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User Benefits Application News Increasing Reporting Confidence in Metabolomics; Reversed-Phase and HILIC LC/QTOF Untargeted Analysis in Multiple Tissue Studies Emily Armitage, Alan Barnes, Neil Loftus Shimadzu Corporation, Manchester, UK; High Performance Liquid Chromatograph Mass Spectrometer Flexible workflows for untargeted metabolomics using LC/QTOF (applicable to LCMS-9030 and 9050). A single data independent acquisition (DIA)-MS/MS method for untargeted metabolic profiling of tissues and biofluids. Application of reversed-phase and HILIC chromatography increases metabolite coverage and reporting confidence in untargeted metabolomics. Introduction The chemical and physical diversity of the metabolome creates challenges in developing methods that generate high metabolite coverage and reporting confidence with a sufficiently high analytical throughput. Reversed-phase high- resolution UHPLC-MS/MS methods are core analytical tools in metabolomic studies of tissues and biofluids. Their strengths include high metabolite coverage, particularly with lipid distributions (for example; phospholipids, fatty acids, fatty acid conjugates and fatty amides) and high reporting confidence for a broad range of polar metabolites (nucleotides, amino acids, purines, pyrimidines and associated derivatives). However, dependent on the study design, tissue type and metabolite class, reporting confidence may be challenged for highly polar metabolite signals. In such cases HILIC can be a powerful complementary technique to help identify metabolites with greater certainty. In this application note, a single data independent acquisition (DIA-MS/MS) method is presented for the untargeted metabolomics analysis of different biological tissues with two alternative chromatography solutions: C18 reversed-phase and HILIC. Figure 1 highlights the reversed phase and HILIC analysis of mouse brain, studied in a C57BL/6 mouse model of ethanol abuse. In this example, several metabolites showed altered brain metabolome perturbations that eluted early in the RP gradient which could be further confirmed using HILIC analysis. 2 4 RT (min) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 RT (min) 2 4 6 8 10 12 14 16 18 20 22 24 26 28 HILIC separation Method specifically developed for highly polar metabolites Note; Lipid distributions LPCs 5.75-7.0 mins LPEs 5-5.5 mins Fatty acids 0.6-0.8 mins HILIC Method Providing further reporting confidence for highly polar metabolites in untargeted analysis Metabolites features found to differ significantly in the brains of chronic ethanol-treated animals in comparison to the controls (HILIC Rt data; Rt range 3.0-9.5 mins) Creatinine; m/z 114.0659; Rt 3.15 mins Proline; m/z 116.0703; Rt 7.00 mins γ-aminobutryic acid; m/z 104.0705; Rt 7.28 mins Adenosine monophosphate; m/z 348.0701; Rt 7.59 mins Glutamine; m/z 148.0605; Rt 7.83 mins Acetylcarnitine; m/z 204.123; Rt 8.77 mins Arginine; m/z 175.1189; Rt 9.39 mins Reversed-Phase Method Highly polar metabolites Nucleotides, amino acids, purines, pyrimidines and associated derivatives Lipid distributions LPCs 6-8 mins LPEs 6-10 mins Fatty acids 7-16 mins GPLs 24-31 mins Note; fatty acids Rt range also includes conjugates and fatty amides Reversed Phase Method Highly polar metabolites Significant metabolite features elute between 0.7-0.85 mins by reversed phase UHPLC-MS/MS Fig. 1 Metabolite features detected by reversed-phase LC-MS/MS (upper plot) for a mouse brain tissue sample as part of a study to determine the effect of ethanol on the metabolome. Highly polar metabolites were further qualified using HILIC (lower plot) to increase reporting confidence in isobaric feature identification and further resolving isobaric features in different tissue types.
