A composite purification filler and a pretreatment method for the detection of sulfonamides

Abstract

This application relates to the technical field of food detection, and particularly relates to a composite purification filler and a pretreatment method for the detection of sulfonamides. The composite purification filler includes 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). Applying the composite purification filler provided by this application to the pretreatment method for the detection of sulfonamide residues in meat samples, especially the pretreatment method for the detection of sulfonamide residues in chicken samples, can simply and quickly complete the efficient purification of the sample to be tested before detection.

Description

Technical Field

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

[0002] With the enhancement of people's food safety awareness, the problem of drug residues in animal-derived foods has attracted more and more extensive attention from the public. Sulfonamide drugs (SAs) are a class of synthetic antibiotics with a sulfanilamide structure, which can inhibit the growth and reproduction of bacteria and have strong antibacterial effects. Due to their excellent characteristics such as stable chemical properties, broad antibacterial spectrum, low cost, and convenient use, they are widely used in the prevention and treatment of livestock diseases. However, the abuse, misuse of sulfonamide drugs and non-compliance with the withdrawal period regulations will cause the sulfonamide residues in animal-derived foods to exceed the standard. Sulfonamide drugs are not easily metabolized, so they will accumulate in the human body and cause various potential hazards to the human body. Moreover, sulfonamide drugs 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 residue limits of sulfonamide drugs in animal-derived foods, and the maximum residue limit of sulfonamide drugs in animal-derived foods is 0.1 mg / kg. To sum up, the development of an effective purification method for sulfonamide drugs has become a research hotspot in this field.

[0003] The detection methods of sulfonamide drugs mainly include enzyme-linked immunosorbent assay, high performance liquid chromatography, ultra-high performance liquid chromatography-tandem mass spectrometry, electrochemical detection method, 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] To ensure accuracy and sensitivity, sample pretreatment before detection is particularly important. Animal-derived foods include chicken, pork, beef, mutton, milk, honey, fish, shrimp, etc., and contain various components such as fat, protein, sugar, phospholipids, etc. Due to the diversity and complexity of the samples, how to select a suitable pretreatment method to remove impurities while fully extracting sulfonamide drugs and ensure the accuracy and sensitivity of detection is a major difficulty in the field of food safety detection.

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

[0006] Traditional liquid-liquid extraction is a common pretreatment method for the confirmatory detection of sulfonamides. At the same time, solid-phase extraction is a more widely used and efficient pretreatment method. The national standard for the determination of sulfonamides mainly combines solvent extraction, liquid-liquid extraction with n-hexane for defatting and solid-phase extraction technology. The solid-phase extraction column mainly uses HLB or MCX columns, and adopts the adsorption-elution mode to minimize the influence of matrix effects on the test.

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

[0008] At present, the pretreatment methods in related technologies have the following disadvantages:

[0009] (1) The effect of removing impurities by traditional liquid-liquid extraction is limited, resulting in a relatively high matrix effect in subsequent liquid chromatography-tandem mass spectrometry, which has a certain interference on the result accuracy and will cause a certain degree of damage to the instrument;

[0010] (2) Liquid-liquid extraction requires multiple extraction steps, with a high proportion of organic solvents used, certain toxicity, and requires rotary evaporation, and the steps are complex;

[0011] (3) Solid-phase extraction requires steps such as activation, loading, washing, and elution, and the operation is complex;

[0012] (4) Dispersive solid-phase extraction requires an additional step - centrifugation, and there is no theoretical plate effect, and the adsorption of impurities is limited.

[0013] Therefore, there is an urgent need in the art for a new purification filler for the detection of sulfonamides in animal-derived foods, so as to simply and quickly complete the efficient purification of the sample to be tested before detection. Summary of the Invention

[0014] This application provides a composite purification filler and a pretreatment method for the detection of sulfonamides. Applying the composite purification filler provided in this application to the pretreatment method for the detection of sulfonamide residues in meat samples, especially the pretreatment method for the detection of sulfonamide residues in chicken samples, can simply and quickly complete the efficient purification of the sample to be tested before detection.

