| Pesticide and veterinary drug residue have been the main factors which influence food safety, and establishing rapid and effective methods for residues detection and analysis has great significance to food quality regulation. Norfloxacin, one synthetic antibacterial of the third generation of quinolones, was widely used to prevent and treat many kinds of infectious diseases in human and animals. The abuse of norfloxacin in veterinary climic may produce its residue in animal-derived foods, which could lead to a public health hazard with the increase of resistant human pathogens from food chain transferring. Capillary electrophoresis based immunoassay (CEIA) is a new type analytical technique developed in recent years which combines the high efficiency separation of CE and the specificity of immunoreaction. Due to the merits of high-throughput, simple sample preparation and little injection, CEIA has been applied in many analysis and application fields of medicine, food and chemical engineering research.In this paper, a capillary electrophoresis based immunoassay method with laser induced fluorescence detection was developed for the determination of norfloxacin. The assay was based on the competitive immunoreaction between fluorescein isothiocyanate labeled norfloxacin tracer and free norfloxacin with a limited amount of antinorfloxacin antibody and achieved the quantitative analysis by monitoring the peak areas of competitors. We synthesised fluorescein isothiocyanate labeled norfloxacin and studied the parameters affecting CE separation efficiency including reaction time, buffer pH, borate concentration, separation voltage and the injection volume.By incubated for5min, the fluorescently labeled antigen and immunocomplex were well separated by CE with good peak shape and high resolution within5min under25kV voltage and0.5psi pressure using30mM sodium borate and10mM sodium phosphate (pH8.0) for background eletrolyte. Good linearity of norfloxacin concentration was achieved in the range of0.08-50ng/mL with detection limit of0.005ng/mL. The reproducibility of the method was investigated by nine test of norfloxacin standard solution (1ng/mL) one day for five consecutive days. The intra-day and inter-day RSDs of immunocomplex migration times were0.17%and0.23%, respectively. The present method was also applied for determination of norfloxacin in chicken, pork, fish and milk with LOD of0.25μg/kg and recoveries ranging of69.7%-92.4%, signifying the application capability for norfloxacin determination. |
