Method for synthesizing cannabinoid ADB-4en-PINACA based on benzoxazolyl probe fluorescence detection

Abstract

The invention provides a method for fluorescence detection of synthetic cannabinoid ADB-4en-PINACA based on a benzoxazolyl probe, a detection reagent in the method is composed of 3-(benzo [d] oxazole-2-yl)-[1, 1 '-biphenyl]-2-alcohol and an organic solvent, after the synthetic cannabinoid ADB-4en-PINACA is added into the obtained probe, the fluorescence color is changed from yellow green to blue, and a ratio fluorescence signal is presented. In order to facilitate on-site actual detection application, a solid-liquid separation portable sensing device is prepared, and the solid-liquid separation portable sensing device has good qualitative analysis capability. The method provided by the invention has the advantages of simplicity in operation, sensitive reaction, good specificity and convenience in visual detection, has a wide application prospect, and can be used for rapid preliminary screening and analysis of the synthetic cannabinoid ADB-4en-PINACA in field drug investigation and drug-related cases.

Description

Technical Field

[0001] The present invention provides a method for detecting synthetic cannabinoid ADB-4en-PINACA based on benzoxazolyl probe fluorescence. Background Art

[0002] Synthetic cannabinoids are a class of chemically synthesized compounds that are the most widely abused and most serious class of new psychoactive substances. These compounds interact with the CB1 and CB2 receptors in the human body to produce hallucinogenic effects similar to those of natural cannabinoids such as tetrahydrocannabinol (THC). ADB-type synthetic cannabinoids are a class of compounds with clear structures, stable properties, and weak activity. They have high marijuana-mimicking activity and are often disguised as seemingly harmless substances such as flavors and e-cigarette liquids, making detection and supervision more complicated. Therefore, it is of great significance to achieve highly sensitive, rapid response, and selective on-site detection of ADB-type synthetic cannabinoids.

[0003] At present, the detection methods of synthetic cannabinoids mainly rely on a variety of large-scale analytical instruments, covering nuclear magnetic resonance, liquid chromatography, gas chromatography-mass spectrometry, surface enhanced Raman scattering and electrochemical methods. However, the above methods have the disadvantages of complex sample pretreatment, long detection time, and professional operation, which limits the scope of application.

[0004] Visual optical sensing methods (fluorescence and colorimetry) have become highly promising detection technologies in the field of chemical sensing due to their rapid response, simple operation and high portability. At present, there are few reports on synthetic cannabinoid fluorescence detection methods based on small molecule probes. This is because synthetic cannabinoids are chemically stable and inert, making them difficult to identify through covalent reactions. Therefore, the development of new visual optical sensing recognition methods is a major challenge facing this field.

[0005] The present invention discloses a method for detecting synthetic cannabinoid ADB-4en-PINACA based on benzoxazolyl probe fluorescence, and a method for detecting synthetic cannabinoid N-(1-carbamoyl-2,2-dimethylpropyl)-1-(4-pentenyl)indazole-3-carboxamide (ADB-4en-PINACA) or N-(1-carbamoyl-2,2-dimethylpropyl)-1-butylindazole-3-carboxamide (ADB-BUTINACA) using the benzoxazolyl probe fluorescence. The method synergistically inhibits the excited state intramolecular proton transfer process of the probe through π-π stacking, hydrophobic interaction and weak hydrogen bond interaction, triggers the intramolecular electron transfer process, and realizes specific and accurate identification of the synthetic cannabinoid ADB-4en-PINACA. At the same time, a detection reagent or a solid-liquid separation sensor device is used to realize high-specificity and high-sensitivity on-site rapid fluorescence detection of the synthetic cannabinoid ADB-4en-PINACA. Summary of the invention

[0006] The present invention aims to provide a method for detecting synthetic cannabinoid ADB-4en-PINACA based on benzoxazolyl probe fluorescence, wherein the 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol probe in the method can realize ratio fluorescence detection of synthetic cannabinoid ADB-4en-PINACA. After adding synthetic cannabinoid ADB-4en-PINACA, the fluorescence color of the probe changes from yellow-green to blue. In order to facilitate actual on-site detection and application, a solid-liquid separation sensor device is prepared. The sensor device is composed of a bottom plug, a bottle body and a bottle cap, and is three-dimensionally modeled using SolidWorks software and printed using an Ultimaker S5 3D printer; the sensor bottom plug is used to store benzoxazolyl solid probes, the bottle body is used to store solvent acetone, and the bottom of the bottle cap is embedded in a silicon substrate. When in use, tear off the sealing ring of the bottom plug, and then press the bottom plug to fully mix the solid probe with the solvent. The mixed solution is dripped onto the silicon substrate through the tip of the bottle, thereby realizing the rapid detection of the synthetic cannabinoid ADB-4en-PINACA, and the detection is not interfered by plants and tobacco oil additives, etc. It has the advantages of high sensitivity, high specificity, rapidity and visualization, and has broad application prospects in on-site analysis.

