Application of a triazine-triphenyltricarboxaldehyde covalent organic framework in enrichment and detection of sulfonamide antibiotics in food samples

Abstract

The invention belongs to the technical field of analytical chemistry and food safety detection, and discloses the application of a triazine-trimethylenediamine covalent organic skeleton in enriching and detecting sulfonamide antibiotics in food samples. The invention adopts a triazine-trimethylenediamine covalent organic skeleton compound as an adsorption material for solid phase extraction, optimizes the sample pretreatment procedure, and establishes a new method for sensitive analysis of sulfonamide antibiotics in food samples based on solid phase extraction and liquid chromatography-mass spectrometry technology, which can effectively separate the target substance from the coexisting matrix, so that the method of the invention has a higher spike recovery rate and a lower detection limit.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry and food safety detection, and specifically relates to the application of a triazine-triphenyltrimaldehyde covalent organic skeleton in enriching and detecting sulfonamide antibiotics in food samples. Background Art

[0002] Sulfonamide antibiotics, as a synthetic antibacterial drug, have the advantages of low cost, high efficiency, stable properties, and broad-spectrum antibacterial activity. They have been widely used in the prevention and treatment of various diseases such as bacterial infections in livestock and poultry. However, due to the existence of irregular drug use such as abuse, misuse, and failure to comply with the withdrawal period, as well as the fact that sulfonamide drugs have a long duration of action in animals and are not easily metabolized, sulfonamide antibiotics are easily accumulated in the human body through animal-derived foods. Long-term consumption of such foods can also cause serious side effects, such as poisoning, allergic reactions, and even have potential carcinogenic, teratogenic, and mutagenic properties. At present, my country and most countries such as Europe and the United States have strictly restricted the use of sulfonamide antibiotics, and listed them as drugs for limited use in animal feeding, limiting the maximum residue limit (MRL) of sulfonamide drugs in animal-derived foods to 0.1 mg / kg.

[0003] Due to the complex matrix, many interfering substances, complex chemical properties and low content of target components in livestock and poultry meat samples, the precision and accuracy of the test results are greatly affected. Therefore, a series of pretreatments such as extraction, purification and concentration of target components are required for livestock and poultry meat samples before testing. At present, the commonly used pretreatment methods for the detection of sulfonamide antibiotic residues include: liquid-liquid extraction, solid phase extraction, solid phase microextraction, magnetic solid phase extraction, etc. Among them, solid phase extraction is simple to operate, has good purification effect, reusable adsorbent, and uses less organic solvent. It is a relatively mature, efficient and reliable pretreatment method. The key to solid phase extraction technology lies in the adsorption material in the solid phase extraction column, because it can filter the interfering substances in the matrix, thereby achieving the enrichment of the target component. Therefore, the selective adsorption performance of the adsorption material is directly related to the efficiency of the pretreatment. At present, the commercially available solid phase extraction adsorption materials include Oasis HLB, MCX, C18, etc., but most of these adsorption materials have the disadvantages of high price and few adsorption types. Therefore, developing a new solid phase extraction material to achieve the detection and enrichment of sulfonamide antibiotics is the best choice to promote the development of solid phase extraction technology.

[0004] Covalent organic frameworks are a new type of porous crystalline material that has emerged in recent years and has attracted the attention of many researchers due to their excellent physical and chemical properties. Covalent organic frameworks are mainly composed of light elements such as C, N, and O connected by strong covalent bonds to form a porous framework with a periodic structure. Therefore, its pore structure is highly ordered, the skeleton density is low, the pore size is adjustable and uniform, and the specific surface area is large. It has the characteristics of diverse synthesis methods, easy functional modification, good thermal stability and chemical stability. At present, covalent organic frameworks have been widely used in various aspects such as gas storage, catalysis, and optoelectronic materials, and have great development potential in the environmental field. It is expected to become an excellent solid phase extraction adsorption material or adsorption coating. Summary of the invention

[0005] In order to overcome the defects and shortcomings of the prior art, the purpose of the present invention is to provide an application of a triazine-trimethylenediamine covalent organic skeleton in the enrichment and detection of sulfonamide antibiotics in food samples; the application utilizes the triazine-trimethylenediamine covalent organic skeleton as a solid phase extraction adsorption material to separate and enrich sulfonamide drugs, which is used to purify food samples, and combines liquid chromatography-mass spectrometry technology to establish a sensitive analysis method for sulfonamide antibiotics.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] Application of a triazine-trimethylenediamine covalent organic framework in the enrichment and detection of sulfonamide antibiotics in food samples.

