GB 23200.60-2016Food safety national standard -- Detection Method of Residue of Acetyl Acetate in Food (English PDF)

4.1 Reagents

4.1.1 Ethyl acetate (C4H8O2, CAS No. 141-78-6). Chromatographic purity.

4.1.2 Cyclohexane (C6H12, CAS No. 110-82-7). Chromatographic purity.

4.1.3 Acetonitrile (C2H3N, CAS No. 75-05-8). Chromatographic Purification.

4.1.4 N-hexane (C6H14, CAS No. 110-54-3). chromatographic purity

4.1.5 Acetone (C3H6O, CAS No. 67-64-1). Chromatographic Purification

4.1.6 anhydrous sodium sulfate (Na2O4S, CAS No. 15124-09-1). 650 °C burning 4 h, stored in a sealed container for use.

4.1.7 Sodium chloride (NaCl, CAS No. 7647-14-5).

4.2 Solution preparation

4.2.1 Ethyl acetate-cyclohexane mixed solvent (1 1, V/V). 500 mL of ethyl acetate and 500 mL of cyclohexane were added and mixed.

4.2.2 N-Hexane - Acetone Mixed Solvent (9 1, V/V). Take 90 mL of n-hexane and 10 mL of acetone.

4.2.3 Cyclohexane-ethyl acetate mixed solvent (6 1, V/V). Take 10 mL of ethyl acetate and 60 mL of cyclohexane.

4.3 Standards

4.3.1 Clenbuterol standard substance. (clodinafop-propargyl; CAS. 105512-06-9; Molecular formula. C17H13ClFNO4 Weight. 349.8) purity >=98.5%.

4.4 Standard solution preparation

4.4.1 standard stock solution (100 mg/L). accurately weighed the appropriate amount of standard material, dissolved with ethyl acetate, prepared into a concentration of 100 mg/L of the standard stock solution, the solution in 0-4 °C refrigerator to save.

4.4.2 standard working solution. if necessary, then diluted with ethyl acetate to the appropriate concentration of standard working solution. Standard working fluid should be used with the current distribution.

4.5 Materials

4.5.1 Graphitized Carbon Black and PSA Mixing Column. 1 mL, 50 mg Graphitized Carbon Black 50 mgPSA, mixed with 3 mL of cyclohexane-ethyl acetate Solvent activation.

4.5.2 HLB solid phase extraction column. 3 mL, 60 mg, or equivalent, with 2 mL of n-hexane-acetone mixed solution, 2 mL of acetone, 5 mL Distilled water pre-leaching.

4.5.3 Organic phase filter. 0.45 m.

6.1 Preparation of the sample

6.1.1 Grain and tea

The sample was divided by a quarter to 500 g, and the whole was ground with an attritor. Mix, are divided into two as a sample, into the clean Sheng Sheng Inside the bottle, sealed, marked with the mark.

6.1.2 Fruits and vegetables

Take 500 g of fruit or vegetable sample (not water wash), cut it, and then use a food mill to process the sample into a slurry shape. Mix, are divided into two as a sample, into the clean Sheng Sheng bag, sealed, marked mark.

6.1.3 Meat and meat products, nuts

A sample of about 1 kg was taken from all the samples taken and crushed evenly by the crusher and divided into two portions Net container as a sample. Sealed and marked.

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

6.2 Sample storage

Cereals, tea, nuts, honey and honey products samples stored at 0-4 °C; other samples in -18 °C below frozen save.

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

7.1 Extraction

7.1.1 Vegetables, fruits, grain

Vegetables, fruit, cereal weighed 10 g (accurate to 0.01 g) homogeneous sample, placed in 50 mL with a plug centrifuge tube, add 20 mL of acetic acid B Ester-cyclohexane mixture solvent, 10000 r/min homogenate 60 s, centrifuged at 4000 r/min for 3 min, the upper organic phase was collected and the residue was washed with 20 mL Ethyl acetate-cyclohexane, and the combined organic phases were combined and dehydrated over anhydrous sodium sulfate column. The mixture was collected in 150 mL of concentrated Bottle, concentrated in a 40 ° C water bath to near dry, accurately add 10.0 mL of ethyl acetate - cyclohexane dissolved residue, and over 0.45 m filter Membrane, to be gel chromatographic purification.

7.1.2 Walnut, rabbit meat, chicken liver, shrimp, chicken

Weigh 10 g (accurate to 0.01 g) homogeneous sample, placed in 50 mL stoppered plastic centrifuge tube, add 20 mL of acetonitrile, 10 mL distillation Water, 3 g sodium chloride, 10000 r/min homogenate 60 s, 4000 r/min centrifugation 3 min, the upper organic phase was collected and the residue was washed with 20 mL of acetonitrile the organic phase was combined and dehydrated over anhydrous sodium sulfate column. The mixture was collected in a 150 mL concentration flask and rotated in a 40 ° C water bath Concentrated to near dry, accurately add 10.0 mL of ethyl acetate - cyclohexane dissolved residue, and over 0.45 m filter, gel chromatography to be purified.

7.1.3 Tea, onions

Weigh 2.5 g (accurate to 0.01 g) homogeneous sample, placed in 50 mL stoppered plastic centrifuge tube, add 15 mL of distilled water, put it aside for 1 h, Into the 20 mL ethyl acetate-cyclohexane mixture solvent, 10000 r/min homogenate 60 s, 4000 r/min centrifugation 3 min, collecting the upper organic phase, residual the residue was extracted once again with 20 mL of ethyl acetate-cyclohexane mixed solvent. The combined organic phases were combined and dehydrated over anhydrous sodium sulfate.

In 150 mL concentrated flask, in 40 °C water bath spin concentrated to near dry, with ethyl acetate - cyclohexane mixed solvent accurate volume to 1.0 ML, over 0.45 m filter, to be solid phase extraction purification.

7.2 Purification

7.2.1 Vegetable, fruit, grain, walnut, rabbit meat, chicken liver, shrimp, chicken gel chromatography

7.2.1.1 Gel Chromatographic Purification Conditions

A) gel cleaning column. 300 mm × 10 mm (id), Bio Beads S-X3, 60-100 mesh, or equivalent;

B) Mobile phase. cyclohexane-ethyl acetate (1 1);

C) flow rate. 4.7 mL/min;

D) Sample loop. 5 mL;

G) Collection time. 7.5 min ~ 12.5 min.

7.2.1.2 Gel Chromatographic Purification Procedure

10mL of the solution to be purified according to 7.2.1 conditions, the collection of all the collection liquid in the nitrogen blowing tube, in the 35 °C water bath blowing to the dry, Ethyl acetate to 1.0 mL, determined by gas chromatograph, and confirmed by gas chromatography-mass spectrometry.

7.2.2 Vinegar, honey

Weigh 2.5 g (accurate to 0.01 g) homogeneous sample, add 5 mL water vortex oscillation mix for 30 s, the solution all through HLB solid phase extraction Column, the flow rate control in the 1 mL/min, and then 2mL distilled water leaching column, discard the eluent, vacuum drying 2 min, then 4 mL Alkane-acetone mixed solution, the eluate was collected in a centrifuge tube, dried at 40 ° C under nitrogen and 0.5 mL of ethyl acetate.

7.2.3 Tea, onions

Take 0.5 mL of the solution to be purified on the graphitized carbon black and PSA mixed column, eluted with 1.5 mL of ethyl acetate. cyclohexane mixed solvent, flow rate Control at 1 mL/min, collect all effluent in a centrifuge tube, dry at 40 ° C, 0.5 mL of ethyl acetate.

7.3 Determination

7.3.1 Gas Chromatographic Reference Conditions

A) Column. DB-1301 quartz capillary column, 30m × 0.25 mm (id) × 0.25 m, or equivalent performance;

B) Column temperature. 50 ° C 20 ° C/min.200 ° C 5 ° C/min 260 ° C (10 min);

C) Inlet temperature. 260 ° C;

D) detector temperature. 300 ° C;

E) Carrier gas. nitrogen, purity greater than or equal to 99.999%, column flow 2 mL/min;

F) Injection method. no shunt, 0.75 min after the opening of the diversion valve;

G) Injection volume. 1 µL.

7.3.2 Gas Chromatography - Mass Spectrometry Reference Conditions

A) Column. HP-5MS quartz capillary column, 30m × 0.25 mm (id) × 0.25 m, or equivalent performance;

B) Column temperature. 50 ° C (1 min) 10 ° C/min 280 ° C (10 min);

C) Inlet temperature. 250 ° C;

D) chromatographic - mass spectrometer interface temperature. 280 ° C;

E) ionization mode. EI;

F) Ionization energy. 70 eV;

G) Carrier gas. helium, purity greater than or equal 99.999%, flow rate 1 mL/min;

H) Injection method. no shunt, 0.75 min after the opening of the diversion valve;

I) Injection volume. 1 µL;

J) Measurement method. Select ion monitoring;

K) Select the monitoring ion (m/z). 349,267,238;

L) solvent delay. 5.0 min.

7.3.3 Determination and confirmation of chromatography

According to the content of acetone in the sample solution, the standard working solution with similar peak area is selected, the standard working solution and the solution the values shall be within the linear range of the instrument. Standard working solution and sample solution volume measurement. Under the above chromatographic conditions, the retention time of the herb is about 16.1 min. The chromatogram of the standard is shown in Appendix A, Appendix A.1.

The standard solution and sample solution are measured according to the conditions of 7.3.1, if the sample solution with the standard solution of the same retention time there is a peak, then the gas chromatography-mass spectrometry was confirmed.

The results show that the retention time of the chromatogram is consistent with that of the standard sample, and in the sample spectrum after subtracting the background, the selected ions are present; the abundance of the selected ions is consistent with the relative abundance of the ions associated with the standard sample, and the similarity is allowed Difference (see Table 1), you can determine the sample for the ciprofloxacin positive detection. Gas Chromatography - Mass Spectrometry Ion Chromatography and the spectrum shown in Appendix A.2 and Appendix A.3.

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 standard of peach, pear, asparagus, potatoes, corn, buckwheat, honey, vinegar, walnut, shrimp, chicken, rabbit meat, green onions, tea Leaf and chicken liver were limited to 0.01 mg/kg.

10.2 Recovery rate

When the levels were 0.01 mg/kg, 0.05 mg/kg, 2.0 mg/kg. The recovery of the addition of caffeine in different matrices is given in attached Record Relative abundance (base) 50% 20% to 50% 10% to 20% <=10% Allowable relative deviation ± 20% ± 25% ± 30% ± 50% Appendix A (Informative) Acetyl Acetate Standard Chromatogram

Figure A.1 Acetyl Acetate Standard Gas Chromatography

Figure A.2 Acetyl Acetate Gas Chromatography-Mass Spectrometry Ion Chromatography

Figure A.3 Ethyl Acetate Standard Mass Spectrum

Appendix B

(Informative)

Addition Recovery of Acetyl Acetate in Different Substrates

Table D.1 Recovery of Acetyl Acetate in Different Substrates

Sample added level mg/kg Recovery range (%) 0.01 82.6 to 97.2 0.05 82.6 to 96.4 0.10 84.6 to 98.1 0.01 84.2 to 97.8 0.05 88.2 to 97.6 0.10 85.5 to 98.7 asparagus 0.01 80.3 to 96.5 0.05 86.4 to 96.2 0.10 87.3 to 99.3 Green onions 0.01 71.8 ~ 104 0.05 72.8 to 96.5 0.10 82.4 to 103.4 potato 0.01 80.6 to 96.3 0.05 83.0 to 95.2 0.10 85.1 to 96.8 honey 0.01 76.8 to 95.4 0.05 77.4 to 91.4 0.10 84.6 ~ 97.8 Buckwheat 0.01 81.9 to 101.1 0.05 82.6 to 96.0 0.10 81.3 to 97.9 tea 0.01 78.2 to 103.4 0.05 78.4 to 98.8 0.10 85.6 ~ 104.9 corn 0.01 77.5 to 101.6 0.05 78.6 ~ 98.6 0.10 84.6 to 99.3 Vinegar 0.01 80.2 to 94.6 0.05 83.0 to 93.2 0.10 84.1 to 96.5 Walnut 0.01 79.8 ~ 98.1 0.05 81.0 to 96.2 0.10 85.3 ~ 97.6 rabbit 0.01 81.1 to 99.2 0.05 82.2 ~ 98.8 0.10 86.1 to 99.8 Chicken liver 0.01 76.3 to 102.2 0.05 79.2 to 102.3 0.10 83.4 to 99.3 Shrimp 0.01 77.9 to 104.1 0.05 81.0 ~ 98.0 0.10 82.4 to 97.8 chicken 0.01 81.9 to 98.1 0.05 82.2 to 97.8 0.10 81.6 to 98.9 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