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.

Department of Analytical Chemistry Faculty of Chemical Technology University of Pardubice Studentská 573 53210 Pardubice Czech Republic

Institute of Physiology of the Czech Academy of Sciences Videnska 1083 14200 Prague Czech Republic

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McCullagh J, Probert F. New analytical methods focusing on polar metabolite analysis in mass spectrometry and NMR-based metabolomics. Curr Opin Chem Biol. 2024. 10.1016/j.cbpa.2024.102466. PubMed DOI

Rakusanova S, Cajka T. Tips and tricks for LC–MS-based metabolomics and lipidomics analysis. TrAC Trends Anal Chem. 2024. 10.1016/j.trac.2024.117940. DOI

Mahmud I, Wei B, Veillon L, Tan L, Martinez S, Tran B, Raskind A, de Jong F, Liu Y, Ding J, Xiong Y, Chan WK, Akbani R, Weinstein JN, Beecher C, Lorenzi PL. Ion suppression correction and normalization for non-targeted metabolomics. Nat Commun. 2025. 10.1038/s41467-025-56646-8. PubMed DOI PMC

Spalding JL, Naser FJ, Mahieu NG, Johnson SL, Patti GJ. Trace phosphate improves ZIC-pHILIC peak shape, sensitivity, and coverage for untargeted metabolomics. J Proteome Res. 2018;17:3537–46. 10.1021/acs.jproteome.8b00487. PubMed DOI PMC

Alseekh S, Aharoni A, Brotman Y, Contrepois K, D’Auria J, Ewald JC, Ewald J, Fraser PD, Giavalisco P, Hall RD, Heinemann M, Link H, Luo J, Neumann S, Nielsen J, Perez de Souza L, Saito K, Sauer U, Schroeder FC, Schuster S, Siuzdak G, Skirycz A, Sumner LW, Snyder MP, Tang H, Tohge T, Wang Y, Wen W, Wu S, Xu G, Zamboni N, Fernie AR. Mass spectrometry-based metabolomics: a guide for annotation, quantification and best reporting practices. Nat Methods. 2021;18:747–56. PubMed DOI PMC

Brookhart A, Arora M, McCullagh M, Wilson ID, Plumb RS, Vissers JP, Tanna N. Understanding mobile phase buffer composition and chemical structure effects on electrospray ionization mass spectrometry response. J Chromatogr A. 2023. 10.1016/j.chroma.2023.463966. PubMed DOI

Zhou B, Xiao JF, Tuli L, Ressom HW. LC-MS-based metabolomics. Mol Biosyst. 2012;8:470–81. PubMed DOI PMC

Johnson CH, Ivanisevic J, Siuzdak G. Metabolomics: beyond biomarkers and towards mechanisms. Nat Rev Mol Cell Biol. 2016;17:451–9. 10.1038/nrm.2016.25. PubMed DOI PMC

Schuhmacher R, Krska R, Weckwerth W, Goodacre R. Metabolomics and metabolite profiling. Anal Bioanal Chem. 2013;405:5003–4. PubMed DOI

Wernisch S, Pennathur S. Evaluation of coverage, retention patterns, and selectivity of seven liquid chromatographic methods for metabolomics. Anal Bioanal Chem. 2016;408:6079–91. 10.1007/s00216-016-9716-4. PubMed DOI PMC

Nováková L, Havlíková L, Vlčková H. Hydrophilic interaction chromatography of polar and ionizable compounds by UHPLC. TrAC - Trends in Analytical Chemistry. 2014;63:55–64. DOI

Sheng Q, Liu M, Lan M, Qing G. Hydrophilic interaction liquid chromatography promotes the development of bio-separation and bio-analytical chemistry. TrAC Trends Anal Chem. 2023. 10.1016/j.trac.2023.117148. DOI

Buszewski B, Noga S. Hydrophilic interaction liquid chromatography (HILIC)-a powerful separation technique. Anal Bioanal Chem. 2012;402:231–47. PubMed DOI PMC

Cubbon S, Antonio C, Wilson J, Thomas-Oates J. Metabolomic applications of HILIC-LC-MS. Mass Spectrom Rev. 2010;29:671–84. 10.1002/mas.20252. PubMed DOI

Zhao S, Li L. Chemical derivatization in LC-MS-based metabolomics study. TrAC Trends Anal Chem. 2020. 10.1016/j.trac.2020.115988. DOI

Assress HA, Hameed A, Pack LM, Ferruzzi MG, Lan RS. Evaluation of ion source parameters and liquid chromatography methods for plasma untargeted metabolomics using orbitrap mass spectrometer. J Chromatogr B Analyt Technol Biomed Life Sci. 2025. 10.1016/j.jchromb.2025.124564. PubMed DOI

