A method for detecting glyphosate and its metabolites in fruits

Abstract

The present invention provides a method for detecting glyphosate and its metabolites in fruits, which comprises S1, preparing an extract; S2, purifying the extract; S3, derivatizing the extract; S4, determining the glyphosate content by isotope dilution internal standard-high performance liquid chromatography tandem mass spectrometry; and adding glyphosate, aminomethylphosphonic acid and glyphosate GLY- to a blank extract of the above sample (without internal standard). 13 C2, 15 A mixed standard working solution is prepared using N as the internal standard; steps S3-S4 are performed simultaneously to generate a standard curve and calculate the content of glyphosate and its metabolites in the sample. The present invention utilizes an isotope internal standard method for quantification, effectively improving recovery, reducing matrix effects, and enhancing the accuracy of quantitative results. The derivatization reagent used in the present invention has strong specificity, requires minimal dosage, and features a simple derivatization step, short processing time, good reproducibility, and high sensitivity. It is suitable for the simultaneous determination of glyphosate and its metabolites in complex fruit matrix samples and possesses strong practicality.

Description

Technical Field

[0001] The invention belongs to the technical field of pesticide residue detection, and particularly relates to a method for detecting glyphosate and its metabolites in fruits. Background Art

[0002] Glyphosate (GLY) is a lethal herbicide widely used worldwide for orchard weed control. Recent studies, both domestically and internationally, have found glyphosate and its metabolite, aminomethylphosphonic acid (AMPA), to be detected in fruits and vegetables such as grapes and mushrooms, as well as their products. Therefore, there is a need to develop rapid methods for the detection of glyphosate and its metabolites in complex matrices.

[0003] Due to the strong water solubility of glyphosate and its metabolites and their lack of colorimetric and fluorescent groups, their detection has always been a challenge. Currently reported pretreatment methods for glyphosate and its metabolites often utilize derivatization with 9-fluorenylmethylchloroformate (FMOC-Cl), followed by detection of the derivatives using techniques such as gas chromatography, liquid chromatography, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry. However, these methods suffer from complex procedures, long derivatization times (requiring overnight), poor reproducibility, and low sensitivity. Therefore, there is a need for a derivatization reagent with enhanced specificity, a simple derivatization step, rapidity, and excellent stability to accurately determine the content of glyphosate and its metabolites. Summary of the Invention

[0004] In order to solve the problems existing in the above-mentioned existing detection technologies, the present invention provides a method for detecting glyphosate and its metabolites in fruits. The method has the advantages of being simple, rapid, specific, highly sensitive, and highly applicable, and can meet the needs of detecting trace amounts of glyphosate and its metabolites.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for detecting glyphosate and its metabolites in fruit comprises the following steps:

[0007] S1. extracting glyphosate and its metabolites from the fruit sample to be tested with water to prepare an extract;

[0008] S2. Purification of the extract;

[0009] S3, adding a derivatization reagent to the purified extract for derivatization; the derivatization reagent is 6-aminoquinoline-N-hydroxysuccinimide carbamate;

[0010] S4, glyphosate content determination, quantification by isotope dilution internal standard-high performance liquid chromatography tandem mass spectrometry;

[0011] At the same time, the blank extract of the fruit sample without internal standard was added with glyphosate, aminomethylphosphonic acid and glyphosate GLY-13 C2, 15 N internal standard is used to prepare a mixed standard working solution; steps S3-S4 are performed simultaneously to perform detection, with the ratio of the peak area of ​​glyphosate derivatives and aminomethylphosphonic acid derivatives to the peak area of ​​the internal standard derivative as the vertical coordinate, and the ratio of the glyphosate and aminomethylphosphonic acid concentrations to the internal standard concentration as the horizontal coordinate, to prepare a standard curve, and obtain a linear regression equation. The ratio of the measured glyphosate and aminomethylphosphonic acid peak areas to the internal standard peak area in the test sample is substituted into the linear regression equation to obtain the concentration of glyphosate and its metabolites in the extract, and then the content of glyphosate and its metabolites in the corresponding sample is calculated based on the mass of the sample represented by the extract.

