Determination of plant hormones in bacterial fermentation products of plant growth-promoting rhizobacteria by liquid－liquid extraction high-performance liquid chromatography

Plant hormones secreted by plant growth-promoting rhizobacteria play an important role in plant growth, as they regulate the various processes related to plant growth, development, and stress resistance. Understanding the content of plant hormones in bacterial fermentation products of plant growth-promoting rhizobacteria is crucial for the screening of excellent growth-promoting bacterial resources. Therefore, this study aims to develop a simple high-performance liquid chromatographic method for the simultaneous detection of gibberellin A₃, indole-3-acetic acid, and trans-Zeatin. The samples were extracted with ultrapure water and ethyl acetate (1 ꞉ 2, by volume) and then separated by Agilent ZORBAX Eclipse Plus C18 chromatographic column. The mobile phase was 45% methanol and 55% ultrapure water (containing 0.2% glacial acetic acid) and the detection wavelength was set at 254 nm. The flow rate was 0.8 mL·min⁻¹, the column temperature of 30 ℃ and the injection volume of 10 μL were adopted. Results indicated that the linear relationship of the three plant hormones is good within their linear range and the correlation coefficients were not less than 0.997. The average recoveries of this method ranged from 88.02% to 108.40% with relative standard deviation of 0.02%～6.51%. This method is simple, rapid, and suitable for simultaneous determination of several plant hormones in plant growth-promoting rhizobacteria, which could provide a good reference and bacterial strain resources for the development and application of microbial fertilizer in the future.