GB 23200.32-2016Food safety national standard -- Determination of butyryl hydrazine residues in foodstuffs by gas chromatography-mass spectrometry (English PDF)

4.1 Reagents

4.1.1 n-hexane (C6H14). chromatographic purity.

4.1.2 Sodium hydroxide (NaOH).

4.1.3 Sodium chloride (NaCl).

4.1.4 glacial acetic acid (C2H4O2).

4.1.5 Salicylaldehyde (C7H6O2).

4.1.6 Ethyl acetate (C4H8O2). Chromatographic purity.

4.2 Solution preparation

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

4.2.2 acetic acid solution (10%, v/v). Remove 10 mL of glacial acetic acid by adding 90 mL of water and mix well.

4.2.3 Sodium hydroxide solution (50%, m/v). Weigh 500 g of sodium hydroxide gradually dissolved in 1 L of water, cooled and mixed thoroughly.

4.3 Standards

4.3.1 1,1-dimethyl hydrazine, C2H8N2, CAS. 57-14-7). purity >=98%.

4.4 Standard solution preparation

4.4.1 1,1-dimethyl hydrazine reserve solution. accurately weighed the appropriate amount of 1,1-dimethyl hydrazine standard 0.1 g (accurate to 0.1 mg), Was added to a small beaker containing 2 mL of acetic acid solution and transferred to 1 L brown volumetric flask with a standard concentration of 100 µg/mL.

Preparation of liquid. The standard stock solution is diluted with water to the standard working solution at the appropriate concentration. The stock solution was stored at 4 ° C in the dark. each 6 months to prepare once.

6.1 Preparation of the sample

6.1.1 Fruits and vegetables

Take apples, parsley and other representative samples of about 500 g, chopped, with a crusher to sample processed into a slurry. Mix well into a clean capacity Inside, sealed and marked. -18 °C below the preservation.

6.1.2 Grains and nuts

Take about 500 g of representative samples of rice, soybeans, peanuts, etc., crushed with a pulverizer and passed through a 20 mesh round hole. Mix well into the clean Net inside the container, sealed and marked. 4 °C below the preservation.

6.1.3 Animal source food

Take a typical sample of chicken, fish, etc. Approximately 500 g minced with a meat grinder, mix well, fit into a clean container, sealed and marked.

-18 °C below the preservation.

6.1.4 Honey and tea

Take about 500 g of representative samples of honey and tea, into a clean container, sealed and marked. 4 °C below the preservation.

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

7.1 Extraction

Grain, Nuts. Weigh the sample 25 g (accurate to 0.1 g) In a 250 mL stoppered Erlenmeyer flask, add 100 mL of water, Oscillator oscillates for 30 min. Centrifuge at 4 000 r/min for 10 min, pour the supernatant into a distillation flask, add 50 mL of sodium hydroxide solution, A small amount of water to wash the bottle wall, connect the steam distillation device, slowly heated to boiling. Add 3 mL of acetic acid solution in advance, 25 mL of salicylaldehyde 50 µL with a plug test tube to receive distillate, collecting distillate about 15 mL.

Vegetables, fruit, aquatic products, muscle tissue, tea, honey. weighed 25 g (accurate to 0.1 g) at 250 mL Bottle, add 100 mL of water, cover the plug, shaking on the oscillator for 30 min. The extract was poured directly into a distillation flask and the sodium hydroxide solution 50 was added ML, rinse the bottle with a small amount of water, connect the water vapor distillation unit, slowly heated to boiling. With the addition of acetic acid solution in advance 3 mL, salicylaldehyde 50 µL of the 25 mL stopper tube to receive distillate, collecting distillate about 15 mL.

7.2 Derived

The tube was received at 50 ° C for 30 min. After cooling to room temperature, add about 3 g of sodium chloride, 5 mL of n-hexane and shake for 1 min. Static stratification, the upper n-hexane phase was purified or GC-MS (vegetable, fruit, tea, honey samples were purified by 7.3 and other samples were directly injected).

The appropriate volume of 1,1-dimethylaminide standard solution was derivatized as described above for GC-MS determination.

