GB 23200.109-2018National food safety standard -- Determination of clopyralid residues in plant-derived foods -- Liquid chromatography-mass spectrometry (English PDF)

1 Scope

This standard specifies a liquid chromatography-mass spectrometry method for glufosinate residues in plant-derived foods:

This standard is applicable to the determination of glufosinate ammonium residues in plant-derived foods:

2 Normative reference documents

The following documents are essential for the application of this document: For dated references, only the dated version applies to this document:

For undated referenced documents, the latest version (including all amendments) applies to this document:

GB 2763 National Food Safety Standard Maximum Residue Limits of Pesticides in Foods GB/T 6682 Analytical laboratory water specifications and test methods

3 Principles

The glufosinate ammonium in the sample was extracted with water and methanol, and then dispersed and purified by solid phase materials: The purification solution was mixed with 9-fluorenylmethyl chloroformate:

The derivative glufosinate-FMOC (glufosinate-FMOC) generated after the reaction of (9-fluorenylmethyl chloroformate) was analyzed by liquid chromatography Detection by spectrometry-mass spectrometry and quantification by external standard method:

4 Reagents and materials

Unless otherwise stated, only analytical grade reagents and first-grade water specified in GB/T 6682 were used in the analysis:

4.1 Reagents

4.1.1 Acetonitrile (CH3CN, CAS number: 75-05-8): chromatographically pure:

4.1.2 Methanol (CH3OH, CAS number: 67-56-1): chromatographically pure:

4.1.3 Ammonium acetate (CH3COONH4, CAS number: 631-61-8):

4.1.4 Sodium borate (Na2B4O2·10H2O, CAS number: 1303-96-4):

4:1:5 9-Fluorenylmethyl chloroformate (CisHCIO2, CAS number: 28920-43-6): purity 99.0%, stored at 0°C~4°C:

4.2 Solution preparation

4.2.1 Borate buffer solution (50:0g/L, pH=9): Weigh 5g of sodium borate (Na2B1O2·10H2O), dissolve it in water and dilute to 100mL:

4.2.2 Acetonitrile solution of 9-fluorenylmethyl chloroformate (10:0g/L): Weigh 1g of 9-fluorenylmethyl chloroformate, dissolve it in acetonitrile and adjust the volume to 100mL:

4.2.3 Ammonium acetate aqueous solution (5 mmol/L): Weigh 0:385g of ammonium acetate and dissolve it in an appropriate amount of water, and adjust the volume to 1000mL with water:

4.3 Standard products Glufosinate ammonium (C3H8N3O4P, CAS number: 77182-82-2) standard product: purity >=99.0%:

4.4 Preparation of standard solution

4.4.1 Glufosinate ammonium standard stock solution (100 mg/L): Accurately weigh 10:0 mg of glufosinate ammonium standard solution in a 50 mL beaker, dissolve it in water, transfer it to a 100 mL volumetric flask, and dilute to volume with water: Stored in a refrigerator at 0°C~4°C, it is valid for 6 months:

4.4.2 Glufosinate ammonium standard intermediate solution (10 mg/L): Pipette 5:0 mL of glufosinate ammonium standard stock solution into a 50 mL volumetric flask, and dilute to volume with water: Store in 0°C~4°C refrigerator, valid for 1 month:

4.5 Materials

4.5.1 Neutral alumina (Al2O2, CAS number: 1344-28-1): 0:075mm~0:15mm:

4.5.2 Multi-walled carbon nanotubes (MWCNTs): particle size range 10nm~20nm; particle length 5µm~15µm; specific surface area (225±25)m²/g:

