Quantification of chlorogenic acid and hyperoside directly from crude blueberry (Vaccinium angustifolium) leaf extract by NMR spectroscopy analysis: Single-laboratory validation

Abstract

A single-laboratory-validated NMR spectroscopy method was established for determining the quantity of chlorogenic acid and hyperoside from crude extract material of blueberry leaves of the species Vaccinium angustifolium var. laevifolium House. The calibration curve of chlorogenic acid showed a highly linear regression, R = 0.99998. NMR spectroscopy identification and quantification of the constituents directly from the mixture, within the error of HPLC-diode array detector analysis, were determined as 7.53 mM chlorogenic acid (64.0 mg chlorogenic acid/g powdered leaf) and 0.77 mM hyperoside (8.58 mg hyperoside/g powdered leaf). The LOD was calculated to be 0.01 mM and the LOQ 0.01 mM by the 9 min and 13 s NMR spectroscopy experiment utilized. The assay showed no significant interference from different field strengths, extraction mesh size, gravimetric scale precision, NMR spectroscopy tube type, pulse program, amount of starting dry material, or day-to-day operation. The robustness of NMR spectroscopy as a means of definitively monitoring chlorogenic acid and hyperoside content directly from crude extracts was demonstrated by Youden statistical analysis.