GB 23200.63-2016Food safety national standard -- Determination of thiazolylamine residues in food by liquid chromatography-mass spectrometry / mass spectrometry (English PDF)

4.1 Reagents

4.1.1 Cyclohexane (C6H12,110-82-7). Chromatographic Purification.

4.1.2 Ethyl acetate (C4H8O2,141-78-6). Chromatographic pure.

4.1.3 acetonitrile (C2H3N, 75-05-8). pure chromatography.

4.1.4 Methanol (CH4O, 67-56-1)). Chromatographic pure.

4.1.5 anhydrous sodium sulfate (Na2SO4,15124-09-1). 650 °C burning 4 h, in the dryer to cool to room temperature, stored in a sealed bottle spare.

4.1.6 Sodium chloride (NaCl, 7647-14-5).

4.2 Standards

4.2.1 tiadinil standard (C11H10ClN3OS, CAS. 223580-51-6). purity >= 97.0%.

4.3 Standard solution preparation

4.3.1 Thiamidylamine Pesticide Standard stock solution. accurately weighed the appropriate amount of thiadiazole pesticide standard substance, with acetonitrile prepared into a concentration of 1.0 Mg/mL standard stock solution.

4.3.2 Thiamidylamine Standard Intermediate Solution. Accurately absorb the appropriate amount of standard stock solution, diluted with acetonitrile to a concentration of 10.0 ug/mL Inter-solution.

4.3.3 Thiamidose standard working solution. Before use, the standard intermediate solution is diluted with a blank matrix of various samples to the appropriate concentration of the standard working fluid.

4.4 Materials

4.4.1 Amino solid phase extraction column..200 mg, 3 mL and 500 mg, 3 mL, or equivalent. Before use, use acetonitrile to the amino column (200 Mg) was activated twice a day for 2 mL. In the amino column (500 mg) filled with 0.5 g anhydrous sodium sulfate, before use with ethyl acetate activated 2 times, each time 2 mL.

4.4.2 CHROMABOND XTR Solid phase extraction column (large pore diatomaceous earth filler). 3000 mg, 15 mL, or equivalent.

4.4.3 Microporous membrane. 0.2 µm and 0.45 µm, organic phase.

6.1 Preparation of the sample

6.1.1 Lettuce, carrots, cabbage, citrus, grapes

Take a representative sample of about 500 g, chopped it, and processed into a slurry with a mashed machine. Mix well, fit into clean container, seal, mark mark.

6.1.2 Rice, chestnut, tea

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

6.1.3 Beef, chicken, sheep liver, tilapia

Take a representative sample of about 500 g, minced with a meat grinder, mix well, into a clean container, sealed, marked 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 a closed situation Conditions, the sample bottle placed in not more than 60 °C in the water bath in the warm, shaking, until the sample all melt and stir well, quickly cooled to 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 to Frozen under the preservation. 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 A 50 mL centrifuge tube, add 10.0 mL of ethyl acetate to wash the homogenizer tip, merge the homogeneous solution. Add 10 g of anhydrous sodium sulfate, shake 10 min. Extract 10 r/min centrifugal 5 min, draw 10.0 mL supernatant, the refrigerator below 0 °C placed 10 h, over 0.45 µm Machine 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 10.0 mL of ethyl acetate wash homogenizer head, combined homogeneous solution.

Add 10 g of anhydrous sodium sulfate, shake 10 min. The sample extract was centrifuged at 10000 r/min for 5 min, and 10.0 mL supernatant was taken Box below 0 °C for 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 a 50 mL centrifuge tube, add 10.0 mL of water, soak for 20 min, add 1 g Water sodium sulfate, vortex oscillation 1 min. Add 10.0 mL of ethyl acetate, vortex for 2 min, centrifuge the sample extract at 4000 r/min Min, learn supernatant. Then add 10.0 mL of ethyl acetate, vortex oscillation 2 min, 4000 r/min centrifugation 5 min, repeated extraction time, Combined with the supernatant, 45 °C blowing nitrogen concentrated to about 2 mL, constant volume to 10 mL, over 0.45 µm organic filter, to be gel permeation color Spectrum purification.

7.1.4 Honey

Weigh 2.0 g (accurate to 0.01 g) sample in 50 mL centrifuge tube, add 3 mL of water, 0.5 g sodium chloride, shock mix, over XTR Solid phase extraction column. After loading, the sample solution was kept for 5 min, eluted with 35 mL of ethyl acetate several times, the flow rate was 1.0 mL/min, the filtrate was collected in a 50 mL chicken heart and concentrated under reduced pressure at 45 ° C to about 0.5 mL to be purified.

7.2 Purification

7.2.1 lettuce, carrots, cabbage, citrus, grapes, chestnut, beef, sheep liver, chicken, tilapia, tomato sauce, rice, tea 10 mL of the obtained solution of 7.1.1, 7.1.2 and 7.1.3, and the supergel permeation chromatography was used to collect the effluent from 7 to 14 min.

Shrink the device, concentrated at 45 °C to near dry, 2 mL acetonitrile volume, as the initial purification solution.

After the primary cleaning solution was passed through the amino column (200 mg), the flask was washed three times with acetonitrile, 1 mL each time, and the lotion was the same.

System for 1 mL/min, collecting all the column solution in the blowing nitrogen tube, at 45 °C blowing nitrogen concentrated to near dry, with methanol volume to 1.0 mL, then Add 1.0 mL of deionized water, vortex oscillation uniform, over 0.2 µm organic filter, to be tested.

