A validated H qNMR method for direct and simultaneous quantification of esculin, fraxin and (-)-epicatechin in Hippocastani cortex.

A fast and precise qNMR method was developed for quantification of major bioactive constituents in the bark of horse chestnut and dry extracts prepared thereof. The method was optimised using 600 MHz spectrometer, and the final acquisition parameters (90°-pulse, acquisition time - 3.0 s, relaxation delay - 27 s, number of transients - 16) allowed for performing of quantitative experiments in under 15 min. The contents of three analytes were determined using specific H resonances at δ7.45 ppm for esculin, δ5.00 ppm for fraxin, and δ5.94 ppm for (-)-epicatechin. The validation showed good precision (RSD < 1.5%) and accuracy (95-103%), and adequate sensitivity (LODs in the range of 3.3-5.9 µg) of the measurements. The determined levels in commercial samples of Hippocastani cortex were in the range of 25.89-38.94 mg/g dry weight (dw) of the bark for esculin, 12.58-17.13 mg/g dw for fraxin and 10.42-13.96 mg/g dw for (-)-epicatechin, and in the dry extracts prepared thereof 97.02-143.51 mg/g, 45.78-58.92 mg/g and 28.07-46.29 mg/g, respectively. The obtained results were cross-validated by a HPLC-PDA method with the use of a fused-core column, and no statistical differences were found between the results obtained by both methodologies, but with the advantage of higher precision of the qNMR assay. The relevant variability in quantitative composition of the commercial samples emphasise the need to introduce quality control studies in production of preparations containing horse chestnut bark and the developed method was proved suitable for this purpose.