CN111896660A - Detection method of glyphosate and glufosinate in plant food - Google Patents
Detection method of glyphosate and glufosinate in plant food Download PDFInfo
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Abstract
The invention provides a method for detecting glyphosate and glufosinate-ammonium in plant food, which is characterized by comprising the following steps: 1: preparing a sample; 2: extracting; 3: purifying; 4: derivatization; 5: preparing a quality control sample; 6: analyzing by an instrument: 7: calculating the analysis result, and calculating the pesticide residue in the sample according to the following formula (1):
Description
Technical Field
The invention relates to a method for detecting glyphosate and glufosinate-ammonium in plant food.
Background
The existing methods for detecting glyphosate and glufosinate in plant food comprise a gas chromatography and gas chromatography combined method, a liquid chromatography and liquid chromatography-mass spectrometry combined method. Gas phase and liquid phase chromatography have the advantages of stable method and accurate quantification, but because glyphosate and glufosinate-ammonium are strong polar compounds, are insoluble in most organic solvents, have no ultraviolet absorption, are not easy to volatilize, and are not retained on a conventional C18 chromatographic column, the gas phase chromatography and the high performance liquid chromatography are difficult to be used for separation and determination; on the other hand, due to the lack of chromophore, it is difficult to directly detect with uv and fluorescence detectors, and therefore, pre-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 values. 9-fluorenylmethyl chloroformate is a desirable derivatizing agent because it reacts rapidly with glyphosate and glufosinate, and the resulting derivative is relatively stable. At present, the traditional pretreatment method for detecting glyphosate and glufosinate-ammonium is to extract a sample with water and then purify the sample by using a solid phase extraction column, the process is complex, and the detection of a large quantity of samples in a laboratory is difficult.
Disclosure of Invention
The invention provides a method for detecting glyphosate and glufosinate-ammonium in plant food, which aims to overcome the defects of the prior art, improve the purification effect of a sample extracting solution, establish a rapid analysis method for determining the residual quantity of the glyphosate and the glufosinate-ammonium in the plant food, improve the working efficiency and reduce the toxicity of a preparation method to a human body.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a method for detecting glyphosate and glufosinate in plant food is characterized by comprising the following steps:
1: sample preparation
Sampling a sample to obtain a test 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
3.1: vegetable, fruit, nut, edible fungus, spice, tea and grain samples:
accurately sucking 1mL of extracting solution into a 2mL centrifuge tube weighed with 5mg of multi-walled carbon nano-tubes, whirling for 1min, centrifuging for 3min at 10000r/min, taking supernate, passing through a 0.22 mu m organic filter membrane, centrifuging in another 2mL centrifuge, and waiting for derivatization;
3.2: oil crop type samples:
accurately sucking 4mL of extracting solution into a 5mL centrifuge tube by using a pipette, placing the centrifuge tube in a refrigerator at the temperature of-18 ℃ for freezing overnight, taking 1mL of supernatant into a 2mL centrifuge tube weighed with 5mg of multi-walled carbon nanotubes and 50mg of neutral alumina, carrying out vortex for 1min, centrifuging for 3min at the speed of 10000r/min, taking the supernatant, passing the supernatant through a 0.22 mu m organic system filter membrane into another 2mL centrifuge tube, and waiting 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, and placing in another 2mL centrifugal tube for derivatization;
4: derivatization
Accurately sucking 0.5mL of purified extracting solution into a 2mL centrifuge tube, adding 0.5mL of borate buffer solution, uniformly mixing, adding 0.5mL of chloroformic acid-9-fluorenylmethyl acetonitrile solution, vortex for 1min, deriving for 1h in a water bath at 40 ℃, centrifuging for 1min at 10000r/min after deriving, taking supernate, passing through a 0.22 mu m organic filter membrane, and waiting for detection on a computer; accurately sucking 0.5mL of standard intermediate solution, and performing derivatization simultaneously according to the step;
repeating the step 2, the step 3 and the step 4, and preparing a sample of a parallel sample in each batch;
