John H. Robertson

Improved high-performance liquid chromatographic method for polypeptide antibiotics and its application to study the effects of treatments to reduce microbial levels in bacitracin powder.

Improvements were made in the high-performance liquid chromatographic (HPLC) method to obtain baseline separation of chromatographic peaks of structurally similar polypeptide components in bacitracin. The improved method uses a 30-cm-long stainless-stell column packed with muBondapak C18. The theoretical plates of the column are approximately 140,000 per meter for the bacitracin A peak. The resolution function between bacitracins B1 and B2 and that between bacitracins A and B2 have been improved 418 and 225%, respectively. The components of bacitracin, bacitracins A, B, C, D, E, F, and G, were fractionated by the countercurrent distribution technique. These components, together with Compound X, a compound separated on a carboxymethylcellulose column, and bacitracin F, obtained by degrading bacitracin A sample at neutral pH, were used to identify peaks in the HPLC chromatogram. Effects of processing methods used to reduce microbial contamination levels in bacitracin powders were evaluated. Heat treatment caused a significant loss of antimicrobial activity (35% reduction), bacitracins A, B1, and B2 were reduced by 37, 22, and 21%, respectively. A significant increase (2.8 times) of bacitracin F, an oxidative degradation compound, was show. Irradiation by 60Co at 1.8 Mrad caused no loss of potency nor change in any of the bacitracin components. Ethylene oxide treatment, on the other hand, caused considerable (46%) reduction of potency. Substantial reduction of areas under the peak of bacitracins A, B1, and B2 (50, 24 and 37%, respectively) were noted. The chromatograms showed numerous unresolved peaks around bacitracins A, B1 and B2,; however, no significant increase in the bacitracin F peak, nor appearance of non-UV absorbing peaks were observed. Peptide antibiotics of the polymyxin group, circulin, colistin, and polymyxin, were also analyzed using the muBondapak C18 column with a linear-gradient elution. A UV monitor was used for polymyxin. A moving-wire flame ionization detector was used to monitor circulin and colistin. A sample of polymyxin, circulin, and colistin may be analyzed in less than 20 min of chromatographic time.

Quantitative high-pressure liquid chromatographic analysis of bacitracin, a polypeptide antibiotic

Abstract A high-pressure liquid chromatographic (HPLC) method for the analysis of bacitracing is described. The method uses as one-meter-long stainless-steel column packed with Bondapak C18/Corasil with a programmed convex gradient elution of decreasing polarity, from 5% methanol in pH 4.5 buffer to 40% methanol and 20% acetonitrile in pH 4.5 buffer, at a flow-rate of 0.90 ml/min. More than 22 components of bacitracin have been separated and analyzed in less than 40 min of chromatography. The number and proportions of the components contained in bacitracin powders varies greatly between manufacturers but remains relatively constant for each manufacturer. The antimicrobial activity of bacitracins B and C againstMicrococcus flavus, a standard test microorganism for the quantitation of bacitracin, is 87% and 30% of bacitracin A respectively. The relative standard deviation of the HPLC method is approximately 1%. Acute toxicity of bacitracin powder containing different proportions of the components and the effect of pharmaceutical manufacturing on sterile bacitracin are described.

High-pressure liquid chromatographic analysis of novobiocin

A high-pressure liquid chromatographic method for the analysis of novobiocin is described. The method uses a 1 -m-long Zipax HCP column with a mobile phase of 15% methanol in 0.02 M phosphate buffer, pH 7.0, at a flow-rate of 0.85 ml/min (68 atm). Novobiocin, isonovobiocin, dihydronovobiocin, descarbamylnovobiocin, desmethyldescarbamylnovobiocin, novobiocic acid, and novenamine are separated in approximately 30 min. The relative standard deviation for the analysis of novobiocin is less than 1%.

[9] Gas-liquid chromatography of antibiotics

Publisher Summary This chapter discusses the gas-liquid chromatography (GLC) methods for chromatography of intact antibiotic molecules—mainly by derivatization. The chapter discusses some of the difficulties associated with quantitative derivatization and chromatography and describes precautionary measures for the successful GLC determination of antibiotics. Spectinomycin is chromatographed intact as the tetrakistrimethylsilyl derivative using an SE-52 column. Kanamyein is chromatographed intact as the silyl ether-silyl amine derivative using an OV-1 column. Neomycin is ehromatographed intact as the silyl ether-silyl amine derivative using an 0V-1 column. The procedure for the GLC analysis of paromomycin is analogous to that of neomycin powder. It follows the procedure as neomycin powder, using paromomycin in place of neomycin. Griseofulvin is chromatographed intact without derivatization using an OV-1 column. Cycloheximide is derivatized as the monotrimethylsilyl ether by treatment with isopropanol after silylation.