SOLID-PHASE CLEANUP AND THIN-LAYER CHROMATOGRAPHIC DETECTION OF VETERINARY AMINOGLYCOSIDES

Chemical methods are needed to confirm the presence of antibiotics det ected by microbial inhibition assays in fluids and tissues of farm ani mals. We have optimized the conditions for the isolation of hygromycin B with a copolymeric bonded solid-phase silica column followed by thi n-layer chromatography (TLC) separation and detection of its fluoresce nce derivative after reaction with fluorescamine. The detection limit of the drug was 50 ng. Serum and plasma samples fortified with hygromy cin B were acidified and passed through the copolymerized solid-phase columns previously conditioned with phosphate buffer. Hygromycin B was trapped in the columns and eluted with diethylamine-methanol and anal yzed by TLC using acetone-ethanol-ammonium hydroxide as the developing solvent. Hygromycin B bands were derivatized at acidic pH with fluore scamine and visualized under ultraviolet light. Hygromycin B added to bovine plasma was detectable at 25, 50, 100, 250 and 500 ng/ml (ppb). Hygromycin B added to swine serum was detected at 50 ng/ml. However, t he serum had to be deproteinized with trichloroacetic acid or acetonit rile prior to solid-phase extraction to gain accurate values. Neomycin and gentamicin (100 ng/ml aqueous solutions) could also be isolated w ith copolymeric solid-phase columns at a level of 50 ng. Gentamicin, n eomycin, gentamicin, spectinomycin, hygromycin B and streptomycin coul d be separated by TLC, allowing multiresidue detection of these aminog lycosides. The respective R(F) values of 0.64, 0.56, 0.52, 0.33 and 0. 20 indicate the separation of these five compounds. This procedure pro vides a rapid and sensitive method for the semi-quantitative estimatio n of aminoglycosides.