A method for detecting benzovinflumizone in animal-derived food

Abstract

The present invention belongs to the technical field of food detection, and specifically discloses a detection method of benzovinflumizone in animal-derived food, comprising the following steps: S1, extraction: mixing the animal-derived food sample with an extraction solvent, mixing and shaking, and centrifuging to obtain a supernatant; S2, purification: treating the supernatant of step S1 with a solid phase extraction column to obtain a test liquid; S3, detection: using a liquid chromatography-mass spectrometer to detect the test liquid of S2; the animal-derived food is one or more of poultry and livestock meat, aquatic products, fat, viscera, eggs, milk and casings. The present invention expands the scope of application of benzovinflumizone determination to animal-derived foods of different matrices such as poultry and livestock meat, aquatic products, fat, viscera, eggs, milk and casings for the first time, and has the advantages of being fast, easy to operate, low cost, accurate, reliable, effective and highly sensitive.

Description

Technical Field

[0001] The invention belongs to the technical field of food detection, and particularly relates to a method for detecting benzovindiflupyr in animal-derived food. Background Art

[0002] Food safety comes first, and the food safety of animal-derived foods has always been a hot topic of concern. According to the safety tracking and tracing of animal-derived foods, their food safety comes from three aspects: one is the breeding process, the second is the processing process, and the third is the transportation and storage process. Among them, the breeding process, which ranks first, is the most important issue in the safety traceability of animal-derived foods. my country's breeding industry has developed rapidly and has broad prospects. Its sustainable development requires the import of a large amount of feed raw materials such as soybeans every year and then processed into animal feed to feed animals. However, soybean cultivation involves pest and disease control, so the use of pesticides is inevitable. Among them, benzovinyl flubacill has strong systemic absorption and penetration properties, can tightly bind to the wax layer of plants, slowly penetrate into plant tissues, affect the respiratory chain electron transfer system of pathogens, thereby blocking the energy metabolism of pathogens, inhibiting the growth of pathogens, and causing their death, and ultimately achieving the purpose of preventing and controlling plant diseases. Therefore, benzovinyl flubacill has a good preventive and therapeutic effect on major diseases of crops such as soybeans and corn, such as soybean rust, and is widely used by the planting industry in the planting process of such feed raw materials. Specifically, soybean meal after soybean processing is the raw material for feed processing, and the pesticides remaining in soybean meal and other raw materials will partially be brought into feed products. After the residual pesticides in feed products are eaten by animals, they can be transferred to animal tissues as the feed is consumed, and may then exist in animal-derived foods such as meat, milk, and eggs, and ultimately cause harm to human health through the food chain.

[0003] By consulting the limit standards of Japan, the United States, the European Union and GB2763-2021 "National Food Safety Standard Maximum Residue Limits of Pesticides in Food", it is found that all countries have limit requirements for benzovindiflupyr. Among them, on December 6, 2016, the European Food Safety Authority (EFSA) revised the maximum residue limit of benzovindiflupyr based on the risk assessment results of benzovindiflupyr in imported animal and plant foods, stipulating that the recommended maximum limit for beans is 0.2 mg / kg, and the recommended minimum limit for animal-derived foods such as beef liver, goat liver, sheep liver, and horse liver is 0.01 mg / kg; the relevant limits in the United States have increased the types of species, such as the minimum limit for milk is 0.01 mg / kg, and the minimum limit for goat liver is 0.01 mg / kg. Meat 0.01mg / kg, etc.; GB2763-2021 stipulates the maximum limits for soybeans and various animal-derived foods, such as mammalian meat (excluding marine mammals) ≤0.03*mg / kg, mammalian viscera (excluding marine mammals) ≤0.1*mg / kg, animal poultry meat ≤0.01*mg / kg, poultry viscera ≤0.01*mg / kg, poultry fat ≤0.01*mg / kg, eggs ≤0.01*mg / kg, etc. However, GB2763-2021 does not specify or recommend corresponding detection methods, which makes it difficult for testing laboratories to carry out relevant benzovindiflupyr detection.

[0004] At present, a large number of detection experts have conducted research on the detection of benzovinflumizone in animal-derived foods, such as the method published by Sui Chengcheng et al. in the Journal of Pesticide Science for the rapid detection of benzovinflumizone residues in animal-derived products by combining QuEChERS with high-performance liquid chromatography-tandem mass spectrometry (HPLC–MS / MS). This article uses the QuEChERS pretreatment method combined with high-performance liquid chromatography-tandem mass spectrometry to establish a detection method for benzovinflumizone in four animal-derived products (pork, chicken, beef and eggs). However, this detection method still has the disadvantages of a small matrix range for animal-derived products and a long detection time. Therefore, obtaining a method that can quickly detect the content of benzovinflumizone in animal-derived foods has become one of the research directions in the field of analytical detection. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for detecting benzovintriazole in animal-derived foods, which realizes the detection of benzovintriazole in animal-derived foods with different matrices and has the advantages of being fast, easy to operate, low cost, accurate, reliable and highly sensitive.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] A method for detecting benzovinflumizone in animal-derived food, the detection method comprising the following steps:

[0008] S1. Extraction: Mix the animal-derived food sample with the extraction solvent, shake the mixture, and centrifuge to obtain the supernatant;

[0009] S2, purification: taking the supernatant obtained in step S1 and treating it with a solid phase extraction column to obtain a test solution;

[0010] S3, detection: the test solution of S2 is detected by liquid chromatography-mass spectrometry;

[0011] The animal-derived food is one or more of poultry and livestock meat, aquatic products, fat, viscera, eggs, milk or casings.

[0012] The present invention expands the applicable scope of benzovinfluazide determination to animal-derived foods of different matrices such as poultry meat, aquatic products, fat, viscera, eggs, milk and casings for the first time, mixes animal-derived food samples treated by different methods with extraction solvents for purification pre-treatment, the supernatant obtained in step S1 is purified by solid phase extraction column treatment, and a liquid chromatography-mass spectrometry is used to detect the residual amount of benzovinfluazide in animal-derived foods. The method provided by the present invention has a quantitative limit of 5.0 μg / kg, correlation coefficients greater than 0.995, an overall average recovery rate range of 84.2% to 110.4%, a relative standard deviation range of 0.81% to 8.61%, and is less than 10.0%. Statistical results show that the verification result of the method meets the requirements of the relevant provisions of SN / T0001-2016 "Basic Provisions for the Preparation of Physical and Chemical Determination Methods for Exported Foods and Cosmetics", and has the advantages of being fast, easy to operate, low cost, accurate, reliable, effective and highly sensitive.

