GB 23200.51-2016Food safety national standard -- Determination of dormone residues in food by liquid chromatography-mass spectrometry / mass spectrometry (English PDF)

4.1 Reagents

4.1.1 Acetonitrile (C2H3N). Chromatographic pure.

4.1.2 n-hexane (C6H14). pure chromatography.

4.1.3 Acetone (C3H6O). Chromatographic pure.

4.1.4 ether (C4H10O). chromatographic purity.

4.1.5 sodium chloride (NaCl). excellent grade pure.

4.1.6 anhydrous sodium sulfate (Na2SO4). analysis of pure, 650 °C burning 4 h, after natural cooling stored in a sealed bottle in reserve.

4.1.7 Potassium hydrogen phosphate (K2HPO4). analytical grade.

4.1.8 Potassium dihydrogen phosphate (KH2PO4). Analytical purity.

4.1.9 Sodium hydroxide (NaOH). Analytical purity.

4.1.10 hydrochloric acid (HCl). excellent grade pure.

4.2 Solution preparation

4.2.1 1 mol/L Sodium hydroxide. Weigh 40 g of sodium hydroxide and dissolve in 1 L of water.

4.2.2 1 mol/L hydrochloric acid. Weigh 36.5 g of pure hydrochloric acid and dissolve in 1 L of water.

4.2.3 N-Hexane Saturated Alice. 100 mL of acetonitrile and 100 mL of n-hexane were added to the 250 mL separatory funnel, the bottom layer is n-hexane saturated acetonitrile.

4.2.4 Acetone-n-Hexane (1 1, V/V). Mix an equal volume of acetone and n-hexane to shake well.

4.2.5 Phosphate buffer. 0.5 mol/L (pH = 7.0), weigh 52.7 g of dipotassium hydrogen phosphate and 30.2 g of potassium dihydrogen phosphate, add about 500 mL Water dissolved, with 1 mol/L sodium hydroxide or 1 mol/L hydrochloric acid to adjust pH7.0, add water to volume 1 L.

4.3 Standards

4.3.1 Dinotefuran reference substance. (dinotefuran, C7H14N4O3), CAS NO. 165252-70-0, purity greater than or equal to 98%.

4.4 Standard solution preparation

4.4.1 Dipyrazole standard stock solution. Weigh the appropriate amount of dinotefuran standard, with acetone - n-hexane (1 1, V/V) prepared into 1.0 mg/mL standard Quasi-stock solution; 0 ~ 4 0C save. The shelf life is one year.

4.4.2 Dipyrazole standard working solution. Prepare the stock solution with acetone-n-hexane (1 1, V/V) to form a suitable concentration Working fluid. 0 ~ 4 0C save. The shelf life is one month.

4.5 Materials

4.5.1 Envi-Carb/LC-NH2 solid phase extraction column. 500 mg/500 mg. Supelclean ENVI-Carb SPE9 (Graphitized non-porous Carbon) in series with Supelclean LC-NH2 SPE (amide propylsilylated silica gel).

4.5.2 Filtration. 0.2 µm.

6.1 Preparation of the sample

6.1.1 Wheat, corn

Approximately 500 g of representative sample, crushed with a pulverizer and passed through a 2.0 mm round hole screen. Mix well, into a clean container, Sealed, marked.

6.1.2 Fruit, vegetables, fish, meat

Approximately 500 g of representative sample was taken and the sample was processed into a slurry with a food masher. Mix well, into a clean container, Sealed, marked.

6.1.3 Peanuts

Take a representative sample 500 g, all grinding with an attritor. Mix well, into a clean container, sealed, marked mark.

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

6.2 Sample storage

Wheat, corn, peanut samples stored at 0 ~ 4 °C; fish, meat and fruits and vegetables samples frozen at -18 °C below the sample.

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

7.1 Extraction

7.1.1 Wheat, corn, peanuts

Weigh 5 g of sample (accurate to 0.01 g) in a 100 mL centrifuge tube and add 20 mL of water for 5 min. Add 30 mL of acetonitrile to 10,000 R/min homogeneous extraction 1 min, centrifugation 3 min, the extract was filtered to 250 mL separatory funnel, the residue and then 20 mL of acetonitrile repeated extraction of a Times. The combined extracts were added to a separatory funnel and 20 g of sodium chloride and 60 mL of phosphate buffer were added successively to shake for 3 min. After standing, discard the water Floor. Acetonitrile layer by adding 5 g of anhydrous sodium sulfate dehydration at 40 ° C after rotation concentrated to near dry.

7.1.2 Fruits, vegetables

Weigh 10 g sample (accurate to 0.01 g) in 100 mL centrifuge tube, add 30 mL of acetonitrile, at 10000 r/min homogeneous extraction 1 min, After centrifugation for 3 min, the extract was filtered into a 250 mL separatory funnel and the residue was extracted again with 20 mL of acetonitrile. The combined extracts were separated Funnel, followed by adding 20 g of sodium chloride and 60 mL of phosphate buffer, shaking 3 min. After standing, discard the water layer. Acetonitrile layer added 5 g no Sodium sulfate dehydration after 40 ° C rotation concentrated to near dry.

