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: 10mM, pH 7.3 phosphate buffer, a fused-silica capillary with a total length of 49 cm x 75 microm ID, 375 microm OD with an effective length of 40 cm, 25 kV, 13 mbar 10s pressure sample injection and 25 degrees 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 microg mL(-1) with high correlation coefficient (r=0.9997 and 0.9993, n=7), low limits of detection (0.47 and 0.38 microg mL(-1)) and quantification (1.56 and 1.28 microg 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.
Liu
Hai-Ming
HM
Key Laboratory of Microbial Metabolism, Ministry of Education and College of Life Science and Biotechnology, Shanghai Jiaotong University, Dongchuan Road 800, Minhang District, Shanghai 200240, PR China.
Zhang
Xue-Hong
XH
Huang
Xian-Qing
XQ
Cao
Cheng-Xi
CX
Xu
Yu-Quan
YQ
D003470:Culture Media / Q000032:analysis; D019075:Electrophoresis, Capillary / Q000379:methods* / Q000592:standards; D005285:Fermentation; D008722:Methods; D010619:Phenazines / Q000032:analysis; D011549:Pseudomonas / Q000378:metabolism*; D015203:Reproducibility of Results
D003470:Culture Media; D010619:Phenazines; C037165:1-phenazinecarboxylic acid; C088013:2-hydroxyphenazine