Purification and structural analysis of pyocyanin and 1-hydroxyphenazine

Pyocyanin and related members of the phenazine family are produced by Pseudomonas aeruginosa and have been associated with events of pathophysiological importance. Pyocyanin and its base hydrolysis product 1-hydroxyphenazine were purified to homogeneity by reverse-phase high-pressure liquid chromatography. Their mass spectrometric behaviour was examined with a view to evaluating the use of high-resolution chromatography/mass spectrometry in studying phenazine-mediated effects in man. The molecular mass of naturally derived pyocyanin was determined as 210 Da by thermospray liquid chromatography/mass spectrometry and confirmed by desorption electron-impact mass spectrometry. Mass spectrometric data could not be obtained by fast-atom bombardment or desorption chemical ionisation, techniques commonly used to determine molecular mass of polar or thermally labile species. The thermal lability of underivatised pyocyanin precluded analysis by gas chromatography/mass spectrometry. In contrast to pyocyanin, mass spectrometric data were readily obtained for 1-hydroxyphenazine, using direct probe analysis as well as with gas and liquid chromatography inlet systems.