Identification of synthetic cannabinoids in herbal incense blends in the United States

Abstract: Synthetic cannabinoid agonists are chemically diverse with multiple analogs gaining popularity as drugs of abuse. We report on the use of thin layer chromatography, gas chromatography mass spectrometry, high‐performance liquid chromatography, and liquid chromatography time of flight mass spectrometry for the identification and quantitation of these pharmacologically active chemicals in street drug dosage forms. Using these approaches, we have identified the synthetic cannabinoids JWH‐018, JWH‐019, JWH‐073, JWH‐081, JWH‐200, JWH‐210, JWH‐250, CP47,497 (C=8) (cannabicyclohexanol), RCS‐4, RCS‐8, AM‐2201, and AM‐694 in various commercially available products. Other noncannabinoid drugs including mitragynine have also been detected. Typical concentrations of drug in the materials are in the range 5–20 mg/g, or 0.5–2% by weight for each compound, although many products contained more than one drug.     

Rapid Identification of Synthetic Cannabinoids in Herbal Incenses with DART‐MS and NMR

The usage of herbal incenses containing synthetic cannabinoids has caused an increase in medical incidents and triggered legislations to ban these products throughout the world. Law enforcement agencies are experiencing sample backlogs due to the variety of the products and the addition of new and still‐legal compounds. In our study, proton nuclear magnetic resonance (NMR) spectroscopy was employed to promptly screen the synthetic cannabinoids after their rapid, direct detection on the herbs and in the powders by direct analysis in real time mass spectrometry (DART‐MS). A simple sample preparation protocol was employed on 50 mg of herbal sample matrices for quick NMR detection. Ten synthetic cannabinoids were discovered in fifteen herbal incenses. The combined DART‐MS and NMR methods can be used to quickly screen synthetic cannabinoids in powder and herbal samples, serving as a complementary approach to conventional GC‐MS or LC‐MS methods.     

Identification of Eight Synthetic Cannabinoids,Including 5F‐AKB48 in Seized Herbal Products Using DART‐TOF‐MS and LC‐QTOF‐MS as Nontargeted Screening Methods

Synthetic cannabinoids are sprayed onto plant material and smoked for their marijuana‐like effects. Clandestine manufacturers modify synthetic cannabinoid structures by creating closely related analogs. Forensic laboratories are tasked with detection of these analog compounds, but targeted analytical methods are often thwarted by the structural modifications. Here, direct analysis in real time coupled to accurate mass time‐of‐flight mass spectrometry (DART‐TOF‐MS) in combination with liquid chromatography quadruple time‐of‐flight mass spectrometry (LC‐QTOF‐MS) are presented as a screening and nontargeted confirmation method, respectively. Methanol extracts of herbal material were run using both methods. Spectral data from four different herbal products were evaluated by comparing fragmentation pattern, accurate mass and retention time to available reference standards. JWH‐018, JWH‐019, AM2201, JWH‐122, 5F‐AKB48, AKB48‐N‐(4‐pentenyl) analog, UR144, and XLR11 were identified in the products. Results demonstrate that DART‐TOF‐MS affords a useful approach for rapid screening of herbal products for the presence and identification of synthetic cannabinoids.     

Synthetic cannabinoids in "spice-like" herbal blends: First appearance of JWH-307 and recurrence of JWH-018 on the German market

Herbal smoking blends, available on the German market were analyzed and several known synthetic cannabinoids were identified (JWH-122 and JWH-018). In addition, we isolated a new active ingredient by silica gel column chromatography and elucidated the structure by nuclear magnetic resonance (NMR) methods. The compound was identified as JWH-307, a synthetic cannabinoid of the phenyl-pyrrole subclass with known in vitro binding affinities for cannabinoid receptors. To date, this is the first appearance of this subclass of cannabimimetics in such products. JWH-307 has been further characterized by gas chromatography accurate mass spectrometry (GC-HRMS), electrospray tandem mass spectrometry (ESI-MS/MS), ultraviolet (UV) and infrared (IR) spectroscopy. JWH-018 was among the first compounds banned by many countries world-wide including Germany. The identification of JWH-018 was striking, since this is the first report where JWH-018 recurred on the German market thus violating existing laws. A generic method was established to quantify synthetic cannabinoids in herbal smoking blends. Quantification was achieved using an isotopically labeled standard (JWH-018-D(3)). JWH-018 was found at a level of 150mg/g while JWH-122 and JWH-307 occurred as a mixture at a total level of 232mg/g.     

