A purification material for pre-detection treatment of cathinone and its degradation products in plant samples, and its preparation method and application

Abstract

The present invention discloses a purification material for pre-treatment of cathinone and its degradation products in plant samples, and its preparation method and application. The purification material is GCB / Fe-NH2, which is prepared by bonding graphitized carbon black GCB and amino groups with nano-iron tetroxide as a magnetic carrier. The purification material has both adsorption and ion exchange functions, and the purification material and the extract can be directly magnetically separated without centrifugation or filtration, and the operation is simple. It can be used in the pre-treatment of cathinone and metabolites of norpseudoephedrine and norephedrine detection in plant samples, and has a good purification effect on different types of plant samples.

Description

Technical Field

[0001] The invention belongs to the field of analytical chemistry and proposes a purification material for pre-detection treatment of cathinone and its degradation products in plant samples and a preparation method and application thereof. Technical Background

[0002] Chat khat ) is also known as Qiaocha ( Catha edulis Forssk ), Arabic tea, Yemen tea, Ethiopian tea, Bushman tea, etc., are evergreen shrubs of the Celastraceae family of the Sapindus order. Khat is known as the "East African poppy". It can be chewed or dried and ground into powder. It has a stimulating effect on the human central nervous system. Long-term chewing can cause addiction. The World Health Organization classifies it as a Class II soft drug.

[0003] The cathinone contained in khat during the picking and drying process is mainly decomposed into norpseudoephedrine and a small amount of norephedrine. Cathinone, norpseudoephedrine and norephedrine are central nervous system stimulants and energy depletion agents. These chemical drugs have side effects to varying degrees, including high blood pressure, palpitations, arrhythmias, delusions, hallucinations, etc. Consumers who take such drugs for a long time without knowing it will cause serious harm to their health.

[0004] The roasted branches and leaves of khat look very similar to tea leaves and can easily be disguised as plant samples. Therefore, it is very important to carry out research on relevant detection technologies and monitor possible illegal activities. Summary of the invention

[0005] The purpose of the present invention is to propose an efficient pretreatment method for detecting cathinone and its degradation products (norephedrine and norephedrine) in plant samples, which combines high performance liquid chromatography, gas chromatography-mass spectrometry, and high performance liquid chromatography-mass spectrometry technology to better extract and separate cathinone, norephedrine and norephedrine from plant samples, remove the interference of endogenous substances such as pigments and organic acids, and is accurate and reliable.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing a purification material GCB / Fe-NH2 for pre-treatment of cathinone and its degradation products in plant samples, comprising the following steps:

[0008] 1) Mix nano-ferroferric oxide and 20 vol% ethanol at a ratio of 1:60 m / v, ultrasonicate at 50°C for 10 min, and then adjust the pH to 3.2 with 0.012 mol / L hydrochloric acid;

[0009] 2) magnetically separating nano-iron tetroxide from the mixture obtained in step 1), and washing with deionized water and anhydrous ethanol in turn;

[0010] 3) Add 100 mL of 50 vol% ethanol to the nano-iron tetroxide washed in step 2), adjust the pH to 4.0 with glacial acetic acid, and ultrasonicate at 50°C for 10 min;

[0011] 4) Add 2 mL of silane coupling agent KH792 to the mixture obtained in step 3), stir at 60°C for 13 h, separate the solid by magnetic separation, wash the obtained solid with deionized water, anhydrous ethanol and anhydrous ether in sequence, and then vacuum dry at 80°C for 12 h to obtain Fe3O4-NH2 dry solid;

[0012] 5) Take 700 mg of the Fe3O4-NH2 dry solid obtained in step 4), add 300 mg of graphitized carbon black (GCB) and 10 mL of N,N-dimethylformamide (DMF), ultrasonicate at 50°C for 1 h, let stand for 1 h, and then magnetically separate the solid. Wash the obtained solid with anhydrous ethanol and deionized water in turn, and then vacuum dry at 80°C for 12 h to obtain the purification material GCB / Fe-NH2;

[0013] The degradation products are norpseudoephedrine and norephedrine.

