Determination of four cephalosporins in milk and milk powder by liquid chromatography-tandem mass spectrometry

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1 Scope of application

Determination of Cefpirin, Cephalexin, Ceflonine and Cefquinome Residues in Milk and Milk Powder by Liquid Chromatography-Tandem Mass Spectrometry
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The detection limit of milk method: cefpirin, cephalexin, cefalornin, and cefquinoxime is 4.

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2 Principle of the method

Residues of cefpirin, cephalexin, cefalonine and cefquinoxime in milk and milk powder were extracted with acetonitrile and phosphate buffer solution, purified by solid phase extraction column, determined by liquid chromatography-tandem mass spectrometry, and quantified by external standard method
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3 Reagents and materials

Methanol, acetonitrile: chromatographically pure; sodium dihydrogen phosphate (NaH ₂ PO ₄ ), sodium hydroxide, acetic acid, n-hexane : excellent grade pure
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Acetonitrile-water solution (3+1, v/v): Measure 60 mL of acetonitrile and 20 mL of water and mix well
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Acetonitrile-saturated n-hexane: Take 100 mL of n-hexane and 50 mL of acetonitrile in a 250 mL separatory funnel, shake for 1 min, stand still for layering, discard the acetonitrile

The purity of the standard substances of Cefpirin, Cephalexin, Ceflonine and Cefquinome ≥99%
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1.
0mg/mL four cephalosporin standard stock solutions: accurately weigh an appropriate amount of each standard substance and prepare a standard stock solution with a concentration of 1.
0mg/mL with water
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The stock solution is stored in a -18°C freezer

Four cephalosporin standard mixed working solutions: draw an appropriate amount of each cephalosporin standard stock solution as needed, and make a mixed standard working solution of appropriate concentration with water
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OasisHLB solid phase extraction column or equivalent: 500mg, 6mL
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Before use, it was pretreated with 5 mL methanol, 5 mL water and 10 mL sodium dihydrogen phosphate buffer solution in sequence to keep the column moist

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4 Apparatus and equipment

Liquid chromatography-tandem quadrupole mass spectrometer, equipped with electrospray ion source; analytical balance: sensitivity 0.
1mg, 0.
01g; solid phase extraction vacuum device; liquid reservoir: 50mL; micro syringe: 25μL, 100μL; homogenization High-speed refrigerated centrifuge: with a 50mL centrifuge tube with stopper, the rotation speed is above 10000r/min; scale sample tube: 5mL, accuracy of 0.
1mL; rotary concentrator; nitrogen concentrator
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5 Sample pretreatment

(1) Sample preparation

Take out a representative sample of about 1kg from all the samples, mix them well, divide them into two parts, and put them into clean containers
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After being sealed, it is used as a sample and marked with a mark

(2) Extraction of sample solution

a.
Milk: Weigh 5g of sample (accurate to 0.
01g) in a 50mL centrifuge tube, add 20mL of acetonitrile, homogenize with a homogenizer for 1min, and centrifuge the extract with a high-speed refrigerated centrifuge at 10℃ for 10min at 10000r/min.
Transfer the upper extract to another centrifuge tube
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The extraction was repeated once with 15 mL of acetonitrile -water solution, the two extracts were combined, and 10 mL of acetonitrile-saturated n-hexane was added, shaken for 1 min, and the n-hexane was discarded

b.
Milk powder: Weigh 0.
5g sample (accurate to 0.
01g) in a 50mL centrifuge tube, add 4.
0mL water to fully dissolve the milk powder, add 20mL acetonitrile, homogenize with a homogenizer for 1 min, and freeze the extract at high speed The centrifuge was centrifuged at 10°C 10000r/min for 10 minutes, and the upper extract was transferred to another centrifuge tube
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Repeat the extraction once with 15 mL of acetonitrile aqueous solution, combine the two extracts, and add 10 mL of acetonitrile-saturated n-hexane, shake for 1 min, and discard the n-hexane

(3) Purification of sample solution

Add 20 mL of sodium dihydrogen phosphate buffer solution to the sample solution from which the acetonitrile has been removed, and then adjust the pH to 8.
5 with sodium hydroxide solution
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Transfer the sample extract to the reservoir connected to the OasisHLB solid phase extraction column, and pass it through the solid phase extraction column at a flow rate of 3mL/min.

(4) Preparation of standard mixed working solution for blank sample

a.
Pipette appropriate amounts of four cephalosporin standard mixed working solutions from the milk and add them to 5.
0g sample.
Follow the above steps to prepare cefpirin, cephalexin, cefalonine, and cefquinoxime with a concentration of 4.
0.
Four samples of μg/kg, 8.
0μg/kg, 16ug/kg, and 40μg/kg were added with standard mixed working solution for determination by liquid chromatography-tandem mass spectrometer
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b.
Milk powder accurately pipet appropriate amounts of four cephalosporin standard mixed working solutions, add them to 0.
5g sample, follow the above extraction and purification steps to prepare cefpirin, cephalexin, cefalonine, and cefquinome concentration respectively Add standard mixed working solution to four samples of 32ug/kg, 64ug/kg, 128μg/kg, and 320μg/kg for determination by liquid chromatography-tandem mass spectrometer
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