GB 23200.46-2016Food safety national standard -- Determination of residues of pyrimethanil, azoxystrobin, myclobutanil and azoxystrobin in foodstuffs by gas chromatography-mass spectrometry (English PDF)

4.1 Reagents

4.1.1 Acetone (C3H6O). Pesticide level.

4.1.2 Ethyl acetate (C4H8O2).

4.1.3 Acetonitrile (CH3CN). HPLC grade. ;

4.1.4 Toluene (C7H8).

4.1.5 n-hexane (C6H14).

4.1.6 Sodium chloride (NaCl).

4.1.7 anhydrous sodium sulfate (Na2SO4). at 650 °C for 4 h, stored in a sealed container for use.

4.2 Solution preparation

4.2.1 Sodium chloride solution (300 g/L). 300 g of sodium chloride was weighed and dissolved in distilled water and fixed to 1 000 mL.

4.2.2 acetonitrile - toluene (3 2, volume ratio). take 30 mL of acetonitrile and 20 mL of toluene, mixed evenly.

4.2.3 acetonitrile - toluene (3 1, volume ratio). take 30 mL of acetonitrile and 10 mL of toluene, mixed evenly.

4.2.4 acetone - n-hexane (1 1, volume ratio). take 50 mL of acetone and 50 mL of n-hexane, mixed evenly.

4.2.5 Acetonitrile saturated n-hexane.

4.2.6 n-hexane saturated acetonitrile.

4.3 Standards

4.3.1 Pyrimethamine standard. Molecular formula, C12H13N3; CAS 53112-28-0; purity >= 99%.

4.3.2 pyrimethamine standard. Molecular formula, C14H13N3; CAS 110235-47-7; purity >= 99%.

4.3.3 Mildenazole standard. Molecular formula, C15H17ClN4; CAS 88671-89-0; purity >= 99%.

4.3.4 azoxystrobin standard. Molecular formula, C22H17N3O5; CAS 131860-33-8; purity >= 99%.

4.4 Standard solution preparation

4.4.1 pyrimethamine, azoxystine, nitrobenzazole, azoxystrobin standard solution. accurately weighed the right amount of pyrimethamine, azoxystrobin, Ester standard, with acetone prepared into 100 µg/mL standard stock solution, 5 °C below the storage, within 6 months of use. And then diluted with acetone into the appropriate when the concentration of the standard working solution, 5 °C below the storage, within 3 months of use.

4.5 Materials

4.5.1 graphitized carbon black column/ammonia column. 500 mg, 6 ml; or graphitized carbon black column (500mg, 6ml) and amino column (500 mg, 3 ml) were used in series from top to bottom.

4.5.2 anhydrous sodium sulfate column. 150 mm × 10 mm glass column, from bottom to top in turn into the cotton, 5 cm high anhydrous sodium sulfate.

5.7 Rotary Evaporator

5.8 Nitrogen Drying Machine.

5.9 Centrifuge tube. glass, 50 mL.

5.10 Horizontal roundabout shaker.

6.1 Preparation of the sample

6.1.1 Fruits, vegetables, nuts

Take about 500 g, crushed with a pulverizer, into a clean container as a sample, sealed and do a good job.

6.1.2 Animal-derived food

6.1.2.1 Meat and aquatic products. take a representative part of the sample about 500 g, crushed with a pulverizer, into a clean container as a sample, Sealed and identified.

6.1.2.2 Honey. take a representative sample of about 500 g, the amorphous sample will be forced to stir evenly, there are crystallization of samples can be samples After the cap is tightened, it is placed in a water bath of not more than 60 ° C. After all the samples have been dissolved and stirred, they are rapidly cooled to room temperature. Prepare the prepared sample Into the clean container sealed and do a good job.

6.1.3 Grain, tea

Take about 500 g of the sample and crush it all through a 20 mesh sieve into a clean container as a sample, seal and mark it.

Note. The above sample sampling site according to GB 2763 Appendix A implementation.

6.2 Sample storage

Fruits, vegetables, nuts, livestock, poultry and aquatic products and other samples frozen at -18 °C below; grain, tea, bee products and other samples Keep at light at room temperature.

During the operation of the sample preparation, the sample should be protected from contamination or changes in the content of the residue.

7.1 Extraction

7.1.1 Fruits and vegetables

Approximately 10 g (accurate to 0.01 g) was weighed in a 100 mL Erlenmeyer flask, 25 mL of acetone and 15 g of sodium chloride were added, Swing on shaker for 30 min. Filtered into a 250 mL separatory funnel, and the Erlenmeyer flask and the residue were washed twice with 20 mL of acetone.

Liquid in the separatory funnel, add 25 mL of sodium chloride solution and 30 mL of ethyl acetate in the same separatory funnel, shaking 2 min, standing stratified, Set organic phase. The lower aqueous phase was extracted again with 20 mL of ethyl acetate and the combined organic phases were passed through an anhydrous sodium sulphate column to a vial. At 45 ° C the water bath is evacuated and evaporated to dryness.

