Method for detecting sulfadimidine in meat product

Abstract

The invention provides a method for detecting sulfadimidine in meat products. The method comprises the following steps: pretreating an object to be detected; specifically, the method comprises the following steps: dissolving a to-be-detected object to obtain a dissolved solution, adding a PSA adsorbent into the dissolved solution for adsorption to obtain an adsorption solution, carrying out nitrogen blowing volatilization on the adsorption solution, and redissolving with a methanol aqueous solution to obtain a sample test solution; sampling the sample test solution to obtain an on-machine test solution; and detecting the loading test solution through a liquid chromatograph-tandem mass spectrometer, and determining the content of the sulfadimidine bromide. According to the method, the sample is pretreated and detected through liquid chromatography-tandem mass spectrometry, so that the market blank is filled, the content of sulfadimidine in the meat product can be detected, and the method has the advantages of excellent repeatability and reproducibility, simplicity and convenience in operation, high speed, easiness in obtaining used equipment and high generalizability.

Description

Technical Field

[0001] The present application relates to the technical field of analytical chemistry, and in particular to a method for detecting sulfabromide in meat products. Background Art

[0002] Sulfonamides (SAs) are a class of sulfonamides that are a general term for drugs with a p-aminobenzenesulfonamide structure. They are a class of widely used antibiotics for preventing and promoting animal growth (often used as feed additives). SAs all contain a p-aminobenzoyl ring, an aromatic amino acid at the N4 position, and different substituents at the N1 position. There are thousands of types of SAs, which have the advantages of a wide antibacterial spectrum, stable properties, and low prices. In particular, the discovery of antibacterial enhancers such as trimethoprim and dimethoprim has expanded the antibacterial spectrum of SAs after being used in combination with antibacterial enhancers, and the antibacterial activity can be greatly enhanced, which can be changed from an antibacterial effect to a bactericidal effect. Therefore, SAs are widely used in veterinary clinics, animal feed additives, aquaculture and other fields. Frequent consumption of animal-derived foods containing SAs residues can cause the gradual accumulation of SAs in the body, and its harmfulness is mainly manifested in allergic reactions, bacterial resistance, teratogenic effects, mutagenic effects, carcinogenic effects, and hormone-like effects. Due to the irrational use, misuse and even abuse of SAs, SAs residues in animal-derived foods have occurred frequently, mainly in pork, followed by beef and poultry. Sulfadiazine, as a sulfonamide drug, is also widely used in the production of meat products.

[0003] Currently, there are no relevant testing standards, nor are there any testing methods for sulfamethoxazole in meat products. Meat products may be contaminated by raw materials during the production process, or sulfamethoxazole may be illegally added to improve product effects, posing a hidden danger to human health. Summary of the invention

[0004] In view of the above problems, the present application is proposed to provide a method for detecting sulfabromide in meat products that overcomes the above problems or at least partially solves the above problems, comprising:

[0005] A method for detecting sulfadiazine in meat products comprises the steps of:

[0006] Pre-treating the object to be tested; specifically, dissolving the object to be tested to obtain a solution, adding a PSA adsorbent to the solution for adsorption to obtain an adsorption solution, blowing the adsorption liquid nitrogen to volatilize it and re-dissolving it in methanol and aqueous solution to obtain a sample test solution;

[0007] Sampling the sample test solution to obtain an on-machine test solution;

[0008] The test solution is detected by liquid chromatography-tandem mass spectrometry to determine the content of sulfabromide; wherein the mass spectrometry parameters are: parent ion 358.2m / z; daughter ions: 108m / z, 92m / z, quantitative ion 156m / z; cone voltage 32V; collision energy: 30eV, 20eV, 35eV.

[0009] Furthermore, the step of detecting the on-machine test solution by liquid chromatography-tandem mass spectrometry uses a chromatographic column model of C18 column, 50mm×2.1mm, and a particle size of 1.7μm.