DART‐MS as a Preliminary Screening Method for “Herbal Incense”: Chemical Analysis of Synthetic Cannabinoids

Direct analysis in real time mass spectrometry (DART‐MS) served as a method for rapid high‐throughput screening of six commercially available “Spice” products, detecting various combinations of five synthetic cannabinoids. Direct analysis in real time is an ambient ionization process that, along with high mass accuracy time‐of‐flight (TOF)‐MS to 0.0001 Da, was employed to establish the presence of cannabinoids. Mass spectra were acquired by simply suspending a small portion of sample between the ion source and the mass spectrometer inlet. The ability to test minute amounts of sample is a major advantage when very limited amounts of evidentiary material are available. In addition, reports are widespread regarding the testing backlogs that now exist because of the large influx of designer drugs. This method circumvents time‐consuming sample extraction, derivatization, chromatographic, and other sample preparative steps required for analysis by more conventional mass spectrometric methods. Accordingly, the synthetic cannabinoids AM‐2201, JWH‐122, JWH‐203, JWH‐210, and RCS‐4 were identified in commercially available herbal Spice products, singly and in tandem, at concentrations within the range of 4–141 mg/g of material. Direct analysis in real time mass spectrometry decreases the time necessary to triage analytical evidence, and therefore, it has the potential to contribute to backlog reduction and more timely criminal prosecution.     

Driving Under the Influence of Synthetic Cannabinoid Receptor Agonist XLR‐11

The case of a 22‐year‐old male Caucasian driver is presented. He was involved in a traffic collision. At the roadside, he displayed blank stare and mellow speech with a barely audible voice. A DRE found low body temperature, rigid muscle tone, normal pulse, lack of horizontal and vertical gaze nystagmus, nonconvergence of the eyes, dilated pupil size, and normal Pupillary reaction to light. A standard toxicology DUID protocol was performed on the driver's whole blood including ELISA and GC‐MS drug screens with negative results. Additional drug screening was undertaken for bath salts and synthetic cannabinoid receptor agonists by LC‐MS/MS by a commercial laboratory and identified the synthetic cannabinoid receptor agonist XLR‐11 in the driver's blood. XLR‐11 was subsequently quantified at 1.34 ng/mL. This is the first documented case involving a driver operating a motor vehicle under the influence of the synthetic cannabinoid receptor agonist XLR‐11.     

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.     

Near‐fatal Intoxication with the “New” Synthetic Opioid U‐47700: The First Reported Case in the Czech Republic

Recreational use of the potent synthetic opioid 3,4‐ dichloro‐N‐(2‐(dimethylamino)cyclohexyl)‐N‐methylbenzamide (U‐47700) is rising, accompanied by increasingly frequent cases of serious intoxication. This article reports a case of near‐fatal U‐47700 intoxication. A man was found unconscious (with drug powder residues). After 40 h in hospital (including 12 h of supported ventilation), he recovered and was discharged. Liquid chromatography/high‐resolution mass spectrometry (LC/HRMS) or gas chromatography/mass spectrometry (GC/MS) were used to detect and quantify substances in powders, serum and urine. Powders contained U‐47700 and two synthetic cannabinoids. Serum and urine were positive for U‐47700 (351.0 ng/mL), citalopram (<LOQ), tetrahydrocannabinol (THC: 3.3 ng/mL), midazolam (<LOQ) and a novel benzodiazepine, clonazolam (6.8 ng/mL) and their metabolites but negative for synthetic cannabinoids. If potent synthetic opioids become cheaper and more easily obtainable than their classical counterparts (e.g., heroin), they will inevitably replace them and users may be exposed to elevated risks of addiction and overdose.     

合成大麻素JWH-122的高效液相色谱分析方法研究

目的研究被我国《非药用类麻醉药品和精神药品列管办法》列入管制的合成大麻素JWH-122的高效液相色谱分析方法。方法以甲醇-去离子水(50%-50%)为流动相进行梯度洗脱,考查有机相初始浓度、梯度陡度、柱温、流速等色谱条件及检测波长,确定最优实验条件,在优化条件下对线性范围及专属性进行实验,通过实际样本检测对所建方法进行验证。结果紫外光谱检测波长221nm、有机相初始浓度为70%、梯度陡度为0.5%/min、流速1.2ml/min、柱温30℃条件下JWH-122在0.002mg/ml-0.1mg/ml范围内线性良好,检出限(S/N≥3)为0.1μg/ml;实际样本检测表明,优化条件下JWH-122能与样本中其它组分很好分离。结论该方法具有快速、灵敏、准确、分离效果好的优点,适用于新型香料毒品中合成大麻素JWH-122的分析检测。     

