GB 23200.45-2016Food safety national standard -- Determination of difluomide residues in food by liquid chromatography-mass spectrometry (English PDF)

4.1 Reagents

4.1.1 Acetonitrile (CH3CN). Chromatographically pure.

4.1.2 n-hexane (C6H14).

4.1.3 glacial acetic acid (CH3COOH).

4.1.4 anhydrous sodium acetate (CH3COONa).

4.1.5 sodium chloride (NaCl). 450 °C burning 4 h, sealed standby.

4.1.6 anhydrous sodium sulfate (Na2SO4). 650 °C burning 4 h, storage dryer in reserve.

4.1.7 anhydrous magnesium sulfate (MgSO4. 650 °C burning 4 h, storage dryer in the spare.

4.1.8 Primary secondary amine (PSA). PSA filler or equivalent, particle size 40 µm.

4.1.9 Octadecyl silane bonded phase (C18). C18 filler or equivalent, particle size 50 µm.

4.2 Solution preparation

4.2.1 glacial acetic acid - acetonitrile solution (0.1 99.9, V/V). take 0.1 mL of glacial acetic acid by adding 99.9 mL of acetonitrile, mix and spare.

4.3 Standards

4.3.1 Pesticide standard. CAS No.. 35367-38-5, purity greater than or equal to 96%.

4.4 Standard solution preparation

4.4.1 Pesticide standard stock solution. accurately weighed appropriate amount of insecticides (accurate to 0.1 mg), with acetonitrile prepared into a concentration of 1.0 Mg/mL standard stock solution, set -18 °C refrigerator to save, the shelf life of 6 months.

4.4.2 Deicerurea standard working solution. according to the need to use acetonitrile standard stock solution diluted to the appropriate concentration of standard working fluid, temporary use with 4.4.3 0.005 mol/L ammonium acetate aqueous solution. Weigh 0.3850 g ammonium acetate, dissolved in water to 1 L, over 0.45 µm filter preparation use.

4.5 Materials

4.5.1 Microporous membrane. 0.22 µm, organic.

6.1 Preparation of the sample

6.1.1 Rice, corn, soybeans, wheat, mushrooms (dry), tea

500 g of the sample was taken and crushed to make it all through the sample sieve, mixed into a clean container, sealed and identified.

6.1.2 Peanuts (peanuts), oranges, apples, celery, onions, mushrooms (fresh)

Take a representative sample 500 g, crushed with a pulverizer. Mix, fit into a clean container, sealed and marked.

6.1.3 Chicken, beef, pork, liver

The samples were mashed with tissue mills and mixed thoroughly. Take 250 g mashed samples with high-speed tissue homogenizer to paste, if not immediately measured You need to cryopreservation.

6.1.4 Beef, beef liver, fish, chicken

Take a representative sample of 500 g, take the edible part of the crusher fully mashed evenly, into the clean sample container, sealed and marked mark.

6.1.5 Honey

To replace the sample 500 g, for non-crystalline honey samples, stir evenly. For a crystalline sample, in a closed case , Below 60 °C in the water bath in the warm, shaking, until the sample all melt and stir well, quickly cooled to room temperature, stored at room temperature.

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

6.2 Sample storage

Cereals, nuts, tea and other dehydration samples can be sealed at room temperature; fruit, vegetable samples should be sealed cold storage, such as If you do not carry out immediate testing, you need to cryopreservation; livestock and poultry meat and their products need to be frozen.

During sample preparation and sample preservation, the contamination of the sample or the change in the content of the analyte should be prevented. All prepared samples should be placed in a clean, sealed Container and make a mark. Water samples should be thoroughly mixed after thawing.

7.1 Extraction

7.1.1 Rice, corn, soybeans, wheat, peanuts (kernel)

Weigh 5g sample (accurate to 0.01 g) in 50 mL stoppered centrifuge tube, add 5 mL of water to mix for 30 min. Add 10 mL Glacial acetic acid - acetonitrile solution, add 1 g of anhydrous sodium acetate and 2 g sodium chloride, vortex oscillation 2 min, 30 °C constant temperature water bath ultrasonic extraction 30 Min after 5000 r/min centrifugation 10 min, the supernatant was filled with an appropriate amount of anhydrous sodium sulfate funnel collected in 50 mL with a plug centrifuge tube, adding 10 mL n-hexane saturated with acetonitrile, shaking 5 min, standing stratification, discarded n-hexane layer, to be purified.

