Combined HPLC-MS and HPLC-NMR on-line coupling for the separation and determination of lutein and zeaxanthin stereoisomers in spinach and in retina
Combined HPLC-MS and HPLC-NMR on-line coupling for the separation and determination of lutein and zeaxanthin stereoisomers in spinach and in retina
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DOI：
10.1021/ac000635g
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发表时间：
2001-02-01
影响因子：
7.4
通讯作者：
Albert, K
中科院分区：
文献类型：
--
作者：
Dachtler, M;Glaser, T;Albert, K
生物组织中类胡萝卜素立体异构体的测定和明确鉴定，避免由于提取过程引起的异构化和氧化，仍然是一个重大挑战。特别地，叶黄素和玉米黄质立体异构体的分析是非常重要的，因为它们是人视网膜的中心部分黄斑的主要成分，并且在预防和治疗年龄相关性黄斑变性（AMD）中充当可能的药剂。通过将温和且快速的提取技术（例如基质固相分散）与高效液相色谱（HPLC）结合，对光和氧极其敏感的叶黄素和玉米黄质立体异构体直接从固体植物或组织样品中提取、富集和分离，不包括人工制品的制备。HPLC分离是用C-30相进行的，因为与C-18相相比，它们具有更高的形状选择性，并且与质谱（RIS）和核磁共振（NMR）光谱在线耦合。采用HPLC-MS大气压化学电离，一次色谱可在皮克浓度范围内区分叶黄素和玉米黄质的立体异构体，而HPLC-NMR联用则可在纳克浓度范围内明确鉴别每种立体异构体。直接从固体生物组织中自由测定叶黄素和玉米黄质立体异构体，菠菜作为类胡萝卜素的来源，视网膜作为AMD的活性范围。此外，通过采用联用分析技术明确阐明了这些立体异构体的结构。
The determination and unambiguous identification of carotenoid stereoisomers 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 retina, and act as possible agents in the prevention and treatment of age-related macular degeneration (AMD), By combining a mild and quick extraction technique such as matrix solid-phase dispersion together with high-performance 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 artifacts. HPLC separations are performed with C-30 phases due to their enhanced shape selectivity compared to C-18 phases and on-line coupled to mass spectrometry (RIS) and nuclear magnetic resonance (NMR) spectroscopy. By using HPLC-MS with atmospheric pressure chemical ionization, the lutein stereoisomers can be distinguished from the zeaxanthin stereoisomers within one chromatographic run in the upper picogram range,whereas HPLC-NMR coupling allows the unequivocal identification of each stereoisomer with a concentration 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 carotenoids and retina as the sphere of activity for AMD. In addition, the structures of these stereoisomers were unambiguously elucidated by employing hyphenated analytical techniques.