Polar microcystins or arginine methyl ester can serve as sensitive reference materials for system suitability tests in untargeted metabolomics using reversed-phase LC-MS: A case study

Polar microcystins or arginine methyl ester can serve as sensitive reference materials for system suitability tests in untargeted metabolomics using reversed-phase LC-MS: A case study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Polar microcystins or arginine methyl ester can serve as sensitive reference materials for system suitability tests in untargeted metabolomics using reversed-phase LC-MS: A case study Tommy Melzer, Georg Pohnert, Nico Ueberschaar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7806340/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Mar, 2026 Read the published version in Metabolomics → Version 1 posted 9 You are reading this latest preprint version Abstract Introduction LC-MS system suitability test (SST) is crucial for reliable data acquisition especially in untargeted metabolomics. Objectives Identification of best reference materials (RMs) to improve best quality assurance (QA) and quality control (QC) practices. Methods Investigations were performed using a C 18 reversed-phase (RP) column LC-MS approach. Results Targeted cyanotoxin analysis revealed a performance loss of the used C 18 RP column although the SST confirmed a fit for purpose instrument which prompted to test several additional RMs. Conclusion QA procedures for LC-MS can be improved by incorporating polar microcystins or arginine methyl ester as RMs for SST. LC-MS Quality assurance System suitability test Reference materials Untargeted metabolomics Figures Figure 1 Figure 2 1 Introduction Liquid chromatography-mass spectrometry (LC-MS) using reversed-phase (RP) columns is the predominant technique in the ever-growing field of targeted analyses and untargeted metabolomics (Fisher et al. 2025 ; Kirwan et al. 2025 ). To obtain reliable experimental data and assure reproducibility, quality assurance (QA) and quality control (QC) are of paramount importance. While many guidelines exist for targeted mass spectrometric analysis, a uniform consensus for untargeted analysis is still lacking (Beger et al. 2019 ). The metabolomics quality assurance and quality control consortium (mQACC) encourage scientists to share their experience and recommendations to establish best QA/QC practices (Evans et al. 2020 ; Mosley et al. 2024 ). QC is defined as the activities that a laboratory does during or immediately after data analysis, while QA processes are performed independently of data acquisition including staff training, creating standard operating procedures (SOPs), performing system suitability tests (SSTs), audits and more (Dudzik et al. 2018 ; Evans et al. 2020 ). Part of the SST in LC-MS based methods is the regular analysis of reference material (RM) or test mixtures and the examination of correct retention times, peak shape, signal intensity, mass accuracy and system pressure to qualify the instrument as “fit for purpose”(Bearden et al. 2014 ; Broadhurst et al. 2018 ). One of the priority endeavours of the mQACC is to engage the community to identify key characteristics of sustainable and widely applicable RMs that the community can afford (Beger et al. 2019 ; Lippa et al. 2022 ). Although there are commercially available standard mixtures in complex matrices, preparing in-house mixtures from chemical reference standards represents a cost-effective and flexible alternative (Lippa et al. 2022 ; Phinney et al. 2013 ; Simon-Manso et al. 2013 ) which we like to present here. In this report, we describe a weekly performed SST procedure and the in-house mixture used to verify the LC-MS device as fit for purpose. Furthermore, we emphasize the use of arginine-derivatives as RMs for SSTs, which proved particularly sensitive in detecting column aging compared to other well-established reference materials. 