[0007] The method for detecting the synthetic cannabinoid ADB-4en-PINACA based on the fluorescence of the benzoxazolyl probe of the present invention is carried out according to the following steps:

[0008] Preparation of detection reagents:

[0009] a. Mix polyphosphoric acid, 3-bromo-2-hydroxybenzoic acid and 2-aminophenol in a 100 ml three-necked flask, stir overnight at 180° C., then add ice water, filter, and obtain a solid product. Extract the solid product with ethyl acetate, collect the organic phase, remove the solvent in vacuo, and purify by silica gel column chromatography using petroleum ether:ethyl acetate in a volume ratio of 40:1 as eluent to obtain 3-(benzo[d]oxazol-2-yl)-[1,1′-biphenyl]-2-ol solid;

[0010] b. Dissolve the 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol solid obtained in step a in acetone, dissolve it by ultrasonication, and prepare a 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol probe solution with a concentration of 0.1 mmol / L to obtain a detection reagent;

[0011] Detection of the synthetic cannabinoid ADB-4en-PINACA:

[0012] c. Preparation of synthetic cannabinoid ADB-4en-PINACA standard solution: Use methanol as solvent to prepare a synthetic cannabinoid standard solution with a concentration of 1 mmol / L;

[0013] d. Place 180 μL of the detection reagent 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol probe solution obtained in step b into a test tube, then add 20 μL of the synthetic cannabinoid ADB-4en-PINACA solution to be detected, stir thoroughly, and record the fluorescence spectrum and optical photographs under 365 nm ultraviolet light excitation to observe the signal changes of the fluorescence characteristic emission peak before and after the reaction;

[0014] Or use sensor devices to detect:

[0015] Preparation of sensor device:

[0016] The sensor device is composed of a bottom plug (1), a bottle body (2) and a bottle cap (3), and is three-dimensionally modeled using SolidWorks software and printed using an Ultimaker S5 3D printer; the bottom plug (1), the bottle body (2) and the bottle cap (3) in the sensor device are connected by means of a buckle;

[0017] Detection of the synthetic cannabinoid ADB-4en-PINACA:

[0018] The detection reagent and acetone solution obtained in step b are placed in a solid-liquid separation sensor device, the bottom plug (1) is designed to store the detection reagent, the bottle body (2) is used to store the solvent acetone, and the bottom of the bottle cap (3) is embedded in a silicon substrate. When in use, the sealing ring of the bottom plug (1) is torn open, and then the bottom plug (1) is pressed to fully mix the detection reagent and the acetone solvent in the bottle body (2), and the mixed solution is dripped onto the silicon substrate at the bottom of the bottle cap (3) through the tip of the bottle body (2); 10 μL of the synthetic cannabinoid ADB-4en-PINACA solution to be detected is drawn and dripped onto the silicon substrate, and the fluorescence change is observed under 365 nm illumination conditions.

[0019] The present invention discloses a method for fluorescently detecting synthetic cannabinoid ADB-4en-PINACA based on a benzoxazolyl probe. The chemical name of the benzoxazolyl probe in the method is 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol, which can be obtained by reaction. Its corresponding chemical structure (I) is:

[0020]

[0021] The present invention discloses a method for fluorescently detecting the synthetic cannabinoid ADB-4en-PINACA based on a benzoxazolyl probe, wherein the probe in the method can realize ratio fluorescence detection of the synthetic cannabinoid N-(1-carbamoyl-2,2-dimethylpropyl)-1-(4-pentenyl)indazole-3-carboxamide (ADB-4en-PINACA). The detection limit of the probe for the synthetic cannabinoid N-(1-carbamoyl-2,2-dimethylpropyl)-1-(4-pentenyl)indazole-3-carboxamide (ADB-4en-PINACA) can reach 24.3nM, and the linear detection range is 0-250μM. Within 1s of dripping the test object, its fluorescence color changes from yellow-green to blue, and other common structural analogs and electronic cigarette oil additives have no obvious interference with the detection of synthetic cannabinoids.