[0008] Preferably, the triazine-trimethylene glycol covalent organic framework is prepared according to the following method: 2,4,6-tris(4-aminophenyl)-1,3,5-triazine and trimesaldehyde are used as raw materials, placed in 1,4-dioxane, glacial acetic acid is added dropwise, and synthesized at room temperature under the catalytic action of acetic acid. The obtained product is washed, vacuum dried, and ground to obtain a triazine-trimethylene glycol covalent organic framework (TAPT-BTCACOF).

[0009] Preferably, the molar ratio of the 2,4,6-tris(4-aminophenyl)-1,3,5-triazine to trimesaldehyde is 1:1; the volume ratio of 1,4-dioxane to glacial acetic acid is 9:2; the amounts of the 2,4,6-tris(4-aminophenyl)-1,3,5-triazine and 1,4-dioxane are in a molar volume ratio of 1 mmol:15 mL; and the washing is performed sequentially using tetrahydrofuran and methanol.

[0010] Preferably, the sulfonamide antibiotic is sulfadiazine, sulfadimethoxine, sulfadoxine, sulfamethoxazole or sulfapyridine.

[0011] Preferably, the enrichment and detection of sulfonamide antibiotics in food samples includes the following steps: using triazine-trimethylenediamine covalent organic skeleton as adsorption material to prepare a solid phase extraction column, pre-treating and enriching the food analyte through the solid phase extraction column to obtain a sample, and then detecting the obtained sample using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry.

[0012] Preferably, the specific operation steps of the pre-treatment are as follows:

[0013] (1) 40-60 mg of triazine-trimethylenediamine covalent organic framework is loaded into a solid phase extraction column; 1-3 mL of methanol and water are used to activate the solid phase extraction column in sequence;

[0014] (2) Adding acetic acid and acetonitrile with a volume percentage concentration of 1% to the food to be tested, extracting and centrifuging to obtain an extract; the amount of acetic acid and acetonitrile with a volume percentage concentration of 1% and the food to be tested is in a volume mass ratio of (3-5) mL: 1 g;

[0015] (3) The extract obtained in step (2) is concentrated and dried by nitrogen blowing, diluted with water, and then degreased twice by adding a saturated acetonitrile n-hexane solution. After centrifugation, the lower layer of the extract is passed through the solid phase extraction column activated in step (1);

[0016] (4) eluting the solid phase extraction column with an eluent, wherein the eluent is an aqueous solution containing 5% by volume of methanol;

[0017] (5) The solid phase extraction column is then eluted with methanol eluent; the eluent is dried with nitrogen gas, re-dissolved with the initial mobile phase, and filtered through a 0.22 μm filter membrane to obtain a sample.

[0018] Preferably, the lower layer extract in step (3) is driven through the column by a vacuum pump.

[0019] Preferably, the specific operation of step (2) is as follows: add 6-10 mL of acetic acid and acetonitrile with a volume percentage concentration of 1% to 2 g of the food to be tested, ultrasonicate for 10 minutes, centrifuge to obtain the supernatant, repeatedly add acetic acid and acetonitrile with a volume percentage concentration of 1% to extract once, and combine the extracts.

[0020] Preferably, the liquid phase detection conditions in the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry are as follows: the chromatographic column is a Waters BEH C18 chromatographic column of 2.1 mm×50 mm×1.7 μm, the chromatographic column temperature is 40°C, and the sample chamber temperature is 10°C; the mobile phase A is an aqueous solution containing 0.1% formic acid, and B is acetonitrile containing 0.1% formic acid; the gradient elution program is: 0-1 min, 10% B; 1-6 min, 10-30% B; 6-8 min, 30-100% B; 8-9 min, 100% B; 9.1-10 min, 10% B; the flow rate is 0.3 mL / min, and the injection volume is 2 μL.

[0021] Preferably, the mass spectrometry detection conditions in the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry are as follows: electrospray ionization, primary mass spectrometry scanning in positive ion mode, a scanning range of m / z=60-650Da, a scanning rate of 0.5spectra / s, and quadrupole time-of-flight mass spectrometry monitoring and analysis; ion source temperature of 120°C; desolvation gas temperature of 350°C; desolvation gas flow rate of 650L / h; cone gas flow rate of 50L / h; collision voltage of 30V; cone body voltage of 40V; capillary voltage of 2500V.

[0022] Compared with the prior art, the present invention has the following advantages and effects:

[0023] (1) The triazine-trimethylenediamine covalent organic framework used in the present invention is simple to prepare and has good thermal stability, chemical stability and crystallinity.

