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

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.

Phenolome of Asian Agrimony Tea (Agrimonia asiatica Juz., Rosaceae): LC-MS Profile, α-Glucosidase Inhibitory Potential and Stability

“…Among plant polyphenols with antidiabetic properties, ellagitannins have attracted a high degree of interest. These compounds involve structurally complex plant phenolics that are widespread in higher plants, especially in the Rosaceae family, and can reach high molecular masses [ 19 , 20 ].…”

Chemical plasticity in the fine root construct of Quercus spp. varies with root order and drought

“…12, first + second-order roots; 3, third-order roots; 4, fourth-order roots. Ellagitannins were identified following the targeted reporter-ion trigger method described in Bowers et al (2018). (Patterson et al, 2019).…”

“…Detailed mass spectrometric parameters are provided in Methods S1. Metabolites were classified as ellagitannins following the unique fragmentation pattern generated by the reporter-ion triggered MS 2 (Bowers et al, 2018).…”

Nitrogen Fertilization Influences the Quantity, Composition, and Tissue Association of Foliar Phenolics in Strawberries

“…Isotopelabeled resveratrol ( 13 C 6 ; 0.5 ng/µl) was added as the internal standard. The samples were analyzed on an Ultimate 3000 HPLC (ultrahigh-pressure liquid chromatography) coupled to an Orbitrap Fusion Tribrid mass spectrometer equipped with electrospray ionization (ESI; Thermo Scientific, Waltham, MA, United States) as per Bowers et al (2018). Separation of the metabolites was carried out using an HSS T3 column (Waters Corp., Milford, MA, United States; 150 mm × 2.1 mm, 1.8 µm) at 30 • C. The following gradient program utilizing water with 0.1% formic acid as mobile phase A and acetonitrile as mobile phase B was employed: 0 min, 5% B; 2 min, 5% B; 24 min, 60% B; 27 min, 90% B; followed by a 3-min washing step at 90% B and a subsequent re-equilibration for 6 min at 5% B.…”

“…The flow rate was set to 0.22 ml/min, and the injection volume was set as 2 µl. The mass spectrometer was operated as per Bowers et al (2018) in negative ionization mode with a data-dependent MS 2 HCD-CID method for compound confirmation. The interface conditions were as follows: emitter voltage, −2,600 V; vaporizer temperature, 325 • C; ion transfer tube, 325 • C; sheath gas, 55 (arb); aux gas, 10 (arb); and sweep gas, 1 (arb).…”