The Study Of Detection Methods Of Mult-quinolones Residual In Aquatic Products

Quinolones were synthetic antibiotics for people and animals, acting on Gram-positive and Gram-negative bacterias, always used in preventing diseases in aquaculture. Well now the problems of food safety were paid close attention by people, the problem of fish drug residual was focused on by relative department, and also the important society issues. The detective methods used in drug residual of aquatic products during recent years were: enzymoimmunoassay, Capillary Electrophoresis CE, gas chromatography, high performance liquid chromatography and coupling technique. High performance liquid chromatography and coupling technique were universal used, but there were methods for one or a few quinolones test. This paper set up a method for 6 quinolones test used HPLC and a method for 19 quinolones test used HPLC-MS/MS in aquatic products. The study included 4 sections:1) Set up a method of HPLC for 6 quinolones test at the same time in aquatic products. On the basis of the characteristic of HPLC, compared the separation effects of different chromatographic columns, investigated the conditions of analysis effects such as the composition and proportion of mobile phase, pH, velocity of flow, column temperature, optimum emission and excitation wavelength; contrasted and optimized the steps of extraction and purification during the preliminary treatment process; investigated the stability, accuracy rating and the scope of application through testing and verifying the limit of detection and adding recovery of the method. After the repeated experiment and exploration, determined to use acidized acetonitrile to extract the residual enrofloxacin, ciprofloxacin, norfloxacin, ofloxacin, oxolinic acid and flumequine, normal hexane used to degrease, Varian Bond Elut C₁₈ SPE column to purify the samples. Choosing the Zorbax Elipse XDB- C₁₈(150 mm×4.6 mm,5μm)column, using the HPLC-FLU to test, external standard method to quantify. The results showed that, the linear relation of standards, the adding recovery of all drugs were above 75.0%, the limit of detection of enrofloxacin, ciprofloxacin, norfloxacin and ofloxacin were 5μg/kg, well the oxolinic acid and flumequine were 10μg/kg, and the relative standard deviation were less than 10.00%.2) Set up a method of HPLC-MS/MS for 19 quinolones test at the same time in aquatic products. The residual of 19 quinolones in the samples were extracted by formic acid-acetonitrile(1:99,V/V), acetonitrile saturation nomal hexane used to purify, the mobile phase was 0.2% formic acid-acetonitrile containing 2 mmol/L ammonium acetate, gradient elution, SRM scanning mode, external standard method to quantify. Adding recovery experiment was achieved under adding 1,5,20μg/kg, repeated 6 measurement. And the results showed that, average recovery was 76.1%¹16.0%, the relative standard deviation was 3.20%¹3.10%. After adding to the blank samples, obtained the limit of detection for 17 quinolones were 1μg/kg, well for fleroxacin and pipemidic acid were 2μg/kg. In the end, adding 5μg/kg to blue crab, weever, shrimp, eel, grass carp, abalone, turtle and oyster, determine the recovery were 77.6%¹02.0%, and the RSD were less than 13.00%.3) Investigated the source of the uncertainty of the two detected methods then analyzed and evaluate them, received an intuitions tic description of the experiment results, gave the quality of the results a more integrated rational description. We found the main factors which impacted the results'veracity during the process of experiment and chose some measures to minish or avoid the influence. After the uncertainty evaluation, we should focus on the confection of the standards and adding recovery, which would impact the results significantly.4) Now the method of detecting 6 quinolones in aquatic products by HPLC has been set up to the industry standard, waiting for examination and ok by the administration section of agriculture department, the method of HPLC-MS/MS would be set up to be the located standard.This issue set up two methods of multil-quinolones test, the preliminary treatment process was simple, convenient and precise, saving the costs and time. Based on the numerous verified experiments, provided practical reference and application value to the work of daily drug residual test.