GB 23200.57-2016Food safety national standard -- Determination of Acetochlor Residues in Foods (English PDF)

4.1 Reagents

4.1.1 n-hexane (C6H14).

4.1.2 Ethyl acetate (C4H8O2).

4.1.3 Cyclohexane (C6H12).

4.1.4 Acetonitrile (C2H3N).

4.1.5 Acetone (C3H6O).

4.1.6 anhydrous sodium sulfate (Na2SO4). analytical pure; 650 °C burning 4 h, stored in a sealed container for use.

4.2 Solution preparation

4.2.1 Ethyl acetate-n-hexane (3 97, V/V). Take 30 mL of ethyl acetate, add 970 mL of n-hexane and shake well.

4.2.2 Ethyl acetate - n-hexane (5 95, V/V). Take 50 mL of ethyl acetate, add 950 mL of n-hexane and shake well.

4.2.3 Ethyl acetate - cyclohexane (50 50, V/V). Take 500 mL of ethyl acetate, add 500 mL of n-hexane and shake well.

4.3 Standards

4.3.1 Acetochlor standards (acetochlor, C14H20ClNO2, CAS No..34256-82-1). purity >= 98%.

4.4 Standard solution preparation

4.4.1 Acetochlor standard solution. accurately weighed the amount of acetochlor standards, with acetone dubbed the concentration of 100 µg/mL standard stock solution.

The standard stock solution is diluted with n-hexane to the standard working solution at the appropriate concentration. Standard stock solution and standard working fluid are protected from 0 ° C to 0 ° C 4 °C under the conditions of storage. Valid for 6 months.

5.6 High speed homogenizer

5.7 silica gel solid phase extraction column. 6 mL/1 000 mg, or equivalent; before use with 10 mL n-hexane rinse.

5.8 Centrifuge tubes. 100 mL, plastic and glass.

5.9 Filtration. 0.45 µm, organic phase.

5.10 Analysis of balance. 0.01 g and 0.0001 g.

6.1 Vegetables, edible fungi

6.1.1 Take about 500 g of representative spicy vegetable samples onions and cut them into small pieces of about 4 cm2. Into a clean container, sealed and mark the mark. -18 °C below the preservation.

6.1.2 Take representative samples of vegetables (except spicy vegetables such as onions and other spicy vegetables) and pine velvet and other representative edible fungus samples about 500 g, the sample is processed into a slurry. Mix, fit into a clean container, sealed and marked. -18 °C below the preservation.

6.2 Seaweed

Take representative samples of seafood about 500 g, chopped, with a crusher to sample processed into a slurry. Mix well into a clean container Closed mark. -18 °C below the preservation.

6.3 Grains and nuts

Take about 500 g of representative samples of peanuts, corn, wheat, soybeans, cashew nuts, etc., crushed and mixed with a pulverizer, packed in clean containers Inside, sealed and marked. 4 °C below the preservation

6.4 Animal source class

Take about 500 g of representative samples of chicken, pork, etc., crushed with a meat grinder, mix well, into a clean container, sealed and marked.

-18 °C below the preservation.

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

7.1 Extraction

Grain, Nuts. Weigh 10 g (accurate to 0.1 g) in a 100 mL plastic centrifuge tube, add 50 mL of acetonitrile, Homogenize 2 min. Centrifuge at 4 000 r/min for 10 min. The supernatant was poured into a vial, concentrated to dryness, washed with 10 mL of ethyl acetate-Cyclohexane solution dissolves the residue, membrane, for gel permeation chromatography (GPC) purification.

Poultry tissue. Weigh the sample 10 g (accurate to 0.1 g) in a 100 mL plastic centrifuge tube, add 5 g of anhydrous sodium sulfate, stir , Add 50 mL of acetonitrile and homogenize on a homogenizer for 2 min. Centrifuge at 4 000 r/min for 10 min, and supernatate the supernatant with anhydrous sodium sulphate After pouring into the concentrated flask, concentrated to dry, with 10 mL of ethyl acetate - cyclohexane solution to dissolve the residue, membrane, for gel permeation chromatography (GPC) Purification.

Onion. Weigh the sample 5 g (accurate to 0.1 g) in a 100 mL glass centrifuge tube, digested at 700 wd digestion for 40 s.

After returning to room temperature, add 50 mL of ethyl acetate and homogenize on a homogenizer for 2 min. Centrifuge at 4 000 r/min for 10 min, pour the supernatant Into the concentrated bottle, concentrated to dry, with 1 mL n-hexane dissolved residue, for solid phase extraction column purification.