[0010] Furthermore, the step of dissolving the test object comprises:

[0011] Dissolving the analyte with a first volume of acetonitrile solution containing 0.1% methanol, and homogenizing and centrifuging to obtain a first supernatant and analyte residue;

[0012] Dissolving the residue of the substance to be tested with a second volume of acetonitrile solution containing 0.1% methanol, and obtaining a second supernatant and the residue of the substance to be tested after homogenization and centrifugation;

[0013] The first supernatant and the second supernatant are mixed to obtain the dissolved solution.

[0014] Furthermore, the first volume is 4 ml / g; and the volume ratio of the second volume to the first volume is 1:2.

[0015] Furthermore, the step of homogenizing and centrifuging comprises:

[0016] The mixture of the residue of the substance to be tested and 0.1% methanol in acetonitrile solution was homogenized at 10000 r / min for 2 min by a homogenizer; and centrifuged at 4000 r / min for 5 min.

[0017] Furthermore, the step of adding a PSA adsorbent to the dissolving solution to obtain an adsorbed solution comprises:

[0018] Adding a PSA adsorbent to the dissolved solution, wherein the mass ratio of the adsorbent to the analyte is 1:5-10;

[0019] Vortex mix for 2 minutes, centrifuge at 10000r / min for 10 minutes after mixing, and take the lower layer of solution as the adsorption solution.

[0020] Furthermore, the step of volatilizing the adsorbed liquid nitrogen and re-dissolving it in methanol-water solution to obtain a sample test solution comprises:

[0021] The adsorption solution was concentrated by nitrogen blowing at 40° C. until the solution was blown dry to obtain a reconstituted sample.

[0022] Dissolve the reconstituted sample in 20% methanol aqueous solution and adjust the volume to 1 ml to obtain a primary sample solution;

[0023] The primary sample solution is filtered through a 0.22 μm filter membrane to obtain a sample solution.

[0024] Furthermore, in the step of detecting the on-machine test solution by liquid chromatography-mass spectrometry, the mobile phase is 0.1% formic acid aqueous solution and 0.1% formic acid acetonitrile solution.

[0025] Furthermore, in the step of detecting the test solution on the machine by liquid chromatography-tandem mass spectrometry, the elution gradient is:

[0026] 0-0.2 minutes, 95% formic acid aqueous solution, 5% formic acid acetonitrile solution; 0.2-2.1 minutes, 95%-5% formic acid aqueous solution, 5%-95% formic acid acetonitrile solution; 2.1-3.5 minutes, 5% formic acid aqueous solution, 95% formic acid acetonitrile solution; 3.5-3.51 minutes, 5%-95% formic acid aqueous solution, 95%-5% formic acid acetonitrile solution; 3.51-5.0 minutes, 95% formic acid aqueous solution, 5% formic acid acetonitrile solution.

[0027] Furthermore, the method further includes performing a spike recovery test on the analyte to determine a spike recovery rate.

[0028] This application has the following advantages:

[0029] In the embodiments of the present application, in view of the fact that there is no further detection scheme for meat products in the prior art, the present application provides a detection method for sulfabromide in meat products, comprising the steps of: pre-treating the analyte; specifically, dissolving the analyte to obtain a dissolving solution, adding a PSA adsorbent to the dissolving solution for adsorption to obtain an adsorbed solution, blowing the adsorbed liquid nitrogen to evaporate and re-dissolving it with a methanol-water solution to obtain a sample test solution; sampling the sample test solution to obtain a machine test solution; detecting the machine test solution by a liquid chromatography-tandem mass spectrometer to determine the content of sulfabromide; wherein the mass spectrometry parameters are: parent ion 358.2m / z; daughter ions: 108m / z, 92m / z, quantitative ion 156m / z; cone voltage 32V; collision energy: 30eV, 20eV, 35eV. The present application fills the market gap by pre-treating the samples and detecting the samples by liquid chromatography-tandem mass spectrometry, and can detect the content of sulfabromide in meat products, has excellent repeatability and reproducibility, is easy to operate, fast, and the equipment used is easy to obtain, so it is highly scalable. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solution of the present application, the drawings required for use in the description of the present application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0031] Figure 1 This is a flow chart of the steps of a method for detecting sulfadiazine in meat products provided in one embodiment of the present application;