[0012] In the above detection method, preferably, in step S1, the extract is prepared by freeze-drying the fruit with a freeze dryer and then grinding it into a fine powder; weighing the fine powder and adding glyphosate GLY- 13 C2, 15 N was used as the internal standard, and water was added for ultrasonic extraction. After centrifugation, the upper layer of the extract was collected.

[0013] In the detection method described above, preferably, the concentration of the internal standard is 50 μg / L, the amount of water added is 1:20 g:mL, ultrasonication is performed for 15 to 30 minutes, and centrifugation is performed at 8000 r / min for 3 minutes.

[0014] In the above-mentioned detection method, preferably, in step S2, the extract is added with dichloromethane, mixed, and centrifuged, and supernatant I is taken, cross-linked polyvinyl pyrrolidone (PVPP) is added, mixed, and centrifuged to obtain supernatant II;

[0015] The MCX column was activated with methanol and water respectively, and the supernatant II was drawn through the column, the filtrate was discarded, and the supernatant II was loaded again. The filtrate was collected and centrifuged to obtain supernatant III for derivatization.

[0016] In the detection method described above, preferably, the ratio of the extract to dichloromethane is 3:1, the amount of supernatant I to PVPP added is 1 mL:5 mg, and the centrifugation is performed at 8000 r / min for 5 min.

[0017] In the detection method described above, preferably, in step S3, the supernatant III is added to a sodium borate buffer solution with a pH of 8.8, mixed, and then 6-aminoquinoline-N-hydroxysuccinimide carbamate is added, mixed, and then sample injection detection is performed.

[0018] In the detection method described above, preferably, 6-aminoquinoline-N-hydroxysuccinimide carbamate is added to acetonitrile to prepare a solution with a concentration of 2.85 mg / mL, and the supernatant III, sodium borate buffer and 6-aminoquinoline-N-hydroxysuccinimide carbamate solution are added in a ratio of 1:7:2.

[0019] In the detection method described above, preferably, in step S4, the chromatographic conditions of liquid chromatography tandem mass spectrometry are as follows: chromatographic column: ACQUITY UPLC HSS T3, specification: 100×2.1 mm, particle size: 1.8 μm;

[0020] Mobile phase A: 5 mmol / L ammonium formate, 0.3% formic acid in water;

[0021] Mobile phase B: pure acetonitrile;

[0022] Column temperature: 50°C; flow rate: 0.45 mL / min;

[0023] Gradient elution ratio:

[0024] 0-0.5min, 2.0% B;

[0025] 0.5-5.0min, 2.0%-8.0% B;

[0026] 5.0-6.0min, 8.0%-20.0% B;

[0027] 6.0-7.0min, 20.0%-60.0% B;

[0028] 7.0-8.0 min, 60.0% B;

[0029] 8.0-10.0 min, 2.0% B;

[0030] The mass spectrometry conditions of the liquid chromatography tandem mass spectrometry are:

[0031] Ion source: electrospray ionization (ESI); Scan mode: positive ion; Monitoring mode: multiple reaction monitoring (MRM); Capillary voltage: 2.7 kV (positive ion mode, ESI+); Nebulizer gas temperature: 500 °C; Desolvation gas flow rate: 1000 L·h -1 ; Ion source temperature: 150℃;

[0032] The mass spectrometry parameters for glyphosate derivatives were as follows: retention time 2.00 min, parent ion 339.83 m / z, product ions 115.95 m / z and 170.90* m / z, cone voltage 15 V, collision energies 55 and 15 eV;

[0033] Glyphosate- 13C2, 15 The mass spectrometry parameters of the N derivative were as follows: retention time 2.00 min, parent ion 342.83 m / z, product ions 115.95 m / z and 170.90* m / z, cone voltage 15 V, collision energies 55 and 15 eV;

[0034] The mass spectrometry analysis parameters of aminomethylphosphonic acid derivatives were as follows: retention time 2.56 min, parent ion 281.86 m / z, product ions 116.00 m / z and 170.90* m / z, cone voltage 15 V, collision energies 55 and 25 eV.

[0035] In the above detection method, preferably, the concentrations of glyphosate and aminomethylphosphonic acid in the mixed standard working solution are 5, 10, 20, 50, and 100 μg / L, respectively. 13 C2, 15 The internal standard concentration of N was 50 μg / L.

