Molecularly Imprinted Polymer-based voltammetric sensor for amino acids/indazole derivatives synthetic cannabinoids detection.

BACKGROUND: Synthetic cannabinoids (SCs) are a broad class of illicit drugs that are classified according to the chemical structure of the aromatic core that they present (i.e., indole, imidazole, pyrrole) and their detection is still a challenge, despite their widespread diffusion. The identification of a specific class of SC in complex matrices, such as real samples with a rapid, economic analytical device useable directly in the field, is highly desirable, as it can provide immediate and reliable information that eventually addresses more targeted analyses. RESULTS: The present paper proposes a Molecularly Imprinted Polymer (MIP)-based voltammetric sensor for the rapid and selective detection of indazole-type SCs. In this context, a polyacrylate-based MIP was used to functionalize a Pt electrode. The MIP composition was optimized through a Design of Experiments approach, and for the sake of safety, a non-psychotropic compound structurally related to the selected SCs was employed as the template in the MIP formulation. A complete characterization of the electrochemical behavior of the selected SCs was performed, and differential pulse voltammetry (DPV) in acetonitrile/lithium perchlorate 0.1 M was the technique applied for their quantification. LOD around 0.01 mM and linearity up to 0.8 mM were found. Comparison with the non-imprinted (NIP) modified and bare electrodes showed better selectivity and reproducibility of the MIP-based sensor. Recovery tests (in the 70-115 % range) were performed on simulated pills and smoking mixtures to test the reliability of the proposed method. SIGNIFICANCE: The method proposed allows the identification and quantification of indazole-based SCs as a class in complex matrices. Due to the selectivity of the obtained device, no clean-up of the sample before analyses is needed. For the same reason, the interference of cutting substances and natural cannabinoids was negligible.

Toxic effects of AB-CHMINACA on liver and kidney and detection of its blood level in adult male mice.

BACKGROUND: AB-CHMINACA is a cannabimimetic indazole derivative. In 2013, it was reported in different countries as a substance of abuse. PURPOSE: This study evaluated the subacute toxic effects of AB-CHMINACA on the liver and kidneys and measured its blood level in adult male mice. METHODS: The histological and biochemical subacute toxic effects on the liver and kidneys were assessed after four weeks of daily intraperitoneal injections of one of the following doses: 0.3 mg/kg, 3 mg/kg, or 10 mg/kg as the highest dose in adult male albino mice. In addition, the blood concentration level of AB-CHMINACA was determined by GC-MS-MS. RESULTS: The histological effects showed congestion, hemorrhage, degeneration, and cellular infiltration of the liver and kidney tissues. Considering the control groups as a reference, biochemical results indicated a significant increase in the serum AST only in the highest dose group, while the ALT and creatinine levels did not significantly change. The mean values of AB-CHMINACA blood levels were 3.05 ± 1.16, 15.08 ± 4.30, and 54.43 ± 8.70 ng/mL for the three treated groups, respectively, one hour after the last dose of intraperitoneal injection. The calibration curves were linear in the 2.5-500 ng/mL concentration range. The intra-assay precision and accuracy of the method were less than 7.0% (RSD) and ± 9.2% (Bias). CONCLUSION: This research supports the available case reports on AB-CHMINACA toxicity that it has low lethality; still, the chronic administration causes evident liver and kidney histotoxic effects even at low doses with unnoticeable clinical effects in mice.

[Determination of four amide synthetic cannabinoid isomers by ultra-high performance liquid chromatography-high resolution mass spectrometry].

