supervised multivariate statistical analysis was performed to reveal variety-specific profiles of phytocannabinoids with surprisingly remarkable levels of phytocannabinoids even in chemotype V samples. Furthermore, a targeted HPLC-HRMS analysis was carried out for the quantitative determination of the major phytocannabinoids including CBDA, CBD, CBGA, CBG, CBCA, CBC, THCA, and trans-Δ 9 -THC. The last part of the study was focused on the evaluation of the enantiomeric composition of CBCA in hemp seeds, sprouts and microgreens in the different varieties by HPLC-CD (HPLC with online circular dichroism). Chiral analysis of CBCA showed a wide variability of its enantiomeric composition in the different varieties, thus contributing to the understanding of the intriguing stereochemical behavior of this compound in an early growth stage. However, further investigation is needed to determine the genetic factors responsible for the low enantiopurity of this compound., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Phytocannabinoids occurring in Cannabis Sativa L. are unique secondary metabolites possessing interesting pharmacological activities. In this study, the dynamics of thermally induced (60 and 120 °C) phytocannabinoid reactions in four cannabis varieties were investigated. Using UHPLC-HRMS/MS, 40 phytocannabinoids were involved in target analysis, and an additional 281 compounds with cannabinoid-like structures and 258 non-cannabinoid bioactive compounds were subjected to suspect screening. As expected, the key reaction was the decarboxylation of acidic phytocannabinoids. Nevertheless, the rate constants differed among cannabis varieties, documenting the matrix-dependence of this process. Besides neutral counterparts of acidic species, ́neẃ bioactive compounds such as hydroxyquinones were found in heated samples. In addition, changes in other bioactive compounds with both cannabinoid-like and non-cannabinoid structures were documented during cannabis heating at 120 °C. The data document the complexity of heat-induced processes and provide a further understanding of changes in bioactivities occurring under such conditions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Cannabidiol (CBD) is the main non-psychoactive phytocannabinoid derived from Cannabis sativa L. It is now an active pharmaceutical ingredient (API), given its usage in treating some types of pediatric epilepsy. For this reason, this compound requires a deep characterization in terms of purity and origin. Previous research work has shown two impurities in CBD samples from hemp inflorescences, namely, cannabidivarin (CBDV) and cannabidibutol (CBDB), while abnormal-cannabidiol (abn-CBD) has been described as the primary by-product that is generated from CBD synthesis. Both natural and synthetic CBD samples exhibit the presence of Δ 9 -tetrahydrocannabinol (Δ 9 -THC) and Δ 8 -THC. This study aimed to develop a new analytical method based on high-performance liquid chromatography (HPLC) with different detection systems to study the purity of CBD and to define its origin based on the impurity profile. In addition to the above-mentioned cannabinoids, other compounds, such as cannabigerovarin (CBGV), cannabigerol (CBG), cannabichromevarin (CBCV), and cannabichromene (CBC), were examined as potential discriminating impurities. Qualitative and quantitative analyses were carried out by UHPLC-HRMS and HPLC-UV/Vis, respectively. Principal component analysis was applied for statistical exploration. Natural CBD samples exhibited purities ranging between 97.5 and 99.7%, while synthetic samples were generally pure, except for three initially labeled as synthetic, revealing natural-derived impurities. To further confirm the origin of CBD samples, the presence of other two minor impurities, namely cannabidihexol (CBDH) and cannabidiphorol (CBDP), was assessed as unequivocal for a natural origin. Finally, an enantioselective HPLC analysis was carried out and the results confirmed the presence of the (-)-trans enantiomer in all CBD samples. In conclusion, the HPLC method developed represents a reliable tool for detecting CBD impurities, thus providing a clear discrimination of the compound origin., (© 2024. The Author(s).)

