A combined liquid chromatography - trapped ion mobility - tandem high-resolution mass spectrometry and multivariate analysis approach for the determination of enzymatic reactivity descriptors in biomass hydrolysates

Intermediate products such as oxygenated compounds may interfere with bioconversion kinetics of lignocellulosic biomass into bioethanol. This work presents a multidimensional approach, based on liquid chromatography (LC), trapped ion mobility spectrometry (TIMS), tandem high-resolution mass spectrometry (HRMS/MS), and multivariate analysis, for the identification of enzymatic reactivity descriptors in 22 industrial biomass samples, called hydrolysates. The first part of the study is dedicated to the improvement of the chemical diversity assessment of the hydrolysates through an original three-dimensional Van Krevelen diagram displaying the double bond equivalent (DBE) as third dimension. In a second part, the evaluation of data by multivariate data analysis allowed the discrimination of sample according to the biomass type and the level of enzymatic reactivity. In the last part, a potential descriptor of low enzymatic reactivity was selected and used in a case study. An in-depth structural analysis was performed on the feature annotated as carbohydrate derivative. Considering the intricate fragmentation spectrum exhibited by the selected feature, trapped ion mobility was employed to enhance separation prior to the HRMS/MS experiments. This final step improved data interpretation and increased the identification confidence level leading to the characterization of xylotriose, 3,5-dimethoxy-4-hydroxybenzaldehyde and 4-hydroxy-3-methoxy-cinnamaldehyde. This is the first study to present an untargeted multidimensional approach for the identification of enzymatic hydrolysis inhibitors in industrial hydrolysate samples.