Fluorescence Asymmetry Ratio as an Optical Index of Antioxidant Activity of RP-HPLC Fractions of Fulvic Acids

Khreptugova Anna, Gorbunov Danila, Volkov Dmitry, Perminova Irina

Analytical Methods, 2025, , doi: 10.1039/d5ay00986c

Abstract

Fulvic acids (FA) have recently gained substantial attention as potential biostimulants within the category of complex carbon-based plant stimulants. However neither preparative technique for isolation of highly active FA components nor quality control demands are formu-lated yet for FA-based biostimulants. The study aims to evaluate antioxidant capacities (AOC) of the FA fractions obtained from the commercial FA material with a use of the preparative RP-HPLC technique and to establish relationships between the AOC values, molecular composi-tion and optical properties in search of the reliable quality control parameters indicating en-hanced AOC values of FA. ABTS assay was used to measure AOC values, high resolution mass spectrometry (HRMS) was applied for molecular composition analysis. More hydrophobic fractions comprised more than 50% of the eluted FA material. They exhibited significantly higher AOC values (0.9-1.4 mmol TE/g) as compared to the more hydrophilic fractions. They were dominated with conjugated tannins – major contributors to the bathochromic shift of the fluorescence spectra. The found close correlation (R2 = 0.96) between fluorescence asymmetry value (ASM350) and the AOC values highlights the potential of simple one-dimensional fluorescence measurements as a rapid, non-destructive alternative to labor-intensive ABTS assays for evaluating antioxidant properties of FA.