7.1.2 Oranges, apples, celery, onions, mushrooms (fresh), honey

Weigh 5 g of sample (accurate to 0.01 g) in a 50 mL stoppered centrifuge tube. Add 5 mL of water to mix and place for 30 min. Add 10 mL of ice Acetic acid-acetonitrile solution, add 1 g of anhydrous sodium acetate and 2 g sodium chloride, vortex oscillation 2 min, 30 °C constant temperature water bath ultrasonic extraction 30 min 5000 r/min centrifugal 10 min, the supernatant was filled with an appropriate amount of anhydrous sodium sulfate funnel collected in 50 mL with a plug centrifuge tube, to be purified.

7.1.3 Mushrooms (dry), tea

Weigh 2.5 g of sample (accurate to 0.01 g) in 50 mL stoppered centrifuge tube, add 10 mL of water to mix for 60 min. Add 10 mL Glacial acetic acid - acetonitrile solution, add 1 g of anhydrous sodium acetate and 2 g sodium chloride, vortex oscillation 2 min, 30 °C constant temperature water bath ultrasonic extraction 30 Min after 5000 r/min centrifugal 10 min, the supernatant was filled with an appropriate amount of anhydrous sodium sulfate funnel collected in 50 mL with a plug centrifuge tube, to be net the

7.1.4 Chicken, beef, pork, liver

Weigh 2.5 g of the sample (accurate to 0.01 g) in a 50 mL stoppered centrifuge tube. Add 10 mL of glacial acetic acid-acetonitrile solution and add 1 g Anhydrous sodium acetate and 2 g sodium chloride, homogenized 2 min, 30 °C constant temperature water bath ultrasonic extraction 30 min after 5000 r/min centrifugal 10 min, the upper the supernatant was filled with an appropriate amount of anhydrous sodium sulfate funnel collected in a 50 mL stoppered centrifuge tube, 10 mL of n-hexane saturated with acetonitrile, Min, standing stratified, discarded n-hexane layer, to be purified.

7.2 Purification

Weigh 0.05 g PSA, 0.1 g C18, 0.15 g anhydrous magnesium sulfate in a 10 mL glass stopper centrifuge tube, accurately absorb 7.1 Extract 2.0 mL to the centrifuge tube, the exact vortex oscillation 1min, to 5000 r/min speed centrifugation 2 min. Take 1 mL supernatant 0.22 µm organic microporous membrane, for liquid chromatography - tandem mass spectrometry.

7.3 Determination

7.3.1 Liquid Chromatographic Reference Conditions

A) Column. C18 column, 150 mm × 2.1 mm (inner diameter), particle size 5 µm, or equivalent;

B) mobile phase. A (acetonitrile), B (0.005 mol/L ammonium acetate aqueous solution). The gradient elution conditions are given in Table A.1 in Appendix A;

C) Injection volume. 10 µL;

D) Column temperature. 30 ° C;

E) Flow rate. 0.2 mL/min.

7.3.2 Mass spectrometry reference conditions

A) ion source. electrospray ion source (ESI);

B) scanning mode. negative ion scanning;

C) Detection method. Multiple reaction monitoring (MRM);

D) monitoring of ion pair. parent ion 308.9 (m/z), quantitative ion pair 308.9/288.9 (m/z); qualitative ion pair of 308.9/288.9 (M/z), 308.9/92.9 (m/z);

E) Other mass spectrometry reference conditions are given in Tables A.2 and A.3 of Appendix A.

7.3.3 Quantitative determination

According to the sample in the sample content of the selected concentration of similar standard working solution together for chromatographic analysis. Diazepam in the sample solution to be tested the response value should be within the linear range of the instrument detection. The standard working solution and the sample solution were measured by volume injection. In the above instrument strips , The retention time of the insecticide was about 7.51 min. Liquid Chromatography - Tandem Mass Spectrometry Total Ion Flow and Mass Chromatogram See Appendix B Figure B.1.

7.3.4 Qualitative determination

According to the above conditions to determine the sample and the standard sample, the sample to be measured in the chromatographic peak retention time and the standard corresponding to the retention time deviation should be the allowable deviation is less than ± 2.5%; and in the sample spectrum after subtracting the background, the relative abundance of each qualitative ion is close to the concentration Under the same conditions obtained under the standard solution spectrum, the maximum allowable relative deviation does not exceed the range specified in Table 1, you can determine the sample There is a corresponding measured object.

