Analysis of condensed tannins in Populus spp. using reversed phase UPLC-PDA-(-)esi-MS following thiolytic depolymerisation.

Abstract

Condensed tannins (CTs) are proanthocyanidin heteropolymers that are widely distributed among plants. Their biochemical properties are determined by molecular structure (e.g. polymer size, hydroxylation, stereochemistry). In Populus, genetically and environmentally-determined CT concentrations have been related to ecological effects, while the potential role of CT molecular structure has received little attention. Evaluate CT polymerisation, major constituent monomers, stereochemistry and overall content in Populus tremuloides foliage using ultra-high-performance liquid chromatography with photodiode array and mass spectrometry (UPLC-PDA-(-)esi-MS) detection following thiolytic depolymerisation of the CTs. CTs were extracted from dried foliage of six P. tremuloides genotypes into methanol and thiolytically depolymerised into constituent monomers. Calibration standards were prepared by thiolysis of CT mixtures isolated from P. tremuloides foliage on Sephadex LH-20, followed by preparative high-performance liquid chromatography (HPLC). Populus tremuloides CTs contained predominantly repeating subunits of three putative stereoisomers each of catechin and gallocatechin. Linear calibrations for standards of these subunits and their thioethers (purities 44-87%, UPLC-(-)esi-MS) were generally stable over the course of 10months. CT polymer size, hydroxylation, stereochemistry and concentrations differed among genotypes. This thiolysis-UPLC-PDA-(-)esiMS method was optimised for analysis of CT polymer size, hydroxylation, stereochemistry, and total concentration in Populus foliage. It revealed significant variation in each of these properties among P. tremuloides genotypes, and will facilitate evaluation of how environmental factors affect CT molecular structures.