[0013] Furthermore, the solid phase extraction column is a SPE purification column.

[0014] Furthermore, the SPE purification column is one of an HLB column (200 mg / 3 mL), a phospholipid-free PPR column (200 mg / 3 mL) and a Florisil (200 mg / 3 mL) column, preferably a phospholipid-free PPR (200 mg / 3 mL).

[0015] In the present invention, when a divinylbenzene and N-vinylpyrrolidone composite column HLB (200 mg / 3 mL), a phospholipid-free PPR column (200 mg / 3 mL) and a Florisil (200 mg / 3 mL) column are used as solid phase extraction columns for purification, the recovery rate of benzovintriazole meets the requirement of 80% to 120%. The phospholipid-free PPR column is preferably used because the use of the phospholipid-free PPR column for purification not only has a high recovery rate of benzovintriazole, but also does not require the consumption of other reagents and greatly shortens the operation time.

[0016] Furthermore, the extraction solvent is an aqueous solution of acetic acid and acetonitrile with a mass fraction of 1%.

[0017] In the present invention, although the simplest acetic acid acetonitrile aqueous solution with a mass fraction of 1% is used as the extraction solvent, this extraction method can effectively precipitate impurities.

[0018] Furthermore, when the animal-derived food is poultry meat and / or aquatic products, the specific steps of extraction in S1 are: the specific steps of extraction in S1 are: weigh the sample and place it in a centrifuge tube A, add a 1% mass fraction of acetic acid acetonitrile solution and place it in a homogenizer for homogenization, then take another centrifuge tube B and add a 1% mass fraction of acetic acid acetonitrile solution to it, clean the homogenizer head, and then merge the washing liquid in centrifuge tube B into centrifuge tube A, mix evenly, add anhydrous magnesium sulfate, sodium chloride, sodium citrate dihydrate and disodium citrate-salt sesquihydrate, mix evenly, oscillate and extract, and then centrifuge to obtain a supernatant.

[0019] Preferably, when the animal-derived food is poultry meat and / or aquatic products, 3-7g of the sample (accurate to 0.01g) is weighed and placed in a 50mL centrifuge tube A, 5-15mL of 1% acetic acid acetonitrile solution is added and placed in a homogenizer, and homogenized at 9000-11000r / min for 10-20s, another 50mL centrifuge tube B is taken, 5-10mL of 1% acetic acid acetonitrile solution is added, and the homogenizer blade is cleaned for 5-15s, and then the washing liquid in the centrifuge tube B is combined with the centrifuge tube A, mixed evenly for 15-25s, and then 3-5g of anhydrous magnesium sulfate, 0.5-1.5g of sodium chloride, 0.5-1g of sodium citrate dihydrate and 0.5-1g of disodium citrate-salt sesquihydrate are added. After mixing evenly, oscillate and extract for 5-15min, and centrifuge at 3000-5000r / min for 5-10min to obtain a supernatant.

[0020] More preferably, when the animal-derived food is poultry meat and / or aquatic products, 5 g of the sample (accurate to 0.01 g) is weighed and placed in a 50 mL centrifuge tube A, 10 mL of 1% acetic acid acetonitrile solution is added and placed in a homogenizer, and homogenized at 10000 r / min for 15 s. Another 50 mL centrifuge tube B is taken, 10 mL of 1% acetic acid acetonitrile solution is added, and the homogenizer blade is cleaned for 10 s. The washing liquid in the centrifuge tube B is then combined with the centrifuge tube A, and 4 g of anhydrous magnesium sulfate, 1 g of sodium chloride, 1 g of sodium citrate dihydrate and 0.5 g of disodium citrate sesquihydrate are added after mixing evenly for 20 s. After mixing evenly, the mixture is shaken and extracted for 10 min, and centrifuged at 4000 r / min for 5 min to obtain a supernatant.

[0021] In the present invention, since the samples of poultry and livestock meat and aquatic products are easily adhered to the blade when being homogenized, the cleaning of the blade is an important factor that cannot be ignored in order to make the samples uniform, so it is necessary to add 1% acetic acid acetonitrile solution for cleaning and merging.

[0022] Furthermore, when the animal-derived food is one or more of fat, viscera, milk or casings, the specific steps of extraction in S1 are: weigh the sample and place it in a centrifuge tube, add acetic acid acetonitrile solution with a mass fraction of 1%, mix well, add anhydrous magnesium sulfate, sodium chloride, sodium citrate dihydrate and disodium citrate-salt sesquihydrate, mix well, oscillate for extraction, and then centrifuge to obtain a supernatant.

[0023] Preferably, when the animal-derived food is one or more of fat, viscera, milk or casings, the specific steps of extraction in S1 are: weigh 3-7 samples (accurate to 0.01g) and place them in a 50mL centrifuge tube, add 5-15mL of 1% acetic acid acetonitrile solution by mass, mix evenly for 1-3min, then add 3-5g anhydrous magnesium sulfate, 0.5-1.5g sodium chloride, 0.5-1g sodium citrate dihydrate and 0.5-1g disodium citrate-salt sesquihydrate, mix evenly, oscillate and extract for 5-15min, centrifuge at 3000-5000r / min for 5-10min to obtain a supernatant.

[0024] More preferably, when the animal-derived food is one or more of fat, viscera, milk or casings, the specific steps of extraction in S1 are: weigh 5 g of the sample (accurate to 0.01 g) and place it in a 50 mL centrifuge tube, add 10 mL of 1% acetic acid acetonitrile solution by mass, mix evenly for 1 minute, then add 4 g of anhydrous magnesium sulfate, 1 g of sodium chloride, 1 g of sodium citrate dihydrate and 0.5 g of disodium citrate-salt sesquihydrate, mix evenly, oscillate and extract for 10 minutes, centrifuge at 4000 r / min for 5 minutes to obtain a supernatant.

