GB 23200.52-2016Food safety national standard -- Determination of residues of cyclic azole in food by gas chromatography-mass spectrometry (English PDF)

4.1 Reagents

4.1.1 n-hexane (C6H14). chromatographic purity.

4.1.2 Acetone (C3H6O). Chromatographic pure.

4.1.3 Cyclohexane (C6H12). Chromatographic Purification. The

4.1.4 ethyl acetate (C4H8O2). chromatography pure.

4.1.5 Methanol (CH3OH). Chromatographic Purification.

4.1.5 Ammonia (NH3 · H2O). Analytical purity.

4.1.7 Hydrochloric acid (HCl). Analytical purity. The

4.1.8 Sodium chloride (NaCl). Analytical purity.

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

4.2 Solution preparation

4.2.1 0.1 moL/L hydrochloric acid solution. the amount of 9 mL of hydrochloric acid, add appropriate amount of water diluted to 1000 mL.

4.3 Standards

4.3.1 Cyprodinil standard substance. purity > 99%, molecular formula. C14H15N3, molecular weight. 225.3 CAS Registry Number. 121552-61-2.

4.4 Standard solution preparation

4.4.1 azoxystine standard solution. accurately weighed the appropriate amount of azoxicillin amine standard material, with acetone dosing concentration of 100 g/mL standard Stock solution. According to the need to use acetone diluted to the appropriate concentration of standard working fluid. The standard stock solution is stored in 0 °C ~ 4 °C refrigerator, valid period for 12 months, the standard working fluid in 0 °C ~ 4 °C refrigerator to save, valid for 6 months.

4.5 Materials

4.5.1 Propanesulfonylsilyl Silicone Cation Exchange Column. 3 mL, 500 mg or equivalent.

6.1 Preparation of the sample

6.1.1 Tea, grain category

Approximately 500 g of the representative sample was ground and pulverized by a pulverizer and passed through a 2.0 mm round hole sieve, mixed and sealed in a clean container.

mark.

6.1.2 Vegetables, fruits and nuts

Replace the sample of about 500 g, chopped, the multi-functional food mixer fully mashed evenly, into a clean container sealed, marked the standard Remember.

6.1.3 Livestock, poultry, aquatic products

Replace the sample of about 500 g, chopped, with a multi-functional food mixer fully mashed uniform, sealed into a clean container, marked mark.

6.1.4 Bee products

To replace the sample about 500 g, the crystallization of the sample will be forced to stir evenly, the crystallization of the sample can be sealed after the sample cap, Placed in a water bath of not more than 60 ° C warm, and so the sample all melted and then stir, quickly cooled to room temperature. In the melting must pay attention to prevent water Points volatile. Sealed in a clean container and marked with a mark.

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

6.2 Sample storage

Tea, cereals, bee products, nuts and other samples stored below 4 °C; vegetables, fruits, livestock, poultry, aquatic products such as samples at -18 °C Save the following. In the process of sample preparation, should prevent the sample contamination or the occurrence of azoxystine residues in the amount of change.

7.1 Extraction

7.1.1 Shrimp

Weigh 20 g (accurate to 0.01 g) sample, add 10 mL of water to mix, add 30 mL of n-hexane - acetone (1 1, volume ratio) Solution, homogenized at 10 000 r/min for 0.5 min, 5 g sodium chloride was added, shaken and centrifuged at 4 000 r/min for 3 min. Learn the upper layer the solution was mixed with 30 mL of n-hexane-acetone (1 1, volume ratio) mixture, and the mixture was extracted once again.

The organic phase was concentrated in a 45 ° C water bath under reduced pressure to near dryness. Accurately add 10.0 mL of cyclohexane-ethyl acetate (1 + 1, by volume) to dissolve the residue, for gel chromatography.