[0036] In the detection method described above, preferably, the calculation formula is as follows:

[0037]

[0038] X i —The content of glyphosate and aminomethylphosphonic acid in the sample, in milligrams per kilogram (mg·kg) -1 ;

[0039] C i —The mass concentrations of glyphosate and aminomethylphosphonic acid in the sample calculated from the standard working curve, in milligrams per liter;

[0040] V—the volume of the extract added, in milliliters;

[0041] m—sample mass, in grams.

[0042] Extensive experimental research has revealed that, compared to the commonly used aqueous acetonitrile-acetic acid solution and acetonitrile-ammonium acetate solution methods in the prior art, the use of ammonium formate and formic acid as the aqueous phase and pure acetonitrile as the organic phase effectively improves the ionization efficiency and sensitivity of glyphosate and its metabolite aminomethylphosphonic acid derivatives. Furthermore, the use of an ACQUITY UPLC HSS T3 column demonstrates improved peak symmetry compared to an ACQUITY BEH C18 column of the same specifications. Studies have shown that using these optimized conditions, glyphosate and its metabolite aminomethylphosphonic acid derivatives exhibit good peak shape, high resolution, and high sensitivity.

[0043] The beneficial effects of the present invention are:

[0044] (1) The present invention provides a method for detecting glyphosate and its metabolites in fruit, specifically targeting the special properties of grapes, which contain a variety of water-soluble substances such as sugars, vitamins, polyphenols, and pigments. When freeze-dried grape powder is extracted with water, in addition to extracting the target compound glyphosate and its metabolite aminomethylphosphonic acid, water-soluble interfering substances such as pigments and polyphenols, which are much higher in content than the target compound, are also extracted, causing great difficulties in detection. The detection method of the present invention combines three purification methods: liquid-liquid extraction, dispersed solid-phase extraction, and solid-phase extraction, which can effectively remove interference from a large amount of matrix in the extract and improve detection sensitivity.

[0045] (2) The detection method provided by the present invention uses a new derivatization reagent 6-aminoquinoline-N-hydroxysuccinimide carbamate (AQC) to derivatize the purified liquid, thereby greatly improving the derivatization efficiency, greatly saving pretreatment time, improving the recovery rate and sensitivity, and solving the problems of long derivatization time, poor stability and reproducibility in the existing detection of trace glyphosate and its metabolites.

[0046] (3) The present invention adds an internal standard before sample extraction and adopts the internal standard method for quantification. Compared with the traditional external standard method, it can effectively correct the loss in the pretreatment process and improve the recovery rate; at the same time, it can also reduce the matrix effect and improve the accuracy of the quantitative results.

[0047] (4) The detection method provided by the present invention further improves the accuracy of detecting trace amounts of glyphosate and its metabolites by optimizing chromatographic conditions and mass spectrometry conditions.

[0048] (5) The detection method provided by the present invention has a wide range of applications and can meet the needs of detecting glyphosate and its metabolites in large quantities of berries such as grapes and other small fruits. It can also be used to detect glyphosate and its metabolites in other fruits.

[0049] (6) The detection method provided by the present invention is simple, rapid, highly sensitive, highly precise and accurate, and highly applicable. Furthermore, it uses a small amount of derivatization reagent, is environmentally friendly, has low limits of detection and quantification, and is highly reproducible. The present invention utilizes an isotope internal standard method, resulting in more accurate quantitative results. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 It is the MRM total ion current chromatogram of glyphosate, aminomethylphosphonic acid and glyphosate derivatives;

[0051] Figure 2 The effect of different PVPP addition amounts on the recovery of glyphosate and aminomethylphosphonic acid;

[0052] Figure 3 The effect of different MWCNT addition amounts on the recovery of glyphosate and aminomethylphosphonic acid;

[0053] Figure 4 The effect of different addition amounts of GCB on the recovery of glyphosate and aminomethylphosphonic acid;

[0054] Figure 5 Flow chart of the extraction process. DETAILED DESCRIPTION

[0055] The present invention provides a method for detecting glyphosate and its metabolites in fruit. This method, which utilizes 6-aminoquinoline-N-hydroxysuccinimide carbamate (AQC) as a derivatization reagent for the first time, demonstrates strong specificity, stable derivatives, and the absence of byproduct interference. This method effectively eliminates impurity interference, improves sensitivity, and reduces the method's detection limit. The derivatization operation can be completed within one minute. This method is simple, rapid, reproducible, and efficient, requiring minimal time and equipment, and is easy to operate. This method overcomes the time constraints and poor reproducibility associated with existing derivatization methods. This method is suitable for detecting glyphosate and its metabolites in large batches of samples, providing a more convenient, rapid, and accurate method for detecting glyphosate and its metabolites in berries, such as grapes, and other small fruits.