7.1.2 Livestock, poultry, aquatic products

Approximately 10 g (accurate to 0.01 g) was weighed into a 50 mL centrifuge tube and 25 mL of ethyl acetate, 15 g of sodium chloride Quality 1 min, centrifuged at 4000 r/min for 5 min. Take the upper layer of organic phase through anhydrous sodium sulfate column into the heart-shaped bottle. And then with 20 mL of ethyl acetate the residue was extracted twice in the same manner as described above, and the organic phase was combined in a vial. In 45 °C water bath on the vacuum rotation to dry.

7.1.3 Grain, tea, chestnut

Weigh about 10 g (accurate to 0.01 g) in the mixed flask and add 25 mL of ethyl acetate (chestnut sample plus 5 g of sodium chloride) Shaking on a horizontal swing shaker for 30 min, filtering into a 250 mL separatory funnel, washing the Erlenmeyer flask with 20 mL of ethyl acetate twice the filtrate was combined and the filtrate was added to the same separatory funnel and 30 mL of sodium chloride solution was added. The mixture was shaken for 1 min, the liquid was extracted, the layers were separated, and the ethyl acetate Layer of anhydrous sodium sulfate into the heart of the bottle; the water phase by adding 25 mL of ethyl acetate liquid extraction, standing stratification, abandoned the water phase, combined the ethyl acetate layer was in the same heart flask as described above. In 45 °C water bath on the vacuum rotation to dry.

7.1.4 Honey

Weigh 10 g (accurate to 0.01 g) honey sample in a Erlenmeyer flask, add 20 mL of sodium chloride solution and 5 mL of acetone to dissolve, Rotary shaker shaking 30 min, into the 250 mL separatory funnel, and then with 30 mL of sodium chloride solution in two, 50 mL of ethyl acetate Wash the original two bottles, are transferred to the same separatory funnel, shaking 2 min, standing stratification (if the occurrence of emulsification, the upper and the emulsion layer Centrifuge at 4000 r/min for 5 min, take the upper layer into the heart-shaped bottle), collect the organic phase to another separatory funnel. The aqueous phase was further washed with 20 m of ethyl acetate Extracted twice, the organic phase to the same separatory funnel, the separation funnel by adding 40 mL of sodium chloride solution shaking 1 min, standing (if hair Raw milk, the upper layer and the emulsion layer can be centrifuged at 4000r/min for 5 min), the ethyl acetate layer passed through the anhydrous sodium sulfate column into the heart-shaped bottle, 45 °C water bath on the vacuum rotary to dry.

7.2 Purification

7.2.1 Livestock, poultry, aquatic products, grain, tea, chestnut

For the sample residue obtained in 7.1.2 and 7.1.3, 40 mL of acetonitrile saturated n-hexane was added to dissolve twice and transferred to the same 250 mL Funnel, respectively, with 50 mL of n-hexane saturated acetonitrile in two, acetonitrile saturated n-hexane 10 mL heart wash bottle, are transferred to the above-mentioned liquid leakage Bucket. Oscillate layer, acetonitrile layer through anhydrous sodium sulfate column into the heart of the bottle, n-hexane layer each time again with n-hexane saturated acetonitrile 15mL wash two Times, n-hexane layer abandoned, acetonitrile layer of anhydrous sodium sulfate column into the heart of the bottle, in the 45 °C water bath vacuum rotary to dry.

7.2.2 Graphite carbon black column/ammonia column purification

The sample residue obtained by dissolving 7.1.1, 7.1.4 and 7.2.1 with 1 mL of acetonitrile-toluene (3 1, volume ratio) was transferred to graphitized carbon black Column -. And then 1 mL of acetonitrile - toluene (3 1, volume ratio) two times wash heart flask, into the above graphitized carbon black column -Discard all the effluent. The graphitized carbon black column-amino composite column was eluted with 10 mL of acetonitrile-toluene (3 2, by volume) to receive complete elution liquid. The nitrogen stream was blown dry on a 45 ° C water bath. And 1.0 mL with acetone-n-hexane (1 1, volume ratio). Analysis of gas chromatographic mass spectrometry.

7.3 Determination

7.3.1 Gas Chromatography - Mass Spectrometry Reference Conditions

7.3.1.1 Chromatographic conditions

A) Column. DB-5MS, 30 m x 0.25 mm (inner diameter), 0.25 µm, or equivalent;

B) Column temperature programmed. 210 ° C (2 min) 30 ° C/min 280 ° C 10 ° C/min 290 ° C (6 min);

C) Inlet temperature. 250 ° C;

D) Carrier gas. helium, purity 99.999% E) Carrier gas flow rate. constant current mode 1 mL/min;

F) Injection method. no shunt;

G) Injection volume. 2 µL;

H) Opening time. 1 min.

7.3.1.2 Mass spectrometry conditions

A) Interface temperature. 280 ° C;

B) ion source. electron bombardment source (EI);

C) ionization voltage. 70 eV;

D) ion source temperature. 230 ° C;

E) Detection method. SIM;

F) solvent delay time. 2.5 min;

G) Select the ion and relative abundance. see Table 1.

