Usally known metabolites are identified on the basis of metabolite databases. For the structure elucidation of unknown metabolites several techniques are availbable. First of all, multivariate data analysis can propose peaks, which belong to the same unknown metabolites. Then advanced 2-dimensional NMR experiment can be performed to analyze these peaks (for example COSY and TOCSY experiments). In addition information about connected carbon atoms is accessible by HSQC and HNBC experiments. Compared to routine NMR measurements these 2-D experiments need long measurement time (up to several days per experiment). If the structure cannot be determined by 2-D experiments or if the concentration of the metabolite of interest is too low, a hyphenated instrument similar to the setup shown in the figure can be used as ultimate tool. Thereby, liquid chromatograpyh helps to separeate the metabolites, which can be concentrated on a solid phase extraction catridge. Then, NMR spectra and MS spectra of the metabolites can be obtained in a parallel mode. Examples of structure elucidations are shown later in this tutorial.
Hyphenated LC-MS-SPE-NMR instrument as ultimate tool for structure elucidation.