7.1.3 Fish and meat

Weigh 10g sample (accurate to 0.01 g) in 100 mL centrifuge tube, add 30 mL of acetonitrile, whirlpool mixed evenly after ultrasonic extraction 15 min, centrifuged 3 min, the extract was filtered into a 250 mL separatory funnel, and the residue was extracted again with 20 mL of acetonitrile. Merge extraction Liquid in a separatory funnel, followed by adding 20 g of sodium chloride and 60 mL of phosphate buffer, shaking 3 min. After standing, discard the water layer. Acetonitrile layer plus Into 5 g anhydrous sodium sulfate after dehydration at 40 ° C rotation concentrated to near dry.

7.2 Purification

7.2.1 Wheat, corn, peanuts, fish, meat

The extract was dissolved in 20 mL of n-hexane and transferred to a 150 mL separatory funnel and dissolved with 20 mL of n-hexane saturated acetonitrile Liquid extraction 2 times, take the next solution to join the extract, 40 °C under reduced pressure concentrated dry, add 2 mL of acetonitrile dissolved.

Envi-carb/LC-NH2 (500 mg/500 mg) was pre-eluted with 10 mL of acetonitrile and the effluent was discarded. The above obtained solution was poured, and rinsed with 20 mL of acetonitrile. The eluate was collected and concentrated to near dryness at 40 ° C. The residue was dissolved in 2.0 mL of acetonitrile and passed through 0.2 µm Membrane for liquid chromatography - mass spectrometry.

7.2.2 Vegetables, fruits

Envi-carb/LC-NH2 (500 mg/500 mg) was pre-eluted with 10 mL of acetonitrile and the effluent was discarded. The above obtained solution was poured, and rinsed with 20 mL of acetonitrile. The eluate was collected and concentrated to near dryness at 40 ° C. The residue was dissolved in 2.0 mL of acetonitrile and passed through 0.2 µm Membrane for liquid chromatography - mass spectrometry.

7.3 Determination

7.3.1 Gas Chromatography - Mass Spectrometry Reference Conditions

A. Column. Waters Acquity Uplc BEH C8 1.7 µm B. Mobile phase. water (A) acetonitrile (B) gradient

Table 1 Mobile phase gradient

Time/min flow rate/mL/min% A% B 0.00 0.25 60 40 0.30 0.25 60 40 2.00 0.25 30 70 2.50 0.25 60 40 C. Column temperature. 30 ° C.

D. Flow rate. 0.2 mL/min.

E. Injection volume. 10 µL.

F. Running time. 3.5 min G. Mass spectrometry conditions See Appendix A.

7.3.2 Determination and confirmation of chromatography

According to the content of the sample in the sample, the selected concentration of similar standard working solution, the standard working solution and sample solution volume into the sample Determination of the standard working solution and the sample solution of dindue in the response of the instrument should be within the linear range.

If the sample is in the mass chromatogram of the standard working solution, the chromatographic peaks appear at the same retention time, and the selected ions appear.

The abundance ratio of the selected ions is proportional to the abundance ratio of the corresponding ions of the standard, and the value is within the allowable range (see Table 1 for the allowable range). In condition 7.3.1 , The retention time of dinotefuran is 0.6 min. Under the condition of 7.3.1, the spectrum of the dinotefuran standard is shown in Appendix B.

Table 2 Use Qualitative Liquid Chromatography - Mass Spectrometry Relative Ion Abundance Maximum Allowable Deviation

Relative ion abundance > 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 this method was 10 µg/kg.

10.2 Recovery rate

The experimental data on the concentration and recovery of the sample are given in Appendix C.

Appendix A

(Informative)

Table A.1 Mass spectrometry conditions

Table A.2 Multi-reaction monitoring conditions

Note. Add "*" ions for quantitation.

Non-Commercial Notices. The parameters listed in Appendix A are performed on a Waters Quattro Premier mass spectrometer, where the test instrument model Only to provide a reference, does not involve commercial purposes, to encourage standard users to try to use different manufacturers or models of equipment.

Ionization mode ESI

Capillary voltage 3.0 kV the source temperature is 120 ° C the solvent temperature was 350 ° C Taper hole air nitrogen, 50L/h Solvent gas stream nitrogen, 600 L/h Collision air pressure argon, 3.10 × 10-6 Pa Monitoring mode multiple reaction monitoring Compound ion ion ion residence time cone hole voltage collision energy Dinoprams 202.9 128.8 * 0.10 s 20 V 12 eV 156.8 0.10 s 20 V 10 eV Appendix B (Informative) Standard substance chromatogram

Figure B.1 Damp Fungus Liquid Chromatography-Mass Spectrometry/Mass Spectrometry Multi-Reaction Monitoring Chromatogram

Appendix C

(Informative)

Sample concentration and recovery of the experimental data

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

sample name

Add concentration

(Mg/kg)

Recovery rate(%)

peanut

10 67.57%

20 74.17%

40 82.68%

corn

10 73.27%

20 80.33%

40 83.19%

wheat

10 68.50%

20 74.23%

40 81.81%

Basil

10 76.55%

20 83.03%

40 89.17%

pork

10 70.77%

20 79.76%

40 84.73%

spinach

10 79.83%

20 83.40%

40 88.16%

Salmon

10 72.20%

20 80.18%

40 87.94%

apple

10 78.53%

20 82.36%

40 87.07%

Appendix D

(Normative appendix) Laboratory repeatability requirements

Table D.1 Laboratory repeatability requirements

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

Table E.1 Inter-laboratory reproducibility requirements

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