7.2.2 Honey

The concentrated solution was passed through an ammonia column (500 mg) and 4 mL of ethyl acetate was eluted three times at a flow rate of 1 mL/min.

Blowing nitrogen tube, at 45 ° C blowing nitrogen concentrated dry, with methanol volume to 1.0 mL, then add 1.0 mL of deionized water, vortex oscillation uniform 0.2 µm organic filter, to be tested.

7.3 Determination

7.3.1 Gel chromatographic purification conditions

7.3.1.1 Gel purification column. Bio-Beads, S-X3, 300 mm × 25 (inner diameter) mm; 38 µm to 75 µm.

7.3.1.2 Concentration temperature. 45 ° C.

7.3.1.3 Mobile phase. cyclohexane-ethyl acetate (50 50, volume ratio).

7.3.1.4 constant volume reagent. acetonitrile.

7.3.1.5 Flow rate. 4.7 mL/min.

7.3.1.6 Injection volume. 5 mL.

7.3.2 LC-MS/MS mass spectrometry reference conditions

7.3.2.1 Reference conditions for liquid chromatography

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 (90 10, volume ratio).

D) Flow rate. 0.30 mL/min.

E) Injection volume. 10 µL.

7.3.2.2 Reference conditions for mass spectrometry determination

See Appendix A.

7.3.3 Determination and confirmation of chromatography

According to the content of thiamizole in the sample solution, the standard working solution with similar peak area is selected. Standard working solution and sample solution of thiamylamine the response value shall be within the linear range of the instrument detection. Standard working solution and sample solution volume measurement. Under the above chromatographic conditions, thiophene the retention time of the amylamine is about 1.01 min. The selected component selects one parent ion and two or more daughter ions. Under the same experimental conditions, the sample in the sample to be tested and the standard solution corresponding to the retention time deviation within ± 2.5%; and the sample spectrum of each component of the qualitative ions Relative abundance and concentration of the standard solution in the standard solution corresponding to the relative abundance of qualitative ions were compared, the deviation does not exceed the provisions of Table 1 the samples were confirmed to be positive for thiamylamine detection. The spectrum of the standard is shown in Appendix B, Figures B.1, B.2.

Table 1 Maximum allowable deviation of relative ion abundance when qualitative confirmation

7.4 Blank experiment

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

10.1 Quantitation limits

The limit of quantification of the method is less than 10 µg/kg.

10.2 Recovery rate

When adding levels of 10 µg/kg, 20 µg/kg, 50 µg/kg, the addition rates of thiazolylamine in different matrices are given in Appendix C.

Appendix A

(Informative)

Reference to mass spectrometry conditions Reference to mass spectrometry conditions A) ion source. electrospray ion source (ESI).

B) Scan mode. negative ion scanning.

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

D) Electrospray voltage (IS). 4500V.

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 flow So that the mass spectrometry sensitivity reaches the detection requirement.

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

G) ion source temperature. 500 ° C.

H) Qualitative ion pairs, quantitative ion pairs, acquisition time, deblocking voltage and collision energy are shown in Table A.1.

Table A.1 thiamidonamine monitoring of ion pairs, quantitative ion pairs, deblocking 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) Thiamidomide 265.9/70.9 265.9/70.9 100 -61-35 265.9/237.9 -15 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 declaration. The reference mass spectrometry conditions listed in Appendix A are done on the API3000 LC/MS, where the test instrument model is provided for Test, does not involve commercial purposes, to encourage standard users to try different manufacturers or models of equipment.

Appendix B

(Informative)

Liquid samples of LC/MS-MS

Figure B.1 Thiadiazole standard ion full-scan mass spectrometry

Figure B.2 Thiamidamine Standard Multi-Reaction Monitoring (MRM) Chromatogram (10 µg/L)

Appendix C

(Informative)

Table C, 1 The recovery rate of thiamidomide pesticides in different matrices

Sample Substrate Add Concentration (µg/kg) Recovery (%) grape 10 92.4 ~ 108.0 20 78.5 ~ 95.0 50 71.8 ~ 91.0 lettuce 10 78.6 ~ 91.6 20 88.5 to 105.0 50 76.0 ~ 95.8 Cabbage 10 100.0 ~ 109.0 20 89.5 to 108.0 50 97.8 ~ 107.8 Tangerine 10 99.4 ~ 109.0 20 89.0 ~ 107.5 50 98.6 ~ 110.2 Chestnut 10 87.7 ~ 110.0 20 76.0 ~ 104.5 50 90.6 ~ 105.8 carrot 10 93.4 ~ 110.0 20 91.0 ~ 117.0 50 87.4 ~ 109.4 ketchup 10 92.0 to 102.0 20 88.0 to 104.5 50 90.8 ~ 101.6 chicken 10 90.7 to 103.0 20 87.5 ~ 104.5 50 87.6 ~ 97.4 Tilapia 10 88.4 to 104.0 20 90.0 to 99.0 50 86.6 ~ 94.4 400 82.0 ~ 105.8 beef 10 67.7 ~ 87.4 20 86.5 ~ 100.0 50 96.8 ~ 105.6 Sheep liver 10 72.8 ~ 95.7 20 78.5 ~ 109.5 50 68.2 to 101.4 Rice 10 90.7 to 109.0 50 90.0 to 104.0 100 93.2 ~ 104.4 1000 103.8 ~ 111.0 tea 10 77.4 ~ 93.4 20 83.0 ~ 116.0 50 79.6 ~ 99.2 honey 10 85.0 to 105.0 20 75.5 to 104.5 50 79.8 ~ 87.2 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