[0032] Figure 2 This is an operation flow chart of a method for detecting sulfadiazine in meat products provided in one embodiment of the present application;

[0033] Figure 3 This is a standard curve diagram of a method for detecting sulfadiazine in meat products provided in one embodiment of the present application;

[0034] Figure 4 It is a chromatogram of a standard curve of a method for detecting sulfabromide in meat products provided in one embodiment of the present application at a concentration point of 5 μg / L;

[0035] Figure 5 It is a chromatogram of a standard curve of a method for detecting sulfabromide in meat products provided in one embodiment of the present application at a concentration point of 10 μg / L;

[0036] Figure 6 It is a chromatogram of a standard curve of a method for detecting sulfabromide in meat products provided in one embodiment of the present application at a concentration point of 20 μg / L;

[0037] Figure 7 It is a chromatogram of a standard curve of a method for detecting sulfabromide in meat products provided in an embodiment of the present application at a concentration point of 50 μg / L;

[0038] Figure 8 It is a chromatogram of a standard curve of a method for detecting sulfadiazine in meat products provided in one embodiment of the present application at a concentration point of 100 μg / L;

[0039] Fig. 9 A method for detecting sulfadiazine in meat products provided in one embodiment of the present application is provided, which is a graph for determining the content of sulfadiazine in a spiked meat sample;

[0040] Fig.10 The present invention provides a method for detecting sulfamethoxazole in meat products provided in one embodiment of the present application, which is a graph for determining the content of sulfamethoxazole in a meat sample. DETAILED DESCRIPTION

[0041] In order to make the objects, features and advantages of the present application more obvious and understandable, the present application is further described in detail below in conjunction with the accompanying drawings and specific implementation methods. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present application.

[0042] By analyzing the existing technology, the inventors found that if antibiotics are improperly used during the treatment of animals, residues of antibiotics such as sulfonamides are likely to remain in the body, causing the raw materials of meat products to be contaminated. Veterinary drug residues are also easily absorbed by the human body through food, posing a potential risk to human health and may also cause antibiotic resistance problems.

[0043] Reference Figure 1-2 , showing a step flow chart and an operation flow chart of a method for detecting sulfabromide in meat products provided in one embodiment of the present application:

[0044] The method comprises:

[0045] S110, pre-treating the object to be tested; specifically, dissolving the object to be tested to obtain a solution, adding a PSA adsorbent to the solution for adsorption to obtain an adsorption solution, blowing the adsorption liquid nitrogen to volatilize it, and re-dissolving it in a methanol-water solution to obtain a sample test solution;

[0046] S120, sampling the sample test solution to obtain an on-machine test solution;

[0047] S130. The test solution is tested by liquid chromatography-tandem mass spectrometry to determine the content of sulfabromide; wherein the mass spectrometry parameters are: parent ion 358.2m / z; daughter ions: 108m / z, 92m / z, quantitative ion 156m / z; cone voltage 32V; collision energy: 30eV, 20eV, 35eV.

[0048] In the embodiments of the present application, in view of the fact that there is no further detection scheme for meat products in the prior art, the present application provides a detection method for sulfabromide in meat products, comprising the steps of: pre-treating the analyte; specifically, dissolving the analyte to obtain a dissolving solution, adding a PSA adsorbent to the dissolving solution for adsorption to obtain an adsorbed solution, blowing the adsorbed liquid nitrogen to evaporate and re-dissolving it with a methanol-water solution to obtain a sample test solution; sampling the sample test solution to obtain a machine test solution; detecting the machine test solution by liquid chromatography-tandem mass spectrometry to determine the content of sulfabromide; wherein the mass spectrometry parameters are: parent ion 358.2m / z; daughter ions: 108m / z, 92m / z, quantitative ion 156m / z; cone voltage 32V; collision energy: 30eV, 20eV, 35eV. The present application fills the market gap by pre-treating the samples and detecting the samples by liquid chromatography-tandem mass spectrometry, and can detect the content of sulfabromide in meat products, has excellent repeatability and reproducibility, is easy to operate, fast, and the equipment used is easy to obtain, so it is highly scalable.