[0015] In the first aspect, this application provides a composite purification filler for reducing residual sulfonamides in meat foods, adopting the following technical solution:

[0016] A composite purification filler for reducing residual sulfonamides in meat products, 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).

[0017] In the composite purification filler provided by this application, the role of the carbonyl polymer particles is not only a support, but also the particles themselves have the ability to adsorb sulfonamides. The carbonyl groups carried by the particles can firmly bind to the amino groups of sulfonamides in an acidic environment, thereby achieving the purification of sulfonamides. On the other hand, the zinc ions of zinc chloride in the composite purification filler of this application can bind to the sulfonyl (SO2) groups of sulfonamides, so that under the "first layer" binding of the carbonyl groups of the carbonyl polymer particles and sulfonamides, the "second layer" binding of sulfonamides can be further achieved through the zinc ions of zinc chloride. 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 sulfonamides.

[0018] 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).

[0019] 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.

[0020] Optionally, the carbonyl polymer particles include any one or two of polyvinylpyrrolidone and polyimide.

[0021] Optionally, the carbonyl polymer particles are polyvinylpyrrolidone.

[0022] Optionally, the carbonyl polymer particles are polyimide.

[0023] Optionally, the carbonyl polymer particles are polyvinylpyrrolidone and polyimide.

[0024] Optionally, the weight ratio of the polyvinylpyrrolidone and the polyimide is 1:(0.5 - 4).

[0025] Optionally, the weight ratio of the polyvinylpyrrolidone and the polyimide is 1:(2 - 3).

[0026] In a specific embodiment, the weight ratio of the polyvinylpyrrolidone to the polyimide can be 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:4.

[0027] In some specific embodiments, the weight ratio of the polyvinylpyrrolidone to the 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).

[0028] Optionally, the particle size range of the carbonyl polymer particles is 20 - 100 μm.

[0029] Optionally, the particle size range of the carbonyl polymer particles is 60 - 80 μm.

[0030] In a second aspect, the present application provides a pretreatment method for sulfonamide drug detection, adopting the following technical solution:

[0031] A pretreatment method for sulfonamide drug residue detection, the pretreatment method specifically includes the following steps: preparing a sample extract, and purifying the sample extract by using a solid-phase column filled with the above-mentioned composite purification filler, so as to obtain a purified sample extract.

[0032] In summary, the present application includes at least one of the following beneficial technical effects:

[0033] Applying the composite purification filler provided by the present application to the pretreatment method for sulfonamide drug residue detection in meat samples, especially the pretreatment method for sulfonamide drug residue detection in chicken samples, can efficiently purify the sample to be detected before detection simply and quickly. Specific Embodiments

[0034] Before describing the embodiments of the present application in detail, it should be understood that the terms used herein are only for the purpose of describing specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this term belongs.

[0035] It should be noted that the terms "first" and "second" are only used for descriptive purposes and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present application, unless otherwise specified, the meaning of "a plurality" is two or more.

[0036] In the ranges disclosed in the present application, the endpoints and any values of the ranges are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0037] In the present application, the term "comprising" or "including" is an open expression, that is, it includes the content specified in the present application, but does not exclude other aspects of the content.

[0038] The present application provides a composite purification filler for reducing residual sulfonamides in meat products. The composite purification filler includes 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). Applying the composite purification filler provided by the present application to the pretreatment method for detecting residual sulfonamides in meat samples, especially the pretreatment method for detecting residual sulfonamides in chicken samples, can simply and quickly complete the efficient purification of the sample to be detected before detection.

[0039] To make the purpose, technical solutions, and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of the present application.

