Enhanced characterization of lignin oligomers and phenolic compounds using CID-MSn combined with HCD-MS2

Abstract

Introduction Structural elucidation of lignin oligomers and lignin-derived phenolic compounds is a prerequisite to characterize processed biomass. Lignin oligomer is composed of three monomeric units (H, G, and S) with various linkage types, which complicates the structural elucidation. Conventional trap-type CID-MS2 is useful for linkage analysis, however, it suffers from the lack of unit-specific ions when analyzing oligomers larger than trimer. In the other hand, HCD could provide efficient fragmentation against stronger bonds compared to trap-type CID. Here, a combined use of CID-MSn with HCD-MS2 is suggested for characterization of the lignin linkages and units. The complementary properties of trap-type CID and HCD was employed to enhance reliability and size-coverage of lignin oligomer identification. Methods The fragmentation patterns of three dimer standards with different linkage types (8-5, 8-8, and 8-O-4) were compared using CID and HCD by varying collision energy on LTQ Orbitrap XL mass spectrometer. Based on the fragmentation rule observed from the standards, an LC-MSn method combining CID-MSn with HCD-MS2 within a single run was developed. CID-MSn was used for the positional analysis of linkage, while HCD-MS2 was designed to elucidate types of each unit. The method was applied to a randomly synthesized mixture of H-type lignin oligomer. The chromatographic and mass spectrometric conditions were optimized to maximize the MSn sensitivity. Finally, the optimized method was evaluated with an acid-pretreated oak wood as a real sample. Preliminary Data or Plenary Speakers Abstract The fragmentation patterns of three dimer standards with different linkage types (8-5, 8-8, and 8-O-4) were compared using trap-type CID and HCD. Increasing normalized collision energy from 40 to 70 (stepped by 10) did not show significant spectral changes on CID, however, HCD showed a significant increase of low m/z ions. HCD produced the even-electron ions including C-O bond cleavage and water (H2O)/formaldehyde (HCHO)/formic acid (HCOOH) losses. In contrast, HCD additionally produced unique odd-electron radical fragment ions including C-C bond cleavage and methyl loss from the methoxy group (-OCH3). As a result, HCD could reveal additional linkages and units of the oligomer. However, HCD caused multiple fragmentation events, which may complicate the fragment ion interpretation. By taking advantage of a single fragmentation event of trap-type CID, the identity of HCD-produced ions was confirmed using CID-MSn. From a randomly synthesized mixture of H-type lignin oligomer, 9 lignin oligomers were identified and their CID/HCD fragmentation pathways were proposed. The fragmentation rule established for lignin dimers was confirmed with larger oligomers up to tetramer. to be applicable for larger oligomers up to tetramer. Finally, the method was applied for the characterization of acid pretreated oak wood biomass. The phenolic compounds from oak wood biomass were confirmed by CID-MSn spectrum of standard compounds, and lignin oligomers were confirmed by matching the retention time and HCD-MS2 spectral library of randomly synthesized mixture of H+G+S lignin oligomer. Novel Aspect Complementary structural elucidation of lignin oligomer using both trap-type CID-MSn and beam-type HCD-MS2