Advanced ultra high pressure liquid chromatography–tandem mass spectrometric methods for the screening of red wine anthocyanins and derived pigments

Anthocyanins are responsible for the colour of red grapes and wine. In addition to their contribution to the sensory properties of wine, these compounds are also of interest due to their beneficial biological properties. Wine anthocyanins exhibit a large structural diversity due to variations in glycosylation and acylation patterns, which is further exacerbated by the diverse reactions involving grape-derived anthocyanins during wine ageing. Chromatographic as well as mass spectrometric resolution of wine anthocyanins is often precluded due to the complexity of these compounds. In this paper we report a rapid, high-efficiency ultra high pressure liquid chromatography (UHPLC) procedure with tandem mass spectrometric (MS/MS) detection for the in-depth screening of wine pigments. Selective detection of wine anthocyanins and derived pigments was achieved utilizing MS/MS in neutral loss scanning mode to observe the loss of dehydrated sugar moieties. This facilitated tentative compound identification based on molar mass information as well as the structured elution order of these compounds. In a second experiment, product ion spectra were recorded to allow identification of the anthocyanidin base using characteristic fragmentation patterns. The proposed methodology therefore involves two analyses for the sensitive and accurate identification of anthocyanins and their derived products in red wines. Mass spectra of wine anthocyanins under high energy collision induced dissociation (CID) conditions are reported, some for the first time. Significantly, chemical alteration of anthocyanins during wine ageing results in an off-set of the predominant fragments for each anthocyanidin base, whilst maintaining similar relative intensities. This allows unambiguous assignment of the derived products of anthocyanidin-glycosides. Using this approach, a total of 121 anthocyanins and derived compounds were identified in wines based on their relative reversed phase elution order as well as mass spectral information.