[0040] The test methods in the following embodiments are all conventional methods unless otherwise specified. The test materials used in the following embodiments are all purchased from regular reagent stores unless otherwise specified. It should be noted that the above-mentioned invention content part and the following detailed description are only for the purpose of specifically explaining the present application and are not intended to limit the present invention in any way.

[0041] The present application will be further described in detail below in combination with preparation examples, examples, and test results.

[0042] Preparation Examples 1 - 25

[0043] Preparation Examples 1-25 respectively provide a composite purification filler for reducing residual sulfonamides in meat products. The specific formulation of the composite purification filler is shown in Table 1 as follows.

[0044] Table 1 Dosages of each component in the composite purification filler in the above Preparation Examples

[0045]

[0046] The preparation method of the above composite purification filler specifically includes the following steps:

[0047] According to the dosages shown in Table 1, mix carbonyl polymer particles (polyvinylpyrrolidone, polyimide (CAS: 62929-02-6; purchased from Hubei Qifei Pharmaceutical Chemical Co., Ltd.)), polylactic acid (CAS: 26100-51-6; purchased from Wuhan Karnos Technology Co., Ltd.), and zinc chloride, add half of the volume of acetone, and place it in a shaker at 28 °C and 60 r / min for treatment until the acetone completely volatilizes, thus obtaining the composite purification filler.

[0048] Examples 1-25

[0049] Examples 1-25 respectively provide a pretreatment method for the detection of sulfonamide residues. The test samples are chicken samples. The composite purification fillers used in Examples 1-25 respectively correspond to the composite purification fillers provided by Preparation Examples 1-25.

[0050] The above pretreatment method specifically includes the following steps:

[0051] (1) Sample extraction: Weigh 5 g of the chicken sample to be detected, 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.

[0052] (2) Solid-phase column purification: First, pack the purification column. Take 2 g of the above composite purification filler and add it to the column tube of the purification column. Add 5 mL of the sample extract to a 15 mL centrifuge tube. Insert the plunger of the purification column from the top of the centrifuge tube into the centrifuge tube, and press the purification column to the bottom of the centrifuge tube at a relatively uniform speed (10 mL / min) from top to bottom. Pour out the purified sample extract in the purification column into the sample bottle to obtain the purified sample extract.

[0053] Comparative Examples 1-6

[0054] Comparative Examples 1-6 respectively provide a pretreatment method for the detection of sulfonamide residues. The difference between the above Comparative Examples and Example 15 is that: the components added in the purification filler used in the above pretreatment method are different, as shown in Table 1 specifically. The rest are the same as Example 15.

[0055] Performance detection test

[0056] (1) Using the pretreatment methods provided in Examples 1 - 25 and Comparative Examples 1 - 6 to process chicken samples, and detecting the residue of sulfonamides (sulfamerazine) in the chicken samples. The specific process is as follows:

[0057] (1) Preparation of standard solution: Add sulfamerazine standard to negative samples, with an addition level of 20 ppb, and repeat 6 times.

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

[0059] (3) Using the method provided in Example 15 to purify the sample extract to obtain the purified sample extract.

[0060] (4) UPLC - MS / MS detection: Take 200 μL of the purified sample extract, blow it dry with nitrogen, re - dissolve 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.

[0061] The chromatographic and mass spectrometric conditions of UPLC - MS / MS are as follows:

[0062] Instrument: UPLC - MS / MS (Triple Quad TM 5500+);

[0063] a. Chromatographic conditions

[0064] Chromatographic column: Waters ACQUITY UPLC BEH C18 column (100×2.1 mm, 1.7 μm);

[0065] 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, and the elution conditions are shown in Table 2 specifically.

[0066] 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)

[0067]

[0068] b. Mass spectrometric conditions

[0069] Ion source: ESI (electrospray ionization source); Scanning mode: positive ion; Detection mode: multiple reaction monitoring; Electrospray voltage: 5500 V; Curtain gas pressure: 0.241 MPa; Nebulizer pressure: 0.379 MPa; Auxiliary gas pressure: 0.379 MPa; Ion source temperature: 5500 °C.

