Purification and structural analysis of pyocyanin and 1-hydroxyphenazine

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Abstract
1 Pyocyanin and related members of the phenazine family are produced by Pseudomonas aeruginosa and have been associated with events of pathophysiological importance. 2 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. 3 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. 4 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.