Determination of Quinolones Residues in Milk

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Application range

This method specifies the determination of quinolones residues in milk by HPLC.

This standard applies to the determination of residues of dafloxacin, enfloxacin and cyprofloxacin in milk. (This test sample is milk).

Reference standard: GB 29692-2013 Determination of quinolones in Milk HPLC

Extraction steps

Accurately weigh 1.0g milk sample in 15mL centrifuge tube, then add 5.0mL acetonitrile-10% trichloroacetic acid solution. Mix well and centrifuge at 8000r/min with ultrasound for 5min. Take the supernatant to use.

Purification steps

SPE column: Welchrom® BRP column, specification: 60mg/3mL.

Activation: 3mL methanol, 3mL water, discard;

Sample loading: all the supernatant, discard;

Washing: 3mL10% methanol aqueous solution, drain and discard;

Elution: 3mL methanol, drain and discard;

Blow the collected eluent to dry with nitrogen at 60 ℃, then dissolve the residue with 500 μL acetonitrile-citric acid/ammonium acetate buffer. Mix well with vortex, and centrifuge at 8000r/min for 10min. Take the supernatant to filter through 0.22μm filter membrane to be tested by machine.

Chromatographic conditions

Column: Ultisil® XB-C18,4.6 × 250mm, 5μm.

Flow rate: 1.5mL/min

Column temperature: 50 ℃

Injecton volume: 20 μL

Fluorescent detector

Time/minEx/nmEm/nm
0278465
23.4312366
32.0278465

Elution program

Time/minAcetonitrile/%Citric acid / ammonium acetate
Buffer solution /%
0.01892
305545
32892
35892

Chromatograph or spike recovery rate

Fig 1. Chromatogram of darfloxacin 10ng/mL, enfloxacin, ciprofloxacin 50ng/mL

Peak nameRtAreaHeightAreaTailng factorPlates
 _minmV*minmV%EPEP
Ciprofloxacin9.8603598045000219.1081.11342506
Danofloxacin10.56173843810161639.2171.11848636
Enrofloxacin11.35478473210522541.6751.12353569

Fig 2. Blank chromatogram of milk sample

Fig 3. Chromatogram of milk sample floloxacin spiked at 5 μg/kg, enfloxacin, cyloprofloxacin spiked at 25 μg/kg

Tab 1. Spike recovery rate

SubstanceSpike levelAverage recovery rateRSD
μg/kg%%(n=2)
Ciprofloxacin578.470.79
Danofloxacin2575.360.26
Enrofloxacin2579.012.88

Ordering Information

P/NProductSpecification
00522-20009SPE columnWelchrom® BRP, 60mg/3mL, 50pk
00837-0500215mL centrifuge tube with screw mouth and conical bottomDisposable centrifuge tube, flat cap, conical bottom, RCF 12000xg, 15 mL, 50/ pk without sterilization
00824-31001Welch SPE instrument12 ports square cylinder
00821-32291Caps and septaPre-slit red PTFE/white Silicone septa, 9mm blue short screw-thread polypropylene cap, 6mm centre hole, 100/pk    
00821-40927Vials2ml wide opening short screw-thread vial with write-on spot, clear, 11.6×32mm, 100/pk  
00201-31043HPLC columnUltisil® XB-C18, 4.6×250mm,5μm
00802-02201Syringe filterImport NY, 13mm×0.22μm, 100pk

If you have any problem or require further information, please contact info@welchmat.com.


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