CN111896660B - Method for detecting glyphosate and glufosinate in plant food - Google Patents
Method for detecting glyphosate and glufosinate in plant food Download PDFInfo
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- CN111896660B CN111896660B CN202010797219.4A CN202010797219A CN111896660B CN 111896660 B CN111896660 B CN 111896660B CN 202010797219 A CN202010797219 A CN 202010797219A CN 111896660 B CN111896660 B CN 111896660B
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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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- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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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
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
Abstract
The invention provides a method for detecting glyphosate and glufosinate in plant food, which is characterized by comprising the following steps: 1: preparing a sample; 2: extracting; 3: purifying; 4: derivatizing; 5: preparing a quality control sample; 6: instrument analysis: 7: calculating analysis results, wherein the pesticide residue in the sample is calculated according to the following formula (1):
Description
Technical Field
The invention relates to a method for detecting glyphosate and glufosinate in plant food.
Background
The current methods for detecting glyphosate and glufosinate in vegetable food include gas chromatography and gas chromatography-mass spectrometry, liquid chromatography and liquid chromatography-mass spectrometry. The gas chromatography and the liquid chromatography have the advantages of stable method and accurate quantification, but because glyphosate and glufosinate are both strong polar compounds, the glyphosate and glufosinate are insoluble in most organic solvents, have no ultraviolet absorption and are not easy to volatilize, are not reserved on a conventional C18 chromatographic column, and are difficult to separate and measure by utilizing gas chromatography and high performance liquid chromatography; on the other hand, due to the lack of chromophore, it is difficult to directly detect with ultraviolet and fluorescence detectors, and therefore, pre-column or post-column derivatization methods are often used to improve the chromatographic behavior of gas chromatography and high performance liquid chromatography and to increase the detection response. The 9-fluorenylmethyl chloroformate is an ideal derivatizing agent because the derivatizing reaction with glyphosate and glufosinate is fast, and the generated derivative is relatively stable. The traditional pretreatment method for detecting glyphosate and glufosinate is that samples are purified by a solid phase extraction column after being extracted by water, the process is complex, and the detection of a large amount of samples is difficult to carry out in a laboratory.
Disclosure of Invention
The invention provides a method for detecting glyphosate and glufosinate in vegetable food, which aims to solve the defects of the prior art, improve the purification effect of a sample extracting solution, establish a rapid analysis method for measuring the residual quantity of glyphosate and glufosinate in vegetable food, improve the working efficiency and reduce the toxicity of the preparation method to human bodies.
The technical scheme adopted for solving the technical problems is as follows:
the method for detecting the glyphosate and the glufosinate in the plant food is characterized by comprising the following steps of:
1: sample preparation
Sampling a sample to obtain a sample;
2: extraction of
Weighing 10g of sample in a 50mL centrifuge tube, supplementing water, adding 10mL of methanol, carrying out vortex oscillation for 2min, and centrifuging at 3800r/min for 5min to be purified;
3: purification of
3.1: vegetables, fruits, nuts, mushrooms, spices, tea leaves, cereal samples:
accurately sucking 1mL of the extracting solution into a 2mL centrifuge tube weighing 5mg of multi-wall carbon nanotubes, swirling for 1min, centrifuging for 3min at 10000r/min, taking supernatant, and centrifuging in another 2mL centrifuge with 0.22 μm organic filter membrane to obtain derivatization;
3.2: oil crop samples:
accurately sucking 4mL of the extracting solution into a 5mL centrifuge tube by using a pipette, placing the extracting solution into a refrigerator at the temperature of minus 18 ℃ for freezing overnight, then taking 1mL of supernatant into a 2mL centrifuge tube weighing 5mg of multiwall carbon nanotubes and 50mg of neutral alumina, centrifuging for 3min at 10000r/min after swirling for 1min, taking the supernatant, and putting the supernatant into another 2mL centrifuge tube through a 0.22 mu m organic filter membrane for derivatization;
3.3: vegetable oil samples:
accurately sucking 1mL of extracting solution, directly passing through a 0.22 mu m organic system filter membrane in another 2mL centrifuge tube, and derivatizing;
4: derivatization
Accurately sucking 0.5mL of the purified extracting solution into a 2mL centrifuge tube, adding 0.5mL of borate buffer solution, uniformly mixing, adding 0.5mL of chloroformate-9-fluorenylmethylene acetonitrile solution, swirling for 1min, derivatizing for 1h in a water bath at 40 ℃, centrifuging for 1min at 10000r/min after derivatization, taking supernatant, and passing through a 0.22 mu m organic filter membrane for on-machine detection; accurately sucking 0.5mL of standard intermediate solution, and simultaneously carrying out derivatization according to the step;
repeating the steps 2, 3 and 4, and preparing a parallel sample in each batch;
5: quality control sample preparation
5.1: the method is blank: weighing 10mL of methanol and 10mL of ultrapure water in a 50mL centrifuge tube without weighing the sample, and repeating the steps 2, 3 and 4;
5.2: quality control solution: weighing 21 mu L of glufosinate-ammonium and glyphosate standard solution with the concentration of 100mg/L in a 50mL centrifuge tube, and repeating the steps 2, 3 and 4 to obtain a sample standard adding solution with the standard adding concentration of 70 mu g/L of the object to be detected;
4.10.3: sample addition of the labeling solution: weighing a sample, taking 21 mu L of glufosinate-ammonium and glyphosate standard solution with the concentration of 100mg/L in a 50mL centrifuge tube, and repeating the steps 2, 3 and 4 to obtain a sample standard adding solution with the standard adding concentration of 70 mu g/L of the object to be detected;
6: instrument analysis:
6.1: conditions of liquid chromatography
6.1.1: chromatographic column: poroshell 120EC-C18, 150 mm. Times.3.0 mm, particle size 2.7 μm;
6.1.2: mobile phase: a is 0.1% formic acid 5mmol/L ammonium acetate aqueous solution, B is 0.1% formic acid 5mmol/L ammonium acetate methanol solution;
6.1.3: flow rate: 0.4mL/min;
6.1.4: column temperature: 30 ℃;
6.1.5: sample injection volume: 1 μl;
6.2: mass spectrum reference conditions
6.2.1: ion source: an electrospray ion source;
6.2.2: ionization source polarity: a positive mode;
6.2.3：Gas Temp：300℃；
6.2.4：Gas Flow：9L/min；
6.2.5：Nebulizer：40psi；
6.2.6：Sheath Gas Temp：350℃；
6.2.7：Sheath Gas Flow：11L/min；
6.2.8：Capillary：4000V；
6.2.9：Nozzle Voltage：500V；
6.2.10: the detection mode is as follows: multiple Reaction Monitoring (MRM);
6.3.0: running a solvent blank containing only methanol, and checking whether an instrument baseline is stable;
6.4.0: running a standard working curve solution; establishing a calibration curve by using the peak area and the concentration, wherein the linear correlation coefficient is not less than 0.995;
6.5.0: the test method is blank to check whether pollution exists;
6.6.0: testing the quality control solution to check recovery;
6.7.0: testing the sample solution and parallel samples;
6.8.0: the test sample is added with a standard solution for qualitative and recovery rate checking;
6.9.0: the mass concentration of the object to be detected in the sample is in the mass concentration range of the standard working curve, and the sample exceeding the upper limit of the mass concentration of the standard working curve is diluted and injected and quantified by an external standard method;
6.3: qualitative and quantitative determination
6.3.1: retention time
Comparing the retention time of the chromatographic peak of the object to be detected in the detected sample with the retention time of the corresponding standard chromatographic peak, wherein the relative error is within +/-2.5%;
6.3.2: quantitative ion, qualitative ion, and ion abundance ratio
When the sample is tested under the same experimental condition, if the retention time of chromatographic peaks of the to-be-tested substances in the sample is consistent with that of a standard sample, and after background subtraction, qualitative ions of the target compound in a mass spectrum of the sample must appear, wherein the qualitative ions at least comprise 1 parent ion and 2 child ions, and the same detection batch is used for judging whether glyphosate or glufosinate exists in the sample or not according to the comparison of the relative abundance ratio of the 2 child ions of the target compound in the sample with that of the standard solution with the equivalent concentration of the same compound;
6.4: quality control
7: analysis result calculation
7.1: the pesticide residue in the sample is calculated according to the following formula (1):
wherein:
X i pesticide residue in the sample, unit mg/kg;
C i the concentration of the measured object in the sample injection liquid is in the unit of mug/L;
v—total volume of extraction solution, unit mL, v=20;
m-mass of sample, unit g;
1000—unit conversion coefficient.
