This standard specifies HPLC methods for drug residue detection of ciprofloxacin, danofloxacin, and enrofloxacin in animal derived food.
This standard applies for the detection of drug residues of ciprofloxacin, danofloxacin, and enrofloxacin in the muscles, liver and kidneys of pigs and chickens. (The sample of this experiment is chicken)
Reference Standard: Method for the Detection of Fluoroquinolone Drug Residues in Animal Derived Food, No. 1025 Announcement -14-2008 by Ministry of Agriculture, HPLC
Accurately weigh 2.0g chicken sample in the centrifuge tube, then add 10.0mL phosphate buffer solution. Mix well with ultrasound for 10min, and centrifuge at 8000r/min for 10min. Transfer the supernatant into another centrifugal tube, and add 10.0mL phosphate buffer solution in residue. Repeat the above extraction steps mentioned above, and combine all the supernatant.
SPE column: Welchrom® BRP column, specification: 200mg/6mL.
Activation: 2mL methanol, 2mL phosphate buffered solution, discard;
Sample loading: 5mL supernatant, discard;
Washing: 1mLwater water,drain and discard;
Elution: elute with 5mL mobile phase,then drain and collect.
Let the collected eluent through 0.22 μm microporous filter membrane for machine detection.
Column: Ultisil ® XB-C18, 4.6*250mm, 5μm.
Mobile phase: 0.05mol/L phosphoric acid solution/triethylamine: acetonitrile =82: 18, gradient elution;
Flow rate: 0.8mL/min;
Column temperature: 25 ℃;
Wavelength: fluorescent detector, excitation wavelength: 280nm, emission wavelength:450nm.
Injection volume: 20 μL.
Chromatogram or Spike Recovery Rate
Fig 1. Chromatogram of dacloxacin 5 μg/L, ennofloxacin, cyprofloxacin 25 μg/L
Fig 2. Chromatogram of Blank Chicken Sample
Fig 3. Chromatogram of chicken sample added flufloxacin 0.05mg/kg, ennofloxacin 0.25mg/kg and cyprofloxacin 0.25mg/kg
|Substance||Spike Level||Average Recovery Rate||RSD（n=2）|
Table: Spike Recovery Rate
|00522-20014||SPE column||Welchrom® BRP,60mg/3mL,50pk|
|00837-05006||50mL centrifuge tube with screw mouth and apical bottom||Disposable centrifuge tube, flat cap,conical bottom, RCF12000xg, 15 mL, 50/ pk without sterilization|
|00824-31001||Welch SPE instrument||12 ports square cylinder|
|00821-32291||Caps and septa||Pre-slit red PTFE/white Silicone septa, 9mm blue short screw-thread polypropylene cap, 6mm centre hole, 100/pk|
|00821-40927||Vials||2ml wide opening short screw-thread vial with write-on spot, clear, 11.6*32mm , 100/pk|
|00201-31043||HPLC column||Ultisil® XB-C18，4.6*250mm，5μm|
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