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 experiment

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

10.1 Quantitation limits

The method of insecticides in rice, corn, soybeans, wheat, peanuts (peanuts), oranges, apples, celery, onions, mushrooms (fresh) the quantitative limit of honey is 10.0 µg/kg, chicken, beef, pork, liver, mushroom (dry), tea limit of 20.0 µg/kg.

10.2 Recovery rate

The recovery of this method in the concentration of 10 ~ 500 µg/kg in different substrates is 65% ~ 115%. See Appendix C.

Appendix A

(Informative)

Reference Liquid Chromatography - Tandem Mass Spectrometry Conditions

Table A.1 Liquid Chromatography Elution Conditions

Time/min A /% B /%

0 ~ 1.0 40 60

3.0 to 8.0 70 30

8.1 to 10.0 95 5

10.1 to 15.0 40 60

Table A.2 Mass spectrometry conditions

Instrument parameter value Electromagnetic spray voltage (IS) -4500 V Atomization gas pressure (GS1) 45 psi (nitrogen) Air curtain gas pressure (CUR) 10 psi (nitrogen) Auxiliary gas pressure (GS2) 50 psi (nitrogen) Ion source temperature (TEM) 500 ° C

Table A.3 Multi-reaction monitoring conditions

Compounds

Mother ion

Ion

To cluster voltage

Collision voltage

Collision chamber

Port voltage /

Collision room out

Port voltage /

Dwell time /

(Msec)

Dexamethasone 308.9

288.9 *

-6.200

92.9 -70 -8.200

* Ions are used for quantification.

1) Non-commercial statements. The parameters listed in Appendix A are done on the API4000 mass spectrometer, where the test instrument model is only listed is to provide a reference, does not involve commercial purposes, to encourage standard users to try to use different manufacturers or models of equipment.

Appendix B

(Informative)

Liquid Chromatography - Mass Spectrometry/Mass Spectrometry

Figure B.1 Multiple reactivity monitoring (MRM) chromatograms of deferir urea reference material (0.01 ng/mL)

Appendix C

(Informative)

The recovery of diflubenzuron in different substrates

Table C.1 Add level and recovery range

Sample Name Add Concentration/(µg/kg) Recovery Rate Range /% Rice 10 73.3 ~ 82.6 20 70.5 to 101.5 50 77.2 ~ 107.1 corn 10 72.4 ~ 81.5 20 75.5 to 106.4 50 71.0 ~ 95.1 Soybeans 10 68.5 to 81.2 20 67.2 ~ 90.5 50 70.5 to 95.7 wheat 10 77.3 ~ 87.5 20 71.5 ~ 90.6 50 75.3 ~ 94.1 peanut kernel) 10 70.1 to 85.5 20 72.0 ~ 98.7 50 73.5 ~ 86.2 orange 50 85.6 ~ 107.6 100 87.2 ~ 115.4 500 79.5 ~ 109.5 apple 50 86.3 ~ 115.5 100 82.4 ~ 107.6 500 85.0 ~ 109.2 Celery 10 75.3 ~ 89.6 20 85.0 ~ 103.5 50 82.6 ~ 105.5 onion 10 81.2 ~ 94.8 20 75.0 ~ 112.8 50 72.4 ~ 102.5 Mushrooms (fresh) 10 72.1 to 101.5 20 76.0 ~ 87.6 50 71.2 ~ 94.6 honey 10 75.0 to 109.2 20 81.0 to 106.8 50 80.5 to 105.5 chicken 20 90.0 to 108.2 40 85.2 ~ 96.0 80 82.0 to 105.7 beef 20 87.5 ~ 102 40 89.0 ~ 98.5 80 80.0 to 103.7 pork 20 92.0 to 100.0 40 93.8 ~ 107.5 80 82.5 to 102.7 Liver 20 85.0 ~ 114.2 40 87.5 ~ 105.3 80 78.6 ~ 108.5 Mushrooms (dry) 20 65.4 to 92.1 50 77.2 ~ 87.6 100 70.3 ~ 81.6 20 70.0 to 85.2 50 68.8 ~ 87.4 100 72.3 ~ 85.0 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