CN113341016A - Method for detecting residual bactericide in animal food - Google Patents

Method for detecting residual bactericide in animal food Download PDF

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CN113341016A
CN113341016A CN202110635932.3A CN202110635932A CN113341016A CN 113341016 A CN113341016 A CN 113341016A CN 202110635932 A CN202110635932 A CN 202110635932A CN 113341016 A CN113341016 A CN 113341016A
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bactericide
solution
animal food
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汤苗苗
张晓莉
侯赛
郭良起
孙晶
范玲
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Henan Grg Metrology & Test Co ltd
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N30/02—Column chromatography
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
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    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
    • G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate

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Abstract

The invention discloses a method for detecting residual bactericides in animal food, which comprises the following steps: s1: mixing and shaking animal food, a dispersing agent and sodium chloride, and centrifuging to obtain a supernatant A; s2: centrifuging the centrifuged precipitate by using a dispersing agent in a shaking way, and mixing the supernatant with the supernatant A to obtain a solution A; s3: carrying out solid phase extraction treatment on the solution A to obtain a solution to be detected; s4: preparing a standard solution of a target bactericide, detecting the chromatographic peak retention time and the chromatographic peak area of the target bactericide by using a liquid chromatography-mass spectrometer, and drawing a standard working curve by taking the mass concentration of the standard solution as a horizontal coordinate and the corresponding chromatographic peak area as a vertical coordinate to obtain a linear equation of the target bactericide; s5: and (4) carrying out qualitative and quantitative determination on the sample to be tested of S3 by using a liquid chromatography-mass spectrometer. The detection method can simultaneously detect the residual quantity of the 21 bactericides in the animal food, and has higher addition recovery rate of the 21 bactericides in the animal food.

Description

Method for detecting residual bactericide in animal food
Technical Field
The invention belongs to the technical field of detection, and particularly relates to a method for detecting residual bactericides in animal food.
Background
Animal-derived food is an important part consumed in daily life of people, and mainly comprises various edible animal tissues, eggs, raw milk and the like. With the improvement of the living standard of people, the proportion of animal-derived food in the living standard of people is increasing day by day, and the safety problem of animal-derived food becomes the focus of global attention, wherein the quality of raw milk directly influences the safety of dairy products.
With the development of science and technology, the quality safety requirements can be met in the aspects of scale management and cultivation, quality control, technical equipment and the like, and factors really influencing the animal derived safety are mostly from the production and cultivation links. On the other hand, whether the environment of the culture area is polluted or not, whether the feed is polluted by pesticide residues or not, and the quality and the safety of the animal origin are determined.
The bactericide is a pesticide generally used for preventing and controlling plant diseases caused by various pathogenic microorganisms, and is mainly applied to crops such as fruit trees and economic crops. The development of disease resistance requires the continuous development of new bactericides with action sites, the bactericides become one of hot spots of pesticide development in recent years, with the development and use of more and more varieties, residues of the bactericides can be transferred into animal tissues along with the consumption of grains and feeds, and then the residues can possibly exist in animal-derived products such as raw milk, animal muscles and the like, and finally the bactericides can cause harm to human health through a food chain. Therefore, it is necessary to intensively study a method for detecting the residual amount of the bactericide in the food of animal origin to monitor the residual amount so as to ensure the physical health of the consumer.
GB 2763 and 2019 maximum limit of pesticide residues in national food standards for food safety are formally implemented in 2, 15 and 2020. There are 140 items of 483 common pesticides in 356 foods, wherein 28 items of foods of animal origin are involved, 16 items have no corresponding detection standard, and the rest reference standard contains a small range of matrix and a small range of pesticide in animal muscle; or the detection standards of fruits, vegetables and honey are referred, so that the method is not suitable for use. The method comprises the steps of liquid chromatography-tandem mass spectrometry for measuring the residual amounts of 461 pesticides and related chemicals in GB/T20772-2008 animal muscles, the gas chromatography-mass spectrometry for measuring the residual amounts of 478 pesticides and related chemicals in GB/T19650-2006 animal muscles, the liquid chromatography-tandem mass spectrometry for measuring the residual amounts of 486 pesticides and related chemicals in GB/T20771-2008 honey, the gas chromatography-mass spectrometry for measuring the residual amounts of 500 pesticides and related chemicals in GB 23200.8-2016 national food safety standard fruits and vegetables, and the liquid chromatography-tandem mass spectrometry for measuring the residual amounts of 450 pesticides and related chemicals in GB/T20769-2008 fruits and vegetables.
