Composite purification filler and pretreatment method for sulfonamides detection

Abstract

The invention relates to the technical field of food detection, in particular to a composite purification filler and a pretreatment method for sulfonamide drug detection. The composite purification filler comprises carbonyl polymer particles, zinc chloride and polylactic acid, the weight ratio of the carbonyl polymer particles to the zinc chloride to the polylactic acid is 1: (0.1-0.5): (0.05-0.25). The composite purification filler provided by the invention is applied to a pretreatment method for detecting the sulfonamide residues in a meat sample, particularly a pretreatment method for detecting the sulfonamide residues in a chicken sample, so that efficient purification of the sample to be detected before detection can be simply and quickly completed.

Description

Technical Field

[0001] The present application relates to the technical field of food detection, and in particular to a composite purification filler and a pretreatment method for sulfonamide drug detection. Background Art

[0002] As people's awareness of food safety increases, the problem of drug residues in animal-derived foods has attracted more and more attention from the public. Sulfonamides (SAs) are a class of synthetic antibiotics with a p-aminobenzenesulfonamide structure that can inhibit the growth and reproduction of bacteria and have strong antibacterial effects. They are widely used to prevent and treat livestock diseases because of their excellent characteristics such as stable chemical properties, broad antibacterial spectrum, low price, and ease of use. However, the abuse and misuse of sulfonamides and failure to comply with the withdrawal period regulations will cause excessive sulfonamide residues in animal-derived foods. Sulfonamides are not easily metabolized, so they will accumulate in the human body and cause various potential hazards to the human body. In addition, sulfonamides also have serious side effects and potential carcinogenicity, and their drug resistance problem is becoming increasingly serious. Most countries have clear regulations on the maximum residual amount of sulfonamides in animal-derived foods, and the maximum residual amount of sulfonamides in animal-derived foods is 0.1 mg / kg. In summary, the development of effective purification methods for sulfonamides has become a research hotspot in this field.

[0003] The main detection methods for sulfonamides include enzyme-linked immunosorbent assay, high performance liquid chromatography, ultra-high performance liquid chromatography-tandem mass spectrometry, electrochemical detection, etc. Among them, ultra-high performance liquid chromatography-tandem mass spectrometry has the characteristics of specificity, reliability, high sensitivity and high throughput, and has now become the golden method for confirmation and determination at home and abroad.

[0004] In order to ensure accuracy and sensitivity, sample pretreatment before testing is particularly important. Animal-derived foods include chicken, pork, beef, mutton, milk, honey, fish, shrimp, etc., which contain fat, protein, sugar, phospholipids and other ingredients. Due to the diversity and complexity of samples, how to choose the appropriate pretreatment method to fully extract sulfonamides while removing impurities and ensure the accuracy and sensitivity of the test is a major difficulty in the field of food safety testing.

[0005] At present, the pretreatment methods for the detection of sulfonamide residues include traditional liquid-liquid extraction, solid phase extraction (SPE), solid phase microextraction (SPME), magnetic solid phase extraction (MSPE), matrix dispersed solid phase extraction (MSPD), molecular imprinting technology (MIPs), dispersive liquid-liquid microextraction (DLLME), QuEChERS, immunoaffinity chromatography, ultrasound-assisted extraction, microwave-assisted extraction and other technologies.

[0006] Traditional liquid-liquid extraction is a common pretreatment method for confirmatory detection of sulfonamides. my country's national food safety standard GB / T 20759-2006 "Determination of 16 sulfonamide residues in livestock and poultry meat by liquid chromatography-tandem mass spectrometry" and Announcement No. 1077-1-2008 both use liquid-liquid extraction, first using acetonitrile or acidified acetonitrile for extraction, then using n-hexane for extraction and fat removal, and then testing on the machine. At the same time, solid phase extraction is a relatively common and efficient pretreatment method. my country's national food safety standard GB 29694-2013 "Determination of 13 sulfonamide residues in animal food by high performance liquid chromatography" uses MCX as a solid phase extraction column for solid phase extraction. The sample is extracted with ethyl acetate and the extract after degreasing with n-hexane is passed through MCX, and the eluent is obtained after adsorption and elution. After nitrogen blowing and drying, it is re-dissolved and the sulfonamide residues are determined by high performance liquid chromatography. The national standard GB / T 22966-2008 "Determination of 16 sulfonamide residues in milk and milk powder by liquid chromatography-tandem mass spectrometry" uses perchloric acid solution to precipitate the protein in dairy products, then uses HLB solid phase extraction column for purification, methanol elution, and liquid chromatography-tandem mass spectrometry for determination. The national standard GB / T 21316-2007 "Determination of sulfonamide residues in animal-derived foods by liquid chromatography-mass spectrometry / mass spectrometry" directly mixes the sample with C18 filler, adds acetonitrile-water extract solution, extracts under the assistance of microwave irradiation, and then uses acetonitrile-saturated n-hexane liquid-liquid distribution for purification, and uses liquid chromatography-mass spectrometry / mass spectrometry for determination. The national standard mainly uses solvent extraction, n-hexane liquid-liquid extraction degreasing and solid phase extraction technology. The solid phase extraction column mainly uses HLB or MCX column, and the adsorption elution mode is adopted to minimize the influence of matrix effect on the test.