Application News Results To increase the metabolome coverage in untargeted tissue metabolic profiling studies and to ensure high reporting confidence requires more than one analytical approach. Reversed-phase based DIA-MS/MS methods are powerful, robust tools for highly positive metabolite identification. However, identifying highly polar metabolites with high confidence is challenging. As highlighted in Figure 1, the application of a generic analytical reversed-phase LC-MS/MS method results in a high metabolome coverage and has been applied to the untargeted metabolomics analysis of biofluids and tissues. However, in metabolomics studies which report early eluting features as significant, a HILIC method was specifically developed to further increase reporting confidence using the same precursor and data-independent MS/MS acquisition (DIA) methods as applied with a reversed-phase UHPLC separation. The HILIC method resulted in the separation of highly polar features with minimal variance in retention time throughout a metabolomics batch analysis (retention time variance was typically less than 0.25% within a batch of tissue sample analysis n=55; pooled QC and tissue extracts, Table 2 and graphically represented in Figure 2). UHPLC: Nexera X2 Reversed phase: Column C18 (2.1x100 mm, 1.7 µm); column maintained at 50 ˚C Flow Rate 0.4 mL/min Mobile Phase A: Water + 0.1% formic acid B: Acetonitrile + 0.1% formic acid Rinse (R0/R1/R2) IPA/MeOH/MeCN/H 2 O(1:1:1:1) + 1% formic acid Gradient 2% B (0-1min); 40% B (2 min); 90% B (25 min, curvi-linear gradient); 100% B (27.5-31 min); 2% B (31.01-35 min) Injection 0.5 µL; autosampler temperature 4 ˚C HILIC: Column Shim-pack Velox HILIC (2.1x100 mm 2.7 µm, PN: 227-32025-03); column maintained at 40 ˚C Flow Rate 0.3 mL/min (0-13 min); 0.4 mL/min (13-17 min); 0.3 mL/min (17-18 min) Mobile Phase A: Water + 10 mM ammonium formate + 0.1% formic acid B: Acetonitrile:water [92:8] + 10 mM ammonium formate + 0.1% formic acid Rinse (R0/R1/R2) IPA/MeOH/MeCN/H 2 O + 1% formic acid Gradient 98% B (0-1.5 min); 65% B (11 min); 10% B (12.5-14 min); 98% B (14.25-18 min) Injection 0.5 µL; autosampler temperature 4 ˚C Mass Spectrometer: LCMS-9030 Interface 300 ˚C, 1.5 kV (ESI+) / -3.0 kV (ESI-) Heat block, DL 400 ˚C, 250 ˚C Gas flow rates Nebulizing gas 3 L/min; Heating gas 10 L/min; Drying gas 15 L/min CID gas pressure 230 kPa MS TOF-MS (m/z 60-1000) MS/MS 27 DIA-MS/MS (m/z 40-1000; precursor isolation 35 Da; collision energy spread 5-55 V Total cycle time (MS and MS/MS) 0.991 seconds Table 1. Analytical conditions Materials and Methods Multiple tissue types from different metabolomics studies were analysed using a standardised reversed-phase LC/QTOF method and a HILIC LC/QTOF method. Tissue extract samples were reconstituted in 90% acetonitrile. Tissues included brain, liver, pancreas, gut, caecal tissue and faecal gut content from several murine metabolomics studies. The HILIC method was developed to selectively analyse highly polar metabolites. Samples were analysed using the conditions detailed in Table 1. Data were processed using LabSolutions Insight software to consider a panel of 225 metabolite targets including lipid distributions (LPCs, LPEs, MGs, fatty acids, fatty acid conjugates and fatty amides) and polar metabolites (nucleotides, amino acids, purines, pyrimidines). Data were compared against an in- house metabolomics library (acquired using authentic reference material, research use only) for high reporting confidence. Library MS/MS spectra were generated using targeted MS/MS with unit mass resolution and a collision energy spread of 5-55 V. Product ion spectra were corrected to theoretical accurate mass values using the Insight Assign application. Spectra were acquired in positive and negative ion modes, first using the reversed-phase method, then retention time mapped to a Shim-pack Velox HILIC separation. Metabolite Rt variance %RSD HILIC Rt and m/z (positive ion) Adenine Creatinine Tryptophan Phenylalanine Trimethylglycine Arginine 0.81% 1.18% 0.58% 0.52% 0.20% 0.12% 1.875 mins; m/z 136.0618 