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METHOD ABSTRACTS205 / AMINOGLYCOSIDE ANTIBIOTICS IN FEEDAPRAMYCIN, GENTAMICIN, NEOMYCINAminoglycoside antibiotics are among those commonly used in animal feeds to manage intestinal microorganisms. Thebenecial effects include improved growth and generally healthier animal populations. Use of antibiotics creates a demandfor analytical procedures to verify concentrations in pre-mixes and feeds and in some instances for residue analysis in animalproducts.This note describes a simple, robust analytical method for the family of Aminoglycoside antibiotics in feeds and animalproducts. The sample is homogenized with a generic extraction solution and the crude soluble portion is directly injectedinto an HPLC ion-exchange column. The column efuent is then mixed with an OPA/Thiouor reagent which formshighly uorescent derivatives with the primary amine moieties of the antibiotics.Call if you need to analyze other Aminoglycosides.Extraction Procedure:Take one part feed:10 parts Extraction solution (w/v)Catalog No. 1700-1118 and homogenize for ve minutes.Centrifuge for 10 minutes. Three layers will form: the pellet,a supernatant emulsion and a soft layer of oating fat. Carefullylift the oating fatty layer with a spatula and discard.Transfer the emulsion to a sealable vial. Coagulate theemulsion by placing the vial in a boiling water bath for 15minutes. Centrifuge for 10 minutes. The clear supernate isltered (0.45 um Nylon) and placed in an autosampler vial.METHODAnalytical ConditionsColumn: ALKION cation-exchange, K+ form, 4 x 150 mm,Catalog No. 9410917ALKION Guard column, 3 x 20 mm,Catalog No. 9493020ApramycinNeomycinGentamicinComplexTemperature: 40 °CFlow Rate: 0.8 mL/minMobile Phase:1700-1101, Potassium buffer, K011700-1102, Potassium titrant, K021700-1103, Potassium ionic strength adjuster, K03Post-column ConditionsPost-column System: Pinnacle PCXReactor Volume: 0.15 mLTemperature: 45 °CReagent: o-Phthalaldehyde/Thiouor + Brij 35®Flow Rate: 0.3 mL/minDetection: Fluorometer ex: 330 nm, em: 465 nm“ALKION” and Thiofl uor are trademarks of Pickering Laboratories, Inc.“Brij 35” is a registered trademark of ICI Americas, Inc.05 10 15 20 25 30minCONDITIONS FOR SEPARATION OF APRAMYCINGENTAMICIN AND NEOMYCINTIME (Min) 1700-1101 % 1700-1102 % 1700-1103 %0 60 40 015 13.2 8.8 7815.1 12 10 7830 12 10 7830.1 0 22 7831 0 22 7831.1 60 40 037 60 40 0FLOW RATE: 0.8 mL/min,COLUMN TEMP.: 40 ºC,INJECTION VOLUME: 10 LREFERENCES:Driver, J. L., Thex, N., Raynie, D., Ofi tserova, M., Pickering, M. Single-Laboratory Validation forthe Quantifi cation of Neomycin B and Neomycin C in Animal Feeds by Liquid ChromatographyFluorescence Detection with Post-column Derivatization.(2009) J.AOAC Int, 92, 34-41