DEVELOPMENT AND OPTIMIZATION OF A LIQUID-CHROMATOGRAPHIC METHOD FOR THE DETERMINATION OF GENTAMICIN IN CALF TISSUE

A simple and sensitive method for the determination of gentamicin in calf tissues (muscle, liver, kidney and fat) based on liquid chromatography (LC) is described. A 5% (w/v) trichloroacetic acid solution was used to precipitate proteins. After centrifugation of the sample, the pH and ionic strength were adjusted for further purification by liquid-solid ion-exchange extraction using CM-Sephadex gel. A 100-mul aliquot of the eluate from the ion-exchange cartridge (in 0.05 M NaOH) was injected on to the LC column after adjustment of the pH and counter ion concentration. The chromatography consisted of an ion-pairing system with a mobile phase containing camphorsulphonate as the counter ion, a reversed-phase column (LiChrospher 100 RP-18 end-capped) and postcolumn derivatization with o-phthalaldehyde and 2-mercaptoethanol followed by fluorimetric detection. The three major components of gentamicin were eluted as a single peak. Recoveries ranged from 68 to 98% in tissue samples fortified with gentamicin at 0.05-3.2 mug g-1 levels. The limit of quantification was 50 ng g-1 in muscle and fat and 100 ng g-1 in liver and kidney.