[0007] Combining the above pretreatment methods, in addition to organic solvent extraction and dispersed liquid-liquid extraction, solid phase extraction, matrix solid phase extraction, dispersed solid phase extraction, etc. all require adsorption and elution. Although QuEChERS omits the two steps of adsorption and elution, it requires an additional centrifugation step.

[0008] At present, the pre-treatment method in the related art has the following disadvantages: (1) Traditional liquid-liquid extraction has limited effect in removing impurities, resulting in a high matrix effect in subsequent liquid chromatography-tandem mass spectrometry, which interferes with the accuracy of the results and causes a certain degree of damage to the instrument; (2) Liquid-liquid extraction requires multiple extraction steps, a high proportion of organic solvents are used, which are toxic to a certain extent, and rotary evaporation is required, which is complicated; (3) Solid phase extraction requires activation, sample loading, elution, and other steps, which makes the operation complicated; (4) Dispersive solid phase extraction requires an additional step - centrifugation, and there is no theoretical plate effect, so the adsorption of impurities is limited.

[0009] Therefore, there is an urgent need in the art for a new purification filler for the detection of sulfonamide drugs in animal-derived foods, which can simply and quickly complete the efficient purification of the sample to be tested before detection. Summary of the invention

[0010] The present application provides a composite purification filler and a pretreatment method for sulfonamide drug detection. The composite purification filler provided by the present application is applied to the pretreatment method for sulfonamide drug residue detection in meat samples, especially the pretreatment method for sulfonamide drug residue detection in chicken samples, which can simply and quickly complete the efficient purification of the sample to be tested before detection.

[0011] In the first aspect, the present application provides a composite purification filler for reducing residual sulfonamide drugs in meat food, using the following technical solution: A composite purification filler for reducing residual sulfonamide drugs in meat food, the composite purification filler comprising carbonyl polymer particles, zinc chloride and polylactic acid; the weight ratio of the carbonyl polymer particles, the zinc chloride and the polylactic acid is 1: (0.1-0.5): (0.05-0.25).

[0012] The carbonyl polymer particles in the composite purification filler provided by the present application not only serve as a support, but also have the ability to adsorb sulfonamide drugs. The carbonyl groups carried by the carbonyl polymer particles can be firmly combined with the amino groups of sulfonamide drugs in an acidic environment, thereby achieving the purification of sulfonamide drugs. On the other hand, the zinc ions of zinc chloride in the composite purification filler of the present application can be combined with the sulfonyl (SO2) groups of sulfonamide drugs, thereby achieving the "secondary" combination of sulfonamide drugs through the zinc ions of zinc chloride under the condition of the "first-level" combination of the carbonyl groups of the carbonyl polymer particles and sulfonamide drugs. At the same time, polylactic acid can improve the connection between the carbonyl polymer particles and zinc chloride, thereby improving the purification ability of the composite purification filler for sulfonamide drugs.

[0013] Optionally, the weight ratio of the carbonyl polymer particles, the zinc chloride and the polylactic acid is 1:(0.2-0.4):(0.1-0.2).

[0014] In a specific embodiment, the weight ratio of the carbonyl polymer particles, the zinc chloride and the polylactic acid is 1:0.1:0.2, 1:0.2:0.2, 1:0.3:0.2, 1:0.4:0.2, 1:0.5:0.2, 1:0.3:0.05, 1:0.3:0.1, 1:0.3:0.15, 1:0.3:0.25.

