Characterization of high-molar-mass fractions in a Scots pine (Pinus sylvestris L.) knotwood ethanol extract

The identification of compounds in Scots pine (Pinus sylvestris L.) knotwood (KnW), obtained by extracting with hydrophilic organic solvents, has been performed previously almost exclusively by gas chromatographymass spectrometry (GS-MS) equipped with long GC columns (>= 25 m). That means that the molar mass (MM) of the majority of the identified compounds was below 500-600 Da, and the analytical data accounted for only about half of the dry extract weight. In the present work, high-molar-mass (HMM) fractions in a Scots pine KnW-EtOH extract were isolated and chemically characterized by means of several advanced analytical techniques, such as high performance size-exclusion chromatography-evaporative light scattering detector (HPSEC-ELSD), high performance liquid chromatography (HPLC)-electrospray ionization-ion trap-mass spectrometry [(HPLC) ESI-IT-MS], ESI-quadrupole time of flight-mass spectrometry (QTO-FMS), pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS), thermally assisted hydrolysis and methylation-gas chromatography-mass spectrometry (THM-GCMS), nuclear magnetic resonance (NMR) and GC-MS. The results indicate that the MM maxima of the HMM fractions ranged from approximately 500 to 2200 Da, and that the compounds consist mainly of oligomers of hydroxylated resin acids (RAs), especially dehydroabietic acid, but also of fatty acids (FAs), stilbenes and sterols. A large number of RA dimers were tentatively identified in the HMM fractions. However, it remains unknown how the monomer units are linked together, as it was not possible to isolate a RA dimer fraction pure enough for NMR characterization. RA dimers in native KnW have not been identified previously.