CN114858928A - Method for detecting residual quantity of kresoxim-methyl in dairy product - Google Patents
Method for detecting residual quantity of kresoxim-methyl in dairy product Download PDFInfo
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 40
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 20
- 229910052757 nitrogen Inorganic materials 0.000 claims description 20
- 238000001514 detection method Methods 0.000 claims description 18
- FKHIFSZMMVMEQY-UHFFFAOYSA-N talc Chemical compound [Mg+2].[O-][Si]([O-])=O FKHIFSZMMVMEQY-UHFFFAOYSA-N 0.000 claims description 16
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- 150000002500 ions Chemical class 0.000 claims description 11
- 238000004817 gas chromatography Methods 0.000 claims description 8
- 238000004587 chromatography analysis Methods 0.000 claims description 5
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 claims description 4
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
- G01N30/8634—Peak quality criteria
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Abstract
The invention provides a method for detecting the residual amount of kresoxim-methyl in a dairy product, which comprises the following steps: mixing a sample to be detected with a first solvent to obtain a first mixture so as to extract residual kresoxim-methyl in the sample to be detected; adding sodium chloride into the first mixture, uniformly mixing, centrifuging, and collecting supernatant; performing a first solid phase extraction on the supernatant and collecting a first eluate; drying the first eluent to obtain first residues, and redissolving the first residues by using a second solvent to obtain a second mixture; performing a second solid phase extraction on the second mixture and collecting a second eluate; drying the second eluent to obtain second residues, and redissolving the second residues by using a third solvent to obtain a third mixture; the third mixture is measured by gas chromatography-tandem mass spectrometry. The method can realize the full separation of the kresoxim-methyl and the matrix interferent in the dairy products, adopts the solid-phase extraction and purification of impurity adsorption and elution, and can quickly and accurately detect the residual amount of the kresoxim-methyl in the dairy products.
Description
Technical Field
The invention belongs to the technical field of food detection, and particularly relates to a method for detecting the residual amount of kresoxim-methyl in a dairy product.
Background
Kresoxim-methyl is a compound developed on the basis of natural beta-methoxy acrylate derivatives, and belongs to methoxy acrylate bactericides. The action mechanism is mainly to act on cytochrome bcl compound in the mitochondrial respiratory chain of the fungus, prevent electron transfer and inhibit the growth of the fungus. Kresoxim-methyl is widely used as a high-efficiency broad-spectrum bactericide.
The quality safety problem of dairy products is strongly concerned by society, and in order to avoid the harm caused by pesticide residues, in the maximum pesticide residue limit in food safety national standard food (GB2763-2019) issued in 2019, the state stipulates a series of limits of pesticide residues in raw milk. Wherein, the temporary limit of the kresoxim-methyl in the raw milk is regulated to be 0.01 mg/kg.
The existing kresoxim-methyl detection methods comprise a gas chromatography, a gas chromatography-mass spectrometry method, a liquid chromatography-series connection method and the like (Wujishan et al, pesticide 2011, 11(10): 754; Zhangyao et al, environmental chemistry, 2019, 38(5): 1204; Zhouyao et al, chromatography, 2017, 35(9):970), but the methods mainly aim at detecting the kresoxim-methyl residue in crops such as vegetables, fruits, grains and the like. Aiming at dairy products such as milk powder, yoghourt, cheese and the like, the detection difficulty is high and the research is less due to the complex matrix, and the investigation finds that the method for detecting the kresoxim-methyl is provided in the gas chromatography-mass spectrometry for detecting the residual quantity of 511 pesticides and related chemicals in milk and milk powder (GB/T23210-ion 2008), but the method is a screening method, is not aiming at a single target compound of the kresoxim-methyl, and has long adopted pretreatment steps and complicated operation. Therefore, a new detection method needs to be optimized or developed to meet the detection of the residual amount of kresoxim-methyl in the dairy product.
Disclosure of Invention
The invention aims to provide a method for detecting the residual amount of kresoxim-methyl in a dairy product, which overcomes the defects of long steps, difficult operation and poor separation effect in a complex matrix of the existing detection method.