The matrix of the standard is only 4 detection methods, is suitable for detecting raw milk and animal muscle, the rest of the detection methods refer to the detection of agricultural products such as fruits, vegetables and honey, the combination of gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry is mainly used, the pretreatment methods comprise acetonitrile extraction, Florisil diatomite or amino SPE column solid phase extraction purification, repeated nitrogen blowing concentration and the like, and no method is specifically used for detecting the pesticide residue such as bactericide. In the existing effective standard detection method, the detection items of the gas chromatography-mass spectrometry for determining the residual amount of boscalid in GB/T22979-2008 milk and milk powder are too single to be limited, and the gas chromatography-mass spectrometry for determining the residual amounts of 511 pesticides and related chemicals in GB/T23210-2008 milk and milk powder and the liquid chromatography-tandem mass spectrometry for determining the residual amounts of 493 pesticides and related chemicals in GB/T23211-2008 milk and milk powder are also mainly related to conventional pesticide residue detection, and are also rarely related to the detection of bactericides.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for detecting residual bactericides in animal food. The detection method can simultaneously detect the residual quantity of the bactericide 21 in the animal food, and the detection result of the detection method has high precision, stability and reproducibility.
In order to achieve the purpose, the invention adopts the technical scheme that: a method for detecting residual bactericides in animal food comprises the following steps:
s1: mixing and shaking animal food, a dispersing agent and sodium chloride, and centrifuging to obtain supernatant A;
s2: repeatedly shaking and centrifuging the centrifuged precipitate by using a dispersing agent, and mixing the supernatant with the supernatant A to obtain a solution A;
s3: treating the solution A through a solid phase extraction column to obtain a solution to be detected;
s4: preparing a standard solution of a target bactericide, detecting chromatographic peak retention time and chromatographic peak area of the target bactericide by using a liquid chromatography-mass spectrometer, and drawing a standard working curve by taking the mass concentration of the standard solution as a horizontal coordinate and the corresponding chromatographic peak area as a vertical coordinate to obtain a linear equation of the target bactericide;
s5: and (4) combining the standard curve, and performing qualitative and quantitative determination on the target bactericide in the sample to be detected of S3 by using a liquid chromatography-mass spectrometer.
The invention combines the extraction process of animal food, dispersant and sodium chloride solvent with solid phase extraction and purification to carry out purification pretreatment on the animal food, and adopts a liquid phase mass spectrometer to simultaneously detect the residual quantity of the bactericide in the animal food 21. The concentration of the target bactericide is in the range of 0.5-20 mug/L, the linearity is good, the detection limit of the method is 0.7 mug/kg, the quantitative limit of the method is 2.0 mug/kg, the correlation coefficients are all larger than 0.995, the average recovery rate is 70.0% -119.7%, and the relative deviation is smaller than 10.0%.
In a preferred embodiment of the present invention, the dispersant is one of acetonitrile, an aqueous solution containing 0.2% vol formic acid and 80% vol acetonitrile, and an acetonitrile solution containing 0.1% to 1% vol formic acid.
A great deal of experiments prove that the dispersant in the 5 can improve the recovery rate of the bactericide in the animal food.
As a preferred embodiment of the present invention, the dispersant is an acetonitrile solution containing 0.2% vol formic acid.
A great deal of experiments prove that the recovery rate of extracting the bactericide from the animal food is the highest when the dispersing agent is acetonitrile solution containing 0.2 percent of vol formic acid.
In a preferred embodiment of the present invention, the ratio of the animal food, the sodium chloride and the dispersant in S1 is 1g to 5 g: 1g-10 g: 2mL-20 mL; the amount of the dispersant in S1 was the same as that in S2.
As a preferred embodiment of the present invention, the target fungicide is pyraclostrobin, kresoxim-methyl, azoxystrobin, difenoconazole, propiconazole, flusilazole, triadimenol, triadimefon, prothioconazole, benzovindiflupyr, penthiopyrad, isopyrazam, fluopicolide, prochloraz, cyprodinil, pyrimethanil, azinam, metrafenone, pyrimethanil-5-hydroxy, thioprothioconazole, difenoconazole.
