Ehrlich chromogenic reaction-based merging-zone flow injection analysis for accurate and rapid determination of total indole phytohormones in plant resources

Indole phytohormones play key regulatory roles in plant growth, development, and stress response. Accurate quantification of these hormones in plant resources, such as safflower seed and Peganum harmala L. (characteristic resources of Xinjiang and traditional medicinal plants) and banana peel (agricultural wastes with bioactive potential), is crucial for exploiting their medicinal value and promoting efficient resource utilization. An automated flow injection-spectrophotometric method was developed for rapid and accurate quantification of total indole phytohormones (TIPH) in plant extracts to overcome the limitations of traditional techniques. A merging-zone flow injection spectrophotometric method was developed based on the characteristic colorimetric reaction of Ehrlich's reagent with indole. Improved sensitivity and high throughput were achieved through systematic optimization of the parameters of the FIA method. The optimum parameters were as follows: detection wavelength: 551 nm, reference wavelength: 697 nm, reaction coil length: 7 m, Ehrlich's reagent concentration and volume: 250 g L^-1 and 20 µL, respectively, sample volume: 200 µL, reaction coil and carrier temperature: 70 °C and 85 °C and total flow rate: 1.5 mL min^-1. The sample throughput was 30 h^-1, with an LOD of 2.40 mg L^-1, an LOQ of 8.00 mg L^-1 and an RSD of 0.79%. The spiked recoveries in the plant extract matrix were in the range of 90.1-104.2%. The determination of TIPH content in seven plant extracts was achieved, and the results were consistent with those obtained using the HPLC method. The developed method has the advantages of automation, easy operation, fast detection, and low consumption of Ehrlich's reagent and sample. It provides a new means for the determination of TIPH in plant extracts.