Further:
sampling a certain amount according to the relevant standard, and executing a sample sampling part according to the specification of GB 2763; the obtained sample is chopped, fully mixed, sampled by a quartering method or directly put into a tissue masher to be mashed into homogenate. Homogenizing in polyethylene container; the cereal sample was sampled at 500g and crushed to pass through a standard 425 μm mesh screen and placed in a polyethylene bottle or bag; 500g of oil crop, tea, nut and spice samples respectively, pulverizing, mixing well, and placing into polyethylene bottles or bags; vegetable oils are stirred uniformly; the obtained sample was stored at a temperature of-18℃or lower.
The invention has the advantages that:
the method adopts the multiwall carbon nanotubes with specific specifications as the sample purifying material, thereby improving the purifying effect of the sample extracting solution.
The method adopts the multiwall carbon nano tube to purify the extracting solution, adopts the ultra-high performance liquid chromatography-tandem mass spectrometry to detect, and establishes a rapid analysis method for measuring the residual quantity of glyphosate and glufosinate in the vegetable food.
The method avoids the use of high-toxicity reagents such as dichloromethane and the like, and the toxicity of the sample preparation method to human bodies is reduced.
The method uses the C18 chromatographic column to carry out qualitative and quantitative analysis of the glyphosate and the glufosinate, improves the working efficiency, and avoids the need of hours for balancing the chromatographic column after using the HILIC chromatographic column.
The method overcomes the defect of the liquid chromatography-mass spectrometry combination method for measuring the residual quantity of the glufosinate in the plant source food of the national food safety standard GB 23200.108-2018, and the national standard can not detect the glyphosate and the glufosinate at the same time.
Detailed Description
1: application range
The method is suitable for measuring the residual quantity of glufosinate-ammonium in the plant-derived food, and is a liquid chromatography-mass spectrometry combined method.
2: principle of the method
The sample is extracted by water and methanol, then the sample is subjected to dispersion and purification treatment by a solid phase material, and the purified liquid reacts with 9-fluorenylmethyl chloroformate to generate derivatives of Glyphosate-FMOC (Glyphosate-FMOC) and Glufosinate-FMOC (Glufosinate-FMOC), and the derivatives are quantified by an external standard method.
3: reference standard
Liquid chromatography-mass spectrometry combined method for measuring residual quantity of glufosinate in GB 23200.108-2018 food safety national standard plant-derived food
Liquid chromatography-mass spectrometry/mass spectrometry method for detecting residual quantity of glyphosate in SN/T1923-2007 imported and exported foods
4: program
4.1: instrument and equipment
4.1.1: liquid chromatograph-mass spectrometer: is provided with an electrospray ion source (ESI).
4.1.2: analytical balance: the sensing amount is 0.0001g and the sensing amount is 0.01g.
4.1.3: a tissue masher.
4.1.4: multitube vortex mixer (MS 200).
4.1.5: centrifuge: the rotating speed is not lower than 10000r/min.
4.1.6: and (5) a constant-temperature water bath kettle.
4.2: reagent(s)
4.2.1: acetonitrile (CH) 3 CN, CAS number: 75-05-8): chromatographic purity.
4.2.2: methanol (CH) 3 OH, CAS number: 67-56-1): chromatographic purity.
4.2.3: ammonium acetate (CH) 3 COONH 4 CAS number: 631-61-8).
4.2.4: sodium borate (Na) 2 B 4 O 7 ·10H 2 O, CAS number: 1303-96-4).
4.2.5: chloroformic acid 9-fluorenylmethyl ester (C) 15 H 11 ClO 2 CAS number: 28920-43-6); the purity is 99.0 percent, and the product is preserved at 0-4 ℃.
