Improving Separation of Stereochemical and Positional Isomers: A Combined HILIC-IM-MS Approach

The fundamental building blocks of life consist of lipids, carbohydrates, proteins, and nucleic acids which are assembled from small repeating monomer subunits. Specifically monosaccharides are the precursors of carbohydrates and amino acids are the building blocks of proteins. These two monomers are chiral (except glycine) and can exist in multiple stereochemical forms making their characterization complex. 

While monosaccharides and amino acids can be the precursor to glycan analysis, further research is critical to understanding their function in the biopharmaceutical industry. These different areas of research present as an analytical challenge. We have developed an approach that allows us to identify the absolute configuration of these biopolymer building blocks. In this method, the analyte is derivatized with an optically pure compound that introduces an additional stereochemical center, enabling separation and identification via ion mobility mass spectrometry (IM-MS).

 In addition to the identification of stereoisomers, this work will also include an investigation of improving characterization of positional isomers during glycan characterization. We have explored the effects of pore size and particle size on resolution in zwitterionic hydrophilic interaction chromatography (Z-HILIC) and its impact on analysis time. While exploring chromatographic tools to aid in distinguishing glycan positional isomers, which can be a challenge by mass spectrometry alone. We present the approach of utilizing chromatography as a tool to help identify glycans by retention times. While existing retention time databases assist in glycan identification, they remain limited to known standards. To overcome this limitation, we are developing a predictive model for N-glycan retention times across various stationary phases, thereby expanding analytical capabilities to previously uncharacterized glycans.