High-performance liquid chromatographic analysis of Pseudomonas aeruginosa phenazines

An HPLC method is presented for the analysis of the major phenazine pigments of Pseudomonas aeruginosa: pyocyanin, 1-hydroxyphenazine (a product of degradation of pyocyanin) and phenazine-1-carboxylic acid. These compounds were determined in the supernatant of bacterial cultures with almost no need of any treatment. Samples were analyzed employing a C-18 column varying the mixture of solvents from water-trifluoroacetic acid (100:0.04, v/v) to acetonitrile-water-trifluoroacetic acid (90:10:0.04, v/v/v) in several steps. The technique proved to be reproducible and highly sensitive. The calibration curves showed a linear response for ranges from 25 ng to 2.5 mu g. Limits of quantification were: 2.1 ng for phenazine-1-carboxylic acid, 7.5 ng for 1-hydroxyphenazine and 13 ng for pyocyanin.