According to one aspect of the invention, the invention provides a method for detecting the residual amount of kresoxim-methyl in a dairy product, which comprises the following steps:
mixing a sample to be detected with a first solvent to obtain a first mixture so as to extract residual kresoxim-methyl in the sample to be detected;
adding sodium chloride into the first mixture, uniformly mixing, centrifuging, and collecting supernatant;
performing a first solid phase extraction on the supernatant and collecting a first eluate;
drying the first eluent by blowing to obtain first residues, and redissolving the first residues by using a second solvent to obtain a second mixture;
subjecting the second mixture to a second solid phase extraction and collecting a second eluate;
drying the second eluent to obtain second residues, and redissolving the second residues by using a third solvent to obtain a third mixture;
the resulting third mixture was measured by gas chromatography-tandem mass spectrometry.
Preferably, the detection method further comprises concentrating the supernatant before the first solid-phase extraction, preferably to 0.5-2.0 mL.
The first solid-phase extraction adopts a C18 solid-phase extraction column, and the second solid-phase extraction adopts a Florisil solid-phase extraction column. Preferably, the C18 solid phase extraction column is pre-activated with acetonitrile. The first solid phase extraction step was eluted with acetonitrile and the volume of acetonitrile eluted in this step was 3.0-5.0 mL.
In the detection method, for each gram of the sample to be detected, the dosage of the first solvent is 5.0-10.0mL, and the dosage of the sodium chloride is 0.5-1.0 time of the mass of the sample to be detected.
In the second solid phase extraction step, the Florisil solid phase extraction column is activated in advance by using a mixed organic solvent, wherein the organic mixed solvent comprises n-hexane and acetone in a volume ratio of 9:1-5: 5.
Preferably, the second solid phase extraction step is eluted with the mixed organic solvent, and the volume of elution of the mixed organic solvent in this step is 3.0-5.0 mL.
Preferably, the first solvent is acetonitrile; the second solvent and the third solvent are n-hexane; the first eluent and the second eluent are respectively dried by nitrogen at 40 ℃.
Preferably, the parameter conditions of the gas chromatography-tandem mass spectrometry are as follows:
gas chromatography conditions:
a chromatographic column: 5% phenyl-methyl polysiloxane quartz capillary chromatography column, chromatographic temperature rising procedure: maintaining at 40 deg.C for 1.5min, programming at 25 deg.C/min to 90 deg.C, programming at 15 deg.C/min to 300 deg.C, and maintaining for 6 min;
tandem mass spectrometry conditions:
the quantitative ion pair is 116.0>89.0m/z, the first qualitative ion pair is 130.9>130.1m/z, the second qualitative ion pair is 116.0>62.9m/z, and the collision voltage is 14/10/24V.
The determination method simplifies the operation steps, is easy to operate, improves the separation effect in a complex matrix, and has quick and accurate detection results.
Drawings
Further objects, features and advantages of the present invention will become apparent from the following description of embodiments of the invention, with reference to the accompanying drawings, in which:
figure 1 shows a Selective Reaction Monitoring (SRM) chromatogram of kresoxim-methyl in a milk base according to example 1.
Figure 2 shows the kresoxim-methyl recovery for different volumes of supernatant concentrate.
Figure 3 shows the kresoxim-methyl recovery for different elution volumes of acetonitrile.
Fig. 4 shows the kresoxim-methyl recovery rate corresponding to mixed organic solvents of different volume ratios.
Detailed Description
Technical features, objects and advantages of the present invention will be more clearly understood and appreciated by those skilled in the art. It should be understood that the following detailed description is merely exemplary, and the technical solution of the present invention is not limited to the specific embodiments listed below.
The method for detecting the residual amount of the kresoxim-methyl in the dairy product comprises an extraction step, a centrifugal separation step, a purification step and a determination step, wherein the purification step is carried out in two steps, and each step respectively comprises a solid-phase extraction step and a redissolution step.
The dairy products detected by the invention can comprise liquid milk, milk powder, ice cream, yoghourt, cheese and the like.
The extraction step comprises the step of mixing a sample to be detected with a first solvent to obtain a first mixture so as to extract the residual kresoxim-methyl in the sample to be detected. The first solvent may be acetonitrile. Specifically, a sample to be detected is mixed with acetonitrile and extracted by vortex oscillation. The amount of the first solvent used is 5.0-10.0mL, preferably 7.5mL, per gram of sample to be tested.
