Activity-based detection of consumption of synthetic cannabinoids in authentic urine samples using a stable cannabinoid reporter system

Synthetic cannabinoids (SCs) continue to be the largest group of new psychoactive substances (NPS) monitored by the European Monitoring Center of Drugs and Drugs of Abuse (EMCDDA). The identification and subsequent prohibition of single SCs has driven clandestine chemists to produce analogues of increasing structural diversity, intended to evade legislation. That structural diversity, combined with the mostly unknown metabolic profiles of these new SCs, poses a big challenge for the conventional targeted analytical assays, as it is difficult to screen for "unknown" compounds. Therefore, an alternative screening method, not directly based on the structure but on the activity of the SC, may offer a solution for this problem. We generated stable CB1 and CB2 receptor activation assays based on functional complementation of a split NanoLuc luciferase and used these to test an expanded set of recent SCs (UR-144, XLR-11, and their thermal degradation products; AB-CHMINACA and ADB-CHMINACA) and their major phase I metabolites. By doing so, we demonstrate that several major metabolites of these SCs retain their activity at the cannabinoid receptors. These active metabolites may prolong the parent compound's psychotropic and physiological effects and may contribute to the toxicity profile. Utility of the generated stable cell systems as a first -line screening tool for SCs in urine was also demonstrated using a relatively large set of authentic urine samples. Our data indicate that the stable CB reporter assays detect CB receptor, activation by extracts of urine in which SCs (or their metabolites) are present at low- or subnanomolar (ng/mL) level. Hence, the developed assays do not only allow activity profiling of SCs and their metabolites, it may also serve as a screening tool, complementing targeted and untargeted analytical assays and preceding analytical (mass spectrometry based) confirmation

Detection of AP-237 and synthetic cannabinoids on an infused letter sent to a German prisoner

In the past years, new psychoactive substances (NPS) started circulating in prisons, leading to health risks and challenges for the criminal justice system. Seizures of papers and cards impregnated with synthetic cannabinoid (SCs) have been reported. In November 2021, a letter suspected to be drug-infused was sent from a German prison to this laboratory. Toxicological analyses were performed by means of gas chromatography-mass spectrometry (GC-MS) for drug screening and liquid chromatography-tandem mass spectrometry (LC-MS/MS) as well as high-performance (HP) LC with diode-array detection (DAD) for semi-quantification of the compounds. The novel synthetic opioid (NSO) AP-237 was detected on the letter, with an estimated concentration of 1.2mug/cm2 , together with the SCs MDMB-4en-PINACA (77mug/cm2 ) and 5F-ADB (6.5mug/cm2 ). To the best of the authors' knowledge, this is the first time an NSO was detected on a drug-infused paper seized in a prison. Highly potent NSOs could easily be dissolved in organic solvents to produce impregnated papers and textiles, and this might represent a serious threat to the health of people in prison. Given the inhomogeneity in drug concentrations, health risks might in particular arise from the consumption of highly concentrated areas of the paper-so-called "hot spots"-especially when highly potent NSOs are used for infusion. Laboratories engaged in analyzing such impregnated papers should be aware of the potential presence of NSOs and adapt the respective methods accordingly

Post-Mortem Toxicology: A Systematic Review of Death Cases Involving Synthetic Cannabinoid Receptor Agonists

BackgroundSynthetic cannabinoid receptor agonists (SCRAs) have become the largest group of new psychoactive substances monitored by the European Union Early Warning System. Despite the wide diffusion on the market, data regarding effects, toxicities, and mechanisms as well as toxic/lethal doses are still scarce.MethodsA comprehensive literature search for articles published up to January 2019 was performed in multiple electronic databases. Only cases of death in which toxicological analyses revealed the presence of SCRAs in blood or urine and at least an external examination was performed, including those occurred in emergency departments, were included.ResultsOf 380 studies identified, 354 were excluded, while 8 additional manuscripts were included through the screening of relevant references cited in the selected articles. A total number of 34 manuscripts (8 case series and 26 case reports) were included.ConclusionsTypical toxic ranges for SCRAs have not been so far identified, and the results of toxicological analyses should be interpreted with caution. In death cases involving SCRAs, a thorough post-mortem examination is a prerequisite to assess the role of the substance use in the deceased and to identify a probable mechanism of death. Even after a comprehensive analysis of clinical, circumstantial, toxicological, and autoptic data, the cause and manner of death remain unclear in some cases

Development and validation of a rapid LC-MS/MS method for the detection of 182 novel psychoactive substances in whole blood