[0049] Next, the detection method of sulfadiazine in a meat product in this exemplary embodiment will be further described.

[0050] As described in step S110, the object to be tested is pre-treated; specifically, the object to be tested is dissolved to obtain a solution, a PSA adsorbent is added to the solution for adsorption to obtain an adsorption solution, the adsorption liquid is evaporated by nitrogen and re-dissolved in a methanol-water solution to obtain a sample test solution.

[0051] In one embodiment of the present application, the specific process of "pre-treating the object to be tested; specifically, dissolving the object to be tested to obtain a solution, adding a PSA adsorbent to the solution to obtain an adsorption solution, volatilizing the adsorbed liquid nitrogen and re-dissolving it through a methanol-water solution to obtain a sample test solution" described in step S110 can be further explained in combination with the following description.

[0052] Dissolve the analyte as described in the following steps.

[0053] In one embodiment of the present application, the specific process of the step of “dissolving the object to be tested” can be further explained in combination with the following description.

[0054] Dissolving the analyte with a first volume of acetonitrile solution containing 0.1% methanol, and homogenizing and centrifuging to obtain a first supernatant and analyte residue;

[0055] Dissolving the residue of the substance to be tested with a second volume of acetonitrile solution containing 0.1% methanol, and obtaining a second supernatant and the residue of the substance to be tested after homogenization and centrifugation;

[0056] The first supernatant and the second supernatant are mixed to obtain a dissolved solution, and the dissolved solution is fixed to a volume equal to the sum of the first volume and the second volume.

[0057] In one embodiment of the present application, the first volume is 4 ml / g; and the volume ratio of the second volume to the first volume is 1:2.

[0058] In one embodiment of the present application, the homogenization and centrifugation step includes:

[0059] The mixture of the test substance and 0.1% methanol in acetonitrile solution was homogenized by a homogenizer at a rate of 10000 r / min for 2 min; and centrifuged at a rate of 4000 r / min for 5 min.

[0060] In a specific implementation, about 5 g of the sample (accurate to 0.01 g) was weighed into a homogenizing tube, 20 mL of 0.1% formic acid acetonitrile solution was added, homogenized with a homogenizer at 10000 r / min for 2 min, centrifuged at 4000 r / min for 5 min, the supernatant was taken out, and the residue was repeatedly extracted once with 10 mL of 0.1% formic acid acetonitrile solution, and the supernatants were combined and fixed to 30 mL.

[0061] As described in the following steps, a PSA adsorbent is added to the dissolving solution for adsorption to obtain an adsorbed solution.

[0062] In one embodiment of the present application, the specific process of the step of "adding a PSA adsorbent to the dissolving solution for adsorption to obtain an adsorbed solution" can be further described in combination with the following description:

[0063] As described in the following steps, a PSA adsorbent is added to the dissolved solution, wherein the mass ratio of the adsorbent to the analyte is 1:5-10;

[0064] As described in the following steps, vortex mixing was performed for 2 minutes, and after mixing, centrifugation was performed at 10,000 r / min for 10 minutes, and the lower layer of solution was taken as the adsorption solution.

[0065] In a specific implementation, 30 ml of the supernatant of the test object, i.e., the dissolved solution, is obtained by using 5 g of the sample. 10 ml is taken into a centrifuge tube, and 0.5 g of PSA adsorbent is added. The sample is vortexed and mixed.

[0066] As described in the following steps, the adsorbed liquid nitrogen is evaporated and re-dissolved in methanol and aqueous solution to obtain a sample test solution.