5: quality control sample preparation
5.1: the method is blank: weighing a sample, putting 10mL of methanol and 10mL of ultrapure water into a 50mL centrifuge tube, and repeating the step 2, the step 3 and the step 4;
5.2: quality control solution: weighing a sample, putting 21 mu L of glufosinate ammonium and glyphosate standard solution with the concentration of 100mg/L into a 50mL centrifuge tube, and repeating the steps 2, 3 and 4 to obtain a sample standard solution with the standard concentration of 70 mu g/L of the object to be measured;
4.10.3: adding a standard solution to a sample: weighing a sample, putting 21 mu L of glufosinate ammonium and glyphosate standard solution with the concentration of 100mg/L into a 50mL centrifuge tube, and repeating the step 2, the step 3 and the step 4 to obtain a sample standard solution with the standard concentration of 70 mu g/L of the object to be detected;
6: analyzing by an instrument:
6.1: conditions of liquid chromatography
6.1.1: a chromatographic column: poroshell 120EC-C18, 150mm × 3.0mm, particle size 2.7 μm;
6.1.2: mobile phase: a is 0.1% formic acid 5mmol/L ammonium acetate water solution, B is 0.1% formic acid 5mmol/L ammonium acetate methanol solution;
6.1.3: flow rate: 0.4 mL/min;
6.1.4: column temperature: 30 ℃;
6.1.5: sample introduction volume: 1 mu L of the solution;
6.2: reference conditions for mass spectrometry
6.2.1: an 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 only containing methanol, and checking whether the baseline of the instrument is stable;
6.4.0: operating 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: test method blank to check if there is contamination;
6.6.0: testing the quality control solution to check recovery;
6.7.0: testing the sample solution and the parallel sample;
6.8.0: adding a standard solution to the test sample for qualitative and checking recovery rate;
6.9.0: the mass concentration of the substance to be measured in the sample is within the mass concentration range of the standard working curve, the sample exceeding the mass concentration upper limit of the standard working curve is diluted and then is injected, and an external standard method is adopted for quantification;
6.3: qualitative and quantitative
6.3.1: retention time
Comparing the retention time of chromatographic peak of the substance to be detected in the detected sample with that of corresponding standard chromatographic peak, and the relative error is within +/-2.5%;
6.3.2: quantitative ion, qualitative ion and daughter ion abundance ratio
When the sample is determined under the same experimental conditions, if the retention time of the chromatographic peak of the substance to be determined in the sample is consistent with that of the standard sample, and after the background is subtracted, the qualitative ions of the target compound in the mass spectrogram of the sample must appear, and at least comprise 1 parent ion and 2 daughter ions, and for the same compound, the relative abundance ratio of the 2 daughter ions of the target compound in the sample is compared with the standard solution with the same concentration in the same detection batch, and whether glyphosate or glufosinate exists in the sample is judged;
6.4: quality control
7: calculation of analysis results
7.1: the amount of pesticide residue in the sample was calculated according to the following formula (1):
in the formula:
Xithe pesticide residue in the sample is mg/kg;
Ci-the concentration of the test substance in the sample solution in μ g/L;
v is the total volume of the extraction solution, unit mL, and V is 20;
m is the mass of the sample in g;
1000-unit conversion factor.
Further:
sampling a certain amount according to related standards, and executing a sampling part of the sample according to the regulation of GB 2763; cutting the obtained sample, mixing, and sampling by quartering method or directly placing into tissue mashing machine to pulverize into homogenate. Placing the homogenate in a polyethylene container; sampling the grain sample at 500g, pulverizing, passing through 425 μm standard mesh screen, and placing into polyethylene bottle or bag; respectively crushing 500g of oil crops, tea leaves, nuts and spice samples, fully and uniformly mixing, and putting into a polyethylene bottle or bag; stirring vegetable oil uniformly; the obtained sample was stored at a temperature of-18 ℃ or lower.
The invention has the advantages that:
the method adopts the multi-walled carbon nano-tube with specific specification as the sample purifying material, and improves the purifying effect of the sample extracting solution.