Seafood, Tricholoma matsuter. Weigh 5 g (accurate to 0.1 g) in a 100 mL glass centrifuge tube, add 50 mL of ethyl acetate, the slurry was homogenized for 2 min. Centrifuge at 4 000 r/min for 10 min. The supernatant was poured into a vial, concentrated to dryness, washed with 1 mL n-hexane the residue is purified by solid phase extraction column.

7.2 Purification

7.2.1 Gel permeation chromatography Purification conditions

A) Column. S-X3 20 × 2 cm (inner diameter), or equivalent;

B) Mobile phase. cyclohexane-ethyl acetate (1 1, V/V);

C) Flow rate. 5.0 mL/min;

D) Injection volume. 5.0 mL;

E) elution volume. 40 mL;

F) Collecting volume. 30 mL;

G) Cleaning volume. 10 mL.

7.2.2 Gel permeation chromatography purification

The filters of the extract of grain, nuts and poultry tissues in Section 7.1 were injected into a gel permeation chromatograph to collect 40 to 70 mL of the effluent, 40 °C water bath under reduced pressure to dry, with 1 mL n-hexane dissolved residue, for solid phase extraction column purification.

7.2.3 Silica gel solid phase extraction column purification

Mix the onion, seaweed, truffle extract and 7.2.2 purifying solution into the silica gel solid phase extraction column in section 7.1, then use 2 × 1 mL the flask was run in a silica gel column, washed with 10 mL of n-hexane, 5 mL of ethyl acetate-n-hexane solution and 5 mL of ethyl acetate - n-hexane solution eluted with 15 mL of ethyl acetate-n-hexane solution and the eluate was collected and concentrated to dryness under reduced pressure at 40 ° C.

Hexane volume to 5 mL, for gas chromatography and gas chromatography-mass spectrometry.

7.3 Determination

7.3.1 Gas Chromatographic Reference Conditions

A) Column. DB-5 quartz capillary column, 30 m x 0.32 mm (inner diameter), film thickness 0.25 µm, or equivalent;

15 ° C/min

B) column temperature. 60 °C (1min) 260 °C (5min), 300 °C (after running 15min);

C) Inlet temperature. 250 ° C;

D) detector temperature. 325 ° C;

E) Carrier gas. helium, purity >=99.995%, 1.8 mL/min;

F) Injection volume. 1 µL;

G) Injection method. no split injection, 0.75 min after the opening valve;

7.3.2 Gas Chromatography - Mass Spectrometry Reference Conditions

A) Column. DB-5MS quartz capillary column, 30 m × 0.25 mm (inner diameter), film thickness 0.25 µm, or equivalent;

B) column temperature. 60 °C (1min) 260 °C (5min), 300 °C (after running 15min);

C) Inlet temperature. 250 ° C;

D) chromatographic - mass spectrometer interface temperature. 280 ° C;

E) Carrier gas. helium, purity >=99.995%, 1.0 mL/min;

F) Injection volume. 1 µL;

G) Injection method. no split injection, 0.75 min after the opening valve;

H) ionization mode. EI;

I) ionization energy. 70 eV;

J) Measurement method. Select the ion monitoring mode (SIM);

K) monitoring of ions (m/z). quantitation 162, qualitative 174,234,269;

L) solvent delay. 3 min.

7.3.3 Determination of gas chromatography

According to the sample solution of acetochlor content, selected concentrations similar to the standard working solution. Standard Working Solution and Acetochlor Response in Samples should be within the linear range of the instrument. Standard working solution and sample solution volume measurement. Under the above chromatographic conditions, acetochlor the retention time is about 12.3 min. The chromatogram of the standard is shown in Appendix A, Figure A.1.

7.3.4 Gas Chromatography - Mass Spectrum Confirmation

The standard solution and sample solution of acetochlor are determined according to the conditions specified in Section 7.3.2. If the same retention time as the sample solution is the same as that of the standard solution Appears, then it is confirmed by mass spectrometry. The sample retention time of the measured sample was consistent with that of the standard sample, and the background After the selected spectra, the selected ions appear; the ions m/z162, 174, 234, 269 (the abundance ratio of 100. 54. 24. 8) the relative abundance of the standard sample-related ions is consistent and the similarity is within the allowable deviation (see Table 1), then the acetochlor is present in the sample.

The total ion chromatogram, selective ion spectrum and full scan mass spectrum of the acetochlor standard are shown in Appendix A, Figures A.2, A.3 and A.4.

