Determination of Zearalenol, Diethylstilbestrol, Diethylstilbestrol and Diethylstilbestrol Residues in Cow Urine by Liquid Chromatography-Tandem Mass Spectrometry

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1 Scope of application

In case of bovine urine zeranol , diethylstilbestrol , hexoestrol , dienes female phenol residues Liquid Chromatography - Tandem Mass Spectrometry
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Detection limit: zearalenone alcohols hexoestrol was 0.

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2 Principle of the method

The specific antibodies contained in the immunoaffinity column selectively combine with diethylstilbestrol, diethylstilbestrol, diethylstilbestrol, and zearalenol in the cow urine sample to form an antibody-antigen complex.
Wash the column with water to remove impurities , Use the eluent to elute the target substance adsorbed on the column, and collect the eluent
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The test solution was determined by liquid chromatography-tandem mass spectrometry, the retention time and ion abundance ratio were qualitatively, and the internal standard method was used for quantification

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3 Reagents and materials

Methanol , ethanol , acetonitrile : chromatographically pure
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Eluent: Dilute the stock solution provided in the Randox kit with deionized water at a ratio of 1:20
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Eluent: ethanol + water (70+30, v/v)
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Measure 70 mL of ethanol and mix with 30 mL of deionized water

Stock solution: Dilute the stock solution provided in the Randox kit 1:5 with deionized water
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Hormone standard substance: Zearalenol (including α-zearalenol and β-zearalenol , 50% each) purity ≥97%; diethylstilbestrol, purity ≥99%; hexestrol, purity ≥98%; double Diethylstilbestrol, purity ≥98%
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Hormone standard solution: Weigh appropriate amounts of zearalenol, diethylstilbestrol, diethylstilbestrol and diethylstilbestrol standard substances, and prepare 1.
0mg/mL standard stock solution with methanol
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According to needs, mixed standard solutions of different concentrations are prepared with methanol as standard working solutions, stored in a refrigerator at 4°C, and can be used for 3 months

Internal standard standard substance: Zearalenol-4 deuterated (including Q-zearalenol-4 deuterated and β-zearalenol-4 deuterated, 50% each), purity ≥99%; Diethylstilbestrol-8 Deuterated, purity ≥98%
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Internal standard standard solution: accurately weigh appropriate amounts of zearalenol-4 deuterated and diethylstilbestrol-8 deuterated standard materials, and prepare 1.
0mg/mL standard stock solutions with methanol
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Dilute it with methanol to a mixed internal standard standard working solution of suitable concentration, store it in a refrigerator at 4°C, and can be used for 3 months

Immunoaffinity chromatography column: Randox (UK) or equivalent; zearalenol immunoaffinity chromatography column: the maximum capacity of the affinity column is 100ng zearamol; stilbene immunoaffinity chromatography column: affinity The maximum capacity of the column is 50ng diethylstilbestrol, 50ng diethylstilbestrol, and 50ng diethylstilbestrol
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4 Instruments and equipment

Liquid chromatography-tandem mass spectrometer: equipped with atmospheric pressure chemical ionization source; centrifuge: rotating speed greater than 3000r/min; nitrogen concentrator; vortex oscillator
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5 Sample preparation

(1) Sample preparation

Take representative samples and make laboratory samples
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The sample is divided into two parts, placed in a sample bottle, sealed, and marked

(2) Sample weighing

Take 5 negative cow urine samples, each 10mL, in a 50mL centrifuge tube, centrifuge at 3000r/min for 15min
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To 5 10mL test tubes, add different amounts of mixed standard working solution to make the concentration of zearalenol and hexestrol 1.

(3) Purification

Pre-wash the immunoaffinity chromatography column twice with 15 mL eluent at a flow rate not greater than 3 mL/min
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Sample loading, natural flow rate

(4) Preparation of sample solution

After centrifugation at 3000r/min for 15min, 3mL of the sample to be tested was placed in a 50mL centrifuge tube, and the internal standard working solution was added to make the content all 2.
0μg/kg
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Follow the steps above
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(5) Preparation of blank matrix solution.

Take 3mL of the negative sample after centrifugation at 3000r/min for 15min in a 50mL centrifuge tube, and operate according to the above steps
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6 Determination

(1) Liquid chromatography conditions

Chromatographic column: ZORBAXEclipseSB-C ₈ , 3.
5μm, 150mm×4.
6mm, or equivalent; column temperature; 25°C; mobile phase: acetonitrile-water (70+30, v/v); flow rate: 1.
0mL/min; Sample volume: 50μL
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(2) Mass spectrometry conditions

Ionization method: atmospheric pressure chemical ionization; scanning method: negative ion scanning; detection method: multiple reaction monitoring (MRM); ion source temperature: 325℃; atomizing gas pressure: 15psi; curtain gas pressure: 10psi; auxiliary gas 1 pressure: 35psi ; Nebulizer current: -5μA; Electrospray voltage: -450V; See Table 9-4 for qualitative ion pair, quantitative ion pair, collision energy and declustering voltage
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