The centrifugation step comprises adding sodium chloride to the first mixture, wherein the amount of sodium chloride is 0.75 times the mass of the sample to be tested. In the case of a solid dairy product, it is preferred to add a certain amount of water. For example, the first mixture and sodium chloride and optionally water are vortexed, e.g., for 5min, and then vortexed at 4 deg.C for 10 min 4 r/min for 5min, collecting the supernatant, e.g. 5mL, and then concentrating the supernatant, e.g. to 0.5-2.0mL, preferably 0.5-1.0mL, at 40 deg.C by nitrogen-blowing. The volume range of the concentrated supernatant is wide, the experimental conditions are loose, and the tolerance is good.
The extraction step of the invention is carried out in two steps, which respectively comprise a solid phase extraction step and a redissolution step.
According to one embodiment, the first solid phase extraction step is performed using a C18 solid phase extraction column, and the C18 solid phase extraction column may be a commercially available column commonly used in the art, such as a 500mg, 3mL format C18 solid phase extraction column. Preferably, the C18 solid phase extraction column is pre-activated with acetonitrile. This first solid phase extraction step is eluted with an appropriate volume of solvent. The solvent usable for this step may be, for example, acetonitrile. The eluate is collected, preferably in a volume of 3.0-5.0mL, preferably 4.0mL, which can be collected. Further, the collected eluate is blow-dried, for example, at 40 ℃ with nitrogen to near dryness, to obtain a first residue. The first residue is redissolved in a solvent, such as n-hexane, to provide a second mixture.
The second solid phase extraction step is carried out on the second mixture using a Florisil solid phase extraction column. The Florisil solid phase extraction column may be any commercially available one commonly used in the art, for example, a 500mg, 3mL size Florisil solid phase extraction column may be used. Preferably, the florisil solid phase extraction column is previously activated with a mixed organic solvent. The second mixture was applied to a florisil solid phase extraction column activated with a mixed organic solvent and eluted with the mixed organic solvent. The mixed organic solvent usable in this step may include, for example, n-hexane and acetone in a volume ratio of 9:1 to 5:5, preferably 7: 3. The eluate is collected, preferably in a volume of 3.0-5.0mL, for example 4.0 mL. Further, the collected eluate is blow-dried, for example, at 40 ℃ with nitrogen to near dryness, to obtain a second residue. The second residue is redissolved in a solvent, such as n-hexane, to provide a third mixture.
The purification step adopts an impurity adsorption elution principle, can well separate the target from the matrix interferent by two times of solid phase extraction column purification, firstly removes the residual weak polar impurities in the concentrated solution by using a C18 solid phase extraction column, and then removes the residual strong polar impurities in the concentrated solution by using a Florisil column, thereby realizing the separation of the target. Compared with the classical solid phase extraction mode, the solid phase extraction purification mode omits the steps of washing, drying and the like, shortens the time and saves the reagent.
The measuring step comprises measuring the third mixture by gas chromatography-tandem mass spectrometry.
The gas chromatography conditions may include:
a chromatographic column: 5% phenyl-methyl polysiloxane quartz capillary chromatography column (e.g. HP-5MS) 30m long, 0.25mm internal diameter, 0.25 μm membrane thickness; chromatographic temperature-raising procedure: maintaining at 40 deg.C for 1.5min, programming at 25 deg.C/min to 90 deg.C, programming at 15 deg.C/min to 300 deg.C, and maintaining for 6 min; and (3) sample introduction mode: no split-flow sample introduction.
Tandem mass spectrometry conditions included:
an ionization mode: an electron impact source (EI);
the monitoring mode is as follows: multiple Reaction Monitoring (MRM);
the quantitative ion pair, the qualitative ion pair and the collision voltage of the kresoxim-methyl pesticide residue are respectively shown in table 1:
TABLE 1
In conclusion, the method for measuring the residual quantity of the kresoxim-methyl in the dairy product is simple and easy to implement, has good separation effect with a complex matrix, and has the advantages of cheap and easily-obtained test reagents and equipment, high tolerance of experimental conditions and accurate detection result.
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments.
The methods used in the following examples are conventional methods unless otherwise specified, and the reagents used are commercially available reagents unless otherwise specified.
Examples
Example 1
(1) Reagent:
water: primary water according with GB/T6682;
organic reagents: pesticide residue grade.