The detection method can be used for qualitatively and quantitatively detecting less than 21 bactericides in animal food.
The detection method can be used for qualitatively and quantitatively detecting 21 bactericides in animal food.
In a preferred embodiment of the present invention, the animal food is animal food such as cow milk, beef, milk powder, pure milk, or the like.
More preferably, the animal food is raw milk, beef, milk powder or pure milk.
As a preferred embodiment of the present invention, in S1, when the animal food is milk powder, the milk powder is soaked in water and then mixed with the dispersant and sodium chloride and shaken; when the animal food is beef, the beef is homogenized, mixed with the dispersing agent and sodium chloride and vibrated.
The milk powder is soaked in water to be dissolved and then is mixed with the dispersing agent and the sodium chloride to be vibrated and extracted, so that the phenomenon that the milk powder is directly mixed with the dispersing agent and the sodium chloride to be caked can be avoided, and the milk powder is soaked in water to be dissolved and then is mixed with the dispersing agent and the sodium chloride to be vibrated and extracted, so that the recovery rate of extracting the bactericide in the animal food can be improved by 20%.
As a preferred embodiment of the present invention, the preparation method of the standard solution of the target bactericide specifically comprises: (1) dissolving a target bactericide with acetonitrile to prepare a stock solution with the mass concentration of 100 mg/L; (2) diluting the stock solution into a mixed standard stock solution with the mass concentration of 10mg/L by using acetonitrile; (3) diluting the mixed standard stock solution into standard solutions of the target bactericide, wherein the mass concentrations of the standard solutions of the target bactericide are 0.5ng/mL, 1.0ng/mL, 2.0ng/mL, 5.0ng/mL, 10.0ng/mL and 20.0ng/mL respectively, by using a blank matrix solution; the blank matrix solution is the solution to be detected after the animal food without the bactericide is processed by the steps S1-S3.
In a preferred embodiment of the present invention, the detection conditions of the liquid chromatography-mass spectrometer are as follows:
column temperature: 40 ℃; temperature of the sample chamber: 4 ℃; sample introduction volume: 10 mu L of the solution; flow rate: 0.3 mL/min;
the mobile phase A is an aqueous solution containing 0.1% vol formic acid and 2mmol/L ammonium acetate, and the mobile phase B is acetonitrile for gradient elution;
the gradient is: 0min, 95% mobile phase a, 5% mobile phase B; 2min, 95% mobile phase a, 5% mobile phase B; 4min, 0% mobile phase a, 100% mobile phase B; 7min, 0% mobile phase A, 100% mobile phase B; 7.1min, 95% mobile phase a, 5% mobile phase B; 8min, 95% mobile phase A, 5% mobile phase B;
electrospray positive ion mode (ESI +), electrospray voltage 2.8kV, multi-reaction monitoring mode (MRM) collection, ion source temperature of 150 ℃, desolvation temperature of 500 ℃, desolvation gas flow of 800L/h, and cone orifice gas flow of 50L/h.
As a preferred embodiment of the method for detecting the residual bactericide in the animal food, the target bactericide is qualitatively determined by using a liquid chromatography-mass spectrometer and mainly comprises the following steps:
the retention time of a chromatographic peak of the sample to be detected of S3, which is measured by a liquid chromatogram-mass spectrometer, is within +/-2.5% of the retention time of the chromatographic peak of the corresponding standard solution, and the ion pair of the target bactericide appears in the mass spectrogram of the sample to be detected after the background is deducted, namely the target bactericide is judged to exist in the sample to be detected.
As a preferred embodiment of the method for detecting the residual bactericide in the animal food, the target bactericide is quantitatively determined by using a liquid chromatography-mass spectrometer and mainly comprises the following steps:
and substituting the corresponding chromatographic peak area determined by the liquid chromatogram-mass spectrometer according to the sample to be detected of S3 into the linear equation of each target bactericide in S4 to obtain the content of each target bactericide.
