Combined HPLC-MS and HPLC-NMR on-line coupling for the separation and determination of lutein and zeaxanthin stereoisomers in spinach and in retina

The determination and unambiguous identification of carotenoid stereoisomer s from biological tissues, avoiding isomerization and oxidation due to the extraction process, is still a major challenge. Particularly, the analysis of lutein and zeaxanthin stereoisomers is of great importance, as these are the main constituents of-the macula lutea, the central part of the human r etina, and act as possible agents in the prevention and treatment of age-re lated macular degeneration (AMD), By combining a mild and quick extraction technique such as matrix solid-phase dispersion together with high-performa nce liquid chromatography (HPLC), the extremely light and oxygen sensitive lutein and zeaxanthin stereoisomers are extracted, enriched, and separated directly from the solid plant or tissue samples, excluding preparation of a rtifacts. HPLC separations are performed with C-30 phases due to their enha nced shape selectivity compared to C-18 phases and on-line coupled to mass spectrometry (RIS) and nuclear magnetic resonance (NMR) spectroscopy. By us ing HPLC-MS with atmospheric pressure chemical ionization, the lutein stere oisomers can be distinguished from the zeaxanthin stereoisomers within one chromatographic run in the upper picogram range,whereas HPLC-NMR coupling a llows the unequivocal identification of each stereoisomer with a concentrat ion in the upper nanogram range, This article provides an analytical method for the artifact;free determination of lutein and zeaxanthin stereoisomers directly from the solid biological tissue spinach as a source of carotenoi ds and retina as the sphere of activity for AMD. In addition, the structure s of these stereoisomers were unambiguously elucidated by employing hyphena ted analytical techniques.