Determination of metronidazole, ronidazole, dimetronidazole and their metabolites in milk and milk powder by liquid chromatography tandem mass spectrometry

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2.
1.
1 Scope of application

It is suitable for the determination of metronidazole, ronidazole , dimetronidazole and their metabolites in milk and milk powder by high performance liquid chromatography-tandem mass spectrometry
.

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2 Principle of the method

The residues of metronidazole, ronidazole, dimetronidazole and their metabolites in milk and milk powder were extracted with acetonitrile - ethyl acetate , purified by cationic solid phase extraction column, determined by high performance liquid chromatography-tandem mass spectrometry, and quantified by internal standard method
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3 Reagents and materials

Acetonitrile, ethyl acetate, methanol, acetone, acetic acid, ammonia: chromatographically pure; anhydrous sodium sulfate , analytically pure: burned at 650℃ for 4h, placed in a desiccator for later use; ammonia-acetonitrile (1+19, v/v ): Measure 5mL ammonia water and dilute to 100mL with acetonitrile
.

Standard products: metronidazole, ronidazole, dimetronidazole, hydroxy metronidazole, 1-methyl-2-hydroxymethyl-5-nitroimidazole, deuterated ronidazole (RNZ-D ³ ), deuterated hydroxy metronidazole (MZNOH-D ₂ ) and deuterated 1-methyl-2-hydroxymethyl-5-nitroimidazole (HMMNI-D ³ ): the purity is greater than or equal to 98%
.

Standard stock solution: Weigh the appropriate amount of standard substance accurately and prepare a 100μg/mL standard stock solution with methanol
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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 methanol, and prepare a mixed standard working solution with a concentration of 1μg/mL
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Internal standard standard stock solution: Weigh an appropriate amount of deuterated internal standard standard and dissolve it with methanol into a stock solution with a concentration of 200μg/mL
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Internal standard mixed standard intermediate working solution: Measure an appropriate amount of standard stock solution, and use methanol to make an internal standard mixed standard intermediate working solution with a concentration of 1μg/mL
.

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High performance liquid chromatography-tandem mass spectrometer: equipped with electrospray ionization source (ESI); centrifuge; analytical balance: sensitivity 0.
1mg and 0.
01g; vortex mixer; rotary evaporator; nitrogen blowing instrument; solid phase extraction device
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5 Sample pretreatment

(1) Sample preparation

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

Weigh 5g (accurate to 0.
01g) of milk sample in a 50mL centrifuge tube with stopper, and weigh 1g (accurate to 0.
01g) of milk powder sample
.

First add 5mL acetonitrile to the centrifuge tube to precipitate the protein, then add 20mL ethyl acetate, shake for 2min, centrifuge at 3000r/min for 10min, remove the supernatant and filter through 5g anhydrous sodium sulfate to the chicken heart In the bottle
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(3) Purification

Rotary evaporate the extract to about 2 mL at 45°C, then transfer it to the activated solid phase extraction column, and then wash the core bottle with 5 mL ethyl acetate twice, and transfer the washing liquid to the solid phase extraction column.
Drop at a flow rate of less than 2 mL/min
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(4) Preparation of blank matrix solution

Take 5g of negative milk sample and 1g (accurate to 0.

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