Determination of a-Trenbolone, β-Trenbolone, 19-Ethylene Nortestosterone and Epi-19-Ethylene Nortestosterone Residues in Milk and Milk Powder

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1 Scope of application

Milk and milk powder suitable for a- trenbolone , trenbolone [beta], 19- nortestosterone ethylene and ethylene-epi-19- nor-testosterone residues liquid chromatography - tandem mass spectrometry
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2 Principle of the method

The sample was hydrolyzed by enzyme at pH 5.
0, extracted with acetonitrile - ethyl acetate , purified by gel chromatography, determined by high performance liquid chromatography-tandem mass spectrometry, and quantified by external standard method
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3 Reagents and materials

Acetonitrile, ethyl acetate, cyclohexane , methanol, and acetic acid are all pure for liquid chromatography; sodium acetate: analytically pure; anhydrous sodium sulfate : analytically pure: burned at 650°C for 4 hours and placed in a desiccator for later use
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β-glucuronidase/arylsulfatase: β-glucuronidase/arylsulfatase, 100000 units/mL
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0.
02mol/L sodium acetate buffer solution (pH=5.
0): Weigh 0.
82g of anhydrous sodium acetate and dissolve it in 500mL water, adjust the pH=5.
0 with acetic acid
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a-Trenbolone, β-trenbolone (β-trenbolone, CAS: 10161-33-8), 19-vinylnortestosterone (CAS: 434-22-0) and epi-19-vinylnortestosterone (CAS: 434-22-0) Testosterone (epi-nortestosterone, CAS: 4409-34-1) standard product: purity greater than or equal to 98%
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Standard stock solution: Weigh the appropriate amount of standard substance accurately and prepare a 100μg/mL standard stock solution with acetonitrile
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Mixed standard intermediate working solution: Take each 1mL to 100mL volumetric flask of the standard stock solution, dilute to the mark with acetonitrile, and prepare a mixed standard working solution with a concentration of 1ug/mL
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4 Instruments and equipment

High performance liquid chromatography-tandem mass spectrometer: equipped with electrospray ion source (ESI)
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5 Sample preparation

(1) Sample preparation

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Take a uniform sample of about 250g of milk into a clean container as a sample, seal it and store it at 4°C, and mark it with a mark
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Take a uniform sample of about 250g of milk powder into a clean container as a sample, seal it, and mark it with a mark
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(2) Extraction

Weigh about 5g (accurate to 0.
01g) of milk sample in a 50mL centrifuge tube with stopper, weigh about 1g (accurate to 0.
01g) of milk powder sample and add 5mL of water in a 50mL centrifuge tube with stopper, and mix well
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Add 5mL sodium acetate buffer and 20μL β-glucuronidase/arylsulfatase into the centrifuge tube where the sample has been weighed, shake well, cover the stopper and place it on a constant temperature water bath shaker, shake and hydrolyze overnight at 37°C
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(3) Purification

1) Gel chromatography conditions

Purification column: 22gS-X3 Bio-Beads packing, 200mm×25mm (inner diameter) or equivalent: mobile phase: ethyl acetate-cyclohexane, flow rate: 5.
0mL/min; quantitative loop: 5mL; purification procedure: 0～10min Discard the eluent, and collect the eluent for 10 to 15.
5 min
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2) Gel chromatography purification

The extract was evaporated to nearly dryness at 45°C, and the volume was filled with ethyl acetate-cyclohexane (50+50, v/v) into a 10 mL glass test tube, and purified according to the above conditions
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(4) Preparation of blank matrix solution

Take milk negative sample Sg, milk powder negative sample 1g (accurate to 0.