[0025] In the present invention, for animal-derived foods such as fat, viscera, milk and casings, since the liver tissue is loose, after adding the extraction solvent, the liver tissue in the extraction system can be dispersed and suspended in the solvent. Therefore, the homogenization step can be omitted in the extraction process, and the milk can be stirred evenly without homogenization. For casings, the experimental process found that the casings were cut into pieces (5mm or less) and homogenized in a homogenizer at 13500r / min for 3min, and there was still no tissue breakage, and the cutter head was easily stuck, the solvent splashed, etc. According to the analysis of the sample morphological properties, it is because the casing tissue is tough. Therefore, there is no homogenization step before the extraction of the casing in this method, and a dynamic immersion and oscillation extraction method for 10min is used instead. For fat, the sample is already in a uniform state after mincing, and no homogenization is required. If homogenization is insisted on, it will be found that the fat sticks to the cutter head and is difficult to wash off. Therefore, for animal-derived foods such as fat, viscera, milk and casings, no homogenization is required during the extraction process.

[0026] Furthermore, when the animal-derived food is eggs, the specific steps of extraction in S1 are: weigh the eggs and place them in a centrifuge tube, add water, mix evenly, add 1% acetic acid acetonitrile solution by mass, mix evenly, then add sodium chloride, oscillate and extract, and then centrifuge to obtain a supernatant.

[0027] Preferably, when the animal-derived food is eggs, the specific steps of extraction in S1 are: weigh 3-7 g of eggs and place them in a 50 mL centrifuge tube, add 5-15 mL of water, mix evenly for 1 min, add 5-10 mL of 1% acetic acid acetonitrile solution, mix evenly for 1-3 min, then add 2-3 g of sodium chloride, mix evenly, oscillate and extract for 10 min, centrifuge at 4000 r / min for 5-10 min, and obtain a supernatant.

[0028] More preferably, when the animal-derived food is eggs, the specific steps of extraction in S1 are: weigh 5 g of eggs (accurate to 0.01 g) and place them in a 50 mL centrifuge tube, add 5 mL of water, mix evenly for 1 min, add 10 mL of 1% acetic acid acetonitrile solution, mix evenly for 1 min, then add 3 g of sodium chloride, mix evenly, oscillate and extract for 10 min, centrifuge at 4000 r / min for 5 min, and obtain a supernatant.

[0029] In the present invention, adding water to eggs can relieve purification pressure when the sensitivity is met, so water is added during extraction.

[0030] Furthermore, when the animal-derived food is one or more of poultry and livestock meat, aquatic products, fat, milk, casings or eggs, the specific purification steps of step S2 are: aspirating the supernatant into a solid phase extraction column to pass through the column, collecting the effluent into a collection tube, mixing evenly and passing through an organic filter membrane, bottling, and obtaining the test liquid.

[0031] Preferably, when the animal-derived food is one or more of poultry and livestock meat, aquatic products, fat, milk, casings or eggs, the specific purification steps of step S2 are: aspirating 2-5 mL of the supernatant into a solid phase extraction column, passing through the column, collecting the effluent into a collection tube, mixing evenly, passing through a 0.22 μm organic filter membrane, bottling, and obtaining the test solution.

[0032] In the present invention, when the animal-derived food is one or more of poultry and livestock meat, aquatic products, fat, milk, casings or eggs, the purification result is ideal without adding water, so no water needs to be added after purification through the column, and the quantitative limit can meet the requirements, so there is no need to consider the nitrogen blowing concentration link.

[0033] Furthermore, when the animal-derived food is viscera, the specific purification steps of step S2 are: aspirate the supernatant into a solid phase extraction column, collect the effluent into a collection tube, mix evenly, take 1-3 mL into a test tube, add 0.5-1.5 ml of water and mix evenly, then pass through an organic filter membrane, bottle, and obtain the test liquid.

[0034] Preferably, when the animal-derived food is viscera, the specific purification steps of step S2 are: aspirate 2 mL of the supernatant into a solid phase extraction column, collect the effluent into a collection tube, mix evenly, take 1 mL into a test tube, add 1 mL of water and mix evenly, pass through a 0.22 μm organic filter membrane, and bottle to obtain the test solution.

[0035] In the present invention, when the animal-derived food is viscera, adding water can further remove impurities while satisfying the detection sensitivity.

[0036] Furthermore, the detection conditions of the liquid chromatography-mass spectrometry are:

[0037] The chromatographic conditions of the liquid chromatography are as follows: chromatographic column: AccucoreaQ (150 mm×2.1 mm2.6 μm), injection volume 2 μL, column temperature 40°C, mobile phase A is methanol, mobile phase B is an aqueous solution containing 0.1% vol formic acid and 5 mmol / L ammonium formate for gradient elution.

[0038] The mass spectrometry conditions are: electrospray positive ion mode, multiple reaction monitoring mode acquisition, ion source temperature of 350°C, and evaporation temperature of 300°C.

[0039] Compared with the prior art, the present invention has achieved beneficial technical effects:

[0040] (1) The present invention is the first to expand the applicable scope of benzovinflumizone determination to different matrices of animal-derived foods such as poultry and livestock meat, aquatic products, fat, viscera, eggs, milk and casings.

[0041] (2) The extraction solvent of the present invention is an acetic acid acetonitrile solution with a mass fraction of 1%. Although the simplest extraction solvent is used, this extraction method can effectively precipitate impurities.

[0042] (3) The present invention is the first to apply the dephospholipidated PPR column purification method to the determination of benzovindiflupyr in animal-derived food; the use of this purifier can further remove impurity interference, and the operation is simple and convenient, which is suitable for large-scale sample testing; and the use of the dephospholipidated PPR column purification does not require the consumption of other reagents, and the operation time is greatly shortened, saving reagent costs and time costs.

