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

A rapid and direct low micromolar H-1 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 H-1 NMR signal of H2O2 at similar to 10.30 ppm in DMSO-d(6) 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 mu mol L-1. Application of an array of NMR experiments, including 2D H-1-C-13, 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.