Quantitative determination of biphenyls and their metabolites in cell cultures of Comamonas thiooxydans N1 using high-performance liquid chromatography

Abstract

Biphenyl-degrading bacteria were isolated from contaminated soil and identified as Comamonas species. Among these strains, C. thiooxydans N1 was the most suitable for biphenyl degradation. When 1200 µg/mL of biphenyl was used, it was completely degraded within 24 h. A simple, rapid, and precise, high-performance liquid chromatography (HPLC) method for quantifying biphenyls and their metabolites in the culture medium was optimised and validated. The proposed method was performed using reverse-phase HPLC equipped with a Luna C18 column. The mobile phase was water and acetonitrile (30/70, v/v) under isocratic conditions, and biphenyls and their metabolites were quantified on a SHIMADZU HPLC system with a diode-array detection detector at 254 nm. Peak identity was confirmed by ultraviolet spectroscopy and gas chromatography-mass spectrometry. Validation was performed according to European Union requirements (2002/657/EC) for linearity, accuracy, repeatability, reproducibility, limit of detection, limit of quantification, and detection capability. Linearity was observed in the concentration range of 0.04-20 µg/mL with a high coefficient of determination (R² ≥ 0.999). The validated method was applied to determine the content of biphenyl and its metabolites in the culture medium.

Keywords:
biphenyl-degrading bacteria; 2,3-dihydroxy-biphenyl; high-performance liquid chromatography; method validation; Comamonas thiooxydans