[0022] The method of the present invention has no special restrictions when used. Qualitative detection can be quickly completed at room temperature without pre-treatment of the analyte. It is simple to operate, economical and practical, has high specificity and sensitivity, and the results are stable and repeatable. The above advantages make the present invention easy to be practically applied and promoted in the field of on-site instant detection.

[0023] In the presence of synthetic cannabinoids, the probe exhibits an obvious ratio fluorescence change, that is, yellow-green turns to blue; after dropping the synthetic cannabinoid solution, an obvious fluorescence response change can occur within 1 second; the probe has good recognition ability for the synthetic cannabinoid N-(1-carbamoyl-2,2-dimethylpropyl)-1-(4-pentenyl) indazole-3-carboxamide (ADB-4en-PINACA), and the detection is not interfered by other common types of synthetic cannabinoids, traditional drugs and electronic cigarette oil additives. The present invention provides a good research basis and effective means of investigation for rapid on-site analysis.

[0024] The detection principle of the method described in the present invention is: the benzoxazole of the probe forms a π...π interaction with the indazole, amide and phenol of the synthetic cannabinoid N-(1-carbamoyl-2,2-dimethylpropyl)-1-(4-pentenyl) indazole-3-carboxamide (ADB-4en-PINACA), a hydrophobic interaction is generated between the phenyl group and the tert-butyl group, and a weak hydrogen bond interaction is formed between the phenol oxygen and the tert-butyl group. These non-covalent forces synergistically inhibit the excited state intramolecular proton transfer process of the probe, trigger the intramolecular electron transfer process, and realize the ratio fluorescence change of the synthetic cannabinoid N-(1-carbamoyl-2,2-dimethylpropyl)-1-(4-pentenyl) indazole-3-carboxamide (ADB-4en-PINACA).

[0025] The specifically designed detection reagent has the characteristics of simple preparation, rapid detection, and easy operation. It has highly specific recognition ability for the synthetic cannabinoid ADB-4en-PINACA and can be expanded to be used in on-site detection of synthetic cannabinoid drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The detection effect of the benzoxazolyl probe in Example 1 of the present invention on the synthetic cannabinoid ADB-4en-PINACA;

[0027] Figure 2 The detection effect of the series concentration probe molecule in Example 2 of the present invention on the synthetic cannabinoid ADB-4en-PINACA; wherein a is the fluorescence spectrum of the series concentration probe solution; b is the fluorescence spectrum of the series concentration probe solution added with the synthetic cannabinoid ADB-4en-PINACA; c is 453nm-530nm (I 453 / I 530 ) at the intensity ratio;

[0028] Figure 3 The detection effect of the benzoxazolyl probe in Example 3 of the present invention on the synthetic cannabinoid ADB-BUTINACA;

[0029] Figure 4 The fluorescence changes before and after the reaction of the benzoxazolyl probe molecule in Example 4 of the present invention to detect the synthetic cannabinoid ADB-4en-PINACA with a series of concentrations, and the correlation between the fluorescence changes and the concentration of the synthetic cannabinoid; wherein a is the optical response image of the probe detecting the synthetic cannabinoid ADB-4en-PINACA with a series of concentrations; b is the fluorescence spectrum of the probe detecting the synthetic cannabinoid ADB-4en-PINACA with a series of concentrations; c is 453nm-530nm (I 453 / I 530 ) and the corresponding correlation between the emission intensity ratio at 400 nm and the concentration of the synthetic cannabinoid ADB-4en-PINACA;

[0030] Figure 5 The detection effect of the benzoxazolyl probe molecule in Examples 5-6 of the present invention in detecting the synthetic cannabinoid ADB-4en-PINACA and other common interferents;

[0031] Figure 6 This is a response time effect diagram of the benzoxazolyl probe molecule in Example 7 of the present invention for detecting the synthetic cannabinoid ADB-4en-PINACA;

[0032] Figure 7The scanning electron microscope effect diagram of the silicon wafer substrate loaded probe in Example 8 of the present invention detecting the synthetic cannabinoid ADB-4en-PINACA; wherein a is a scanning electron microscope effect diagram of the probe; b is a scanning electron microscope effect diagram of the probe detecting the synthetic cannabinoid ADB-4en-PINACA;

