Profiling and accurate quantification of Rubus ellagitannins and ellagic acid conjugates using direct UPLC-Q-TOF HDMS and HPLC-DAD analysis.

Accurate quantification and structural characterization of ellagitannins and ellagic acid conjugates in food, beverages, and food supplements are essential starting points for studying their effect on human health. However, accuracy is hindered both by the lack of pure standard compounds and by methods that maintain the compounds in their native form, avoiding any chemical modification of the structure. The objective of this work was to develop a new method for the purification, chromatographic separation, and accurate quantification of ellagitannins and ellagic acid conjugates to provide thorough characterization of the diversity in composition of 11 Rubus cultivars grown in Trentino, Italy. As such, two major steps were required: (i) the isolation and purification (with associated detailed structural characterization and determination of their molar extinction coefficients) of sanguiin H-6 and lambertianin C, providing essential data for their use, together with ellagic acid, as external standards, and (ii) the determination of the chemical structure of 20 novel minor ellagitannins and 4 ellagic acid conjugates on the basis of their Q-TOF-HDMS and DAD spectra. This survey of ellagitannins and ellagic acid conjugates provides evidence for the existence of significant differences in the pattern between and within blackberry and raspberry cultivars. To our knowledge, this is the first paper that has combined detailed metabolite profiling with accurate quantification of the main ellagitannins in Rubus using their respective standards.