2 Materials and methods LC-MS analysis was conducted on a Dionex UltiMate 3000 UHPLC system equipped with a Thermo Accucore® C 18 RP column (100×2.1 mm; particle size 2.6 µm) coupled to a QExactive plus orbitrap mass spectrometer. Details about instrumental setup, applied parameters and supplier of chemicals are provided in Supplementary Information. Weekly maintenance of the LC-MS instrument is routinely performed; the full maintenance protocol is summarized in Supplementary Information. As part of the maintenance, system suitability was verified by analysing an in-house standard mixture containing the reference standards p -fluoro- l -phenylalanine, p -fluoro-benzoic acid and decanoic acid-D 19 (10 µg/mL each). The system's suitability was confirmed by checking the proper retention time, peak shape, mass accuracy and signal intensity of the reference standards. Details of cyanotoxins analysis is described in Otto et al . (Otto et al. 2023 ). All solvents were used in LC-MS-grade, RMs were purchased from commercial suppliers and analysed using the same LC-MS conditions as applied for the in-house mixture on both, the deteriorated column and a new C 18 RP column ( Table S1 ). (NOTE: in part we used enantio-pure chemicals and write their absolute configuration in this manuscript even if the methods are not capable to separate enantiomers.) 3 Results and discussion The present laboratory is equipped with a high-resolution mass spectrometer (HRMS) for the purpose of conducting targeted and untargeted analyses in the domain of metabolomics, in addition to associated research projects. During setup of our instrument, we developed and implemented an in-house mixture for SSTs that meet the following requirements. The standards must be easily available and stable in solution preferably over years without reacting with each other. The compounds must be ionisable in both polarities and cover a broad retention time range eluting from polar to unpolar conditions on a standard C 18 RP column. Furthermore, the compounds should be suitable for the use as internal standards. Also, these are said to be non-natural substances that are chemically similar to natural substances. A mixture of p -fluoro- l -phenylalanine, p -fluoro-benzoic acid and decanoic acid-D 19 meets all our prerequisites. The results by analysing the in-house standard mixture over a 2-year period on the C 18 RP column are depicted (Fig. 1 ). Two prominent retention time shifts occurred during this period: one due to a leaking injection valve from the autosampler (marked with * in Fig. 1 a), and the other due to the use of a different batch of formic acid to prepare the aqueous eluent (indicated with ** in Fig. 1 a). Both discrepancies were noticed during the weekly maintenance and could be corrected. Besides the utilization in untargeted metabolomics experiments, the C 18 RP column was used in another project for targeted cyanotoxin analysis. In this study, eight microcystins (MCs) and nodularin-R were quantified from aqueous samples (Melzer et al. 2025 ). MCs are cyclic peptides consisting of 7 amino acids (AA) and often abbreviated as MC-[AA2][AA4], where AA2 and AA4 denote the amino acids in the one-letter code in position 2 and 4 that exhibit the largest variability (Bouaïcha et al. 2019 ; Ortiz et al. 2017 ). Regarding their polarity, MCs differ mostly by the presence of the polar amino acid arginine, allowing to categorize them based on this characteristic. By measuring the MCs on the C 18 RP column, we recognised, that the arginine containing MCs are subjected to a huge retention time shift of more than two minutes compared to previous measurements. This retention time shift co-occurred with a very strong tailing and peak broadening, while the non-arginine containing MCs showed only very slightly decreased retention times compared to previous measurements (< 0.2 min Fig. 2 a and b ). This behaviour was observed in several repeated measurements and was still present after the exchange of the guard column. After using a new C 18 RP column (including new guard column) all peak shapes and retention times were restored. This observation indicated a performance loss of the “used” C 18 RP column probably due to a deteriorated column material. To recognize this change in properties is particularly critical, since the reference standards from our in-house mixture injected into the identical column showed no retention time shifts or changes in the peak area/ signal intensities (*** dashed frame in Fig. 1 a and b ). This led to the fact that the LC-MS device was approved as “fit for purpose” during the weekly maintenance despite this shortcoming. This observation prompted further investigation to determine how those critical column changes can be tracked in subsequent SSTs. A recent review by Lippa et al. summarized a variety of in-house standard mixtures employed by different research groups for SST of LC-MS in untargeted metabolomics (Lippa et al. 2022 ). The reported RMs included amino acids, organic acids, sugars and nucleosides. To evaluate whether our used reference compounds were sufficient for reliable SSTs, we tested the commonly applied RMs on both, the deteriorated and an identical, new C 18 RP column. All tested compounds exhibited slightly increased retention times on the deteriorated column (0.02–0.21 min), corresponding to deviations of up to 15% ( Table S1 ) . However, none of the analysed RMs showed retention time shift comparable to those of the polar, arginine containing MCs. Interestingly, the amino acid arginine and structurally related reference standards such as creatine or creatinine, which contain a guanidine unit, displayed only minor shifts ( 1 min) co-occurring with a peak broadening similar to that observed for MC-RR ( Fig. S1 ). 