Currently, there is a significant demand in forensic toxicology for biomarkers of cannabis exposure that, unlike ∆ 9 -tetrahydrocannabinol, can reliably indicate time and frequency of use, be sampled with relative ease, and correlate with impairment. Oral fluid (OF) and exhaled breath condensate (EBC) are alternative, non-invasive sample matrices that hold promise for identifying cannabis exposure biomarkers. OF, produced by salivary glands, is increasingly utilized in drug screening due to its non-invasive collection and is being explored as an alternative matrix for cannabinoid analysis. EBC is an aqueous specimen consisting of condensed water vapor containing water-soluble volatile and non-volatile components present in exhaled breath. Despite potential advantages, there are no reports on the use of EBC for cannabinoid detection. This study developed a supported liquid extraction approach and LC-QqQ-MS dMRM analytical method for quantification of 25 major and minor cannabinoids and metabolites in OF and EBC. The method was validated according to the ANSI/ASB 036 standard and other published guidelines. LOQ ranged from 0.5 to 6.0 ng/mL for all cannabinoids in both matrices. Recoveries for most analytes were 60-90%, with generally higher values for EBC compared to OF. Matrix effects were observed with some cannabinoids, with effects mitigated by use of matrix-matched calibration. Bias and precision were within ± 25%. Method applicability was demonstrated by analyzing ten authentic OF and EBC samples, with positive detections of multiple analytes in both matrices. The method will facilitate comprehensive analysis of cannabinoids in non-invasive sample matrices for the development of reliable cannabis exposure biomarkers., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Cannabis sativa , otherwise known as hemp, is discussed to highlight the various problems and prospects associated with its use as an herbal ingredient. The chemical composition of hemp, with classification based on cannabinoid contents, its biological activities, current global scenarios and legality issues, economic importance, and future prospects, are discussed.

Cannabinoids can be detected in breath after cannabis use, but different breath matrices need to be explored as studies to date with filter-based devices that collect breath aerosols have not demonstrated that breath-based measurements can reliably identify recent cannabis use. Exhaled breath condensate (EBC) is an unexplored aqueous breath matrix that contains condensed volatile compounds and water vapor in addition to aerosols. EBC was collected from participants both before and at two time points (0.7 ± 0.2 h and 1.7 ± 0.3 h) after observed cannabis use. Eleven different cannabinoids were monitored with liquid chromatography tandem mass spectrometry. Five different cannabinoids, including Δ 9 -tetrahydrocannabinol (THC), were detected in EBC collected from cannabis users. THC was detected in some EBC samples before cannabis use, despite the requested abstinence period. THC was detected in all EBC samples collected at 0.7 h post use and decreased for all participants at 1.7 h. Non-THC cannabinoids were only detected after cannabis use. THC concentrations in EBC samples collected at 0.7 h showed no trend with sample metrics like mass or number of breaths. EBC sampling devices deserve further investigation with respect to modes of cannabis use (e.g, edibles), post use time points, and optimization of cannabinoid recovery., (Creative Commons Attribution license.)

Studies with secretory cavity contents and air-dried inflorescence extracts of the CBD-rich hemp strain, Cannabis sativa cv. 'Cherry Wine', were conducted to compare the decarboxylation rates of acidic cannabinoids between two groups. The secretory cavity contents acquired from the capitate-stalked glandular trichomes by glass microcapillaries, and inflorescence samples air-dried for 15 days of storage in darkness at room temperature were analysed by high-pressure liquid chromatography. The ratio of acidic cannabinoids to the total cannabinoids was ranging from 0.5% to 2.4% lower in the air-dried inflorescence samples compared to the secretory cavity samples as follows. In the secretory cavity content, the percentage of acidic cannabinoids to the total cannabinoids was measured as 86.4% cannabidiolic acid (CBDA), 6.5% tetrahydrocannabinolic acid (THCA), 4.3% cannabichromenic acid (CBCA), 1.4% cannabigerolic acid (CBGA), and 0.6% cannabidivarinic acid (CBDVA), respectively. In the air-dried inflorescence, however, the acidic cannabinoids were detected with 84% CBDA, 4.8% THCA, 3.3% CBCA, 0.8% CBGA, and 0.3% Δ 9 -tetrahydrocannabivarinic acid (Δ 9 -THCVA), respectively. The ratio of cannabidiol (CBD) to cannabidiolic acid (CBDA) was close to 1:99 (w/w) in secretory cavity contents, however, it was roughly 1:20 (w/w) in the air-dried inflorescence. In addition, Δ 9 -tetrahydrocannabivarin (Δ 9 -THCV) and Δ 9 -tetrahydrocannabivarinic acid (Δ 9 -THCVA) were only detected in the air-dried inflorescence sample, and the ratio of Δ 9 -THCV to Δ 9 -THCVA was about 1:20 (w/w). Besides, cannabidivarinic acid (CBDVA) was only observed in the secretory cavity content., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

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 C 18 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.