Isomerization commonly occurs in synthetic cannabinoids (SCs). Owing to the few differences in their structure and properties, it is difficult to simultaneously separate and identify them. Thus, the identification of synthetic cannabinoids is challenging, posing a threat to public security. This study aims to separate and identify four SCs, which are 2-[1-(5-fluoropentyl)-1H-indole-3-formylamino]-3,3-dimethylbutyrate methyl ester (5F-MDMB-PICA), 2-[1-(5-fluoropentyl)-1H-indole-3-formylamino]-3-methylbutyrate ethyl ester (5F-EMB-PICA), N-(1-amino-2,2-dimethyl-1-oxobutyl-2-yl)-1-butyl-1H-indazole-3-formamide (ADB-BINACA), N-(1-carbamoyl-2-methylpropyl)-1-pentyl indazole-3-formamide (AB-PINACA).Supercritical fluid chromatography-mass spectroscopy (SFC-MS) can realize the effective separation of some cannabinoid isomers. However, most laboratories are not equipped with SFC-MS systems. Ultra-high performance liquid chromatography-high resolution mass spectroscopy (UHPLC-HRMS) effectively combines the excellent efficient separation characteristics of liquid chromatography and the powerful qualitative ability of mass spectrometry. It is a commonly used technical method for the detection of amide synthetic cannabinoids and their metabolites in vivo and in vitro because of its advantages of high accuracy and efficiency. Liquid chromatography allows the separation of tested components by exploiting the difference in the partition coefficients between the mobile and stationary phases. When the two phases are in relative motion, the tested components are divided between the two phases, facilitating the separation and analysis of each component. Although the difference in the polarities of the tested amide synthetic cannabinoid isomeric substances is extremely small, liquid chromatography can induce a strong separation effect. The advantages of UHPLC-HRMS include high resolution imparted by mass spectrometry and high sensitivity, allowing its application in the qualitative analysis of various substances. Through UHPLC-HRMS, trace analytes at the nanogram scale as well as pure drugs and their metabolites in biosamples can be detected. This study proposed a method for the determination of two pairs of amide synthetic cannabinoid isomers-5F-EMB-PICA and 5F-MDMB-PICA, ADB-BINACA and AB-PINACA-through UHPLC-HRMS. A Hypersil GOLD C18 column (100 mm×2.1 mm, 1.9 µm) was selected for separation via liquid chromatography, and gradient elution was performed with methanol containing 0.1% formic acid and a 0.1% formic acid aqueous solution containing 10 mmol/L ammonium formate. Full scan/data-dependent secondary mass spectrometry (Full MS/dd-MS2) was conducted in the positive ion mode for detection. The results indicated that the four synthetic cannabinoid isomers could be accurately detected under the abovementioned conditions. The resolution between 5F-EMB-PICA and 5F-MDMB-PICA was 2.06, while that between ADB-BINACA and AB-PINACA was 1.22, indicating the effective separation and detection of both pairs. Furthermore, method validation was conducted to ensure the accuracy of the proposed method. The relationship of the four amide synthetic cannabinoid isomers exhibited excellent linearity. The correlation coefficients (R2) were >0.99. Moreover, the matrix effects of the four SCs in hair samples were between 88.67% and 111.76% and the recoveries were 96.23%-105.11%. The intra-day and inter-day precisions (RSDs) were <10%. The proposed method was used to identify the case materials. AB-PINACA was detected in a hair sample at a content of 0.73 µg/g. 5F-MDMB-PICA was detected in a tobacco sample at a content of 11.3 mg/g. The results indicate that the proposed method can be used for the examination of practical samples conducted by public security organizations. This study provides a reference method for the identification of synthetic cannabinoid isomers.

ADB-HEXINACA-a novel synthetic cannabinoid with a hexyl substituent: phase I metabolism in authentic urine samples, a case report and prevalence on the German market.