The method adopts multi-walled carbon nanotubes to purify the extracting solution, adopts ultra-high performance liquid chromatography tandem mass spectrometry to detect, and establishes a rapid analysis method for determining the residual quantity of glyphosate and glufosinate-ammonium in the plant food.
The method avoids using high-toxicity reagents such as dichloromethane and the like, and the preparation method of the sample has low toxicity to human bodies.
The method uses the C18 chromatographic column to perform qualitative and quantitative analysis on glyphosate and glufosinate-ammonium, improves the working efficiency, and avoids the need of hours for chromatographic column balance after using the HILIC chromatographic column.
The method makes up the defects of the liquid chromatography-mass spectrometry combined usage method for measuring the residual amount of glufosinate-ammonium in plant-derived food of national standard GB 23200.108-2018 for food safety, and the national standard cannot simultaneously detect glyphosate and glufosinate-ammonium.
Detailed Description
1: application scope
The method is suitable for measuring the residual quantities of glufosinate-ammonium and glyphosate in plant-derived food, and adopts a liquid chromatography-mass spectrometry combined method.
2: principle of method
A sample is extracted by water and methanol, then is dispersed and purified by a solid phase material, and the purified liquid reacts with 9-fluorenylmethyl chloroformate (9-fluoromethylenemethyl chloride) to generate derivatives Glyphosate-FMOC (Glyphosate-FMOC) and Glufosinate-FMOC (Glufosinate-FMOC) which are detected by liquid quality and quantified by an external standard method.
3: reference standard
Liquid chromatography-mass spectrometry combined use method for determining residual amount of glufosinate-ammonium in plant-derived food of national standard for GB 23200.108-2018 food safety
Detection method of glyphosate residue in SN/T1923-2007 import and export foods by liquid chromatography-mass spectrometry/mass spectrometry
4: procedure for measuring the movement of a moving object
4.1: instrumentation and equipment
4.1.1: liquid chromatography-mass spectrometer: an electrospray ion source (ESI) was provided.
4.1.2: analytical balance: 0.0001g for sensory, 0.01g for sensory.
4.1.3: tissue triturator.
4.1.4: a multi-tubular vortex mixer (MS 200).
4.1.5: a centrifuge: the rotating speed is not less than 10000 r/min.
4.1.6: a constant temperature water bath kettle.
4.2: reagent
4.2.1: acetonitrile (CH)3CN, CAS number: 75-05-8): and (4) carrying out chromatographic purification.
4.2.2: methanol (CH)3OH, CAS number: 67-56-1): and (4) carrying out chromatographic purification.
4.2.3: ammonium acetate (CH)3COONH4CAS number: 631-61-8).
4.2.4: sodium borate (Na)2B4O7·10H2O, CAS number: 1303-96-4).
4.2.5: chloroformate-9-fluorenylmethyl ester (C)15H11ClO2CAS number: 28920-43-6); the purity is 99.0%, and the product is preserved at 0-4 ℃.
4.3: solution preparation
4.3.1: borate buffer solution (50.0g/L, pH 9): 5g of sodium borate (Na) are weighed out2B4O7·10H2O) (4.2.4 (i.e., sodium borate (Na) of reference numeral 4.2.4 in the reagent of 4.2 above)2B4O7·10H2O, CAS number: 1303-96-4), the following meaning of such reference numerals is to select the reagent of the corresponding reference numeral already described), dissolved with water and made to 100 mL.
4.3.2: chloroformic acid-9-fluorenylmethyl ester acetonitrile solution (10.0 g/L): 1g of 9-fluorenylmethyl chloroformate (4.2.5) was weighed out, dissolved in acetonitrile (4.2.1) and made up to 100 mL.
4.3.3: ammonium acetate aqueous solution (5 mmol/L): 0.385g of ammonium acetate (4.2.3) was weighed out and dissolved in an appropriate amount of water, and the volume was adjusted to 1000mL with water.
4.4: standard article
4.4.1: the glufosinate-ammonium standard solution has the concentration of 100mg/L and the validity period of 12 months.
