Liquid chromatography-tandem mass spectrometry for the determination of nitrofuran metabolites residues in food of animal origin

7.
2.
1.
5 Sample pretreatment

(1) Pork, beef, chicken, pork liver, fish, shrimp, crab and shellfish samples

1) Sample preparation

The laboratory samples were minced with a tissue masher, and 0.
5 kg was divided into samples
.

2) Sample weighing and degreasing

Weigh 2g of the sample (accurate to 0.
01g), place it in a 50mL brown centrifuge tube, add 10mL methanol -water mixed solution (2+1, v/v), homogenize for 1min, and then wash with 5mL methanol-water mixed solution.
Centrifuge at 4000r/min for 5min with the mass spectrometer cutter head, draw the supernatant and discard it
.

3) Hydrolysis and derivatization

Add 10mL 0.
2mol/L hydrochloric acid solution to the above centrifuge tube and homogenize for 1min, wash the homogenizer blade with 10mL 0.
2mol/L hydrochloric acid solution, add 0.
3mL derivatizing agent after the two are combined, and mix with a liquid mixer.
Place in a constant temperature shaking water bath at 37°C for 16 hours to avoid light
.

4) Purification

After the above-mentioned derivative solution is placed at room temperature, add 5mL 0.
1mol/L dipotassium hydrogen phosphate solution, adjust the pH of the solution to about 7.
4 with 1mo/ L sodium hydroxide solution, centrifuge at 4000r/min for 10 minutes, and supernatant (if the sample to be tested contains There is more fat, add 5mL n-hexane to the supernatant , shake for 2min, centrifuge at 4000r/min for 10min to absorb and discard the n-hexane) pour it into the reservoir connected to the OasisHLB solid-phase extraction column, and use it on the solid-phase extraction device.
The sample solution passes through the OasisHLB column at a flow rate of less than 2 mL/min.
After the sample solution passes through the solid phase extraction column, the solid phase extraction column is washed with 10 mL of water, and all the effluent is discarded
.

5) Preparation of mixed matrix standard calibration solution

Weigh five negative samples, each weighing 2g (accurate to 0.
01g), put them in a 50mL brown centrifuge tube, add 10mL methanol-water mixed solution (2+1, v/v), homogenize for 1min, and then use 5mL methanol-water mixed solution was used to wash the homogenizer blade, the two were combined at 4000r/min and centrifuged for 5min, and the supernatant was sucked and discarded
.

(2) Milk or milk powder samples

1) Sample preparation

The laboratory samples are mixed evenly, and 0.
5kg is separated as the sample
.

2) Deproteinization, hydrolysis and derivatization of milk samples

Weigh 8g of milk sample (accurate to 0.
01g), place it in a 50mL brown centrifuge tube, and add 15mL of trichloroacetic acid solution
.

3) Deproteinization, hydrolysis and derivatization of milk powder samples

Weigh 1g of milk powder sample (accurate to 0.
01g), place it in a 50mL brown centrifuge tube, add 8mL water, vortex to mix, and then add 15mL trichloroacetic acid solution
.

4) Sample solution purification

Add 5mL 0.
1mol/L dipotassium hydrogen phosphate solution to the above derivative solution, adjust the pH of the solution to about 7.
4 with 4mol/L sodium hydroxide solution, centrifuge at 10000r/min for 10min, pour the supernatant into the next OasisHLB solid phase extraction column In the liquid reservoir of the solid phase extraction device, the sample solution is passed through the OasisHLB solid phase extraction column at a flow rate of less than 2 mL/min.
After the sample solution passes through the solid phase extraction column, the solid phase extraction is rinsed with 20 mL of 20% methanol water.
Column, discard all effluent
.

5) Preparation of milk mixed matrix standard calibration solution

Weigh five negative milk samples, each sample weighing 8g (accurate to 0.
01g)
.
Add appropriate amount of mixed standard solutions to prepare four nitrofuran metabolites content of 0.
1μg/kg, 0.
2μg/kg, 0.
5μg/kg, 1.
0μg/kg and 2.
0μg/kg matrix standard solutions, and then respectively Add an appropriate amount of mixed internal standard solution, the content is 2.
0ng/mL
.
Others are operated according to the hydrolytic derivatization and purification steps of the sample
.

6) Preparation of standard calibration solution for milk powder mixed matrix

Weigh five negative milk powder samples, each weighing 1g (accurate to 0.
01g)
.
Add appropriate amounts of mixed standard solutions to prepare four nitrofuran metabolites content of 0.
8μg/kg, 1.
6μg/kg, 4.
0μg/kg, 8.
0μg/kg and 16.
0μg/kg matrix standard solutions, and then separately Add an appropriate amount of mixed internal standard solution, the content is 2.
0ng/mL
.
Others are operated according to the hydrolytic derivatization and purification steps of the sample
.

(3) Honey sample

1) Sample preparation

For laboratory samples without crystals, stir them evenly
.
For samples with crystals, place them in a water bath not exceeding 60°C under airtight conditions, and shake them.
After all the samples have melted, stir them evenly and quickly cool to room temperature
.
Separate 0.
5 kg as a sample
.
The prepared sample is placed in a sample bottle, sealed, and marked
.
Store the sample at room temperature
.

2) Hydrolysis and derivatization

Weigh 4g sample (accurate to 0.
01g) into a 50mL brown centrifuge tube, add 5mL 0.
2mol/L hydrochloric acid solution and 0.
15mL derivatizing agent, vortex and mix for 1min, and place it in a 37°C constant temperature oven for 16h
.

3) Purification

Take out the above-mentioned derivative solution and place it at room temperature, add 3mL 0.
1mol/L dipotassium hydrogen phosphate solution, adjust the pH of the solution to about 7.
4 with 1mol/L sodium hydroxide solution, and pour it into the reservoir connected to the OasisHLB solid phase extraction column.
On the solid phase extraction device, the sample solution was passed through the OasisHLB column at a flow rate of less than 2 mL/min.
After the sample solution passed through the solid phase extraction column, the solid phase extraction column was washed with 6 mL of water, and all the effluent was discarded
.
Use a vacuum pump to drain the OasisHLB solid phase extraction cartridge for 10 minutes under 65kPa negative pressure
.
Then use 4mL ethyl acetate to elute the analyte, collect all the eluate in a 25mL brown centrifuge tube, and dry it in a 40℃ water bath on a nitrogen dryer, and make 1mL sample constant volume solution to a constant volume
.
After mixing the constant volume solution, pass through a 0.
45um filter membrane, and the filtrate is used for liquid chromatography-tandem mass spectrometry
.

4) Preparation of mixed matrix standard calibration solution

Weigh five negative samples, each sample weighing 4g (accurate to 0.
01g), put them in a 50mL brown centrifuge tube, add appropriate amount of mixed standard solution, and make four nitrofuran metabolites with 0.
2μg/kg , 0.
5μg/kg, 1.
0ug/kg, 2.
0μg/kg and 8.
0μg/kg matrix standard solutions, and then add an appropriate amount of mixed internal standard solution, the content is 2.
0ng/mL
.
Others follow the sample hydrolysis derivatization and purification steps
.

Related Links: Determination of Nitrofuran Metabolite Residues in Food of Animal Origin by Liquid Chromatography-Tandem Mass Spectrometry