A pretreatment method for detecting cathinone-type new psychoactive substances in tea

Abstract

The present invention provides a pretreatment method for detecting cathinone-type new psychoactive substances in tea. First, the present invention studied the extraction method and found that the pH value of the extraction solution significantly affects the results, requiring it to be within the range of 5.0 to 6.0. Second, the present invention utilizes a purification combination of anhydrous calcium chloride + N-propylethylenediamine (PSA) + nano-ferroferric oxide-loaded graphitized carbon black (GCB@Fe3O4). This method not only leverages the superparamagnetic properties, large specific surface area, and numerous adsorption sites of magnetic nano-adsorbent materials, but also effectively adsorbs interfering substances in the tea matrix. These methods include: anhydrous calcium chloride can form chelate precipitates with the main matrix components of tea leaves, such as polyphenols, flavonoids, pigments, and some alkaloids; GCB and PSA can chelate metal ions, further removing organic acids, pigments, and metal ions. This method is more efficient and rapid than other methods.

Description

Technical Field

[0001] The invention belongs to the field of analytical chemistry and proposes an efficient pretreatment method for detecting multiple cathinone-type new psychoactive substances in tea. Background Art

[0002] Current detection methods for cathinone compounds mostly target biological samples such as blood, urine, and hair (e.g., CN113109491, CN 104833743, CN 113804746, CN 115060562, etc.), or focus on the suspected drugs themselves (e.g., CN107345946, CN106324149, CN 115144489, etc.). These methods aim to detect users or identify suspected drugs, but none involve the detection methods and pretreatment research of illegally added cathinones in food. Currently, cathinones are being synthesized. In addition to adding khat to tea, more synthetic cathinones are being added to tea and other foods through methods like spraying and soaking. Direct drug detection methods for these samples cannot achieve sufficient sensitivity and detection limits. Furthermore, detection methods for biological samples differ significantly from the tea matrix and are easily affected by the pigments, polyphenols, inorganic mineral elements, and secondary metabolites present in tea. This results in significant matrix effects and can easily cause interference in detection. Therefore, developing new processing technologies to enable rapid screening of tea samples is of paramount importance.

[0003] The present invention proposes an efficient pretreatment method for multiple cathinone-type new psychoactive substances in tea, which mainly solves the problem of removing interfering matrices from tea samples. The method can be combined with high-performance liquid chromatography, gas chromatography, liquid chromatography-mass spectrometry / mass spectrometry, gas chromatography / mass spectrometry and other methods to carry out detection. It can complete the detection of more than 15 cathinone compounds in tea within 1 hour, which is simple, rapid, accurate and reliable.

[0004] Patent CN 113680335A proposes a method for preparing nanomagnetic microspheres, Patent CN 115144489A proposes a method for preparing molecularly imprinted polymers, and Patents CN 114042440A and CN 113731381A propose methods for preparing magnetic solid-phase extraction adsorbents and magnetic nanomaterials, respectively. The main steps of these patents are: (1) preparing ferroferric oxide nanospheres. (2) coating the surface of ferroferric oxide nanospheres with silica to form silica microspheres. (3) wrapping the outer layer with functional materials such as C18 and PDA to form an adsorbent. (4) using the standard curve method for quantitative analysis by liquid chromatography-tandem mass spectrometry. The principle of this method is to use the prepared material to adsorb drugs in sewage and urine, and then elute the target from the material with a special solvent. However, when it was applied to tea samples, it was found that the adsorbent was affected by pigments and inorganic minerals, and at the same time brought in a large number of interfering impurities, making it unsuitable for use in tea samples.

