GB 23200.62-2016Food safety national standard -- Determination of fulvic acid residues in foodstuffs by gas chromatography-mass spectrometry (English PDF)

4.1 Reagents

4.1.1 Acetone (C3H6O). Pesticide level.

4.1.2 Ethyl acetate (C4H8O2).

4.1.3 Cyclohexane (C6H12).

4.1.4 Acetonitrile (CH3CN). liquid chromatography grade.

4.1.5 n-hexane (C6H14).

4.1.6 Sodium chloride (NaCl).

4.1.7 anhydrous sodium sulfate. at 650 °C for 4 h, stored in a sealed container in reserve.

4.2 Solution preparation

4.2.1 Acetonitrile saturated n-hexane.

4.2.2 n-hexane saturated acetonitrile.

4.2.3 Ethyl acetate - cyclohexane (1 1, V/V). Mix the ethyl acetate and cyclohexane in equal volume and mix.

4.2.4 sodium chloride solution..200 g/L, take.200 gNaCl, constant volume to 1 L.

4.3 Standards

4.3.1 Fenvalerate standard. English name. flumiclorac-pentyl, CAS. 87546-18-7, purity >= 99%.

4.4 Standard solution preparation

4.4.1 standard solution of fenvalerate. accurately weighed the appropriate amount of fluoroacetic acid standard, formulated with acetone 100 µg/mL standard reserve liquid.

4.4.2 Fenvalerate standard intermediate solution. 1 µg/mL. Accurately absorb 1 mL of the standard stock solution into a 100 mL volumetric flask with acetone Constant volume to the scale, the solution stored at 0 ~ 4 °C.

4.4.3 Matrix standard working solution. If necessary, use a blank sample according to the sample treatment steps of the extract, with different concentrations of the base Quality standard working solution, matrix standard working solution to be used with the current distribution.

4.5 Materials

4.5.1 Gel Permeation Column. The filler is S-X3 BioBeads, 22g.

4.5.2 anhydrous sodium sulfate column. in the 150 mm × 10 mm (diameter) column, followed by adding absorbent cotton and 5 cm anhydrous sodium sulfate.

6.1 Fruit, vegetables

Take about 500 g of the sample, crush with a pulverizer, into a clean container as a sample, sealed and marked, stored in -18 °C refrigerator.

6.2 Nuts

Take about 500 g, crushed with a pulverizer, into a clean container as a sample, sealed and well marked, stored in the refrigerator at 4 °C.

6.3 Grain, tea

Take about 500 g of the sample, crushed into a whole by a pulverizer through a 20 mesh sieve, into a clean container as a sample, sealed and marked, Store at room temperature.

6.4 Meat and aquatic products

Take about 500 g of the sample, crush with a pulverizer, into a clean container as a sample, sealed and marked, stored in -18 °C refrigerator.

6.5 Honey

Take a representative sample of about 500 g, the amorphous sample will be forced to stir evenly, there are crystallization of the sample can be sealed after the sample cap, Placed in a water bath of not more than 60 °C, until the sample all dissolved and stir well, quickly cooled to room temperature. The prepared sample is packed in a clean container Sealed and well marked, room temperature preservation.

6.6 Milk

Take about 500 g sample, mix well, install clean container as sample, seal and make sure, save at -18 °C.

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

7.1 Extraction

7.1.1 Fruits and vegetables

Weigh the sample about 10 g (accurate to 0.01 g) in a 50 mL centrifuge tube, add 10 g of anhydrous sodium sulfate and 20 mL of ethyl acetate,.20000 r/min Homogenized extraction 2 min, 4000 r/min centrifugal 10 min, the upper organic phase through the anhydrous sodium sulfate column into the heart of the bottle. The residue was washed with 20 mL Ethyl acetate repeated extraction, centrifugal filtration, the combined secondary extract, the extract in 45 °C water bath with a rotary evaporator concentrated to near dry, to be purified by GPC.

