Quantitative 13C-IS pyrolysis-GC-MS lignin analysis: Overcoming matrix effects in animal feed and faeces

Recently, a pyrolysis-GC-MS methodology for specific lignin quantification and structural characterisation was developed, relying on the use of uniformly ¹³C-labeled polymeric lignin isolate as internal standard (IS). The ¹³C-IS py-GC-MS method has been validated for grasses, woods, and applied in various showcases. To study the fate of lignin in animals, this method still requires careful validation in animal feeds and, especially complex faecal samples, hence the aim of this work. Hereto, faecal material was collected from pigs fed with wheat straw as lignin source and subjected to the py-GC-MS analytical platform for thorough examination of IS pyrolysis behaviour in terms of response and structural features. Next, ^13C-ISpy-lignin contents and corrected Klason lignin contents were compared. Most importantly, we revealed that pyrolysis behaviour of ¹³C-IS lignin and ¹²C-sample lignin was differently affected in the faecal matrix, resulting in the ultimate underestimation of ^13C-ISpy-lignin contents. In-depth examination and evaluation of matrix constituents showed that predominantly matrix ash was responsible for the effects observed. We further demonstrated that said matrix effects can be overcome by water extraction of the samples prior to analysis. Our validation and approach extend the use of the specific ¹³C-IS py-GC-MS methodology for accurate quantitative lignin analysis to biomass samples with complex matrices like pig faeces, and now call for application in future digestibility studies.