4 Conclusion A fundamental aspect of QA in LC-MS based methods is the regular execution of SSTs by analysing RMs to confirm the adequate performance of the analytical instrument. In this case study, we report a pronounced decline in the performance of a C 18 RP column during LC-MS analysis, which remained undetected by the routinely performed weekly SST but revealed by targeted cyanotoxin analysis. Commonly used RMs for SST in untargeted metabolomics were investigated for their capability to indicate the column performance loss and the results indicated that they proved ineffective in detecting such deterioration. To address this gap, we propose the integration of arginine-containing microcystins or l -arginine methyl ester as stable, cost-effective RMs that are particularly sensitive to column performance decline. Their inclusion in SST mixtures could substantially improve QA procedures for validating LC-MS-based untargeted metabolomics using RP columns. Declarations Compliance with ethical standards Not applicable Conflict of interest: The authors declare that they have no conflict of interest. Author Contribution TM and NU conceptualized the content of the manuscript. NU and GP supervised and acquired the funding. TM and NU performed LC-measurements including data analysis and figure drafts for this manuscript. TM and NU prepared the draft of this manuscript, TM and NU finalized the manuscript. All authors reviewed, edited and approved the final version of the manuscript. Acknowledgement This work was funded as a Cluster 4 Future initiative by the German Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung, BMBF, Förderkennzeichen 03ZU1214BB). The project is part of the Thuringian Water Innovation Cluster (ThWIC). This work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy “Balance of the Microverse” - EXC 2051 - Project-ID 390713860 and the DFG CRC 1127 Project number 239748522 “ChemBioSys” for funding of the Q-Exactive plus mass spectrometer. Data Availability Data is provided within the manuscript or supplementary information files. References Bearden, D. W., Beger, R. D., Broadhurst, D., Dunn, W., Edison, A., Guillou, C., Trengove, R., Viant, M., & Wilson, I. (2014). The New Data Quality Task Group (DQTG): ensuring high quality data today and in the future. Metabolomics , 10 , 539–540. 10.1007/s11306-014-0679-1 Beger, R. D., Dunn, W. B., Bandukwala, A., Bethan, B., Broadhurst, D., Clish, C. B., Dasari, S., Derr, L., Evans, A., Fischer, S., Flynn, T., Hartung, T., Herrington, D., Higashi, R., Hsu, P. C., Jones, C., Kachman, M., Karuso, H., Kruppa, G., et al. (2019). Towards quality assurance and quality control in untargeted metabolomics studies. Metabolomics , 15 , 4–8. 10.1007/s11306-018-1460-7 Bouaïcha, N., Miles, C. O., Beach, D. G., Labidi, Z., Djabri, A., Benayache, N. Y., & Nguyen-Quang, T. (2019). Structural diversity, characterization and toxicology of microcystins. Toxins , 11 , 714–753. 10.3390/toxins11120714 Broadhurst, D., Goodacre, R., Reinke, S. N., Kuligowski, J., Wilson, I. D., Lewis, M. R., & Dunn, W. B. (2018). 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A., & Ueberschaar, N. (2023). Tracking a broad inventory of cyanotoxins and related secondary metabolites using UHPLC-HRMS. Journal of Hazardous Materials Advances , 12 , 100370–100382. 10.1016/j.hazadv.2023.100370 Phinney, K. W., Ballihaut, G., Bedner, M., Benford, B. S., Camara, J. E., Christopher, S. J., Davis, W. C., Dodder, N. G., Eppe, G., Lang, B. E., Long, S. E., Lowenthal, M. S., McGaw, E. A., Murphy, K. E., Nelson, B. C., Prendergast, J. L., Reiner, J. L., Rimmer, C. A., Sander, L. C., et al. (2013). Development of a Standard Reference Material for metabolomics research. Analytical Chemistry , 85 , 11732–11738. 