GB 23200.75-2016Food safety national standard -- Determination of Residual Potassium Amide Residues in Foods (English PDF)

4.1 Reagents

4.1.1 Cyclohexane (C6H12, CAS No. 110-82-7).

4.1.2 Ethyl acetate (C4H8O2, CAS No. 141-78-6). Chromatographic purity.

4.1.3 Methanol (CH3OH, CAS No. 67-56-1). Chromatographic Purification.

4.1.4 Sodium chloride (NaCl, CAS No. 7647-14-5).

4.1.5 anhydrous sodium sulfate (Na2SO4, CAS No. 15124-09-1). 650 °C burning 4 h, in the dryer to cool to room temperature, stored in Sealed in a bottle.

4.2 Solution preparation

4.2.1 Ethyl acetate-cyclohexane solution (1 1). Take.200 mL of ethyl acetate, add.200 mL of cyclohexane and shake well.

4.2.2 methanol - water solution (9 1). take 900 mL of methanol, add 100 mL of water, shake back.

4.3 Standards

4.3.1 Flonicamid (standard) (C9H6F3N3O, CAS number. 158062-67-0). purity >=98.8%.

4.4 Standard solution preparation

4.4.1 Fluoride insect amide standard stock solution (1.0 mg/mL). accurately weighed amount of fluoxetine amide, with ethyl acetate prepared into the concentration A standard stock solution of 1.0 mg/mL. The solution was stored in a refrigerator at -18 ° C.

4.4.2 Fluoride insect amide standard intermediate solution (100 µg/mL). Accurate to absorb the appropriate amount of standard stock solution, diluted with ethyl acetate to the concentration A standard intermediate solution of 100 µg/mL. The solution was stored in a refrigerator at -18 ° C.

4.4.3 Flutamide Amide Standard Working Solution. Standard intermediate solution is diluted with a blank matrix of various samples as appropriate before use.

When the concentration of standard working fluid.

4.5 Materials

4.5.1 Amino solid phase extraction column..200 mg, 3 mL and 500 mg, 3 mL, or equivalent. Before use with 2 mL of ethyl acetate live 2 times.

4.5.2 XTR Diatomite Solid Phase Extraction Column. 3000 mg, 15 mL, or equivalent.

4.5.3 Microporous membrane. 0.45 µm, organic phase.

6.1 Preparation of the sample

6.1.1 Lettuce, carrots, cabbage, citrus, grapes, chestnut

Take a representative sample of about 500 g, chopped, processed with a crusher into a slurry or fine particles. Mix well into a clean container, close Seal, mark.

6.1.2 Rice, tea

Approximately 500 g of representative sample was pulverized and pulverized through a pulverizer and passed through a 1.2 mm round hole. Mix well into a clean container, close Seal, mark.

6.1.3 Beef, chicken, sheep liver, tilapia

Take a representative sample of about 500 g, minced with a meat grinder, mix well, fit into a clean container, seal, mark the mark.

6.1.4 Tomato sauce

Take a representative sample of about 500 g, mix, mix into a clean container, seal, mark the mark.

6.1.5 Honey

To replace the sample of about 500 g, the crystallization of honey samples will be evenly stirred; on the crystallization of honey samples, in the dense in the case of closed, the vials were placed in a water bath of not more than 60 ° C and warmed and shaken until the samples were completely melted and stirred and cooled rapidly Room temperature, in the melting must pay attention to prevent moisture evaporation. Load a clean container, seal, mark the mark.

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

6.2 Sample storage

Tea, honey, grain and nuts and other samples stored at 0 ~ 4 °C; fruits and vegetables and animal-derived food samples at -18 °C the following frozen storage. During sample and sample preparation, the sample should be protected from contamination or changes in the residue content.

7.1 Extraction

7.1.1 lettuce, carrots, cabbage, citrus, grapes, chestnuts, beef, sheep liver, chicken, tilapia, tomato sauce Weigh 10 g (accurate to 0.01 g) sample in 50 mL centrifuge tube, add 15.0 mL ethyl acetate, homogeneous extraction 1 min;

Another 50 mL centrifuge tube, add 10 mL of ethyl acetate wash homogenizer knife head, combined homogeneous solution. Add 10 g of anhydrous sodium sulfate, Cover, shake for 10 min. Centrifuge the extract at 10,000 r/min for 5 min, draw 10 mL supernatant, place the refrigerator below 0 ° C.

H, then 0.45 µm organic filter, to be gel permeation chromatography purification.

7.1.2 Rice

Weigh 10 g (accurate to 0.01 g) sample in a 50 mL centrifuge tube, add 10.0 mL of water, soak for 20 min, add 15.0 mL B Ethyl acetate, homogeneous extraction 1 min, another take a 50 mL centrifuge tube, add 10 mL of ethyl acetate wash homogenizer knife head, combined homogeneous solution.

Add 10 g of anhydrous sodium sulfate, cover, shake 10 min. Centrifuge the sample at 10,000 r/min for 5 min, draw 10 mL supernatant, fridge 0 °C below placed 10 h, then 0.45 µm organic filter, to be gel permeation chromatography purification.