[0015] Optionally, the carbonyl polymer particles include any one or both of polyvinyl pyrrolidone and polyimide.

[0016] Optionally, the carbonyl polymer particles are polyvinyl pyrrolidone.

[0017] Optionally, the carbonyl polymer particles are polyimide.

[0018] Optionally, the carbonyl polymer particles are polyvinyl pyrrolidone and polyimide.

[0019] Optionally, the weight ratio of the polyvinyl pyrrolidone to the polyimide is 1:(0.5-4).

[0020] Optionally, the weight ratio of the polyvinyl pyrrolidone to the polyimide is 1:(2-3).

[0021] In a specific embodiment, the weight ratio of polyvinyl pyrrolidone to polyimide can be 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, or 1:4.

[0022] In some specific embodiments, the weight ratio of polyvinyl pyrrolidone to polyimide can be 1: (0.5-1), 1: (0.5-1.5), 1: (0.5-2), 1: (0.5-3), 1: (1-1.5), 1: (1-2), 1: (1-2.5), 1: (1-3), 1: (1-4), 1: (1.5-2), 1: (1.5-2.5), 1: (1.5-3), 1: (1.5-4), 1: (2-2.5), 1: (2-3), 1: (2-4), 1: (3-4).

[0023] Optionally, the carbonyl polymer particles have a particle size range of 20-100 μm.

[0024] Optionally, the carbonyl polymer particles have a particle size range of 60-80 μm.

[0025] In the second aspect, the present application provides a pretreatment method for sulfonamide drug detection, which adopts the following technical scheme: A pretreatment method for detecting sulfonamide drug residues, the pretreatment method specifically comprising the following steps: preparing a sample extract, and purifying the sample extract using a solid phase column filled with the composite purification filler, thereby obtaining a purified sample extract.

[0026] In summary, the present application includes at least one of the following beneficial technical effects: The composite purification filler provided in the present application is applied to the pretreatment method for detecting sulfonamide drug residues in meat samples, especially the pretreatment method for detecting sulfonamide drug residues in chicken samples, which can simply and quickly complete the efficient purification of the samples to be tested before testing. DETAILED DESCRIPTION

[0027] Before describing the embodiments of the present application in detail, it should be understood that the terms used herein are only used for the purpose of describing specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by ordinary technicians in the field to which the terms belong.

[0028] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of this application, unless otherwise specified, the meaning of "plurality" is two or more.

[0029] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0030] In the present application, the terms "comprise" or "include" are open expressions, that is, including the contents specified in the present application but not excluding other contents.

[0031] The present application provides a composite purification filler for reducing residual sulfonamide drugs in meat food. The composite purification filler comprises carbonyl polymer particles, zinc chloride and polylactic acid; the weight ratio of the carbonyl polymer particles, the zinc chloride and the polylactic acid is 1: (0.1-0.5): (0.05-0.25). The composite purification filler provided by the present application is applied to the pretreatment method for the detection of sulfonamide drug residues in meat samples, especially the pretreatment method for the detection of sulfonamide drug residues in chicken samples, which can simply and quickly complete the efficient purification of the sample to be tested before the detection.

[0032] To make the purpose, technical solutions and advantages of this application clearer, the technical solutions in the embodiments of this application are described clearly and completely below. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of this application.

[0033] The test methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are purchased from conventional reagent stores unless otherwise specified. It should be noted that the above summary of the invention and the detailed description below are only for the purpose of specifically illustrating the present application and are not intended to limit the present invention in any way.

[0034] The present application is further described in detail below in combination with preparation examples, embodiments and test results.

[0035] Preparation Example 1-25

[0036] Preparation Examples 1-25 respectively provide a composite purification filler for reducing residual sulfonamide drugs in meat food. The specific formula of the composite purification filler is shown in Table 1.

[0037] Table 1 The amount of each component added in the composite purification filler in the above preparation examples

[0038] The preparation method of the composite purification filler specifically comprises the following steps: According to the dosage shown in Table 1, carbonyl polymer particles (polyvinyl pyrrolidone, polyimide (CAS: 62929-02-6; purchased from Hubei Qifei Pharmaceutical Chemical Co., Ltd.)), polylactic acid (CAS: 26100-51-6; purchased from Wuhan Kanos Technology Co., Ltd.), and zinc chloride were mixed, and half volume of acetone was added, and the mixture was placed in a shaking table at 28°C and 60 r / min for treatment until the acetone was completely volatilized to obtain a composite purification filler.