[0005] The pretreatment method described in the present invention differs significantly from the aforementioned patents. First, the present invention studied the extraction method and found that the pH value of the extraction solution significantly affects the results, requiring it to be within the range of 5.0-6.0. Second, the present invention utilizes a purification combination of anhydrous calcium chloride + N-propylethylenediamine (PSA) + nano-ferroferric oxide-loaded graphitized carbon black (GCB@Fe3O4). This not only leverages the superparamagnetic properties, large specific surface area, and numerous adsorption sites of magnetic nano-adsorbent materials, but also effectively adsorbs interfering substances in the tea matrix. These include: anhydrous calcium chloride forms chelate precipitates with the main tea matrix components, polyphenols, flavonoids, pigments, and some alkaloids; GCB and PSA chelate metal ions, further removing organic acids, pigments, and metal ions. This method is more efficient and rapid than other methods. Summary of the Invention

[0006] The purpose of the present invention is to provide a pretreatment method for detecting cathinone-type new psychoactive substances in tea.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A pretreatment method for detecting cathinone-type new psychoactive substances in tea comprises the following steps:

[0009] (1) Preparation of GCB@Fe3O4: Nano-Fe3O4 with a particle size of 10-50 nm and graphitized carbon black GCB were mixed in a weight ratio of 1-3:1, and then anhydrous ethanol 5 times the weight of the solid was added. After vortex mixing, the mixture was ultrasonicated at a power of 1000-2000 W for 1 h. After being taken out, it was allowed to stand at room temperature for more than 2 h, and then centrifuged at 3000-5000 r / min for 1-3 min. The supernatant was removed and the product was placed in a vacuum dryer at 80 °C for 4 h. After that, the centrifuge tube was taken out, the obtained product was crushed and passed through a 10-mesh sieve to prepare the magnetic nano-adsorbent material GCB@Fe3O4.

[0010] (2) Extraction: Weigh the tea sample, add saturated sodium chloride solution, vortex for 30 seconds to mix, soak for 5 minutes, add methanol, and then add an appropriate amount of 20g / L sodium hydroxide solution to adjust the pH value of the system to between 5.0 and 6.0. High-speed homogenization extraction for 1 minute, centrifuge at 4000 r / min for 3 minutes, and collect the supernatant for purification;

[0011] (3) Purification: Take the extract obtained above, add anhydrous calcium chloride, GCB@Fe3O4, ethylenediamine-N-propylsilane PSA, and anhydrous sodium sulfate, vortex mix for 30 seconds, use a magnet to adsorb the solid matter to the bottom and side wall of the centrifuge tube or centrifuge at 4000 r / min for 3 minutes, take 1 mL of the upper organic phase solution and filter it through a 0.22 μm filter membrane for testing.

[0012] Furthermore, in step (2), the amount of saturated sodium chloride solution added is based on the tea sample and is added at a material-liquid ratio of 2:2~5 g / ml.

[0013] Furthermore, in step (2), the amount of methanol added is based on the tea sample and is added at a material-liquid ratio of 1:5~10 g / ml.

[0014] Furthermore, in step (3), the mass ratio of anhydrous calcium chloride to tea leaves is 0.1-0.5:1. The mass ratio of GCB@Fe3O4 to tea leaves is 0.05-0.4:1. The mass ratio of ethylenediamine-N-propylsilane PSA to tea leaves is 0.1-0.3:1. The mass ratio of anhydrous sodium sulfate to tea leaves is 1-2:1.

[0015] Furthermore, the test liquid obtained by the pretreatment method is analyzed by gas chromatography-tandem mass spectrometry.

[0016] The advantages of the present invention are:

[0017] First, there is a lack of research and literature specifically targeting cathinone-like new psychoactive substances in tea. Second, most of the methods described in the literature target biological samples (blood, urine, hair, etc.) or suspected drugs, and cannot address the issue of illegally added cathinones in tea. Third, using methods developed for biological samples to test matrices such as tea can significantly impact the quality of the material, particularly extraction and purification. Fourth, optimization has uncovered the impact of pH on extraction. Fifth, a purification material has been developed that effectively targets cathinones in tea. Results demonstrate that the above approach achieves excellent results. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Electron microscope images of GCB (A), nano-Fe3O4 (B) and the Fe3O4@GCB mixture of the present invention (C);

[0019] Figure 2 This is the GCB@Fe3O4 separation effect diagram under the action of an external magnetic field;

[0020] Figure 3 This is the matrix effect diagram of tea samples;

[0021] Figure 4 This is the total ion current chromatogram of 15 cathinone substances;

[0022] Figure 5 Total ion chromatogram of tea sample spiked at the limit of quantification. DETAILED DESCRIPTION

[0023] To make the above features and advantages of the present invention more clearly understood, the following embodiments are given for detailed description. The methods of the present invention are all conventional methods in the art unless otherwise specified.

