Liquid chromatography-tandem mass spectrometry for the determination of metabolite residues of metabolites in cattle and pigs

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2.
1.
1 Scope of application

Suitable for bovine and porcine muscle 4-amido methamphetamine antipyrine, 4-acetamido-antipyrine , 4-amino antipyrine and 4-aminoantipyrine residues Billing Liquid Chromatography - Tandem mass spectrometry
.

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2.
1.
2 Principle of the method

The residual metabolites of Analgin in the muscle were extracted with sodium sulfate solution (pH=7), filtered, purified by Bond Elut C ₁₈ solid phase extraction column or equivalent, eluted with methanol, and dried with nitrogen
.

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3 Reagents and materials

Methanol, acetonitrile , ammonium acetate : chromatographically pure; sodium sulfate, sodium sulfite : analytically pure
.

Sodium sulfate + sodium sulfite extraction solution: accurately weigh 14.
20g of anhydrous sodium sulfate and 2.
52g of sodium sulfite, dissolve in water, and make the volume reach about 950mL, then use 0.
5mol/L dilute sulfuric acid to adjust the pH of the solution to 7.
0, set with water Volume to 1000mL; eluent: methanol + water (1+19, v/v); sample constant volume: methanol + water (1+9, v/v)
.

Standard substances of 4-methylaminoantipyrine, 4-acetamidoantipyrine, 4-formylaminoantipyrine and 4-aminoantipyrine: purity ≥99%
.

Standard stock solution: 100mg/L
.

Mixed standard stock solution: 1.

Internal standard stock solution: Accurately weigh an appropriate amount of internal standard standard material and use methanol to prepare a standard stock solution with a concentration of 100mg/L, store it at -18°C under light, and can be used for 3 months
.

Mixed standard stock solution: 1.
0mg/L
.

Internal standard stock solution: Accurately weigh an appropriate amount of internal standard standard material and prepare a standard stock solution with a concentration of 100 mg/L in methanol.
Store it in the dark at -18°C.
It can be used for 3 months
.

Internal standard working solution: 0.
20mg/L
.

Matrix mixed standard working solution: draw an appropriate amount of mixed standard stock solution and an appropriate amount of internal standard working solution, and use the blank sample extract to make the concentration of 1.
0μg/L, 5.
0μg/L, 10.
0ug/L, 20.
0μg/L, 40.
0 ug/L and a matrix mixed standard working solution with an internal standard concentration of 0.
20mg/L
.

Bond Elut C ₁₈ solid phase extraction column or equivalent: 500mg, 3mL
.
Before use, treat with 5mL methanol and 5mL water respectively to keep the column moist
.

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4 Apparatus and equipment

Liquid chromatography-tandem mass spectrometer: equipped with electrospray ion source (ESI); solid phase extraction device; nitrogen concentrator; vortex mixer; analytical balance: sensitivity 0.
1mg and 0.
01g; vacuum pump; homogenizer; transfer Liquid container: 10-100μL and 100-1000μL; polypropylene centrifuge tube: 50mL with stopper; pH meter: measurement accuracy ±0.
02; low-temperature centrifuge: can be cooled to 4°C; glass centrifuge tube: 15mL
.

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5 Sample pretreatment

(1) Sample preparation

Take representative bovine or pig muscle tissue to make laboratory samples
.
The sample is divided into two parts, placed in a sample bottle, sealed, and marked
.
The prepared samples are stored in a -18°C freezer and protected from light
.

(2) Extraction

Weigh 5g sample, accurate to 0.
01g
.
Place the above sample in a 50mL polypropylene centrifuge tube, add 0.
25mL of the internal standard working solution with a concentration of 2.
0mg/L, and then add 15mL of sodium sulfate + sodium sulfite extraction solution, homogenize for 1 min, and centrifuge at 10°C and 4000r/min for 5 min
.
Transfer the supernatant to another clean 50mL volumetric flask, add 15mL, 10mL sodium sulfate + sodium sulfite extraction solution to the residue and extract twice.
After centrifugation, combine the supernatants, and dilute to the volume with sodium sulfate + sodium sulfite extraction solution 50mL, mix well, then filter with glass filter paper, to be purified
.

(3) Purification

Take 25mL of the extract and put it into the _C18 solid phase extraction column, and let the sample liquid pass through the solid phase extraction column at a flow rate of about 1mL/min.
After all the sample liquid has passed, it is rinsed with 5mL water and 5mL methanol + water eluent in sequence.
Wash the solid phase extraction column, discard all the effluent, and drain the solid phase extraction column under reduced pressure for 10 minutes
.
Finally, it was eluted with 5mL methanol, and the eluate was collected in a 15mL glass centrifuge tube, placed on a nitrogen concentrator and blown to near dryness at 55°C, and the volume was adjusted to 1.
0mL with the sample volumetric solution
.
After mixing the constant volume solution, pass through a 0.
45um filter membrane for liquid chromatography-tandem mass spectrometry determination
.

At the same time, a negative sample is taken, and a blank sample extract is prepared according to the above extraction and purification steps for preparing a matrix mixed standard working solution
.