A sodium-containing polymyxin derived from polymyxin-complex during chromatography

Commercial colistin (polymyxin E)-complex was separated into two major components (colistins A and B) on a preparative scale by HPLC (alkyl bonded silica and aqueous-organic mobile phase containing 0.2 M NaCl-HCl buffer (pH 2.0]. Desalting of the colistins A and B fractions was completed by reversed-phase adsorption and elution using methyl alcohol. In these experiments, it was inadvertently found that prolonged elution with water gave two hydrophilic peptides, which were tentatively named colistins AH and BH, respectively. Further elution with methyl alcohol produced two lipophilic peptides which were named colistins AL and BL. Colistin BH showed higher potency than colistin AH, AL or BL, and it was also effective in vivo. The fatty acid and amino acid composition of colistin BH was identical with that of colistin BL, but colistin BH contained a relatively large amount of nonionic sodium which scarcely responded to the sodium ion-selective electrode of an ion meter; colistin BH had a slightly lower molar extinction coefficient than the colistin BL which contained negligible amount of nonionic sodium. Colistin AH also contained nonionic sodium. Potassium containing colistins could also be derived from colistin-complex. These compounds could not be formed merely by adding sodium chloride or potassium chloride to colistin-complex solution. To obtain the sodium-containing compounds, contact with some hydrophobic stationary phase, such as alkyl-bonded silica or styrene-divinylbenzene copolymer, was necessary. It is postulated that one of the antibacterial mechanisms of polymyxin is associated with its complex-forming action on monovalent cations after contact with the lipid layer of the bacterial outer membrane.