Determination of Residual Nitrofuran Metabolites in Food of Animal Origin by Liquid Chromatography-Tandem Mass Spectrometry

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1 Scope of application

This method is suitable for pork, beef, chicken, pork liver, fish (fish, shrimps and crabs and shellfish), milk, honey and milk powder furaltadone metabolite (5-methyl-3-morpholin - 2-oxazolidinone, AMOZ), nitrofuran metabolite (semicarbazide, SEM), nitrofurantoin metabolite (1-amino-2-lactam, AHD) and furazolidone metabolite (3-amino-2-oxazole) Alkyl ketone, AOZ) residual content determination by liquid chromatography-tandem mass spectrometry
.

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2 Principle of the method

The residual nitrofuran metabolites in pork, beef, chicken, liver, and aquatic products (fish, shrimp, crab, and shellfish) are derivatized with 2-nitrobenzaldehyde under acidic conditions , and OasisHLB or Equivalent solid phase extraction column purification
.

Milk or milk powder samples are hydrolyzed with trichloroacetic acid and deproteinized.
Nitrofuran metabolites are derivatized with 2-nitrobenzaldehyde under acidic conditions and purified with OasisHLB or a solid phase extraction column with equivalent performance
.

The residual nitrofuran antibiotic metabolites in the honey sample are derivatized with 2-nitrobenzaldehyde under acidic conditions and purified with OasisHLB or equivalent solid phase extraction column
.

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3 Reagents and materials

Methanol, acetonitrile, ethyl acetate : chromatographically pure
.

Dipotassium hydrogen phosphate solution: 0.
1mol/L
.

Hydrochloric acid: 0.
2mol/L
.

Sodium hydroxide solution: 1mol/L
.

Derivative: Containing 0.
05mol/L of 2-nitrobenzaldehyde
.
Weigh 0.
075g of 2-nitrobenzaldehyde, dissolve it in 10mL of dimethyl sulfoxide, and make it ready for use
.

Sample constant volume solution: 10mL acetonitrile, 0.
3mL acetic acid, diluted with water to 100mL
.

Four standard nitrofuran metabolites: purity ≥99%
.

Four nitrofuran metabolites internal standard reference materials: 5-morpholinomethyl-3-amino-2-xantazolidinone internal standard material, Ds-AMOZ; internal standard material of semicarbazide, ¹³ C ¹⁵ N- SEM; internal standard of 1-amino-2-lactam, 13C ₃ -AHD; internal standard of ₃ -amino-2-oxazolidinone, D4-AOZ
.
The purity of the four internal standard reference materials is ≥99%
.

Standard stock solutions of four nitrofuran metabolites: 1.
0 mg/mL
.
Weigh appropriate amounts of the four nitrofuran metabolites standard materials, and dilute them with methanol into 1.
0 mg/mL standard stock solutions
.
Stored in a -18℃ freezer, protected from light, can be used for 6 months
.

Mixed standard solution of four nitrofuran metabolites: 0.
1μg/mL
.
Draw an appropriate amount of the standard stock solution of the four nitrofuran metabolites, dilute it with methanol to a mixed standard solution of 0.
1μg/mL, store it in a freezer at -18°C, protected from light, and can be used for 3 months
.

Internal standard stock solution of four nitrofuran metabolites: 1.
0mg/mL
.
Weigh appropriate amounts of the four nitrofuran metabolites internal standard standard materials, and use methanol to prepare 1.
0 mg/mL standard stock solutions
.
Stored in a -18℃ freezer, protected from light, can be used for 6 months
.

Four nitrofuran metabolites internal standard standard solutions: 0.
1μg/mL pipette an appropriate amount of four nitrofuran metabolites internal standard stock solutions, dilute with methanol to a 0.
1ug/mL mixed internal standard standard solution, protected from light Stored in -18℃ freezer, it can be used for 3 months
.

OasisHLB solid phase extraction column: 60mg, 3mL
.
Before use, pretreated with 5mL methanol and 10mL water respectively to keep the column moist
.

5-Methylmorpholine-3-amino-2-oxazolidinone, semicarbazide, 1-amino-2-lactone and 3-amino-2-oxazolidinone mixed standard working solution: 1.
0ug/mL
.
Draw appropriate amount of standard stock solutions of four nitrofuran metabolites, mix with methanol to prepare a 1.
0μg/mL mixed standard working solution.
Store the standard working solution in a freezer at -18℃ in the dark.
The shelf life is 3 months.
Return before use.
Warm to room temperature
.

5-Methylmorpholine-3-amino-2-oxazolidinone, semicarbazide, 1-amino-2-lactone and 3-amino-2-oxazolidinone mixed matrix standard calibration solution: Weigh 7 A honey blank sample (weighing 4g) was placed in a 50mL brown centrifuge tube, and 6 blank samples were added with appropriate amounts of mixed standard working solutions of four nitrofuran metabolites, and operated simultaneously according to the sample operation steps
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Make the concentration of 5-methylmorpholine-3-amino-2-oxazolidinone and 3-amino-2-oxazolidinone in the final sample solution respectively 0ng/mL, 0.
5ng/mL, 1.
0ng/ mL, 2.
0ng/mL, 5.
0ng/mL, 10.
0ng/mL, 20.
0ng/mL; the concentrations of semicarbazide and 1-amino-2-lactone are 0ng/mL, 1.
0ng/mL, 2.
0ng, respectively /mL, 5.
0ng/mL, 10.
0ng/mL, 20.
0ng/mL, 50.
0ng/mL as the matrix standard calibration solution, the matrix standard calibration solution is ready to use
.

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4 Instruments and equipment

Liquid chromatography-tandem quadrupole mass spectrometer, equipped with electrospray ion source
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Analytical balance: each with a sensitivity of 0.
1mg and 0.
01g
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Liquid mixer
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Solid phase extraction device
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Nitrogen drying instrument
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Constant temperature shaking water bath
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Vacuum pump: the vacuum degree should reach 80kPa
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Micro syringe: 25μL, 100μL
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Brown centrifuge tube with stopper: 25mL, 50mL
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pH meter: measurement accuracy ±0.
02pH unit
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Reservoir: 50mL
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Centrifuge: The speed is above 4000r/min
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