Improving LC-MSn sensitivity for Structural Characterization of Lignin Oligomers Using Acetic Acid as the Mobile Phase Additive

Abstract

Novel aspect Acetic acid mobile phase additive significantly improves the sensitivity of LC-(−)-ESI-MSn analysis of lignin compared to the ammonium salts.

Introduction Lignin is the aromatic biopolymer deposited in the secondary plant cell wall. There are emerging interests in the characterization of lignin oligomers, for industrial application of degradation products and plant physiological study on phenolic compound metabolism. Reversed-phase liquid chromatography-negative mode electrospray ionization mass spectrometry (RPLC-(−)-ESI-MS) is commonly applied to characterize a complex mixture of lignin oligomers since they are moderately polar and favor deprotonation. Still, the mass spectrometric structural elucidation of lignin is challenging because of relatively low product ion intensity of negative ion mode ESI-MS/MS compared to the positive ion mode. Here, we report significant enhancement of MS/MS signal intensity of lignin oligomers leading to a larger number of compounds structurally identified by using acetic acid as an additive.

Experimental Four different mobile phase additives (acetic acid, formic acid, ammonium acetate, ammonium formate) were evaluated by analyzing lignin standard compounds with RPLC-(−)-ESI-Q-TOF. To assess the effect of the different mobile phase additives on ionization efficiency of standards, precursor peak area was compared. To assess the effect on chromatographic separation, peak intensity and the tailing factor of the EIC peak were compared. The effect of the precursor ion intensity on the structural elucidation was studied for both trap-type CID-MS3 based and beam-type CID-MS2 based analytical system. A combinatorial model mixture of lignin oligomer was produced by enzymatic radical synthesis. The model mixture was analyzed with HPLC-Q-TOF by untargeted MS2 and nanoLC-ion trap by untargeted MS3. On four different additives, the number of identified compounds was compared.

Preliminary data Several articles on negative mode ESI-MS of lipids and amino acids reported the “wrong-way-round” ionization, meaning higher ionization efficiency in mobile phase having lower pH, which is opposite to the common idea of the pH-dependency on deprotonation of the liquid-phase analytes. On lignin standards and a synthetic mixture of lignin oligomers, we observed a similar phenomenon of wrong-way-round ionization. Acetic acid additive showed significant improvement of both ionization efficiency and chromatographic separation compared to the ammonium acetate and ammonium formate. Analysis of lignin standard compounds with acetic acid additive showed average signal enhancement of the precursor ion over 5-fold compared to the ammonium acetate and ammonium formate, and 2-fold compared to the equal concentration (%, w/w) of formic acid. Reversed-phase chromatographic separation of the standards with acetic acid additive was improved compared to the ammonium salts, especially for standards with low pKa value containing the carboxylic functional group. For instance, the USP tailing factor of caffeic acid was halved by using acetic acid instead of the ammonium acetate. The product ion peak intensity also has been increased by using acetic acid, resulting in increased number of detectable product ion peaks per precursor and number of structurally identified compounds.