10.1021/ac402689t Simon-Manso, Y., Lowenthal, M. S., Kilpatrick, L. E., Sampson, M. L., Telu, K. H., Rudnick, P. A., Mallard, W. G., Bearden, D. W., Schock, T. B., Tchekhovskoi, D. V., Blonder, N., Yan, X., Liang, Y., Zheng, Y., Wallace, W. E., Neta, P., Phinney, K. W., Remaley, A. T., & Stein, S. E. (2013). Metabolite profiling of a NIST Standard Reference Material for human plasma (SRM 1950): GC-MS, LC-MS, NMR, and clinical laboratory analyses, libraries, and web-based resources. Analytical Chemistry , 85 , 11725–11731. 10.1021/ac402503m Additional Declarations No competing interests reported. Supplementary Files 20251008QCSI.docx Cite Share Download PDF Status: Published Journal Publication published 07 Mar, 2026 Read the published version in Metabolomics → Version 1 posted Editorial decision: Revision requested 07 Feb, 2026 Reviews received at journal 06 Feb, 2026 Reviews received at journal 03 Feb, 2026 Reviewers agreed at journal 18 Jan, 2026 Reviewers agreed at journal 16 Jan, 2026 Reviewers invited by journal 09 Oct, 2025 Editor assigned by journal 09 Oct, 2025 Submission checks completed at journal 09 Oct, 2025 First submitted to journal 08 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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11.07.2024. The intensity of the three standards is normalized to 100. The eluent gradient is depicted in the background in yellow, using eluent A: (water +2 % acetonitrile +0.1% formic acid) and B: 100 % acetonitrile. Box plots above each peak illustrate the variances of the retention time. Additionally, anomalies due to *Leaking injection valve of autosampler, ** formic acid added from bottle not from an ampoule, ***time frame of column performance loss without significant Rt-shifts of the three analytes) are indicated. \u003cstrong\u003eb\u003c/strong\u003e: Absolute values of the peak intensities (lighter colour) and peak area (darker colour) of the analytes over a 2-years period; errors are given with p=95%. \u003cstrong\u003eb1\u003c/strong\u003e: \u003cem\u003ep\u003c/em\u003e-fluoro-l-phenylalanine (positive ionisation [M+H]\u003csup\u003e+\u003c/sup\u003e), \u003cstrong\u003eb2\u003c/strong\u003e: \u003cem\u003ep\u003c/em\u003e-fluoro-l-phenylalanine (negative ionisation [M−H]\u003csup\u003e−\u003c/sup\u003e), \u003cstrong\u003eb3\u003c/strong\u003e: \u003cem\u003ep\u003c/em\u003e-fluoro benzoic acid (negative ionisation [M-H]\u003csup\u003e−\u003c/sup\u003e) and \u003cstrong\u003eb4\u003c/strong\u003e: decanoic acid-D\u003csub\u003e19\u003c/sub\u003e (negative ionisation [M−H]\u003csup\u003e−\u003c/sup\u003e)\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7806340/v1/65ccc4ed1d0165059d06b84e.png"},{"id":94140473,"identity":"a085bf2c-8e1b-4d29-af4b-08e57a8b6547","added_by":"auto","created_at":"2025-10-22 19:41:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":491895,"visible":true,"origin":"","legend":"\u003cp\u003eIllustration of microcystin (MC) measurements: \u003cstrong\u003ea:\u003c/strong\u003e Chromatogram of six analysed microcystins (MC-RR,MC-LR,MC-YR,MC-LA,MC-LF and MC-LY, c = 70 µg/L) with habitual retention times and peak shapes;\u003cbr\u003e\n\u003cstrong\u003eb: \u003c/strong\u003eChromatogram measured under identical parameters but using the deteriorated column where MC-LR and MC-YR showed increased retention times of over two minutes co-occurring by a strong tailing, MC-RR elutes very broad over a period of 6-12 minutes, while MC-LA, MC-LF and MC-LY have slightly decreased retention times.\u003cbr\u003e\n\u003cstrong\u003ec: \u003c/strong\u003ePlotting the critical MC-RR (bad peak shape in \u003cstrong\u003eFig. 2b\u003c/strong\u003e) over a period of nine months after using a new C\u003csub\u003e18\u003c/sub\u003e RP column, analysed at c= 1 µg/L; box plot on left side represents the variations in signal intensity and area, on the right side represents the distribution of retention times within the analysed time frame.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7806340/v1/f47c01601ec4f8ce732718da.png"},{"id":104251446,"identity":"d52519f1-f43f-4fac-8a85-5b511c1d9bac","added_by":"auto","created_at":"2026-03-09 16:13:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1846299,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7806340/v1/5a25ccad-ae44-4d46-9104-e7cc47a4dd85.pdf"},{"id":94139370,"identity":"63c13882-0c64-4d61-adba-257b46bf03e4","added_by":"auto","created_at":"2025-10-22 19:33:11","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":709995,"visible":true,"origin":"","legend":"","description":"","filename":"20251008QCSI.docx","url":"https://assets-eu.researchsquare.com/files/rs-7806340/v1/923d624806322b480cdc3311.