INTRODUCTION: The analysis of novel psychoactive substances (NPS) represents a challenge in forensic toxicology, due to the high number of compounds characterized by different structures and physicochemical properties both among different subclasses and within a single subclass of NPS. The aim of the present work is the development and validation of a targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the detection of NPS in whole blood.MATERIALS AND METHODS: A protein-precipitation based LC-MS/MS method for the detection of more than 180 NPS was developed and validated by assessing the following parameters: selectivity, linearity, accuracy, precision, limit of detection (LOD) and of quantification (LOQ) recovery, and matrix effect. Then, the method was applied to real forensic samples.RESULTS: The method allowed the identification of 132 synthetic cannabinoids, 22 synthetic opioids, and 28 substances among synthetic cathinones, stimulants, and other drugs. Validation was successfully achieved for most of the compounds. Linearity was in the range of 0.25-10ng/ml for synthetic cannabinoids and 0.25-25ng/ml for other drugs. Accuracy and precision were acceptable according to international guidelines. Three cases tested positive for fentanyl and ketamine, in the setting of emergency room administration.CONCLUSIONS: The present methodology represents a fast, not expensive, wide-panel method for the analysis of more than 180 NPS by LC-MS/MS, which can be profitably applied both in a clinical context and in postmortem toxicology

Detection and activity profiling of synthetic cannabinoids and their metabolites with a newly developed bioassay

Synthetic cannabinoids (SCs) are the largest group of compounds currently monitored in Europe by the EU Early Warning System on new psychoactive substances. Emerging recreational use of these products has led to multiple cases of adverse health effects and even death. In contrast to marijuana, where Delta(9)-tetrahydrocannabinol (Delta(THC)-T-9) is metabolized to only one major active metabolite, it has been reported that several major phase I metabolites of SCs remain biologically active, exerting cannabinoid (CB) receptor affinity, potency, and efficacy greater than those of Delta(THC)-T-9. It is therefore reasonable that more SCs can also be biotransformed into molecules with various levels of CB activity. Here, we developed and applied a new G-protein coupled receptor (GPCR) activation assay based on NanoLuc binary technology (Promega). More specifically, by demonstrating CB1 and CB2 receptor activation by JWH-018 and a selection of its metabolites, we are the first to show the suitability of the newly developed bioassay for monitoring GPCR-mediated activity. We also successfully applied this reporter system to evaluate the in vitro activity of JWH-122, JWH-210, and PB-22, their S-fluoro analogues (MAM-2201, EAM-2201, and SF-PB-22, respectively), and their main phase I metabolites. By doing so, we demonstrate that several major metabolites of these SCs retain their activity at cannabinoid receptors. All of these active metabolites may prolong the parent compound's psychotropic and physiological effects and may contribute to its toxicity profile. We also demonstrate a proof of concept of the applicability of the newly developed bioassay for screening urine for CB receptor activity exerted by SCs

CONCENTRATION OF FATTY ACID ETHYL ESTERS IN HAIR OF ALCOHOLICS: COMPARISON TO OTHER BIOLOGICAL STATE MARKERS AND SELF REPORTED-ETHANOL INTAKE

Aims: In a variety of clinical and forensic situations long term use of alcohol must be monitored. In this project we explore the utility of fatty acid ethyl esters (FAEE) in this regard. Additionally, we propose a cut-off value of FAEE to distinguish teetotallers/moderate/social drinkers from alcoholics or individuals drinking at harmful levels. Patients and methods: FAEE levels from 18 alcohol-dependent patients in detoxification were contrasted with those of 10 social drinkers and 10 teetotallers. FAEE in hair were determined, using headspace solid phase microextraction and gas chromatography mass spectrometry. CFAEE, as sum of the concentrations of four esters, was compared to a major FAEE, ethyl palmitate. PEth was measured in heparinized whole blood with a high pressure liquid chromatography (HPLC) method. Drinking validation criteria include self reports, phosphatidyl ethanol (PEth) in whole blood as well as the traditional markers of heavy drinking, gamma glutamyl transpeptidase (GGT), mean corpuscular volume (MCV) and carbohydrate deficient transferrin (CDT). Results: Receiver-operating characteristic (ROC) curve analysis for CFAEE, indicated a sensitivity of 100% and a specificity of 90% for a cut-off of 0.29 ng/mg. By using a cut-off of 0.4 ng/mg, CFAEE identified 94.4% correctly. CFAEE and ethyl palmitate were significantly associated (r = 0.945; P < 0.001) as were CFAEE and PEth (r = 0.527; P = 0.025). No significant correlation was found between CFAEE and total grams of ethanol consumed last month, blood-alcohol concentration at admission to the hospital, CDT, MCV, or GGT. Among the serum and blood markers, %CDT identified 47.1%, MCV 38.8% and GGT 72.2% of patients with chronic intake of higher amounts of ethanol correctly, whereas PEth achieved 100% accuracy. Conclusions: The data suggest that CFAEE is a potentially valuable marker of chronic intake of high quantities of ethanol. Furthermore, the results indicate that a reasonable and provisional FAEE cut-off to distinguish between social/moderate and heavy drinking/alcoholism in hair is 0.4 ng/m