[0024] (2) The triazine-trimethylenediamine covalent organic framework used in the present invention is a microporous material with a large specific surface area and a good pore structure. It has a good adsorption effect, a high enrichment efficiency and a good spike recovery rate for sulfonamide antibiotics in food samples, and can be reused for more than 5 times.

[0025] (3) The present invention simplifies the pretreatment process for detecting sulfonamide antibiotic residues in food samples, is simple to operate, reduces the workload of pretreatment, reduces the amount of organic solvent used, and reduces toxic hazards.

[0026] (4) The detection method adopted by the present invention has high sensitivity, a wide linear range, and a good linear relationship. It can efficiently detect the content of sulfonamide antibiotic residues in food samples, has good reproducibility, a low detection limit, and a high spike recovery rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the infrared spectrum of TAPT-BTCA COF and its monomer.

[0028] Figure 2It is the XRD pattern of TAPT-BTCA COF.

[0029] Figure 3 It is the thermogravimetric curve of TAPT-BTCA COF.

[0030] Figure 4 This is the SEM image of TAPT-BTCA COF.

[0031] Figure 5 This is the nitrogen adsorption-desorption curve of TAPT-BTCA COF.

[0032] Figure 6 It is the pore size distribution diagram of TAPT-BTCA COF.

[0033] Figure 7 These are the results of solid phase extraction optimization in Example 2, where (a) is an optimization diagram of adsorbent dosage, (b) is an optimization diagram of sample pH value, (c) is an optimization diagram of elution solvent type, (d) is an optimization diagram of elution solvent type, (e) is an optimization diagram of elution solvent dosage, and (f) is a diagram of the number of times TAPT-BTCA COF can be recycled. DETAILED DESCRIPTION

[0034] The present invention is further described below with reference to specific embodiments, but they should not be construed as limiting the present invention.

[0035] Example 1: Preparation of solid phase extraction adsorbent based on TAPT-BTCA COF material

[0036] 531.6 mg of 2,4,6-tris(4-aminophenyl)-1,3,5-triazine and 243.2 mg of trimesic acid were added to 22.5 ml of 1,4-dioxane, and 5 mL (0.6 mol / L) of glacial acetic acid was added after ultrasonic treatment for 15 min. The mixture was allowed to stand at room temperature for 3 days, and washed with tetrahydrofuran and methanol three times respectively. The mixture was dried in vacuo at 60°C for 12 hours, and ground to obtain a yellow solid powder, which was the triazine-trimesic acid covalent organic framework (TAPT-BTCA COF material).

[0037] Characterization of the obtained TAPT-BTCA COF materials:

[0038] The functional groups of the synthesized TAPT-BTCA COF materials were confirmed by infrared spectroscopy (FT-IR). Figure 1 As shown, the raw material BTCA is at 1697cm -1 A strong and sharp absorption peak appeared at 1634 cm, which was attributed to the terminal aldehyde group in its structure. However, in the synthetic COF material, this absorption peak disappeared and turned to 1634 cm -1A strong absorption peak appears at 3322 cm-1, which is attributed to the C=N bond, indicating the successful formation of the imine bond. -1 、3210cm -1 The absorption peak at 1470 nm belongs to the amino group, while there is no such absorption peak in TAPT-BTCA COF, which further confirms that the amino and aldehyde groups in the two raw materials undergo Schiff base reaction to form an imine bond.

[0039] The crystallinity of the obtained TAPT-BTCA COF material was characterized by X-ray diffraction (XRD). Figure 2 From the above, we can see that TAPT-BTCA COF is a material with high crystallinity.

[0040] The thermal stability of the obtained TAPT-BTCA COF material was characterized by thermogravimetric curve (TGA). Figure 3 From the results, it can be seen that the material is relatively stable within the range of 500°C, and the quality is always maintained above 95%.

[0041] The morphology of the obtained TAPT-BTCA COF material was characterized by scanning electron microscopy (SEM). Figure 4 From the perspective of nanostructured particles, the average diameter of the synthesized powder is about 200 nm, and it is a flower-shaped particle polymer composed of rod-like structure clusters.

[0042] The specific surface area of ​​the obtained TAPT-BTCA COF material was characterized by nitrogen adsorption-desorption curve. Figure 5 The specific surface area of ​​the synthesized material is 176.0 cm 3 ·g -1 .

[0043] The pore structure composition of the obtained TAPT-BTCACOF material was characterized by pore size distribution. Figure 6 From the above, it can be seen that the synthesized material is a microporous material with a good pore structure.

