Determination of zearalenol, zearalenol, diethylstilbestrol, diethylstilbestrol and diethylstilbestrol residues in puffer fish, eels and grilled eels by liquid chromatography-tandem mass spectrometry

9.

Suitable fugu, eel and eel are zeranol , Zearalanone , diethylstilbestrol , hexoestrol , residual amounts of phenol diene female liquid chromatography - tandem mass spectrometry

9.

Residues of zearalenol, zearalenone, diethylstilbestrol, diethylstilbestrol, diethylstilbestrol in puffer fish, eels and grilled eels are extracted with tert-butyl methyl ether and acetate buffer plus enzymatic hydrolysing agent.

9.

Tert-butyl methyl ether, methanol , acetonitrile, ethyl acetate, n-hexane, dichloromethane: chromatographically pure; acetic acid, sodium hydroxide: superior grade; sodium acetate (CH ₃ COONa·3H ₂ O) analytically pure

3mol/L sodium hydroxide solution: weigh 120g sodium hydroxide and dissolve in 1000mL deionized water; 0.

β-glucuronidase/sulfatase complex enzyme: contains β-glucuronidase 124400units/mL, sulfatase 3610units/mL

Hormone and metabolite standard substances: Zearalenol (including α-zearalenol and β-zearalenol, 50% each) purity ≥97%; zearalenone, purity ≥97%; Diethylstilbestrol, purity ≥99 %; Hexestrol, purity ≥98%; Diethylstilbestrol, purity ≥98%

Standard solution: accurately weigh appropriate amounts of zearalenol, zearalenone, diethylstilbestrol, diethylstilbestrol and hexestrol standard substances, and prepare 1mg/mL standard stock solution with methanol

Internal standard standard material: a-zearalenol-4 deuterated, purity ≥99%; Diethylstilbestrol-8 deuterated, purity ≥98%

Internal standard standard solution: Accurately weigh appropriate amounts of a-zearalenol-4 deuterated and diethylstilbestrol-8 deuterated standard materials, and prepare 1mg/mL standard stock solutions with methanol

Silica gel solid phase extraction column: 500mg, 3mL

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Liquid chromatography-tandem mass spectrometer: equipped with electrospray ionization source; analytical balance: sensitivity 0.

9.

(1) Sample preparation

Take 500g of a representative sample, mince it and stir it evenly to make a laboratory sample
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The sample is divided into two parts, placed in the sample box, sealed, and marked
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Store the sample at -18°C
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(2) Sample weighing

Weigh 5 negative samples, each sample is 5g (accurate to 0.
1g), put the above samples in a 50mL centrifuge tube and add appropriate amount of mixed standard working solution to make each test component zearalenol and zearalenol The concentrations of ketone, diethylstilbestrol, diethylstilbestrol and diethylstilbestrol are 2.
5ng/mL, 5.
0ng/mL, 10ng/mL, 25ng/mL, 50ng/mL, respectively
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Then add an appropriate amount of internal standard standard working solution to make the concentration all 10ng/mL
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(3) Extraction

Add 20mL tert-butyl methyl ether and homogenize at 10000r/min for 1min
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Centrifuge at 3000r/min for 5min, and transfer all the supernatant to another 50mL centrifuge tube with stopper for use
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Place the residue in the centrifuge tube in a fume hood to volatilize for 30 minutes, add 15 mL of acetate buffer, homogenize at high speed for 1 min, centrifuge at 3000 r/min for 5 min, transfer all the supernatant to another 25 mL stoppered test tube, and place it in nitrogen.
After blowing off the residual tert-butyl methyl ether in a water bath at 40°C on the blowing instrument, add 80 μL β-glucuronidase to mix evenly, and place in an oven at 52°C overnight
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Add sodium hydroxide solution to the buffer solution to adjust the pH of the solution to 7, add 10 mL of tert-butyl methyl ether, mix well, and centrifuge at 3000r/min for 2min
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Pipette the tert-butyl methyl ether layer and mix with the aforementioned tert-butyl methyl ether extract, and blow dry on a nitrogen blowing apparatus in a water bath at 40°C
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Add 1mL dissolving solution, vortex for 30s to dissolve, and wait for purification
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(4) Purification

Solid-phase extraction purification conditions: pre-wash the silica gel column twice with 6 mL n-hexane at a flow rate of 4 mL/min
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Load the sample at a flow rate of 2mL/min.
Add 3mL of eluent to the sample tube.
After mixing, pass through the column at a flow rate of 2mL/min.
Use 3mL of eluent to rinse at a rate of 3mL/min.
Add 2mL of air at a rate of 4mL/min.
Blow through the silica gel column at a rate of min
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Elute with 6mL eluent at a flow rate of 2mL/min.
Add 2mL of air and blow through the silica gel column at a speed of 6mL/min to collect the eluent
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The eluate was blown dry in a 40°C water bath on a nitrogen blower, 1 mL of mobile phase was added, and vortexed for 30 seconds to dissolve
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After the solution passes through a 0.
2μm filter membrane, it is used for liquid chromatography-tandem mass spectrometry
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(5) Preparation of measured sample solution

Weigh 5g (accurate to 0.
1g) of the sample to be tested into a 50mL centrifuge tube, add the internal standard working solution to the final constant volume concentration of 10ng/mL
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Follow the steps above
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(6) Preparation of blank matrix solution

Weigh 5g (accurate to 0.
1g) of the negative sample in a 50mL centrifuge tube and follow the above steps
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(7) Matrix standard working solution

The standard working solution and the internal standard working solution are mixed and dried in a nitrogen blowing apparatus, dissolved with the matrix extract, and vortexed for 30 seconds to become the matrix standard working solution
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