Synthetic cannabinoid receptor agonists (SCRAs) are one of the largest groups of new psychoactive substances (NPS). Yet, another novel analog started spreading on the NPS market around 2021. Soon after, the substance could be analytically characterized in herbal material as ADB-HEXINACA, an SCRA containing a hexyl-substituted tail on the indazole core. Here, we present suitable urinary markers to prove the consumption of this analog, a case report of acute polydrug intoxication and data on its prevalence in Germany. Anticipated phase I metabolites were detected in 12 authentic urine samples that were collected for abstinence control and analyzed by ultra-performance liquid chromatography coupled to a time-of-flight mass spectrometer (UPLC-qToF-MS). The results of in vivo samples were confirmed by analysis of in vitro incubates with pooled human liver microsomes (pHLMs). Forensic samples were used to assess the prevalence of ADB-HEXINACA. Thirty-two phase I metabolites were detected in the authentic urine samples. The main metabolites resulted from amide hydrolysis in combination with either monohydroxylation or ketone formation at the hexyl moiety (M15 and M26), the monitoring of which is recommended as a proof of consumption. ADB-HEXINACA was detected in 3.5% of SCRA positive urine samples collected for abstinence control in Freiburg up to December 2022 and in 5.5% of the SCRA positive blood/serum samples. The hexyl substituent of ADB-HEXINACA allows for the detection of specific urinary biomarkers suggested as analytical targets to confirm its prior intake. ADB-HEXINACA had a rather low prevalence in Germany, alternating months of higher prevalence with periods of total absence.

[Simultaneous determination of five indole/indazole amide-based synthetic cannabinoids in electronic cigarette oil by ultra performance liquid chromatography].

Synthetic cannabinoids (SCs), which are considered some of the most widely abused new psychoactive substances available today, are much more potent than natural cannabis and display greater efficacy. New SCs can be developed by adding substituents such as halogen, alkyl, or alkoxy groups to one of the aromatic ring systems, or by changing the length of the alkyl chain. Following the emergence of the so-called first-generation SCs, further developments have led to eighth-generation indole/indazole amide-based SCs. Given that all SCs were listed as controlled substances on July 1, 2021, the technologies used to detect these substances must be quickly improved. Due to the sheer number of SCs, the chemical diversity and the fast update speed, it is challenging to determine and identify the new SCs. In recent years, several types of indole/indazole amide-based SCs have been seized, but systematic research on these compounds remains limited. Therefore, developing rapid, sensitive, and accurate quantitative methods to determine new SCs are of great importance. Compared with high performance liquid chromatography (HPLC), ultra performance liquid chromatography (UPLC) shows higher resolution, better separation efficiency, and faster analysis speeds; thus, it can meet the demand for the quantitative analysis of indole/indazole amide-based SCs in seized materials. In this study, a UPLC method was developed for the simultaneous determination of five indole/indazole amide-based SCs, including N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-butyl-1H-indazole-3-carboxamide (ADB-BUTINACA), methyl 2-(1-(4-fluorobutyl)-1H-indole-3-carboxamido)-3,3-dimethylbutanoate (4F-MDMB-BUTICA), N-(1-methoxy-3,3-dimethyl-1-oxobutan-2-yl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide (5F-MDMB-PICA), methyl 3,3-dimethyl-2-(1-(pent-4-en-1-yl)-1H-indazole-3-carboxamido)butanoate (MDMB-4en-PINACA), and N-(adamantan-1-yl)-1-(4-fluorobutyl)-1H-indazole-3-carboxamide (4F-ABUTINACA) in electronic cigarette oil; these SCs have been detected with increasing frequency in seized materials in recent years. The main factors influencing the separation and detection performance of the proposed method, including the mobile phase, elution gradient, column temperature, and detection wavelength, were optimized. The proposed method successfully quantified the five SCs in electronic cigarette oil via the external standard method. The samples were extracted using methanol, and the target analytes were separated on a Waters ACQUITY UPLC CSH C18 column (100 mm×2.1 mm, 1.7 µm) at column temperature of 35 ℃ and flow rate of 0.3 mL/min. The injection volume was 1 µL. The mobile phase consisted of acetonitrile and ultrapure water, and gradient elution was employed. The detection wavelengths were 290 and 302 nm. The five SCs were completely separated within 10 min under optimized conditions and showed good linear relationships between 1-100 mg/L, with correlation coefficients (r2) of up to 0.9999. The limits of detection (LOD) and quantification (LOQ) were 0.2 and 0.6 mg/L, respectively. Precision was determined using standard solutions of the five SCs at mass concentrations of 1, 10, and 100 mg/L. The intra-day precision (n=6) was <1.5%, and the inter-day precision (n=6) was <2.2%. Accuracy was determined by spiking electronic cigarette oil with low (2 mg/L), moderate (10 mg/L), and high (50 mg/L) levels of the five SCs, with six replicates per determination. The recoveries of the five SCs were 95.5%-101.9%, and their relative standard deviations (RSDs, n=6) were 0.2%-1.5%, with accuracies ranging from -4.5% to 1.9%. The proposed method showed good performance when applied to the analysis of real samples. It is accurate, rapid, sensitive, and effective for the determination of five indole/indazole amide-based SCs in electronic cigarette oil. Thus, it satisfies the requirements for practical determination and provides a reference for the determination of SCs with similar structures by UPLC.