[0033] Figure 8 : The detection effect of the benzoxazolyl probe-silicon wafer substrate on a series of concentrations of the synthetic cannabinoid ADB-4en-PINACA in Example 9 of the present invention; wherein a is the optical response image of the probe-silicon wafer substrate to a series of concentrations of the synthetic cannabinoid ADB-4en-PINACA; b is the functional relationship between the B / R value of the fluorescence image on the substrate and the ADB-4en-PINACA concentration;

[0034] Fig. 9 1 is the detection effect of the portable solid-liquid separation sensor device in Example 10 of the present invention on the synthetic cannabinoid ADB-4en-PINACA mixed in the real sample; wherein a is a schematic diagram of the sensor device, (1) bottom plug, (2) bottle body, (3) bottle cap; b is a schematic diagram of the detection process change; c is an effect diagram of the sensor device for detecting suspicious objects, including: (i) flower petals containing ADB-4en-PINACA; (ii) electronic cigarette oil; (iii) tobacco leaves. DETAILED DESCRIPTION

[0035] The present invention is further described below by means of specific examples, but the invention is not limited to these examples.

[0036] Example 1

[0037] Preparation of detection reagents:

[0038] a. 16 ml of polyphosphoric acid, 5 mmol, 1.0581 g of 3-bromo-2-hydroxybenzoic acid and 5 mmol, 0.5451 g of 2-aminophenol were mixed in a 100 ml three-necked flask, and stirred overnight at a temperature of 180 ° C. Then ice water was added and filtered to obtain a solid product. The solid product was extracted with ethyl acetate, and the organic phase was collected. After removing the solvent in vacuo, it was purified by silica gel column chromatography using petroleum ether: ethyl acetate in a volume ratio of 40: 1 as an eluent to obtain 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol solid;

[0039] b. Dissolve the 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol solid obtained in step a in acetone, dissolve it by ultrasonication, and prepare a 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol probe solution with a concentration of 0.1 mmol / L to obtain a detection reagent;

[0040] Detection of the synthetic cannabinoid ADB-4en-PINACA:

[0041] c. Preparation of synthetic cannabinoid ADB-4en-PINACA standard solution: Use methanol as solvent to prepare a synthetic cannabinoid standard solution with a concentration of 1 mmol / L;

[0042] d. Take 180 μL of the 0.1 mmol / L 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol probe solution of the detection reagent obtained in step b and place it in a test tube. Then add 1 mmol / L of the synthetic cannabinoid ADB-4en-PINACA methanol solution. After sufficient reaction, select the excitation wavelength as 365 nm, record the fluorescence spectrum and take a fluorescence picture. The detection effect of the benzoxazolyl probe molecule on the synthetic cannabinoid in the acetone solution is as follows: Figure 1 As shown; under 365nm ultraviolet light, the fluorescence color of the reagent changes from yellow-green to blue. In addition, when measured by fluorescence spectrometer, the excitation wavelength is selected as 365nm, and the fluorescence characteristic peak is blue-shifted from 530nm to about 453nm. The above phenomenon shows obvious fluorescence changes.

[0043] Example 2

[0044] The fluorescence detection reagent was prepared according to Example 1:

[0045] The detection reagent 0.1 mmol / L 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol probe solution obtained in step b of Example 1 was prepared into solutions with concentrations of 20, 40, 60, 80, and 100 μmol / L, and then 135 μL of the probe solution of the corresponding concentration was taken into the test tube respectively;

[0046] Synthetic cannabinoid ADB-4en-PINACA standard solution preparation: Use methanol as solvent to prepare a synthetic cannabinoid standard solution with a concentration of 1 mmol / L;

[0047] Detection process: 15 μL of the synthetic cannabinoid ADB-4en-PINACA standard solution was added to the prepared detection reagents of various concentrations. After sufficient reaction, the excitation wavelength was selected as 365 nm, the fluorescence spectrum was recorded, and the fluorescence image was taken;

[0048] Detection effect: The detection effect of benzoxazolyl probe solution at different concentrations on the synthetic cannabinoid ADB-4en-PINACA is shown in Figure 2 As shown, the spectra before and after detection were measured by a fluorescence spectrometer. Under 365nm excitation, a new emission peak appeared at 453nm in the fluorescence spectrum, while the peak intensity at 530nm weakened, confirming the presence of the synthetic cannabinoid ADB-4en-PINACA.