Peterka O, Langová A, Jirásko R, Holčapek M. Bioinert UHPLC system improves sensitivity and peak shapes for ionic metabolites. J Chromatogr A. 2025. 10.1016/j.chroma.2024.465588. PubMed DOI

Gilar M, DeLano M, Gritti F. Mitigation of analyte loss on metal surfaces in liquid chromatography. J Chromatogr A. 2021. 10.1016/j.chroma.2021.462247. PubMed DOI

Serafimov K, Lämmerhofer M. Comprehensive coverage of glycolysis and pentose phosphate metabolic pathways by isomer-selective accurate targeted hydrophilic interaction liquid chromatography-tandem mass spectrometry assay. Anal Chem. 2024;96:17271–9. 10.1021/acs.analchem.4c03490. PubMed DOI

Garcia-Contreras R, Sugimoto M, Umemura N, Kaneko M, Hatakeyama Y, Soga T, Tomita M, Scougall-Vilchis RJ, Contreras-Bulnes R, Nakajima H, Sakagami H. Alteration of metabolomic profiles by titanium dioxide nanoparticles in human gingivitis model. Biomaterials. 2015;57:33–40. 10.1016/j.biomaterials.2015.03.059. PubMed DOI

Rojahn AM, Esser D. HILIC analysis of oligonucleotides using bioinert columns. LCGC Europe 2023;36(6):238–239.

Lardeux H, D’Atri V, Guillarme D. Recent advances and current challenges in hydrophilic interaction chromatography for the analysis of therapeutic oligonucleotides. TrAC -Trends Anal Chem. 2024;176. 10.1016/j.trac.2024.117758.

Murisier A, Fekete S, Guillarme D, D’Atri V. The importance of being metal-free: the critical choice of column hardware for size exclusion chromatography coupled to high resolution mass spectrometry. Anal Chim Acta. 2021;1183:338987. 10.1016/j.aca.2021.338987. PubMed

Gilar M, Berthelette KD, Walter TH. Contribution of ionic interactions to stationary phase selectivity in hydrophilic interaction chromatography. J Sep Sci. 2022;45:3264–75. 10.1002/jssc.202200165. PubMed DOI PMC

Partecke LI, Sendler M, Kaeding A, Weiss FU, Mayerle J, Dummer A, Nguyen TD, Albers N, Speerforck S, Lerch MM, Heidecke CD, von Bernstorff W, Stier A. A syngeneic orthotopic murine model of pancreatic adenocarcinoma in the C57/BL6 mouse using the Panc02 and 6606PDA cell lines. Eur Surg Res. 2011;47:98–107. 10.1159/000329413. PubMed DOI

Idkowiak J, Jirásko R, Kolářová D, Bártl J, Hájek T, Antonelli M, Vaňková Z, Wolrab D, Hrstka R, Študentová H, Melichar B, Pešková K, Holčapek M. Robust and high-throughput lipidomic quantitation of human blood samples using flow injection analysis with tandem mass spectrometry for clinical use. Anal Bioanal Chem. 2023;415:935–51. 10.1007/s00216-022-04490-w. PubMed DOI

Wolrab D, Chocholoušková M, Jirásko R, Peterka O, Holčapek M. Validation of lipidomic analysis of human plasma and serum by supercritical fluid chromatography–mass spectrometry and hydrophilic interaction liquid chromatography–mass spectrometry. Anal Bioanal Chem. 2020;412:2375–88. 10.1007/s00216-020-02473-3. PubMed DOI

Wishart DS, Guo AC, Oler E, Wang F, Anjum A, Peters H, Dizon R, Sayeeda Z, Tian S, Lee BL, Berjanskii M, Mah R, Yamamoto M, Jovel J, Torres-Calzada C, Hiebert-Giesbrecht M, Lui VW, Varshavi D, Varshavi D, Allen D, Arndt D, Khetarpal N, Sivakumaran A, Harford K, Sanford S, Yee K, Cao X, Budinski Z, Liigand J, Zhang L, Zheng J, Mandal R, Karu N, Dambrova M, Schiöth HB, Greiner R, Gautam V. HMDB 5.0: the human metabolome database for 2022. Nucleic Acids Res. 2022;50:D622–31. 10.1093/nar/gkab1062. PubMed DOI PMC

Ruttkies C, Schymanski EL, Wolf S, Hollender J, Neumann S. Metfrag relaunched: incorporating strategies beyond in silico fragmentation. J Cheminform. 2016. 10.1186/s13321-016-0115-9. PubMed DOI PMC