Compared with the prior art, the invention has the beneficial effects that: the invention combines the extraction process of the mixed solvent of the animal food, the dispersant and the sodium chloride with the solid-phase extraction and purification to carry out the purification pretreatment on the animal food, and adopts a liquid-phase mass spectrometer to simultaneously detect the residual quantity of the bactericide in the animal food 21. The concentration of the target bactericide is in the range of 0.5-20 mug/L, the linearity is good, the detection limit of the method is 0.7 mug/kg, the quantitative limit of the method is 2.0 mug/kg, the correlation coefficients are all larger than 0.995, the average recovery rate is 70.0-119.7%, the relative deviation is smaller than 10.0%, and the detection result obtained by the detection method is good in precision, stability and repeatability. Therefore, the detection method defined by the invention can provide a corresponding matched detection method for the bactericide in animal-derived food in GB 2763-2019 'maximum residue limit of pesticide in national food Standard for food safety'. The method provides new technical support and data support for national standards of food safety, can provide corresponding matched detection standards for the national standards, and further protects the benefits of consumers.
Drawings
FIG. 1 is a schematic flow chart of the method for detecting residual bactericide in animal food according to the present invention;
FIG. 2 is a graph showing the recovery of 7 bactericides from raw milk measured with the dispersant described in example 2;
FIG. 3 is the result of the dispersant of example 2 to determine the recovery rate of 7 bactericides in milk powder;
FIG. 4 shows the recovery of 7 bactericides in beef measured with the dispersant described in example 2.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.
The detection conditions of the liquid chromatography-mass spectrometer in the embodiments 1-3 of the invention are as follows:
column temperature: 40 ℃; temperature of the sample chamber: 4 ℃; sample introduction volume: 10 mu L of the solution; flow rate: 0.3 mL/min;
the mobile phase A is an aqueous solution containing 0.1 vol% of formic acid and 2mmol/L of ammonium acetate, and the mobile phase B is acetonitrile for gradient elution;
the gradient is: 0min, 95% mobile phase a, 5% mobile phase B; 2min, 95% mobile phase a, 5% mobile phase B; 4min, 0% mobile phase a, 100% mobile phase B; 7min, 0% mobile phase A, 100% mobile phase B; 7.1min, 95% mobile phase a, 5% mobile phase B; 8min, 95% mobile phase A, 5% mobile phase B;
electrospray positive ion mode (ESI +), electrospray voltage 2.8kV, multi-reaction monitoring mode (MRM) collection, ion source temperature of 150 ℃, desolvation temperature of 500 ℃, desolvation gas flow of 800L/h, and cone orifice gas flow of 50L/h.
Fig. 1 is a schematic flow chart of the method for detecting residual bactericide in animal food according to the present invention, and example 1 and example 3 detect and label-add recover residual bactericide in animal food according to the schematic flow chart of fig. 1.
Example 1
The embodiment of the method for detecting the residual bactericide in the animal food comprises the following specific steps:
s1: taking 3 parts of containers containing 5g of raw milk matrix, respectively adding standard solutions containing 21 bactericides into 5g of raw milk matrix to enable the content of the 21 bactericides to be 10 mug/kg, 20 mug/kg and 40 mug/kg, then respectively adding 10mL of 0.2% formic acid acetonitrile solution and 5g of sodium chloride into the three containers, performing vortex oscillation extraction, performing centrifugal layering to obtain supernate a1, a2 and a3, repeatedly oscillating and centrifuging the centrifuged precipitate by using 10mL of 0.2% formic acid acetonitrile solution, and respectively mixing the supernate with supernate a1, a2 and a3 to obtain solution a1, solution a2 and solution a 3;
homogenizing beef, placing 5g of homogenized beef in 3 containers, respectively adding 21 standard solutions containing 21 bactericides to make the content of the 21 bactericides be 10 mug/kg, 20 mug/kg and 40 mug/kg, respectively adding 10mL of 0.2% formic acid acetonitrile solution and 5g of sodium chloride into the three containers, performing vortex oscillation extraction, performing centrifugal layering to obtain supernate b1, b2 and b3, repeatedly oscillating and centrifuging the centrifuged precipitate by using 10mL of 0.2% formic acid acetonitrile solution, respectively mixing the supernate with supernate b1, b2 and b3 to obtain solution b1, solution b2 and solution b 3;