[0056] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the present invention all fall within the scope of the present invention.

[0057] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The materials, reagents, instruments, and equipment used in the examples were obtained from the following sources: acetonitrile (HPLC grade, Fisher, USA); dichloromethane (HPLC grade, Fisher, USA); cross-linked polyvinyl pyrrolidone (PVPP, Aladdin Reagents); multi-walled carbon nanotubes (MWCNT, Aladdin Reagents); GCB (graphitized carbon black, Aladdin Reagents); glyphosate and aminomethylphosphonic acid standard samples (Dr. Ehrenstorfer, Germany); 6-aminoquinoline-N-hydroxysuccinimide carbamate (AQC, Shanghai Yuanye Biotechnology Co., Ltd.); Oasis MCX (60 mg, 3 mL) solid phase extraction cartridges (Waters); ACQUITY ultra-high performance liquid chromatograph, XEVO TQS triple quadrupole mass spectrometer equipped with an ESI ionization source (Waters); 3K30 high-speed refrigerated centrifuge (Sigma, USA); and ultrasonic cleaning machine (Kunshan Ultrasonic Instrument Co., Ltd.).

[0058] Example 1

[0059] A method for detecting glyphosate and its metabolite aminomethylphosphonic acid in grapes, berries, and other small fruits. The specific determination steps are as follows:

[0060] (1) Sample pretreatment

[0061] Pulverization: The grape samples were freeze-dried using a freeze dryer and then ground into fine powder by adding liquid nitrogen.

[0062] Extraction: Weigh 0.5 g of powder sample into a 50 mL centrifuge tube, add 50 μL of 10 μg / mL glyphosate internal standard (GLY- 13 C2, 15 N), 10 mL of water was added, ultrasonic extraction was performed for 30 min, and then centrifuged at 8000 rpm for 3 min, and the upper water extract was retained.

[0063] Purification: Take 6 mL of water extract, add 2 mL of dichloromethane, vortex for 1 min, and centrifuge at 8000 r / min for 3 min to obtain supernatant I; take 3 mL of supernatant I, add 15 mg of PVPP, vortex for 1 min, and centrifuge at 8000 r / min for 3 min to obtain supernatant II; MCX column purification (60 mg / 3 mL) was activated with 3 mL of methanol and 3 mL of water respectively, 1.0 mL of supernatant II was drawn through the column, the filtrate was discarded, and 1.0 mL of supernatant II was taken for sample loading, the filtrate was collected, and centrifuged at 8000 r / min for 5 min to obtain supernatant III for derivatization.

[0064] Derivatization: Pipette 10 μL of supernatant III, add 70 μL of sodium borate buffer (3 g sodium borate is dissolved in pure water and the volume is adjusted to 100 mL, and the pH is adjusted to 8.8 with formic acid), vortex mix, and then quickly add 20 μL of 6-aminoquinoline-N-hydroxysuccinimide carbamate (AQC) derivatization reagent while vortexing again. After vortex mixing for 15 seconds, pour into a liquid phase injection vial.

[0065] The derivation process is as follows:

[0066]

[0067] (2)LC-MS / MS conditions

[0068] Ultra-high performance liquid chromatography conditions Column: ACQUITY UPLC HSS T3 (100 × 2.1 mm, 1.8 μm);

[0069] Column temperature: 50°C;

[0070] Sample chamber temperature: 10°C;

[0071] Injection volume: 5 μL;

[0072] Mobile phase A: 5 mM / L ammonium formate, 0.1% formic acid in water;

[0073] Mobile phase B: acetonitrile; flow rate: 0.45 mL min -1 ;

[0074] Gradient elution conditions: 0-0.5 min, 2.0% B; 0.5-5.0 min, 2.0%-8.0% B; 5.0-6.0 min, 8.0%-20.0% B; 6.0-7.0 min, 20.0%-60.0% B; 7.0-8.0 min, 60.0% B; 8.0-10.0 min, 2.0% B.

