GB 23200.71-2016Food safety national standard -- Determination of residues of diester imide in foodstuffs by gas chromatography-mass spectrometry (English PDF)

4.1 Reagents

4.1.1 Acetone (C3H6O). Residue level.

4.1.2 n-hexane (C6H14). residual grade.

4.1.3 Cyclohexane (C6H12). Residue level.

4.1.4 ethyl acetate (C4H8O2). residual grade.

4.1.5 Sodium chloride (NaCl).

4.1.6 anhydrous sodium sulfate (Na2SO4). 650 °C burning 4 h, set the dryer in the cooling, sealed preservation.

4.2 Solution preparation

4.2.1 Acetone-n-hexane solution (1 4, V1 V2). Take 100 mL of acetone, add 400 mL of n-hexane and shake well.

4.2.2 Acetone-n-hexane solution (1 1, V1 V2). Take 100 mL of acetone, add 100 mL of n-hexane and shake well.

4.2.3 Ethyl acetate + cyclohexane (1 + 1, V1 V2). Take 100 mL of ethyl acetate, add 100 mL of cyclohexane and shake well.

4.3 Standards

(Vinclozolin, C12H9Cl2NO3, CAS NO. 50471-44-8), Bacillus fungus standard (Ethephon, C13H11Cl2NO5, CAS NO. 84332-86-5), Pythiumide standard (Procymidone, C13H11Cl2NO2, CAS NO. 32809-16-8), the purity of the steroid urea standard (iprodione, C13H13Cl2N3O3, CAS NO. 36734-19-7) was > 99%.

4.4 Standard solution preparation

4.4.1 Velveti, Bacteriocarbide, Pythium, Biomurureum Standard stock solution. accurately weighed the amount of vinyl mushroom, Mold, antibiotic urea standard, with acetone preparation of 100µg/mL standard stock solution, and then according to the test requirements with n-hexane diluted to the appropriate concentration Mixed standard working solution. The standard stock solution is protected from 0 to 4°C.

4.4.2 Iodine urea matrix standard working solution. Isozyme matrix standard working solution is prepared with a sample blank solution of different concentrations of the substrate standard Quasi-working solution for standard curve. Substrate standard working solution is now available.

4.5 Materials

4.5.1 Gel purification column. 400mm × 25mm. Filler Bio-Beads S-X3, 38-75um, before use to do the leaching curve.

4.5.2 Graphitized Carbon Black Solid Phase Extraction Column. 250 mg, 3 mL or equivalent. Use the elution curve before use, before use with 3mL acetone - n - hexane (4.2.2) pre-leaching.

6.1 Preparation of the sample

6.1.1 Apple, garlic, spinach, chestnut

Take a representative sample of about 500g (can not be washed), crushed with a pulverizer, into a clean container as a sample, sealed, marked mark.

6.1.2 Tea, rice

Take a representative tea, rice sample about 500 g, crushed to all through a 20 mesh sieve, into a clean container as a test Kind, sealed, marked mark.

6.1.3 Honey

Take a representative sample of about 500g, the crystallization of the sample will be evenly stirred; on the crystallization of the sample, in a closed case, the vials are allowed to warm in a water bath of not more than 60 ° C until all of the samples are melted and then stirred and cooled to room temperature.

Intended to prevent moisture from evaporation. Install the clean container as a sample, seal, mark the mark.

6.1.4 Chicken, fish, pig kidney, pork

To replace the sample of about 500g, crushed by the mashed evenly, into a clean container, sealed, marked mark.

6.1.5 Wine

Replace the sample 500 mL, shake, into a clean container, sealed, marked mark.

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

6.2 Sample storage

Cereals, nuts, tea, honey, alcohol samples stored at 0 ~ 4 °C, other samples were frozen at -18 °C below, in the During the operation of the sample preparation, the sample should be protected from contamination or the change in the content of the residue.

7.1 Extraction

7.1.1 Spinach, apple, garlic, wine, honey, chicken, fish, pig kidney, pork

Accurately weigh 5 g (10 g of honey) sample (accurate to 0.01 g) in a 50 mL centrifuge tube, add 20 mL of acetone-n-hexane, add Into the 2 g sodium chloride to 10000 r/min homogeneous 1min, the extract at 4000 r/min centrifugal 5 min, supernatant by anhydrous sodium sulfate Filtered and transferred to the concentration of the bottle, the residue by adding 20 mL of acetone - n-hexane and then extracted once, the combined extract, the extract at 40 °C Concentrated to near dry, with 10 mL of ethyl acetate - cyclohexane dissolved residue, to be purified.