taking 3 parts of 5g milk powder, adding 5mL of water, uniformly mixing and soaking for 30min, respectively adding standard solutions containing 21 bactericides, enabling the content of the 21 bactericides to be 10 mug/kg, 20 mug/kg and 40 mug/kg, then respectively adding 10mL of 0.2% acetonitrile formate solution and 5g of sodium chloride, carrying out vortex oscillation extraction, carrying out centrifugal layering to obtain supernatant c1, c2 and c3, repeatedly oscillating and centrifuging the centrifuged precipitate by using 10mL of 0.2% acetonitrile formate solution, respectively mixing the supernatant with supernatant c1, c2 and c3 to obtain solution c1, solution c2 and solution c 3;
s2: respectively treating the solution a1, the solution a2, the solution a3, the solution b1, the solution b2, the solution b3, the solution c1, the solution c2 and the solution c3 by a solid phase extraction column, receiving all effluent, and filtering by a 0.22 mu m filter membrane to respectively obtain a solution a1 to be detected, a solution a2 to be detected and a solution a3 to be detected; liquid b1 to be detected, liquid b2 to be detected and liquid b3 to be detected; a to-be-detected liquid c1, a to-be-detected liquid c2 and a to-be-detected liquid c 3;
s3: dissolving 21 target bactericides with acetonitrile respectively to prepare stock solutions with the mass concentration of 100 mg/L; preparing the stock solution into a mixed standard stock solution with the mass concentration of 10mg/L by using acetonitrile; diluting the stock solution into a standard solution of the target bactericide with the mass concentration of 0.5ng/mL, 1.0ng/mL, 2.0ng/mL, 5.0ng/mL, 10.0ng/mL and 20.0ng/mL by using a blank matrix solution; the blank matrix solution is xx; detecting chromatographic peak retention time and chromatographic peak area of the target bactericide by using a liquid chromatography-mass spectrometer, and drawing a standard working curve by taking the mass concentration of a standard solution as a horizontal coordinate and the corresponding chromatographic peak area as a vertical coordinate to obtain a linear equation of each target bactericide;
s4: and (4) performing qualitative and quantitative determination on the 9 solutions to be determined obtained in the step (S2) by using a liquid chromatography-mass spectrometer in combination with the standard curve.
The types of the 21 bactericides and the mass spectrum detection parameters are shown in table 1; the addition recovery rates and precision of 21 bactericides in the solution to be tested are shown in the results in table 2, and the results in table 2 are the results of 6 parallel experiments.
Mass spectrometric detection parameters for 121 fungicides in Table
Figure BDA0003105170190000071
Figure BDA0003105170190000081
Note: quantitation of ion pairs.
TABLE 2 recovery and precision of 21 bactericides in raw milk, milk powder and beef
Figure BDA0003105170190000082
Figure BDA0003105170190000091
Figure BDA0003105170190000101
As can be seen from Table 2, the method for detecting residual bactericides in animal food has the advantages that the normalized recovery rate of the bactericides in 21 animal food reaches 70-119.7%, and the RSD is less than 10%.
At the lowest level of additive recovery, the present invention combines 3-fold and 10-fold signal-to-noise ratios with concentration extrapolation to determine the method detection Limit (LOD) and quantitation Limit (LOQ). The lowest addition concentration meeting the methodological requirements is taken as the quantitative limit, the quantitative limits of the 21 bactericides are all 2.0 mug/kg, and the detection limits are all 0.7 mug/kg.
Example 2
The embodiment of the method for detecting the residual bactericide in the animal food comprises the following specific steps:
s1: dissolving 7 target bactericides with acetonitrile respectively to prepare stock solutions with the mass concentration of 100 mg/L; preparing the stock solution into a mixed standard stock solution with the mass concentration of 10mg/L by using acetonitrile; diluting the stock solution into a standard solution of the target bactericide with the mass concentration of 0.5ng/mL, 1.0ng/mL, 2.0ng/mL, 5.0ng/mL, 10.0ng/mL and 20.0ng/mL by using a blank matrix solution; the blank matrix solution is xx; detecting chromatographic peak retention time and chromatographic peak area of the target bactericide by using a liquid chromatography-mass spectrometer, and drawing a standard working curve by taking the mass concentration of a standard solution as a horizontal coordinate and the corresponding chromatographic peak area as a vertical coordinate to obtain a linear equation of each target bactericide;
s4: respectively adding standard solutions containing 7 bactericides into a blank matrix by a standard adding recovery method to ensure that the concentration of the 7 bactericides is 20 mug/kg, respectively adding 20mL of dispersing agent after standing for 24h, filtering through a 0.22 mu m filter membrane, and carrying out qualitative and quantitative determination on the liquid by using a liquid chromatography-mass spectrometer in combination with a standard curve; the blank matrix is 5g of raw milk, 5g of homogenized beef or 5g of milk powder, and 5mL of water are uniformly mixed and soaked for 30 min.