[0024] 1 Experimental part

[0025] 1.1 Experimental Instruments

[0026] Agilent 7890A / 5975C gas chromatograph-mass spectrometer; electronic balance; centrifuge; nitrogen blowdown apparatus.

[0027] 1.2 Reagents and Materials

[0028] Methanol, sodium chloride, sodium hydroxide, anhydrous sodium sulfate, anhydrous calcium chloride, ferrosoferric oxide (Fe3O4) particles: 50nm; ethylenediamine-N-propylsilane (PSA); graphitized carbon black (GCB).

[0029] 1.3 Preparation of Fe3O4-loaded graphitized carbon black magnetic nano-adsorbent material (GCB@Fe3O4)

[0030] Nano-Fe3O4 with a particle size of 10-50 nm and graphitized carbon black (GCB) were mixed in a weight ratio of 2:1, and then anhydrous ethanol 5 times the weight of the solid was added. After vortex mixing, the mixture was ultrasonicated at 1500 W power for 1 hour. After being taken out, it was allowed to stand at room temperature for more than 2 hours, and then centrifuged at 4000 r / min for 2 minutes. The supernatant was removed and the product was placed in a vacuum dryer at 80 °C for 4 hours. The centrifuge tube was taken out, the resultant was crushed and passed through a 10-mesh sieve to prepare the magnetic nano-adsorbent material GCB@Fe3O4.

[0031] 1.4 Extraction

[0032] Weigh 2 g of tea sample, add 5 mL of saturated sodium chloride solution (the material-liquid ratio to tea is 2:2~5 g / ml), vortex for 30 s to mix, soak for 5 min, add 10 mL of methanol, add an appropriate amount of 20 g / L sodium hydroxide solution to adjust the pH value of the system to between 5.0 and 6.0, extract with high-speed homogenization for 1 min, centrifuge at 4000 r / min for 3 min, and collect the supernatant for purification.

[0033] 1.5 Purification

[0034] Take 10 mL of the extract obtained above, add 0.2 g of anhydrous calcium chloride, 0.1 g of GCB@Fe3O4, 0.2 g of ethylenediamine-N-propylsilane (PSA), and 2 g of anhydrous sodium sulfate, vortex mix for 30 s, and use a magnet to adsorb the solid matter to the bottom and side wall of the centrifuge tube or centrifuge at 4000 rpm for 3 min.

[0035] Take 1 mL of the above organic phase solution and pass it through a 0.22 μm filter membrane for testing.

[0036] 1.6 Quantitative Analysis

[0037] (1) Perform sample extraction, purification and other pre-treatment steps as described above.

[0038] (2) Select tea samples that do not contain the target substance, and perform pretreatment according to steps 1.3-1.5 to obtain a blank matrix solution. Use the blank matrix solution to prepare cathinone standard solutions into mixed standard solutions with concentrations of 10.00 μg / L, 20.00 μg / L, 50.00 μg / L, 100.00 μg / L, 200.00 μg / L, and 300.00 μg / L. Measure under the selected chromatographic conditions and mass spectrometry parameters, and establish a linear standard curve with the target compound quantitative ion peak area as the ordinate and the concentration as the abscissa;

[0039] (3) The test solution of cathinone compounds in tea obtained in step (1) is analyzed by gas chromatography-tandem mass spectrometry, and the types of target substances are determined according to the peak time and qualitative and quantitative ions, and the peak areas are respectively substituted into the corresponding cathinone mixed standard curve to obtain the content of various target substances in the test solution.

