Study On Detection Methods Of Six Kinds Of Animal Origin Fluoroquinolone Residues In Honey Product

Objective: To establish a method for determining six kinds of fluoroquinolones norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, sarafioxacin, and difloxacin in honey. Methods: 1.The detection method of six kinds of fluoroquinolone residues above in honey product has been researched by using thin layer chromatography(TLC). Vortex extract the sample by the solution of 5% acetic acid in acetonitrile, centrifugal extract the supernatant and purify with dispersivesolid phase extraction(Qu ECh ERS). Using the lower solution of trichloromethanemethyl alcohol-ethyl acetate-ammonia-water(15∶10∶4 ∶2∶1) as a solvent to extract and dry, and be sprained with chromogenic agent 3%Terbium(III) chloride hexahydrate. 2. We use high performance liquid chromatography with fluorescence(HPLC- FLD) to study the detection method of six fluoroquinolone residues in honey products. Solutions are obtained by pretreatment of samples.We use the Ultimate XB-C18(250×4.6 mm, 5 μm) chromatographic column, with isocratic elution using acetonitrile and 0.2% formic acid solution(13∶87) as mobile phase at a flow rate of 1.0 m L/min.The column temperature is 30℃. High performance liquid chromatography-fluorescence detection, excitation wavelength 280 nm, emission wavelength 450 nm. 3. We used ultra performance liquid chromatography combined with mass spectrometry(UPLC/MS/MS) to study the qualitative methods of six fluoroquinolones that residue in honey products. Sample solution was tested in sequence by UPLC/MS/MS analysis, chromatographic column, agilent ZORBAX SB-C18(2.1 mm×50 mm, 1.8 μm).The acetonitrile as the mobile phase A, ammonium formate solution with 0.1% methanoic acid for B using the gradient elution mode with column flow rate 0.5 m L/min. Temperature 45℃. Injection volume 2 μL, ESI as ionization source, Capillary voltage 3000-3500 k V. Temperature of drying gas, 300℃. Gas flowing rate 5 m L/min, atomizing air pressure 50 psi. Results:1. The better separating degree for norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, sarafioxacin and difloxacin were obtained under the UV 365 nm by using thin layer chromatography(TLC) with Rf ranged from 0.2 to 0.8. 2. The results of high performance liquid chromatography determination showed that six fluoroquinolone’s separating degree is clear and a good linear relationship was obtained in the range of 0.02 to 0.5 μg/m L, r= 0.9999, the detection limit ranged from 0.97 to 3.91 μg/kg. The average recovery rate was between 72.08% and 109.83.%.The relative standard deviation(RSD) ranged from 1.59% to12.16%.3.Ultra performance liquid chromatography combined with mass spectrometry(UPLC/MS/MS)determination results showed that six kinds of fluoroquinolones had a good linear relationship in the range of 2.00 to 100 ng/m L r= 0.9968. The average recovery rate was between 93.36% and 129.75% while the relative standard deviation(RSD) ranged from 2.15% to 12.23%. Conclusion: The method of TLC is applied to primary screening test of fluoroquinoloness in the honey due to its simple operation and application in basic unit. HPLC-FLD is suitable for large scale detection for institution because of low detection. Due to high sensitivity,UPLC-MS/MS can be used for confirmation analysis of positive samples.