Determination of Zearabinol Residues in Food of Animal Origin by Liquid Chromatography-Tandem Mass Spectrometry

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It is suitable for the determination of zearalenol, β-zearalenol and zearalenone residues in animal muscle, liver, fish meat, eggs and milk by high performance liquid chromatography-tandem mass spectrometry

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After the sample was hydrolyzed by β-glucuronide /sulfate compound enzyme, it was extracted with ether .

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Methanol, acetonitrile : chromatographic purity; anhydrous ether, chloroform , sodium hydroxide , sodium acetate (NaAc·3H ₂ O), glacial acetic acid : analytically pure; phosphoric acid: purity greater than 85%

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Phosphoric acid solution (1+4, v/v): Mix 10 mL of phosphoric acid and 40 mL of water

Methanol-water solution (1+1, v/v): Mix 50 mL of methanol and 50 mL of water

β-glucuronidase/sulfatase: 96000 unit/mL β-glucuronidase; 390 unit/mL sulfatase (H-2, Formhelix pomatia)

Standard materials of zearalenol, β-zearalenol and zearalenone: purity ≥99%

100μg/mL standard stock solution: Weigh appropriate standards (accurate to 0.

10μg/mL and 1.

Matrix mixed standard working solution: Take appropriate amount of mixed standard intermediate solution, and use the blank sample extract to prepare matrix mixed standard solutions of different concentrations

OasisHLB solid phase extraction column or equivalent: 500mg/6mL

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4 Apparatus and equipment

Liquid chromatography-tandem mass spectrometer: equipped with electrospray ionization source (ESI); tissue masher; analytical balance: sensitivity 0.
1mg and 0.
01g; pipette: 10-100μL, 100-1000μL; homogenizer: 10000r/min; vortex mixer; constant temperature oscillator; centrifuge: 4000r/min; pH meter: measurement accuracy ±0.
02pH unit; nitrogen blowing instrument; solid phase extraction device; centrifuge tube with stopper: 50mL; graduated test tube: 10mL
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5 Sample preparation

(1) Sample preparation

The laboratory samples were minced with a tissue masher, and 0.
5 kg was divided into samples
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The samples are stored in a refrigerator at -18°C and protected from light
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(2) Hydrolysis

Weigh 5g sample (accurate to 0.
01g) into a 50mL centrifuge tube with stopper, add 10mL sodium acetate buffer solution and 0.
025mL β-glucuronide/sulfate complex enzyme, vortex to mix, and shake in a 37℃ water bath for 12h
.

(3) Extraction

After hydrolysis, add 15mL anhydrous ether, shake and extract for 5min, centrifuge at 4000r/min for 2min, transfer the supernatant to a concentrating bottle, then repeat the extraction with 15mL anhydrous ether, combine the supernatants, spin and concentrate below 40℃ Nearly dry
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The residue is dissolved by 1.
0mL of chloroform, transferred to a 10mL centrifuge tube, then rinsed with 3mL of sodium hydroxide solution and transferred to the same centrifuge tube, vortexed to mix, centrifuged at 4000r/min for 2min, and the upper layer was sucked Sodium hydroxide solution
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Then use 3mL of sodium hydroxide solution to rinse and extract once again, combine the sodium hydroxide solution, add 1mL of phosphoric acid solution, mix well and wait for purification
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(4) Purification

Transfer the sample extract to the HLB solid phase extraction column
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Rinse with 5 mL of water and 5 mL of methanol-water solution and discard; then use 10 mL of methanol for elution, and collect the eluate
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The flow rate of the entire solid phase extraction purification process is controlled not to exceed 2 mL/min
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The eluent was blown dry with nitrogen below 40°C
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The residue was dissolved in 1.
0 mL of acetonitrile, vortexed to mix, and passed through a 0.
2 μm filter membrane for instrument measurement
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