7.1.2 Rice, chestnut, tea

Accurately weighed 10 g (2 g of tea) sample (accurate to 0.01 g) in 50 mL centrifuge tube, add 5 mL of water, soak for 30 min, Add 20 mL of acetone-n-hexane, add 2 g sodium chloride, homogenize at 10,000 r/min for 1 min, extract at 4000 r/min centrifugal 5 min, the supernatant was filtered through anhydrous sodium sulfate and transferred to a concentrated flask. The residue was added with 20 mL of acetone-n-hexane and extracted again.

The extract was concentrated at 40 ° C under reduced pressure to near dryness and the residue was dissolved with 10 mL of ethyl acetate-cyclohexane.

7.2 Purification

7.2.1 Gel chromatography (GPC) purification

A) Gel purification column. Bio Beads S-X3, 400 mm × 25 mm (inner diameter), or equivalent;

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

C) Flow rate. 5 mL/min;

D) Injection volume. 5 mL;

E) Collection time. 1100 s ~ 1800 s.

7.2.2 Spinach, tea

The purified liquid (spinach, tea) of the above sample is purified according to the conditions specified in 7.2.1, and the collected fraction is below 40 ° C Pressure to near dry, with 3 mL of acetone - n-hexane dissolved residue, and added to the graphitized carbon black solid phase extraction column, the total extract to be out and then eluted with 5 mL of acetone-n-hexane to maintain a flow rate of 1.0 mL/min. The whole effluent was collected and the whole effluent was blown with a nitrogen blower Close to dry, with n-hexane volume to 2.0 mL, gas chromatography-mass spectrometry.

7.2.3 Apple, garlic, honey, rice, chestnut, wine, chicken, fish, pig kidney, pork

(Apple, garlic, honey, wine, chicken, fish, pig kidney, pork, rice, chestnut) Purified according to the conditions specified in 7.2.1, the collected fractions were concentrated under reduced pressure at 40 ° C to near dryness and fixed to 2.0 mL with n-hexane.

Determination by Gas Chromatography - Mass Spectrometry.

7.3 Determination

7.3.1 Gas Chromatography - Mass Spectrometry Reference Conditions

A) Column. DB-5MS quartz capillary column, 30 mx 0.25 mm (id) x 0.25 m, or equivalent;

B) Column temperature. initial temperature 70 °C, keep 2 min, 20 °C/min up to 230 °C for 10 min;

C) Inlet temperature. 220 ° C;

D) chromatographic-mass spectrometer interface temperature. 250 ° C;

E) Carrier gas. helium, purity greater than or equal to 99.995%, 1.0 mL/min;

F) Injection volume. 1 µL;

G) Injection method. no split injection;

H) ionization mode. EI;

I) ionization energy. 70 eV;

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

K) Select ion (m/z).

(M/z 187,285,287 *), Bacillus sp. (M/z 188,259,331 *), Pythium (m/z 255, 283, 285 *), isobacteria (m/z 187,314,316 *), with * as a quantitative ion.

7.3.2 Quantitative determination

According to the sample of the target content of the situation, select the peak area similar to the standard working solution compared quantitative, Significantly, with a standard working solution. The target response values in the standard working solution and the sample solution shall be within the linear range of the instrumentation, Quasi - working solution and sample solution. Under the above chromatographic conditions, the retention time of each target is about. vinblastin (10.97 min), Bacteriocarbide (12.30 min), Pythium (12.63 min), Isoconazole (18.11 min), Total ion current of standard See Figure A-1 in Appendix A for chromatograms.

7.3.3 Qualitative determination

The standard solution and the sample solution are tested according to the conditions specified in 7.3.1. If the sample solution is in the same retention time as the standard solution Peak appears, then it is confirmed by mass spectrometry, after deducting the background of the sample spectrum, the selected ions all appear, while the selected ion from the the abundance ratio is consistent with the relative abundance of the ions associated with the standard, and the fluctuation range is within the maximum allowable deviation of Table 1.

In the corresponding target. The validated sample can be judged positive for detection. The ion abundance ratio of the fragment ions of the positive detectors under the condition of 7.3.1 (187. 285. 287 = 95. 100. 64), Bacillus sp. (188. 259. 331 = 100. 94. 75), Pythium (255. 283. 285 = 13. 100. 65), asparagine (187. 314. 316 = 33. 100. 64). Vibrio, A-2.3; A-2.3; A-2.4. A-2.2; A-2.3;

Table 1 Maximum allowable error for relative ion abundance using gas chromatography-mass spectrometry

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 quantification limit of the method was 0.025 mg/kg, 0.05 mg/kg of mycobacterium, the limit of quantification of staphylococcus aureus, Bacillus fungus and Pythium in rice, honey, chestnut was 0.005 mg/kg, 0.01% Wine, garlic, apple, spinach, fish, chicken, pig kidney, pork, 0.01 mg/kg, and.200 mg/kg of isoflavone.