[0075] (3) Mass spectrometry conditions

[0076] Ion source: electrospray ionization (ESI);

[0077] Scan mode: positive ion;

[0078] Monitoring mode: multiple reaction monitoring mode (MRM);

[0079] Capillary voltage: 2.7 kV (positive ion mode, ESI + );

[0080] Atomizing gas temperature: 500℃;

[0081] Desolvation gas flow rate: 1000L·h -1 ;

[0082] Ion source temperature: 150°C.

[0083] The mass spectrometry analysis parameters are shown in Table 1.

[0084] Table 1 Mass spectrometry analysis parameters

[0085]

[0086] Note: * in the table represents quantitative ions.

[0087] (4) Establishment of quantitative equation

[0088] Grape samples without glyphosate and aminomethylphosphonic acid (without internal standard) were pretreated as described above and used as blank matrix solutions. Accurately pipette 25, 50, 100, 250, and 500 μL of a 1 μg / mL glyphosate and aminomethylphosphonic acid mixed standard solution into a 5 mL volumetric flask, and then add 1 μg / mL GLY- 13 C2, 15 250 μL of N internal standard solution was diluted to volume with blank matrix solution to prepare 5 different concentrations of glyphosate and aminomethylphosphonic acid standard solutions of 5, 10, 20, 50, and 100 μg / L, of which GLY- 13 C2, 15The internal standard concentration of N was 50 μg / L, and a standard curve was prepared for quantification by internal standard method, that is, the mixed standard working solution contained glyphosate, aminomethylphosphonic acid and GLY- 13 C2, 15 Mixed standard solution of N.

[0089] Secondly, the linear range, detection limit and quantification limit of the method

[0090] According to the above conditions, different concentrations of glyphosate, aminomethylphosphonic acid and GLY- 13 C2, 15 After the N mixed standard solution was derivatized, it was measured on an instrument. The ratio of the peak area of ​​the glyphosate derivative and aminomethylphosphonic acid derivative obtained after derivatization to the peak area of ​​the internal standard derivative was used as the vertical coordinate, and the ratio of the glyphosate and aminomethylphosphonic acid concentrations to the internal standard concentration was used as the horizontal coordinate. A standard curve was prepared to obtain a linear regression equation.

[0091] The detection limit was 3 times the signal-to-noise ratio, and the quantification limit was 10 times the signal-to-noise ratio. The specific results are shown in Table 2.

[0092] Table 2 Linear equations, correlation coefficients, detection limits, and quantification limits of glyphosate and aminomethylphosphonic acid

[0093]

[0094] As can be seen from the above, the detection limit of the method of the present invention for glyphosate is 0.02 μg·kg -1 The detection limit of aminomethylphosphonic acid was 0.19 μg·kg -1 .

[0095] (5) Determination of glyphosate and its metabolite aminomethylphosphonic acid in fruit samples

[0096] Substitute the ratio of the peak area of ​​glyphosate and its metabolite aminomethylphosphonic acid in the sample to be tested, measured by S4, to the peak area of ​​the internal standard into the linear regression equation to obtain the concentration of glyphosate and its metabolites in the extract. Then, calculate the content of glyphosate and its metabolites in the corresponding sample based on the mass of the sample represented by the extract. The calculation formula is as follows:

[0097]

[0098] X i —The content of glyphosate and aminomethylphosphonic acid in the sample, in milligrams per kilogram (mg·kg) -1 ;

[0099] C i —The mass concentrations of glyphosate and aminomethylphosphonic acid in the sample calculated from the standard working curve, in milligrams per liter (mg / L);

[0100] V—the volume of the extract added, in milliliters (mL);

[0101] m—sample mass, in grams (g).

[0102] The chromatogram obtained by the above method is as follows Figure 1 As shown, the horizontal axis is time (min) and the vertical axis is the response value. Figure 1 It can be seen that the retention time of glyphosate derivatives, i.e., the peak time, is 2 min. 13 C2, 15 The peak elution time of the N derivative is 2 min, and the peak elution time of the aminomethylphosphonic acid derivative is 2.56 min.