7.1.2 Honey

Weigh 20 g (accurate to 0.01 g) sample, add 20 mL of water to mix, add 30 mL n-hexane - acetone (1 1, volume ratio) vortex Spin mixed for 1 min, add 5 g of sodium chloride, shake, and centrifuged at 4 000 r/min for 3 min. Absorb the upper organic phase in the concentrated bottle, the residue Was added 30 mL of n-hexane-acetone (1 1, volume ratio) mixed solution, repeated extraction once, combined with the upper organic phase, in a 45 ° C water bath Concentrated to near dryness. Accurately add 10.0 mL of cyclohexane-ethyl acetate (1 + 1, volume ratio) to dissolve the residue for gel chromatography.

7.1.3 Rice, soybeans, peanuts, tea, small cabbage, sweet peas, pears, citrus, beef, chicken

Weigh 5 g (tea weighing 2 g) (accurate to 0.01 g) sample, add 5 mL of water to mix (tea, rice and peanuts need to place 0.5 h) Add 15 mL of n-hexane - acetone (1 1, volume ratio) mixed solution, 10 000 r/min homogenization 0.5 min, adding 5 g sodium chloride, shake and centrifuged at 4 000 r/min for 3 min. Absorb the upper organic phase in the concentrated flask, add 15 mL of the hexane - acetone (1 1, Volume ratio) mixed solution, repeated extraction once, combined with the upper organic phase, in a water bath at 45 °C under reduced pressure to near dry. Accurately add 10.0 mL Cyclohexane-ethyl acetate (1 + 1, by volume) to dissolve the residue for gel chromatography.

7.2 Purification

7.2.1 Gel chromatography purification

7.2.1.1 Gel Chromatographic Purification Conditions

A) Gel purification column. 400 mm x 25 mm (id); Filler. Bio-Beads, S-X3, 38 m ~ 75 m, or equivalent Need to do before the leaching curve).

B) mobile phase. cyclohexane-ethyl acetate (1 1, volume ratio);

C) Flow rate. 5.0 mL/min;

D) Sample loop. 5 mL;

E) Collection time. 20 min ~ 25 min.

7.2.1.2 Gel Chromatographic Purification Procedure

The extract was transferred to a centrifuge tube and centrifuged at 4 000 r/min for 3 min. The supernatant was transferred to a vial of the gel permeation chromatograph , According to the conditions of 7.2.1.1 purification, the collection of water below 45 °C water bath concentrated to near dry, add 5 mL of methanol dissolved, add 5 mL Water mix well.

7.2.2 Propane sulfonyl silyl silica gel cation exchange column purification

The propanesulfonylsilyl silica gel cation exchange column was mounted on a solid phase extraction apparatus, 5 mL of methanol was added, the effluent was discarded, Add 5 mL 0.1 mol/L hydrochloric acid solution, discard the effluent. Add 7.2.1 of the solution, add 10 mL of methanol - water (1 1, volume), Discard the effluent, evacuate to dry, add 10 mL of ammonia - methanol (5 95, volume ratio), and collect 10 mL of the effluent. in 45 °C water bath concentrated under reduced pressure to near dry. Accurately add 0.5 mL of acetone to dissolve the residue and set the volume for GC-MS detection.

7.3 Determination

7.3.1 Gas Chromatography - Mass Spectrometry Reference Conditions

A) Column. DB-5ms quartz capillary column, 30 m × 0.25 mm (id) × 0.25 µm (film thickness), or equivalent.

B) Column temperature. 50 ° C (1 min) 20 ° C/min 300 (10 min).

C) Inlet temperature. 250 ° C.

D) Interface temperature. 280 ° C.

E) Carrier gas. helium, purity greater than or equal to 99.999%. Flow rate. 1.0 mL/min.

F) Injection volume. 2 µL.

G) Injection mode. pulse splitless injection, pulse pressure 25 psi, delay 0.75 min, 0.75 min after the valve.

H) ion source. electron bombardment source (EI).

I) Ionization energy. 70 eV.

J) solvent delay time. 8 min.

K) Detection method. Select the ion monitoring mode.