7.1.2 Tea, grain, soybeans, peanuts

Weigh the sample about 10 g (accurate to 0.01 g) in 150 mL with a triangular flask, add 50 mL of acetonitrile,.20000 r/min homogeneous extraction 2 min, the upper organic phase is filtered into the separatory funnel. The residue was extracted once again with 50 mL of acetonitrile and filtered, and the combined extracts were separated in a separatory funnel.

7.1.3 Livestock, poultry, aquatic products

Weigh the sample about 10 g (accurate to 0.01 g) in a 50 mL centrifuge tube, 10 g sodium chloride, mix, add 20 mL acetonitrile,.20000 r/min Homogenized 2 min, centrifuged at 4000 r/min for 5 min. The upper organic phase was passed through an anhydrous sodium sulfate column and transferred to a separatory funnel. And then with 20 mL of acetonitrile each time the residue was extracted twice in the same manner as described above, and the organic phases were combined in a separatory funnel.

7.1.4 Honey

Weigh 10 g (accurate to 0.01 g) honey sample in 150 mL Erlenmeyer flask, add 20 ml of water to dissolve, shake for 30 min on a flat shaker, Transferred to 250ml separatory funnel, and then were 30 mL of water and 50 mL of ethyl acetate in two times the original prune bottle, are transferred into the separatory funnel, vibration Shake 2min, standing stratification (if the occurrence of emulsification, the upper layer and the emulsion layer can be centrifuged at 4000r/min 5min, take the upper transfer into the liquid Funnel), the organic phase was passed through an anhydrous sodium sulfate column in a heart-shaped bottle. The lower aqueous phase was added twice every 20 ml of ethyl acetate, gently shaken for 1 min, Placed in the heart of the bottle, in 45 °C water bath with a rotary evaporator concentrated to near dry, to be GPC purification.

7.1.5 Milk

Weigh 10 g (accurate to 0.01 g) milk sample in 50 mL stoppered centrifuge tube, add 20 mL of acetonitrile, vortex for 1 min, White, 4000 r/min centrifugal 5 min, the supernatant filter into the heart of the bottle, in the 45 °C water bath on the vacuum slowly evaporate to about 10 mL, the solution was transferred to a 250 mL separatory funnel and 10 mL of sodium chloride solution was added, mixed, 20 mL of ethyl acetate was added, shaken for 2 min, allowed to stand Layered. The ethyl acetate layer was transferred to a 150 mL heart flask and the lower aqueous phase was further added with 20 mL of ethyl acetate solution to extract the aqueous phase, Ester layer into the heart of the bottle, in 45 °C water bath rotary evaporator concentrated to near dry, to be GPC purification.

7.2 Purification

7.2.1 Livestock, poultry, aquatic products, peanuts, grain, tea, soybeans

The organic phase solution obtained in 7.1.2 and 7.1.3 was added with 40 ml of acetonitrile saturated n-hexane and shaken for 2 min. The layers were allowed to stand and the acetonitrile layer was transferred Heart-shaped bottles, n-hexane layer each time again with n-hexane saturated acetonitrile 15 mL wash twice, acetonitrile layer into the same heart bottle, in the 45 °C water bath Vacuum Rotate to dry. With ethyl acetate - cyclohexane volume 10 mL, to be GPC purification.

7.2.2 Fruit, vegetables, honey, milk

The residue obtained by dissolving 7.1.1, 7.1.4 and 7.1.5 with ethyl acetate-cyclohexane was fixed at 10 mL and operated as follows.

7.2.3 Gel permeation chromatography purification

7.2.3.1 Conditions

A) Purification column. 400 mm (length) × 25 mm (inner diameter), built with BIO-Beads S-X3 packing or equivalent.

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

C) Flow rate. 4.7 mL/min.

D) Injection volume. 5 mL.

E) Start collection time. 9.5 min.

F) End of collection time. 14.0 min.

7.2.3.2 Purification

The solutions obtained in 7.2.1 and 7.2.2 were injected with 5 mL of the sample and purified by gel permeation chromatography. A fraction of 9.5 to 14 min was collected in a heart and evaporated to dryness in a water bath at 45 ° C, fixed to 1 mL with acetone and determined by gas chromatography-mass spectrometry.