Objective: Many young adults report frequent cannabis use and are at risk for cannabis harms. Knowledge of the tetrahydrocannabinol (THC) and cannabidiol (CBD) concentrations of cannabis products may promote harm reduction, but few studies have characterized cannabinoid concentration knowledge in this population. This study used day-level data to examine predictors of cannabinoid concentration knowledge and associations of cannabinoid concentration knowledge with substance-related consequences among young adults., Method: Participants ( n = 131; mean age = 22.11 years; 64.12% female) from a larger study of cannabis and alcohol co-use completed daily surveys over 21 days assessing knowledge of the cannabinoid concentrations of cannabis used, forms of cannabis used, motives for cannabis use (medicinal, nonmedicinal, both), and substance-related consequences., Results: On average, participants reported at least some knowledge of the THC and CBD concentrations of their cannabis on 48% and 32% of their cannabis use days, respectively. Generalized linear mixed models revealed that participants with a greater propensity to use nonflower (relative to flower) cannabis products and to report medicinal (relative to exclusively nonmedicinal) motives for cannabis use reported greater cannabinoid concentration knowledge overall across days, controlling for sociodemographic factors and level of cannabis involvement. Participants with greater overall cannabinoid concentration knowledge reported positive substance-related consequences more often. In addition, participants were more likely to report negative substance-related consequences on days during which cannabinoid concentrations were known versus unknown., Conclusions: Findings suggest that cannabinoid concentration knowledge may be higher among young adults who report primarily nonflower and medicinally motivated cannabis use, although cannabinoid concentration knowledge, alone, may not protect against negative substance-related consequences at the day level.

Background and Objectives: As cannabis use increases among reproductive-aged women, there is a growing need to better understand the presence of cannabinoids in milk produced by women using cannabis. It is unclear how concentrations of cannabinoids such as delta-9-tetrahydrocannabinol (Δ 9 -THC) persist in milk after cannabis use and what factors contribute to variation in milk Δ 9 -THC concentrations. Our objectives were to measure cannabinoids in human milk following cannabis abstention, after single and repeated instances of cannabis use, and identify factors contributing to concentration variation. Methods: The Lactation and Cannabis (LAC) Study prospectively observed 20 breastfeeding participants who frequently used cannabis (≥1/week), had enrolled <6 months postpartum, were feeding their infant their milk ≥5 times/day, and were not using any illicit drugs. Participants collected a baseline milk sample after ≥12 hours of abstaining from cannabis and five milk samples at set intervals over 8-12 hours after initial cannabis use. Participants completed surveys and recorded self-directed cannabis use during the study period. Results: Δ 9 -THC peaked 120 minutes after a single instance of cannabis use (median, n = 9). More instances of cannabis use during the study period were associated with greater Δ 9 -THC area-under-the-curve concentrations (ρ = 0.65, p = 0.002), indicating Δ 9 -THC bioaccumulation in most participants. Baseline Δ 9 -THC logged concentration was positively associated with self-reported frequency of cannabis use ( b = 0.57, p = 0.01). Conclusions: Cannabinoids are measurable in human milk following cannabis use, and concentrations remain elevated with repeated cannabis use over a day. Substantial variation in Δ 9 -THC milk concentrations reflects individual differences in characteristics and behavior, including average postpartum frequency of cannabis use.

