Determination of testosterone, epitestosterone and progesterone residues in beef liver and beef by liquid chromatography-tandem mass spectrometry

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1 Scope of application

It is suitable for the determination of testosterone, epitestosterone and progesterone residues in beef liver and beef by liquid chromatography-tandem mass spectrometry
.
Detection limit of the method: testosterone, epitestosterone , and progesterone in the liver are 0.

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2 Principle of the method

The testosterone, epitestosterone, and progesterone drug residues in beef liver and beef were extracted with methanol tert-butyl methyl ether after enzymatic hydrolysis .
The extract was purified with a C ₁₈ solid phase extraction column, and the liver sample was purified by a silica gel solid phase extraction column.
After deliquation, concentration and constant volume, it is used for liquid chromatography-tandem mass spectrometry determination
.

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3 Reagents and materials

Acetonitrile, methanol, tert-butyl methyl ether , n-hexane : chromatographically pure; chloroform , ammonium formate : analytically pure
.

β-Glucuronidase/sulfatase: H-2 type, Helixpomatia
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Phosphate buffer: 1.

Testosterone, epitestosterone, and progesterone standard materials: purity ≥99%
.
Deuterated testosterone (d ₅ -testosterone), deuterated progesterone (d ₉ -progesterone) internal standard reference materials: purity ≥98%

Standard stock solution: 100μg/mL
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Accurately weigh the appropriate amount of testosterone, epitestosterone, and progesterone standard materials, prepare 100μg/mL standard stock solutions with methanol, store them at -18°C, and use them for 6 months

Standard working solution: 5μg/mL
.
Draw 5mL testosterone, epitestosterone, and progesterone standard stock solutions into a 100mL volumetric flask, dilute with methanol and make the volume constant.

Mixed standard working solution: 50ug/L
.
Draw 1.

Internal standard stock solution: 100μg/mL
.
Accurately weigh the appropriate amount of deuterated testosterone (ds-testosterone) and deuterated progesterone (d9-progesterone) standards, respectively prepare 100μg/mL internal standard stock solution with methanol, store at -18℃, and use for 6 months

Internal standard working solution: 5μg/mL
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Separately draw 5 mL of deuterated testosterone (d5-testosterone) and deuterated progesterone (d9-progesterone) internal standard stock solutions into a 100 mL volumetric flask, dilute with methanol and make the volume constant

Internal standard mixed working solution: 25μg/L
.
Dilute 0.
50 mL of deuterated testosterone (d5-testosterone) and deuterated progesterone (d9-progesterone) internal standard working solutions into a 100 mL volumetric flask, dilute with methanol and make the volume constant
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Stored at -18℃, it can be used for 3 months
.

Matrix mixed standard working solution: According to the sensitivity of each standard and the linear range of the instrument, draw a certain amount of mixed standard working solution and internal standard working solution, and use the blank sample extract to prepare a series of concentrated matrix mixed standard working solutions
.
Prepared on the same day
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C ₁₈ solid phase extraction column: 500mg, 6mL
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Activate with 6mL methanol and 6mL water in sequence before use
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Silica gel solid phase extraction column: 200mg, 6mL
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Activate with 6mL n-hexane before use
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4 Instruments and equipment

Liquid chromatography-tandem mass spectrometer, equipped with electrospray ion source; analytical balance: sensitivity 0.
1mg and 0.
01g; homogenizer; high-speed tissue homogenizer; low-temperature high-speed centrifuge: speed greater than 4000r/min; high-speed centrifuge : Rotation speed is greater than 12000r/min; ultrasonic; vortex oscillator; oscillating water bath; nitrogen concentrator; solid phase extraction device; rotary evaporator; KD concentrating bottle
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5 Sample preparation

(1) Sample preparation

Take about 500g of the sample and mash it with a tissue masher, put it into a clean container as a sample, seal it, and mark it
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Store the sample at -18°C
.

(2) Enzymatic hydrolysis

Accurately weigh 5g sample, accurate to 0.
01g, place it in a 50mL centrifuge tube, add 200μL internal standard mixed working solution and 10mL phosphate buffer in sequence, homogenize at 14000r/min for 30s, add 60μL glucuronidase /sulfate Enzyme, mix well for 30s, put in 37℃ constant temperature shaking water bath for enzymolysis for 16h
.

(3) Extraction

Add 10mL methanol to the sample solution after enzymolysis , mix well for 2min, place it in an ice bath for 5min, centrifuge at 4000r/min at 5℃ for 10min, transfer the supernatant to a 50mL centrifuge tube, and repeat the above operation with 5mL methanol for the lower precipitation Once, combine the supernatants into the above 50mL centrifuge tube, add 15mL tert-butyl methyl ether to the centrifuge tube, shake for 5min, centrifuge at 4000r/min for 5min, transfer the upper solution to a 125mL separatory funnel, and use 15mL for the lower solution in turn , 10mL tert-butyl methyl ether extract twice, combine the upper layer solution into the above separatory funnel, add 10mL saturated sodium chloride aqueous solution, shake for 30s, stand for layering, the upper layer solution is transferred to a pear-shaped bottle, and rotary evaporated at 40 ℃ To dryness, add 1.
5 mL of methanol to the residue, vortex for 1 min, sonicate for 5 min, add 20 mL of water, mix well, and wait for purification
.

(4) Purification

Transfer the sample extract to the activated C18 solid phase extraction column, wash the pear-shaped flask with 5 mL of water, combine the washing liquid into the C18 solid phase extraction column, and pass the sample liquid through the solid phase extraction column at a flow rate of ≤ 2 mL/min.
After the sample solution has passed through the column, rinse the C18 solid phase extraction column with 5 mL of water, discard the eluent, use 10 mL of methanol to elute the component to be tested from the C18 solid phase extraction column, and transfer the eluent to the pear shape In the bottle, rotary evaporate to dryness at 40°C
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Dissolve the residue of the muscle sample with 0.
5 mL methanol, vortex for 1 min, add 0.
5 mL water to mix, and pass through a 0.
2um filter membrane for liquid chromatography tandem mass spectrometry
.

After the eluent of the liver sample is evaporated to dryness, add 0.
5 mL of chloroform to the pear-shaped flask, vortex for 1 min, add 5 mL of n-hexane, sonicate for 1 min, and then transfer to an activated silica gel solid phase extraction column, with a concentration of 1 mL/ Min flow rate makes the sample liquid pass through the solid phase extraction column.
After the sample liquid passes through the column, rinse the silica gel solid phase extraction column with 5 mL n-hexane, discard the eluent, and elute the components to be tested with 6 mL water-saturated ethyl acetate.
The eluent was transferred to the KD receiving flask, rotary evaporated to dryness at 40°C, 0.
5 mL methanol was added, vortexed for 1 min, 0.
5 mL water was added to mix, and it was passed through a 0.
2um filter membrane for liquid chromatography-tandem mass spectrometry determination
.

Weigh the negative samples, and prepare the blank sample extract according to the above extraction and purification steps to prepare a series of matrix mixed standard working solutions
.

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