Determination of zearalenol, zearalenone, diethylstilbestrol, hexestrol and diethylstilbestrol residues in bovine pig liver, kidney and muscle tissue by liquid chromatography-tandem mass spectrometry

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1 Scope of application

Pig liver and bovine muscle tissue zeranol, Zearalanone, diethylstilbestrol, Hexoestrol , dienes female phenol residues Liquid Chromatography - Tandem Mass Spectrometry
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2 Principle of the method

Residues of zearalenol, zearalenone, diethylstilbestrol, diethylstilbestrol and diethylstilbestrol in the sample were extracted with tert-butyl methyl ether and acetate buffer solution plus enzymatic hydrolysis agent, and purified by silica gel solid phase extraction column After concentration, it is determined by liquid chromatography-tandem mass spectrometer, the retention time and ion abundance ratio are qualitative, and the internal standard method is used for quantification
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3 Reagents and materials

Methanol, acetonitrile, ethyl acetate, n-hexane, dichloromethane, tert-butyl methyl ether: chromatographically pure; acetic acid, sodium acetate (CH ₃ COONa: 3H ₂ O): top grade pure
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Acetate buffer solution: 0.
2mol/L, pH=5.
2
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Sodium hydroxide: pure superior grade; sodium hydroxide solution: 3mol/L
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Dissolving solution: n-hexane+dichloromethane (60+40, v/v)
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Eluent: ethyl acetate + n-hexane (6+94, v/v)
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Eluent: ethyl acetate-n-hexane (60+40, v/v)
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β-Glucuronidase: H-2 type, containing β-glucuronidase 124400unit/mL, sulfatase 3610units/mL
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Hormone and metabolite standard substances: zearalenol (including α-zearalenol and β-zearalenol, 50% each), zearalenol: purity ≥97%; diethylstilbestrol, purity ≥99%; hexane Estrol, diethylstilbestrol: purity ≥98%
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Hormone and metabolite standard solutions: accurately weigh appropriate amounts of zearalenol, zearalenone, diethylstilbestrol, diethylstilbestrol and hexestrol standard substances, and prepare 1.
0mg/mL standard stock solution with methanol
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Internal standard standard solution: accurately weigh out appropriate amounts of zearalenol-4 deuterated and diethylstilbestrol-8 deuterated standard materials, and prepare 1.

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Liquid chromatography-tandem mass spectrometer: equipped with atmospheric pressure chemical ionization source; analytical balance: sensitivity 0.
1mg and 0.
01g; automatic solid phase extraction instrument or solid phase extraction device; high-speed homogenizer: rotation speed greater than 10000r/min; Nitrogen concentrator; vortex oscillator; centrifuge: rotation speed greater than 3000r/min; pH meter: measurement accuracy ±0.
2pH unit; test tube with stopper: 25mL, 50mL; micro syringe: 25μL, 100μL
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5 Sample pretreatment.

(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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The prepared samples are stored in a -18°C freezer
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(2) Weigh the sample

Weigh 5 negative samples, each sample weighing 5g (accurate to 0.
01g), put them in a 50mL centrifuge tube, and add different amounts of mixed standard working solution to make zearalenol, zearalenol and hexestrol Concentrations are 1.
25ng/mL, 2.
5ng/mL, 5.
0ng/mL, 12.
5ng/mL, 25ng/mL; the concentrations of diethylstilbestrol and diethylstilbestrol are 2.
5ng/mL, 5.
0ng/mL, 10ng/mL, 25ng /mL, 50ng/mL
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Then add an appropriate amount of internal standard standard working solution to make the internal standard concentration all 10ng/mL
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(3) Extraction

Add 20mL tert-butyl methyl ether and homogenize at high speed for 1 min at a speed of 10000r/min
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Centrifuge at 3000r/min for 5min, and transfer all the supernatant to another 50mL test 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 test tube with a stopper, and place it under nitrogen.
After blowing off the residual tert-butyl methyl ether in a 40°C water bath on the concentrator, add 80 μL of β-glucuronidase to mix well, and place it in an oven at 52°C overnight
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Add sodium hydroxide solution to the buffer solution to adjust the pH value 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 concentrator 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

Transfer the sample solution to a silica gel solid phase extraction column with a flow rate of 2 mL/min.
Add 3 mL of eluent to the sample tube, and pass through the column after mixing.
The flow rate is 2 mL/min.
Use 3 mL of eluent at a rate of 3 mL/min.
Wash, add 2mL of air and blow through the silica gel column at a rate of 4mL/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 eluent was blown dry on a nitrogen concentrator in a 40°C water bath, 1 mL of mobile phase was added, and vortexed for 30 seconds to dissolve
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After the solution is filtered by a filter membrane, it is subjected to liquid chromatography-tandem mass spectrometry
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(5) Preparation of sample solution.

Weigh 5g (accurate to 0.
01g) of the sample to be tested in a 50mL centrifuge tube, add the internal standard working solution to make the content of the internal standard both 2.
0μg/kg, and operate according to the above steps
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(6) Preparation of blank matrix solution

Weigh 5g (accurate to 0.
01g) of the negative sample into a 50mL brown centrifuge tube, and operate according to the above steps
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