Novel psychoactive substances (NPS) are everchanging and plague forensic laboratories who must identify an unending variety of emerging substances and evolve current methodologies to detect these substances. Identifying potential regional NPS targets and timely examining trends in seized drug data could help mitigate the burden laboratories face. Over 17 months, NPS seized drug data were processed and categorized from three laboratories located across the United States to determine any NPS regional similarities and prevalent NPS drug categories: the South Carolina Law Enforcement Division (SLED), the Sedgwick County Regional Forensic Science Center (SCRFSC), and the Orange County Crime Laboratory (OCCL). Seized drug materials, including pills, powders, and plant material, were primarily analyzed for NPS via gas chromatography-mass spectrometry and Fourier transform infrared spectroscopy. From June 2022 to October 2023, 1940 NPS seized drug identifications were reported by these laboratories with 63 different NPS reported. Novel synthetic opioids (NSO) were the most prevalent NPS class across all three laboratories (55%), with fluorofentanyl accounting for 74% of NSO identifications. This is unsurprising given the fentanyl epidemic in the United States. Furthermore, these data highlighted varying regional NPS seized drug trends: eutylone, a synthetic cathinone, was one of the most frequently identified NPS in SLED, SCRFSC observed the most diverse set of synthetic cannabinoids, and OCCL observed an increased prevalence in the designer benzodiazepine, bromazolam. NPS scope recommendations are a valuable resource for forensic laboratories; however, most focus on a national perspective. Timely analysis and reporting of NPS seized drug data may help to develop regional NPS scope recommendations laboratories may employ., (© 2024 American Academy of Forensic Sciences.)

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., (© 2024. The Author(s).)

Introduction: Organic molecules that bind to cannabinoid receptors are known as cannabinoids. These molecules possess pharmacological properties similar to those produced by Cannabis sativa L. High-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC, also known as ultra-high-performance liquid chromatography, UHPLC) have become the most widely used analytical tools for detection and quantification of phytocannabinoids in various matrices. HPLC and UPLC (or UHPLC) are usually coupled to an ultraviolet (UV), photodiode array (PDA), or mass spectrometric (MS) detector., Objective: To critically appraise the literature on the application of HPLC and UPLC (or UHPLC) methods for the analysis of phytocannabinoids published from January 2020 to December 2023., Methodology: An extensive literature search was conducted using Web of Science, PubMed, and Google Scholar and published materials including relevant books. In various combinations, using cannabinoid in all combinations, cannabis, hemp, hashish, C. sativa, marijuana, analysis, HPLC, UHPLC, UPLC, and quantitative, qualitative, and quality control were used as the keywords for the literature search., Results: Several HPLC- and UPLC (or UHPLC)-based methods for the analysis of phytocannabinoids were reported. While simple HPLC-UV or HPLC-PDA-based methods were common, the use of HPLC-MS, HPLC-MS/MS, UPLC (or UHPLC)-PDA, UPLC (or UHPLC)-MS, and UPLC (or UHPLC)-MS/MS was also reported. Applications of mathematical and computational models for optimization of protocols were noted. Pre-analyses included various environmentally friendly extraction protocols., Conclusion: During the last 4 years, HPLC and UPLC (or UHPLC) remained the main analytical tools for phytocannabinoid analysis in different matrices., (© 2024 John Wiley & Sons Ltd.)

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., (© 2024. The Author(s).)

Cannabis sativa L. has been the most discussed medicinal plant in recent years. In particular, the dynamic shift from a formerly illicit and tightly controlled substance to a plant recognized for both medicinal and recreational purposes has brought C. sativa into the global spotlight. Due to the ongoing international legalization processes, fast and convenient analytical methods for the quality control of C. sativa flowers for medicinal and recreational purposes are of tremendous interest. In this study, we report the development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method applying atmospheric pressure chemical ionization (APCI) to fully quantify 16 terpenes and 7 cannabinoids including their acidic forms by a single chromatographic method. The method presented here is unique and simple, as it eliminates the need for derivatization reactions and includes the unconventional analysis of volatile compounds by liquid chromatography. Samples were prepared by a simple and fast ethanolic extraction. Separation was accomplished within 25 min on a reversed-phase C18 column. Method validation was conducted according to international guidelines regarding selectivity, accuracy, precision, robustness, and linearity. Detection was done in multiple reaction monitoring, which allowed the simultaneous quantification of co-eluting analytes applying two selective mass transitions. In addition, due to reproducible in-source decarboxylation, the acidic forms of cannabinoids were reliably quantified using mass transitions of the neutral forms. The accuracy given as the bias was below 15% for all analytes. Matrix effects for cannabinoids were studied by spiking Humulus lupulus extracts with the analytes at varying concentrations. APCI did not show susceptibility toward ion suppression or enhancement. In addition, the recovery effect after spiking was between 80 and 120% for terpenes. Further, 55 authentic C. sativa extracts were fully quantified, and the obtained results for the terpene profiles were compared to state-of-the-art gas chromatography coupled to flame ionization detection. Comparable results were achieved, emphasizing the method's applicability for cannabinoids and terpenes. Further, acquired metabolite patterns for C. sativa samples were studied, identifying a relationship between cannabinoid and terpene patterns, as well as the abundance of myrcene in CBD-dominant C. sativa strains., (© 2024. The Author(s).)

