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Effect of Mobile Phase Composition on Retention Behavior of Metabolites in Bioinert LC Analysis

Authors: Langová Alena | Jirásko Robert | Peterka Ondřej | Holčapek Michal

Year: 2024

Type of publication: ostatní - přednáška nebo poster

Page from-to: nestránkováno

Titles:

Language	Name	Abstract	Keywords
eng	Effect of Mobile Phase Composition on Retention Behavior of Metabolites in Bioinert LC Analysis	Metabolomics is defined as the large-scale study of all metabolites within an organism and represents the frontline in the 'omics' approaches, providing direct information on the metabolic responses and perturbations in metabolic pathways. Changes in the composition and concentration of metabolites can indicate various serious diseases, such as cancer. Metabolites are small molecules that often contain polar functional groups, such as phosphate and sulphate, which can interact with the metal surface of the instrument, resulting in unfavorable chromatographic peak shapes and reduced sensitivity. A fully bioinert system should not contain any metal surfaces, which should remove problems with peak tailing caused by interactions of ionic metabolites with metal ions. In metabolomics, two commonly used modes for separating metabolites are hydrophilic interaction liquid chromatography (HILIC) and reversed-phased liquid chromatography (RP-LC) coupled with mass spectrometry (MS). The diverse polarities, structures, and concentrations of metabolites pose a challenge in establishing ideal conditions for their comprehensive separation. In liquid chromatography, the composition of the mobile phase plays a crucial role in numerous aspects of the separation process, especially for peak shape and resolution. For example, nucleotides containing from one to three phosphate groups (e.g., AMP, ADP, ATP) or coenzymes, require different separation conditions compared to amino acids. The best separation of these nucleotides can be achieved at pH around 9, while organic acids usually require lower pH, typically around 5, for optimal separation. In this work, the bioinert Premier UHPLC system with the Premier BEH Amide column (150×2.1 mm; 1.7 µm) coupled to MS was used for the optimization of metabolomic analyses. Two new separation methods with different pH levels were developed and compared using 90 standards, with a total run time of 20 minutes, including the column equilibration.	metabolomics