Liquid chromatography–mass spectrometry study towards the pH and temperature-induced N-acyl migration in polymyxin B

In the course of the synthesis and purification of new polymyxins and analogues, formation of a by-product with identical mass was observed and it was believed that this might be the result of acyl migration from the Nα- to the Nγ-position of residue α,γ-diaminobutyric acid 1 (Dab1) under acidic conditions. Therefore, a LC–MS/MS study was initiated to establish the stability of polymyxin B 3 in aqueous solution at room temperature and 60 °C, as well as different pH values (i.e. 1.4, 4.4 and 7.4). It was shown that the by-product, which is actually formed in the course of the purification of polymyxin B 3 after evaporation in acidic medium, has a retention time similar to Nγ-polymyxin B 3. Acyl-migration occurred most rapidly at 60 °C and pH 7.4. Furthermore, it was established that migration of the acyl from the Nα- to the Nγ-position of residue Dab1 is reversible and that the equilibrium seems to be in favor of the Nα-acylated compound.