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

Extraction Steps

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.

Purification Steps

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.

Chromatographic Conditions

HPLC conditions

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

NameRtAreaHeightAreaTailing factorPlates

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

SubstanceSpike LevelAverage Recovery RateRSD(n=2)

Table: Spike Recovery Rate

Ordering Information

00522-20014SPE columnWelchrom® BRP,60mg/3mL,50pk
00837-0500650mL centrifuge tube with screw mouth and apical bottomDisposable centrifuge tube, flat cap,conical bottom, RCF12000xg, 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

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