DETERMINATION OF GENTAMICIN IN BOVINE-MILK USING LIQUID-CHROMATOGRAPHY WITH POSTCOLUMN DERIVATIZATION AND FLUORESCENCE DETECTION

A method capable of separating and quantifying the three major and one minor components of gentamicin in milk has been developed. The method is capable of detecting 15 ng/ml gentamicin, based on a total of the four components. Milk samples are centrifuged at 4 degrees C, the fat layer removed, and the samples deproteinated with 30% trichloracetic a cid. After a second centrifugation, the supernatant is passed through a C-18 solid-phase extraction column. The column is washed with water, water-methanol (50:50) and methanol. Ammonium hydroxide (16%) in meth anol is used to elute the gentamicin. The eluent is evaporated to near dryness and taken up with water. An aliquot of the sample is then mix ed with an ion-pairing reagent for chromatography. Separation is achie ved using pentanesulfonic acid in a water-methanol mobile phase on a C ,, reversed-phase column. The o-phthalaldehyde fluorescence derivative s of gentamicin are formed post-column and are detected with excitatio n at 340 nm and emission at 430 nm. The percent recovery of gentamicin averaged 72, 78 and 88% from milk samples fortified at 15, 30 and 60 ng/ml, respectively.