[0043] (4) The method for detecting benzovindiflupyr in animal-derived food provided by the present invention has the advantages of being rapid, easy to operate, low cost, accurate, reliable, effective and highly sensitive. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is the positive ion secondary mass spectrum of benzovinflumizole, wherein the mobile phase ratio is 90% methanol-10% 5mmol / L ammonium formate (0.1% formic acid);

[0045] Figure 2 This is the negative ion secondary mass spectrum of benzovinflumizone, where the mobile phase is 100% methanol;

[0046] Figure 3 This is the negative ion secondary mass spectrum of benzovinflumizole, wherein the mobile phase ratio is 90% methanol-10% 5mmol / L ammonium formate (0.1% formic acid);

[0047] Figure 4 The effect of different extraction solvents on the recovery of benzovinflumizole;

[0048] Figure 5 The effects of extraction reagent volume and extraction times on the recovery of benzovinflumizone;

[0049] Figure 6 The effect of different purification agents on the recovery of benzovindiflupyr in QuEChERS.

[0050] Figure 7 Selection of cartridges for SPE cleanup. DETAILED DESCRIPTION

[0051] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. However, the scope of protection claimed in the present invention is not limited to the following specific embodiments, and the reagents or instruments used in the detection method of the present invention can be purchased from the market.

[0052] Example 1

[0053] A method for detecting benzovintriazole in animal-derived food (in this embodiment, the animal-derived food is beef), the detection method comprising the following steps:

[0054] S1. Extraction: Weigh 5 g of sample (accurate to 0.01 g) and place it in a 50 mL centrifuge tube A, add 10 mL of 1% acetic acid acetonitrile solution, and homogenize it in a homogenizer at 10,000 r / min for 15 seconds. Take another 50 mL centrifuge tube B, add 10 mL of 1% acetic acid acetonitrile solution, clean the homogenizer blade for 10 seconds, then combine the washing liquid in centrifuge tube B into centrifuge tube A, mix well for 20 seconds, add 4 g of anhydrous magnesium sulfate, 1 g of sodium chloride, 1 g of sodium citrate dihydrate and 0.5 g of disodium citrate-salt sesquihydrate, mix well, shake and extract for 10 minutes, centrifuge at 4,000 r / min for 5 minutes to obtain the supernatant;

[0055] S2, purification: draw 2mL of supernatant into a phospholipid-free PPR column (200mg / 3mL with divinylbenzene, pyrrolidone, silanol and other groups) and pass through the column, collect the effluent into a collection tube, mix evenly and pass through a 0.22μm organic phase microporous filter membrane, bottle and obtain the test solution;

[0056] S3, detection: the test solution of S2 is detected by liquid chromatography-mass spectrometry.

[0057] The detection conditions of the liquid chromatography-mass spectrometry are:

[0058] The chromatographic conditions of the liquid chromatography are as follows: chromatographic column: AccucoreaQ (150mm×2.1mm2.6μm), injection volume 2μL, column temperature 40°C, mobile phase A is methanol, mobile phase B is an aqueous solution of methanol containing 0.1% vol formic acid and 5mmol / L ammonium formate for gradient elution.

[0059] The mass spectrometry conditions are: electrospray positive ion mode, multiple reaction monitoring mode acquisition, ion source temperature of 350°C, and evaporation temperature of 300°C.

[0060] Example 2

[0061] The detection method of Example 2 is the same as that of Example 1, except that the animal-derived food to be detected is replaced by chicken.

[0062] Example 3

[0063] The detection method of Example 3 is the same as that of Example 1, except that the animal-derived food to be detected is replaced by tilapia.

[0064] Example 4

[0065] A method for detecting benzovintriazole in animal-derived food (in this embodiment, the animal-derived food is pig fat), the detection method comprising the following steps:

[0066] S1. Extraction: Weigh 5 g of sample (accurate to 0.01 g) and place it in a 50 mL centrifuge tube, add 10 mL of 1% acetic acid acetonitrile solution, mix evenly for 1 min, then add 4 g of anhydrous magnesium sulfate, 1 g of sodium chloride, 1 g of sodium citrate dihydrate and 0.5 g of disodium citrate monohydrate, mix evenly and shake for 10 min, centrifuge at 4000 r / min for 5 min to obtain the supernatant.

[0067] S2. Purification: After the supernatant is frozen at -18°C for 1 hour, 2 mL of the supernatant is pipetted into a phospholipid-free PPR column, passed through the column, and the effluent is collected into a collection tube. After mixing evenly, the supernatant is filtered through a 0.22 μm organic phase microporous filter membrane and bottled to obtain the test solution.

[0068] S3, detection: the test solution of S2 is detected by liquid chromatography-mass spectrometry.

[0069] The detection conditions of the liquid chromatography-mass spectrometry are the same as those in Example 1.

[0070] Example 5

[0071] A method for detecting benzovindiflupyr in animal-derived food (in this embodiment, the animal-derived food is pig casings), the detection method comprising the following steps:

[0072] S1. Extraction: Accurately weigh 5 g of sample (accurate to 0.01 g) and place it in a 50 mL centrifuge tube, add 10 mL of 1% acetic acid acetonitrile solution, mix evenly for 1 min, then add 4 g of anhydrous magnesium sulfate, 1 g of sodium chloride, 1 g of sodium citrate dihydrate and 0.5 g of disodium citrate monohydrate, mix evenly and shake for 10 min, centrifuge at 4000 r / min for 5 min to obtain the supernatant.

[0073] S2. Purification: Pipette 2 mL of supernatant into a phospholipid-free PPR column, pass through the column, collect the effluent into a collection tube, mix evenly, pass through a 0.22 μm organic phase microporous filter membrane, and bottle to obtain the test solution.

[0074] S3, detection: the test solution of S2 is detected by liquid chromatography-mass spectrometry.

[0075] The detection conditions of the liquid chromatography-mass spectrometry are the same as those in Example 1.

[0076] Example 6

[0077] The detection method of Example 6 is the same as that of Example 5, and is different from Example 1 in that the animal-derived food to be detected is replaced by milk.