[0014] A purification material GCB / Fe-NH2 prepared by the above preparation method.

[0015] The above-mentioned purification material GCB / Fe-NH2 is used in the pretreatment of the detection of cathinone and its degradation products in plant samples, wherein the degradation products are norpseudoephedrine and norephedrine.

[0016] A pretreatment method for detecting cathinone and its degradation products in plant samples, the pretreatment method comprising: crushing the plant sample to be tested, adding methanol, ultrasonically extracting at 40°C, adding the above-mentioned purification material GCB / Fe-NH2 to the extract, shaking evenly, magnetically separating, and collecting the supernatant.

[0017] The significant advantages of the present invention are:

[0018] The current detection methods for cathinones in plant samples mainly include direct analysis after reagent extraction with organic solvents, liquid-liquid distribution methods, etc. Such methods are difficult to effectively remove the interference of endogenous components such as pigments and organic acids in plant samples. It is difficult to identify khat mixed in plant samples (such as tea leaves), and the effect is not good. Since the psychoactive substance cathinone and metabolites of norpseudoephedrine and norephedrine in khat are monoamine alkaloids with strong polarity, a large amount of pigments, organic acids and other substances will be extracted when polar organic solvents are used to extract plant samples, which brings difficulties to subsequent detection and affects the analysis effect. The purification material GCB / Fe-NH2 synthesized by the present invention utilizes the adsorption function of graphitized carbon black and the weak anion exchange function of amino groups, and achieves retention through adsorption and weak anion exchange, so it has a dual purification effect. At the same time, in order to further facilitate the operation, the present invention uses nano-ferroferric oxide as a magnetic carrier to bond GCB and amino groups, so that the prepared purification material GCB / Fe-NH2 has both adsorption and ion exchange functions, and the purification material and the extract can be directly magnetically separated without centrifugation or filtration, and the operation is simpler. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : UV-visible absorption spectra of sodium copper chlorophyllin, curcumin, quercetin and β-carotene before and after purification. A: Sodium copper chlorophyllin. B: Curcumin. C: Quercetin. D: β-carotene.

[0020] Figure 2 :The adsorption amount of pigments and organic acids by different purification materials. A: Pigments. B: Organic acids.

[0021] Figure 3 :Comparison of purification effects of different purification materials on tea extract. A: UV-visible full wavelength scan. B: Liquid chromatography-mass spectrometry full scan. C: Comparison of appearance color of the purified sample. DETAILED DESCRIPTION

[0022] In order to further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the present invention.

[0023] Example 1

[0024] 1 Experimental part

[0025] 1.1 Reagents and Materials

[0026] Cathinone standard solution, pseudoephedrine hydrochloride standard solution, norephedrine hydrochloride standard solution, Tianjin Alta Technology Co., Ltd.); nano-ferroferric oxide (Fe3O4), N-[3-(trimethoxysilyl)propyl]ethylenediamine (KH792), Shanghai Aladdin Reagent Co., Ltd.; anhydrous ethanol, 95% ethanol, methanol, glacial acetic acid, N,N-dimethylformamide (DMF), hydrochloric acid (analytical grade), China National Pharmaceutical Group Chemical Reagent Company; acetonitrile (chromatographic grade), ethyl acetate chromatographic grade, ThermoFisher, USA; graphitized carbon black (GCB), N-propylethylenediamine (PSA), Agilent, USA.

[0027] Experimental instruments

[0028] Nexera X2 LC-30A ultra-high performance liquid chromatograph (Shimadzu, Japan), AB SCIEX 3200 QTRAP mass spectrometer (AB SCIEX, USA), CR22G high-speed centrifuge; Vacucell vacuum drying oven (MMM, Germany); S300H ultrasonic cleaner (Elma, Germany); VORTEX4digital vortex oscillator (IKA, Germany); Milli-Q ultrapure water system (Millipore, USA).

