Quantification of Lignin and Its Structural Features in Plant Biomass Using 13C Lignin as Internal Standard for Pyrolysis-GC-SIM-MS

Understanding the mechanisms underlying plant biomass recalcitrance at the molecular level can only be achieved by accurate analyses Of both the content and structural features of the molecules involved. Current quantification of lignin is, however, majorly based on unspecific gravimetric analysis after sulfuric acid hydrolysis. Hence, our research aimed at specific lignin quantification with concurrent Characterization of its structural features. Hereto, for the first time, a polymeric C-13 lignin was used as internal standard (IS) for lignin quantification via analytical pyrolysis coupled to gas chromatography with mass-spectrorrietric detection in selected ion monitoring mode (py-GC-SIM-MS). In addition, relative response factors (RRFs) for the various pyrolysis products obtained were determined and applied. First, C-12 and C-13 lignin were isolated from nonlabeled and uniformly C-13 labeled wheat straw, respectively, and characterized by heteronuclear single quantum coherence (HSQC), nuclear magnetic resonance (NMR), and py-GC/MS. The two lignin isolates were found to have identical structures. Second, C-13-IS based lignin quantification by py-GC-SIM-MS was validated in reconstituted biomass model systems with known contents of the C-12 lignin analogue and was shown to be extremely accurate (>99.9%, R-2>0.999) and precise (RSD < 1.5%). Third, C-13-IS based lignin quantification was applied to four common poaceous biomass sources (wheat straw, barley straw, corn stover, and sugar Cane bagasse), and lignin contents were in good agreement with the total gravimetrically determined lignin contents. Our robust method proves to be a promising alternative for the high-throughput quantification of lignin in milled biomass samples directly and simultaneously provides a direct insight into the structural features of lignin.