Detailed knowledge of the chemical content of organisms, organs, tissues, and cells is needed to fully characterize complex biological systems. The high chemical variety of compounds present in biological systems is illustrated by the presence of a large variety of compounds, ranging from apolar lipids, semi-polar phenolic conjugates, toward polar sugars. A molecules’ chemical structure forms the basis to understand its biological function. The chemical identification process of small molecules (i.e., metabolites) is still one of the major focus points in metabolomics research. Actually, no single analytical platform exists that can measure and identify all existing metabolites. In this thesis, two analytical techniques that are widely used within metabolite identification studies have been combined, i.e. mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR). MS was used to ionize the metabolites and to record their molecular weight and to provide substructure information based on fragmentation in the mass spectrometer. NMR gave the comprehensive structural information on the chemical environment of protons and their linkage to other protons within the molecule. The additional structural information as compared to MS is at the cost of an increased amount of compound needed for NMR detection and spectra generation. Here we combined both analytical methods into a liquid chromatography (LC)-based platform that concentrated compounds based on their specific mass; thereby providing a direct link between MS and NMR data.
Another platform was developed that generated robust multistage MSn data, i.e., the systematic fragmentation of metabolites and subsequent fragmentation of resulting fragments.
This thesis aims to accelerate metabolite identification of low abundant plant and human derived compounds by following a systematic approach. The acquired structural information from MSn and 1D-1H-NMR spectra resulted in the complete elucidation of phenolic metabolites in microgram scale from both plant and human origin.