[0103] Example 2 Method Spiked Recovery

[0104] Add 0.01, 0.02, and 0.05 mg kg -1 A total of three mass fractions of mixed standard solutions of glyphosate and aminomethylphosphonic acid were measured according to the experimental method in Example 1, and the recovery rate was calculated according to the following formula (2). The measurement was repeated 6 times for each addition level.

[0105]

[0106] ω0—theoretical added glyphosate and aminomethylphosphonic acid content, in milligrams per kilogram (mg / kg);

[0107] ω1—The measured glyphosate and aminomethylphosphonic acid content in the sample after addition, in milligrams per kilogram (mg / kg).

[0108] The results are shown in Table 3. The recoveries of glyphosate and aminomethylphosphonic acid were between 72.0% and 81.5%, and the relative standard deviations were between 3.7% and 7.1%.

[0109] Table 3 Spiked recovery and precision

[0110]

[0111] The results show that the accuracy of the method established by the present invention meets the requirement of recovery rate between 70% and 120%.

[0112] Comparative Example

[0113] The present invention also considers the key technical points of the detection method, mainly the type and amount of adsorbent, as follows:

[0114] Add 0.01 mg kg -1A mixed standard solution of glyphosate and aminomethylphosphonic acid with different mass fractions was prepared using the same detection steps as in Example 1, except that the amount of adsorbent added was different, namely, PVPP (15, 30, and 50 mg). Other conditions remained unchanged, and the recovery was calculated.

[0115] MWCNT was used as the adsorbent instead of PVPP, and the addition amounts were 1, 2, and 5 mg, respectively. Other operations were the same as those in Example 1, and the recovery rate was detected and calculated.

[0116] GCB was used as the adsorbent instead of PVPP, and the addition amounts were 1 mg and 2 mg, respectively. Other operations were the same as in Example 1, and the recovery was detected and calculated.

[0117] The results are as follows Figure 2-4 As shown in the figure, when the PVPP addition amount was 15 mg, the recoveries of AMPA and GLY were both above 80%, but the recoveries decreased with increasing PVPP addition amount. When the MWCNT addition amount was 1 mg, the GLY recovery was low, only 51.2%, and the recovery rate decreased with increasing MWCNT addition amount, reaching only 3.8% at 5 mg. When the GCB addition amount was 1 mg, the GLY recovery rate was low, only 54.0%, and the recovery rate decreased with increasing GCB addition amount. Considering that the GLY and AMPA recoveries both met the requirements, a PVPP addition amount of 15 mg was preferred.