[0039] Examples 1-25 Examples 1-25 provide a pretreatment method for detecting sulfonamide drug residues. The test sample is a chicken sample. The composite purification fillers used in Examples 1-25 correspond to the composite purification fillers provided in Preparation Examples 1-25.

[0040] The above-mentioned pre-treatment method specifically includes the following steps: (1) Sample extraction: Weigh 5 g of the chicken sample to be tested, add 20 mL of 80% acetonitrile aqueous solution containing 0.2% formic acid, and vortex at high speed for 3 min to obtain the sample extract.

[0041] (2) Solid phase column purification: First, fill the purification column, take 2g of the above-mentioned composite purification filler and add it to the column tube of the purification column, and add 5mL of sample extract to a 15mL centrifuge tube. Insert the plunger of the purification column into the centrifuge tube from the top of the centrifuge tube, and press the purification column from top to bottom at a relatively uniform speed (10mL / min) to the bottom of the centrifuge tube. Pour the purified sample extract in the purification column into the sample bottle to obtain the purified sample extract.

[0042] Comparative Examples 1-6

[0043] Comparative Examples 1-6 respectively provide a pretreatment method for detecting sulfonamide drug residues. The difference between the above comparative examples and Example 15 is that the components added to the purification filler used in the above pretreatment method are different, as shown in Table 1. The rest are consistent with Example 15.

[0044] Performance testing

[0045] (I) The chicken samples were treated using the pre-treatment methods provided in Examples 1-25 and Comparative Examples 1-6 to detect the residual sulfonamides (sulfamethazine) in the chicken samples. The specific process is as follows: (1) Preparation of standard solution: Add sulfamethazine standard to negative samples at a level of 20 ppb and repeat 6 times.

[0046] (2) Sample extraction: Weigh 5 g of sample to which sulfamethoxazole standard has been added, add 20 mL of 80% acetonitrile aqueous solution containing 0.2% formic acid, and vortex at high speed for 3 min to obtain the sample extract.

[0047] (3) Purify the sample extract using the method provided in Example 15 to obtain a purified sample extract.

[0048] (4) UPLC-MS / MS detection: Take 200 μL of the purified sample extract and blow it dry with nitrogen, reconstitute it with 1 mL of the initial mobile phase, filter it through a 0.22 μm filter membrane, and finally perform UPLC-MS / MS analysis.

[0049] The chromatographic and mass spectrometric conditions of UPLC-MS / MS are as follows: Instrument: UPLC-MS / MS (Triple Quad TM 5500+); a. Chromatographic conditions Chromatographic column: Waters ACQUITY UPLC BEH C18 column (100×2.1 mm, 1.7 μm); Mobile phase: A: 0.1% formic acid water; B: methanol; flow rate: 0.35 mL / min; column temperature: 40°C; injection volume: 10 μL; elution method: gradient elution, the elution conditions are shown in Table 2.

[0050] Table 2 Gradient elution program (A represents mobile phase A, B represents mobile phase B, and the corresponding percentages of A and B represent the proportions of mobile phase A and mobile phase B in the mobile phase system)

[0051] b. Mass spectrometry conditions Ion source: ESI (electrospray ion source); scanning mode: positive ion; detection mode: multiple reaction monitoring; electrospray voltage: 5500V; curtain gas pressure: 0.241MPa; nebulizer pressure: 0.379MPa; auxiliary gas pressure: 0.379MPa; ion source temperature: 5500℃.

[0052] Judgment criteria: According to the provisions of the national standard for sulfonamide drugs, if the spike recovery rate ranges from 60-120%, the intra-batch RSD is ≤15%, and the inter-batch RSD is ≤20%, the recovery method is considered qualified.

[0053] The test results are shown in Table 3.

[0054] Table 3 Detection results corresponding to the pretreatment method provided in the above embodiment (repeatability RSD is the RSD within the batch)

[0055] As shown in Table 3, the chicken samples were pre-treated with the composite purification filler provided by the present application. At the standard concentration, the average recovery rate was above 86%, and the RSD was less than 10%, which was within the prescribed range of the national standard for sulfonamide drugs. It can be seen that the pre-treatment method is a qualified pre-treatment method for sulfonamide drugs.