Advances in urinary biomarker research of synthetic cannabinoids.

New psychoactive substances (NPS) are chemical compounds designed to mimic the action of existing illicit recreational drugs. Synthetic cannabinoids (SCs) are a subclass of NPS which bind to the cannabinoid receptors, CB1 and CB2, and mimic the action of cannabis. SCs have dominated recent NPS seizure reports worldwide. While urine is the most common matrix for drug-of-abuse testing, SCs undergo extensive Phase I and Phase II metabolism, resulting in almost undetectable parent compounds in urine samples. Therefore, the major urinary metabolites of SCs are usually investigated as surrogate biomarkers to identify their consumption. Since seized urine samples after consuming novel SCs may be unavailable in a timely manner, human hepatocytes, human liver microsomes and human transporter overexpressed cell lines are physiologically-relevant in vitro systems for performing metabolite identification, metabolic stability, reaction phenotyping and transporter experiments to establish the disposition of SC and its metabolites. Coupling these in vitro experiments with in vivo verification using limited authentic urine samples, such a two-pronged approach has proven to be effective in establishing urinary metabolites as biomarkers for rapidly emerging SCs.

Synthetic Pathways to Non-Psychotropic Phytocannabinoids as Promising Molecules to Develop Novel Antibiotics: A Review.

Due to the rapid emergence of multi drug resistant (MDR) pathogens against which current antibiotics are no longer functioning, severe infections are becoming practically untreatable. Consequently, the discovery of new classes of effective antimicrobial agents with novel mechanism of action is becoming increasingly urgent. The bioactivity of Cannabis sativa, an herbaceous plant used for millennia for medicinal and recreational purposes, is mainly due to its content in phytocannabinoids (PCs). Among the 180 PCs detected, cannabidiol (CBD), Δ8 and Δ9-tetrahydrocannabinols (Δ8-THC and Δ9-THC), cannabichromene (CBC), cannabigerol (CBG), cannabinol (CBN) and some of their acidic precursors have demonstrated from moderate to potent antibacterial effects against Gram-positive bacteria (MICs 0.5-8 µg/mL), including methicillin-resistant Staphylococcus aureus (MRSA), epidemic MRSA (EMRSA), as well as fluoroquinolone and tetracycline-resistant strains. Particularly, the non-psychotropic CBG was also capable to inhibit MRSA biofilm formation, to eradicate even mature biofilms, and to rapidly eliminate MRSA persiter cells. In this scenario, CBG, as well as other minor non-psychotropic PCs, such as CBD, and CBC could represent promising compounds for developing novel antibiotics with high therapeutic potential. Anyway, further studies are necessary, needing abundant quantities of such PCs, scarcely provided naturally by Cannabis plants. Here, after an extensive overture on cannabinoids including their reported antimicrobial effects, aiming at easing the synthetic production of the necessary amounts of CBG, CBC and CBD for further studies, we have, for the first time, systematically reviewed the synthetic pathways utilized for their synthesis, reporting both reaction schemes and experimental details.

Overview of Synthetic Cannabinoids ADB-FUBINACA and AMB-FUBINACA: Clinical, Analytical, and Forensic Implications.