(2) Preparing a standard solution:
kresoxim-methyl standard solution: the concentration is 0.1 mg/mL;
kresoxim-methyl standard working solution: and diluting the kresoxim-methyl standard solution into a standard working solution with the concentration of 1 mu g/mL by using n-hexane in a stepwise dilution mode. Storing at 4 deg.C or below.
(3) Sample pretreatment:
weighing 2.0g of pure milk in a 30mL centrifuge tube with a plug, adding 10mL of acetonitrile, mixing, and performing vortex oscillation extraction for 1 min; adding 1.0g sodium chloride into the mixture, sequentially vortexing and shaking for 5min at 4 deg.C for 10 min 4 Centrifuging at r/min for 5min, collecting 5mL of supernatant, and concentrating the supernatant to 0.5-1.0mL at 40 deg.C by nitrogen blowing; loading the obtained concentrated solution to a C18 solid phase extraction column activated by acetonitrile in advance, eluting by 4.0mL of acetonitrile, and blowing the collected eluent to be nearly dry by nitrogen at 40 ℃; redissolving the obtained residue with 0.5mL of n-hexane, loading the residue to a Florisil solid phase extraction column activated in advance with a mixed organic solvent (the volume ratio of n-hexane to acetone is 7:3), eluting with 5.0mL of the same mixed organic solvent, collecting the eluate, blowing nitrogen to near dryness at 40 ℃, redissolving with 1.0mL of n-hexane, and measuring with a gas chromatography tandem mass spectrometer.
(4) The measurement conditions were as follows:
chromatographic conditions are as follows:
a chromatographic column: 5% phenyl-methyl polysiloxane quartz capillary chromatography column (HP-5MS), column length: 30m, inner diameter: 0.25mm, film thickness: 0.25 μm; chromatographic temperature-raising procedure: maintaining at 40 deg.C for 1.5min, programming at 25 deg.C/min to 90 deg.C, programming at 15 deg.C/min to 300 deg.C, and maintaining for 6 min; and (3) sample introduction mode: no split-flow sample introduction.
Mass spectrum conditions:
an ionization mode: an electron impact source (EI); the monitoring mode is as follows: the Multiple Reaction Monitoring (MRM), the quantitative ion pair, the qualitative ion pair and the collision voltage of the kresoxim-methyl pesticide residue are shown in table 1 above. The measured Selective Reaction Monitoring (SRM) chromatogram of kresoxim-methyl in the fresh milk substrate is shown in figure 1.
(5) Drawing of Standard Curve
Blowing the blank substrate solution with nitrogen, adding a certain volume of mixed standard substance working solution, fixing the volume to 1mL by using normal hexane, preparing a series of substrate mixed standard working solutions with the concentrations of 5.0ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL and 250ng/mL in sequence, measuring by using a gas chromatography-mass spectrometer, and drawing a standard curve by using the peak area of the target compound quantitative ion as a vertical coordinate and the target compound mass concentration as a horizontal coordinate.
(6) Sample assay
And sampling the sample liquid to be detected to obtain the peak area of the target object, and obtaining the corresponding on-machine concentration according to the standard curve.
(7) Calculation of results
In the formula:
x < - > -the residual quantity of the tested components in the sample, mu g/kg;
c- - - -concentration of the measured component solution, ng/mL, obtained from the standard working curve;
v- - - -sample solution volume, mL;
m- - - -mass of sample represented by the sample solution, g;
f- - -dilution factor;
1000-a scaling factor.
(8) Method quantitative limit verification
Adding standard solutions with different mass concentrations into a blank matrix sample, measuring the concentration corresponding to the signal-to-noise ratio equal to 10 by using a gas chromatography-tandem mass spectrometer, and finally determining the limit of quantitation to be 5 mu g/kg.
(9) Standard curve and linearity verification
The standard curve and the correlation coefficient of kresoxim-methyl are obtained by drawing the standard curve in the step (5) of the example 1, and are shown in table 2.
TABLE 2
| Compound (I) | Standard curve | Correlation coefficient |
| Kresoxim-methyl | Y＝1.904e 5 X-3.517e 4 | 0.9999 |
As can be seen from Table 2, the concentration of kresoxim-methyl and the peak area have a good linear relationship, and the correlation coefficient is greater than 0.9900, which meets the requirements.