7.1.3 Tea

Weigh 2 g (accurate to 0.01 g) sample in 50 mL centrifuge tube, add 10 mL of water soak for 20 min; add 1 g of anhydrous sulfuric acid sodium. Add 10.0 mL of ethyl acetate, shock extraction 1 0 min, extraction 2 times. Samples were centrifuged at 4000 r/min for 5 min Liquid, constant volume to 20 mL; take 10 mL of 0.45 µm organic phase filter, to be gel permeation chromatography purification.

7.1.4 Honey

Weigh 2 g sample (accurate to 0.01 g) in 50 mL centrifuge tube, add 3 mL of water, 0.5 g sodium chloride, shake and mix the solid phase extraction column was allowed to stand for 5 min and rinsed with 35 mL of ethyl acetate. The flow rate was controlled at 1 mL/min. The filtrate was collected in 50 mL Concentrated in the bottle, spin at 45 °C below the concentration of about 1 mL, to be purified.

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

7.2 Purification

7.2.1 lettuce, carrots, cabbage, citrus, grapes, chestnut, rice, beef, sheep liver, chicken, tilapia, tomato sauce, tea the solution obtained in 7.1.1, 7.1.2 and 7.1.3 was taken into 10 mL supergel permeation chromatography to collect 7 to 14 min effluent, 2 mL Ethyl acetate constant volume, as the primary purification solution.

After the primary purification solution was passed through the amino column, the flask was washed three times with 1 mL of ethyl acetate, and the washing solution was also passed through the column and the flow rate was controlled to 1 ML/min, the whole eluate was collected in a test tube, concentrated at 45 ° C to near dryness, and the residue was swollen with ethyl acetate to dissolve the residue.

Constant volume 1.0 mL, to be tested.

7.2.2 Honey

0.5 g of anhydrous sodium sulfate was filled on the amino column before activation. After the column was purified, the residue was eluted with 2 mL of ethyl acetate and the flow rate was controlled to 1 mL/min. The whole eluate was collected in a test tube and concentrated at about 45 ° C to about 0.5 mL.

Ester volume to 1.0 mL, to be tested.

7.3 Determination

7.3.1 Gel Chromatographic Purification Reference Conditions

A) gel cleaning column. 300 mm × 25 (id) mm; filler. Bio-Beads, S-X3, 38 µm to 75 µm.

B) Concentration temperature. 45 ° C.

C) mobile phase. cyclohexane-ethyl acetate (1 1, volume ratio).

D) constant volume reagent. ethyl acetate.

E) Flow rate. 5 mL/min.

F) Injection volume. 5 mL.

7.3.2 Gas Chromatographic Reference Conditions

A) Column. DB-1701 capillary column, 30 m × 0.32 mm (id), 0.25 µm, or equivalent.

B) heating process. the initial temperature of 80 °C, keep 1 min, to 15 °C per minute rose to 240 °C, keep 1 min, Clock 10 °C up to 260 °C, keep 10 min.

C) Inlet temperature. 260 ° C.

D) Detector temperature. 320 ° C.

E) Carrier gas. nitrogen (purity 99.999%), flow rate 2.5 mL/min.

F) Injection mode. Splitless injection.

G) Injection volume. 1.0 µL.

7.3.3 Determination of gas chromatography

According to the content of fluoride in the sample solution, the standard working solution with similar peak area was selected. Standard working solution and sample solution the value of the fluphenamide response should be within the linear range of the instrument. Standard working solution and sample solution volume measurement. above Under the conditions of chromatographic conditions, the retention time of flutamide was about 13.1 min. The chromatogram of the standard is shown in Figure A.1 in Appendix A.

7.3.4 LC-MS/MS mass spectrometry reference conditions

7.3.4.1 Liquid Chromatographic Reference Conditions

A) Column. Waters Atlantis Hilic Silica Column, 3 µm, 3.0 mm (id) x 50 mm, or equivalent;

B) Column temperature. 40 ° C;

C) mobile phase. methanol - water (9 1, volume ratio);

D) Flow rate. 0.30 mL/min;

E) Injection volume. 10 µL.

7.3.4.2 Mass Spectral Reference Conditions See Appendix B.

7.3.5 Determination by liquid chromatography mass spectrometry

Ethyl acetate volume of the sample solution for solvent conversion to methanol - water (9 1, volume ratio) and then on the machine. According to the sample in the fluorine the content of the solution was similar to that of the standard working solution. Standard working solution and sample solution of fluoride acid amide reaction value are should be within the linear range of the instrument. Standard working solution and sample solution volume measurement. Under the above chromatographic conditions, the retention time of the amide was about 1.03 min.