4.4.2: the glyphosate standard solution has the concentration of 100mg/L and the validity period of 12 months.
4.5: standard solution preparation
4.5.1: standard intermediate solution
A standard intermediate solution was prepared according to Table 1 below, and the matrix-matched standard curve intermediate solution was diluted with a sample blank extract to a constant volume. The standard curve intermediate solution was diluted with aqueous methanol (V/V ═ 1: 1) to volume.
Storage conditions are as follows: storage at-18 ℃, pot life: 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 substrate-matched standard solutions having concentrations of 10. mu.g/L, 20. mu.g/L, 50. mu.g/L, 100. mu.g/L, and 200. mu.g/L, and the measurement was performed by an instrument. And drawing a curve by taking the mass concentration of the glufosinate-ammonium or the glyphosate as an abscissa and taking the peak area of the derivative of the glufosinate-ammonium or the glyphosate as an ordinate.
The validity period is as follows: and 7 days.
TABLE 2 preparation of Standard working Curve solutions
4.6: sample preparation
Vegetable, fruit and edible fungus samples are taken according to relevant standards, and sampling positions of the samples are executed according to the regulation of GB 2763. For smaller individual samples, all treatment was done after sampling; for larger basically uniform samples of individuals, the samples can be divided or cut into small blocks on a symmetry axis or a symmetry plane for post-treatment; for samples which are slender, flat or have different component contents in each part, small pieces can be cut at different positions or cut into small pieces for post-treatment; cutting the obtained sample, mixing, and sampling by quartering method or directly placing into tissue mashing machine to pulverize into homogenate. The homogenate was placed in a polyethylene container. A sample of cereal 500g was taken, crushed and passed through a standard 425 μm mesh screen and placed in a polyethylene bottle or bag. Taking 500g of oil crops, tea leaves, nuts and spice samples respectively, crushing, fully mixing uniformly, and putting into a polyethylene bottle or bag. The vegetable oil is stirred uniformly. The samples were stored at temperatures below-18 ℃.
4.7: extraction of
4.7.1: vegetables, fruits and edible fungi
Weigh 10g (exactly to 0.01g) of sample into a 50mL centrifuge tube, supplement water as in appendix A, then add 10mL of methanol, vortex for 2min, centrifuge at 3800r/min for 5min, and allow to purify.
4.7.2: spices and tea leaves
Weigh 2g (exactly to 0.01g) of sample into a 50mL centrifuge tube, supplement water as in appendix A, then add 10mL of methanol, vortex for 2min, centrifuge at 3800r/min for 5min, and allow to purify.
4.7.3: cereals, nuts, oil crops, vegetable oils
Weigh 5g (exactly to 0.01g) of sample into a 50mL centrifuge tube, supplement water as in appendix A, then add 10mL of methanol, vortex for 2min, centrifuge at 3800r/min for 5min, and allow to purify.
Appendix A
TABLE 1 water content, sample weighing and water supplement before extraction information table of selected substrate
4.8: purification
4.8.1: vegetables, fruits, nuts, edible fungi, spices, tea, and cereals
Accurately sucking 1mL of the extract into a 2mL centrifuge tube weighed with 5mg of multi-walled carbon nanotubes, vortexing for 1min, centrifuging for 3min at 10000r/min, taking the supernatant, passing through a 0.22 mu m organic filter membrane, centrifuging in another 2mL centrifuge, and waiting for derivatization.
4.8.2: oil crops
Accurately sucking 4mL of extracting solution into a 5mL centrifuge tube by using a pipette, placing the centrifuge tube in a refrigerator at the temperature of-18 ℃ for freezing overnight, taking 1mL of supernatant into a 2mL centrifuge tube weighed with 5mg of multi-walled carbon nanotubes and 50mg of neutral alumina, whirling for 1min, centrifuging for 3min at 10000r/min, taking the supernatant, passing the supernatant through a 0.22 mu m organic system filter membrane into another 2mL centrifuge tube, and waiting for derivatization.