ADB-FUBINACA and AMB-FUBINACA are two synthetic indazole-derived cannabinoid receptor agonists, up to 140- and 85-fold more potent, respectively, than trans-∆9-tetrahydrocannabinol (∆9-THC), the main psychoactive compound of cannabis. Synthesised in 2009 as a pharmaceutical drug candidate, the recreational use of ADB-FUBINACA was first reported in 2013 in Japan, with fatal cases being described in 2015. ADB-FUBINACA is one of the most apprehended and consumed synthetic cannabinoid (SC), following AMB-FUBINACA, which emerged in 2014 as a drug of abuse and has since been responsible for several intoxication and death outbreaks. Here, we critically review the physicochemical properties, detection methods, prevalence, biological effects, pharmacodynamics and pharmacokinetics of both drugs. When smoked, these SCs produce almost immediate effects (about 10 to 15 s after use) that last up to 60 min. They are rapidly and extensively metabolised, being the O-demethylated metabolite of AMB-FUBINACA, 2-(1-(4-fluorobenzyl)-1H-indazole-3-carboxamide)-3-methylbutanoic acid, the main excreted in urine, while for ADB-FUBINACA the main biomarkers are the hydroxdimethylpropyl ADB-FUBINACA, hydroxydehydrodimethylpropyl ADB-FUBINACA and hydroxylindazole ADB-FUBINACA. ADB-FUBINACA and AMB-FUBINACA display full agonism of the CB1 receptor, this being responsible for their cardiovascular and neurological effects (e.g., altered perception, agitation, anxiety, paranoia, hallucinations, loss of consciousness and memory, chest pain, hypertension, tachycardia, seizures). This review highlights the urgent requirement for additional studies on the toxicokinetic properties of AMB-FUBINACA and ADB-FUBINACA, as this is imperative to improve the methods for detecting and quantifying these drugs and to determine the best exposure markers in the various biological matrices. Furthermore, it stresses the need for clinicians and pathologists involved in the management of these intoxications to describe their findings in the scientific literature, thus assisting in the risk assessment and treatment of the harmful effects of these drugs in future medical and forensic investigations.

Acute Pharmacological Effects and Oral Fluid Biomarkers of the Synthetic Cannabinoid UR-144 and THC in Recreational Users.

Synthetic cannabinoids (SCs) are one of the most frequent classes of new psychoactive substances monitored by the EU Early Warning System and World Health Organization. UR-144 is a SC with a relative low affinity for the CB1 receptor with respect to that for the CB2 receptor. As with other cannabinoid receptor agonists, it has been monitored by the EU Early Warning System since 2012 for severe adverse effects on consumers. Since data for UR-144 human pharmacology are very limited, an observational study was carried out to evaluate its acute pharmacological effects following its administration using a cannabis joint as term of comparison. Disposition of UR-144 and delta-9-tetrahydrocannibinol (THC) was investigated in oral fluid. Sixteen volunteers smoked a joint prepared with tobacco and 1 or 1.5 mg dose of UR-144 (n = 8) or cannabis flowering tops containing 10 or 20 mg THC (n = 8). Physiological variables including systolic and diastolic blood pressure, heart rate and cutaneous temperature were measured. A set of Visual Analog Scales (VAS), the Addiction Research Centre Inventory (ARCI)-49-item short form version and the Evaluation of the Subjective Effects of Substances with Abuse Potential (VESSPA-SSE) were administered to evaluate subjective effects. Oral fluid was collected at baseline, 10, 20, 40 min and 1, 2, 3 and 4 h after smoking, for UR-144 or THC concentration monitoring. Results showed significant statistical increases in both systolic and diastolic blood pressure and heart rate after both UR-144 and cannabis smoking. Both substances produced an increase in VAS related to stimulant-like and high effects, but scores were significantly higher after cannabis administration. No hallucinogenic effects were observed. Maximal oral fluid UR-144 and THC concentrations appeared at 20 and 10 min after smoking, respectively. The presence of UR-144 in oral fluid constitutes a non-invasive biomarker of SC consumption. The results of this observational study provide valuable preliminary data of the pharmacological effects of UR-144, showing a similar profile of cardiovascular effects in comparison with THC but lower intensity of subjective effects. Our results have to be confirmed by research in a larger sample to extensively clarify pharmacological effects and the health risk profile of UR-144.