Metabolic Pathways and Potencies of New Fentanyl Analogs

Up to now, little is known about the metabolic pathways of new fentanyl analogs that have recently emerged on the drug markets worldwide with high potential for producing addiction and severe adverse effects including coma and death. For some of the compounds, limited information on the metabolism has been published, however, for others so far no information is available. Considering the well characterized metabolism of the pharmaceutically used opioid fentanyl and the so far available data, the metabolism of the new fentanyl analogs can be anticipated to generally involve reactions like hydrolysis, hydroxylation (and further oxidation steps), N- and O-dealkylation and O-methylation. Furthermore, phase II metabolic reactions can be expected comprising glucuronide or sulfate conjugate formation. When analyzing blood and urine samples of acute intoxication cases or fatalities, the presence of metabolites can be crucial for confirmation of the uptake of such compounds and further interpretation. Here we present a review on the metabolic profiles of new fentanyl analogs responsible for a growing number of severe and fatal intoxications in the United States, Europe, Canada, Australia, and Japan in the last years, as assessed by a systematic search of the scientific literature and official reports

Cannabinoid Receptor 2 Signaling Does Not Modulate Atherogenesis in Mice

BACKGROUND:Strong evidence supports a protective role of the cannabinoid receptor 2 (CB(2)) in inflammation and atherosclerosis. However, direct proof of its involvement in lesion formation is lacking. Therefore, the present study aimed to characterize the role of the CB(2) receptor in Murine atherogenesis. METHODS AND FINDINGS:Low density lipoprotein receptor-deficient (LDLR(-/-)) mice subjected to intraperitoneal injections of the selective CB(2) receptor agonist JWH-133 or vehicle three times per week consumed high cholesterol diet (HCD) for 16 weeks. Surprisingly, intimal lesion size did not differ between both groups in sections of the aortic roots and arches, suggesting that CB(2) activation does not modulate atherogenesis in vivo. Plaque content of lipids, macrophages, smooth muscle cells, T cells, and collagen were also similar between both groups. Moreover, CB(2) (-/-)/LDLR(-/-) mice developed lesions of similar size containing more macrophages and lipids but similar amounts of smooth muscle cells and collagen fibers compared with CB(2) (+/+)/LDLR(-/-) controls. While JWH-133 treatment reduced intraperitoneal macrophage accumulation in thioglycollate-elicited peritonitis, neither genetic deficiency nor pharmacologic activation of the CB(2) receptor altered inflammatory cytokine expression in vivo or inflammatory cell adhesion in the flow chamber in vitro. CONCLUSION:Our study demonstrates that both activation and deletion of the CB(2) receptor do not relevantly modulate atherogenesis in mice. Our data do not challenge the multiple reports involving CB(2) in other inflammatory processes. However, in the context of atherosclerosis, CB(2) does not appear to be a suitable therapeutic target for reduction of the atherosclerotic plaque

Messunsicherheit und Grenzwerte

Um ein Bild von der aktuellen Messunsicherheit bei der Bestimmung von Tetrahydrocannabinol (THC) in Serum zu erhalten, wurden von sieben forensisch-toxikologischen Laboren rechtsmedizinischer Institute Daten der letzten Jahre erhoben. Zur Abschätzung der Messunsicherheit wurde eine Einteilung in zufällige Fehler (Präzision) und systematische Fehler (Richtigkeit) vorgenommen. Ergebnisse zur Richtigkeit wurden aus Daten von Ringversuchen ermittelt. Es ergab sich eine mittlere erweiterte Messunsicherheit für die THC-Bestimmung von ca. 33 %, das heisst, bei einem Messwert von 1 ng/ml liegt der "wahre Wert" mit circa 95 prozentiger Wahrscheinlichkeit im Intervall zwischen 0,67 und 1,33 ng/ml. Dieses Ergebnis bestätigt, dass die verwendete Analytik geeignet ist, bei Konzentrationen um 1 ng/ml einen sicheren Substanznachweis zu führen. Es ist erkennbar, dass bei der THC-Bestimmung im Serum in der Regel der Richtigkeitsanteil an der Messunsicherheit größer ist als der Präzionsanteil