7.3 Purification

For vegetables, fruits, tea, honey samples, with silica gel solid phase extraction column (SPE) purification. The solid phase extraction column was pre-leached with 10 mL of n-hexane Wash, accurately remove the hexane solution 4 mL to the solid phase extraction column, rinse with 10 mL of n-hexane, and then 5 mL of ethyl acetate n-hexane solution leaching, 10 mL of ethyl acetate n-hexane solution. The eluate was collected and concentrated to dryness under reduced pressure at 50 ° C and set to 4 mL with n-hexane for GC-MS Determination.

7.4 Determination

7.4.1 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. 70 ° C 270 ° C (5 min);

C. Inlet temperature. 240 ° C;

D. Chromatography - 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 valve;

H Ionization mode. EI;

I Ionization energy. 70 eV;

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

K Monitoring ions (m/z). quantification 164, qualitative 149,163,165;

L. Solvent delay. 6 min.

7.4.2 Determination and confirmation of chromatography

7.4.2.1 Quantitative determination

According to the content of butyryl hydrazine in the sample solution, the selective concentration of 1,1-dimethyl-labeled standard solution was similar. 1,1-dimethyl the reaction value of the 1,1-dimethylaminobenzene derivative in the standard solution and the sample solution should be within the linear range of the instrument. The 1,1-15 ° C/min Dimethylsilane standard working solution derivatives and sample volumes were measured by volume injection.

Under the above chromatographic conditions, the retention time of 1,1-dimethylaminide derivatives was about 8.17 min. 1,1-dimethylaminide standard derived the chromatogram of the material SIM is shown in Appendix A, Figure A.1.

7.4.2.2 Gas Chromatography-Mass Spectrum Confirmation

Standard solution derivatives and sample solutions are determined according to the conditions specified in 7.4.1, if the sample solution with the standard solution derivatives of the same retention Between the emergence of a peak, then its mass spectrometry confirmed. It was confirmed that the retention time of the SIM chromatographic peak was consistent with that of the standard sample, in addition to the background of the sample spectrum, the selected ions appear; select ion m/z 164, 149,163,165 (its abundance ratio 100. 8. 7. 14) the relative abundance of the ions associated with the standard sample derivative is consistent and the similarity is within the allowable deviation (see Table 1). 1,1-dimethyl-linked standard the spectrum of the quasi-derivative SIM is shown in Figure A.2 in Appendix A.

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

7.5 Blank test

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

10.1 Quantitation limits

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

10.2 Recovery rate

The sample addition concentration and recovery data are given in Appendix B.

Relative abundance (base) 50% 20% to 50% 10% to 20% <=10% Allowable relative deviation ± 20% ± 25% ± 30% ± 50% X = Appendix A (Informative) Standard chromatogram

Figure A.1 1,1-Dimethylaminide Derivative Selective Ion Monitoring Mode (S IM) Total Ion Flow Diagram (T IC)

144 150 156 162 168 174

Figure A.2 1,1-Dimethylaminide Derivative Selective Ion Monitoring Mode (S IM) Mass Spectrometry

1,1-dimethylbisamine derivatives TR/min Abundance Abundance M/z Appendix B (Informative) Sample concentration and recovery of the experimental data

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

sample

name

0.01 mg/kg addition

Recovery rate range (%) Precision (RSD%) 0.02 mg/kg add Recovery rate range (%) Precision (RSD%) 0.05 mg/kg add Recovery rate range (%) Precision (RSD%) Peanut 90.5 100.3 7.8 89.2 95.6 4.6 88.6 92.4 3.4 Rice 91.2100.7 7.0 86.595.6 6.8 86.292.2 3.4 Soybean 90.7 97.8 5.6 91.9 99.8 5.8 87.8 94.4 3.8 Parsley 85.796.2 8.0 81.392.6 6.0 82.790.2 6.4 Apple 84.695.1 7.4 85.695.7 6.6 85.093.2 4.2 Fish 86.794.9 4.8 87.896.7 3.0 90.298.0 3.4 Chicken 88.094.6 4.0 92.397.8 3.8 93.198.2 2.2 Tea 96.0104.7 5.8 97.1 103.5 5.2 98.9 106.8 6.0 Honey 94.3103.7 6.4 80.489.2 6.8 85.492.7 4.8 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