[0029] Purification material preparation

[0030] Step 1: Mix nano-ferroferric oxide with 20 vol% ethanol aqueous solution at a ratio of 1:60 m / v, and ultrasonicate at 50°C for 10 min, with an ultrasonic frequency of 40-50 KHZ and an ultrasonic power of 1 W / cm 2 , and then adjust the pH to 3.2 with 0.01 mol / L hydrochloric acid.

[0031] Step 2: Nano-ferroferric oxide is removed from the mixture obtained in step 1 by magnetic separation, and washed with deionized water and anhydrous ethanol for 3 to 5 times, each time for about 0.5 minutes.

[0032] Step 3: Add 100 mL of 50 vol% ethanol aqueous solution to the nano-iron tetroxide after cleaning in step 2, adjust the pH to 4.0 with glacial acetic acid, and ultrasonicate at 50°C for 10 min, with an ultrasonic frequency of 40-50 KHZ and an ultrasonic power of 1 W / cm 2 .

[0033] Step 4: Add 2 mL of KH792 to the mixture obtained in step 3, stir at 60 °C for 13 h, and magnetically separate the solid. Wash the solid with deionized water, anhydrous ethanol, and anhydrous ether for 3 to 5 times, each time for about 0.5 min, and then vacuum dry at 80 °C for 12 h to obtain Fe3O4-NH2 dry solid.

[0034] Step 5: Take 700 mg of the Fe3O4-NH2 dry solid obtained in step 4, add 300 mg of GCB and 10 mL of DMF, and ultrasonicate at 50°C for 1 h, with an ultrasonic frequency of 40-50 KHZ and an ultrasonic power of 1 W / cm 2 , then let it stand at room temperature for 1 hour, then magnetically separate the solid, wash the solid with anhydrous ethanol and deionized water for 3 to 5 times, each time for about 0.5 min, and then vacuum dry at 80°C for 12 hours to obtain the purification material GCB / Fe-NH2.

[0035] Sample preparation

[0036] Weigh 2 g of homogenized plant sample, add 10 mL of methanol, and ultrasonicate at 40 °C (ultrasonic frequency 40-50 KHZ, ultrasonic power 1 W / cm 2 ) for 20 min, take 5 mL of supernatant and add 500 mg of purification material GCB / Fe3O4-NH2 to the supernatant, vortex at 3000 r / min for 30 s and then perform magnetic separation, accurately transfer 200 μL of supernatant and add it to 800 μL of deionized water, filter through 0.22 μm filter membrane and wait for machine testing.

[0037] Instrument conditions

[0038] Chromatographic conditions:

[0039] Chromatographic column: Waters T3 column, column temperature: 40℃; injection volume: 10 uL, mobile phase: A is 5% methanol aqueous solution (containing 0.1% formic acid), B is 95% methanol aqueous solution; flow rate: 0.4 mL / min, gradient elution. 0~1.4min, mobile phase A is 98%; 1.4~1.6min, mobile phase A is 90%; 1.6~3.0min, mobile phase A is 90%; 3.0~4.0min, mobile phase A is 2%.

[0040] Mass spectrometry conditions

[0041] Ionization source: positive ion electrospray ionization source (ESI+); detection mode: multiple reaction monitoring mode (MRM); nebulizer voltage (IS): 5500 V; ion source temperature: 500°C; nebulizer gas pressure: 50 kPa, auxiliary gas pressure: 50 kPa. The mass spectrometry analysis parameters of cathinone and metabolites are shown in Table 1.

[0042] Table 1 Mass spectrometry parameters for cathinones and their metabolites

[0043]

[0044] * indicates quantification product ion

[0045] 2 Results and Discussion

[0046] 2.1 Comparison of purification effects

[0047] The current detection methods for cathinones in plant samples mainly include direct analysis after extraction with organic solvents and liquid-liquid distribution. These methods are difficult to effectively remove the interference of endogenous components such as pigments and organic acids in plant samples. They are difficult to identify khat mixed in plant samples (such as tea), and the effect is not good.