Development of a specific fragmentation pattern-based quadrupole-Orbitrap™ mass spectrometry method to screen drugs in illicit products

Over the past decade, illicit drugs have been founded in marketed products, which pose a risk to public health. In particular, newly designed analogues synthesized by chemical modification of parent compounds to avoid detection by authorities are frequently detected worldwide. Although many analytical methods for determination of drugs have been reported, analytical methods using high-resolution mass spectrometry, which has the advantage of rapid screening and accurate identification of new substances, are necessary to control illicit drugs in marketed products. In this study, a rapid analytical method using an Orbitrap™ mass spectrometer for identification of illicit drugs in marketed products was developed. The 32 drugs were classified as benzodiazepine-, synthetic cannabinoid-, amphetamine- and benzylpiperazine-type drugs according to their chemical structures, and from their fragmentation patterns in tandem mass spectrometry spectra of an established method. The method validation gave a limit of detection of 0.06–5.30 ng/mL and a limit of quantification of 0.18–16.50 ng/mL, high linearity (R² > 0.994) and mean recoveries of spiked matrix-blank samples ranging from 83.7% to 117.1%. Approximately 71% of 21 samples collected over 3 years were found to individually contain one of four types of benzodiazepines or two different synthetic cannabinoids. In one case, levels as high as 827.2 mg/g were measured suggesting adulteration at high levels, which suggests that potential illicit products containing drugs should be regularly screened to protect public health.     

Species Identification of Cannabis sativa Using Real‐Time Quantitative PCR (qPCR),

Most narcotics‐related cases in the United States involve Cannabis sativa. Material is typically identified based on the cystolithic hairs on the leaves and with chemical tests to identify of the presence of cannabinoids. Suspect seeds are germinated into a viable plant so that morphological and chemical tests can be conducted. Seed germination, however, causes undue analytical delays. DNA analyses that involve the chloroplast and nuclear genomes have been developed for identification of C. sativa materials, but they require several nanograms of template DNA. Using the trnL 3′ exon‐trnF intragenic spacer regions within the C. sativa chloroplast, we have developed a real‐time quantitative PCR assay that is capable of identifying picogram amounts of chloroplast DNA for species determination of suspected C. sativa material. This assay provides forensic science laboratories with a quick and reliable method to identify an unknown sample as C. sativa.     

Quantitative Determination of Cannabinoids in Cannabis and Cannabis Products Using Ultra‐High‐Performance Supercritical Fluid Chromatography and Diode Array/Mass Spectrometric Detection

Ultra‐high‐performance supercritical fluid chromatography (UHPSFC ) is an efficient analytical technique and has not been fully employed for the analysis of cannabis. Here, a novel method was developed for the analysis of 30 cannabis plant extracts and preparations using UHPSFC /PDA ‐MS . Nine of the most abundant cannabinoids, viz . CBD , ?⁸‐THC , THCV , ?⁹‐THC , CBN , CBG , THCA ‐A, CBDA , and CBGA , were quantitatively determined (RSD s < 6.9%). Unlike GC methods, no derivatization or decarboxylation was required prior to UHPSFC analysis. The UHPSFC chromatographic separation of cannabinoids displayed an inverse elution order compared to UHPLC . Combining with PDA ‐MS , this orthogonality is valuable for discrimination of cannabinoids in complex matrices. The developed method was validated, and the quantification results were compared with a standard UHPLC method. The RSD s of these two methods were within ±13.0%. Finally, chemometric analysis including principal component analysis (PCA ) and partial least squares‐discriminant analysis (PLS ‐DA ) were used to differentiate between cannabis samples.     

气相色谱/质谱检验合成大麻素K3中AKB48

目的建立较为快速准确的合成大麻素K3中AKB48的气相色谱/质谱检验方法。方法对进样口温度、初始柱温、柱流速及质谱采样率等4项色谱及质谱实验参数进行考察优化。结果 GC/MS检验合成大麻素K3中AKB48的优化条件为:进样口温度280℃,柱初始温度80℃,柱流速为2.0ml/min,质谱采样率为2。结论该方法具有快速、准确、灵敏等优点,可用于K3中AKB48的定性检验鉴定。     

Determination of Herbicides Paraquat,Glyphosate, and Aminomethylphosphonic Acid in Marijuana Samples by Capillary Electrophoresis

