Studies On Detecting Five Classes Of Antibiotic Residues In Honey Simultaneously Using HPLC

In the apiculture, antibiotics are often used for the treatment of buzzers diseases. And antibiotics residues are often found when buzzers are overdosed or the drugs are used in improper ways, which will cause serious harm to human body health. Many countries in the world have been paying high attention to the supervision and detection of antibiotics residues in honey. At present, many countries including China have proposed strict limitation standard for antibiotics residues in animal derived food.Regarding five kinds of quinolones, three kinds of tetracyclines, four kinds of sulfonamides, two kinds of nitromidazoles and chloramphenicol as the object of research, this study set up a method of extracting, purifying and detecting 5 classes of antibiotics in honey at one time. The main research content includes:1) Setting up the method of detecting 5 classes of antibiotics residues in honey at one time by high performance liquid chromatography (HPLC). In the experiment, a UV detector was used. And the ultraviolet absorption band was determined at 274 nm and 315 nm. The effect of chromatographic column, mobile phase composition and pH, gradient, flow rate and column temperature on the retention and separation of 15 kinds of antibiotics was discussed. At last, it was determined that the chromatographic column was Welch Ultimate AQ-C18 column (250 mm×4.6 mm, 5μm), the mobile phase was acetonitrile-phosphoric acid solution (pH2.50), the flow rate of mobile phase was 1 mL/min and the column temperature was 35℃. In this method, the limits of detection (LOD) of 15 kinds of antibiotics were 9.70-12.54μg/kg, and the limits of quantization (LOQ) of 15 kinds of antibiotics were 32.34-41.80μg/kg. At 0.2-20.0μg·mL-1 concentration range, the linear relations of 15 kinds of antibiotics were good, and the correlation coefficient were higher than 0.99.2) The extracting and purifying process were explored repeatedly. The best preprocessing way was at last determined and the conditions of preprocessing were optimized. In the experiment, the solid phase extraction (SPE) column, the extracting solution pH, the composition and the amount of eluent, and the elution rate were optimized. As a result, the phosphate solution at pH2.0 was used to dissolve the honey sample, the Cleanert PCX SPE column was chosen as the purifying column, 9 mL amonium methanol which contains 5% aqueous ammonia was used in the elution, and the elution rate was 1 mL/min. The standard added recovery experiments were conducted to find that the average recoveries of 15 kinds of antibiotics were 60.1%-105.2%, and the relative standard deviation (RSD) were less than 10%, which met the requirements of the multi-residual analysis.3) The method that had been established before, which contains the processes of acid aqueous dissolving, SPE column purifying and HPLC detection, was applied in the detection of 3 different brands of honey to determine whether they have antibiotic residues. And the mass spectrometer (MS) was used to confirm the detection result. It was found that there were residues of tetracyclines in one brand of honey. The average recoveries of 15 kinds of antibiotics in three different brands of honey were 65.5%-100.8%, 60.1%-98.7% and 64.7%-105.1%, and the RSD were all less than 10%. 4) The resource of uncertainty of this method was discussed and evaluated, through which the factors which affected the uncertainty in the experiment t can be found and avoided by taking some measures. It was found in the evaluation of uncertainty that the uncertainty of this method mainly came from the concentration of standard solution and the repeatability of standard added recovery. Thus we should pay attention to the two steps in the process of detection.