Enhancing metabolite coverage using dedicated mobile phases for individual polarity modes in HILIC-MS

Sep 30, 2026

A HILIC-MS method is developed with mobile phases optimized for high metabolite coverage in individual polarity modes, using a dual mobile-phase strategy to expand annotated metabolites and increase identification confidence. A bioinert chromatographic system improves sensitivity further. Among three chromatographic columns, the BEH amide column provides the strongest performance for metabolomic analysis. Mobile-phase composition, pH, and a medronic acid additive are evaluated for MS response, highlighting alkaline pH for sensitive detection of polyphosphorylated metabolites and showing chelating redundancy in a fully bioinert setup. The optimized workflow is applied to mouse plasma, pancreas, and liver for reliable results in complex matrices.

LanguageEnglish

CategoryHealthcare

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Pages11

Reading Time28 min

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Abstract

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Introduction

Analytical and Bioanalytical Chemistry (2025) 418:747–757 [https://doi.org/10.1007/s00216-025-06189-0](https://doi.org/10.1007/s00216-025-06189-0) Enhancing metabolite coverage using dedicated mobile phases for individual polarity modes in HILIC-MS Alena Langová1 · Malena Manzi1 · Jana Brejchová2 · Ondřej Kuda2 · Michal Holčapek1 · Robert Jirásko1 Received: 29 August 2025 / Revised: 8 October 2025 / Accepted: 13 October 2025 / Published online: 19 November 2025 © The Author(s) 2025 Abstract We have developed a new hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) method with mobile phases optimized for high metabolite coverage in individual polarity modes. This dual mobile phase strategy expands the range of annotated metabolites and improves identification confidence, providing broader and more accurate metabolic profiles. The incorporation of a bioinert chromatographic system further enhances sensitivity. The bridged ethyl hybrid (BEH) amide column yields the best results for metabolomic analysis among the three chromatographic columns in this comparison. The method development involves investigating the effects of mobile phase composition, pH, and a medronic acid additive on the MS response under bioinert chromatographic conditions. The results highlight the important role of alkaline pH for the sensitive detection of polyphosphorylated metabolites, while demonstrating the redundancy of chelating additives in a fully bioinert system. Finally, the optimized method is applied to mouse plasma, pancreas, and liver samples to demonstrate its versatility and reliable performance in complex biological matrices, establishing it as a powerful tool for confident and reproducible metabolomics studies. Keywords HILIC · Metabolomics · Mass spectrometry · Bioinert chromatography · Metabolite annotation Introduction A major analytical challenge in MS-based metabolomicsis the comprehensive analysis of metabolites with varying polarities, concentration ranges, and diverse physicochemical properties [1 , 2] . These complexities are intensified by challenges in the MS ion source, including ionization suppression and in-source fragmentation, particularly for highly polar or unstable compounds like phosphates [3–6] . In metabolomics, ultrahigh-performance liquid chromatography (UHPLC) combined with electrospray (ESI) quadrupole-time-of-flight (QTOF) MS is commonly used due to Published in the topical collection featuring Promising EarlyCareer (Bio)Analytical Researchers in 2026 with guest editors Antje J. Baeumner, Soledad Cárdenas, and Alberto Cavazzini. * Robert Jirásko [robert.jirasko@upce.cz](robert.jirasko@upce.cz) 1 Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Studentská 573, 53210 Pardubice, Czech Republic 2 Institute of Physiology of the Czech Academy of Sciences, Videnska 1083, 14200 Prague, Czech Republic its high chromatographic resolution, rapid data acquisition, good sensitivity, and precise mass accuracy in MS and MS/ MS mode, making it one of the best alternatives for holistic metabolite profiling [7–9] . Reversed-phase liquid chromatography (RP-LC) and hydrophilic interaction liquid chromatography (HILIC) are commonly used for liquid chromatography-mass spectrometry (LC/MS) metabolomic analyses. While RP-LC/MS is primarily applied to less polar metabolites, HILIC-MS remains a versatile technique for the comprehensive analysis of small, polar, and ionizable molecules within biological systems [10] . The core principle of HILIC involves partitioning between an aqueous layer partially immobilized on a hydrophilic stationary phase and a more hydrophobic mobile phase, with electrostatic interactions and hydrogen bonding also contributing to analyte retention depending on the stationary phase properties [11] . The primary advantage of HILIC in metabolomics is its ability to enhance the retention of polar metabolites, enabling the s

FAQs

What is the main technical goal of the presented HILIC-MS method?

To improve metabolite coverage by optimizing mobile phases for individual polarity modes, thereby increasing the number of annotated metabolites and improving identification confidence.

How does the bioinert chromatographic system contribute to performance?

It enhances sensitivity and supports more reliable detection during chromatographic-MS analysis.

Which factor is emphasized for sensitive detection of polyphosphorylated metabolites?

Alkaline pH is highlighted as important for sensitive detection, while chelating additives are shown to be redundant under fully bioinert conditions.

Sep 30, 2026

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