3.206 mins; m/z 114.0662 5.259 mins; m/z 205.0972 5.460 mins; m/z 166.0863 7.766 mins; m/z 118.0863 9.401 mins; m/z 175.1190 Table 2. Variance of retention time for metabolites detected in tissue matrix extracts using HILIC. Data include extracts from brain, caecal, gut, liver, pancreas, faecal gut content and plasma. The RT stability with this method is high despite the wide variance in biological matrix. RT (min) 0 2 4 6 8 10 12 14 RT (min) 0 2 4 6 8 10 12 14 Adenine Arginine Creatinine Tryptophan Phenylalanine Trimethylglycine HILIC separation Reversed-phase separation Fig.2 Chromatographic separation of adenine, creatinine, tryptophan, phenylalanine, trimethylglycine and arginine in pancreas tissue extract by HILIC and reversed-phase. Peaks are displayed to a ±5ppm mass tolerance, and the same colours are used for each metabolite in HILIC or reversed-phase.
Application News RT (min) 0 2 4 6 8 10 12 14 m/z 146.1176; [C7H15NO2+H]+ Acetylcholine; Rt 6.64 mins 4-Trimethylammoniobutanoate; Rt 9.20 mins [4-Trimethylammoniobutanoate is also known as 3-deoxycarnitine. Involved in the lysine degradation pathway] m/z 132.1019; [C6H13NO2+H]+ Leucine; Rt 5.71 mins Isoleucine; Rt 5.88 mins [Valine, leucine, and isoleucine biosynthesis pathway] m/z 118.0863; [C5H11NO2+H]+ Valine; Rt 6.42 mins 5-aminopentanoic acid; RT 6.99 mins Norvaline; RT 7.28 mins Trimethlyglycine; Rt 7.79 mins [Trimethlyglycine is also known as betaine part of the methionine cycle] m/z 90.0550 [C3H7NO2+H]+ L-alanine; Rt 7.07 mins Beta-alanine; Rt 7.73 mins [Alanine has a key role in protein biosynthesis and is a marker for a number of disease states] Acetylcholine 43.0178 87.0441 146.1176 4-Trimethylammoniobutanoate 45.0335 60.0808 87.0441 146.1176 44.0495 86.0964 132.1019 56.0495 69.0699 86.0964 132.1019 Leucine Iso-Leucine 53.0386 55.0542 72.0808 118.0863 59.0729 58.0651 118.0863 Valine Trimethylglycine 0 100 200 m/z 44.04948 90.05463 0 100 200 m/z 44.9971 72.0444 90.0550 L-alanine Beta-alanine Fig. 3 HILIC was applied to the separation of several isobaric metabolite features which elute between 0.7-0.85 mins on a reversed-phase UHPLC method. Using HILIC, the isobaric metabolite features could be selectively separated despite the complexity of the faecal gut content matrix. The sample was part of a gut microbiome disruption model following treatment with the antibiotic metronidazole. HILIC is particularly powerful for the separation of isobaric compounds that elute in the first minute of a reversed-phase gradient developed for a high metabolite and lipid coverage. Figure 3 highlights the application of HILIC to the separation of several isobaric metabolite features which elute between 0.7- 0.85 mins on a reversed-phase UHPLC method in faecal gut content. Using HILIC the isobaric metabolite features could be selectivity separated despite the complexity of the faecal gut content matrix. The sample was part of a gut microbiome disruption model following treatment with the antibiotic metronidazole. Applying HILIC can also reduce the false discovery rate in highly polar metabolite analysis. Positive identification of highly polar metabolites such as nucleotides, amino acids, purines, pyrimidines is highly dependent on the selectivity of the precursor ion and the chimeric nature of the MS/MS spectra. As examples of reducing the false discovery rate, ion signals at m/z 146.1176 successfully separated acetylcholine and 4- trimethylammoniobutanoate, and at m/z 118.0863 resolved valine, 5-aminopentanoic acid, norvaline and trimethylglycine. The isomers leucine/isoleucine and alanine/beta-alanine were also resolved. HILIC separation was used to confirm metabolite identification with authentic reference materials (with the exception of 5-aminopentanoic acid and norvaline). These examples are highlighted in Fig. 3. Figure 4 shows the separation of valine, 5-aminopentanoic acid, norvaline, and trimethylglycine (also known as betaine) at m/z 118.0863 in different biological tissues highlighting the need to apply HILIC for increased reporting confidence for highly polar features.
