Rapid quantitative analysis of phenazine-1-carboxylic acid and 2-hydroxyphenazine from fermentation culture of Pseudomonas chlororaphis GP72 by capillary zone electrophoresis

Natural phenazines in secondary metabolites of bacteria have been receiving increasing attention in recent years due to their potential usage as antibiotics. In the present study, a rapid and reliable capillary zone electrophoresis (CZE) method was developed and validated for monitoring for the first time dynamic phenazine-1-carboxylic acid (PCA) and the 2-hydroxyphenazine (2-OH-PHZ) production of Pseudomonas chlororaphis GP72 during the entire fermentation cycle. The paper begins with the optimization of separate conditions for 2-OH-PHZ and PCA together with phenazine (PHZ), which is used as internal standard. The optimized conditions are: 10 mM, pH 7.3 phosphate buffer, a fused-silica capillary with a total length of 49 cm × 75 μm ID, 375 μm OD with an effective length of 40 cm, 25 kV, 13 mbar 10 s pressure sample injection and 25 °C air-cooling. The three compounds could be separated within 2 min under optimized conditions. The validation of the newly developed study shows the linear response of 2-OH-PHZ and PCA ranging from 10 to 250 μg mL−1 with high correlation coefficient (r = 0.9997 and 0.9993, n = 7), low limits of detection (0.47 and 0.38 μg mL−1) and quantification (1.56 and 1.28 μg mL−1), respectively. Good precision values for intra- and inter-day detection and acceptable individual recovery ranges for 2-OH-PHZ and PCA are indicated. The newly developed method was also validated through monitoring dynamic PCA and 2-OH-PHZ production of P. chlororaphis GP72 during an 84 h growth cycle.