GB 23200.105-2016Food safety national standard -- Meat and meat products -- Determination of carbazate residues -- Liquid chromatography -- Post-column derivatization fluorescence detection (English PDF)

4.1 Reagents

4.1.1 Acetonitrile (CH3CN). Chromatographically pure.

4.1.2 Ethyl acetate (C4H8O2). Chromatographic purity.

4.1.3 Cyclohexane (C6H12). Chromatographic Purification.

4.1.4 Acetone (CH3COCH3). Chromatographic pure.

4.1.5 petroleum ether. boiling range 30 °C ~ 60 °C.

4.1.6 anhydrous sodium sulfate (Na2SO4). 650 °C burning 4 h, in the dryer to cool to room temperature, stored in a sealed bottle in reserve.

4.1.7 Sodium chloride (NaCl).

4.1.8 post-column derivatization reagents.

4.1.9 o-phthalaldehyde (C8H602, O-Phthaladehyde, OPA).

4.1.10 mercaptoethanol (C2H6OS, Thiofluor).

4.2 Solution preparation

4.2.1 NaOH solution (0.2%, m/v). take 2 g NaOH, the volume of water to 1000 mL.

4.2.2 O-phthalaldehyde diluent. borax solution (0.4%, m/v).

4.2.3 O-phthalaldehyde test solution. 945 ml of OPA diluent was injected into the solvent tank and purged with inert gas (nitrogen) for at least 10 min, 100 mg of OPA solids were dissolved in about 10 mL of chromatographic pure methanol and the OPA solution was added to the deoxygenated OPA diluent to dissolve 2 g of mercaptan Base ethanol solids in 5 mL OPA diluent, added to the tank, covered with cap, open airflow, and then continue to purge for a few minutes, the valve is gently agitated to make it completely mixed.

4.2.4 Ethyl acetate-cyclohexane mixed solution (1 1, v/v). Take 100 mL of ethyl acetate, add 100 mL of cyclohexane and shake well.

4.3 Standards

4.3.1 Carbaryl. Molecular formula C12H11NO2, CAS 63-25-2, purity > 99.5%.

4.4 Standard solution preparation

4.4.1 Monazalose standard stock solution. accurately weighed the right amount of methacon standard, dissolved in acetonitrile, 4 °C refrigerator, valid for 6 month. Store in 4 °C refrigerator.

4.4.2 Carbaryl standard working solution. take a certain amount of standard stock solution, diluted with acetonitrile to the appropriate concentration, 4 °C refrigerator, valid for 1 week. Store in 4 °C refrigerator.

4.5 Materials

4.5.1 0.45 µm nylon filter.

6.1 Preparation of the sample

6.1.1 Meat

All the samples taken, fully mixed and mixed, take a representative sample, the total amount of not less than 500 g, into the cleaning container, sealed and mark the mark.

6.1.2 Canned

The whole sample of the whole pot can be poured out, fully blended mix, take a representative sample, the total amount of not less than 500 g, into the cleaning container Sealed and marked.

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

6.2 Sample storage

Samples should be stored below -18 ° C. In the operation of sampling and sample preparation, the sample should be protected from contamination or changes in content.

The test sample can represent the overall sample.

7.1 Extraction

Weigh the sample 20 g (accurate to 0.01 g), add 100 mL of water in a 100 mL stopper, add water 6 mL (depending on the moisture content of the sample Water about 20 g, meat is usually about 70%, add water 6 mL), add 40 mL of acetone, homogenate 1 min, add sodium chloride 6 g, shake well, Plus 30 mL of petroleum ether, shake for 30 min. Take 35 mL of the organic layer supernatant, filtered through anhydrous sodium sulfate in a rotary vial and concentrated to About 1 mL, add 2 mL of ethyl acetate-cyclohexane solution and re-concentrate, so repeat 3 times. Ethyl acetate - cyclohexane volume of 5 mL, 0.45 µ M filter, to be purified.

7.2 Gel Chromatography (GPC) purification

7.2.1 Gel Chromatographic Purification Reference Conditions

A) Purification column. 400 mm × 30 mm, Bio Beads S-X3, or equivalent.

B) Mobile phase. ethyl acetate-cyclohexane.

C) Flow rate. 5.0 mL/min.

D) Sample loop. 5 mL.

E) Distillate collection section. 10.0 min ~ 15.0 min.

F) purification column balance time. 5 min.

7.2.2 Gel Chromatography Concentration Reference conditions

A) Sample concentration conditions are shown in Table 1.

B) Concentration end point determination. Level sensing mode.

C) Solvent Replacement. 2 mL acetonitrile, repeated twice.

D) constant volume. 1 mL.

7.2.3 Gel Chromatography Purification Procedure

5 mL of the purified liquid according to the conditions specified in 7.2.1 and 7.2.2 purification and concentration, and finally get 1 mL of the solution to be determined.

Table 1 Fraction Concentration Conditions

Concentration Period Concentration Cup Area Temperature (° C) Vacuum (Torr) Sample concentration process Zone 1 50 280 Zone 2 52 220 Zone 3 54 Concentration end point arrival process Zone 1 52 220 Zone 2 53 220

7.3 Determination

7.3.1 Liquid Chromatographic Reference Conditions

A) Column. C18,250 mm x 4.6 mm x 5 µm, or equivalent;

B) Column temperature. 42 ° C;

C) Fluorescence detector. lambdaex 330 nm, lambdaem 465 nm;

D) mobile phase. acetonitrile water (40 60, volume ratio);

E) Flow rate. 1.0 mL/min;

F) Injection volume. 20 L.

7.3.2 Post-column derivatization

A) Derivative reagent 1. Hydrolysis reagent, 0.4% sodium hydroxide solution, flow rate 0.4 mL/min;

B) Derivative reagent 2. OPA reagent at a flow rate of 0.4 mL/min;

C) Reactor temperature. hydrolysis temperature is 100 ° C and the temperature is room temperature.

7.3.3 Chromatographic determination

According to the content of kaempferol measured in the sample solution, the standard working fluid with similar concentration should be selected. The response value should be in the linear range Inside. Under the above liquid chromatographic conditions, the retention time of Carbaryl is 11.1 min, and the chromatogram is shown in Figure A.1 in Appendix A.

7.3.4 Blank test

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

10.1 Quantitation limits

The limit of quantification of this method is 0.005 mg/kg.

10.2 Recovery rate

The recovery rate of norethium is shown in Appendix B.

Appendix A

(Informative)

Monochloride standard chromatogram

Figure A.1 Liquid Chromatography (0.1 µg/mL)

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

Add level

(Mg/kg)

Recovery rate range

sample

Add level

(Mg/kg)

Recovery rate range

Fish

0.005 82.0 ~ 110.0

Ham

0.005 90.0 to 96.0

0.010 89.0 to 107.0 0.010 100.0 to 114.0 0.020 94.0 to 112.5 0.020 97.0 to 115.5 0.050 90.0 to 105.6 0.050 101.6 to 113.6 0.100 92.0 to 111.2 0.100 92.5 to 107.3 beef 0.005 82.0 ~ 98.0 Shrimp 0.005 86.0 ~ 100.0 0.010 86.0 to 99.0 0.010 85.0 to 96.0 0.020 84.5 to 109.5 0.020 87.0 to 105.0 0.050 90.6 ~ 98.0 0.050 88.6 ~ 94.6 0.100 88.4 ~ 98.0 0.100 86.7 ~ 95.1 chicken 0.005 86.0 to 96.0 0.010 89.0 to 97.0 0.020 95.5 to 112.5 0.050 92.0 to 97.2 0.100 85.9 ~ 95.0 5.000 87.7 to 102.5 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