The 7 bactericides include azoxystrobin, prothioconazole, isopyrazam, prochloraz, cyprodinil, fluazinam and metrafenone.
The dispersant is acetonitrile, an aqueous solution containing 0.2% vol formic acid and 80% vol acetonitrile, an acetonitrile solution containing 0.2% vol formic acid, an acetonitrile solution containing 0.1% vol acetic acid, or an acetonitrile solution containing 1% vol formic acid.
TABLE 3 recovery of the dispersant on the biocides in animal food
Figure BDA0003105170190000111
In FIGS. 2-4 80% acetonitrile (containing 0.2% formic acid) is an aqueous solution containing 0.2% vol formic acid and 80% vol acetonitrile; 1% formic acid acetonitrile is acetonitrile solution containing 1% vol formic acid; 0.1% formic acid acetonitrile is acetonitrile solution containing 0.1% vol acetic acid; 0.2% formic acid acetonitrile is acetonitrile solution containing 0.2% vol formic acid.
As can be seen from Table 3 and FIGS. 2 to 4, the 5 dispersants of the present invention showed 41.6% to 130.2% recovery of the bactericide from the animal food, and the highest recovery was obtained by extraction with 0.2% vol formic acid in acetonitrile.
Example 3
The repeatability of the detection method for the residual bactericide in the animal food is realized.
S1: adding a standard solution containing 21 bactericides into a raw milk matrix to ensure that the content of the 21 bactericides is 20 mug/kg, adding 10mL of 0.2% acetonitrile formate solution and 5g of sodium chloride, performing vortex oscillation extraction, performing centrifugal layering to obtain a supernatant a, repeatedly oscillating and centrifuging the centrifuged precipitate by using 10mL of 0.2% acetonitrile formate solution, and mixing the supernatant with the supernatant a to obtain a solution a;
s2: treating the solution a by a solid phase extraction column, receiving all effluent liquid, and filtering by a 0.22 mu m filter membrane to respectively obtain a liquid a to be detected;
s3: dissolving 21 target bactericides with acetonitrile respectively to prepare stock solutions with the mass concentration of 100 mg/L; preparing the stock solution into a mixed standard stock solution with the mass concentration of 10mg/L by using acetonitrile; diluting the stock solution into a standard solution of the target bactericide with the mass concentration of 0.5ng/mL, 1.0ng/mL, 2.0ng/mL, 5.0ng/mL, 10.0ng/mL and 20.0ng/mL by using a blank matrix solution; the blank matrix solution is xx; detecting chromatographic peak retention time and chromatographic peak area of the target bactericide by using a liquid chromatography-mass spectrometer, and drawing a standard working curve by taking the mass concentration of a standard solution as a horizontal coordinate and the corresponding chromatographic peak area as a vertical coordinate to obtain a linear equation of each target bactericide;
s4: and (4) carrying out qualitative and quantitative determination on the liquid a to be determined obtained in the step (S2) by using a liquid chromatography-mass spectrometer in combination with the standard curve.
The kinds of the 21 bactericides are shown in table 4.
In this example, 5.1g, 5.2g, 5.3g and 5.4g of raw milk of the same batch and the same product are weighed respectively, and then the qualitative and quantitative analysis of 21 bactericides is carried out according to the test method of example 3, and the bactericides are respectively counted as No. 1, No. 2, No. 3 and No. 4; the results of the tests are shown in Table 4, and the results of the recovery in Table 4 are obtained in 6 parallel experiments.
