As part of our efforts to investigate the biosynthesis of proferrioxamines in Erwinia amylovora, it was of interest to develop a methodology with which a large number of basic amino acids, di- and polyamines, and C- and N-hydroxy derivatives thereof could be monitored simultaneously. Towards this end, the on-line coupling of electrospray mass spectrometry with ion chromatography or reversed-phase chromatography was explored. Tandem mass spectrometry was found to be an excellent method for obtaining relevant structural information on underivatized polyamines and basic amino acid, including N-hydroxy derivatives. However, the coupling of ion chromatography with electrospray mass spectrometry for on-line extraction and separation of the underivatized compounds is hampered by compatibility problems. For most target compounds, on-line reversed-phase liquid chromatography/electrospray mass spectrometry was the method of choice but required derivatization and in this respect dansylation was preferred over benzoylation. Neither derivatization method is suitable for N-hydroxy compounds, which are basesensitive.