Background: Since late 2019, fortification of 'regular' cannabis plant material with synthetic cannabinoid receptor agonists (SCRAs) has become a notable phenomenon on the drug market. As many SCRAs pose a higher health risk than genuine cannabis, recognizing SCRA-adulterated cannabis is important from a harm reduction perspective. However, this is not always an easy task as adulterated cannabis may only be distinguished from genuine cannabis by dedicated, often expensive and time-consuming analytical techniques. In addition, the dynamic nature of the SCRA market renders identification of fortified samples a challenging task. Therefore, we established and applied an in vitro cannabinoid receptor 1 (CB 1 ) activity-based procedure to screen plant material for the presence of SCRAs., Methods: The assay principle relies on the functional complementation of a split-nanoluciferase following recruitment of β-arrestin 2 to activated CB 1 . A straightforward sample preparation, encompassing methanolic extraction and dilution, was optimized for plant matrices, including cannabis, spiked with 5 µg/mg of the SCRA CP55,940., Results: The bioassay successfully detected all samples of a set (n = 24) of analytically confirmed authentic Spice products, additionally providing relevant information on the 'strength' of a preparation and whether different samples may have originated from separate batches or possibly the same production batch. Finally, the methodology was applied to assess the occurrence of SCRA adulteration in a large set (n = 252) of herbal materials collected at an international dance festival. This did not reveal any positives, i.e. there were no samples that yielded a relevant CB 1 activation., Conclusion: In summary, we established SCRA screening of herbal materials as a new application for the activity-based CB 1 bioassay. The simplicity of the sample preparation, the rapid results and the universal character of the bioassay render it an effective and future-proof tool for evaluating herbal materials for the presence of SCRAs, which is relevant in the context of harm reduction., (© 2024. The Author(s).)

The diversity of cannabinoid isomers and complexity of Cannabis products pose significant challenges for analytical methodologies. In this study, we developed a method to analyze 14 different cannabinoid isomers in diverse samples within milliseconds by leveraging the unique adduct-forming behavior of silver ions in advanced cyclic ion mobility spectrometry-mass spectrometry. The developed method achieved the separation of isomers from four groups of cannabinoids: Δ3-tetrahydrocannabinol (THC) ( 1 ), Δ8-THC ( 2 ), Δ9-THC ( 3 ), cannabidiol (CBD) ( 4 ), Δ8-iso-THC ( 5 ), and Δ(4)8-iso-THC ( 6 ) (all MW = 314); 9α-hydroxyhexahydrocannabinol ( 7 ), 9β-hydroxyhexahydrocannabinol ( 8 ), and 8-hydroxy-iso-THC ( 9 ) (all MW = 332); tetrahydrocannabinolic acid (THCA) ( 10 ) and cannabidiolic acid (CBDA) ( 11 ) (both MW = 358); Δ8-tetrahydrocannabivarin (THCV) ( 12 ), Δ8-iso-THCV ( 13 ), and Δ9-THCV ( 14 ) (all MW = 286). Moreover, experimental and theoretical traveling wave collision cross section values in nitrogen ( TW CCS N2 ) of cannabinoid-Ag(I) species were obtained for the first time with an average error between experimental and theoretical values of 2.6%. Furthermore, a workflow for the identification of cannabinoid isomers in Cannabis and Cannabis-derived samples was established based on three identification steps ( m / z and isotope pattern of Ag(I) adducts, TW CCS N2 , and MS/MS fragments). Afterward, calibration curves of three major cannabinoids were established with a linear range of 1-250 ng·ml -1 for Δ8-THC ( 2 ) ( R 2 = 0.9999), 0.1-25 ng·ml -1 for Δ9-THC ( 3 ) ( R 2 = 0.9987), and 0.04-10 ng·ml -1 for CBD ( 4 ) ( R 2 = 0.9986) as well as very low limits of detection (0.008-0.2 ng·ml -1 ). Finally, relative quantification of Δ8-THC ( 2 ), Δ9-THC ( 3 ), and CBD ( 4 ) in eight complex acid-treated CBD mixtures was achieved without chromatographic separation. The results showed good correspondence ( R 2 = 0.999) with those obtained by gas chromatography-flame ionization detection/mass spectrometry.