(10) Verification of recovery and precision
And (3) taking a blank pure milk sample, and adding three levels of a quantitative limit, a critical limit and a 10-time critical limit respectively to perform a standard adding recovery experiment. The results of the measurement, recovery and precision of the spiked samples are shown in Table 3 below.
TABLE 3
As can be seen from Table 3, when the addition amount is 5. mu.g/kg, 10. mu.g/kg and 100. mu.g/kg, the recovery rate ranges of kresoxim-methyl are 77.4-90.5%, 80.9-90.7% and 87.7-98.0%, and the recovery rate of the method meets the requirement; the RSD of the kresoxim-methyl is 5.3 percent, 3.9 percent and 3.8 percent, and the precision of the method meets the requirement.
Example 2
In this embodiment, a commercially available milk powder of a certain brand is selected and used for the kresoxim-methyl residue addition recovery rate experiment. The specific method comprises the following steps:
weighing 2.0g of milk powder into a 30mL centrifuge tube with a plug, adding the standard solution of the example 1 into the centrifuge tube, and fully and uniformly mixing to obtain a milk powder sample to be detected with the mass concentration of 10 mug/kg. Adding 5mL of pure water, mixing with 10mL of acetonitrile, and extracting for 1min by vortex oscillation; to the above mixture was added 1.5g of chlorineDissolving sodium, sequentially vortex and shake for 5min at 4 deg.C for 10 min 4 Centrifuging at r/min for 5min, collecting 5mL of supernatant, and concentrating the supernatant to 0.5-1.0mL at 40 deg.C by nitrogen blowing; loading the obtained concentrated solution to a C18 solid phase extraction column activated by acetonitrile in advance, eluting by 4.0mL of acetonitrile, and blowing the collected eluent to be nearly dry by nitrogen at 40 ℃; redissolving the obtained residue with 0.5mL of n-hexane, loading the residue to a Florisil solid phase extraction column activated by a mixed organic solvent (the volume ratio of n-hexane to acetone is 7:3), eluting with 5.0mL of the same mixed organic solvent, collecting the eluate, blowing nitrogen to near dryness at 40 ℃, redissolving with 1.0mL of n-hexane, and measuring by a gas chromatography-tandem mass spectrometer.
The chromatographic conditions and mass spectrometric conditions were specifically the same as in example 1.
Matrix standard curve a blank milk powder matrix was used and the formulation procedure was the same as in example 1.
The results of measurement by gas chromatography-tandem mass spectrometry are shown in table 4.
TABLE 4
Example 3
In this example, a commercially available yogurt of a certain brand was selected and an experiment of the kresoxim-methyl residue addition recovery rate was performed. The specific method comprises the following steps:
weighing 2.0g of yoghourt in a 30mL centrifuge tube with a plug, adding the standard solution in the embodiment 1, and fully and uniformly mixing to obtain a yoghourt sample to be detected with the mass concentration of 10 mug/kg. Adding 5mL of pure water, mixing with 10mL of acetonitrile, and extracting for 1min by vortex oscillation; adding 1.5g sodium chloride into the mixture, sequentially vortexing and shaking for 5min at 4 deg.C for 10 min 4 Centrifuging at r/min for 5min, collecting 5mL of supernatant, and concentrating the supernatant to 0.5-1.0mL at 40 deg.C by nitrogen blowing; loading the obtained concentrated solution to a C18 solid phase extraction column activated by acetonitrile in advance, eluting by 4.0mL of acetonitrile, and blowing the collected eluent to be nearly dry by nitrogen at 40 ℃; the obtained residue was redissolved with 0.5mL of n-hexane, and then loaded on a Florisil solid previously activated with a mixed organic solvent (volume ratio of n-hexane to acetone was 7:3)And (3) carrying out phase extraction on the column, eluting with 5.0mL of the same mixed organic solvent, blowing the collected eluent to be nearly dry at 40 ℃ by nitrogen, redissolving with 1.0mL of n-hexane, and measuring by using a gas chromatography tandem mass spectrometer.
The chromatographic conditions and mass spectrometric conditions were specifically the same as in example 1.
Matrix standard curve a blank yogurt matrix was used and the formulation procedure was the same as in example 1.
The results of measurement by gas chromatography-tandem mass spectrometry are shown in table 5.