[0070] Judgment criteria: According to the regulations in the national standard for sulfonamides, if the addition recovery rate ranges from 60% to 120%, the within-batch RSD ≤ 15%, and the between-batch RSD ≤ 20%, then the recovery method is considered qualified.

[0071] The test results are shown in Table 3.

[0072] Table 3 Test results corresponding to the pretreatment method provided by the above embodiment (the repeatability RSD is the within-batch RSD)

[0073]

[0074] As can be seen from Table 3, when using the composite purification filler provided by this application to pretreat chicken samples, at the standard concentration, the average recovery rate obtained is above 86%, and the RSDs are all less than 10%, within the scope specified by the national standard for sulfonamides. It can be seen that this pretreatment method is a qualified pretreatment method for sulfonamides.

[0075] (2) Using the pretreatment method provided in Example 15 to process chicken samples and detecting the residue of sulfonamides in the chicken samples. The process is as follows:

[0076] Chicken samples added with different concentrations of standard products were used as samples, and a solid-phase purification column filled with the composite purification filler provided in Example 15 was used for the pretreatment of chicken samples. Take 200 μL of the purified sample extract, blow it dry with nitrogen, re-dissolve it with 1 mL of the initial mobile phase, then filter it through a 0.22 μm filter membrane, and finally perform UPLC-MS / MS analysis.

[0077] The analysis method is as follows:

[0078] (1) Preparation of the mixed standard solution: Add a mixed standard of 20 sulfonamides (sulfapyridine, sulfathiazole, sulfamerazine, sulfadimethoxine, sulfaquinoxaline, sulfaphenazole, sulfamethizole, sulfisoxazole, benzoyl sulfanilamide, sulfamethazine, sulfamethoxydiazine, sulfadimethoxine, trimethoprim, sulfamethoxazole, sulfadiazine, sulfadimidine, sulfamethoxypyridazine, sulfamonomethoxine, sulfachloropyridazine, and sulfamethizole) to a negative sample, with addition levels of 5 ppb, 10 ppb, 20 ppb, and 50 ppb, and each addition level is repeated 6 times.

[0079] (2) Sample extraction: Weigh 5 g of the sample added with sulfonamide standard, 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.

[0080] (3) Purify the sample extract using the method provided in Example 15 to obtain the purified sample extract.

[0081] (4) UPLC-MS / MS detection: Take the purified sample extract, dilute it in a certain proportion or reconstitute it by nitrogen blowing, then filter it through a 0.22 μm filter membrane, and finally perform UPLC-MS / MS analysis. The analysis method is as described in Performance Detection Test (1).

[0082] The detection results are shown in Table 4-6.

[0083] Table 4 Recovery Accuracy and Precision of Sulfonamides in Chicken (1)

[0084]

[0085] Table 5 Recovery Accuracy and Precision of Sulfonamides in Chicken (2)

[0086]

[0087] Table 6 Recovery Accuracy and Precision of Sulfonamides in Chicken (3)

[0088]

[0089] As can be seen from Table 4-6, at different standard concentrations, the average recovery rate obtained is above 90%, and the RSD is less than 10%, within the scope specified by the national standard for sulfonamides. It can be seen that this pretreatment method is a qualified pretreatment method for sulfonamides.

[0090] In the description of this specification, the description referring to terms such as "one embodiment", "some embodiments", "example", "specific example" or "some examples" means that the specific features, structures, materials or characteristics described in connection 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 can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0091] 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 them; although the present application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the various 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); The carbonyl polymer particles are a mixture of polyvinyl pyrrolidone and polyimide, and the weight ratio of the polyvinyl pyrrolidone to the polyimide is 1:(2-3).

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 particle size of the carbonyl polymer particles is in the range of 20-100 μm.

4. 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.

5. 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 4, thereby obtaining a purified sample extract.

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