Application News www.shimadzu.com/an/ Shimadzu Corporation Copyright © 2026 Shimadzu Corporation and/or its affiliates. All rights reserved. The analysis method described is intended solely to illustrate the potential application opportunities. For Research Use Only. Not for use in diagnostic procedures. This publication may contain references to products that are not available in your country. Please contact us to check the availability of these products in your country. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. See http://www.shimadzu.com/about/trademarks/index.html for details. Third party trademarks and trade names may be used in this publication to refer to either the entities or their products/services, whether or not they are used with trademark symbol “TM” or “”. Shimadzu disclaims any proprietary interest in trademarks and trade names other than its own. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. SHIMADZU Europa GmbH, www.shimadzu.eu First Edition: Jul. 2026 05-SCA-210-088-EN Conclusion To increase the metabolome coverage in untargeted tissue metabolic profiling studies and to ensure high reporting confidence requires more than one analytical approach. Reversed-phase based DIA-MS/MS methods are powerful, robust tools for highly positive metabolite identification with a high metabolite coverage. However, identifying highly polar metabolites with high reporting confidence is challenging given near zero chromatography resolution and isobaric mass spectra. Applying HILIC methods to highly polar metabolite analysis resulted in increased confidence in metabolite identification and enabled resolution of isobaric features. The metabolite identification was further verified using authentic standards analysed with the HILIC method. HILIC Method Selective separation of metabolite features at m/z 118.0853 (positive ion mode) Applied to faecal, caecal, pancreas and brain tissue extracts. Valine; Rt 6.4 mins 5-Aminopentanoic acid; Rt 6.99 mins Norvaline; Rt 7.28 mins Trimethylglycine; Rt 7.81 mins Norvaline Rt 7.28 mins RT (min) 0 1 2 3 Reversed-Phase Method Feature co-elution Co-elution of ion signal at m/z 118.0853 (positive ion mode) in a faecal extract. RT (min) 2 3 4 5 6 7 8 9 Faecal extract Multiple metabolite features detected. Identification confirmed by authentic reference standards or correspondence with third party MS/MS data repositories. Pancreas tissue extract Valine and trimethlyglycine detected. (Authentic standards used). Brain tissue extract Valine and trimethlyglycine detected. (Authentic standards used). Caecal tissue extract Multiple metabolites detected which are common to the faecal sample. Trimethylglycine Rt 7.81 mins Valine Rt 6.4 mins 5-Aminopentanoic acid Rt 6.99 mins Fig. 4 Identification of components eluting at 0.67 mins at m/z 118.0853 (±5 ppm) by reversed-phase UHPLC was compromised by co-elution. HILIC separation was used to confirm metabolite identification of valine and trimethylglycine with authentic reference materials. In the case of faecal and caecal samples valine, 5- aminopentanoic acid, norvaline, and trimethylglycine (also known as betaine) were detected. In other tissues such as pancreas and brain, only valine and trimethylglycine were detected. Nexera and LabSolutions Insight are trademarks of Shimadzu Corporation or its affiliated companies in Japan and/or other countries.
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