[0078] Example 7

[0079] A method for detecting benzovindiflupyr in animal-derived food (in this embodiment, the animal-derived food is chicken liver), the detection method comprising the following steps:

[0080] S1. Extraction: Accurately weigh 5 g of sample (accurate to 0.01 g) and place it in a 50 mL centrifuge tube, add 10 mL of 1% acetic acid acetonitrile solution, vortex mix for 1 min, then add 4 g of anhydrous magnesium sulfate, 1 g of sodium chloride, 1 g of sodium citrate dihydrate and 0.5 g of disodium citrate monohydrate sesquihydrate, quickly shake by hand to mix, then place on a shaker for 10 min, centrifuge at 4000 r / min for 5 min to obtain the supernatant.

[0081] S2. Purification: Pipette 2 mL of supernatant into a phospholipid-free PPR column, pass through the column, collect the effluent into a collection tube, mix evenly, take 1 mL into a test tube, add 1 mL of water and mix evenly, pass through a 0.22 μm organic phase microporous filter membrane, and bottle to obtain the test solution.

[0082] S3, detection: the test solution of S2 is detected by liquid chromatography-mass spectrometry.

[0083] The detection conditions of the liquid chromatography-mass spectrometry are the same as those in Example 1.

[0084] Example 8

[0085] A method for detecting benzovintriazole in animal-derived food (the animal-derived food in this embodiment is eggs), the detection method comprising the following steps:

[0086] S1. Extraction: Accurately weigh 5 g of eggs (accurate to 0.01 g) and place them in a 50 mL centrifuge tube, add 5 mL of water, vortex mix for 1 min, add 10 mL of 1% acetic acid acetonitrile solution, mix evenly for 1 min, then add 2 g of sodium chloride, mix evenly, shake and extract for 10 min, centrifuge at 4000 r / min for 5 min to obtain the supernatant.

[0087] S2, purification: draw 2 mL of supernatant into a phospholipid-free PPR column, pass through the column, collect the effluent into a collection tube, mix evenly, pass through a 0.22 μm organic phase microporous filter membrane, bottle, and wait for determination;

[0088] S3, detection: the test solution of S2 is detected by liquid chromatography-mass spectrometry.

[0089] The detection conditions of the liquid chromatography-mass spectrometry are the same as those in Example 1.

[0090] The following examples investigate the effects of different factors on the detection of benzovindiflupyr in animal-derived foods.

[0091] The standard working solution of benzovintriazole is prepared as follows:

[0092] (1) dissolving benzovintril in acetonitrile to prepare a stock solution with a mass concentration of 100 mg / L;

[0093] (2) Dilute the stock solution with acetonitrile to a standard stock solution with a mass concentration of 1.0 mg / L for later use.

[0094] (3) The 1.0 mg / L standard stock solution was diluted with acetonitrile to prepare standard solutions of 1 μg / L, 2 μg / L, 5 μg / L, 10 μg / L, 20 μg / L, 50 μg / L, and 100 μg / L of benzovindiflupyr for later use.

[0095] Example 9

[0096] This example is about the optimization of liquid chromatography and mass spectrometry conditions.

[0097] 1. Optimization of liquid phase conditions

[0098] 1.1 Choice of mobile phase

[0099] The chromatographic column was AccucoreaQ (150 mm × 2.1 mm 2.6 μm), the injection volume was 2 μL, and the column temperature was 40 °C. Two mobile phases were used to perform chromatographic analysis and compare their peak areas. The specific results are shown in Table 1.

[0100] Table 1 Comparison of mobile phase selection

[0101]

[0102] As shown in Table 1, by comparing the response peak areas of 2.00 μg / L, 5.00 μg / L, 10.0 μg / L, and 20.0 μg / L standard solutions in different mobile phases, it is found that the peak areas of the corresponding concentrations of benzovindiflupyr (positive ion) in the mobile phase A of methanol and the mobile phase B of an aqueous solution containing 0.1% vol formic acid and 5 mmol / L ammonium formate are larger. Therefore, the mobile phase A of methanol and the mobile phase B of an aqueous solution containing 0.1% vol formic acid and 5 mmol / L ammonium formate are selected as the mobile phases for chromatographic analysis.

[0103] 1.2 Selection of chromatographic columns

[0104] The Accucorea QC18 (150 mm × 2.1 mm 2.6 μm) and Agilent XDBC18 (150 mm × 2.1 mm 2.6 μm) columns were compared. The injection volume was 2 μL, the column temperature was 40°C, the mobile phase A was methanol, and the mobile phase B was an aqueous solution containing 0.1% vol formic acid and 5 mmol / L ammonium formate. The chromatographic analysis was performed. The results are shown in Table 2.

[0105] Table 2 Selection of chromatographic columns

[0106]

[0107] As shown in Table 2, by comparing the response signal-to-noise ratios of 1.00μg / L, 5.00μg / L, 10.0μg / L, 50.0μg / L and 100.0μg / L standard solutions, the two sets of data were analyzed and found that under the same gradient conditions, the signal-to-noise ratio of each standard solution concentration was AccucoreaQC18>AgilentXDBC18, so AccucoreaQC18 (150mm×2.1mm2.6μm) was selected as the chromatographic column for this method.

[0108] 1.3 Optimization of mobile phase ratio

[0109] In liquid chromatography, under the condition of fixed chromatographic column, the separation of the tested compound and other components in the sample is completed by mobile phase. The composition and proportion of mobile phase have an important influence on the chromatographic peak shape and separation degree of the separated components. The composition of mobile phase is related to the chromatographic column and the separated components. The elution ability of mobile phase is determined by its polarity. The mobile phase with strong polarity has a strong ability to occupy the adsorption center and has a strong elution ability, which makes the k value of the component small and the retention time short. In actual operation, mixed solvents with more than two components are often used as mobile phases. Because the strength of the mixed solvent changes continuously with its composition, a mobile phase with suitable solvent strength can be found, and the low viscosity of the solvent can be maintained to reduce the column pressure and improve the column efficiency, while also improving the selectivity of separation. An aqueous solution containing 0.1% vol formic acid and 5mmol / L ammonium formate was selected as the aqueous phase of the mobile phase for chromatographic analysis. The results are shown in Table 3.