4.3: solution preparation
4.3.1: borate buffer (50.0 g/L, ph=9): 5g of sodium borate (Na) 2 B 4 O 7 ·10H 2 O) (4.2.4 (i.e., sodium borate (Na) labeled 4.2.4 in the reagent of 4.2 above 2 B 4 O 7 ·10H 2 O, CAS number: 1303-96-4), the following reference numerals are used to select the corresponding reference numerals of the reagents already described), are dissolved in water and the volume is set to 100mL.
4.3.2: 9-fluorenylmethylchloroformate acetonitrile solution (10.0 g/L): 1g of 9-fluorenylmethyl chloroformate (4.2.5) was weighed, dissolved in acetonitrile (4.2.1) and fixed to 100mL.
4.3.3: aqueous ammonium acetate (5 mmol/L): 0.385g of ammonium acetate (4.2.3) was weighed and dissolved in a suitable amount of water, and the volume was fixed with water to 1000mL.
4.4: standard substance
4.4.1: standard glufosinate solution with concentration of 100mg/L and effective period of 12 months.
4.4.2: standard glyphosate solution with concentration of 100mg/L and effective period of 12 months.
4.5: standard solution preparation
4.5.1: standard intermediate solution
Standard intermediate solutions were prepared according to table 1 below, and the matrix-matched standard curve intermediate solutions were diluted with sample blank extract to volume. The standard curve intermediate solution was diluted with aqueous methanol (V/v=1:1) to volume.
Preservation conditions: -18 ℃ for storage, expiration date: for 1 month.
TABLE 1 preparation of standard intermediate solutions
4.5.2: standard working curve solution
The standard intermediate solution (4.5.1) was diluted with the sample blank extract to a series of matrix-matched standard solutions at concentrations of 10. Mu.g/L, 20. Mu.g/L, 50. Mu.g/L, 100. Mu.g/L, 200. Mu.g/L, and subjected to instrumental measurements. And drawing a curve by taking the mass concentration of glufosinate-ammonium or glyphosate as an abscissa and the peak area of the derivative thereof as an ordinate.
Validity period: 7 days.
TABLE 2 preparation of standard working Curve solutions
4.6: sample preparation
The vegetable, fruit and edible fungus samples are taken to a certain amount according to the relevant standard, and the sample sampling part is implemented according to the specification of GB 2763. For smaller samples of individuals, all treatments after sampling; for larger and substantially uniform samples of individuals, the sample can be divided or cut into small pieces on the symmetry axis or symmetry plane for post-treatment; for samples with different slender, flat or component contents in each part, small pieces can be cut at different positions or cut into small sections for post-treatment; the obtained sample is chopped, fully mixed, sampled by a quartering method or directly put into a tissue masher to be mashed into homogenate. The homogenate is placed in a polyethylene container. 500g of cereal samples were taken and crushed and passed through a standard 425 μm mesh screen and placed in polyethylene bottles or bags. 500g of oil crop, tea, nut and spice samples are taken, crushed and fully and uniformly mixed, and put into a polyethylene bottle or bag. Vegetable oils are stirred uniformly. The sample was stored at a temperature below-18 ℃.
4.7: extraction of
4.7.1: vegetables, fruits and edible fungi
10g (to the nearest 0.01 g) of the sample was weighed into a 50mL centrifuge tube, see appendix A for additional water, then 10mL of methanol was added, after vortexing for 2min, centrifuged at 3800r/min for 5min, and the sample was purified.
4.7.2: spice and tea
2g (to the nearest 0.01 g) of the sample was weighed into a 50mL centrifuge tube, see appendix A for additional water, then 10mL of methanol was added, after vortexing for 2min, centrifuged at 3800r/min for 5min, and the sample was purified.
4.7.3: cereal, nut, oil crop and vegetable oils
5g (to the nearest 0.01 g) of the sample was weighed into a 50mL centrifuge tube, see appendix A for additional water, then 10mL of methanol was added, after vortexing for 2min, centrifuged at 3800r/min for 5min, and the sample was purified.
Appendix A
Table 1 list of selected matrix moisture content, sample amount, and Water make-up amount before extraction
4.8: purification of
4.8.1: vegetables, fruits, nuts, edible fungi, spices, tea leaves and grains
Accurately sucking 1mL of the extract into a 2mL centrifuge tube weighing 5mg of multi-wall carbon nanotubes, swirling for 1min, centrifuging for 3min at 10000r/min, taking supernatant, and centrifuging in another 2mL centrifuge with 0.22 μm organic filter membrane to obtain the derivatization product.
4.8.2: oil crops
Accurately sucking 4mL of the extracting solution into a 5mL centrifuge tube by using a pipette, placing the extracting solution into a refrigerator at the temperature of minus 18 ℃ for freezing overnight, then taking 1mL of supernatant into a 2mL centrifuge tube weighing 5mg of multi-walled carbon nanotubes and 50mg of neutral alumina, centrifuging for 3min at 10000r/min after swirling for 1min, taking the supernatant, and putting the supernatant into another 2mL centrifuge tube through a 0.22 mu m organic system filter membrane for derivatization.
4.8.3: vegetable oils
Accurately sucking 1mL of the extracting solution, directly passing through a 0.22 mu m organic system filter membrane in another 2mL centrifuge tube, and carrying out derivatization.
4.9: derivatization
Accurately sucking 0.5mL of the purified extracting solution into a 2mL centrifuge tube, adding 0.5mL of borate buffer solution, uniformly mixing, adding 0.5mL of chloroformic acid-9-fluorenylmethylene acetonitrile solution, swirling for 1min, derivatizing for 1h in a water bath at 40 ℃, centrifuging for 1min at 10000r/min after derivatization, taking supernatant, and passing through a 0.22 mu m organic filter membrane for on-machine detection. Accurately sucking 0.5mL of standard intermediate solution, and carrying out derivatization according to the step.
Repeating the steps 4.7-4.9, and preparing a parallel sample in each batch.
4.10: quality control sample preparation
4.10.1: the method is blank: the sample is not weighed, 10mL of methanol and 10mL of ultrapure water are taken in a 50mL centrifuge tube, and the steps 4.7 to 4.9 are repeated.
4.10.2: quality control solution: samples were not weighed. Taking 21 mu L of glufosinate-ammonium standard solution with the concentration of 100mg/L in a 50mL centrifuge tube, and repeating the steps 4.7-4.9. Obtaining the sample labeling solution with the labeling concentration of the object to be detected being 70 mug/L.
4.10.3: sample addition of the labeling solution: the sample was weighed. Taking 21 mu L of glufosinate-ammonium standard solution with the concentration of 100mg/L in a 50mL centrifuge tube, and repeating the steps 4.7-4.9. Obtaining the sample labeling solution with the labeling concentration of the object to be detected being 70 mug/L.
5, instrument analysis:
5.1: conditions of liquid chromatography
5.1.1: chromatographic column: poroshell 120EC-C18, 150 mm. Times.3.0 mm, particle size 2.7 μm;
5.1.2: mobile phase: a is 0.1% formic acid 5mmol/L ammonium acetate aqueous solution, B is 0.1% formic acid 5mmol/L ammonium acetate methanol solution;
5.1.3: flow rate: 0.4mL/min;
5.1.4: column temperature: 30 ℃;
5.1.5: sample injection volume: 1 mul.
TABLE 3 Mobile phase and gradient elution procedure
5.2: mass spectrum reference conditions
5.2.1: ion source: an electrospray ion source;
5.2.2: ionization source polarity: a positive mode;
5.2.3：Gas Temp：300℃；
5.2.4：Gas Flow：9L/min；
5.2.5：Nebulizer：40psi；
5.2.6：Sheath Gas Temp：350℃；
5.2.7：Sheath Gas Flow：11L/min；
5.2.8：Capillary：4000V；
5.2.9：Nozzle Voltage：500V；
5.2.10: the detection mode is as follows: multiple Reaction Monitoring (MRM), multiple reaction monitoring conditions are shown in table 4.
TABLE 4 retention time and Multiple Reaction Monitoring (MRM) conditions for two pesticide derivatives
5.3.0: the run was run with a solvent blank containing only methanol and checked for a plateau in the instrument baseline.
5.4.0: standard working curve solution (4.5.2) was run. A calibration curve was established using peak area and concentration, and the linear correlation coefficient was not less than 0.995.