10.2 Recovery rate

When the levels were 0.001 mg/kg, 0.05 mg/kg, 0.1 mg/kg, the vasopressin, bacteriocide, See Appendix B for the recovery rate.

Appendix A

(Informative)

The total ion chromatogram and mass spectrum of the bacterium,

Figure A. 1 Ethyl mycotoxin, Bacterial, Pythium, and Isozyme Total ion chromatogram

Figure A.2-1 Mass Spectrometry of Bacteriocin

Figure A.2-2 Spectrum of Bacteriide Standard

Figure A.2-3 Mass Spectrometry of Pythium

Figure A.2-4 Mass Spectrometry of Isoamylurea Standards

Appendix B

(Informative)

Table B.1 Bacteria in cotton, rice, garlic, apple, spinach, chestnut, wine, honey, fish, chicken, pig kidney, pig

Meat accuracy and precision test results (indoor addition recovery, n = 6) (%) sample name Add concentration (Mg/kg) Recovery rate range (%) Precision (%) tea 0.025 87.5 ~ 100.8 4.8 0.1 74.7 to 89.6 6.9 0.2 84.0 ~ 94.5 4.2 Rice 0.005 85.1 to 103.2 7.2 0.02 78.6 ~ 99.5 8.2 0.05 90.4 to 107.2 7.5 0.10 85.2 ~ 98.1 5.0 wine 0.01 77.0 to 89.7 6.0 0.05 82.1 to 87.9 2.7 0.10 95.6 ~ 97.9 0.9 spinach 0.01 77.6 to 89.5 5.3 0.05 71.0 to 90.7 11.1 0.10 78.0 to 107.2 12.8 apple 0.01 74.3 to 90.0 6.8 0.05 84.6 to 99.6 6.1 0.1 92.2 ~ 103.8 4.3 garlic 0.01 93.6 to 107.2 5.1 0.05 87.4 to 102.0 6.3 0.1 91.5 to 99.9 3.6 honey 0.005 87.9 to 106.3 6.9 0.05 92.7 to 109.2 6.2 0.10 93.0 to 101.2 3.0 Chestnut 0.005 75.3 ~ 97.9 11.4 0.05 76.0 ~ 104.8 12.2 0.10 86.1 to 104.6 7.8 chicken 0.01 88.4 to 104.0 6.7 0.05 90.0 to 99.0 3.9 0.10 86.6 ~ 94.4 3.6 Carassius meat 0.01 78.2 to 97.0 7.8 0.05 86.5 to 100.0 5.7 0.10 96.8 to 105.6 3.5 pork 0.01 72.8 to 95.7 10.9 0.05 83.2 ~ 105.2 8.5 0.10 88.2 to 98.1 4.1 0.20 78.4 to 96.8 7.5 Pig kidney 0.01 75.0 to 93.2 8.2 0.05 72.9 to 89.8 7.7 0.10 76.4 ~ 96.9 9.8

Table B.2 Bacteria in tea, rice, garlic, apple, spinach, chestnut, wine, honey, fish, chicken, pig kidney, pork

Accuracy and precision test results (indoor addition recovery, n = 6) (%) sample name Add concentration (Mg/kg) Recovery rate range (%) Precision (%) tea 0.025 79.0 to 104.4 9.0 0.1 81.4 ~ 98.3 7.2 0.2 75.2 to 100.4 10.7 Rice 0.005 93.1 to 100.0 2.7 0.02 76.8 ~ 106.2 11.5 0.05 87.4 ~ 97.8 4.2 0.10 86.8 ~ 106.2 7.8 wine 0.01 72.8 to 91.8 8.6 0.05 71.2 ~ 80.3 4.6 0.10 78.0 to 100.9 9.6 spinach 0.01 83.6 to 96.7 5.6 0.05 75.5 to 95.8 10.3 0.10 84.1 to 108.7 9.8 apple 0.01 74.5 to 81.4 3.2 0.05 70.8 to 96.1 11.1 0.1 81.6 ~ 94.3 5.4 garlic 0.01 92.2 to 105.0 4.8 0.05 85.7 to 97.0 5.1 0.1 93.7 to 100.3 2.7 honey 0.005 81.5 to 97.1 6.7 0.05 72.6 ~ 95.9 11.1 0.10 78.6 ~ 90.6 4.9 Chestnut 0.005 90.2 to 96.2 2.5 0.05 83.1 to 109.2 10.4 0.10 78.9 to 100.2 9.7 chicken 0.01 78.5 to 101.5 9.9 0.05 85.0 to 100.4 6.6 0.10 83.9 to 99.2 6.8 Carassius meat 0.01 71.2 ~ 102.1 12.2 0.05 81.3 ~ 105.2 10.0 0.10 79.1 to 97.0 8.1 pork 0.01 90.2 to 98.2 3.3 0.05 92.4 to 102.6 4.0 0.10 89.0 to 97.1 3.0 0.20 83.1 to 104.1 7.8 Pig kidney 0.01 88.4 to 96.4 3.3 0.05 79.6 to 96.6 8.1 0.10 83.6 to 96.4 5.6

