Kanamycin B Sulfate

Separation of the main components of the nebramycin complex of apramycin, kanamycin B and tobramycin was achieved in the form of their 2,4-dinitrophenyl derivatives. A chromatograph SR 8000 of 'Spectra-Physics" and a column 'Sperisorb C6" (4.6 X 250 mm) with the granule size of 5 micrometers were used in the study with the mobile phase of acetone-trisbuffer, pH 7 in a ratio of 65 to 35, the flow rate of 1 ml/min, a temperature of 35 degrees C and detection at lambda 350 nm. Quantitative determination of the tobramycin purity level was performed with an equation developed for the given conditions. The purity levels of the reference tobramycin base and its sulfate were estimated. The results of the estimation corresponded to the data of biological titration.

The analyses of aminoglycoside antibiotics by capillary electrophoresis utilizing borate complexation and direct UV detection are discussed. Twelve aminoglycosides were studied and separated to demonstrate identification capabilities, with migration time RSDs from 0.21 to 0.44% (n = 6) for individual components. This buffer system permitted the detection of minor impurities such as precursors or closely related fermentation products. Quantification of dihydrostreptomycin and streptomycin was accomplished in 160 mM sodium tetraborate decahydrate with linearity over the range 0.050-1.0 mg ml-1. Determination of the purity of bulk dihydrostreptomycin was possible by the addition of the cationic surfactant myristyltrimethylammonium bromide. This reversed the electroosmotic flow, thereby reversing the migration order, and causing the streptomycin impurity to migrate before the dihydrostreptomycin main peak. Quantification was also demonstrated with the closely related compounds amikacin, bekanamycin, kanamycin A, and tobramycin, using sisomicin as an internal standard. The reproducibility of the method was typically 2-3% over 1 day, and 2% day-to-day. These studies illustrate the use of capillary electrophoresis for the identification and quantification of selected aminoglycosides as potential alternative methods to the assays given by the US Pharmacopeia.

The effects of 50 antibiotics on the detection and determination of bacterial endotoxins by the chromogenic method using a Limulus reagent (Tachypleus hemocyte lysate) and a chromogenic substrate of p-nitroaniline derivatives were tested, and the antibiotic concentration for 50% inhibition of the chromogenic reaction in the presence of 0.5 ng of endotoxin (Escherichia coli 0111:B4) per ml was estimated. All the antibiotic preparations were depyrogenized by ultrafiltration treatment before they were subjected to the test. The reaction was conducted in the presence of a high concentration (0.5 M) of Tris buffer to constantly maintain the pH of the reaction mixture, and liberated p-nitroaniline was determined by high-pressure liquid chromatography. Several aminoglycosides (amikacin, bekanamycin, kanamycin, and streptomycin sulfate), bleomycin hydrochloride, and fosfomycin disodium showed no inhibition of the reaction up to 20 mg/ml. However, other antibiotics, including penicillins, cephalosporins, macrolides, and tetracyclines, inhibited the reaction concentration dependently. Polymyxin B sulfate was the most potent inhibitor, with less than 8 micrograms/ml for 50% inhibition. It was concluded that the chromogenic method can be applied to the detection and determination of endotoxin in most of the antibiotic preparations. An application of this method to carbenicillin disodium preparations was exemplified.