Medina M B, Unruh J J
Eastern Regional Research Center, United States Department of Agriculture, Philadelphia, PA 19118, USA.
J Chromatogr B Biomed Appl. 1995 Jan 6;663(1):127-35. doi: 10.1016/0378-4347(94)00414-z.
Chemical methods are needed to confirm the presence of antibiotics detected by microbial inhibition assays in fluids and tissues of farm animals. We have optimized the conditions for the isolation of hygromycin B with a copolymeric bonded solid-phase silica column followed by thin-layer chromatography (TLC) separation and detection of its fluorescence derivative after reaction with fluorescamine. The detection limit of the drug was 50 ng. Serum and plasma samples fortified with hygromycin 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 analyzed by TLC using acetone-ethanol-ammonium hydroxide as the developing solvent. Hygromycin B bands were derivatized at acidic pH with fluorescamine 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, the serum had to be deproteinized with trichloroacetic acid or acetonitrile prior to solid-phase extraction to gain accurate values. Neomycin and gentamicin (100 ng/ml aqueous solutions) could also be isolated with copolymeric solid-phase columns at a level of 50 ng. Gentamicin, neomycin, gentamicin, spectinomycin, hygromycin B and streptomycin could be separated by TLC, allowing multiresidue detection of these aminoglycosides. The respective RF values of 0.64, 0.56, 0.52, 0.33 and 0.20 indicate the separation of these five compounds. This procedure provides a rapid and sensitive method for the semi-quantitative estimation of aminoglycosides.
需要采用化学方法来确认在农场动物的体液和组织中通过微生物抑制试验检测到的抗生素的存在。我们优化了使用共聚键合固相硅胶柱分离潮霉素B的条件，随后进行薄层色谱（TLC）分离，并在与荧光胺反应后检测其荧光衍生物。该药物的检测限为50 ng。用潮霉素B强化的血清和血浆样品经酸化后通过预先用磷酸盐缓冲液平衡的共聚固相柱。潮霉素B被截留在柱中，用二乙胺 - 甲醇洗脱，并使用丙酮 - 乙醇 - 氢氧化铵作为展开剂通过TLC进行分析。潮霉素B条带在酸性pH下用荧光胺衍生化，并在紫外光下可视化。添加到牛血浆中的潮霉素B在25、50、100、250和500 ng/ml（ppb）时可被检测到。添加到猪血清中的潮霉素B在50 ng/ml时可被检测到。然而，在固相萃取之前，血清必须用三氯乙酸或乙腈进行脱蛋白处理以获得准确值。新霉素和庆大霉素（100 ng/ml水溶液）也可以用共聚固相柱以50 ng的水平进行分离。庆大霉素、新霉素、庆大霉素、壮观霉素、潮霉素B和链霉素可以通过TLC分离，从而实现这些氨基糖苷类药物的多残留检测。这五种化合物各自的比移值分别为0.64、0.56、0.52、0.33和0.20，表明它们能够被分离。该方法为氨基糖苷类药物的半定量估计提供了一种快速且灵敏的方法。
