The fluorescence asymmetry ratio as an optical index of antioxidant activity of RP-HPLC fractions of fulvic acids

Fulvic acids (FAs) have recently gained substantial attention as potential biostimulants within the category of complex carbon-based plant stimulants. However neither preparative techniques for isolation of highly active FA components nor quality control demands are formulated yet for FA-based biostimulants. The study aims to evaluate antioxidant capacities (AOCs) 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 composition and optical properties in search of the reliable quality control parameters indicating enhanced AOC values of FA. ABTS assay was used to measure AOC values, and 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 per 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 (<i>R</i>² = 0.96) between the 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.