Table 1 Select the ion and relative abundance

Measured component Quantitative ion (relative abundance) /% Qualitative ion (relative abundance) /% Pyrimethamine 198 (100).199 (47) 188 (3) 184 (4) Azoxystine 222 (100) 223 (51) 208 (5) 181 (3) (100) 150 (53) 245 (14) 288 (13) Azoxystrobin 344 (100) 388 (28) 372 (14) 403 (13)

7.3.2 Quantitative determination

The volume of the sample solution and the standard working fluid and so on. Practical application of the standard working solution and the sample to be measured, pyrimethamine, azole bacteria the response values of amines, myclobutanil and azoxystrobin should be within the linear range of the instrument. Under the above gas chromatography-mass spectrometry conditions, pyrimethamine, pyrimidine the retention times of the bacteria, the bacteria and the azoxystrobin were 3.42 min, 5.48 min, 5.90 min and 11.63 min, respectively. Standard Gas Chromatography -See Appendix A for the spectrum.

7.3.3 Qualitative determination

When the sample is measured, if the mass retention time of the detected mass is consistent with that of the standard sample, and the sample spectrum after subtracting the background in the figure, the relative abundance of the qualitative ions is close to the standard solution obtained under the same conditions, and the error does not exceed the standard of the range, you can determine the existence of the corresponding sample in the sample.

Table 2 The maximum allowable error of relative ion abundance when qualitative confirmation

Relative abundance (base) 50% 20% to 50% 10% to 20% <=10% Allowable relative deviation ± 20% ± 25% ± 30% ± 50%

7.4 Blank experiment

In addition to the sample, according to the above determination steps.

10.1 Quantitation limits

The limits of quantification of the four pesticides were. 0.01 mg/kg pyrimethamine; 0.01 mg/kg pyruvate; 0.01 mg/kg the strain was 0.005 mg/kg.

10.2 Recovery rate

The addition of pyrimethamine, azoxystrobin, mycelia and azoxystrobin was added when the levels were 0.01 mg/kg, 0.05 mg/kg, 0.1 mg/kg the recovery rate is given in Appendix B.

Appendix A

(Informative)

Standard Gas Chromatography and Gas Chromatography - Mass Spectrometry 1-pyrimethamine;

2 - pyrimethamine;

4-azolyl ester.

Figure A.1 Total ion chromatogram (TIC) for standard mixtures (1 µg/mL for four pesticide concentrations)

Appendix B

(Informative)

Sample concentration and recovery of the experimental data

Table B.1 Experimental data on the concentration and recovery of the sample

sample

name

Add concentration/(mg/kg) Azoxystine azoxystrobin 0.01 0.05 0.1 0.01 0.05 0.1 0.01 0.05 0.1 0.005 0.05 0.1 Recovery rate/(%) Apple 81.0-93.0 88.0-102.0 84.0-96.0 82.0-92.0 84.6-94.7 86.0-98.0 75.0-98.0 82.0-98.0 84.0-94.0 92.0-120.0 84-102.0 85.0-105.

Beef 81.0-92.0 82.0-92.0 82.0-96.0 75.0-97.0 84.0-110 84.0-91.0 82.0-95.0 82.0-102 83.0-97.0 82.0-102 82.0-98.0 88.0-105 Chicken 84.0-110.0 82.0-98.0 81.0-95.0 83.0-104.0 82.0-104 84.0-96.0 84.0-105.82.0-94.0 82-104.0 82.0-102 82.0-108 84.0-96.0 Eggplant 81.0-95.0 84.0-102 84.0-96.0 80.0-95.0 80.0-96.0 84.0-97.0 81.0-116 82.0-96.0 82.0-95.0 84.0-110 84.0-104 87.0-97.0 Rice 81.0-98.0 80-102 83.0-98.0 82.0-103 88.0-98.0 82.0-97.0 80.0-99.0 72.0-92.0 81.0-96.0 76.0-94.0 84.0-110 83.0-98.0 Fish meat 81.0-97.0 80.2-102 86.0-105 82.0-98.0 80.2-96.0 84.0-97.0 81.0-98.0 80.2-106 85.0-97.0 74.0-116 80.2-106 81.0-97.0 Honey 83.0-97.0 80.2-102 84.0-98.0 81.0-96.0 80.2-106 81.0-99.0 73.0-106 80.6-110 82.0-96.0 80.0-120 86.0-94.0 81.0-105 Chestnut 80.0-95.0 80.0-102 85.0-104 82.0-115 82.0-102 80.0-95.0 84.0-107 82.0-100.0 81.0-98.0 82.0-108.0 84.0-110.0 84.0-98.0 Tea 81.0-107.0 80.0-110 81.0-94.0 81.0-98.0 82.0-96.0 81.0-95.0 86..0-120 82.0-108.0 81.0-98.0 76.0-116 82.0-98.0 81.0-97.0 Appendix C (Normative appendix) Laboratory repeatability requirements

Table C.1 Laboratory repeatability requirements

Measured component content Mg/kg Precision <=0.001 36 > 0.01 > 1 14 Appendix D (Normative appendix) Inter-laboratory reproducibility requirements

Table D.1 Inter-laboratory reproducibility requirements

Measured component content Mg/kg Precision <=0.001 54 > 0.01 > 1 19