5.5.0: the test method is blank (4.10.1) to check for contamination.
5.6.0: the quality control solution (4.10.2) was tested to check recovery.
5.7.0: test sample solutions and parallel (4.9).
5.8.0: the test sample is labeled with a solution (4.10.3) for qualitative and check recovery.
5.9.0: the mass concentration of the object to be detected in the sample is in the mass concentration range of the standard working curve, and the sample exceeding the upper limit of the mass concentration of the standard working curve is diluted and injected and quantified by an external standard method.
5.3: qualitative and quantitative determination
5.3.1: retention time
The retention time of the chromatographic peak of the object to be detected in the detected sample is compared with the retention time of the corresponding standard chromatographic peak, and the relative error is within +/-2.5 percent.
5.3.2: quantitative ion, qualitative ion, and ion abundance ratio
When the sample is tested under the same experimental conditions, if the retention time of chromatographic peaks of the to-be-tested substances in the sample is consistent with that of a standard sample, and after background subtraction, qualitative ions of the target compound in a mass spectrum of the sample must appear, at least 1 parent ion and 2 child ions are included, and the relative abundance ratio of the 2 child ions of the target compound in the sample is not more than the range specified in table 5 for the same compound compared with a standard solution with equivalent concentration in the same detection batch, the presence of glyphosate or glufosinate in the sample can be judged.
Table 5 maximum allowable deviation (in percent) of relative ion abundance for qualitative determination
5.4: quality control
Note that: a and b represent the results of the two replicates.
6: analysis result calculation
6.1: the pesticide residue in the sample is calculated according to the following formula (1):
wherein:
X i pesticide residue in the sample, unit mg/kg;
C i the concentration of the measured object in the sample injection liquid is in the unit of mug/L;
v—total volume of extraction solution, unit mL, v=20.
m-mass of sample, unit g;
1000—unit conversion coefficient.
The 2-bit significant number is reserved. When the result is greater than 1mg/kg, the 3 significant digits remain.
7: reporting detection limits
The quantitative limit of the spice and the tea is 0.1mg/kg, and the quantitative limit of other samples is 0.05mg/kg.
The following are chemical structural formulas relating to the materials:
1. chemical structure of glyphosate
2. Chemical structural formula of glufosinate
3. 9-fluorenylmethyl chloroformate (9-fluorenylmethyl chloroformate, FMOC) chemical structural formula
4. Glyphosate-FMOC (Glyphosate-FMOC) chemical formula
5. Glufosinate-FMOC (Glufosinate-FMOC)
6.: cleavage mechanism of glyphosate and glufosinate pesticide residue derivatives in mass spectrometry
6.1: cleavage mechanism of glyphosate pesticide residue derivative in mass spectrum
By systematic analysis and research on the cleavage mechanism of the glyphosate derivative in tandem mass spectrometry, the cleavage mechanism of the glyphosate derivative can be explored to be: first the glyphosate derivative is cleaved into ions m/z 214, and one of the CO is lost during the passage of the ions m/z 214 through the collision cell 2 The molecules become ions m/z 170, and the ions m/z 170 collide effectively with the inert gas nitrogen in the collision chamber, and are again dissociated into ions m/z 88.
6.2: cleavage mechanism of glufosinate residue derivatives in mass spectrometry
By systematic analysis and research on the cleavage mechanism of the glufosinate-ammonium derivative in tandem mass spectrometry, the cleavage mechanism of the glufosinate-ammonium derivative can be explored as follows: first the glufosinate derivative is cleaved into ions m/z 208 and m/z 226, and one CO is lost during the flow of the ions m/z 226 through the collision cell 2 The molecules become ions m/z 182, and the ions m/z 182 are effectively collided with the inert gas nitrogen in the collision chamber and are again split into ions m/z136.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other. The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (2)
1. The method for detecting the glyphosate and the glufosinate in the plant food is characterized by comprising the following steps of:
1: sample preparation
Sampling a sample to obtain a sample;
2: extraction of
Weighing 10g of sample in a 50mL centrifuge tube, supplementing water, adding 10mL of methanol, carrying out vortex oscillation for 2min, and centrifuging at 3800r/min for 5min to be purified;
3: purification of
3.1: vegetables, fruits, nuts, mushrooms, spices, tea leaves, cereal samples:
accurately sucking 1mL of the extracting solution into a 2mL centrifuge tube weighing 5mg of multi-wall carbon nanotubes, swirling for 1min, centrifuging for 3min at 10000r/min, taking supernatant, and centrifuging in another 2mL centrifuge with 0.22 μm organic filter membrane to obtain derivatization;
3.2: oil crop samples:
accurately sucking 4mL of the extracting solution into a 5mL centrifuge tube by using a pipette, placing the extracting solution into a refrigerator at the temperature of minus 18 ℃ for freezing overnight, then taking 1mL of supernatant into a 2mL centrifuge tube weighing 5mg of multiwall carbon nanotubes and 50mg of neutral alumina, centrifuging for 3min at 10000r/min after swirling for 1min, taking the supernatant, and putting the supernatant into another 2mL centrifuge tube through a 0.22 mu m organic filter membrane for derivatization;
3.3: vegetable oil samples:
accurately sucking 1mL of extracting solution, directly passing through a 0.22 mu m organic system filter membrane in another 2mL centrifuge tube, and derivatizing;
4: derivatization
Accurately sucking 0.5mL of the purified extracting solution into a 2mL centrifuge tube, adding 0.5mL of borate buffer solution, uniformly mixing, adding 0.5mL of chloroformate-9-fluorenylmethylene acetonitrile solution, swirling for 1min, derivatizing for 1h in a water bath at 40 ℃, centrifuging for 1min at 10000r/min after derivatization, taking supernatant, and passing through a 0.22 mu m organic filter membrane for on-machine detection; accurately sucking 0.5mL of standard intermediate solution, and simultaneously carrying out derivatization according to the step;
repeating the steps 2, 3 and 4, and preparing a parallel sample in each batch;
5: quality control sample preparation
5.1: the method is blank: weighing 10mL of methanol and 10mL of ultrapure water in a 50mL centrifuge tube without weighing the sample, and repeating the steps 2, 3 and 4;
5.2: quality control solution: weighing 21 mu L of glufosinate-ammonium and glyphosate standard solution with the concentration of 100mg/L in a 50mL centrifuge tube, and repeating the steps 2, 3 and 4 to obtain a sample standard adding solution with the standard adding concentration of 70 mu g/L of the object to be detected;
4.10.3: sample addition of the labeling solution: weighing a sample, taking 21 mu L of glufosinate-ammonium and glyphosate standard solution with the concentration of 100mg/L in a 50mL centrifuge tube, and repeating the steps 2, 3 and 4 to obtain a sample standard adding solution with the standard adding concentration of 70 mu g/L of the object to be detected;
6: instrument analysis:
6.1: conditions of liquid chromatography
6.1.1: chromatographic column: poroshell 120EC-C18, 150 mm. Times.3.0 mm, particle size 2.7 μm;
6.1.2: mobile phase: a is 0.1% formic acid 5mmol/L ammonium acetate aqueous solution, B is 0.1% formic acid 5mmol/L ammonium acetate methanol solution;
6.1.3: flow rate: 0.4mL/min;
6.1.4: column temperature: 30 ℃;
6.1.5: sample injection volume: 1 μl;
6.2: mass spectrum reference conditions
6.2.1: ion source: an electrospray ion source;
6.2.2: ionization source polarity: a positive mode;
6.2.3：Gas Temp：300℃；
6.2.4：Gas Flow：9L/min；
6.2.5：Nebulizer：40psi；
6.2.6：Sheath Gas Temp：350℃；
6.2.7：Sheath Gas Flow：11L/min；
6.2.8：Capillary：4000V；
6.2.9：Nozzle Voltage：500V；
6.2.10: the detection mode is as follows: multiple Reaction Monitoring (MRM);
6.3.0: running a solvent blank containing only methanol, and checking whether an instrument baseline is stable;
6.4.0: running a standard working curve solution; establishing a calibration curve by using the peak area and the concentration, wherein the linear correlation coefficient is not less than 0.995;
6.5.0: the test method is blank to check whether pollution exists;
6.6.0: testing the quality control solution to check recovery;
6.7.0: testing the sample solution and parallel samples;
6.8.0: the test sample is added with a standard solution for qualitative and recovery rate checking;
6.9.0: the mass concentration of the object to be detected in the sample is in the mass concentration range of the standard working curve, and the sample exceeding the upper limit of the mass concentration of the standard working curve is diluted and injected and quantified by an external standard method;
6.3: qualitative and quantitative determination
6.3.1: retention time
Comparing the retention time of the chromatographic peak of the object to be detected in the detected sample with the retention time of the corresponding standard chromatographic peak, wherein the relative error is within +/-2.5%;
6.3.2: quantitative ion, qualitative ion, and ion abundance ratio
When the sample is tested under the same experimental condition, if the retention time of chromatographic peaks of the to-be-tested substances in the sample is consistent with that of a standard sample, and after background subtraction, qualitative ions of the target compound in a mass spectrum of the sample must appear, wherein the qualitative ions at least comprise 1 parent ion and 2 child ions, and the same detection batch is used for judging whether glyphosate or glufosinate exists in the sample or not according to the comparison of the relative abundance ratio of the 2 child ions of the target compound in the sample with that of the standard solution with the equivalent concentration of the same compound;
6.4: quality control
7: analysis result calculation
7.1: the pesticide residue in the sample is calculated according to the following formula (1):
wherein:
X i pesticide residue in the sample, unit mg/kg;
C i the concentration of the measured object in the sample injection liquid is in the unit of mug/L;
v—total volume of extraction solution, unit mL, v=20;
m-mass of sample, unit g;
1000—unit conversion coefficient.
2. The method for detecting glyphosate and glufosinate in a vegetable food product according to claim 1 comprising the steps of:
sampling a certain amount according to the relevant standard, and executing a sample sampling part according to the specification of GB 2763; chopping the sampled sample, fully and uniformly mixing, sampling by a quartering method or directly putting the sample into a tissue masher to be mashed into homogenate; homogenizing in polyethylene container; the cereal sample was sampled at 500g and crushed to pass through a standard 425 μm mesh screen and placed in a polyethylene bottle or bag; 500g of oil crop, tea, nut and spice samples respectively, pulverizing, mixing well, and placing into polyethylene bottles or bags; vegetable oils are stirred uniformly; the obtained sample was stored at a temperature of-18℃or lower.
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| CN103529148A (en) * | 2013-10-21 | 2014-01-22 | 福建出入境检验检疫局检验检疫技术中心 | Method for detecting residual quantity of glyphosate and aminomethyl phosphonic acid metabolite of glyphosate in food |
| CN104730186A (en) * | 2013-12-19 | 2015-06-24 | 勐海茶业有限责任公司 | Precolumn derivatization-UPLC(ultra performance liquid chromatography)-ESI(electronic spray ion)+-MS/MS (mass spectrometry) detection method of glyphosate and glufosinate-ammonium pesticide residue in tea |
| CN107576732A (en) * | 2016-07-04 | 2018-01-12 | 烟台杰科检测服务有限公司 | A kind of assay method of food glyphosate, AminomethylphosphoniAcid Acid and glufosinate-ammonium |
| CN108445133A (en) * | 2018-04-02 | 2018-08-24 | 杨梅 | A kind of method of glufosinate-ammonium and glyphosate drug residue in detection tealeaves |
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| CN103529148A (en) * | 2013-10-21 | 2014-01-22 | 福建出入境检验检疫局检验检疫技术中心 | Method for detecting residual quantity of glyphosate and aminomethyl phosphonic acid metabolite of glyphosate in food |
| CN104730186A (en) * | 2013-12-19 | 2015-06-24 | 勐海茶业有限责任公司 | Precolumn derivatization-UPLC(ultra performance liquid chromatography)-ESI(electronic spray ion)+-MS/MS (mass spectrometry) detection method of glyphosate and glufosinate-ammonium pesticide residue in tea |
| CN107576732A (en) * | 2016-07-04 | 2018-01-12 | 烟台杰科检测服务有限公司 | A kind of assay method of food glyphosate, AminomethylphosphoniAcid Acid and glufosinate-ammonium |
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