Determination Of Novel Synthetic Cannabinoid Metabolic Markers And Study Of Their Testing Methods

Synthetic cannabinoids(SCs)are a class of compounds with pharmacological and physiological effects similar to those of tetrahydrocannabinol,the main active ingredient of natural cannabis,with the difference that tetrahydrocannabinol is only a partial agonist of the cannabinoid receptors.In contrast,most synthetic cannabinoids are full agonists of the cannabinoid receptors and therefore produce stronger pharmacological and physiological effects.At the same time,SCs are relatively simple to synthesize and can change their structure to circumvent legal restrictions without significantly reducing the activity of cannabinoid receptors.Since the first detection of naphthoylindole SCs from "spice" in 2006,criminals who manufacture and sell drugs have made certain modifications to the structure of existing SCs to create new SCs that are temporarily outside the scope of regulation,resulting in an increasing number of SCs,with about 324 SCs reported at home and abroad.China has officially listed the entire class of SCs as new psychoactive substances from July 1,2021.In this context,there is an urgent need to study the metabolic process of new SCs and to identify metabolic markers to provide a basis for identifying SCs and establishing methods for testing such substances in biological samples.Based on the actual work of public security,this paper investigates the metabolic pathways and metabolites of three novel SCs(ADB-BUTINACA,MDMB-4en-PINACA,and 5FMDMB-PICA)commonly found in domestic and foreign cases in recent years through in vivo and in vitro metabolic models as well as authentic human urine to identify metabolic markers.A liquid chromatography-high resolution mass spectrometry(LC-HRMS)method was also established to detect three novel SCs and their metabolic markers in the urine based on the identified metabolic markers.The central studies were as follows:1.In vitro metabolic models of human liver microsomes and in vivo metabolic models of zebrafish were established to simulate the human metabolic environment,study the metabolites of three novel SCs and identify metabolic pathways and metabolic markers,respectively.Authentic human urine samples were also used to verify the feasibility of the metabolic models.The results showed that both the in vivo and in vitro metabolic models could simulate the metabolism of the novel SCs in humans well.2.In vitro metabolic models of human liver microsomes and in vivo metabolic models of zebrafish were established to simulate the human metabolic environment,study the metabolites of three novel SCs and identify metabolic pathways and metabolic markers,respectively.Authentic human urine samples were also used to verify the feasibility of the metabolic models.The results showed that both the in vivo and in vitro metabolic models could simulate the metabolism of the novel SCs in humans well.The intra-day precision of the three SCs and their metabolites ranged from 2.28% to 7.47%,and the inter-day precision ranged from 3.64% to9.84%.The detection limits were 0.1-1 ng/m L,and the limits of quantification were 0.5-2ng/m L,with good linearity in the range of 2-200 ng/m L and correlation coefficients greater than 0.998.3.The pre-treatment methods of urine samples were examined and optimized,and an LCHRMS test for three novel SCs and their metabolic markers in urine was established.The detection limit of the precipitated protein method was 1-3 ng/m L,the quantification limit was2-5 ng/m L,the matrix effect was between 77.6% and 91.2%,the recovery was 77.2% to91.5%,the intra-day precision was 2.1% to 11.6%,and the inter-day precision was 0.7% to10.9%.The limits of detection for the solid phase extraction method were 0.1-1 ng/m L,the limits of quantification were 0.5-2 ng/m L,the recoveries ranged from 78.5% to 91.7%,the matrix effects were 80.6% to 96.4%,the intra-day precision was 1.1% to 9.5%,and the interday precision was 2.1% to 9.8%.4.The established method was used to test the urine of suspected SCs smokers.The target substances were detected in the urine specimens,indicating that the established method can be used for the identification of relevant cases,which can provide a basis for the establishment of standard methods for testing such substances in biological specimens,and is of great significance for the effective monitoring and combating of SCs-related crimes in anti-drug work.