4.8.3: vegetable oils
Accurately draw 1mL of the extract directly through a 0.22 μm organic filter into another 2mL centrifuge tube for derivatization.
4.9: derivatization
Accurately sucking 0.5mL of purified extract into a 2mL centrifuge tube, adding 0.5mL of borate buffer solution, uniformly mixing, adding 0.5mL of chloroformic acid-9-fluorenylmethyl methyl acetonitrile solution, vortexing for 1min, deriving for 1h in a water bath at 40 ℃, centrifuging for 1min at 10000r/min after derivation, taking supernatant, passing through a 0.22 mu m organic filter membrane, and performing detection on a machine. 0.5mL of the standard intermediate solution was aspirated accurately, and derivatization was performed simultaneously according to this procedure.
And (4) repeating the steps 4.7-4.9, and preparing one parallel sample in each batch.
4.10: quality control sample preparation
4.10.1: the method is blank: weighing a sample, taking 10mL of methanol and 10mL of ultrapure water in a 50mL centrifuge tube, and repeating the step 4.7-4.9.
4.10.2: quality control solution: the sample was not weighed. And (3) taking 21 mu L of glufosinate ammonium and glyphosate standard solution with the concentration of 100mg/L into a 50mL centrifuge tube, and repeating the steps of 4.7-4.9. Obtaining the sample standard adding solution with the standard adding concentration of the substance to be detected being 70 mu g/L.
4.10.3: adding a standard solution to a sample: the sample was weighed. And (3) taking 21 mu L of glufosinate ammonium and glyphosate standard solution with the concentration of 100mg/L into a 50mL centrifuge tube, and repeating the steps of 4.7-4.9. Obtaining the sample standard adding solution with the standard adding concentration of the substance to be detected being 70 mu g/L.
5, instrumental analysis:
5.1: conditions of liquid chromatography
5.1.1: a chromatographic column: poroshell 120EC-C18, 150mm × 3.0mm, particle size 2.7 μm;
5.1.2: mobile phase: a is 0.1% formic acid 5mmol/L ammonium acetate water solution, B is 0.1% formic acid 5mmol/L ammonium acetate methanol solution;
5.1.3: flow rate: 0.4 mL/min;
5.1.4: column temperature: 30 ℃;
5.1.5: sample introduction volume: 1 μ L.
TABLE 3 mobile phase and gradient elution procedure
5.2: reference conditions for mass spectrometry
5.2.1: an 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: a solvent blank containing only methanol was run to check if the instrument baseline was stable.
5.4.0: the standard working curve solution (4.5.2) was run. The peak area and the concentration are used to establish a calibration curve, and the linear correlation coefficient is not less than 0.995.
5.5.0: test method 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 replicates (4.9).
5.8.0: the test samples were spiked with a solution (4.10.3) for characterization and to check recovery.
5.9.0: the mass concentration of the substance to be measured in the sample is within the mass concentration range of the standard working curve, and the sample exceeding the mass concentration upper limit of the standard working curve is diluted and then injected, and is quantified by adopting an external standard method.
5.3: qualitative and quantitative
5.3.1: retention time
The retention time of the chromatographic peak of the substance to be detected in the detected sample is compared with that of the corresponding standard chromatographic peak, and the relative error is within +/-2.5%.
5.3.2: quantitative ion, qualitative ion and daughter ion abundance ratio
When the sample is tested under the same experimental conditions, if the retention time of the chromatographic peak of the object to be tested in the sample is consistent with that of the standard sample, and after the background is subtracted, the qualitative ions of the object compound in the mass spectrum of the sample must appear, at least 1 parent ion and 2 daughter ions should be included, and the relative abundance ratio of the 2 daughter ions of the object compound in the sample for the same compound and the standard solution with the same concentration in the same test batch does not exceed the range specified in table 5, the glyphosate or glufosinate can be judged to be present in the sample.
TABLE 5 maximum permissible deviation in relative ion abundance (in percent) for qualitative determination
5.4: quality control
Note: a and b represent the results of two parallel measurements.