[0067] In one embodiment of the present application, the specific process of the step of "evaporating the adsorbed liquid nitrogen and re-dissolving it in methanol and aqueous solution to obtain a sample test solution" can be further described in combination with the following description:

[0068] The eluate was concentrated by nitrogen blowing at 40° C. until the solution was blown dry to obtain a reconstituted sample.

[0069] Dissolve the reconstituted sample in 20% methanol aqueous solution and adjust the volume to 1 ml to obtain a primary sample solution;

[0070] The primary sample solution is filtered through a 0.22 μm filter membrane to obtain a sample solution.

[0071] It should be noted that it can be blown to a nearly dry state according to actual conditions.

[0072] As described in step S120, the sample test solution is sampled to obtain the on-machine test solution.

[0073] As described in step S130, the test solution on the machine is detected by liquid chromatography-tandem mass spectrometry to determine the content of sulfabromide; wherein the mass spectrometry parameters are: parent ion 358.2m / z; daughter ions: 108m / z, 92m / z, quantitative ion 156m / z; cone voltage 32V; collision energy: 30eV, 20eV, 35eV.

[0074] In one embodiment of the present invention, the specific process of step S130 described in "detecting the test solution on the machine by liquid chromatography-tandem mass spectrometry to determine the content of sulfabromide; wherein the mass spectrometry parameters are: parent ion 358.2m / z; daughter ions: 108m / z, 92m / z, quantitative ion 156m / z; cone voltage 32V; collision energy: 30eV, 20eV, 35eV" can be further explained in combination with the following description.

[0075] Liquid chromatography reference conditions:

[0076] Instrument: LC-MS / MS;

[0077] Chromatographic column: C18 column, 50 mm × 2.1 mm (inner diameter), particle size 1.7 μm, or equivalent.

[0078] Mobile phase: A: 0.1% formic acid in water; B: 0.1% formic acid in acetonitrile.

[0079] Flow rate: 0.3mL / min.

[0080] Column temperature: 35°C.

[0081] Injection volume: 10 μL.

[0082] The flow rate gradient elution program is shown in Table 1:

[0083] Time / min A B 0 95 5 0.2 95 5 2.1 5 95 3.5 5 95 3.51 95 5 5.0 95 5

[0084] Table 1

[0085] Mass spectrometry reference conditions:

[0086] Ion source: electrospray ionization (ESI).

[0087] Scanning mode: multiple reaction monitoring (MRM).

[0088] The mass spectrometry reference parameters are shown in Table 2:

[0089]

[0090] Table 2

[0091] refer to Figure 3-8 , is the chromatogram of each concentration point of the sulfamethoxazole standard curve determined by liquid chromatography-tandem mass spectrometry and the linearity of sulfamethoxazole, the linear range of the standard curve is: 5-100 μg / L.

[0092] Fig.10 The chromatographic data of sulfadiazine measured by liquid chromatography-tandem mass spectrometry for a certain meat product. Fig. 9 The spectrum data of sulfadiazine was measured by liquid chromatography-tandem mass spectrometry after the meat product sample was spiked. As shown in the spectrum, the method can achieve high-efficiency separation and stable baseline. The recovery rate was within a satisfactory range of 60%-120% through the spike recovery test of the sample. It has good repeatability and reproducibility, and accurate quantification.

[0093] It can be seen from the above embodiments that the present scheme uses LC-MS / MS (liquid chromatography tandem mass spectrometry) to detect the content of sulfabromide in meat samples, has excellent repeatability and reproducibility, is easy to operate, is fast, the equipment used is easy to obtain, and is highly scalable.

[0094] Although the preferred embodiments of the present application have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the embodiments of the present application.

[0095] Finally, it should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or terminal device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or terminal device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or terminal device including the elements.

[0096] The above is a detailed introduction to the detection method of sulfabromide in meat products provided by the present application. Specific examples are used in this article to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the method of the present application and its core idea; at the same time, for general technical personnel in this field, according to the idea of ​​the present application, there will be changes in the specific implementation method and application scope. In summary, the content of this specification should not be understood as a limitation on the present application.