TABLE 5
Example 4
In this example, an ice cream of a certain brand sold in the market was selected and tested for kresoxim-methyl residue addition recovery rate. The specific method comprises the following steps:
weighing 2.0g of ice cream in a 30mL centrifuge tube with a plug, adding the standard solution of the embodiment 1 into the centrifuge tube, and fully and uniformly mixing to obtain an ice cream sample to be detected with the mass concentration of 10 mug/kg. Adding 5mL of pure water, mixing with 10mL of acetonitrile, and extracting for 1min by vortex oscillation; adding 1.5g sodium chloride into the mixture, sequentially vortexing and shaking for 5min at 4 deg.C for 10 min 4 Centrifuging at r/min for 5min, collecting 5mL of supernatant, and concentrating the supernatant to 0.5-1.0mL at 40 deg.C by nitrogen blowing; loading the obtained concentrated solution to a C18 solid phase extraction column activated by acetonitrile in advance, eluting by 4.0mL of acetonitrile, and blowing the collected eluent to be nearly dry by nitrogen at 40 ℃; redissolving the obtained residue with 0.5mL of n-hexane, loading the residue to a Florisil solid phase extraction column activated in advance with a mixed organic solvent (the volume ratio of n-hexane to acetone is 7:3), eluting with 5.0mL of the same mixed organic solvent, collecting the eluate, blowing nitrogen to near dryness at 40 ℃, redissolving with 1.0mL of n-hexane, and measuring with a gas chromatography tandem mass spectrometer.
The chromatographic conditions and mass spectrometric conditions were specifically the same as in example 1.
Matrix standard curve a blank milk powder matrix was used and the formulation procedure was the same as in example 1.
The results of measurement by gas chromatography-tandem mass spectrometry are shown in table 6.
TABLE 6
Example 5
In this example, a commercially available cheese of a certain brand was selected and tested for the recovery rate of the kresoxim-methyl residue. The specific method comprises the following steps:
weighing 2.0g of cheese in a 30mL centrifuge tube with a plug, adding the standard solution in the example 1 into the centrifuge tube, and fully and uniformly mixing to obtain a cheese sample to be detected with the mass concentration of 10 mug/kg. Adding 5mL of pure water, mixing with 10mL of acetonitrile, and extracting for 1min by vortex oscillation; adding 1.5g sodium chloride into the mixture, sequentially vortexing and shaking for 5min at 4 deg.C for 10 min 4 Centrifuging at r/min for 5min, collecting 5mL of supernatant, and concentrating the supernatant to 0.5-1.0mL at 40 deg.C by nitrogen blowing; loading the obtained concentrated solution to a C18 solid phase extraction column activated by acetonitrile in advance, eluting by 4.0mL of acetonitrile, and blowing the collected eluent to be nearly dry by nitrogen at 40 ℃; redissolving the obtained residue with 0.5mL of n-hexane, loading the residue to a Florisil solid phase extraction column activated in advance with a mixed organic solvent (the volume ratio of n-hexane to acetone is 7:3), eluting with 5.0mL of the same mixed organic solvent, collecting the eluate, blowing nitrogen to near dryness at 40 ℃, redissolving with 1.0mL of n-hexane, and measuring with a gas chromatography tandem mass spectrometer.
The chromatographic conditions and mass spectrometric conditions were specifically the same as in example 1.
Matrix standard curve a blank milk powder matrix was used and the formulation procedure was the same as in example 1.
The results of measurement by gas chromatography-tandem mass spectrometry are shown in table 7.
TABLE 7
Comparative example 1
The recovery rate of kresoxim-methyl residue (added amount of 5. mu.g/kg) was measured in the same manner as in example 1, except that the volume of the supernatant after concentration was different in the case of C18 solid-phase extraction. Specifically, the post-concentration volumes of the supernatant used in this comparative example were 0.5mL, 1.0mL, 1.5mL, and 2.0mL, respectively. The results of the experiment are shown in FIG. 2.
Comparing the data in fig. 2, it can be seen that the recovery rate of the target substance is the best when the volume of the concentrated supernatant is 0.5mL, and the average recovery rate is 85.4%; when the volume was 1.0mL, the recovery rate of the target product was inferior, and the average recovery rate was 80.8%.