The selected component selected one parent ion, two or more child ions, under the same experimental conditions, if the sample to be detected in the sample with the standard the corresponding retention time deviation in the quasi-solution is within ± 2.5%; and the relative abundance of the qualitative ions of each component in the sample spectrum is the relative abundance of the corresponding qualitative ions in the standard solution spectrum is compared with the range specified in Table 1, the samples can be judged to be positive for fluphenectam amide. The spectrum of the standard is shown in Appendix C, C.1, C.2.

Table 1 Maximum allowable deviation 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 test

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

10.1 Quantitation limits

In this method, the fluconamide in lettuce, carrots, cabbage, citrus, grapes, tea, beef, chicken, sheep liver, tomato sauce the limit of quantitation was 20 µg/kg, and the limit of quantitation in rice, chestnut, tilapia, and honey was 10 µg/kg.

10.2 Recovery rate

When the levels were 0.02 mg/kg, 0.04 mg/kg, 0.4 mg/kg, 4 mg/kg, the addition recoveries of flutamide See Appendix D.

Appendix A

(Informative)

Gas Chromatographic Spectrum of Flutamide Amide Standard

Figure A.1 Gas chromatogram of the fluoroacetamide standard (10 µg/kg) (GC-ECD)

Appendix B

(Informative)

Determination of the conditions for the determination of fluoxetine amide mass spectrometry Mass spectrum reference conditions.

A) ion source. electrospray ion source (ESI), temperature 500 ° C;

B) scanning mode. negative ion scanning;

C) Detection method. Multi-reaction selective ion detection (MRM);

D) electrospray voltage (IS). 4500 V;

E) atomization gas, air curtain gas, auxiliary heating gas, collision gas are high purity nitrogen and other suitable gas; before use should adjust the gas Body flow to make the mass spectrometry sensitivity to meet the testing requirements;

F) Auxiliary gas temperature (TEM). 500 ° C;

G) Monitoring of ion pairs, doping ions, dwell time, deblocking voltage and collision energy are shown in Table B.1.

Table B.1 Fluorinated butanamide monitoring ion pair, quantitative ion pair, dwell time, de-cluster voltage and collision energy

Name of the test object Monitor ion pairs / (M/z) Quantitative ion pair / (M/z) Dwell time / (Ms) to cluster voltage / (V) Collision energy / (V) Flutamide amide 228.0/81.1 228.0/81.1 100 -64-14 228.0/146.0 -30 Note. For different mass spectrometry instruments, the instrument parameters may be different, before the determination of the mass spectrometry parameters should be optimized to the best.

Non-commercial statement. The reference mass spectrometry conditions listed in Appendix B are performed on the API3000 LC/MS, where the test instrument model is only Provide reference, does not involve commercial purposes, to encourage standard users to try different manufacturers or models of equipment.

Appendix C

(Informative)

Fluethanone Amide Standard LC/MS-MS Mass Spectrometry

Figure C.1 Fluoride insect amide standard ion ion full scan mass spectrometry

Figure C.2 Fluoride Amide Standard Multi-Reaction Monitoring (MRM) Chromatogram (20 µg/L)

Appendix D

(Informative)

The addition of fluoxetine amide and the recovery rate

Table D.1 Fluorinated butyramide addition levels and added recovery

sample name

Add level

(Mg/kg)

Recovery rate

lettuce

20 92.5 to 112.5

40 87.3 ~ 95.3

400 83.0 ~ 87.8

4000 84.7 to 94.1

Cabbage

20 102.0 to 108.5

40 94.0 ~ 114.5

400 92.9 ~ 100.6

4000 92.5 ~ 106.4

carrot

20 83.0 to 91.5

40 84.3 ~ 95.0

400 90.4 to 95.5

4000 86.7 ~ 100.6

Rice

10 85.0 ~ 99.0

20 82.0 to 90.5

100 90.1 to 98.7

Tangerine

20 101.0 to 116.0

40 90.8 to 107.0

400 101.7 ~ 105.6

grape

20 87.5 to 92.5

40 88.8 ~ 94.3

400 89.4 ~ 106.5

Chestnut

10 89.0 ~ 109.0

20 83.5 ~ 97.0

100 91.1 to 99.3

tea

20 83.0 ~ 99.5

40 83.0 ~ 98.5

500 96.8 ~ 98.7

40000 95.0 ~ 109.9

beef

20 74.5 to 92.5

50 80.2 ~ 95.4

100 87.2 ~ 92.6

Sheep liver

20 78.5 ~ 86.5

80 82.8 to 91.4

200 83.7 to 98.2

chicken

20 80.5 to 91.0

40 80.3 ~ 91.8

100 87.5 ~ 89.6

Tilapia

10 89.0 ~ 97.0

20 80.0 to 85.0

100 84.4 ~ 93.5

ketchup

20 80.5 to 99.5

40 84.8 ~ 98.3

500 84.4 ~ 95.3

2000 96.8 ~ 104.9

honey

10 87.0 to 91.0

20 83.0 to 96.5

100 85.0 to 91.4

Appendix E

(Normative appendix) Laboratory repeatability requirements

Table E.1 Laboratory repeatability requirements

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

Table F.1 Inter-laboratory reproducibility requirements

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