7.3 Preparation of blank matrix solution

Weigh the negative samples of the various substrates 10 g, according to 7.1 and 7.2 operation.

7.4 Determination

7.4.1 Gas Chromatography - Mass Spectrometry Reference Conditions

A) Column. DB-5MS, 30 m (length) × 0.25 mm (inner diameter) × 0.25 µm (film thickness) or equivalent.

B) Column temperature programmed..200 ° C (for 1 min) and ramp up to 280 ° C (for 12 min) at a rate of 10 ° C/min.

C) Inlet temperature. 280 ° C.

D) Carrier gas. helium, purity 99.999%.

E) Carrier gas flow rate. constant current mode 1 mL/min.

F) Injection mode. no shunt.

G) Injection volume. 2 µL.

H) Interface temperature. 280 ° C.

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

J) Ionization voltage. 70eV.

K) ion source temperature. 230 ° C.

L) Detection method. SIM.

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

Table 1 Select the ion and relative abundance

The quantified ion is quantified by the measured component Select ion (m/z) 423 308 318 280 Relative abundance (%) 100 49 26 12

7.4.2 Quantitative determination

The volume of the sample solution and the standard working fluid and so on. The actual application of the standard working fluid and the sample to be measured, the reaction of fluoroacetic acid Values should be within the linear range of the instrument. Under the above conditions of gas chromatography-mass spectrometry, the retention time of fenvalerate was about 15.9 min. standard See Appendix A for gas chromatography-mass spectrometry.

7.4.3 Qualitative determination

When the sample is measured, if the mass retention time of the detected mass is consistent with that of the standard sample, and the sample spectrum after subtracting the background , The relative abundance of the qualitative ions is close to the standard solution obtained under the same conditions as the standard solution spectrum, the error does not exceed the provisions of Table 2 Range, you can determine the existence of the corresponding sample in the sample.

Table 2 The maximum allowable error of relative ion abundance when qualitative confirmation

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

7.5 Blank test

Except for the same conditions and procedures as described above.

10.1 Quantitation limits

The limit of quantification of fenvalerate is 0.005 mg/kg.

10.2 Recovery rate

When the levels were 0.005 mg/kg, 0.01 mg/kg, 0.05 mg/kg, the addition rates for the addition of fenvalerate were given in Appendix B.

Appendix A

(Informative)

Gas Chromatography - Mass Spectrometry of Fluoroally Oxalic Acid Standard

Figure A.1 Gas Chromatography-Mass Spectrometry (TIC) of Fenvalerate Standards

1-fluoro-oxalic acid

Figure A.2 Full scan of the fluoroacetic acid standard

Appendix B

(Informative)

Sample concentration and recovery of the experimental data

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

Substrate addition level/(mg/kg) Recovery/(%) corn 0.005 71.6 ~ 102.0 0.010 78.8 ~ 94.1 0.050 85.6 ~ 101.0 celery 0.005 79.6 ~ 96.8 0.010 82.2 to 100.3 0.050 81.8 ~ 100.2 apple 0.005 77.6 ~ 99.0 0.010 75.4 to 96.3 0.050 81.2 ~ 95.6 peanut 0.005 77.2 ~ 94.2 0.010 78.3 to 96.7 0.050 80.0 ~ 97.8 tea 0.005 79.8 ~ 99.0 0.010 79.5 to 97.2 0.050 79.4 to 97.0 beef 0.005 76.4 to 99.2 0.010 79.6 ~ 95.3 0.050 81.2 ~ 99.4 Chicken liver 0.005 77.4 ~ 93.6 0.010 76.1 to 98.2 0.050 82.2 ~ 94.8 0.005 79.8 ~ 97.0 0.010 79.2 ~ 100.6 0.050 82.6 ~ 99.2 honey 0.005 81.4 ~ 97.6 0.010 86.8 ~ 101.8 0.050 84.4 to 100.2 Soybeans 0.005 77.2 ~ 94.2 0.010 78.3 to 96.7 0.050 80.0 ~ 97.8 milk 0.005 80.4 to 98.2 0.010 82.6 ~ 92.4 0.050 85.6 ~ 95.8 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