Studies On Establishment Of HPLC Determination Methods Of Aminoglycosides Residue In Seafood

Aminoglycosides,such as neomycin, gentamicin, kanamycin, apramycin,etc. are universally applied in animal husbandry because of their broad-spectrum bactericidal effect, but their residues in animal tissues of animal-derived food will bring human beings great harm due to their side effects.Thus,there is an increasing attention to this problem.Some developed countries and the CAC have made very strict rules on the limits of aminoglycosides,so detection technology for which becomes a hot topic. In this article, the determination methods for the residues of neomycin, gentamicin in seafood by using high performance liquid chromatography(HPLC) with pre-column derivatization are initially established,a HPLC detection method for aminoglycoside multiresidues is preliminary explored.1 Determination method of neomycin in seafood by liquid chromatography with pre-column derivatization and fluorescence detection was established. Neomycin in the sample was extracted by a solution contains 10% trichloroacetic acid and 2% EDTA-Na2 monopotassium phosphate, then centrifugated, hexane was empolyed to remove fats, C18 SPE column was for purification. Derivatization reagent:FMOC-Cl. Mobile phase:methyl cyanide:H2O(90:10), Determined by fluorescence detection,ex=260nm, em=315nm, The linear range of neomycin was 0.04μg/mL-4.0μg/mL,the liner correlation(r) was 0.9999. The limit of detection by this method was 0.04μg/mL,and the limit of quantification was 0.2mg/kg. The average recovery of neomycin added in pike eel and flounder were between 55%-81% and 47%-79% respectively with the relative standard deviation(RSD)of 3.8%-38% and 6%-27%, respectively.2 Determination method of gentamicin in seafood by liquid chromatography with pre-column derivatization and fluorescence detection was established. Gentamicin in the sample was extracted by PBS,and TCA was used for protein precipitation, then centrifugated, hexane was used to remove fats, C18 SPE column was for purification. Derivatization reagent:FMOC-Cl. Mobile phase:methylcyanide:H2O(85:15).ex=260 nm,em=315nm.The peak area and concentration showed a good linear relationship while the concentration of gentamicin was between 0.04μg/mL-4.0μg/mL,r=0.9994. The limit of detection by this method was 0.04μg/mL, and the limit of quantification was 0.1mg/kg. The average recovery of gentamicin added in pike eel and flounder were between 75%-83% and 63%-85% respectively with RSD of 2%-12% and 5%-18%, respectively.3 Determination method of aminoglycosides residues in seafood by liquid chromatography with post-column derivatization and fluorescence detection was initially established. C18 column and potassium cation-exchange column were used, and Mobile phase composition and mobile phase ratio of the potassium cation-exchange column were optimized. Kanamycin, apramycin, gentamicin, neomycin in flounder were detected.A mixture of kanamycin, apramycin, gentamicin, neomycin in the sample was extracted by 8mL solution contains 10% trichloroacetic acid and 2% EDTA-Na2 monopotassium phosphate, then centrifugated, hexane and chloroform was empolyed to remove fats, purified by C18 SPE column and separated by potassium cation column.After O-phthalaldehyde(OPA) post column derivatization,the sample was determined by fluorescence detection,ex=330nm,em= 465nm.These four aminoglycosides had been effectively separated through this method.