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Polar microcystins or arginine methyl ester can serve as sensitive reference materials for system suitability tests in untargeted metabolomics using reversed-phase LC-MS: A case study","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eLiquid chromatography-mass spectrometry (LC-MS) using reversed-phase (RP) columns is the predominant technique in the ever-growing field of targeted analyses and untargeted metabolomics (Fisher et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Kirwan et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). To obtain reliable experimental data and assure reproducibility, quality assurance (QA) and quality control (QC) are of paramount importance. While many guidelines exist for targeted mass spectrometric analysis, a uniform consensus for untargeted analysis is still lacking (Beger et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The metabolomics quality assurance and quality control consortium (mQACC) encourage scientists to share their experience and recommendations to establish best QA/QC practices (Evans et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mosley et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). QC is defined as the activities that a laboratory does during or immediately after data analysis, while QA processes are performed independently of data acquisition including staff training, creating standard operating procedures (SOPs), performing system suitability tests (SSTs), audits and more (Dudzik et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Evans et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Part of the SST in LC-MS based methods is the regular analysis of reference material (RM) or test mixtures and the examination of correct retention times, peak shape, signal intensity, mass accuracy and system pressure to qualify the instrument as \u0026ldquo;fit for purpose\u0026rdquo;(Bearden et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Broadhurst et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). One of the priority endeavours of the mQACC is to engage the community to identify key characteristics of sustainable and widely applicable RMs that the community can afford (Beger et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Lippa et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Although there are commercially available standard mixtures in complex matrices, preparing in-house mixtures from chemical reference standards represents a cost-effective and flexible alternative (Lippa et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Phinney et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Simon-Manso et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) which we like to present here.\u003c/p\u003e\u003cp\u003eIn this report, we describe a weekly performed SST procedure and the in-house mixture used to verify the LC-MS device as fit for purpose. Furthermore, we emphasize the use of arginine-derivatives as RMs for SSTs, which proved particularly sensitive in detecting column aging compared to other well-established reference materials.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cp\u003eLC-MS analysis was conducted on a Dionex UltiMate 3000 UHPLC system equipped with a Thermo Accucore\u0026reg; C\u003csub\u003e18\u003c/sub\u003e RP column (100\u0026times;2.1 mm; particle size 2.6 \u0026micro;m) coupled to a QExactive plus orbitrap mass spectrometer. Details about instrumental setup, applied parameters and supplier of chemicals are provided in Supplementary Information. Weekly maintenance of the LC-MS instrument is routinely performed; the full maintenance protocol is summarized in Supplementary Information. As part of the maintenance, system suitability was verified by analysing an in-house standard mixture containing the reference standards \u003cem\u003ep\u003c/em\u003e-fluoro-\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-phenylalanine, \u003cem\u003ep\u003c/em\u003e-fluoro-benzoic acid and decanoic acid-D\u003csub\u003e19\u003c/sub\u003e (10 \u0026micro;g/mL each). The system's suitability was confirmed by checking the proper retention time, peak shape, mass accuracy and signal intensity of the reference standards. Details of cyanotoxins analysis is described in Otto \u003cem\u003eet al\u003c/em\u003e. (Otto et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAll solvents were used in LC-MS-grade, RMs were purchased from commercial suppliers and analysed using the same LC-MS conditions as applied for the in-house mixture on both, the deteriorated column and a new C\u003csub\u003e18\u003c/sub\u003e RP column (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). (NOTE: in part we used enantio-pure chemicals and write their absolute configuration in this manuscript even if the methods are not capable to separate enantiomers.)