Determination of α-Trenbolone and β-Trenbolone Residues in Cattle Muscle, Liver and Kidney by Liquid Chromatography-Tandem Mass Spectrometry

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1 Scope of application

It is suitable for the determination of a-Trenbolone and β-Trenbolone residues in cattle muscle, liver and kidney by liquid chromatography-tandem mass spectrometry
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The detection limit is 2μg/kg

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2 Principle of the method

The sample was hydrolyzed with enzyme under the condition of pH=5.
0, the trenbolone residue was extracted with ethyl acetate , purified by gel chromatography (GPC) and silica gel column, determined by high performance liquid chromatography-tandem mass spectrometry, and quantified by external standard method
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3 Reagents and materials

Methanol , acetic acid , acetonitrile : chromatography; ethyl acetate, cyclohexane , acetone, n-hexane: AR; [beta] -glucuronidase / arylsulfatase ester (beta] - glucuronidase,-/ and Aryl sulfatase): 100000unit / mL
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Ethyl acetate+cyclohexane (50+50, v/v), acetone+n-hexane (10+90, v/v), acetone+n-hexane (30+70, v/v)
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Sodium acetate buffer: 0.
02mol/L, pH=5.
0
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Weigh 0.

a-Trenbolone and β-Trenbolone standard materials: purity ≥98%
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Standard stock solution: accurately weigh out 0.
0100g each of a-Trenbolone and β-Trenbolone standard substances, dissolve them in acetonitrile and dilute to 100mL, and prepare a 100μg/mL standard stock solution
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This solution is stored frozen at -18°C and can be used for 6 months

Mixed standard working solution: Take each 5mL to 50mL volumetric flask of the standard stock solution, dilute with acetonitrile, and prepare a mixed standard working solution with a concentration of 10μg/mL.
This solution can be stored frozen at -18°C and can be used for 3 months
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Silica gel solid phase extraction column: 500mg, 3mL; pre-treated with 5mL acetone + n-hexane before use, keep the column moist
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4 Instruments and equipment

The high performance liquid chromatograph is equipped with an ultraviolet detector
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High performance liquid chromatography-tandem mass spectrometer: equipped with electrospray ion source (ESI)

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5 Sample pretreatment

(1) Sample preparation

The bovine muscle, liver, and kidney tissues were minced with a tissue masher, and 0.
5 kg was separated as a sample for later use
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The prepared samples are stored in a -18°C freezer and protected from light

(2) Weighing of the sample

Weigh 5 negative samples, each weighing 5g (accurate to 0.
01g), put them in a 50mL centrifuge tube with stopper, and then add different amounts of mixed standard working solution to make the concentration of each test component 2.
5ng /mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL
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(3) Extraction

Add 5 mL of sodium acetate buffer solution and 20 μL of β-glucuronidase/aryl sulfate to the above 50 mL stoppered centrifuge tube containing 5 negative samples, shake well, cover the stopper and place it on a constant temperature water bath shaker, 37 Hydrolyze with shaking at ℃ overnight (≥5h)
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After the hydrolyzate is cooled to room temperature, add 20 mL of ethyl acetate, homogenize and extract on a homogenizer at a speed of 10000 r/min for 1 min, centrifuge at 3000 r/min for 10 min, and filter the supernatant into a chicken heart bottle

(4) Purification

1) Gel chromatograph conditions

Purification column: 22g S-X3 Bio-Beads packing, 200～400 mesh, 200mm×25mm(id), or equivalent; mobile phase: ethyl acetate + cyclohexane (50+50, v/v), flow rate: 5.
0mL/min; quantitative loop: 5mL; purification procedure: 0～10.
5min discard the eluate, 10.
5～15.
5min collect the eluate, 15.
5～18min discard the eluate
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2) GPC and silica gel solid phase extraction purification

The extract was evaporated to near dryness at 45°C, and the volume was filled with ethyl acetate + cyclohexane into a 10 mL glass test tube, and purified according to GPC conditions
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After purification, evaporate the collected eluate, vortex the residue with 3 mL of acetone + n-hexane to dissolve the residue, transfer the solution to the reservoir on the pre-washed silica gel column, and then use 3 mL of acetone + n-hexane (10+90, v/v) dissolve once, pass the combined solution through a silica gel column, then rinse the silica gel column with 3 mL acetone + n-hexane (10+90, v/v), and finally use 5 mL acetone + n-hexane The alkane elutes the analyte and collects it

(5) Preparation of sample solution

Weigh 5g (accurate to 0.

(6) Preparation of blank matrix solution

Weigh 5g (accurate to 0.