[0049] Example 3

[0050] The fluorescence detection reagent was prepared according to Example 1:

[0051] The detection reagent 0.1mmol / L 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol probe solution obtained in step b of Example 1 was added, and then 135μL of the probe solution of corresponding concentration was taken into a test tube;

[0052] Preparation of test solution: Using methanol as solvent, prepare a standard solution of synthetic cannabinoid N-(1-carbamoyl-2,2-dimethylpropyl)-1-butylindazole-3-carboxamide (ADB-BUTINACA) with a concentration of 1 mmol / L;

[0053] Detection process: 15 μL of 1 mmol / L synthetic cannabinoid ADB-BUTINACA was added to the prepared detection reagent. After sufficient reaction, the excitation wavelength was selected as 365 nm, and the fluorescence spectrum and optical photos were recorded;

[0054] Detection effect: The effect of benzoxazolyl probe in detecting the synthetic cannabinoid ADB-BUTINACA is as follows Figure 3 As shown, the addition of the synthetic cannabinoid ADB-BUTINACA causes the color of the detection reagent to change from yellow-green to blue, and the characteristic fluorescence peak blue-shifts from 530nm to around 453nm, indicating that the benzoxazolyl probe molecule has good selectivity for ADB-type synthetic cannabinoids.

[0055] Example 4

[0056] Preparation of detection reagents:

[0057] 16 ml of polyphosphoric acid, 5 mmol, 1.0581 g of 3-bromo-2-hydroxybenzoic acid and 5 mmol, 0.5451 g of 2-aminophenol were mixed in a 100 ml three-necked flask, and stirred overnight at a temperature of 180° C., followed by addition of ice water, and filtration to obtain a solid product. The solid product was extracted with ethyl acetate, and the organic phase was collected. After removing the solvent in vacuo, it was purified by silica gel column chromatography using petroleum ether:ethyl acetate in a volume ratio of 40:1 as an eluent to obtain 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol solid;

[0058] The obtained 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol was dissolved in acetone, and after ultrasonic dissolution, a 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol probe solution with a concentration of 60 μmol / L was prepared to obtain a detection reagent;

[0059] The obtained detection reagent 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol probe solution was taken and 135 μL of the probe solution of the corresponding concentration was placed in a test tube;

[0060] Preparation of synthetic cannabinoid ADB-4en-PINACA standard solution: using methanol as solvent, prepare synthetic cannabinoid ADB-4en-PINACA standard solution with concentrations of 10, 20, 40, 60, 80, 100, 150, 200, 250 μmol / L;

[0061] Detection process: Take 15 μL of the prepared synthetic cannabinoid ADB-4en-PINACA standard solution and add it to the detection reagent. After sufficient reaction, select the excitation wavelength of 365 nm and take a fluorescence photo ( Figure 4 a), and the fluorescence spectrum was measured, and the ratio of the fluorescence intensity at the characteristic peak of the complex at 453 nm and the characteristic peak of the probe at 530 nm was used to make a calibration curve corresponding to the ADB-4en-PINACA content ( Figure 4 b);

[0062] Detection effect: Figure 4 As shown, the change in the intensity ratio of the characteristic peak of the complex to the characteristic peak of the probe after the benzoxazolyl probe molecule detected the synthetic cannabinoid ADB-4en-PINACA in the acetone solution system showed a good linear relationship with the concentration change of the synthetic cannabinoid ADB-4en-PINACA in the range of 0-250 μmol / L ( Figure 4 c), therefore, the probe molecule can quantitatively detect the synthetic cannabinoid ADB-4en-PINACA molecule.

[0063] Example 5

[0064] Selective testing of the synthetic cannabinoid ADB-4en-PINACA using a benzoxazolyl-based probe:

[0065] Preparation of detection reagents:

[0066] 16 ml of polyphosphoric acid, 5 mmol, 1.0581 g of 3-bromo-2-hydroxybenzoic acid and 5 mmol, 0.5451 g of 2-aminophenol were mixed in a 100 ml three-necked flask, and stirred overnight at a temperature of 180° C., followed by addition of ice water, and filtration to obtain a solid product. The solid product was extracted with ethyl acetate, and the organic phase was collected. After removing the solvent in vacuo, it was purified by silica gel column chromatography using petroleum ether:ethyl acetate in a volume ratio of 40:1 as an eluent to obtain 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol solid;

[0067] The obtained 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol was dissolved in acetone, and after ultrasonic dissolution, a 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol probe solution with a concentration of 60 μmol / L was prepared to obtain a detection reagent;