[0110] Table 3 Selection of mobile phase ratio

[0111]

[0112] As can be seen from Table 3, the comparison of the two mobile phase gradient ratios of 15 min and 10 min in length shows that the peak area of ​​benzovindiflupyr is not much different when the mobile phase ratio is 90% methanol, and the 10 min length gradient saves more detection time. Therefore, the 10 min length gradient mobile phase ratio with a shorter gradient time is adopted.

[0113] 1.4 Optimization of column temperature

[0114] Take 10 μg / L benzovindiflupyr standard solution and enter the instrument for analysis according to the above chromatographic conditions.

[0115] Table 4 Selection of column temperature

[0116]

[0117] As can be seen from Table 4, the effects of room temperature, 30°C, 35°C, and 40°C on chromatographic separation were compared. The results showed that there was little effect on the chromatographic separation of benzovindiflupyr, but the higher the column temperature, the faster the chromatographic separation. Therefore, in order to save chromatographic analysis time, 40°C was selected as the column temperature in liquid chromatography.

[0118] 2. Optimization of mass spectrometry conditions

[0119] 2.1 Selection of mass spectrometry parameters

[0120] A peristaltic pump was used to continuously inject 1.0 mg / L of benzovinflumazole standard stock solution into the ESI ion source at a flow rate of 10 μL / min. The primary mass spectrometry analysis (Q1 scan) of benzovinflumazole was performed in both negative and positive ion modes to obtain quasi-molecular ion peaks (M-1) and (M+1), and the declustering voltage was optimized. The secondary mass spectrometry analysis (daughter ion scan) of the quasi-molecular ion peak was performed to obtain fragment ion information and optimize the collision energy. The secondary mass spectrum of benzovinflumazole is shown in Figure 1-3 The ion pairs with high response value and low baseline noise were selected as monitoring ion pairs (qualitative ion pairs), and the ion pairs with the strongest signal were selected as quantitative ion pairs. In addition, the parameters such as ion source temperature, sheath gas, and auxiliary gas were optimized. The results are shown in Table 5. The MRM mode was used to collect data, and the multiple reaction monitoring conditions for each ion pair were optimized as shown in Table 6.

[0121] Table 5 Optimization parameters of benzovinflumizone

[0122]

[0123] Table 6 Benzovindiflupyr multi-reaction monitoring conditions

[0124]

[0125] 2.2 Selection of positive and negative ions

[0126] When optimizing mass spectrometry conditions, the benzovinyl fluopicolide positive ion was selected, the mobile phase A was methanol, the mobile phase B was an aqueous solution of 0.1% vol formic acid and 5mmol / L ammonium formate, and the negative ion was selected as mobile phase A of methanol and mobile phase B of 90% methanol-10% 5mmol / L ammonium formate (0.1% formic acid) mobile phase ratio. The experiment found that the response of the benzovinyl fluopicolide negative ion in 100% methanol was higher than that in the mobile phase ratio of 90% methanol-10% 5mmol / L ammonium formate (0.1% formic acid), but both were not as ideal as the response of the positive ion. Similarly, the peak and ion comparison experiment of the ratio of benzovinyl fluopicolide positive ion and negative ion in methanol-5mmol / L ammonium formate (0.1% formic acid) aqueous solution mobile phase found that the benzovinyl fluopicolide positive ion response of a series of different concentrations such as 2.00μg / L, 5.00μg / L, 10.0μg / L, 20.0μg / L was higher, and the compound ion ratio at each concentration level was more stable. Therefore, the benzovinyl fluopicolide positive ion was selected as the experimental ion parameter.

[0127] Example 10

[0128] This example investigates the effect of the extraction solvent on the recovery of benzovindiflupyr.

[0129] 0.05 mL of 1000 μg / L benzovinflumazole standard solution was added to blank tilapia samples and blank milk samples (blank samples refer to samples without benzovinflumazole), respectively. Referring to Example 1, 1% acetic acid acetonitrile aqueous solution was replaced with acetonitrile, 1% formic acid acetonitrile solution and 0.2% formic acid-80% acetonitrile aqueous solution in sequence, and other conditions remained unchanged. After treatment with S1 and S2, liquid chromatography-mass spectrometry was used for detection, and the recovery rates of benzovinflumazole recovery rates were calculated, as shown in FIG. Figure 4 shown.

[0130] Depend on Figure 4 It can be seen from the results that among the four solvents, the extraction effect of 0.2% formic acid 80% acetonitrile aqueous solution on milk and tilapia is the lowest. After comparison, it is found that 1% acetic acid acetonitrile solution is the most ideal. After comprehensive consideration, 1% acetic acid acetonitrile solution is selected as the extraction solvent for the sample.

[0131] Embodiment 11

[0132] This example investigates the effects of extraction reagent volume and extraction times on the recovery of benzovindiflupyr.

[0133] 0.05 ml of 1000 μg / L benzovinflumizole standard solution was added to blank milk samples respectively. Referring to Example 4, when extracting benzovinflumizole from milk samples, 10 ml of 1% acetic acid acetonitrile aqueous solution was mixed with the sample and replaced with 5 mL of 1% acetic acid acetonitrile aqueous solution for extraction once, 5 mL of 1% acetic acid acetonitrile aqueous solution for extraction twice, and 10 mL of 1% acetic acid acetonitrile aqueous solution for extraction twice. Other conditions remained unchanged. After treatment with S1 and S2, the samples were detected by liquid chromatography-mass spectrometry, and the recovery rates of benzovinflumizole were calculated respectively. The specific results are as follows: Figure 5 shown.

[0134] Depend on Figure 5 It can be seen that adding 5mL of acetonitrile to extract twice is better than extracting once with 5mL, the results of adding 10mL of acetonitrile to extract twice are not much different from the results of extracting once with 10mL, and the results of adding 5mL of acetonitrile to extract twice are close to those of extracting once with 10mL. Therefore, considering the operation steps and time consumption, extracting once with 10mL is selected as the extraction volume and number of milk.

[0135] 0.05 ml of 1000 μg / L benzovintril standard solution was added to the blank chicken sample. Referring to Example 4, 20 ml of 1% acetic acid acetonitrile aqueous solution was mixed with the sample and replaced with 10 ml of acetic acid acetonitrile aqueous solution and the extraction was repeated twice. The specific results are shown in FIG. Figure 5 shown.