Garcıa MC, Hogenboom AC, Zappey H, Irth H. Effect of the mobile phase composition on the separation and detection of intact proteins by reversed-phase liquid chromatography-electrospray mass spectrometry. J Chromatogr A. 2002. 10.1016/S0021-9673(02)00345-X PubMed

Zhang J, Dai J, Wang S, Rui K, Dai W, Kang X, Ji J, Wang W. Evaluation of metabolites and biological activities of Areca nut ( DOI

Chan CCY, Gregson DB, Wildman SD, Bihan DG, Groves RA, Aburashed R, Rydzak T, Pittman K, Van Bavel N, Lewis IA. Metabolomics strategy for diagnosing urinary tract infections. Nat Commun. 2025. 10.1038/s41467-025-57765-y. PubMed DOI PMC

Zhao Y, Ma C, Cai R, Xin L, Li Y, Ke L, Ye W, Ouyang T, Liang J, Wu R, Lin Y. NMR and MS reveal characteristic metabolome atlas and optimize esophageal squamous cell carcinoma early detection. Nat Commun. 2024. 10.1038/s41467-024-46837-0. PubMed DOI PMC

McCalley DV. Influence of metals in the column or instrument on performance in hydrophilic interaction liquid chromatography. J Chromatogr A. 2022. 10.1016/j.chroma.2021.462751. PubMed DOI

Wang G, Tomasella FP. Ion-pairing HPLC methods to determine EDTA and DTPA in small molecule and biological pharmaceutical formulations. J Pharm Anal. 2016;6:150–6. 10.1016/j.jpha.2016.01.002. PubMed DOI PMC

Birdsall RE, Kellett J, Yu YQ, Chen W. Application of mobile phase additives to reduce metal-ion mediated adsorption of non-phosphorylated peptides in RPLC/MS-based assays. J Chromatogr B Analyt Technol Biomed Life Sci. 2019. 10.1016/j.jchromb.2019.121773. PubMed DOI

Hsiao JJ, Potter OG, Chu TW, Yin H. Improved LC/MS methods for the analysis of metal-sensitive analytes using medronic acid as a mobile phase additive. Anal Chem. 2018;90:9457–64. 10.1021/acs.analchem.8b02100. PubMed DOI

Xu YF, Lu W, Rabinowitz JD. Avoiding misannotation of in-source fragmentation products as cellular metabolites in liquid chromatography-mass spectrometry-based metabolomics. Anal Chem. 2015;87:2273–81. 10.1021/ac504118y. PubMed DOI PMC

Sumner LW, Amberg A, Barrett D, Beale MH, Beger R, Daykin CA, Fan TWM, Fiehn O, Goodacre R, Griffin JL, Hankemeier T, Hardy N, Harnly J, Higashi R, Kopka J, Lane AN, Lindon JC, Marriott P, Nicholls AW, Reily MD, Thaden JJ, Viant MR. Proposed minimum reporting standards for chemical analysis: Chemical Analysis Working Group (CAWG) Metabolomics Standards Initiative (MSI). Metabolomics. 2007;3:211–21. 10.1007/s11306-007-0082-2. PubMed DOI PMC

Schymanski EL, Jeon J, Gulde R, Fenner K, Ruff M, Singer HP, Hollender J. Identifying small molecules via high resolution mass spectrometry: communicating confidence. Environ Sci Technol. 2014;48:2097–8. PubMed DOI

Domingo-Almenara X, Montenegro-Burke JR, Benton HP, Siuzdak G. Annotation: a computational solution for streamlining metabolomics analysis. Anal Chem. 2018;90:480–9. PubMed DOI PMC

Saarinen MT, Kärkkäinen O, Hanhineva K, Tiihonen K, Hibberd A, Mäkelä KA, Raza GS, Herzig K-H, Anglenius H. Metabolomics analysis of plasma and adipose tissue samples from mice orally administered with polydextrose and correlations with cecal microbiota. Sci Rep. 2020;10:21577. 10.1038/s41598-020-78484-y. PubMed DOI PMC

Goon DE, Ab-Rahim S, Mohd Sakri AH, Mazlan M, Tan JK, Abdul Aziz M, Mohd Noor N, Ibrahim E, Sheikh Abdul Kadir SH. Untargeted serum metabolites profiling in high-fat diet mice supplemented with enhanced palm tocotrienol-rich fraction using UHPLC-MS. Sci Rep. 2021;11:21001. 10.1038/s41598-021-00454-9. PubMed DOI PMC

Giesbertz P, Padberg I, Rein D, Ecker J, Höfle AS, Spanier B, Daniel H. Metabolite profiling in plasma and tissues of ob/ob and db/db mice identifies novel markers of obesity and type 2 diabetes. Diabetologia. 2015;58:2133–43. 10.1007/s00125-015-3656-y. PubMed