Neomycin,separation

Neomycin has been produced by growing the organism, Streptomyces No. 3535, in a suitable nutrient medium under appropriate stationary or submerged aerobic (viz shaken) conditions, and then isolating and purifying the substance, e.g., by procedure of the sort described in the figure including various steps of adsorption, recovery by elution, separation from impurities, and precipitation. 

An iterative procedure using the solid film linear driving force model has been used with a steric mass action isotherm to model displacement chromatography on ion exchange materials and the procedure applied to the separation of horse and bovine cytochrome c using neomycin sulfate as the displacer.4 The solid film linear driving force model is a set of two differential equations imposing mass transfer limitations. 

To determine the relative amounts of neomycins B, C and neamine by a radio chemical method, Kaiser 2 separated the -labelled N-acetyl derivatives by paper chromatography and quantitated the chromatograms by liquid scintillation counting. A coefficient of variation of 3.6% was obtained. 

From a preparative aspect, Peck et alX have employed the counter-current distribution technique with a partition system of water/ butanol/p-toluene sulphonic acid to separate and purify the neamine (neomycin A) present in commercial neomycin. 

Neomycin is a readily ionisable molecule and should thus be separable from other antibiotics by application of an electric field (zone electrophoresis). Various workers have successfully applied this technique to neomycin and Table 10 summarises the conditions reported in the literature. A number of authors described the qualitative separation of neomycin from other chemical-types of antibiotics using paper-electro-phoresis181,185,187 while Ochabl89 described systems specifically designed to separate compounds within the aminoglycoside group of antibiotics. 

Electrophoresis has also been employed to separate neomycin from analytically-interfering substances such as proteins. Hence Brammer and Hemsonl82 have determined the neomycin content of blood serum. Neomycin was separated from the serum proteins by electrophoresis on cellulose acetate and assayed colorimetrically following elution from the support. 

A number of minor components present in commercial neomycin have been separated by column chromatography on a carboxylic cation-exchange resin, Amberlite CG-50 99. The components were eluted from the resin with ammonium hydroxide solution. T.L.C. of the eluent fractions showed the presence of two previously unreported impurities which were then isolated on Dowex 1X2 and tentatively identified using NMR and mass spectrometry. 

Many column chromatographic procedures have been described for the commercial separation and purification of neomycin following fermentation. 

Paper Chromatographic Systems Separating Neomycin from other Antibiotics... 

Separation of neomycin from chloramphenicol, tetracycline and penicillin... 

Chromatographic Systems Separating Neomycin Components and Degradation Products... 

The use of bioautography for the quantitation of chromatograms has been described by many workers. Emilianowicz-Czerska and Herman228 separated neamine from neomycins B and C then quantitatively determined total neomycin B and C by bioautography with B.iubtZZZi. A linear response for concentrations between 0.25 and lOyg of neomycin was obtained. With a similar technique but using a mathematical analysis of the bioauto-... 

Tsuj i and Robertson achieved the separation of neomycin B, neomycin C and neamine as the trimethylsilyl ethers on a 6ft. column of 0.75% OV-1 on Gas Chrom Q at a temperature of 290°C. The same conditions have also been shown to separate neobiosamine B, neosamine and deoxystreptamine from neomycin and neamine. Hence the method could be used to study the stability of neomycin or to monitor the biosynthetic production process. Use of the procedure to assess the stability of neomycin in pharmaceutical formulations has been demonstrated by Van Giessen and Tsuji237 with trilaurin as internal standard. However,these authors recommended a 2ft. column packed with 3%... 

Neomycin has been separated from mixtures of other aminoglycoside antibiotics containing the 2-deoxystreptamine moiety as both the pertrimethylsilyl derivative and the N-trifluoro-acetyl pertrimethylsilyl derivative using a column of 0.75% 0V-1 on Gas Chrom q240. The procedure may be used to estimate the number of sugar moieties bound in the antibiotic as a close relationship exists between the number of rings and the retention time. 

Examination of neomycin B by a combined G.C.-M.S. procedure has been reported by Murata et all5. G.c. was accomplished with the tri-methylsilyl derivative on a lm column of 1% 0V-1 on Chromosorb W, by which means neomycin was separated from kanamycin. The resulting mass spectra of the neomycin derivative exhibited a minute molecular ion peak at m/e 1550 indicating that all active hydrogens of both hydroxy and amine groups were completely silylated. 

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