[0040] 1.7 Gas chromatography conditions

[0041] Chromatographic column: DB-17MS capillary column (30 m × 0.25 mm × 0.25 µm); injection port temperature: 250°C; injection volume: 1.0 μL; injection method: splitless; carrier gas: high-purity helium (99.999%); flow rate: constant flow rate of 1.5 mL / min; temperature program: initial 70°C, hold for 2 min, increase to 180°C at 10°C / min, then increase to 300°C at 15°C / min, hold for 1 min. Solvent delay time: 7.5 min.

[0042] 1.8 Mass spectrometry conditions

[0043] Electron impact ionization (EI source); ionization energy: 70 eV; ion source temperature: 310°C; transfer line temperature: 290°C; solvent delay time: 7.5 min; detection method: selected ion monitoring (SIM).

[0044] 2 Results Analysis

[0045] 2.1 Comparison of purification methods

[0046] The purification combination of anhydrous calcium chloride + N-propylethylenediamine (PSA) + nano-iron tetroxide-loaded graphitized carbon black (GCB@Fe3O4) used in the present invention has the following main advantages compared with the solid phase extraction cartridge and QuEChERS purification method used in literature reports and traditional detection: (1) The pH value of the extraction solution is optimized. A large number of experiments have shown that when the pH of the extract is in the range of 5.0-6.0, it has a better extraction effect on cathinone-type new psychoactive substances. In particular, for different types of tea, adjusting the pH value to the appropriate range has a crucial impact on the stability and accuracy of the results. (2) Anhydrous calcium chloride can form chelate precipitates with the main matrix components of tea, such as polyphenols, flavonoids, pigments, and some alkaloids, effectively reducing the matrix effect and improving the stability of the detection results. (3) GCB and PSA can remove organic acids, pigments and metal ions from tea leaves and are often used for the detection and analysis of tea leaves. By preparing graphitized carbon black magnetic nano-adsorption materials loaded with ferroferric oxide, not only the original purification effect is retained, but also the problems of poor stability, easy oxidation and instability of GCB materials under extreme acid and alkaline conditions are overcome. Compared with the traditional method of simply mixing functional materials with samples, this method has better purification effect and easier operation, and separation and purification can be achieved under the action of an external magnetic field.

[0047] 2.2 Matrix Effect

[0048] This method investigated the matrix effect of various matrices. The slope of the standard curve prepared with methanol (SlopeA) was compared with the slope of the standard curve of the same concentration prepared with blank matrix (SlopeB). The matrix effect (ME) was calculated as (Slope B-Slope A) / Slope A×100%. The results are shown in Figure 3 .

[0049] According to the matrix effect criteria, a weak matrix effect is considered when |ME| < 20%, a moderate matrix effect is considered when 20% < |ME| < 50%, and a strong matrix effect is considered when |ME| > 50%. As can be seen from the figure, most matrix effects are moderate. To further minimize the impact of matrix effects, this method uses a matrix curve quantification method to further correct for matrix effects, thereby improving method stability. The recovery and precision results from the spiked experiments show good stability after using the matrix curve correction, achieving satisfactory recovery and precision specifications.

[0050] 2.3 Recovery and precision

[0051] Following the methods described in 1.3–1.8, tea samples containing no analyte were spiked and subjected to recovery tests. The spiked concentration was 25 μg / kg, and six replicates were performed at each level. The average recovery and relative standard deviation (RSD) were calculated. The results are shown in Table 1.

[0052] Table 1 Recovery and precision table (n=6)

[0053]

[0054] 2.4 Linear range and regression equation of the method

[0055] Fifteen cathinone standards were prepared using blank tea matrix solutions to prepare mixed standard working curves with mass concentrations of 25.0 μg / L, 50.0 μg / L, 100.0 μg / L, 200.0 μg / L, and 400.0 μg / L. After instrumental determination, the linear equations and correlation coefficients of each component were calculated. The results are shown in Table 2.