[0056] (II) The chicken samples were treated with the pretreatment method provided in Example 15 to detect the residual sulfonamide drugs in the chicken samples. The specific process is as follows: Chicken with different concentrations of standard added was used as a sample, and the chicken sample was pre-treated using a solid phase purification column filled with the composite purification filler provided in Example 15. 200 μL of the purified sample extract was blown dry with nitrogen, re-dissolved with 1 mL of the initial mobile phase, filtered through a 0.22 μm filter membrane, and finally analyzed by UPLC-MS / MS.

[0057] The analysis method is as follows: (1) Preparation of mixed standard solution: Add a mixed standard of 20 sulfonamide drugs (sulfapyridine, sulfathiazole, sulfamethazine, sulfamethoxazole, sulfaquinoxaline, sulfaphenazole, sulfadimethoxazole, sulfisoxazole, sulfabenzoyl, sulfadimethoxine, sulfamethoxazole, trimethoprim, sulfamethoxazole, sulfadimethoxine, sulfadimethoxine, sulfamethoxazole, sulfadimethoxine, sulfamethoxypyridazine, sulfamethoxazole, sulfachloropyridazine and sulfamethixazole) to the negative sample at the addition levels of 5 ppb, 10 ppb, 20 ppb and 50 ppb. Repeat 6 times for each addition level.

[0058] (2) Sample extraction: Weigh 5 g of sample to which sulfonamide standard has been added, add 20 mL of 80% acetonitrile aqueous solution containing 0.2% formic acid, and vortex at high speed for 3 min to obtain the sample extract.

[0059] (3) Purify the sample extract using the method provided in Example 15 to obtain a purified sample extract.

[0060] (4) UPLC-MS / MS detection: The purified sample extract is diluted or re-dissolved by nitrogen blowing at a certain ratio, filtered through a 0.22 μm filter membrane, and finally subjected to UPLC-MS / MS analysis. The analysis method is as described in the performance test (I).

[0061] The test results are shown in Table 4-6.

[0062] Table 4 Recovery accuracy and precision of sulfonamides in chicken (I)

[0063] Table 5 Recovery accuracy and precision of sulfonamides in chicken (II)

[0064] Table 6 Recovery accuracy and precision of sulfonamides in chicken (III)

[0065] As shown in Table 4-6, at different concentrations of standard substances, the average recovery rate was above 90%, and the RSD was less than 10%, which was within the prescribed range of the national standard for sulfonamide drugs. It can be seen that this pretreatment method is a qualified pretreatment method for sulfonamide drugs.

[0066] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A composite purification filler for reducing residual sulfonamide drugs in meat food, characterized in that: The composite purification filler comprises carbonyl polymer particles, zinc chloride and polylactic acid; The weight ratio of the carbonyl polymer particles, the zinc chloride and the polylactic acid is 1:(0.1-0.5):(0.05-0.25).

2. The composite purification filler according to claim 1, characterized in that: The weight ratio of the carbonyl polymer particles, the zinc chloride and the polylactic acid is 1:(0.2-0.4):(0.1-0.2).

3. The composite purification filler according to claim 1, characterized in that: The carbonyl polymer particles include any one of polyvinyl pyrrolidone and polyimide.

4. The composite purification filler according to claim 1, characterized in that: The carbonyl polymer particles include polyvinyl pyrrolidone and polyimide.

5. The composite purification filler according to claim 4, characterized in that: The weight ratio of the polyvinyl pyrrolidone to the polyimide is 1:(0.5-4).

6. The composite purification filler according to claim 4, characterized in that: The weight ratio of the polyvinyl pyrrolidone to the polyimide is 1:(2-3).

7. The composite purification filler according to claim 1, characterized in that: The particle size of the carbonyl polymer particles is in the range of 20-100 μm.

8. The composite purification filler according to claim 1, characterized in that: The particle size of the carbonyl polymer particles is in the range of 60-80 μm.

9. A pretreatment method for sulfonamide drug detection, characterized in that: The pretreatment method specifically comprises the following steps: preparing a sample extract, and purifying the sample extract using a solid phase column filled with the composite purification filler according to any one of claims 1 to 8, thereby obtaining a purified sample extract.

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