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Toward Robust and Reproducible Metabolomics: Improving Polar Metabolite Analysis Via LC-MS

Authors: Langová Alena | Holčapek Michal | Jirásko Robert

Year: 2025

Type of publication: ostatní - přednáška nebo poster

Page from-to: nestránkováno

Titles:

Language	Name	Abstract	Keywords
eng	Toward Robust and Reproducible Metabolomics: Improving Polar Metabolite Analysis Via LC-MS	The reliable detection and quantification of polar and ionic metabolites remains a significant analytical challenge in the field of metabolomics, primarily due to their high polarity, low retention, and susceptibility to ion suppression. To overcome these limitations, we developed and validated a novel hydrophilic interaction liquid chromatography coupled with mass spectrometry methodology utilising a bioinert ultra-high pressure liquid chromatography system and polarity optimised mobile phases. The study systematically evaluated the effects of mobile phase composition (ammonium acetate vs. ammonium formate), pH, and gradient slope on chromatographic performance and mass spectrometric signal. A key innovation was a dual polarity HILIC-MS workflow, which combines optimized conditions for both positive and negative electrospray ionization. This approach allowed for the selective enhancement of various metabolite classes including amino acids, nucleotides, sugar phosphates, and carboxylic acids. The bioinert liquid chromatography (LC) configuration significantly reduced peak tailing and signal suppression, particularly for highly polar and ionic analytes1. Comparative analysis with conventional LC setups demonstrated the critical effect of system configuration and column choice on sensitivity and peak shape. Several sample preparation approaches were compared, revealing notable differences in signal quality and method reproducibility. Proper selection and optimization of these steps proved essential for achieving robust metabolomic performance. Comprehensive method validation was performed to confirm linearity across dynamic concentration ranges, determine limits of detection and quantification, assess intra-day precision, and evaluate matrix effects from biological samples. Metabolite detection and identification was achieved using high-resolution quadrupole time-of-flight mass spectrometry, retention time alignment with reference standards, and tandem mass spectr	Metabolomics; Polar Metabolite; LC-MS