Simultaneous HPLC‐DAD‐MS (ESI+) determination of structural and geometrical isomers of carotenoids in mature grapes

Abstract

Carotenoids are uniquely functional polyene pigments ubiquitous in nature; aside from being responsible for the color of a wide variety of vegetables, interest is being focused on food carotenoids due to their likely health benefits. From analytical point of view, it is important to unequivocally identify individual carotenoid compounds in many food stuffs. Therefore, isolation of standards from natural sources must be encouraged for accurate identifications. Like many fruits, mature grape berries contain numerous carotenoid compounds, mostly found in the skin at levels two to three times higher than in the pulp. Carotenoid compounds in a typical wine grape variety (Negroamaro) grown in Apulian region were investigated by reversed-phase C(30) (RP-30) HPLC-DAD-MS (ESI(+)) analysis. As a consequence of an unusual ionization process of carotenoids, their mass spectra registered in the positive ion mode comprised both protonated molecules and molecular ion radicals with little fragmentation. Additionally, selective collision-induced dissociation (CID) experiments, together with fine structures of the UV-vis spectra, were used to differentiate structural and geometrical isomers. This technique allowed the simultaneous determination of regio- and cis-isomers of lutein (zeaxanthin, 9Z and 9'Z-lutein) and a cis-isomer of β-carotene (9Z- β-carotene), 5,6-epoxy xanthophylls (violaxanthin, (9'Z)-neoxanthin, lutein-5,6-epoxide) and 5,8-epoxy xanthophylls diasteroisomers (neochrome, auroxanthin, luteoxanthin, flavoxanthin, chrysanthemaxanthin).