Identification of New Coloured Anthocyanin–Flavanol Adducts in Pressure-Extracted Pomegranate (Punica granatum L.) Juice by High-Performance Liquid Chromatography/Electrospray Ionization Mass Spectrometry

Abstract

Pomegranate (Punica granatum L.) juice, obtained by pressure extraction of the whole fruit, contains coloured flavanol–anthocyanin adducts (the flavanol occupies the upper part of the dimmer) from the direct condensation between anthocyanidins delphinidin, cyanidin and pelargonidin and flavan-3-ols (epi)gallocatechin, (epi)catechin and (epi)afzelechin. The presence of adducts between these same flavanols and anthocyanidins, but belonging to the coloured anthocyanin–flavanol adduct type (anthocyanin occupies the upper part of the dimmer) has been revealed by reversed-phase liquid chromatography coupled to tandem mass spectrometry (ion trap), with positive electrospray ionization (LC–ESI/MSⁿ). These new adducts are isotopic with their corresponding counterparts (flavanol–anthocyanin adducts) and indistinguishable from them by comparison of the mass spectra from the MS² of the isotopic parent ions. However, they can be distinguished by comparison of the mass spectra from the MS³ of their corresponding isotopic aglycons. Hence, the MS³ of the aglycon from a given flavanol–anthocyanin adduct always yields a mass spectrum containing five characteristic ions, the three with smaller m/z being only dependent on the anthocyanidin and the other two on both the anthocyanidin and the flavanol. In contrast, the mass spectrum from the MS³ of the aglycon of its counterpart anthocyanin–flavanol adduct gives only two of the above five characteristic ions, where the ion with smaller m/z only depends on the anthocyanidin and the other on both the anthocyanidin and the flavanol. Ten 3-hexoside derivatives of coloured anthocyanin–flavanol adducts were detected in pomegranate juice being reported for the first time this type of adducts from a natural source.