[0136] Depend on Figure 5 It can be seen that by adding 10mL for repeated extraction for comparison, it was found that the results of the two extraction methods were similar, but repeated extraction was more time-consuming and cumbersome. After comprehensive consideration, 20mL was selected as the extraction volume and number of times for chicken.

[0137] Example 12

[0138] This example investigates the effect of purification methods on the recovery rate of benzovindiflupyr.

[0139] Effects of the commonly used cleanup methods QuEChERS cleanup and SPE cleanup on the recovery of benzovindiflupyr.

[0140] (1) Add 20 mg / kg of benzovinflumizole to a silica gel-bonded octadecyl (C 18 ), aminopropyl powder (NH2), graphitized carbon black (GCB), N-propylethylenediamine (PSA), silica gel powder (Silica), neutral alumina (Al-N) were placed in 6 equal amounts of bullet-shaped centrifuge tubes, shaken and centrifuged, and measured using liquid chromatography-mass spectrometry, and the recovery rate of benzovinflumizone was calculated. The results are shown in Figure 6 .

[0141] Depend on Figure 6 It can be seen that the 5 kinds of purification powders have adsorption effects on benzovindiflupyr to varying degrees, resulting in recovery rates all lower than 80%. There will also be partial recovery losses in the extraction steps. Therefore, this invention will not consider QuEChERS as the purification method for this experiment.

[0142] (2) Add 0.10 ml of 1000 μg / L benzovindiflupyr standard solution to the blank tilapia sample. Referring to Example 1, sequentially replace the dephospholipid PPR small column with a silica-bonded octadecyl (C18, 200 mg / 3 mL) small column, a divinylbenzene and N-vinylpyrrolidone composite column (HLB, 200 mg / 3 mL), Florisil (200 mg / 3 mL), and EMR-LIPID (300 mg / 3 mL). With other conditions unchanged, after treatment with S1 and S2, use a liquid chromatography-mass spectrometry instrument for detection, and calculate the recovery rates of benzovindiflupyr respectively. The specific details are as Figure 7 shown.

[0143] From Figure 7 it can be seen that the small columns of HLB (200 mg / 3 mL), PPR (200 mg / 3 mL), and Florisil (200 mg / 3 mL) have the best recovery rates: EMR < C18 < Florisil < HLB < PPR. The recovery rates of EMR and C18 are slightly lower and can be excluded first. Then, through further comprehensive comparison of the activation equilibrium, elution method steps, reagent usage amounts, and operation times of the three small columns of HLB, PPR, and Florisil, it is found that both HLB and Florisil require five steps: activation, sample loading, elution, washing, and collection, and relatively more solvents are used. The phospholipid PPR small column only needs to directly load the sample and collect it, and the purification step is completed in one step, with simple and convenient operation, which is suitable for the detection of a large number of samples. By comparing the solvents used in the purification process, it is found that about 15 mL or more of reagents are required for activation, elution, and washing with HLB and Florisil, and the single-sample operation time is about 15 minutes or more. However, when using PPR for purification, no other reagents are consumed, and the operation time is greatly shortened. Considering the comprehensive aspects of saving reagent costs and time costs for the detection of a large number of samples, choose the phospholipid PPR as the purification small column for this standard.

[0144] Example 13

[0145] 1. Linear range and regression equation

[0146] In this example, acetonitrile and 8 blank matrix extracts (the blank matrix extract is the test solution after the animal-derived food without benzovinflumizone is treated by steps S1 and S2 of the example) are used to prepare 1 mg / L standard stock solution into solutions with concentrations of 1.0 μg / L, 2.0 μg / L, 5.0 μg / L, 10.0 μg / L, 20.0 μg / L, 50.0 μg / L, and 100.0 μg / L, respectively.

[0147] The method for drawing the standard curve of benzovinflumazole is as follows: the solution concentration (1.0μg / L, 2.0μg / L, 5.0μg / L, 10.0μg / L, 20.0μg / L, 50.0μg / L, 100.0μg / L) is used as the horizontal coordinate, and the ion peak area corresponding to benzovinflumazole is used as the vertical coordinate to draw the standard curve. The results show that within the corresponding mass concentration range of benzovinflumazole, the peak area of ​​the quantitative ion and the concentration have a good linear relationship, and the correlation coefficient r 2 It can reach above 0.999, which can meet the experimental analysis requirements. See Table 7 for details.

[0148] Table 7 Regression equation and correlation coefficient of benzovinflumizone standard working solution

[0149] Matrix Regression equation Linear range μg / L <![CDATA[Correlation coefficient (r 2 )]]> Acetonitrile Y=129900X+11200 1.00～100.0 0.9998 beef Y=83500X-26560 1.00～100.0 0.9998 chicken Y=138900X+12920 1.00～100.0 1.0000 Tilapia Y=113000X-39210 1.00～100.0 0.9999 liver Y=120600X-61000 1.00～100.0 0.9998 milk Y=140900X+13600 1.00～100.0 1.0000 egg Y=161500X-51700 1.00～100.0 0.9997 Fat Y=112100X-3289 1.00～100.0 0.9998 casings Y=86980X-30140 1.00～100.0 1.0000

[0150] 2. Limit of detection and limit of quantification

[0151] According to the above operation, when a blank chicken is used as the matrix and the spiked concentration of benzovinflumizone is 2.0μg / kg, the signal-to-noise ratio of the method is S / N=3, and the detection limit of the method is 2.0μg / kg. Similarly, the confirmation of the quantitative limit is also verified by adding certified reference materials to blank chicken matrix samples. When the added concentration level is 5.0μg / kg, the corresponding signal-to-noise ratio (S / N) is greater than 10 times, and the quantitative limit confirmed by this method is 5.0μg / kg. Its accuracy and precision meet the method and regulatory requirements.