[0068] The obtained detection reagent 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol probe solution was taken and 135 μL of the probe solution of the corresponding concentration was placed in a test tube;

[0069] Preparation of test solution: Using methanol as solvent, 24 potential interfering agents were selected, including traditional drugs (opium, heroin, ecstasy, methamphetamine, ecstasy, etc.), new psychoactive substances (including other types of synthetic cannabinoids, ketamine, fentanyl, etc.) and synthetic cannabinoid precursors (indazole) as interfering agents for the detection of synthetic cannabinoids ADB-4en-PINACA, and a solution with a corresponding concentration of 250 μmol / L was prepared;

[0070] Detection process: Add 15 μL of 250 μmol / L traditional drugs, new psychoactive substances, synthetic cannabinoid precursors or 250 μmol / L synthetic cannabinoid ADB-4en-PINACA to the prepared detection reagent. After sufficient reaction, select the excitation wavelength of 365 nm and record the fluorescence spectrum ( Figure 5 );

[0071] Detection effect: The detection effect of benzoxazolyl probe on the synthetic cannabinoid ADB-4en-PINACA and its interferents is as follows Figure 5 As shown, the addition of synthetic cannabinoid ADB-4en-PINACA causes the color of the detection reagent to change from yellow-green to blue, and the characteristic fluorescence peak shifts from 530nm to around 453nm. The yellow-green fluorescence remains unchanged after the addition of other interfering substances ( Figure 5 ), indicating that the benzoxazolyl probe molecule has good selectivity for the synthetic cannabinoid ADB-4en-PINACA.

[0072] Example 6

[0073] Preparation of detection reagents:

[0074] 16 ml of polyphosphoric acid, 5 mmol, 1.0581 g of 3-bromo-2-hydroxybenzoic acid and 5 mmol, 0.5451 g of 2-aminophenol were mixed in a 100 ml three-necked flask, and stirred overnight at a temperature of 180° C., followed by addition of ice water, and filtration to obtain a solid product. The solid product was extracted with ethyl acetate, and the organic phase was collected. After removing the solvent in vacuo, it was purified by silica gel column chromatography using petroleum ether:ethyl acetate in a volume ratio of 40:1 as an eluent to obtain 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol solid;

[0075] The obtained 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol was dissolved in acetone, and after ultrasonic dissolution, a 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol probe solution with a concentration of 60 μmol / L was prepared to obtain a detection reagent;

[0076] The obtained detection reagent 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol probe solution was taken and 135 μL of the probe solution of the corresponding concentration was placed in a test tube;

[0077] Preparation of test solution: Using methanol as solvent, 24 potential interfering agents were selected, including traditional drugs (opium, heroin, ecstasy, methamphetamine, ecstasy, etc.), new psychoactive substances (including other types of synthetic cannabinoids, ketamine, fentanyl, etc.) and synthetic cannabinoid precursors (indazole) as interfering agents for the detection of synthetic cannabinoids ADB-4en-PINACA, and a solution with a corresponding concentration of 250 μmol / L was prepared;

[0078] Detection process: Add 15 μL of 250 μmol / L traditional drugs, new psychoactive substances, synthetic cannabinoid precursors and 250 μmol / L synthetic cannabinoid ADB-4en-PINACA to the prepared detection reagent. After sufficient reaction, select the excitation wavelength of 365 nm and record the fluorescence spectrum and optical photos ( Figure 5 );

[0079] Detection effect: The detection effect of benzoxazolyl probe on the synthetic cannabinoid ADB-4en-PINACA and its interferents is as follows Figure 5 As shown in the figure, the addition of synthetic cannabinoid ADB-4en-PINACA causes the color of the detection reagent to change from yellow-green to blue, and the characteristic fluorescence peak blue-shifts from 530nm to around 453nm. This change also occurs after other interfering substances and synthetic cannabinoid ADB-4en-PINACA are added together ( Figure 5 ), indicating that the benzoxazolyl probe molecule has good anti-interference ability to the synthetic cannabinoid ADB-4en-PINACA.