[0048] Since the psychoactive substance cathinone and metabolites of norpseudoephedrine and norephedrine in khat grass are monoamine alkaloids, they have strong polarity. When a plant sample is extracted using a polar organic solvent, a large amount of pigments, organic acids and other substances will be extracted together, which brings difficulties to subsequent detection and affects the analysis effect. The purification material GCB / Fe-NH2 synthesized by the present invention utilizes the adsorption function of graphitized carbon black and the amino weak anion exchange function, and the retention effect is achieved by adsorption and weak anion exchange, so there is a dual purification effect. At the same time, in order to further facilitate operation, nano-iron tetroxide is used as a magnetic carrier, bonding GCB and amino groups, so that the prepared purification material GCB / Fe-NH2 has both adsorption and ion exchange functions, and the purification material GCB / Fe-NH2 and the extract can be directly magnetically separated, without centrifugation or filtration, and the operation is easier.

[0049] GCB and PSA are the most commonly used purification materials for removing sample pigments and organic acids. Nano-ferroferric tetroxide has a good complexing effect on compounds with ortho-phenolic hydroxyl structures and is also used for the purification of some samples. This paper selects representative pigments and organic acids and compares the purification effects of several purification materials.

[0050] 2.1.1 Comparison of purification effects on pigments and organic acids

[0051] The purification effects of representative natural pigments in plant samples such as sodium copper chlorophyllin, curcumin, quercetin, and β-carotene, and representative organic acids in plant samples such as tartaric acid, oxalic acid, malic acid, citric acid, gallic acid, and ascorbic acid were compared. 100 mg of GCB or PSA or Fe3O4 or the purification material GCB / Fe-NH2 synthesized in this paper was added to 10 mL of 5 mg / mL standard solution of pigments and organic acids, respectively. After oscillation for 30 s, the solution was allowed to stand for 1 min, and the supernatant was obtained by centrifugation or magnetic separation. The pigment solution before and after adsorption was scanned at full wavelength (200-800 nm) by UV-visible spectrophotometer, and the adsorption amount of each pigment by the four materials was calculated; the adsorption amount of gallic acid by the four materials was determined by Folin phenol colorimetry; the adsorption amount of tartaric acid, oxalic acid, malic acid, citric acid, and ascorbic acid by the four materials was determined by acid-base titration.

[0052] like Figure 1 and Figure 2 As shown in the figure, the absorption spectra of the four pigment solutions after adsorption by the purification material (GCB / Fe-NH2) synthesized in this paper have decreased significantly, and the purification effect is significantly better than PSA and Fe3O4, and the adsorption effect on sodium copper chlorophyllin is also better than GCB (currently the material with the best adsorption effect on pigments). This may be because the diamino group in GCB / Fe-NH2 has both adsorption and ion exchange effects on sodium copper chlorophyllin, which to some extent makes up for the lack of GCB pigment adsorption performance. For the purification of organic acids, GCB / Fe-NH2 also showed a purification effect that was significantly better than GCB and Fe3O4.

[0053] Example of adsorption cleanup of actual samples

[0054] Taking carrots (high pigment content), apples (high organic acid content) and tea leaves (high pigment, organic acid and phenolic substance content) as examples, methanol extraction and liquid chromatography-mass spectrometry were used to analyze cathinone and its metabolites, norephedrine and pseudoephedrine, and the purification effects of the synthesized purification material (GCB / Fe-NH2) in this paper were compared with those of GCB, PSA and Fe3O4.

[0055] Figure 3 The following is a sample matrix background image of tea leaves before and after purification. Figure 3 It can be seen that after purification by GCB / Fe-NH2 and GCB, the appearance of tea methanol extract is basically colorless ( Figure 3 C), which is significantly stronger than the purification effect of PSA and Fe3O4. UV-visible spectrum ( Figure 3 A) and full scan chromatogram of HPLC-MS ( Figure 3 B) It also proves that the matrix background is relatively weakened after GCB / Fe-NH2 and GCB purification.