6: calculation of analysis results
6.1: the amount of pesticide residue in the sample was calculated according to the following formula (1):
in the formula:
Xithe pesticide residue in the sample is mg/kg;
Ci-the concentration of the test substance in the sample solution in μ g/L;
v-total volume of extraction solution, unit mL, V ═ 20.
m is the mass of the sample in g;
1000-unit conversion factor.
2 significant digits are reserved. When the result is more than 1mg/kg, 3 significant digits are retained.
7: limit of report detection
The limit of the amount of the spices and the tea leaves is 0.1mg/kg, and the limit of the amount of other samples is 0.05 mg/kg.
The following are the chemical structural formulas of the involved substances:
1. chemical structural formula of glyphosate
2. Chemical structural formula of glufosinate-ammonium
3. 9-fluorenylmethyl chloroformate (FMOC) chemical structural formula
4. Chemical structural formula of Glyphosate-FMOC (Glyphosate-FMOC)
5. Glufosinate-FMOC (Glufosinate-FMOC)
6.: cracking mechanism of glyphosate and glufosinate pesticide residue derivatives in mass spectrum
6.1: cracking mechanism of glyphosate pesticide residue derivative in mass spectrum
By carrying out systematic analysis and research on the cracking mechanism of the glyphosate derivative in the tandem mass spectrum, the cracking mechanism of the glyphosate derivative can be explored as follows: the glyphosate derivative is first cleaved into ions m/z 214, which lose a CO during the flow through the collision cell2The molecule is changed into ion m/z 170, and the ion m/z 170 is effectively collided with the inert gas nitrogen in the collision chamber and is cracked into ion m/z 88 again.
6.2: decomposition mechanism of glufosinate-ammonium residue derivative in mass spectrum
By carrying out systematic analysis and research on the cracking mechanism of the glufosinate-ammonium derivative in the tandem mass spectrum, the cracking mechanism of the glufosinate-ammonium derivative can be explored as follows: the glufosinate derivative is first split into ions m/z 208 and m/z226, the ions m/z226 losing one CO during the flow through the collision cell2The molecules are changed into ions m/z 182, and the ions m/z 182 are effectively collided with the inert gas nitrogen in the collision chamber and are cracked into ions m/z136 again.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred 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. A method for detecting glyphosate and glufosinate in plant food is characterized by comprising the following steps:
1: sample preparation
Sampling a sample to obtain a test 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
3.1: vegetable, fruit, nut, edible fungus, spice, tea and grain samples:
accurately sucking 1mL of extracting solution into a 2mL centrifuge tube weighed with 5mg of multi-walled carbon nano-tubes, whirling for 1min, centrifuging for 3min at 10000r/min, taking supernate, passing through a 0.22 mu m organic filter membrane, centrifuging in another 2mL centrifuge, and waiting for derivatization;
3.2: oil crop type samples:
accurately sucking 4mL of extracting solution into a 5mL centrifuge tube by using a pipette, placing the centrifuge tube in a refrigerator at the temperature of-18 ℃ for freezing overnight, taking 1mL of supernatant into a 2mL centrifuge tube weighed with 5mg of multi-walled carbon nanotubes and 50mg of neutral alumina, carrying out vortex for 1min, centrifuging for 3min at the speed of 10000r/min, taking the supernatant, passing the supernatant through a 0.22 mu m organic system filter membrane into another 2mL centrifuge tube, and waiting 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, and placing in another 2mL centrifugal tube for derivatization;
4: derivatization
Accurately sucking 0.5mL of purified extracting solution into a 2mL centrifuge tube, adding 0.5mL of borate buffer solution, uniformly mixing, adding 0.5mL of chloroformic acid-9-fluorenylmethyl acetonitrile solution, vortex for 1min, deriving for 1h in a water bath at 40 ℃, centrifuging for 1min at 10000r/min after deriving, taking supernate, passing through a 0.22 mu m organic filter membrane, and waiting for detection on a computer; accurately sucking 0.5mL of standard intermediate solution, and performing derivatization simultaneously according to the step;
repeating the step 2, the step 3 and the step 4, and preparing a sample of a parallel sample in each batch;