[0080] Example 7

[0081] Preparation of detection reagents:

[0082] 16 ml of polyphosphoric acid, 5 mmol, 1.0581 g of 3-bromo-2-hydroxybenzoic acid and 5 mmol, 0.5451 g of 2-aminophenol were mixed in a 100 ml three-necked flask, and stirred overnight at a temperature of 180° C., followed by addition of ice water, and filtration to obtain a solid product. The solid product was extracted with ethyl acetate, and the organic phase was collected. After removing the solvent in vacuo, it was purified by silica gel column chromatography using petroleum ether:ethyl acetate in a volume ratio of 40:1 as an eluent to obtain 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol solid;

[0083] The obtained 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol was dissolved in acetone, and after ultrasonic dissolution, a 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol probe solution with a concentration of 60 μmol / L was prepared to obtain a detection reagent;

[0084] The obtained detection reagent 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol probe solution was taken and 135 μL of the probe solution of the corresponding concentration was placed in a test tube;

[0085] Synthetic cannabinoid ADB-4en-PINACA standard solution preparation: Use methanol as solvent to prepare a synthetic cannabinoid standard solution with a concentration of 250 μmol / L;

[0086] Detection process: 50 μL and 250 μmol / L of the synthetic cannabinoid ADB-4en-PINACA were added to the prepared detection reagent respectively. After sufficient reaction, the excitation wavelength was selected as 365 nm, and the fluorescence spectrum and optical photograph were recorded;

[0087] Detection effect: The detection effect of benzoxazolyl probe on the synthetic cannabinoid ADB-4en-PINACA is as follows Figure 6 As shown, the addition of the synthetic cannabinoid ADB-4en-PINACA caused the color of the detection reagent to change from yellow-green to blue, and the response was rapid, <1s.

[0088] Example 8

[0089] The SEM results of the silicon wafer substrate loading test reagent to detect the synthetic cannabinoid ADB-4en-PINACA:

[0090] Detection reagent SEM sample preparation: 60 μmol / L 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol was dissolved in acetone and mixed, and then the mixture was dropped on the silicon wafer substrate. 10 μL of the synthetic cannabinoid ADB-4en-PINACA solution to be detected was drawn and dropped on the silicon wafer substrate loaded with the detection reagent, and the fluorescence change was observed under 365 nm illumination conditions;

[0091] Preparation of synthetic cannabinoid ADB-4en-PINACA standard solution: Use methanol as solvent to prepare a synthetic cannabinoid ADB-4en-PINACA standard solution with a concentration of 250 μmol / L;

[0092] Detection process: a mixture of 60 μmol / L 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol dissolved in acetone was dropped on a silicon wafer substrate, and 10 μL of the prepared synthetic cannabinoid ADB-4en-PINACA standard solution was added to the substrate. After sufficient contact and reaction, it was irradiated with 365 nm ultraviolet light, and the fluorescence effect was observed and a scanning electron microscope (SEM) image was taken;

[0093] Detection effect: The detection effect of the silicon wafer loaded with detection reagent to detect the synthetic cannabinoid ADB-4en-PINACA solution is as follows Figure 7 As shown in the scanning electron microscope (SEM), the original flake structure changes into spherical aggregates ( Figure 7 ).

[0094] Example 9

[0095] Detection results of the silicon wafer substrate loading test reagent test series concentrated synthetic cannabinoid ADB-4en-PINACA methanol droplets:

[0096] Preparation of silicon wafer substrate for detection reagent: 60 μmol / L 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol dissolved in acetone was dropped on the silicon wafer substrate, 10 μL of the synthetic cannabinoid ADB-4en-PINACA solution to be detected was drawn and dropped on the substrate loaded with the detection reagent, and the fluorescence change was observed under 365 nm illumination;

[0097] Preparation of synthetic cannabinoid ADB-4en-PINACA standard solution: using methanol as solvent, prepare synthetic cannabinoid ADB-4en-PINACA standard solution with concentrations of 10, 20, 40, 60, 80, 100, 150, 200, 250, 300, 350 μmol / L;

[0098] Detection process: Take 10 μL of the synthetic cannabinoid ADB-4en-PINACA standard solution of each concentration and drop it onto the silicon wafer substrate. After sufficient contact and reaction, irradiate with 365nm ultraviolet light, observe the fluorescence effect and take photos ( Figure 8 );

[0099] Detection effect: The detection effect of the silicon wafer loaded with detection reagent to detect the synthetic cannabinoid ADB-4en-PINACA solution is as follows Figure 8 As shown, the fluorescence color changes from yellow-green to blue, and the fluorescence change is obvious.

