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A growing interest in sustainable development has made efficient utilisation of starting materials and, if they occur, by-products become increasingly important. Vast amounts of by-products are generated by the forestry and food industry. Incineration for energy production is one way to make use of these by-products but some of them contain compounds that would have an increased value if they were extracted, so called “high value species”. The by-products are often very complex, so reliable methods for analysis of the high value species are required in the development of processes to utilise them. A wide range of compounds can be analysed using chromatographic separation coupled to mass spectrometry, making it a powerful tool in the evaluation of methods for extracting high value species from industry by-products. This thesis is based on four studies of potential high value species. In the first study, methods were developed to differentiate isobaric flavonoids and then use this knowledge to determine the identity of the flavonoids in three different plant extracts. In the second study, three different methods to extract betulin from birch bark were evaluated regarding extracted amount and purity of betulin. One of the methods was then investigated in industrial scale using a model approach. In the third study, the flavonoid contents of lovage were determined and other major extracted compounds were investigated by high performance liquid chromatography coupled to electrospray ionisation mass spectrometry. Gas chromatography and supercritical fluid chromatography were used to obtain complementary information about major components. In the fourth study, high resolution mass spectrometry utilising two different types of fragmentation was used with the purpose of overcoming the shortcomings of the methods developed in the first study. The results indicated that it would be possible to develop methods compatible with chromatographic separation for differentiating different types of isobaric substituents. The ability of performing sequential fragmentation was used to investigate some isobaric aglycones by creating spectral trees, and unique pathways were found for each of them.

Flavonoids are a class of secondary plant metabolites existing in great variety in nature. Due to this variety, identification can be difficult, especially as overlapping compounds in both chromatographic separations and mass spectrometric detection are common. Methods for distinguishing isobaric flavonoids using MS2 and MS3 have been developed. Chromatographic separation of various plant extracts was done with RP-HPLC and detected with positive ESI-MS operated in information-dependent acquisition (IDA) mode. Two methods for the determination of flavonoid identity and substitution pattern, both featuring IDA criteria, were used together with the HPLC equipment. A third method where the collision energy was ramped utilized direct infusion. With the developed strategies, it is possible to differentiate between many isobaric flavonoids. Various classes of flavonoids were found in all of the plant extracts, in the red onion extract 45 components were detected and for 29 of them the aglycone was characterized, while the substituents were tentatively identified for 31 of them. For the strawberry extract, those numbers were 66, 30 and 60, and for the cherry extract 99, 56 and 71. The great variety of flavonoids, several of them isobaric, found in each of the extracts highlights the need for reliable methods for flavonoid characterization. Methods capable of differentiating between most of the isobars analyzed have been developed.