Systematic Evaluation of HILIC Stationary Phases for Global Metabolomics of Human Plasma

Abstract

Polar hydrophilic metabolites have been identified as important actors in many biochemical pathways. Despite continuous improvement and refinement of hydrophilic interaction liquid chromatography (HILIC) platforms, its application in global polar metabolomics has been underutilized. In this study, we aimed to systematically evaluate polar stationary phases for untargeted metabolomics by using HILIC columns (neutral and zwitterionic) that have been exploited widely in targeted approaches. To do so, high-resolution mass spectrometry was applied to thoroughly investigate selectivity, repeatability and matrix effect at three pH conditions for 9 classes of polar compounds using 54 authentic standards and plasma matrix. The column performance for utilization in untargeted metabolomics was assessed using plasma samples with diverse phenotypes. Our results indicate that the ZIC-c HILIC column operated at neutral pH exhibited several advantages, including superior performance for different classes of compounds, better isomer separation, repeatability and high metabolic coverage. Regardless of the column type, the retention of inorganic ions in plasma leads to extensive adduct formation and co-elution with analytes, which results in ion-suppression as part of the overall plasma matrix effect. In ZIC-c HILIC, the sodium chloride ion effect was particularly observed for amino acids and amine classes. Successful performance of HILIC for separation of plasma samples with different phenotypes highlights this mode of separation as a valuable approach in global profiling of plasma sample and discovering the metabolic changes associated with health and disease.

Methods on LDL particle isolation, characterization, and component fractionation for the development of novel specific oxidized LDL status markers for atherosclerotic disease risk assessment

“…The innovation of this method is the isolation of LDL via two-step sequential precipitation: (1) by heparin, which removes most contaminating serum proteins ( 32 ); (2) by Mg 2+ , combined with an intermediate step of LDL solubilization in 4% NaCl which eliminates any remnant serum proteins ( 32 , 33 ). These steps neutralize matrix effects mostly through ion suppression ( 34 , 35 ). The protocol steps are as follows:…”

Comparison between 5 extractions methods in either plasma or serum to determine the optimal extraction and matrix combination for human metabolomics

“…Despite the precision of our experiment, a limited number of samples could be the reason but for the rest of the study, this consequence was not a bias. Concerning “biologically meaningless noise level” for − ESI HILIC analysis, this phenomenon was totally independent from extraction because this method is known to the high noise level [ 24 , 25 ].…”

Reconstruction of Glutathione Metabolism in the Neuronal Model of Rotenone-Induced Neurodegeneration Using Mass Isotopologue Analysis with Hydrophilic Interaction Liquid Chromatography-Zeno High-Resolution Multiple Reaction Monitoring

“…Chromatographic separation was performed using the SeQuant ZIC-c HILIC HPLC column (2.1 mm × 100 mm, 3.0 μm, Merck, Darmstadt, Germany) on a Shimadzu Nexera Ultra high-performance liquid chromatograph (LC) (Duisburg, Germany). The LC method was adapted from a previously described method . Mobile phase A consists of 90% acetonitrile and 10% water with 5 mM ammonium formate, and mobile phase B consists of 10% acetonitrile and 90% water with 5 mM ammonium formate.…”

“…The LC method was adapted from a previously described method. 35 Mobile phase A consists of 90% acetonitrile and 10% water with 5 mM ammonium formate, and mobile phase B consists of 10% acetonitrile and 90% water with 5 mM ammonium formate. The injection volume was 3 μL.…”