Polymyxin, assay

A culture of Bacillus polymyxa in a tube with Trypticase soybean broth was incubated overnight at 25°C. 5 ml of this culture was transferred to 100 ml of the tank medium in a 500 ml Erlenmeyer flask which was incubated for 48 hours at room temperature. This 100 ml culture served as inoculum for one tank. During the course of fermentation the medium was aerated at the rate of 0.3 volume of air per volume of mash per minute. The temperature was maintained at about 27 C. Samples of mash were taken every 8 hours in order to determine pH and the presence of contaminants and spores. After 88 hours of fermentation the pH was about 6.3 and an assay using Escherichia coll showed the presence of 1,200 units of polymyxin per cubic centimeter. The polymyxin was extracted and purified by removing the mycelia, adsorbing the active principle on charcoal and eluting with acidic methanol. 

United States Pharmacopoeia 28 [1] describes a microbiological method under antibiotics-microbial assays for the analysis of OTC and nystatin capsules, OTC and nystatin for oral suspension, OTC HC1 and hydrocortisone ointment, and OTC HC1 and polymyxin B sulfate ointment. The methods are relative rather than absolute, which are based on the determination of the level of oxytetracycline by a microbiological response to a series of standard oxytetracycline concentrations by a... 

Erythromycin, a macrolide antibiotic, lacks a significant chromophore. Detection sensitivity was enhanced by using a wavelength of 200 nm and selecting an injection solvent of lower conductivity than the BGE. In order to facilitate the separation of erythromycin and its related substances, 35% (v/v) ethanol was incorporated into a 150 mM phosphate buffer pH 7.5. Resolution of all of the compounds was achieved in approximately 45 min. The method was employed as an assay method for erythromycin and for impurity determination. Peptide antibiotics, such as colistin and polymyxin, are mixtures of many closely related compounds. A validated CZE method for impurity analysis of polymyxin B was described, employing 130 mM triethanolamine-phosphate buffer at pH 2.5 to reduce the adsorption of analyte onto the capillary wall. Methyl-/l-cyclodextrin (M-/1-CD) and 2-propanol were found to be necessary for selectivity enhancement. Using similar buffer additives, the same group developed and validated a method for colistin analysis. ... 

DS-96 is the most active compound identified by us to date. The compound behaves in every in vitro and in vivo assay, at least equivalent to, if not better than, polymyxin B (Sil et al., 2007). Both DS-96 and PMB bind LPS with equal affinity (Sil et al., 2007), and inhibit LPS-induced NF-kB transactivation with an IC50 of 30 nM (Fig. 12.16a). DS-96 was also found to be active against LPS isolated from a broad range of Gram-negative bacteria (Fig. 12.16c). That the mechanism of action of DS-96 is that of a true LPS-sequestrant was confirmed not only by its specificity toward LPS in that non-LPS stimuli such as TNF-a, PMA and Tlr-2 agonists such... 

No chemical methods for assaying either polymyxin or colistin are available at the present time. For the determination of potency either the cylinder plate assay using Brucella bronchiseptica ATCC 4617 as test organism or the nephelometric method is used. The current USP standards are 7,850 units/mg for polymyxin B and 20,070 units/mg for colistin. 

A casual examination of the current literature indicates that there is considerable confusion regarding antibiotic assay standards with regard to the terms unit and microgram used to indicate potency. Bacitracin and polymyxin are defined in terms of antimicrobial units which are not directly expressible in terms of weight of the compounds since these antibiotics have not been isolated in pure form penicillin originally fell into this category until its crystallization. For others which have been isolated in pure... 

Based on work carried out at Burroughs Wellcome Laboratories, London, it would appear that pure polymyxin B HCl should assay approximately 10,000 units per milligram. 

Good assay plate methods are available for penicillin, streptomycin, bacitracin, and polymyxin however, the newer broad spectrum antibiotics tend to give poorly defined zone edges on assay plates. 

The lipopolysaccharide (LPS) endotoxin is the most powerful immune stimulant known and a causative agent in the clinical syndrome known as sepsis. Sepsis is responsible for more than 100,000 deaths annually, in large part due to the lack of a rapid, reliable, and sensitive diagnostic technique. An evanescent wave fiber-optic biosensor was developed for the detection of LPS from E. coli at concentrations as low as lOngmL in 30 s (James et al., 1996). Polymyxin B covalently immobilized onto the surface of the fiber-optic probe was able to bind fluorescently labeled LPS selectively. Unlabeled LPS present in the biological samples was detected in a competitive assay format, by displacing the labeled LPS. The competitive assay format worked in buffer and in plasma with similar sensitivities. This method might also be used with other LPS capture molecules, such as antibodies, lectins, or antibiotics, to simultaneously detect LPS and determine the LPS serotype. 

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