基于HPLC-DAD-SPE-CryoNMR-MS技术的代谢物快速定性和结构确定

快速定性并有效地确定代谢物结构是代谢组学、植物化学和天然药物发现等研究的重要内容. 本文以迷迭香的两种溶剂提取物（氯仿/甲醇=3/1和50%含水甲醇）作为模型样品，通过分析其中的一些化学成分，展现了HPLC-DAD-SPE-CryoNMR-MS无缝联仪技术在确定代谢物结构方面的应用潜力. 基于高效液相色谱、紫外吸收、核磁共振波谱及质谱数据，具有代表性的6个迷迭香代谢物的结构得到了确定，它们分别为1个酚类二萜（鼠尾草酚）、2个酚酸（迷迭香酸和咖啡酸）、2个黄酮（6-甲氧基木樨草素-7-葡萄糖苷和高车前甙）和1个香豆酸 (顺-4-香豆酸葡萄糖苷). 该技术使分析型HPLC柱所分离的组分可以获得高质量同核及异核二维核磁共振波谱，从而能够更加便捷地确定微量成分的结构. 这些研究结果不仅提供了上述6种代谢物详细的谱学数据，而且证实HPLC-DAD-SPE-CryoNMR-MS技术是天然产物以及其他复杂体系结构分析的重要工具，即使同一色谱峰中含有浓度相差一个数量级的多个代谢物时，该方法依然有效.

Efficient Identification and Structural Elucidation of Metabolites Using HPLC-DAD-SPE-CryoNMR-MS Method

Rapid identification and efficient structural elucidation of metabolites are vitally important in metabonomics, phytochemistry and drug discoveries from the natural sources. In this work, the potentials of the hyphenated HPLC-DAD-SPE-CryoNMR-MS techniques were demonstrated in characterization and structural elucidation using the complex extracts of rosemary (Rosmarinus of ficinalis L.) as an example. 6 metabolites, including 1 phenolic diterpene (carnosol), 2 phenolic acids (rosmarinic acid and caffeic acid), 2 flavonoids (6-methoxyluteolin-7-glucoside and homoplantaginin) and 1 coumaric acid (cis-4-glucosyloxycinnamic acid), were successfully identified from two rosemary extracts with chloroform-methanol (v/v, 3/1) and 50% aqueous methanol on the basis of HPLC, UV, NMR and MS data. Such structural determination benefited from the capability of acquiring the high-quality homo- and hetero-nuclear 2D NMR spectra with the analytical HPLC separation. The results have not only provided whole set spectroscopic data for these metabolites, but also proved the hyphenated HPLC-DAD-SPE-CryoNMR-MS method to be an important structural elucidation platform for the components in natural products and probably other complex mixtures, even when multiple components in the same chromatographic peak are present with concentration differences in an order of magnitude.