Determination of 9 penicillin residues in milk and milk powder——liquid chromatography-tandem mass spectrometry

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1 Scope of application

It is suitable for the determination of 9 penicillin residues in milk and milk powder by liquid chromatography-tandem mass spectrometry
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Detection limit of the method: 1μg/kg for ampicillin and nafcillin in milk, 2μg/kg for amoxicillin, piperacillin, penicillin G, penicillin V and cloxacillin , and 4μg/ kg for oxacillin and dicloxacillin.

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2 Principle of the method

Amoxicillin, ampicillin, piperacillin, penicillin G, penicillin V, oxacillin, cloxacillin, nafcillin and dicloxacillin residues in milk and milk powder are extracted with acetonitrile-water solution and purified by solid phase extraction column.
High performance liquid chromatography-tandem mass spectrometry determination, external standard method quantification
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3 Reagents and materials

Acetonitrile and acetic acid are both pure liquid chromatography; disodium hydrogen phosphate and sodium hydroxide are both analytical pure
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5mol/L sodium hydroxide solution: Dissolve 100g sodium hydroxide in 450mL water, add water to make the volume up to 500mL
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The purity of the nine penicillin standard substances is greater than or equal to 98%
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Standard stock solution: Weigh appropriate standards (accurate to 0.
0001g), and prepare a 100μg/mL standard stock solution with acetonitrile-water solution
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Mixed standard intermediate working solution: Take each 1mL of the standard stock solution to a 100mL volumetric flask, dilute to the mark with acetonitrile-water solution, and prepare a mixed standard working solution with a concentration of 1μg/mL
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Standard working solution: As needed, draw a certain amount of mixed standard intermediate working solution, dilute it to the required concentration with the blank sample extract, and prepare it for use now
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HLB solid phase extraction column or equivalent: 500mg, 6mL
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Use 3mL methanol , 3mL water and 3mL phosphate buffer solution to activate sequentially before use

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4 Apparatus and equipment

High performance liquid chromatography-tandem mass spectrometer: equipped with an electrospray ion source (ESI); analytical balance: a sensitivity of 0.
01g; centrifuge; vortex mixer; rotary evaporator; solid phase extraction device; pH meter
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5 Sample pretreatment

(1) Sample preparation

Milk sample: 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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Milk powder sample: take a uniform sample of about 250g into a clean container as a sample, seal it, and mark it

(2) Extraction

Weigh about 4g (accurate to 0.
01g) of milk sample in a 50mL centrifuge tube with stopper, weigh about 0.
5g (accurate to 0.
01g) of milk powder sample and add 4mL of water in a 50mL centrifuge tube with stopper, and mix well
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After adding 20 mL of acetonitrile-water solution for high-speed shaking and extracting for 2 min, centrifuge at 3000 r/min for 10 min, transfer the supernatant and filter it into a chicken heart bottle

(3) Purification

Rotate the extract to about 7 mL at 45°C, add 2 mL of phosphate buffer, and after mixing, transfer to the activated HLB solid phase extraction column, and then wash the heart bottle twice with 2 mL of phosphate buffer.
The washing liquid is transferred to the column at the same time, and the flow rate is controlled to be less than 2mL/min
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Rinse and drain the extraction column with 3 mL of water, eluate with 4 mL of acetonitrile-water solution and collect in a 10 mL graduated glass tube (control flow rate less than 2 mL/min)

(4) Preparation of blank matrix solution

Take 4g of negative milk sample and 0.