Table B.3 Pythium in tea, rice, garlic, apple, spinach, chestnut, wine, honey, fish, chicken, pig kidney, pork

Accuracy and precision test results (indoor addition recovery, n = 6) (%) sample name Add concentration (Mg/kg) Recovery rate range (%) Precision (%) tea 0.025 73.1 to 96.9 9.8 0.1 81.9 ~ 93.9 5.2 0.2 80.0 to 94.4 6.3 Rice 0.005 90.4 to 102.0 4.7 0.02 81.5 to 105.8 9.2 0.05 87.1 to 100.2 5.4 0.10 80.6 ~ 105.8 9.7 wine 0.01 77.0 to 89.0 5.2 0.05 76.1 to 86.4 4.8 0.10 91.6 ~ 94.8 1.3 spinach 0.01 78.4 to 92.3 6.4 0.05 71.0 to 86.3 7.8 0.10 74.0 to 102.7 12.6 apple 0.01 81.2 ~ 92.0 4.7 0.05 78.6 to 99.0 8.2 0.1 84.1 to 102.5 6.9 garlic 0.01 80.8 ~ 98.1 6.7 0.05 80.3 to 96.3 7.6 0.1 80.9 to 94.5 5.8 honey 0.005 76.8 to 99.7 9.8 0.05 80.9 to 96.0 6.3 0.10 82.9 ~ 98.9 6.9 Chestnut 0.005 78.5 to 109.5 11.6 0.05 88.2 to 101.4 5.1 0.10 90.7 to 109.0 8.2 chicken 0.01 90.0 to 104.0 5.4 0.05 93.2 ~ 104.4 4.4 0.10 77.4 ~ 93.4 7.3 Carassius meat 0.01 76.1 to 101.2 10.1 0.05 79.6 ~ 99.2 7.9 0.10 85.0 to 105.0 8.0 pork 0.01 75.5 ~ 104.5 11.8 0.05 79.8 ~ 87.2 3.4 0.10 93.1 to 102.1 3.3 0.20 83..4 ~ 99.5 6.2 Pig kidney 0.01 74.7 to 95.8 9.6 0.05 81.7 to 100.7 6.9 0.10 87.8 ~ 98.7 4.5

Table B.4 Iodine in tea, rice, garlic, apple, spinach, chestnut, wine, honey, fish, chicken, pig kidney, pork

Accuracy and precision test results (indoor addition recovery, n = 6) (%) sample name Add concentration (Mg/kg) Recovery rate range (%) Precision (%) tea 0.05 75.7 to 113.0 13.1 0.1 87.5 to 114.5 10.8 0.2 87.6 ~ 97.4 4.0 Rice 0.01 78.1 to 112.6 13.9 0.02 83.4 ~ 105.0 8.7 0.05 92.4 to 106.5 5.6 0.10 96.1 to 105.0 3.3 wine 0.02 92.4 to 108.0 6.3 0.05 78.5 to 95.0 7.9 0.10 71.8 to 91.0 9.3 spinach 0.02 78.6 to 91.6 5.6 0.05 88.5 to 105.0 6.9 0.10 76.0 to 95.8 9.1 apple 0.02 100.0 to 109.0 3.2 0.05 89.5 to 108.0 7.0 0.1 97.8 to 107.8 3.7 garlic 0.02 99.4 to 109.0 3.4 0.05 89.0 to 107.5 8.0 0.1 98.6 ~ 110.2 4.7 honey 0.01 87.7 to 110.0 8.0 0.05 76.0 ~ 104.5 11.2 0.10 90.6 ~ 105.8 6.1 Chestnut 0.01 93.4 to 110.0 6.1 0.05 91.0 to 117.0 9.5 0.10 87.4 to 109.4 9.1 chicken 0.02 92.0 to 102.0 3.9 0.05 88.0 to 104.5 6.9 0.10 90.8 to 101.6 4.2 Carassius meat 0.02 77.4 ~ 107.7 11.7 0.05 90.9 to 108.7 7.7 0.10 92.6 ~ 108.9 6.5 pork 0.02 83.0 to 116.0 11.4 0.05 87.5 to 100.8 5.7 0.10 74.8 to 89.6 6.7 0.20 78.2 ~ 93.4 6.4 Pig kidney 0.02 84.0 ~ 94.5 4.3 0.05 73.8 ~ 112.1 14.2 0.10 81.7 to 106.7 9.3 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