[0100] Example 10

[0101] The prepared portable detection sensor device shows the detection effect of the synthetic cannabinoid ADB-4en-PINACA mixed in the real sample. Prepared sensor device:

[0102] The sensor device is composed of a bottom plug 1, a bottle body 2 and a bottle cap 3, which is 3D modeled using SolidWorks software and printed using an Ultimaker S5 3D printer; the bottom plug 1, the bottle body 2 and the bottle cap 3 in the sensor device are connected by buckles;

[0103] Detection of the synthetic cannabinoid ADB-4en-PINACA:

[0104] 3-(Benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol and acetone solution are placed in a solid-liquid separation sensor device, the bottom plug 1 is used to store 3-(Benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol, the bottle body 2 is used to store the solvent acetone, and the bottom of the bottle cap 3 is embedded in a silicon substrate. When in use, the sealing ring of the bottom plug 1 is torn open, and then the bottom plug 1 is pressed to fully mix the 3-(Benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol and the acetone solvent in the bottle body 2. The mixed solution is dripped onto the silicon substrate at the bottom of the bottle cap 3 through the tip of the bottle body 2; then a suspicious liquid or solid (such as electronic cigarette oil or tobacco leaves, etc.) is added to the silicon substrate, and under the irradiation of an external 365nm ultraviolet lamp, the fluorescence change of the sensing area is observed to qualitatively confirm whether the suspicious liquid contains the synthetic cannabinoid ADB-4en-PINACA( Fig. 9 b);

[0105] When 10 μL of the substance containing the synthetic cannabinoid ADB-4en-PINACA to be tested (such as e-cigarette oil, flower petals and tobacco leaves) was added and the device was used for detection and analysis, the yellow-green fluorescence was observed to turn into blue ( Fig. 9 c), the results show that the prepared sensor device has good ability to qualitatively detect the synthetic cannabinoid ADB-4en-PINACA.

Claims

1. A method for detecting the synthetic cannabinoid ADB-4en-PINACA based on benzoxazolyl probe fluorescence, characterized in that: Follow these steps: Preparation of detection reagents: a. Mix polyphosphoric acid, 3-bromo-2-hydroxybenzoic acid and 2-aminophenol in a 100 ml three-necked flask, stir overnight at 180° C., then add ice water, filter, and obtain a solid product. Extract the solid product with ethyl acetate, collect the organic phase, remove the solvent in vacuo, and purify by silica gel column chromatography using petroleum ether:ethyl acetate in a volume ratio of 40:1 as eluent to obtain 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol solid; b. Dissolve the 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol solid obtained in step a in acetone, dissolve it by ultrasonication, and prepare a 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol probe solution with a concentration of 0.1 mmol / L to obtain a detection reagent; Detection of the synthetic cannabinoid ADB-4en-PINACA: c. Preparation of synthetic cannabinoid ADB-4en-PINACA standard solution: Use methanol as solvent to prepare a synthetic cannabinoid standard solution with a concentration of 1 mmol / L; d. Place 180 μL of the detection reagent 3-(benzo[d]oxazol-2-yl)-[1,1'-biphenyl]-2-ol probe solution obtained in step b into a test tube, then add 20 μL of the synthetic cannabinoid ADB-4en-PINACA solution to be detected, stir thoroughly, and record the fluorescence spectrum and optical photographs under 365 nm ultraviolet light excitation to observe the signal changes of the fluorescence characteristic emission peak before and after the reaction; Or use sensor devices to detect: Preparation of sensor device: The sensor device is composed of a bottom plug (1), a bottle body (2) and a bottle cap (3), and is three-dimensionally modeled using SolidWorks software and printed using an Ultimaker S5 3D printer; the bottom plug (1), the bottle body (2) and the bottle cap (3) in the sensor device are connected by means of a snap-fit ​​connection; Detection of the synthetic cannabinoid ADB-4en-PINACA: The detection reagent and acetone solution obtained in step b are placed in a solid-liquid separation sensor device, the bottom plug (1) is designed to store the detection reagent, the bottle body (2) is used to store the solvent acetone, and the bottom of the bottle cap (3) is embedded in the silicon substrate. When in use, the sealing ring of the bottom plug (1) is torn off, and then the bottom plug (1) is pressed to fully mix the detection reagent and the acetone solvent in the bottle body (2). The mixed solution is dripped onto the silicon substrate at the bottom of the bottle cap (3) through the tip of the bottle body (2); 10 μL of the synthetic cannabinoid ADB-4en-PINACA solution to be detected is drawn and dripped onto the silicon substrate, and the fluorescence change is observed under 365 nm illumination conditions.

Images