In this work, two methods were developed to determine herbicides paraquat, glyphosate, and aminomethylphosphonic acid (AMPA) in marijuana samples by capillary electrophoresis. For paraquat analysis, sample was extracted with aqueous acetic acid solution and analyzed by capillary zone electrophoresis with direct UV detection. The running electrolyte was 50 mmol/L phosphate buffer (pH 2.50). For glyphosate and AMPA, indirect UV/VIS detection was used, as these substances do not present chromophoric groups. Samples were extracted with 5 mmol/L hydrochloric acid. The running electrolyte was 10 mmol/L gallic acid, 6 mmol/L TRIS, and 0.1 mmol/L CTAB (pH = 4.7). The methods presented good linearity, precision, accuracy, and recovery. Paraquat was detected in 12 samples (n = 130), ranging from 0.01 to 25.1 mg/g. Three samples were positive for glyphosate (0.15–0.75 mg/g), and one sample presented AMPA as well. Experimental studies are suggested to evaluate the risks of these concentrations to marijuana user.     

Applicability of Cranial Models in Urethane Resin and Foam as a Substitute for Bone: Are Synthetic Materials Reliable?

As literature is poor in functional synthetic cranial models, in this study, synthetic handmade models of cranial vaults were produced in two different materials (a urethane resin and a self‐hardening foam), from multiple bone specimens (eight original cranial vaults: four human and four swine), in order to test their resemblance to bone structure in behavior, during fracture formation. All the vaults were mechanically tested with a 2‐kg impact weight and filmed with a high‐speed camera. Fracture patterns were homogeneous in all swine vaults and heterogeneous in human vaults, with resin fractures more similar to bone fractures. Mean fracture latency time extrapolated by videos were of 0.75 msec (bone), 1.5 msec (resin), 5.12 msec (foam) for human vaults and of 0.625 msec (bone), 1.87 msec (resin), 3.75 msec (foam) for swine vaults. These data showed that resin models are more similar to bone than foam reproductions, but that synthetic material may behave quite differently from bone as concerns fracture latency times.     

Determination of salvinorin A and salvinorin B in Salvia divinorum-related products circulated in Japan

Two major salvinorins, salvinorin A (SalA) and salvinorin B (SalB), in three Salvia divinorum dried leaf products and nine of its “concentrated extract” products circulated in Japan were determined. These ingredients were extracted twice with acetonitrile and decolored with graphite carbon powder. SalA and SalB were confirmed by liquid chromatography–tandem mass spectrometry in product ion scan mode, and quantified by high-performance liquid chromatography with UV detection (for SalA) and by mass spectrometry in single ion monitoring mode (for SalB). The SalA/SalB contents (μg/mg) were in the range of 3.2–5.0/0.10–0.17 in the dried leaf products and 4.1–38.9/0.26–2.42 in the “concentrated extract” products. These findings would be useful for analysis of S. divinorum-related products circulated in the drug market.     

Determination of Acetamiprid and IM‐1‐2 in PostMortem Human Blood,Liver, Stomach Contents by HPLC‐DAD

An HPLC‐DAD method was developed to detect and quantify a neonicotinoid insecticide acetamiprid (ATP) and its metabolite IM‐1‐2 in autopsy samples of a fatal intoxication case. The postmortem blood and tissue distribution of ATP and IM‐1‐2 was determined for the first time. The method showed acceptable precisions and recoveries with relative standard deviations of <10% for ATP level and 1.38 % for IM‐1‐2. The detection and quantification limits for ATP were 0.015 μg/mL and 0.030 μg/mL for blood and were 0.035 μg/g and 0.050 μg/g for liver samples, respectively. The mean contents of ATP were 0.79 μg/g in the liver, 47.35 μg/g in the stomach contents and 2.7 μg/mL in the blood. IM‐1‐2 content was 17.0 μg/g in the stomach contents. ATP and IM‐1‐2 were not detected in the urine. The presence of ATP and IM‐1‐2 in the samples was confirmed by GC‐MS. The method can be exploited in future forensic casework.     

K2 Toxicity: Fatal Case of Psychiatric Complications Following AM2201 Exposure

Limited forensic and clinical experience and the lack of confirmatory testing strategies for synthetic cannabinoids (SC) prevent adequate characterization of SC toxicity and the potential impact on public health. A statewide surveillance system identified a fatality involving a 23‐year‐old man found with a large stab wound to the neck following use of a SC product suspected of containing AM2201. Analytical testing for common SCs, SC metabolites, routine drugs of abuse, and over‐the‐counter medications was performed on heart blood obtained at autopsy. Additionally, assays were performed on the SC raw material and drug paraphernalia found on the decedent. High concentrations of AM2201 were detected in all samples. AM2201 metabolites were detected in postmortem blood. Other than a trace amount of JWH‐073 found in smoke residue, no other substances were detected. Psychiatric complications including self‐induced, lethal trauma can occur after the use of SC products.