Investigation of metabolites of synthetic cannabinoid 4CN-MDMB-BUTINAC in vitro by liquid-chromatography-quadruple/orbitrap mass spectrometry / 药学学报

To investigate the metabolites of a new synthetic cannabinoid 3,3-dimethyl-2-[1-(4-cyanobutyl)indazole-3-formamimino]methyl butyrate (4CN-MDMB-BUTINACA) in vitro, a human liver microsome incubation model was established to analyze the metabolic biotransformation of synthetic cannabinoids using ultra-high performance liquid chromatography coupled to quadrupole-orbitrap high-resolution mass spectrometry. Nontarget metabolomic results showed that the metabolites of 4CN-MDMB-BUTINACA included hydroxylation, ester hydrolysis, ester hydrolysis with hydroxylation reaction, pentane oxidation and ester hydrolysis with pentane oxidation reaction, among which M1-a, M2 and M4 were potential metabolic markers. The research results provide a theoretical basis and technical support for the biomonitoring and metabolic characterisation of the cannabinoid 4CN-MDMB-BUTINACA.

Detection of Synthetic Cannabinoid CUMYL-PEGACLONE in E-Cigarette Oil and Hair / 法医学杂志

OBJECTIVES@#To establish a combined high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) method to detect the synthetic cannabinoid CUMYL-PEGACLONE in e-cigarette oil and hair.@*METHODS@#HPLC-MS/MS and GC-MS were used to establish the detection method of CUMYL-PEGACLONE, and the hair of drug-involved persons and the seized e-cigarette oil were detected.@*RESULTS@#The main mass spectrometry characteristic ions m/z of CUMYL-PEGACLONE measured by GC-MS were 91, 179, 197, 254 and 372. CUMYL-PEGACLONE had a good linear relationship in the mass concentration range of 2-50 ng/mL, and the linear correlation coefficient (r) was greater than 0.99. The limit of detection (LOD) of CUMYL-PEGACLONE in hair was 0.01 ng/mg, and the limit of quantitation (LOQ) was 0.02 ng/mg. The LOD of CUMYL-PEGACLONE in e-cigarette oil was 1 ng/mg, and the LOQ was 2 ng/mg. The average recoveries of CUMYL-PEGACLONE under the attempt at high, intermediate and low levels in blank human hair and e-cigarette oil matrix were 98.2%-132.4% and 93.5%-110.6%, and the intraday and intraday precision were 1.2%-12.9% and 0.7%-2.9%. CUMYL-PEGACLONE was detected in the hair of 15 drug-involved persons. Except for 1 person who was lower than LOQ, the concentration of CUMYL-PEGACLONE in the hair of other 14 persons was 0.035-0.563 ng/mg. The mass fraction of CUMYL-PEGACLONE in 2 e-cigarette oil were 0.17% and 0.21%, respectively.@*CONCLUSIONS@#The established HPLC-MS/MS and GC-MS methods are applied to the detection of HPLC-MS/MS in drug-related cases, which provides strong evidence support for the handling authority to quickly investigate these cases, and also provides a reference for the identification of such substances in future.

Identification of Synthetic Cannabinoid New Psychoactive Substances 4F-MDMB-BUTINACA and MDMB-4en-PINACA / 法医学杂志

Objective To establish a method that combines a series of techniques including Fourier transform infrared spectrum （FTIR）, gas chromatography-mass spectrometry （GC-MS）, high resolution mass spectrometry and nuclear magnetic resonance spectroscopy （NMR） for identification of unknown substances. Methods The unknown samples （off-white powder and yellow crystal） seized in the actual cases were detected by FTIR, GC-MS （methanol as solvent）, high resolution mass spectrometry （methanol as solvent） and NMR （deuterated methanol as solvent）. Results The mass spectrum characteristic ions m/z of the main components in the samples measured by GC-MS were 219 （base peak）, 363, 307, 304, 275, 145, 131 and 213 （base peak）, 357, 301, 298, 269, 185, 171, 145 and 131, respectively. The accurate mass numbers [M+H]+ measured by high resolution mass spectrometry were 364.203 61 and 358.212 34, respectively. The unknown samples were identified as synthetic cannabinoid new psychoactive substances 4F-MDMB-BUTINACA and MDMB-4en-PINACA after data consultation and database retrieval and comparison, combined with infrared analysis and mass spectrometry data analysis, and their structures were confirmed by 1H-NMR. Conclusion The established multi-technology joint identification method can be used to identify 4F-MDMB-BUTINACA and MDMB-4en-PINACA in unknown samples. This method is fast, convenient, accurate, reliable and practical, and can provide reference for the identification of cases involving such substances in the future.

Analysis and Application of Seven Common New Psychoactive Substances of Synthetic Cannabinoids in Hair / 法医学杂志

Objective To establish a detection method for common new psychoactive substances of synthetic cannabinoids in hair with ultra-high performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS）. Methods In the 1 mL of internal standard methanol solution, 20 mg hair was added. After cryogenic grinding and ultrasonic extraction, the extract was separated by ACQUITY UPLC HSS T3 column （100 mm×2.1 mm, 1.8 μm）. The mobile phase A was aqueous solution that composed of 20 mmol/L ammonium acetate, 0.1% formic acid, and 5% acetonitrile. The mobile phase B was acetonitrile. Electrospray ionization source in positive ion mode was used for data acquisition in multi-reaction monitoring （MRM） mode. Results The seven common new psychoactive substances of synthetic cannabinoids in hair had a good linear relationship within their respective linear ranges （r>0.99）, the limits of detection were 0.5-2 pg/mg, the limits of quantification were 1-5 pg/mg, the intra-day and inter-day precisions were 0.1%-12.6%, the intra-day and inter-day accuracies were 89.2%-110.7%, the recovery rates were 52.3%-93.3%, and the matrix effects were 19.1%-95.2%. Conclusion The established method has a simple sample preparation process and high sensitivity. It is suitable for qualitative and quantitative analysis of common new psychoactive substances of synthetic cannabinoids in hair.

