Determination of a-Trenbolone, β-Trenbolone, 19-Ethylene Nortestosterone and Epi-19-Ethylene Nortestosterone Residues in Cow Urine

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1 Scope of application

In case of bovine urine 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 with enzyme under pH=5.
0, purified by immunoaffinity 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 , acetonitrile , acetic acid: chromatographically pure; β -glucuronidase/aryl sulfatase (B-glucuronidase/aryl sulfatase): 100000 units/mL
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Sodium hydroxide solution: 1mol/L
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Hydrochloric acid solution: 1mol/L
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Immune affinity column elution buffer stock solution and column storage buffer stock solution: attached to the immunoaffinity column
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Column elution buffer solution: Measure 1mL stock solution to be miscible with 19mL water, and prepare it immediately before use
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Column storage buffer solution: Measure 1 mL of stock solution to dissolve in 4 mL of water, and prepare it immediately before use
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a-Trenbolone, β-Trenbolone, 19-ethylene nortestosterone and epi-19-ethylene nortestosterone standard materials: purity ≥98%
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Standard stock solution: accurately weigh out 0.
0100g each of a-Trenbolone, β-Trenbolone, 19-vinyl nortestosterone and epi-19-vinyl nortestosterone standard substances, dissolve it with acetonitrile and dilute to 100mL, Prepare a standard stock solution of 100μg/mL
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Mixed standard working solution: Take each 5mL to 50mL volumetric flask of the standard stock solution, dilute to the mark with acetonitrile, and prepare a mixed standard working solution with a concentration of 10μg/mL.
This solution is stored frozen at -18°C and can be used for three months
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Trenbolone/19-ethylene nortestosterone immunoaffinity column: with column wash buffer stock solution and column storage buffer stock solution, store at 2～8℃
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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

Take fresh cow urine and quickly freeze it as a sample for later use
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(2) Weigh the sample

Take 5 negative samples, melt and mix the frozen urine, take some samples and centrifuge at 4000r/min for 10min
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Then, measure 5mL (accurate to 0.
01mL) of each sample into a 15mL centrifuge tube with stopper, and then add different amounts of mixed standard working solution, so that the concentration of each test component is 2.
5ng/mL, 5ng/mL.
mL, 10ng/mL, 50ng/mL, 100ng/mL
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(3) Hydrolysis

Use 1mol/L hydrochloric acid to adjust the pH of the urine sample to between 4.
9 and 5.
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Then add 40μL β-glucuronidase/aryl sulfate, shake well, cover the stopper, and place it in a constant temperature water bath.
Hydrolyze on the bed with shaking at 37°C for 2h
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After the hydrolyzed solution is cooled to room temperature, 4mL column elution buffer solution is added, and the pH of the urine sample is adjusted between 7-9 with 1mol/L sodium hydroxide solution
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(4) Purification of immunoaffinity column.

First pass the buffer solution stored in the column through the column, and then equilibrate the column with 15 mL column wash buffer solution
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Then, the hydrolyzed urine sample is passed through the column, and then the column is rinsed with 8 mL of column elution buffer solution and 5 mL of water in sequence
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Finally, the analytes were eluted with 4 mL methanol + water solution and collected in a graduated test tube (the flow rate through the column in the above process should be ≤ 2 mL/min)
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The eluent was blown to about 1 mL in a 60°C water bath with a nitrogen concentrator, and the volume was adjusted to 2 mL with mobile phase, mixed with a vortex, and passed through a 0.
45 μm filter membrane for HPLC-MS-MS determination
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(5) Preparation of sample solution

The frozen urine sample was melted and mixed uniformly, and part of the sample was centrifuged at 4000r/min for 10min
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Then, measure 5mL (accurate to 0.
01mL) into a 15mL centrifuge tube with stopper
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Follow the steps above
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(6) Preparation of blank matrix solution

The negative frozen urine samples were melted and mixed uniformly, and part of the samples were centrifuged at 4000r/min for 10min
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Then, measure 5mL (accurate to 0.
01mL) into a 15mL centrifuge tube with stopper
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Follow the steps above
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