Rapid Screening of Ellagitannins in Natural Sources via Targeted Reporter Ion Triggered Tandem Mass Spectrometry

Jeremiah J Bowers,Stéphane Quideau,Denis Deffieux,Antoine Richieu,Anaëlle Cornu,Harsha P Gunawardena,Ashwini S Narvekar,Nishanth Tharayil

doi:10.1038/s41598-018-27708-3

Abstract

Complex biomolecules present in their natural sources have been difficult to analyze using traditional analytical approaches. Ultrahigh-performance liquid chromatography (UHPLC-MS/MS) methods have the potential to enhance the discovery of a less well characterized and challenging class of biomolecules in plants, the ellagitannins. We present an approach that allows for the screening of ellagitannins by employing higher energy collision dissociation (HCD) to generate reporter ions for classification and collision-induced dissociation (CID) to generate unique fragmentation spectra for isomeric variants of previously unreported species. Ellagitannin anions efficiently form three characteristic reporter ions after HCD fragmentation that allows for the classification of unknown precursors that we call targeted reporter ion triggering (TRT). We demonstrate how a tandem HCD-CID experiment might be used to screen natural sources using UHPLC-MS/MS by application of 22 method conditions from which an optimized data-dependent acquisition (DDA) emerged. The method was verified not to yield false-positive results in complex plant matrices. We were able to identify 154 non-isomeric ellagitannins from strawberry leaves, which is 17 times higher than previously reported in the same matrix. The systematic inclusion of CID spectra for isomers of each species classified as an ellagitannin has never been possible before the development of this approach.

Highlights

Ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/ MS) is one of the more efficient approaches to characterize plant metabolites, including phenolics, in complex extracts[18,19,20,21,22]
This was followed by collision-induced dissociation (CID) as it was better suited to generate unique fragmentation spectra of any isomers observed given the inherent specificity of the method since only first-generation product ions were formed[53,54,55,56,57]
Given that precursor classification was driven by the detection of these reporter ions, an approach to maximize the abundance of these reporters within MS2 spectra of any ellagitannin was prudent to maximize method sensitivity

Summary

Introduction

Ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/ MS) is one of the more efficient approaches to characterize plant metabolites, including phenolics, in complex extracts[18,19,20,21,22]. The number of studies focusing on compound-specific fragmentation using multiple reaction monitoring (MRM) methods on triple-quadrupole mass spectrometers (QqQ-MS) has increased[20,21,22,28,29,30,31,32,33,34]. Focused large-scale discovery efforts to detect unknown ellagitannins have generally been unaddressed. This is the first attempt to rapidly screen ellagitannins and systematically catalogue unique fragmentation spectra of isomers for potential inclusion in spectral libraries. We demonstrate how a tandem HCD-CID experiment might be used to screen natural sources for ellagitannins using UHPLC-MS/MS by application of 22 method conditions from which an optimized data-dependent acquisition (DDA) that classified 154 non-isomeric ellagitannins emerged

Methods

Results

Conclusion