L) Select the ion and relative abundance. see Table 1

Table 1 Select the ion and relative abundance

Select the ion

224 (quant.) 225 226 210 Relative abundance (%) 100 61.7 9.0 11.3

7.3.2 Quantitative determination

According to the sample solution of azole ring amine content of the situation, selected peak area similar to the standard working solution. Standard working solution and sample solution in azole ring the amine response value should be within the linear range of the instrument detection. Standard working solution and sample volume and other volume interspersed injection detection. Under the above chromatographic conditions, the retention time of the cypermethrin is about 10.5 mim. The total ion chromatogram of the standard is shown in Figure A.1 in Appendix A.

7.3.3 Qualitative determination

The standard solution and sample solution are subject to the conditions specified in 7.3.1, if the sample solution and the standard solution in the same retention time peak Now, it is confirmed by mass spectrometry, after deducting the background of the sample spectrum, the selected ions all appear, while the selected ion from the the abundance ratio is consistent with the relative abundance of the ions associated with the standard, and the fluctuation range is within the maximum allowable deviation of Table 2 (see Table 2) the presence of azoxycarbonylamine in the sample. The confirmed sample can be judged as azoxicillinamine positive detection. The spectrum of the cypermethrin standard is shown in the Appendix A in Figure A.2.

Table 2 Mass Spectrometry Relative Ion Abundance Maximum Allowable Deviation

7.3.4 Blank test

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

Relative abundance (base) 50% 20% to 50% 10% to 20% <=10% Allowable relative deviation ± 20% ± 25% ± 30% ± 50%

10.1 Quantitation limits

The limits of this method are 0.0004 mg/kg for honey and shrimp and 0.01 mg/kg for other samples.

10.2 Recovery rate

The experimental data on the concentration, recovery and precision of the sample are given in Appendix B.

Appendix A

(Informative)

Chromatograms and spectra

Figure A.1 Total ion chromatogram of azoxicamines

Figure A.2 Mass spectrum of azoxicicyclic amine reference substance

Appendix B

(Informative)

Sample concentration and recovery of the experimental data

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

Add concentration

(Mg/kg)

Recovery rate range% precision degree% Sample added concentration (Mg/kg) Recovery rate Precision% Rice 0.01 77.5 ~ 112 15.4 Tea 0.01 69.8 ~ 114 21.0 0.02 77.5 to 107 12.4 0.02 72.5 to 86.0 10.2 0.05 77.8 to 102 10.4 0.05 77.0 to 106 12.4 Sweet pea 0.01 78.2 ~ 113 13.9 Beef 0.01 78.9 ~ 113 14.7 0.02 77.5 to 110 13.4 0.02 78.8 to 103 10.6 0.05 79.6 ~ 104 10.0 0.05 83.0 ~ 102.2 10.6 Komatsu 0.01 75.1 ~ 102 11.5 Chicken 0.01 78.1 ~ 113 14.2 0.02 79.5 to 110 11.3 0.02 79.5 to 107 12.6 0.05 75.8 to 96.4 9.59 0.05 81.6 to 102 12.3 Pear 0.01 76.7 ~ 112 14.8 Shrimp 0.0004 56.5 ~ 85.5 15.9 0.02 79.5 ~ 107 11.7 0.0008 58.4 ~ 87.9 18.0 0.05 77.6 to 108 12.3 0.002 68.0 to 84.0 7.8 Citrus 0.01 69.1 ~ 112 18.0 Honey 0.0004 54.5 ~ 81.0 14.4 0.02 76.0 ~ 108 15.2 0.0008 56.8 ~ 82.9 14.4 0.05 80.2 to 110 13.2 0.002 62.5 to 81.5 10.8 Peanut 0.01 78.5 to 114 13.5 Soybean 0.01 73.8 to 107 16.4 0.02 79.5 to 111 13.2 0.02 75.0 to 104 12.0 0.05 79.4 to 108 14.0 0.05 79.6 to 110 13.5 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