Sample preparation for residue determination of gentamicin and neomycin by liquid chromatography

Abstract

The effect of sample preparation on the determination of gentamicin and neomycin residues in animal tissues was investigated. The extract was mixed with an ion-pair reagent and applied to an octadecyl cartridge. The cartridges were washed with buffer followed by water, and analytes were eluted with ion-pair buffer–acetonitrile mixture. The aminoglycosides were derivatized with 9-fluorenylmethyl chloroformate prior to liquid chromatography using a reversed-phase column and fluorescence detection. Under the conditions applied neomycin was fully separated from all the gentamicin compounds. The highest recoveries of gentamicin and neomycin from spiked tissues were obtained using trichloroacetic acid after initial extraction with phosphate-buffered saline. No interfering peaks from endogenous compounds of matrix were noted at the elution position of the analytes. An intra-laboratory validation of the whole procedure was performed. The calibration graphs were linear from 0.1 to 1.0 mg/kg for gentamicin, and from 0.2 to 1.0 mg/kg for neomycin. Limits of detection were 0.05 mg/kg and 0.10 mg/kg for gentamicin and neomycin, respectively. Limits of quantitation for gentamicin and neomycin were 0.1 and 0.20 mg/kg muscle, liver or kidney tissue, respectively. Recoveries of gentamicin spiked at levels of 0.1 mg/kg porcine tissues ranged from 76 to 86%. Recoveries of neomycin spiked at levels of 0.2 mg/kg porcine tissues ranged from 77 to 83%. The validated procedure was used to determine gentamicin concentrations in porcine tissue after dosing with gentamicin at a level of 5 mg/kg body mass.