Claims

1. A method for detecting glyphosate and its metabolites in fruits, characterized in that: It includes the following steps: S1. extracting glyphosate and its metabolites from the fruit sample to be tested with water to prepare an extract; S2. Purification of the extract; S3, adding a derivatization reagent to the purified extract for derivatization; the derivatization reagent is 6-aminoquinoline-N-hydroxysuccinimide carbamate; S4, glyphosate content determination, quantification by isotope dilution internal standard-high performance liquid chromatography tandem mass spectrometry; At the same time, glyphosate, aminomethylphosphonic acid and glyphosate GLY- 13 C2, 15 N internal standard to prepare a mixed standard working solution; simultaneously perform steps S3-S4 and perform detection, using the ratio of the peak area of ​​glyphosate derivatives and aminomethylphosphonic acid derivatives to the peak area of ​​the internal standard derivative as the ordinate, and the ratio of the glyphosate and aminomethylphosphonic acid concentrations to the internal standard concentration as the abscissa, to prepare a standard curve, obtain a linear regression equation, substitute the measured ratio of the glyphosate and aminomethylphosphonic acid peak area to the internal standard peak area in the test sample into the linear regression equation, obtain the concentration of glyphosate and its metabolites in the extract, and then calculate the content of glyphosate and its metabolites in the corresponding sample based on the mass of the sample represented by the extract; In step S1, the extract is prepared by freeze-drying the fruit with a freeze dryer and then grinding it into a fine powder; weighing the fine powder and adding glyphosate GLY- 13 C2, 15 N was used as an internal standard, and water was added for ultrasonic extraction. After centrifugation, the upper layer of the extract was collected; In step S2, the extract is added with dichloromethane, mixed, and centrifuged. Supernatant I is taken, cross-linked polyvinyl pyrrolidone is added, mixed, and centrifuged to obtain supernatant II. MCX column purification is activated with methanol and water, respectively. Supernatant II is drawn through the column, the filtrate is discarded, and supernatant II is loaded again. The filtrate is collected and centrifuged to obtain supernatant III for derivatization. In step S3, the supernatant III is added to a sodium borate buffer solution with a pH of 8.8, mixed, and then 6-aminoquinoline-N-hydroxysuccinimide carbamate is added, mixed, and then sample injection detection is performed; Wherein, in step S4, the chromatographic conditions of liquid chromatography tandem mass spectrometry are as follows: chromatographic column: ACQUITY UPLC HSS T3, specification: 100×2.1 mm, particle size: 1.8 μm; Mobile phase A: 5 mmol / L ammonium formate, 0.3% formic acid in water; Mobile phase B: pure acetonitrile; Column temperature: 50 °C; flow rate: 0.45 mL / min; Gradient elution ratio: 0-0.5 min, 2.0% B; 0.5-5.0 min, 2.0%-8.0% B; 5.0-6.0 min, 8.0%-20.0% B; 6.0-7.0 min, 20.0%-60.0% B; 7.0-8.0 min, 60.0% B; 8.0-10.0 min, 2.0% B; The mass spectrometry conditions of the liquid chromatography tandem mass spectrometry are: Ion source: electrospray ionization (ESI); scanning mode: positive ion; monitoring mode: multiple reaction monitoring (MRM); capillary voltage: 2.7 kV, positive ion mode, ESI+; nebulizer temperature: 500 °C; desolvation gas flow rate: 1000 L·h -1 ; Ion source temperature: 150 ℃; The mass spectrometry parameters for glyphosate derivatives were as follows: retention time 2.00 min, parent ion 339.83 m / z, product ions 115.95 m / z and 170.90* m / z, cone voltage 15 V, collision energies 55 and 15 eV; Glyphosate- 13 C2, 15 The mass spectrometry parameters of the N derivative were as follows: retention time 2.00 min, parent ion 342.83 m / z, product ions 115.95 m / z and 170.90* m / z, cone voltage 15 V, collision energies 55 and 15 eV; The mass spectrometry analysis parameters of aminomethylphosphonic acid derivatives are as follows: retention time 2.56 min, parent ion 281.86 m / z, product ions 116.00 m / z and 170.90* m / z, cone voltage 15 V, collision energies 55 and 25 eV, where * represents a quantitative ion.

2. The detection method according to claim 1, wherein The concentration of the internal standard was 50 μg / L, the amount of water added was 1:20 g:mL, ultrasonication was performed for 15–30 min, and centrifugation was performed at 8000 r / min for 3 min.

3. The detection method according to claim 1, wherein The ratio of extract to dichloromethane was 3:1, the amount of supernatant I to cross-linked polyvinyl pyrrolidone added was 1 mL:5 mg, and the centrifugation was carried out at 8000 r / min for 5 min.

4. The detection method according to claim 1, wherein 6-Aminoquinoline-N-hydroxysuccinimide carbamate was added to acetonitrile to prepare a solution with a concentration of 2.85 mg / mL, and the supernatant III was added with sodium borate buffer and 6-aminoquinoline-N-hydroxysuccinimide carbamate solution in a ratio of 1:7:

2.

5. The detection method according to claim 1, wherein The concentrations of glyphosate and aminomethylphosphonic acid in the mixed standard working solution were 5, 10, 20, 50, and 100 μg / L, respectively. 13 C2, 15 The internal standard concentration of N was 50 μg / L.

6. The detection method according to claim 1, wherein The calculation formula is as follows: （1） X i — The content of glyphosate and aminomethylphosphonic acid in the sample, in milligrams per kilogram (mg•kg) -1 ; C i — The mass concentrations of glyphosate and aminomethylphosphonic acid in the sample calculated from the standard working curve, in milligrams per liter; V — Volume of extract added, in milliliters; m — Mass of the sample in grams.

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