Comprehensive two-dimensional liquid chromatographic analysis of anthocyanins

[摘要] ENGLISH ABSTRACT: Anthocyanins are naturally occurring pigments responsible for the colour of many natural products,including grapes and wine. These pigments are important to the food industry and have beenrecognised for their nutritional value since they play an important role in the reduced risk of variouschronic diseases in humans. Anthocyanins also play an important role in the aesthetic perception andquality of red wine. However, due to the large structural diversity of grape-derived anthocyanins andthe many derivatives formed from these during wine ageing, the accurate analysis of wine pigmentsis extremely challenging. Reversed-phase liquid chromatography (RP-LC) is mostly used foranthocyanin analysis, although the technique often provides insufficient resolving power for complexmixtures of anthocyanins. In addition, the lack of commercially available standards and identicalmass spectral characteristics hampers identification of these compounds. The coupling of multipleorthogonal separation systems in comprehensive 2-dimensional liquid chromatography (LC×LC)offers a more powerful approach for the separation of complex mixtures. The current work thereforefocussed on exploring the potential of LC×LC for the improved analysis of anthocyanins and derivedpigments in natural products and wine.The first part of this work focussed on developing a hydrophilic interaction chromatography (HILIC)method as an alternative to RP-LC for the anthocyanin analysis. Following extensive optimisation,the method proved suitable for the analysis of a diverse range of anthocyanins in natural products.Significantly, it also showed alternative selectivity compared to RP-LC. The optimised HILICmethod was then used in combination with RP-LC to develop an off-line LC×LC approach foranthocyanins. For this purpose, half-minute fractions of the HILIC effluent were collected and reinjectedonto a RP-LC column. The off-line HILIC×RP-LC method demonstrated exceptionally highresolving power, as measured in terms of the practical peak capacity, with many compounds separatedin two dimensions that co-eluted in 1-dimensional HPLC. Interestingly, group-type separation wasalso observed based on the degree and/or nature of glycosylation and acylation of anthocyanins. In the final part of the work, a systematic approach was used for the development and optimisation ofand on-line HILIC×RP-LC method by using a 10-port switching valve to automatically transferfractions between the two columns. This method was then coupled to high resolution massspectrometry (MS) to allow the detailed investigation of anthocyanins and derived products in wine.Ninety four pigments were identified in one- and six-year old Pinotage wines based on HILIC×RPLCseparation in combination with accurate mass MS data and fragmentation information. Significantdifferences in especially the content of derived pigments were observed between the wines.In summary, the methods developed in this work provide the means to improve anthocyanin analysis,and therefore also show promise for the detailed investigation of these important compounds and theiralteration in natural products and their derived commodities.

[发布机构] Stellenbosch University