[0044] Example 2: Sample pretreatment and solid phase extraction process

[0045] Weigh 2 g of meat homogenized sample and place it in a 50 mL centrifuge tube, add 6 mL of 1% acetic acid and acetonitrile by volume, vortex and ultrasonicate for 10 min, centrifuge and take the supernatant, repeat the extraction once, combine the extracts, concentrate the extracts to near dryness with a nitrogen blower, then add 5 mL of water and ultrasonicate to re-dissolve, degrease with 3 mL of n-hexane twice; discard the upper layer and pass the lower layer through a solid phase extraction column.

[0046] Take 60 mg of the TAPT-BTCA COF material obtained in Example 1 and put it into a 3 mL solid phase extraction column. Use 3 mL of methanol and 2 mL of water to activate the solid phase extraction filler in sequence; purify the treated sample at a loading rate of 1 mL / min, elute with 2 mL of an aqueous solution containing 5% methanol by volume, discard the eluent, elute with 3 mL of methanol solution, collect the eluent, blow dry with nitrogen, re-dissolve with the initial mobile phase, pass through a 0.22 μm filter membrane, and obtain a sample for machine testing.

[0047] This experiment optimized the extraction conditions: referring to the conditions in Example 2, single factor optimization experiments were performed.

[0048] Different adsorbent dosages (20, 40, 60, 80 mg), different sample pH values ​​(2, 4, 6, 8, 10, 12), different methanol contents in the elution solvent (0%, 5%, 10%,), different elution solvents (methanol, ethanol, acetonitrile, ethyl acetate, methanol containing 5% glacial acetic acid, methanol containing 5% ammonia water), and different eluent dosages (1 mL, 2 mL, 3 mL, 4 mL) were tested. Figure 7 Finally, the following optimal conditions were selected: adsorbent dosage 60 mg, sample pH value 8, methanol content in eluent 5%, elution solvent pure methanol, and elution solvent dosage 3 mL.

[0049] Example 3: LC-MS detection process for five sulfonamide antibiotics

[0050] Waters Xevo G2 QTOF with ACQUITY BEH C 18 The detection was carried out with water containing 0.1% formic acid by volume as phase A, acetonitrile containing 0.1% formic acid by volume as phase B, and the gradient elution program was as follows: 0-1min, 10% B; 1-6min, 10-30% B; 6-8min, 30-100% B; 8-9min, 100% B; 9.1-10min, 10% B; injection 2μL, chromatographic column temperature 40℃, sample chamber temperature 10℃. Electrospray ion source ionization was used, primary mass spectrometry scanning was performed in positive ion mode, the scanning range was m / z=60~650Da, and quadrupole time-of-flight mass spectrometry was used for monitoring and analysis. The ion source temperature was 120℃; the desolvation gas temperature was 350℃; the desolvation gas flow rate was 650L / h; the cone gas flow rate was 50L / h; the collision voltage was 30V; the cone body voltage was 40V; and the capillary voltage was 2500V. Primary mass spectrometry ions [M+H] of five sulfonamide antibiotics + , retention time see Table 1.

[0051] Method establishment and application evaluation:

[0052] Under the optimal conditions, the detection limit, quantification limit, linear range and precision of the method were investigated. The correlation coefficient was between 0.992 and 0.997, and the detection limit was between 0.02 and 0.19 μg / kg. Details are shown in Table 2.

[0053] In order to verify the practicality of the established method, the method was applied to the detection of sulfonamide antibiotics (sulfadiazine SDZ, sulfadimethazine SMZ, sulfadoxine SDX, sulfamethoxazole SMX and sulfapyridine SPD) in three actual samples of chicken, pork and milk. 2g of homogenized blank sample was accurately weighed, and appropriate amounts of mixed standard working solutions of sulfonamide drugs of different concentrations were added to prepare spiked samples of three addition levels of 5μg / kg, 50μg / kg and 100μg / kg. The samples were treated according to the sample pretreatment method and solid phase extraction process of Example 2 above, and the recovery and precision of each concentration addition level were calculated using the linear equation of the matrix matching standard curve. The results are shown in Table 3, and the spiked recovery was 70.5%-105.3%.

[0054] Table 1 Primary mass spectrometry ions of analytes [M+H] + and retention time

[0055]

[0056] Table 2 Detection limit, quantification limit and linear range of the method

[0057]

[0058] Note: The linear range is 1-200 μg·L -1 .

[0059] Table 3 Recovery and precision of sulfonamide antibiotics in food samples

[0060]

[0061]

[0062] Note: The intra-batch recovery rate is the lowest value among the three batches.