Research Progress on Metabolite Identification of Synthetic Cannabinoid New Psychoactive Substances / 法医学杂志

Synthetic cannabinoids are currently a class of new psychoactive substances with the largest variety and most abused. Metabolite identification research can provide basic data for monitoring synthetic cannabinoids abuse, which is the current research hotspot. The main trend of structural modification of synthetic cannabinoid is to replace the fluorine atom on pentyl indole or indazole cyclopentyl with hydrogen atom, which greatly improves the biological activity of the compound. The main metabolic reactions include hydroxylation, fluoropentyl oxidative, ester hydrolyze, amide hydrolysis. Liquid chromatography-high resolution mass spectrometry has become the preferred choice for the structural identification of metabolites. This review mainly summarizes research on metabolism software prediction and human hepatocyte model, human liver microsomes model, rat in vivo model, zebrafish model and fungus C. elegans model in metabolite identification based on the structure and classification of synthetic cannabinoids.

Study on the analytic method of synthetic cannabinoid JWH-122 by the high performance liquid chromatography / 中国法医学杂志

Objective To establish an effective method for determination of synthetic cannabinoid JWH-122 by the high-performance liquid chromatography (HPLC), which is controled by China’s Regulations on non-clinical narcotic and psychoactive drug. Methods Methanol-deionized water (50%-50%) was used as mobile phase for gradient elution. In addition to the initial concentration in organic phase, gradient steepness, column temperature, flow rate and other chromatographic conditions, the determine wavelengths were tested so as to ifnd out optimal experimental conditions. Linearity range and speciifcity were tested under optimal conditions, and actual samples were used to verify the method established. Results Under the condition of ultraviolet spectrum detection wavelength at 221nm, initial concentration of 70%, organic phase gradient steepness of 0.5%/min, lfow rate at 1.2 ml/min and column temperature of 30℃,excellent linearity of JWH-122 was observed at 0.002mg/mL-0.1mg/mL and the detection limit (S/N≥3) was 0.1μg/mL. The test of actual samples suggested that JWH-122 was able to be well separated from the sample under the optimal conditions. Conclusion Our method has advantages of rapidity, sensitivity, accuracy and excellent separation efifciency, and is capable of the detection of synthetic cannabinoid JWH-122 of the novel “spice” drugs.

Neurotoxicity of Synthetic Cannabinoids JWH-081 and JWH-210

Synthetic cannabinoids JWH-018 and JWH-250 in 'herbal incense' also called 'spice' were first introduced in many countries. Numerous synthetic cannabinoids with similar chemical structures emerged simultaneously and suddenly. Currently there are not sufficient data on their adverse effects including neurotoxicity. There are only anecdotal reports that suggest their toxicity. In the present study, we evaluated the neurotoxicity of two synthetic cannabinoids (JWH-081 and JWH-210) through observation of various behavioral changes and analysis of histopathological changes using experimental mice with various doses (0.1, 1, 5 mg/kg). In functional observation battery (FOB) test, animals treated with 5 mg/kg of JWH-081 or JWH-210 showed traction and tremor. Their locomotor activities and rotarod retention time were significantly (p<0.05) decreased. However, no significant change was observed in learning or memory function. In histopathological analysis, neural cells of the animals treated with the high dose (5 mg/kg) of JWH-081 or JWH-210 showed distorted nuclei and nucleus membranes in the core shell of nucleus accumbens, suggesting neurotoxicity. Our results suggest that JWH-081 and JWH-210 may be neurotoxic substances through changing neuronal cell damages, especially in the core shell part of nucleus accumbens. To confirm our findings, further studies are needed in the future.

Dependence Potential of the Synthetic Cannabinoids JWH-073, JWH-081, and JWH-210: In Vivo and In Vitro Approaches

Synthetic cannabinoids (CBs) such as the JWH series have caused social problems concerning their abuse liability. Because the JWH series produces euphoric and hallucinogenic effects, they have been distributed illegally under street names such as "Spice" and "Smoke". Many countries including Korea have started to schedule some of the JWH series compounds as controlled substances, but there are a number of JWH series chemicals that remain uncontrolled by law. In this study, three synthetic CBs with different binding affinities to the CB1 receptor (JWH-073, 081, and 210) and Delta9-tetrahydrocannabinol (Delta9-THC) were evaluated for their potential for psychological dependence. The conditioned place preference test (unbiased method) and self-administration test (fixed ratio of 1) using rodents were conducted. Ki values of the three synthetic cannabinoids were calculated as supplementary data using a receptor binding assay and overexpressed CB1 protein membranes to compare dependence potential with CB1 receptor binding affinity. All mice administered JWH-073, 081, or 210 showed significantly increased time spent at unpreferred space in a dose-dependence manner in the conditioned place preference test. In contrast, all tested substances except Delta9-THC showed aversion phenomenon at high doses in the conditioned place preference test. The order of affinity to the CB1 receptor in the receptor binding assay was JWH-210 > JWH-081 >> JWH-073, which was in agreement with the results from the conditioned place preference test. However, no change in self-administration was observed. These findings suggest the possibility to predict dependence potential of synthetic CBs through a receptor binding assay at the screening level.