Determination of Molecular Structures of Condensed Tannins from Plant Tissues Using HPLC-UV Combined with Thiolysis and MALDI-TOF Mass Spectrometry

Background

Condensed tannins are polyphenolic oligomers made of flavan-3-ol monomeric units that could be extracted from several plant tissues (e.g., softwood barks). They have been recognized as suitable alternatives to synthetic phenolics in resin formulations such as wood adhesives and foamed materials. The most common flavan-3-ol monomers detected in condensed tannins, which differ in the hydroxylation pattern and stereochemistry, are shown in Figure 1.


Figure 1. Most common monomers identified in the structure of condensed tannins

The specific structure of monomeric units in the oligomer and the polymerization degree strongly influence the chemical reactivity and physical properties of the tannins, e.g., condensation reaction rate with aldehydes, heavy metal chelation ability, and viscosity of aqueous solutions (Pizzi and Stephanou, 1994; Yoneda and Nakatsubo, 1998; Garnier et al., 2001). Identification of the molecular structure of the tannins is therefore important to better determine their possible exploitation.

The analysis of the structure of condensed tannins monomers has been performed with different methodologies, e.g., size exclusion chromatography (SEC), normal and reversed-phase high-performance liquid chromatography (HPLC), matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and nuclear magnetic resonance (NMR) (Hümmer and Schreier, 2008).

Depolymerisation of tannins in acidic methanol and in the presence of toluene-α-thiol or cysteamine hydrochloride (thiolysis) followed by reversed-phase HPLC-UV was recognised as a suitable method for the estimation of the mean degree of polymerization of condensed tannins and the configuration of their building units (Matthews et al., 1997; Cheynier et al., 2001; Jerez et al., 2007; Bianchi et al., 2015). As well, MALDI-TOF MS has been shown to be successful in the determination of the structure of condensed tannins (Pasch et al., 2001; Monagas et al., 2010; Bianchi et al., 2014).

The proposed method for the analysis of condensed tannins extracted from plant tissues represents an optimization of the previously published procedures for HPLC-UV after acid thiolysis and MALDI-TOF MS. A careful tuning of the mobile phase gradient in the HPLC analysis was performed in order to reach a sufficient separation between the released flavanols and their corresponding thioethers. The choice of the cationization agent in the preparation of the sample for MALDI-TOF MS was also carefully gauged, especially in consideration of samples containing a relatively high amount of inorganic compounds like bark extracts.

The method was successfully used in the characterization of condensed tannins extracted from the bark of softwood species, e.g., Silver fir, European larch, Norway spruce, Douglas fir and Scots pine (Bianchi et al., 2015).