[0063] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Application of a triazine-trimethylenediamine covalent organic framework in enriching and detecting sulfonamide antibiotics in food samples, characterized in that: The triazine-trimethylenediamine covalent organic skeleton is prepared according to the following method: 2,4,6-tris(4-aminophenyl)-1,3,5-triazine and trimethylenediamine are used as raw materials, placed in 1,4-dioxane, glacial acetic acid is added dropwise, and synthesized at room temperature under the catalytic effect of acetic acid. The obtained product is washed, vacuum dried, and ground to obtain the triazine-trimethylenediamine covalent organic skeleton.

2. The use according to claim 1, characterized in that: The molar ratio of the 2,4,6-tris(4-aminophenyl)-1,3,5-triazine to trimesaldehyde is 1:1; the volume ratio of the 1,4-dioxane to glacial acetic acid is 9:2; the amounts of the 2,4,6-tris(4-aminophenyl)-1,3,5-triazine and 1,4-dioxane are in a molar volume ratio of 1 mmol:15 mL; and the washing is performed sequentially with tetrahydrofuran and methanol.

3. The use according to claim 1, characterized in that: The sulfonamide antibiotic is sulfadiazine, sulfadimethoxine, sulfadoxine, sulfamethoxazole or sulfapyridine.

4. The use according to claim 1, characterized in that: The enrichment and detection of sulfonamide antibiotics in food samples comprises the following steps: using triazine-trimethylenediamine covalent organic skeleton as adsorption material to prepare a solid phase extraction column, pre-treating and enriching the food to be tested through the solid phase extraction column to obtain a sample, and then detecting the obtained sample by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry.

5. The use according to claim 4, characterized in that: The specific steps of the pre-treatment are as follows: (1) 40-60 mg of triazine-trimethylenediamine covalent organic framework was loaded into a solid phase extraction column; 1-3 mL of methanol and water were used to activate the solid phase extraction column in sequence; (2) Add acetic acid and acetonitrile with a volume percentage concentration of 1% to the food to be tested, extract and centrifuge to obtain an extract; the amount of acetic acid and acetonitrile with a volume percentage concentration of 1% and the food to be tested is in a volume mass ratio of (3-5) mL: 1 g; (3) The extract obtained in step (2) is concentrated and dried by nitrogen blowing, diluted with water, and then degreased twice by adding a saturated acetonitrile n-hexane solution. After centrifugation, the lower layer of the extract is passed through the solid phase extraction column activated in step (1); (4) eluting the solid phase extraction column with an eluent, wherein the eluent is an aqueous solution containing 5% by volume of methanol; (5) Elute the solid phase extraction column with methanol as eluent; after the eluent is dried with nitrogen, it is reconstituted with the initial mobile phase and filtered through a 0.22 μm filter membrane to obtain the sample.

6. The use according to claim 5, characterized in that: In step (3), the lower layer extract is driven through the column by a vacuum pump.

7. The use according to claim 5, characterized in that: The specific operation of step (2) is as follows: add 6-10 mL of acetic acid and acetonitrile with a volume percentage concentration of 1% to 2 g of the food to be tested, perform ultrasonic treatment for 10 min, centrifuge and obtain the supernatant, repeatedly add acetic acid and acetonitrile with a volume percentage concentration of 1% to extract once, and combine the extracts.

8. The use according to claim 4, characterized in that: The liquid phase detection conditions in the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry are as follows: the chromatographic column is a Waters BEH C18 chromatographic column of 2.1 mm×50 mm×1.7 µm, the chromatographic column temperature is 40°C, and the sample chamber temperature is 10°C; the mobile phase A is an aqueous solution containing 0.1% formic acid, and B is acetonitrile containing 0.1% formic acid; the gradient elution program is: 0-1 min, 10% B; 1-6 min, 10-30% B; 6-8 min, 30-100% B; 8-9 min, 100% B; 9.1-10 min, 10% B; the flow rate is 0.3 mL / min, and the injection volume is 2 µL.

9. The use according to claim 4, characterized in that: The mass spectrometry detection conditions in the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry method are as follows: electrospray ionization, primary mass spectrometry scanning in positive ion mode, a scanning range of m / z=60~650 Da, a scanning rate of 0.5 spectra / s, and monitoring and analysis by quadrupole time-of-flight mass spectrometry; ion source temperature of 120°C; desolvation gas temperature of 350°C; desolvation gas flow rate of 650 L / h; cone gas flow rate of 50 L / h; collision voltage of 30V; cone body voltage of 40V; and capillary voltage of 2500 V.

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