Comparative example 2
The recovery rate of kresoxim-methyl pesticide residue (addition amount of 100 μ g/kg) was measured in the same manner as in example 1, except that the elution volume of acetonitrile used in the C18 solid-phase extraction step was different. Specifically, the elution volumes of acetonitrile used in this comparative example were 3.0mL, 4.0mL, and 5.0mL, respectively. The results of the experiment are shown in FIG. 3.
Comparing the data in fig. 3, it can be seen that the recovery of the target was optimal with an average recovery of 86.8% at an elution volume of 4.0mL acetonitrile.
Comparative example 3
The recovery rate of kresoxim-methyl pesticide residue (addition amount is 100 mug/kg) was measured in the same manner as in example 1, except that the mixed organic solvent ratio used in the florisil solid-phase extraction step was different. Specifically, the volume ratios of n-hexane to acetone in the mixed organic solvent used in this comparative example were 9:1, 8:2, 7:3, and 5:5, respectively. The results of the experiment are shown in FIG. 4.
Comparing the data in fig. 4, it can be seen that the target recovery was best at a 7:3 volume ratio of n-hexane to acetone, with an average recovery of 94.2%.
The foregoing is only a preferred embodiment of the present invention. It will be appreciated that various modifications, combinations, alterations, and substitutions of the details and features of the invention may be made by those skilled in the art without departing from the spirit and nature of the invention. Such modifications, combinations, alterations and substitutions are also to be understood as being included within the scope of the invention as claimed.
Claims (10)
1. A method for detecting the residual amount of kresoxim-methyl in a dairy product is characterized by comprising the following steps:
mixing a sample to be detected with a first solvent to obtain a first mixture so as to extract residual kresoxim-methyl in the sample to be detected;
adding sodium chloride into the first mixture, uniformly mixing, centrifuging, and collecting supernatant;
performing a first solid phase extraction on the supernatant and collecting a first eluate;
drying the first eluent to obtain first residues, and redissolving the first residues by using a second solvent to obtain a second mixture;
subjecting the second mixture to a second solid phase extraction and collecting a second eluate;
drying the second eluent to obtain second residues, and redissolving the second residues by using a third solvent to obtain a third mixture;
the resulting third mixture was measured by gas chromatography-tandem mass spectrometry.
2. The assay of claim 1, further comprising concentrating the supernatant prior to the first solid phase extraction, preferably to 0.5-2.0 mL.
3. The detection method according to claim 1, wherein the first solid-phase extraction employs a C18 solid-phase extraction column, and the second solid-phase extraction employs a Florisil solid-phase extraction column.
4. The detection method according to claim 1, wherein the amount of the first solvent is 5.0-10.0mL and the amount of the sodium chloride is 0.5-1.0 times the mass of the sample per gram of the sample.
5. The detection method according to claim 1, wherein the C18 solid phase extraction column is activated with acetonitrile in advance.
6. The detection method according to claim 1, wherein the first solid phase extraction step is performed by eluting with acetonitrile, and the volume of acetonitrile eluted in the step is 3.0 to 5.0 mL.
7. The detection method according to claim 3, wherein the Florisil solid-phase extraction column is activated in advance with a mixed organic solvent comprising n-hexane and acetone in a volume ratio of 9:1 to 5: 5.
8. The detection method according to claim 7, wherein the second solid phase extraction step is performed by eluting with the mixed organic solvent, and the volume of the mixed organic solvent eluted in this step is 3.0 to 5.0 mL.
9. The detection method according to claim 1, wherein the first solvent is acetonitrile; the second solvent and the third solvent are n-hexane; the first eluent and the second eluent are respectively dried by nitrogen at 40 ℃.
10. The detection method according to claim 1, wherein the parameter conditions of the gas chromatography-tandem mass spectrometry are as follows:
gas chromatography conditions:
a chromatographic column: 5% phenyl-methyl polysiloxane quartz capillary chromatography column, chromatographic temperature rising procedure: maintaining at 40 deg.C for 1.5min, programming at 25 deg.C/min to 90 deg.C, programming at 15 deg.C/min to 300 deg.C, and maintaining for 6 min;
tandem mass spectrometry conditions:
the quantitative ion pair is 116.0>89.0m/z, the first qualitative ion pair is 130.9>130.1m/z, the second qualitative ion pair is 116.0>62.9m/z, and the collision voltage is 14/10/24V.
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