Concerns have been raised about synthetic cannabinoids (SCs), which are among the most often trafficked and used illegal substances. An analytical method that holds promise for determining illicit drug use in the general population is wastewater-based epidemiology (WBE). Unfortunately, the concentration of SCs in wastewater is often extremely low on account of their hydrophobic nature, thus presenting a significant obstacle to the accurate detection and quantification of SCs using WBE. In this study, we present novel magnetic nanomaterials as amphiphilic adsorbents for pretreatment of wastewater using magnetic solid phase extraction (MSPE). Polydopamine-modified Fe 3 O 4 nanoparticles were used as the magnetic core and further functionalized with poly(divinylbenzene- N -vinylpyrrolidone). Coupled with UHPLC-MS/MS analysis, an analytical method to simultaneously detect nine SCs at trace-levels in wastewater was developed and validated, enriching 50 mL wastewater to 100 μL with limits of detection (LOD) being 0.005-0.5 ng L -1 , limits of quantification (LOQ) being 0.01-1.0 ng L -1 , recoveries ranging from 73.99 to 110.72%, and the intra- and inter-day precision's relative standard deviations less than 15%. In comparison to the time-consuming conventional column-based solid phase extraction, the entire MSPE procedure from sample pre-treatment to data acquisition could be finished in one hour, thus largely facilitating the WBE method for drug surveillance and control.

Introduction: In the dynamic universe of new psychoactive substances (NPS), the identification of multiple and chemically diverse compounds remains a challenge for forensic laboratories. Since hair analysis represents a gold-standard to assess the prevalence of NPS, which are commonly detected together with classical drugs of abuse (DoA), our study aimed at developing a wide-screen method to detect and quantify 127 NPS and 15 DoA on hair., Materials and Methods: A multi-analyte ultra-high performance liquid chromatography mass spectrometry method for the identification and quantification of 127 NPS (phenethylamines, arylcyclohexylamines, synthetic opioids, tryptamines, synthetic cannabinoids, synthetic cathinones, designer benzodiazepines) and 15 DoA in hair samples was developed. A full validation was performed according to the European medicines Agency (EMA) guidelines, by assessing selectivity, linearity, accuracy, precision, limit of quantification (LOQ), limit of detection (LOD), matrix effect and recovery. As a proof of the applicability, the method was applied to 22 authentic hair samples collected for forensic purposes., Results: Successful validation was achieved, by meeting the required technical parameters, for 137 compounds (122 NPS and 15 DoA), with LOQ set at 4 pg/mg for 129 compounds, at 10 pg/mg for 6 and at 40 pg/mg for 2. The method was not considered validated for 5 NPS, as LLOQ resulted too high for a forensic analysis (80 pg/mg). Among authentic forensic samples, 17 tested positive for DoA, and 10 to NPS, most samples showing positivity for both. Detected NPS were ketamine and norketamine, 5-MMPA, ritalinic acid, methoxyacetyl fentanyl, methylone and RCS-4., Conclusion: The present methodology represents an easy, low cost, wide-panel method for the quantification of 122 NPS and 15 DoA, for a total of 137 analytes, in hair samples. The method can be profitably applied by forensic laboratories. Similar multi-analyte methods on the hair matrix might be useful in the future to study the prevalence of NPS and the co-occurrence of NPS-DoA abuse., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)