Isolation, detection, and quantification of hydrolyzable tannins of the biosynthetic pathway by liquid chromatography coupled with tandem mass spectrometry

Hydrolyzable tannins (HTs) are widely distributed complex secondary metabolites with potential bioactivities and health-promoting benefits. A highly sensitive compound-specific ultrahigh-performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) method is required for their successful detection and quantification in order to advance the study of these compounds.

In this study, 36 HTs belonging to the HT biosynthetic pathway covering 13 major branches were extracted by cold extraction and fractionated by Sephadex LH-20 size-exclusion chromatography. Followed fractionation, the HTs were purified by semipreparative HPLC so that they could be used for the development of a UHPLC/QqQ-MS/MS multiple reaction monitoring (MRM) method for their characterization. The cone voltage and collision energy for each HT were extensively optimized during the development of the MRM method.

The developed method was very useful for the detection and quantification of marker tannins with a low limit of detection (LOD) and limit of quantification (LOQ), depending on the size and complexity of the structures of the HTs. Each isolated compound was successfully identified and characterized by UHPLC/ESI-Orbitrap-MS/MS analysis. In addition, a new methodology for cold extraction and fractionation by Sephadex LH-20 chromatography has been developed for the targeted extraction of HTs.

This study has provided a compound-specific MRM method for the detection and quantification of representative HTs from diverse phytochemical samples that can thus be used in large-scale screening tests to pinpoint the major biosynthetic branches of HTs utilized by plants to accumulate specific types of HTs.