| Objective: The aim of this study was to establish an air-assisted dispersive solid-phase extraction combined with high-performance liquid chromatography method for the determination of ofloxacin,norfloxacin,ciprofloxacin and enrofloxacin in honey,and to use the established method for the determination of the four quinolones in real honey samples.Methods: The honey sample was diluted with water and was adjusted to alkaline.Then nickel/aluminum layered double hydroxides were added as the extractant for the extraction of ofloxacin,norfloxacin,enrofloxacin,and ciprofloxacin from honey by air-assisted dispersive solid-phase extraction.The extractant was dissolved in phosphoric acid solution after centrifugation and detected by high-performance liquid chromatographyfluorescence detector.Qualitative analysis of the quinolones was done by retention time and quantitative analysis was conducted by working standard calibration curves.The morphology,size,functional groups and other physical and chemical properties of the selected layered double hydroxides were characterized by scanning electron microscopy,transmission electron microscopy,Fourier transform infrared spectroscopy and X-ray diffraction.Taking the extraction efficiencies of four quinolones as the index,the volume of diluent,p H value of the sample,type and the amount of the extractant,auxiliary extraction method,extraction time,type and the amount of the dissolution solvent were optimized to achieve satisfied extraction of the four quinolones in honey.Meanwhile,taking the resolution,analysis time and peak areas of the target veterinary drugs into consideration,the wavelength,mobile phase,column temperature and flow rate of the high-performance liquid chromatograph method were optimized.The sensitivity,accuracy and precision of the method were evaluated by the linear ranges,detection limits,quantitation limits,recoveries,intra-day and inter-day relative standard deviations.The established method was applied to the determination of ofloxacin,norfloxacin,enrofloxacin and ciprofloxacin residues in 43 honey samples.Results: This study successfully established an air-assisted dispersive solid-phase extraction/high-performance liquid chromatography method for the simultaneous detection of ofloxacin,norfloxacin,ciprofloxacin,and enrofloxacin in honey.The optimal extraction conditions for quinolone veterinary drugs in honey were as follows: 1 g of honey sample was diluted with 6 m L of ultra-pure water,and its p H was adjusted to 8.Then 1 m L of the aqueous solution was taken.Afterward,2 mg of nickel/aluminum layered double hydroxides were added to extract the quinolones with the assistance of air using a syringe to pump the solution for 50 times.At last,250 μL of 0.1% phosphoric acid solution was added to dissolve the precipitation before injection.The optimized high-performance liquid chromatograph conditions were as follows: a C18 column was used.The mobile phase consists of methanol,acetonitrile and phosphoric acidtriethylamine aqueous solution(1 L of water contained 3.4 m L of phosphoric acid and 6 m L of triethylamine),with a volume ratio of 18:6:76.The fluorescence excitation wavelength was 280 nm,and the fluorescence emission wavelength was 460 nm.The column temperature was 35 ℃.The flow rate was 0.9 m L/min.Under the optimized sample pretreatment and high-performance liquid chromatography conditions,the extraction of four quinolone veterinary drugs in honey could be completed in 10 minutes,and the determination of four quinolone veterinary drugs in honey can be completed in 17 minutes.The concentration of the four quinolone veterinary drugs were linear in the range of 1.00～100 ng/g,the correlation coefficients were in the range of 0.9996～0.9999.The detection limits and quantitative limits of the method were 0.25～0.82 ng/g and 0.82～2.73 ng/g,respectively.The recoveries were 81.9～90.4%.The intra-day and inter-day relative standard deviations were 4.39～11.0% and 2.16～17.5%,respectively.Ofloxacin and norfloxacin were not detected in honey samples.Enrofloxacin was detected in three honey sources including one chaste honey,one acacia honey and one creamed honey.Ciprofloxacin was detected in one loquat nectar.Ciprofloxacin was detected in one honey sample from Sichuan province,enrofloxacin was detected in three honey samples from Fujian province,Hubei province and Hunan province,respectively.No quinolone veterinary drug was detected in autumn honey samples.Enrofloxacin was detected in one spring honey sample and two summer honey samples.Enrofloxacin was detected in one spring honey sample and two summer honey samples.Ciprofloxacin was detected in one winter honey sample.The detected amounts of quinolone veterinary drugs in honey samples were all lower than the lowest maximum residue limit of the target 4 quinolone veterinary drug residues in food regulated by the’National Food Safety Standard Maximum Residue Limit of 41 Veterinary Drugs in Food ’(GB 31650.1-2022).Conclusion: The established air-assisted dispersive solid-phase extraction/high-performance liquid chromatography method is convenient,sensitive,accurate,high extraction efficiency,low cost and environmentally friendly.It’s suitable for the determination of quinolone veterinary drug residues in honey. |
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