4.5.3 Filter membrane: 0:22µm, organic system:

5 Instruments and equipment

5.1 Liquid chromatography-mass spectrometer: equipped with electrospray ion source (ESD):

5.2 Analytical balance: sensitivity 0:0001g, sensitivity 0:01g:

5.3 Tissue mashing instrument:

5.4 Centrifuge: the rotation speed is not less than 10000 r/min:

5.5 Vortex oscillator:

5.6 Constant temperature water bath:

6 Sample preparation

Samples of vegetables, fruits and edible fungi are taken in a certain amount according to relevant standards, and the sampling parts are in accordance with the provisions of GB 2763: For small individual samples, all are processed after sampling; for large individual and basically uniform samples, they can be divided or cut into small pieces on the axis or plane of symmetry and then processed; for slender, flat or component content in each part For samples with differences, small pieces or segments can be cut from different parts for processing; the sample should be chopped into small pieces, mixed thoroughly, and sampled using the quartering method or directly put into a tissue masher and mashed into a homogenate: : The homogenate was placed in a polyethylene container:

Take 500g of cereal samples, crush them so that they can all pass through a 425µm standard mesh sieve, and put them into polyethylene bottles or bags: Take 500g each of oil crops, tea leaves, nuts and spice samples, crush them and mix thoroughly, then put them into polyethylene bottles or bags:

Mix the vegetable oil evenly:

Samples should be stored at temperatures below -18°C:

7 Analysis steps

7.1 Extraction

7.1.1 Vegetables, fruits and edible fungi Weigh 10g (accurate to 0:01g) of the sample into a 50mL centrifuge tube, refer to Appendix A to add water, and then add 10mL methanol (4:1:2), after vortexing for 2 minutes, centrifuge at 3800r/min for 5 minutes to be purified:

7.1.2 Spices and tea leaves Weigh 2g (accurate to 0:01g) of the sample into a 50mL centrifuge tube, refer to Appendix A to add water, then add 10mL methanol (4:1:2), vortex for 2 minutes, and centrifuge at 3800 r/min for 5 minutes until purification:

7.1.3 Cereals, nuts, oil crops, vegetable oils Weigh 5g (accurate to 0:01g) of the sample into a 50mL centrifuge tube, refer to Appendix A to add water, then add 10mL methanol (4:1:2), vortex for 2 minutes, and centrifuge at 3800 r/min for 5 minutes until purification:

7.2 Purification

7.2.1 Vegetables, fruits, nuts, edible fungi, spices, tea, and cereals Accurately transfer 1 mL of the extraction solution into a 2 mL centrifuge tube containing 5 mg of multi-walled carbon nanotubes (4:5:2), vortex for 1 min, centrifuge at 10000 r/min for 3 min, and pass the supernatant through a 0:22 µm organic filter membrane ( 4:5:3) in another 2mL centrifuge tube for derivatization:

7.2.2 Oil crops

Use a pipette to accurately transfer 4mL of the extraction solution into a 5mL centrifuge tube, freeze it in a refrigerator at -20°C overnight, and then take 1mL of the supernatant into a mixture of 5mg multi-walled carbon nanotubes (4:5:2) and 50mg neutral into a 2 mL centrifuge tube of aluminum oxide (4:5:1), vortex for 1 min, and then Centrifuge at 10:000 r/min for 3 minutes, pass the supernatant through a 0:22 µm organic filter membrane (4:5:3) and place it in another 2 mL centrifuge tube for derivatization:

7.2.3 Vegetable oils Accurately draw 1 mL of the extraction solution directly through a 0:22 µm organic filter membrane (4:5:3) into another 2 mL centrifuge tube for derivatization:

7.3 Derivatization

Accurately pipette 0:5mL of the purified extraction solution into a 2mL centrifuge tube, add 0:5mL of borate buffer solution (4:2:1), and mix Then add 0:5 mL of 9-fluorenylmethyl chloroformate acetonitrile solution (4:2:2), vortex for 1 min, derivatize in a 40°C water bath for 1 h, centrifuge at 10000 r/min for 1 min after derivatization, and take the supernatant and pass it through 0:22 µm organic filter membrane (4:5:3) for detection: Accurately draw glufosinate ammonium standard solution (7:5)0:5mL was derivatized simultaneously according to this method:

7.4 Instrument reference conditions

7.4.1 Liquid Chromatography Reference Conditions

a) Chromatographic column: Cs, 50mm×2:1mm, particle size 1:7µm, or equivalent;

b) Mobile phase: A is 5 mmol/L ammonium acetate aqueous solution, B is acetonitrile, and the gradient elution procedure is shown in Table 1;

c) Flow rate: 0:2mL/min;

d) Column temperature: room temperature;

e) Injection volume: 5 µL:

7.4.2 Mass spectrometry reference conditions

a) Ion source: electrospray ion source;

b) Scanning method: positive ion scanning;

c) Spray voltage: 3500V;

d) Capillary temperature: 320°C;

e) Gasification temperature: 300°C;

f) Sheath gas (N2): 45 psi;

g) Auxiliary gas (N2):10 psi;

h) Collision gas argon: 1:5mTorr;

i) Detection method: multiple reaction monitoring (MRM): The conditions for multiple reaction monitoring are shown in Table 2:

7.5 Standard working curve Dilute the glufosinate ammonium standard intermediate solution with the blank extract solution to a mass concentration of 0:01mg/L, 0:05mg/L, 0:1mg/L, A series of matrix-matched standard solutions of 0:5 mg/L and 1 mg/L are measured according to reference chromatography and mass spectrometry conditions after derivatization (7:3): Taking the mass concentration of glufosinate ammonium as the abscissa and the peak area integral value of its derivatives as the ordinate, draw a standard curve and find the regression equation and correlation coefficient: Matrix matching standard solutions should be prepared and used immediately:

7.6 Qualitative and quantitative

7.6.1 Retention time Compared with the retention time of the corresponding standard chromatographic peak of the glufosinate-ammonium derivative glufosinate-FMOC chromatographic peak in the test sample, the relative error should be within ±2.5%:

7.6.2 Quantitative ions, qualitative ions and product ion abundance ratios when measuring samples under the same experimental conditions, if the retention time of the detected chromatographic peak is consistent with that of the standard sample, and in the mass spectrum of the sample after subtracting the background, the mass spectrometric qualifier ion of the target compound must appear, and at least one should be included: The parent ion and two product ions, and the same detection batch, for the same compound, the relative abundance ratio of the two product ions of the target compound in the sample is compared with the standard solution of equivalent concentration, and its relative deviation does not exceed the range specified in Table 3: It can be determined that glufosinate is present in the sample:

7.7 Determination

After derivatization, the matrix-matched standard solution and the solution to be tested are injected into the liquid chromatography-mass spectrometer, and the retention time and qualifier ions are used for identification: The mass concentration of glufosinate in the sample should be within the mass concentration range of the standard working curve: Samples that exceed the upper limit of the mass concentration of the standard working curve should be diluted and injected, and the external standard method should be used for quantification: At the same time short self-experiment:

7.8 Blank test

Except that no sample is added, parallel operations shall be carried out in accordance with the provisions of 7:1 to 7:7:

8 Result calculation

The residual amount of glufosinate ammonium in the sample is measured as mass fraction w, expressed in milligrams per kilogram (mg/kg), and calculated according to formula (1):

9 Precision

9.1 Under repeatability conditions, the absolute difference between the results of two independent measurements is not greater than the repeatability limit (r): The data for the repeatability limit (r) is:

a) When the content is 0:05mg/kg, the repeatability limit (r) is 0:01;

b) When the content is 0:1mg/kg, the repeatability limit (r) is 0:02;

c) When the content is 0:5mg/kg, the repeatability limit (r) is 0:09;

d) When the content is 1mg/kg, the repeatability limit (r) is 0:19:

9.2 Under the conditions of reproducibility, the absolute difference between the results of two independent measurements is not greater than the reproducibility limit (R): The data of the reproducibility limit (R) is:

a) When the content is 0:05mg/kg, the reproducibility limit (R) is 0:02;

b) When the content is 0:1mg/kg, the reproducibility limit (R) is 0:04;

c) When the content is 0:5mg/kg, the reproducibility limit (R) is 0:16;

d) When the content is 1mg/kg, the reproducibility limit (R) is 0:24:

10 Others

The quantitation limit for spices and tea is 0:1 mg/kg, and the quantitation limit for other samples is 0:05 mg/kg:

11 Maps

11.1 The full scan mass spectrum of the 5 mg/L glufosinate derivative standard solution is shown in Figure 1: