Rapid and Direct Low Micromolar NMR Method for the Simultaneous Detection of Hydrogen Peroxide and Phenolics in Plant Extracts

Abstract

A rapid and direct low micromolar ¹H NMR method for the simultaneous identification and quantification of hydrogen peroxide and phenolic compounds in plant extracts was developed. The method is based on the highly deshielded ¹H NMR signal of H₂O₂ at ∼10.30 ppm in DMSO-d₆ and the combined use of picric acid and low temperature, near the freezing point of the solution, in order to achieve the minimum proton exchange rate. Line widths of H₂O₂ below 3.8 Hz were obtained for several Greek oregano extracts which resulted in a detection limit of 0.7 μmol L⁻¹. Application of an array of NMR experiments, including 2D ¹H-¹³C HMBC, spiking of the samples with H₂O₂, and variable temperature experiments, resulted in the unequivocal assignment of H₂O₂ precluding any confusion with interferences from intrinsic phenolics in the extract.