Table 1 GC-MS confirms the maximum allowable deviation of relative ion abundance

7.4 Blank test

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

10.1 Quantitation limits

The limit of quantification of this method is 0.01 mg/kg.

10.2 Recovery rate

The addition and recovery of the sample by gas chromatography are shown in Appendix B (Table B.1). Gas Chromatography-Mass Spectrometry Concentration and recovery data are given in Appendix B (Table B.2).

Appendix A

(Informative)

Standard Chromatography and Mass Spectrometry Min 8 9 10 11 12 13 14 15 Hz

Figure A.1 Acetochlor Standard Gas Chromatogram

4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 Time - > Abundance TIC..2007120712.D.

Figure A.2 Acetochlor Standard Total Ion Flow Chromatogram (TIC)

Acetochlor

Acetochlor

160 170 180 190.200 210 220 230 240 250 260 270 M/z - > Abundance Scan 4496 (12.581 m in)..2007120712.D

Figure A.3 Acetochlor Standard Selective Ion (SIM) Mass Spectrometry

50 100 150.200 250 300 350 400 450 M/z - > Abundance Scan 980 (12.591 min)..2007120702.D 242 341 420305 401381360 446

Figure A.4 Acetochlor Standard Full Scanning (SCAN) Mass Spectrometry

Appendix B

(Informative)

Sample concentration and recovery of the experimental data

Table B.1 Determination of the concentration and recovery of samples by gas chromatography

sample name

Add concentration

(Mg/kg)

Recovery rate range

Precision

RSD (%)

sample name

Add concentration

(Mg/kg)

Recovery rate range

Precision

RSD (%)

peanut

0.01 71.3 91.2 9.8

seaweed

0.01 86.795.9 8.3

0.02 77.2 91.5 6.0 0.02 81.8 96.7 4.4 0.05 78.1 96.4 9.1 0.05 79.1 93.2 6.9 Soybeans 0.01 76.2 88.7 5.1 chicken 0.01 82.094.6 7.4 0.02 74.5 86.9 3.1 0.02 80.3 88.8 4.1 0.05 76.0 94.2 8.6 0.05 80.1 97.2 6.3 corn 0.01 75.786.0 6.9 pork 0.01 76.086.7 5.3 0.02 73.9 86.8 4.9 0.02 77.1 85.5 3.5 0.05 76.8 92.3 6.9 0.05 78.9 98.8 6.5 wheat 0.01 75.9 88.2 6.7 Cashew nuts 0.01 74.3 87.7 5.5 0.02 78.3 91.6 3.4 0.02 75.4 89.2 4.9 0.05 76.7 89.1 7.4 0.05 75.4 92.7 7.8 onion 0.01 76.6 93.1 9.4 Matsutake 0.01 81.5 97.6 8.3 0.02 82.6 93.7 4.3 0.02 83.1 96.5 4.4 0.05 80.0 93.2 6.9 0.05 79.3 95.3 6.9

Table B.2 Gas Chromatography-Mass Spectrometry Detection of Sample Concentration and Recovery Data

sample name

Add concentration

(Mg/kg)

Recovery rate range

Precision

RSD (%)

sample name

Add concentration

(Mg/kg)

Recovery rate range

Precision

RSD (%)

peanut

0.01 71.5 88.3 8.6

seaweed

0.01 79.0 93.7 6.8

0.02 77.5 94.0 5.9 0.02 76.1 93.5 5.8 0.05 70.1 90.2 8.3 0.05 81.9 97.8 7.1 Soybeans 0.01 74.9 85.2 6.9 chicken 0.01 80.0 97.7 6.7 0.02 82.5 90.3 4.7 0.02 84.1 95.5 5.2 0.05 76.2 96.2 6.7 0.05 79.9 98.8 7.5 corn 0.01 70.9 85.8 4.4 pork 0.01 77.4 89.2 4.6 0.02 75.988.8 4.0 0.02 74.986.2 4.6 0.05 78.1 96.4 6.0 0.05 80.4 94.2 7.5 wheat 0.01 71.386.2 4.4 Cashew nuts 0.01 76.4 90.2 7.3 0.02 84.392.6 4.1 0.02 77.4 87.2 6.4 0.05 74.7 90.2 6.8 0.05 77.4 97.9 6.7 onion 0.01 82.6 91.1 4.6 Matsutake 0.01 82.0 96.5 6.8 0.02 78.6 89.7 5.5 0.02 83.9 100.3 5.8 0.05 79.0 95.2 8.7 0.05 87.6 101.5 7.1 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