Mass Spectrometry of Biologically Active Small Molecules

The main foci of this dissertation are on advanced liquid chromatography (LC) separation and mass spectrometry (MS) techniques for the analysis of small bioactive molecules. In addition to discussing general aspects of such techniques the results from analyses of polyphenols, alkaloids and amino acids published in five appended studies are presented and discussed. Various MS methods were applied in the studies, and several prior work-up/separation techniques (see below), but LC was used in most cases, since it can often provide valuable corroborative and/or complementary (e.g. quantitative) information to the data generated by emerging MS techniques such as laser desorption/ionization (LDI) MS. High availability, relatively convenient handling and well understood principles make LC the method of choice for separating analytes in many kinds of scientific investigations. LC systems can also be conveniently and robustly coupled to various kinds of detectors. Most importantly (in the context of these studies) they are frequently coupled nowadays to MS instruments to separate and analyze diverse analytes. In LC-MS analytes are separated in two stages: firstly they are separated and pre-concentrated in narrow bands using LC and then separated according to their mass-to-charge (m/z) ratios in an MS instrument. Furthermore, valuable information about their molecular structure can be acquired through MS detection (far more than can be acquired using most other on-line LC detection techniques). Some MS instruments can even provide highly accurate molecular weight measurements and sufficient mass resolution to identify unknown compounds in complex mixtures based purely on MS data, thus making prior separation unnecessary. However, prior separation is essential for analyzing substances in most complex matrices, and in many other applications it greatly improves the analysis – especially if ultra-high performance LC (UHPLC) is used. The advantages of using UHPLC rather than HPLC for the analysis of polyphenols (PPs) in tea and wine were evaluated in one the study this thesis is based upon. The UHPLC methods typically offered higher separation efficiency, together with substantial reductions in run time and solvent consumption compared to HPLC. The phenolic composition of red