Abstract
In metabolomic analysis, one of the most commonly used techniques to support the detection sensitivity and quantitation of mass spectrometry is combining it with liquid chromatography. Recently, we developed a method that enables comprehensive single-run measurement of hydrophilic metabolites using unified-hydrophilic interaction/anion exchange liquid chromatography/high-resolution mass spectrometry (unified-HILIC/AEX/HRMS) with a polymer-based mixed amines column (Gelpack GL-HilicAex). However, the importance of stationary phase functional groups and mobile phase conditions for the separation mechanisms and sensitive detection in unified-HILIC/AEX/HRMS is not yet fully understood. This study aimed to understand the importance of the mobile and stationary phases in unified-HILIC/AEX/HRMS. Two different alkali-resistant polymer-based amines-modified columns (Gelpack GL-HilicAex, primary, secondary, tertiary, and quaternary amine-modified polyglycerol dimethacrylate gel; Asahipak NH2P-50 2D, secondary amine-modified polyvinyl alcohol gel) and two eluents (acetonitrile and ammonium bicarbonate solution, pH 9.8) were used for comparative validation. A comparison of mobile phase conditions using both columns confirmed that the two-step separation from HILIC to AEX characteristic of unified-HILIC/AEX requires a linear gradient condition from acetonitrile to nearly 50% water and AEX with up to 40 mM bicarbonate ions. We found that when alkali-resistant hydrophilic polymer packing materials are modified with amines, unified-HILIC/AEX separation can be reproduced if at least one secondary amine associated with the amine series is present in the stationary phase. Furthermore, the difference in sensitivity in the HILIC and AEX modes owing to the different columns indicates the need for further improvements in the mobile phase composition and stationary phase.