Claims

1. A method for detecting sulfadiazine in meat products, characterized in that: Includes steps: Pre-treating the object to be tested; specifically, dissolving the object to be tested to obtain a solution, adding a PSA adsorbent to the solution for adsorption to obtain an adsorption solution, blowing the adsorption liquid nitrogen to volatilize it and re-dissolving it in methanol and aqueous solution to obtain a sample test solution; Sampling the sample test solution to obtain an on-machine test solution; The test solution is detected by liquid chromatography-tandem mass spectrometry to determine the content of sulfabromide; wherein the mass spectrometry parameters are: parent ion 358.2m / z; daughter ions: 108m / z, 92m / z, quantitative ion 156m / z; cone voltage 32V; collision energy: 30eV, 20eV, 35eV.

2. The detection method according to claim 1, characterized in that: The step of detecting the test solution on the machine by liquid chromatography-tandem mass spectrometry uses a chromatographic column model C 18 Column, 50 mm × 2.1 mm, particle size 1.7 μm.

3. The detection method according to claim 1, characterized in that: The step of dissolving the object to be tested comprises: Dissolving the analyte with a first volume of acetonitrile solution containing 0.1% methanol, and homogenizing and centrifuging to obtain a first supernatant and analyte residue; Dissolving the residue of the substance to be tested with a second volume of acetonitrile solution containing 0.1% methanol, and obtaining a second supernatant and the residue of the substance to be tested after homogenization and centrifugation; The first supernatant and the second supernatant are mixed to obtain the dissolved solution.

4. The detection method according to claim 3, characterized in that: The first volume is 4 ml / g; and the volume ratio of the second volume to the first volume is 1:

2.

5. The detection method according to claim 3, characterized in that: The step of homogenization and centrifugation comprises: The mixture of the residue of the substance to be tested and 0.1% methanol in acetonitrile solution was homogenized at 10000 r / min for 2 min by a homogenizer; and centrifuged at 4000 r / min for 5 min.

6. The detection method according to claim 1, characterized in that: The step of adding a PSA adsorbent to the dissolving solution for adsorption to obtain an adsorbed solution comprises: Adding a PSA adsorbent to the dissolved solution, wherein the mass ratio of the adsorbent to the analyte is 1:5-10; Vortex mix for 2 minutes, centrifuge at 10000r / min for 10 minutes after mixing, and take the lower layer of solution as the adsorption solution.

7. The detection method according to claim 1, characterized in that: The step of volatilizing the adsorbed liquid nitrogen and re-dissolving it in methanol and aqueous solution to obtain a sample test solution comprises: The adsorption solution was concentrated by nitrogen blowing at 40° C. until the solution was blown dry to obtain a reconstituted sample. Dissolve the reconstituted sample in 20% methanol aqueous solution and adjust the volume to 1 ml to obtain a primary sample solution; The primary sample solution is filtered through a 0.22 μm filter membrane to obtain a sample solution.

8. The detection method according to claim 1, characterized in that: In the step of detecting the test solution on the machine by liquid chromatography-tandem mass spectrometry, the mobile phase is 0.1% formic acid aqueous solution and 0.1% formic acid acetonitrile solution.

9. The detection method according to claim 8, characterized in that: The step of detecting the test solution on the machine by liquid chromatography-tandem mass spectrometry, the elution gradient is: 0-0.2 minutes, 95% formic acid aqueous solution, 5% formic acid acetonitrile solution; 0.2-2.1 minutes, 95%-5% formic acid aqueous solution, 5%-95% formic acid acetonitrile solution; 2.1-3.5 minutes, 5% formic acid aqueous solution, 95% formic acid acetonitrile solution; 3.5-3.51 minutes, 5%-95% formic acid aqueous solution, 95%-5% formic acid acetonitrile solution; 3.51-5.0 minutes, 95% formic acid aqueous solution, 5% formic acid acetonitrile solution.

10. The detection method according to claim 1, characterized in that: The method also includes performing a spike recovery test on the analyte to determine a spike recovery rate.

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