TABLE 4 repeatability results for recovery of bactericides in raw milk of the present invention
Figure BDA0003105170190000121
Figure BDA0003105170190000131
As can be seen from the results in Table 4, the recovery rate of the bactericide in the raw milk of the present invention has good repeatability.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims (9)

1. A method for detecting residual bactericides in animal food is characterized by comprising the following steps:
s1: mixing and shaking animal food, a dispersing agent and sodium chloride, and centrifuging to obtain supernatant A;
s2: repeatedly shaking and centrifuging the centrifuged precipitate by using a dispersing agent, and mixing the supernatant with the supernatant A to obtain a solution A;
s3: treating the solution A through a solid phase extraction column to obtain a solution to be detected;
s4: preparing a standard solution of a target bactericide, detecting chromatographic peak retention time and chromatographic peak area of the target bactericide by using a liquid chromatography-mass spectrometer, and drawing a standard working curve by taking the mass concentration of the standard solution as a horizontal coordinate and the corresponding chromatographic peak area as a vertical coordinate to obtain a linear equation of the target bactericide;
s5: and (4) combining the standard curve, and performing qualitative and quantitative determination on the target bactericide in the sample to be detected of S3 by using a liquid chromatography-mass spectrometer.
2. The method of claim 1, wherein the dispersant is one of acetonitrile, an aqueous solution containing 0.2% vol formic acid and 80% vol acetonitrile, and an acetonitrile solution containing 0.1% to 1% vol formic acid.
3. The method according to claim 1, wherein the dispersant is an acetonitrile solution containing 0.2% vol formic acid.
4. The method for detecting the residual bactericide in the animal food according to claim 1, wherein the ratio of the animal food, the sodium chloride and the dispersant in S1 is 1g-5 g: 1g-10 g: 2mL-20 mL; the amount of the dispersant in S1 was the same as that in S2.
5. The method for detecting residual fungicide in animal food according to claim 1, characterized in that the target fungicide is pyraclostrobin, kresoxim-methyl, azoxystrobin, difenoconazole, propiconazole, flusilazole, triadimenol, triadimefon, prothioconazole, benzovindiflupyr, penthiopyrad, isopyrazam, fluopicolide, prochloraz, cyprodinil, pyrimethanil, triforine, metrafenone, pyrimethanil-5-hydroxy, desulfoltiprazole, difenoconazole.
6. The method for detecting the residual bactericide in the animal food according to claim 1, wherein the preparation method of the standard solution of the target bactericide specifically comprises the following steps: (1) dissolving a target bactericide with acetonitrile to prepare a stock solution with the mass concentration of 100 mg/L; (2) diluting the stock solution into a mixed standard stock solution with the mass concentration of 10mg/L by using acetonitrile; (3) diluting the mixed standard stock solution into standard solutions of the target bactericide, wherein the mass concentrations of the standard solutions of the target bactericide are 0.5ng/mL, 1.0ng/mL, 2.0ng/mL, 5.0ng/mL, 10.0ng/mL and 20.0ng/mL respectively, by using a blank matrix solution; the blank matrix solution is the solution to be detected after the animal food without the bactericide is processed by the steps S1-S3.
7. The method for detecting the residual bactericide in the animal food according to claim 1, wherein the detection conditions of the liquid chromatography-mass spectrometer are as follows:
column temperature: 40 ℃; temperature of the sample chamber: 4 ℃; sample introduction volume: 10 mu L of the solution; flow rate: 0.3 mL/min;
the mobile phase A is an aqueous solution containing 0.1% vol formic acid and 2mmol/L ammonium acetate, and the mobile phase B is acetonitrile for gradient elution;
the gradient is: 0min, 95% mobile phase a, 5% mobile phase B; 2min, 95% mobile phase a, 5% mobile phase B; 4min, 0% mobile phase a, 100% mobile phase B; 7min, 0% mobile phase A, 100% mobile phase B; 7.1min, 95% mobile phase a, 5% mobile phase B; 8min, 95% mobile phase A, 5% mobile phase B;
the electrospray positive ion mode has electrospray voltage of 2.8kV, and multiple reaction monitoring mode collection, wherein the ion source temperature is 150 ℃, the desolvation temperature is 500 ℃, the desolvation gas flow is 800L/h, and the cone orifice gas flow is 50L/h.
8. The method of claim 1, wherein the animal food is cow's milk, beef, milk powder or pure milk.
9. The method for detecting residual bactericide in animal food according to claim 8, wherein in S1, when the animal food is milk powder, the milk powder is soaked in water and then mixed with the dispersant and sodium chloride and shaken; when the animal food is beef, the beef is homogenized, mixed with the dispersing agent and sodium chloride and vibrated.
CN202110635932.3A 2021-06-08 2021-06-08 Method for detecting residual bactericide in animal food Pending CN113341016A (en)

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