[0056] Table 2 Linear equations and correlation coefficients of 15 cathinone compounds

[0057]

[0058] 2.5 Method detection limit and quantification limit

[0059] Fifteen cathinone standard solutions at concentrations of 1 to 100 μg / L were added to negative tea matrix samples. The samples were determined according to the optimized pretreatment method and instrument conditions. The limits of detection (LOD) and quantification (LOQ) of the method were defined by the responses at quantitative ion signal-to-noise ratios of 3 and 10. Based on the signal-to-noise ratio results, the LODs of various targets were between 5 and 8 μg / kg, and the LOQs were around 25 μg / kg. The LOQs of some targets could be lower, but considering the ease of operation and use, the LOQs were unified based on the least sensitive compounds. Therefore, the LOQs of various cathinones in tea leaves of this method were determined to be 25 μg / kg. The total ion chromatogram of the spiked samples at the limit of quantification is shown in Figure 2. Figure 5 .

[0060] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A pretreatment method for detecting cathinone-type new psychoactive substances in tea, characterized in that: The following steps are involved: (1) Preparation of GCB@Fe3O4: Nano-Fe3O4 with a particle size of 10-50 nm and graphitized carbon black GCB were mixed in a weight ratio of 1-3:1, and then anhydrous ethanol 5 times the weight of the solid was added. After vortex mixing, the mixture was ultrasonicated at a power of 1000-2000 W for 1 h. After being taken out, it was allowed to stand at room temperature for more than 2 h, and then centrifuged at 3000-5000 r / min for 1-3 min. The supernatant was removed and the product was placed in a vacuum dryer at 80 °C for 4 h. After that, the centrifuge tube was taken out, the obtained product was crushed and passed through a 10-mesh sieve to prepare the magnetic nano-adsorbent material GCB@Fe3O4. (2) Extraction: Weigh the tea sample, add saturated sodium chloride solution, vortex for 30 seconds to mix, soak for 5 minutes, add methanol, and then add an appropriate amount of 20g / L sodium hydroxide solution to adjust the pH value of the system to between 5.0 and 6.

0. High-speed homogenization extraction for 1 minute, centrifuge at 4000 r / min for 3 minutes, and collect the supernatant for purification; (3) Purification: Take the extract obtained above, add anhydrous calcium chloride, GCB@Fe3O4, ethylenediamine-N-propylsilane PSA, and anhydrous sodium sulfate, vortex mix for 30 seconds, use a magnet to adsorb the solid matter to the bottom and side wall of the centrifuge tube or centrifuge at 4000 r / min for 3 minutes, take 1 mL of the upper organic phase solution and filter it through a 0.22 μm filter membrane for testing; The cathinone-type new psychoactive substances include CAS No.1189726-22-4, CAS No.879665-92-6, CAS No.94-09-7, CAS No.5485-65-4, CAS No.186028-80-8, CAS No.1214-15-9, CAS No.1454266-19-3, CAS No.17762-90-2, CAS No.1147-62-2, CAS No.17763-01-8, CAS No.1391052-36-0, CAS No.24622-60-4, CAS No.24622-62-6, and CAS No.850352-11-3, a total of 14 types; In step (2), the amount of saturated sodium chloride solution added is based on the tea sample, and the material-liquid ratio is 2:2~5 g / ml; the amount of methanol added is based on the tea sample, and the material-liquid ratio is 1:5~10 g / ml; The mass ratio of anhydrous calcium chloride to tea leaves is 0.1~0.5:1; The mass ratio of GCB@Fe3O4 to tea leaves is 0.05~0.4:1; The mass ratio of ethylenediamine-N-propylsilane PSA to tea leaves is 0.1~0.3:1; The mass ratio of anhydrous sodium sulfate to tea leaves is 1~2:

1.

2. The pre-treatment method according to claim 1, characterized in that The test liquid obtained by the pretreatment method was analyzed by gas chromatography-tandem mass spectrometry.

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