[0152] 3. Verification of the precision and recovery rate of the detection method

[0153] Referring to the preparation and detection method of the blank matrix spiked sample solution in Example 7, 5.0 μg / kg, 10 μg / kg, and 100 μg / kg of benzovintriazole standard solutions at three concentration levels were added to beef, chicken, tilapia, chicken liver, eggs, milk, pig fat, and pig casings, respectively. The three concentration levels of spiked and tested results, the recovery rate (%) and the relative standard deviation (RSD) are shown in Table 8.

[0154] Table 8 Recovery and relative standard deviation of 8 blank matrix samples added with benzovinflumizone

[0155]

[0156] It can be seen from Table 8 that the overall average recovery rate of the detection method of the present invention is in the range of 84.2% to 110.4%, the relative standard deviation is in the range of 0.81% to 8.61%, and the calculated relative standard deviation is less than 10%. The verification results of the method meet the requirements of the relevant provisions of SN / T0001-2016 "Basic Provisions for the Preparation of Physical and Chemical Determination Methods for Export Food and Cosmetics".

[0157] Embodiment 14

[0158] The present embodiment investigates the influence of matrix effect, and matrix effect (ME) refers to the interference of matrix to the analytical process and the influence on the analytical result, and its calculation formula is ME (%) = (A1-A2) / A2 × 100%, wherein A1, A2 are respectively the curve slopes of the target compound in the matrix curve and the pure solvent curve. The present embodiment uses acetonitrile and 8 kinds of blank matrix extracts, respectively, 10mg / L of mixed standard stock solutions to be prepared into concentrations of 1.0μg / L, 2.0μg / L, 5.0μg / L, 10.0μg / L, 20.0μg / L, 50.0μg / L, 100.0μg / L for comparison, and obtains matrix effect ME (%), and is commonly represented by K value, and is investigated by K value. If K is between -10% and 10%, it indicates that the matrix effect is weak and not obvious, K<-10%, then a stronger negative effect is presented, and K>10%, then a stronger positive effect is presented. Specific results are shown in Table 9.

[0159] Table 9 Matrix effects

[0160] Matrix beef chicken Tilapia Chicken liver Matrix effect (%) -35.7 6.9 -13 -7.2 Matrix milk egg Pig fat Hog casings Matrix effect (%) 8.5 24.3 -13.7 -33

[0161] As shown in Table 9, the matrix effects of chicken, chicken liver and milk were not obvious, the egg matrix showed a strong positive effect, and the other matrices had negative effects to varying degrees. Since the matrix effects of 5 samples were obvious, the blank matrix curve was used for calculation and quantification after comprehensive consideration.

[0162] Test Example 1

[0163] The detection methods of Examples 1-8 were used to determine the benzovintriazole residues in 25 samples of beef, chicken, tilapia, chicken liver, pig fat, milk, eggs, pig casings, etc. purchased from multiple supermarkets and markets in Guangzhou. The results are shown in Table 10.

[0164] Table 10 Determination of benzovinflumizone in actual samples

[0165]

[0166]

[0167] According to the disclosure and teaching of the above description, those skilled in the art to which the present invention belongs may also make changes and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and changes to the invention should also fall within the scope of protection of the claims of the present invention. In addition, although some specific terms are used in this specification, these terms are only for the convenience of description and do not constitute any limitation to the invention.

Claims

1. A method for detecting benzovindiflupyr in animal-derived food, characterized in that: The following steps are involved: S1. Extraction: Mix the animal-derived food sample with the extraction solvent, shake the mixture, and centrifuge to obtain the supernatant; When the animal-derived food is aquatic product, the specific steps of extraction in S1 are: weighing a sample and placing it in a centrifuge tube A, adding a 1% acetic acid acetonitrile solution by mass fraction, and then placing it in a homogenizer for homogenization, and then taking another centrifuge tube B and adding a 1% acetic acid acetonitrile solution thereto, cleaning the blade of the homogenizer, and then combining the washing liquid in the centrifuge tube B into the centrifuge tube A, mixing evenly, adding anhydrous magnesium sulfate, sodium chloride, sodium citrate dihydrate and disodium citrate-salt sesquihydrate, mixing evenly, shaking extraction and then centrifuging to obtain a supernatant. When the animal-derived food is one or more of fat, viscera or casings, the specific steps of extraction in S1 are: weighing the sample and placing it in a centrifuge tube, adding acetic acid acetonitrile solution with a mass fraction of 1%, mixing evenly, adding anhydrous magnesium sulfate, sodium chloride, sodium citrate dihydrate and disodium citrate monohydrate, mixing evenly, shaking and extracting, and then centrifuging to obtain a supernatant. When the animal-derived food is eggs, the specific steps of extraction in S1 are: weighing the eggs and placing them in a centrifuge tube, adding water, mixing evenly, adding 1% acetic acid acetonitrile solution by mass, mixing evenly, then adding sodium chloride, shaking and extracting, and then centrifuging to obtain a supernatant. S2, purification: taking the supernatant obtained in step S1 and treating it with a phospholipid-removing PPR column to obtain a test solution; When the animal-derived food is one or more of aquatic products, fat, casings or eggs, the specific purification steps of step S2 are: sucking the supernatant into a solid phase extraction column, collecting the effluent into a collection tube, mixing evenly, passing through an organic filter membrane, bottling, and obtaining a test solution. When the animal-derived food is viscera, the specific purification steps of step S2 are: aspirate the supernatant into a solid phase extraction column, collect the effluent into a collection tube, mix evenly, take 0.5-1.5 mL into a test tube, add 0.5-1.5 ml of water and mix evenly, then pass through an organic filter membrane, bottle, and obtain the test solution. S3, detection: the test solution of S2 is detected by liquid chromatography-mass spectrometry. The chromatographic conditions of the liquid chromatography are as follows: chromatographic column: Accucore aQ, 150 mm×2.1 mm 2.6 μm, injection volume 2 μL, column temperature 40°C, mobile phase A is methanol, mobile phase B is an aqueous solution containing 0.1% vol formic acid and 5 mmol / L ammonium formate for gradient elution; The mass spectrometry conditions are: electrospray positive ion mode, multiple reaction monitoring mode acquisition, ion source temperature of 350°C, and evaporation temperature of 300°C.

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