5: quality control sample preparation
5.1: the method is blank: weighing a sample, putting 10mL of methanol and 10mL of ultrapure water into a 50mL centrifuge tube, and repeating the step 2, the step 3 and the step 4;
5.2: quality control solution: weighing a sample, putting 21 mu L of glufosinate ammonium and glyphosate standard solution with the concentration of 100mg/L into a 50mL centrifuge tube, and repeating the steps 2, 3 and 4 to obtain a sample standard solution with the standard concentration of 70 mu g/L of the object to be measured;
4.10.3: adding a standard solution to a sample: weighing a sample, putting 21 mu L of glufosinate ammonium and glyphosate standard solution with the concentration of 100mg/L into a 50mL centrifuge tube, and repeating the step 2, the step 3 and the step 4 to obtain a sample standard solution with the standard concentration of 70 mu g/L of the object to be detected;
6: analyzing by an instrument:
6.1: conditions of liquid chromatography
6.1.1: a chromatographic column: poroshell 120EC-C18, 150mm × 3.0mm, particle size 2.7 μm;
6.1.2: mobile phase: a is 0.1% formic acid 5mmol/L ammonium acetate water solution, B is 0.1% formic acid 5mmol/L ammonium acetate methanol solution;
6.1.3: flow rate: 0.4 mL/min;
6.1.4: column temperature: 30 ℃;
6.1.5: sample introduction volume: 1 mu L of the solution;
6.2: reference conditions for mass spectrometry
6.2.1: an 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 only containing methanol, and checking whether the baseline of the instrument is stable;
6.4.0: operating 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: test method blank to check if there is contamination;
6.6.0: testing the quality control solution to check recovery;
6.7.0: testing the sample solution and the parallel sample;
6.8.0: adding a standard solution to the test sample for qualitative and checking recovery rate;
6.9.0: the mass concentration of the substance to be measured in the sample is within the mass concentration range of the standard working curve, the sample exceeding the mass concentration upper limit of the standard working curve is diluted and then is injected, and an external standard method is adopted for quantification;
6.3: qualitative and quantitative
6.3.1: retention time
Comparing the retention time of chromatographic peak of the substance to be detected in the detected sample with that of corresponding standard chromatographic peak, and the relative error is within +/-2.5%;
6.3.2: quantitative ion, qualitative ion and daughter ion abundance ratio
When the sample is determined under the same experimental conditions, if the retention time of the chromatographic peak of the substance to be determined in the sample is consistent with that of the standard sample, and after the background is subtracted, the qualitative ions of the target compound in the mass spectrogram of the sample must appear, and at least comprise 1 parent ion and 2 daughter ions, and for the same compound, the relative abundance ratio of the 2 daughter ions of the target compound in the sample is compared with the standard solution with the same concentration in the same detection batch, and whether glyphosate or glufosinate exists in the sample is judged;
6.4: quality control
7: calculation of analysis results
7.1: the amount of pesticide residue in the sample was calculated according to the following formula (1):
in the formula:
Xithe pesticide residue in the sample is mg/kg;
Ci-the concentration of the test substance in the sample solution in μ g/L;
v is the total volume of the extraction solution, unit mL, and V is 20;
m is the mass of the sample in g;
1000-unit conversion factor.
2. The method of claim 1, comprising the steps of:
sampling a certain amount according to related standards, and executing a sampling part of the sample according to the regulation of GB 2763; cutting the obtained sample, mixing, and sampling by quartering method or directly placing into tissue mashing machine to pulverize into homogenate. Placing the homogenate in a polyethylene container; sampling the grain sample at 500g, pulverizing, passing through 425 μm standard mesh screen, and placing into polyethylene bottle or bag; respectively crushing 500g of oil crops, tea leaves, nuts and spice samples, fully and uniformly mixing, and putting into a polyethylene bottle or bag; stirring vegetable oil uniformly; the obtained sample was stored at a temperature of-18 ℃ or lower.
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