[0056] Six batches of tea, carrot and apple samples without cathinone and metabolites of norpseudoephedrine and norephedrine were taken, crushed and mixed, and 100uL of 10ppm cathinone, 100uL of 10ppm norpseudoephedrine and 100uL of 10ppm norephedrine were added to the samples at the same time, and then 10mL of methanol was added for ultrasonic extraction at 40℃ for 10min. 5mL of extract was taken, and 200mg of GCB or PSA or Fe3O4 or purification material GCB / Fe-NH2 was added respectively. After centrifugation or magnetic separation, the purified liquid was taken, nitrogen was blown to near dryness, and 20% methanol aqueous solution was vortexed and re-dissolved. Liquid chromatography-mass spectrometry was used for detection, and the recovery rate and precision (relative standard deviation) were measured in parallel 6 times. The results are shown in Table 2. The results show that among the four purification materials, the material synthesized in this paper (GCB / Fe-NH2) has satisfactory recovery rate and precision for three different samples.

[0057] Six batches of tea, carrot and apple samples without cathinone and metabolites of norpseudoephedrine and norpseudoephedrine (hereinafter referred to as blank samples) were taken respectively, and GCB / Fe-NH2 synthesized in this paper was used as purification material. The blank sample extract was prepared by the pretreatment steps in 1.4 above, and then the target analyte standard solution was added respectively, so that the content of the target analyte in the sample extract was 20μg / L, and the sample was introduced into the liquid chromatography-mass spectrometry analysis system together with the pure reagent spiked sample of the same concentration for analysis, and the response signals of the blank matrix extract spiked sample and the pure reagent spiked sample were obtained. For each batch of samples, the normalized matrix effect factor was calculated according to the formula ME= A / B (where A: blank matrix extract spiked sample response signal; B: blank reagent spiked sample response signal). The results showed that the ME values ​​of the three samples were between 0.91 and 1.19, all showing weak matrix effects, which were acceptable.

[0058] Table 2 Recovery and precision of target analytes in tea, carrot and apple samples (n=6)

[0059]

[0060]

Claims

1. A purification material GCB / Fe-NH for pre-treatment of cathinone and its degradation products in plant samples 2 The preparation method of Features: The following steps are involved: 1) Mix nano-ferroferric oxide and 20 vol% ethanol at a ratio of 1:60 m / v, ultrasonicate at 50°C for 10 min, and then adjust the pH to 3.2 with 0.012 mol / L hydrochloric acid; 2) magnetically separating nano-iron tetroxide from the mixture obtained in step 1), and washing with deionized water and anhydrous ethanol in turn; 3) Add 100 mL of 50 vol% ethanol to the nano-iron tetroxide washed in step 2), adjust the pH to 4.0 with glacial acetic acid, and ultrasonicate at 50°C for 10 min; 4) Add 2 mL of silane coupling agent KH792 to the mixture obtained in step 3), stir at 60°C for 13 h, separate the solid by magnetic separation, wash the solid with deionized water, anhydrous ethanol, and anhydrous ether in turn, and then vacuum dry at 80°C for 12 h to obtain Fe 3 O 4 -NH 2 Drying solids; 5) Take 700 mg of Fe obtained in step 4) 3 O 4 -NH 2 The solid was dried, 300 mg of graphitized carbon black and 10 mL of N,N-dimethylformamide were added thereto, ultrasonicated at 50 °C for 1 h, then allowed to stand for 1 h, and then the solid was magnetically separated. The obtained solid was washed with anhydrous ethanol and deionized water in turn, and then vacuum dried at 80 °C for 12 h to obtain the purification material GCB / Fe-NH 2 ; The degradation products are norpseudoephedrine and norephedrine.

2. A purification material GCB / Fe-NH prepared by the preparation method according to claim 1 2 .

3. The purification material GCB / Fe-NH as claimed in claim 2 2 Application in pretreatment of cathinone and its degradation products in plant samples, Features: The degradation products are norpseudoephedrine and norephedrine.

4. A pretreatment method for detecting cathinone and its degradation products in plant samples, Features: The plant sample to be tested was crushed, methanol was added, ultrasonic extraction was performed at 40°C, and the purification material GCB / Fe-NH 2 , shake well, separate magnetically, and collect the supernatant.

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