\u003c/p\u003e"},{"header":"3 Results and discussion","content":"\u003cp\u003eThe present laboratory is equipped with a high-resolution mass spectrometer (HRMS) for the purpose of conducting targeted and untargeted analyses in the domain of metabolomics, in addition to associated research projects. During setup of our instrument, we developed and implemented an in-house mixture for SSTs that meet the following requirements. The standards must be easily available and stable in solution preferably over years without reacting with each other. The compounds must be ionisable in both polarities and cover a broad retention time range eluting from polar to unpolar conditions on a standard C\u003csub\u003e18\u003c/sub\u003e RP column. Furthermore, the compounds should be suitable for the use as internal standards. Also, these are said to be non-natural substances that are chemically similar to natural substances. A mixture of \u003cem\u003ep\u003c/em\u003e-fluoro-\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-phenylalanine, \u003cem\u003ep\u003c/em\u003e-fluoro-benzoic acid and decanoic acid-D\u003csub\u003e19\u003c/sub\u003e meets all our prerequisites.\u003c/p\u003e\u003cp\u003eThe results by analysing the in-house standard mixture over a 2-year period on the C\u003csub\u003e18\u003c/sub\u003e RP column are depicted (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Two prominent retention time shifts occurred during this period: one due to a leaking injection valve from the autosampler (marked with * in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), and the other due to the use of a different batch of formic acid to prepare the aqueous eluent (indicated with ** in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Both discrepancies were noticed during the weekly maintenance and could be corrected.\u003c/p\u003e\u003cp\u003eBesides the utilization in untargeted metabolomics experiments, the C\u003csub\u003e18\u003c/sub\u003e RP column was used in another project for targeted cyanotoxin analysis. In this study, eight microcystins (MCs) and nodularin-R were quantified from aqueous samples (Melzer et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). MCs are cyclic peptides consisting of 7 amino acids (AA) and often abbreviated as MC-[AA2][AA4], where AA2 and AA4 denote the amino acids in the one-letter code in position 2 and 4 that exhibit the largest variability (Boua\u0026iuml;cha et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ortiz et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Regarding their polarity, MCs differ mostly by the presence of the polar amino acid arginine, allowing to categorize them based on this characteristic. By measuring the MCs on the C\u003csub\u003e18\u003c/sub\u003e RP column, we recognised, that the arginine containing MCs are subjected to a huge retention time shift of more than two minutes compared to previous measurements. This retention time shift co-occurred with a very strong tailing and peak broadening, while the non-arginine containing MCs showed only very slightly decreased retention times compared to previous measurements (\u0026lt;\u0026thinsp;0.2 min Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and \u003cb\u003eb\u003c/b\u003e). This behaviour was observed in several repeated measurements and was still present after the exchange of the guard column.\u003c/p\u003e\u003cp\u003eAfter using a new C\u003csub\u003e18\u003c/sub\u003e RP column (including new guard column) all peak shapes and retention times were restored. This observation indicated a performance loss of the \u0026ldquo;used\u0026rdquo; C\u003csub\u003e18\u003c/sub\u003e RP column probably due to a deteriorated column material. To recognize this change in properties is particularly critical, since the reference standards from our in-house mixture injected into the identical column showed no retention time shifts or changes in the peak area/ signal intensities (*** dashed frame in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and \u003cb\u003eb\u003c/b\u003e). This led to the fact that the LC-MS device was approved as \u0026ldquo;fit for purpose\u0026rdquo; during the weekly maintenance despite this shortcoming. This observation prompted further investigation to determine how those critical column changes can be tracked in subsequent SSTs.\u003c/p\u003e\u003cp\u003eA recent review by Lippa \u003cem\u003eet al.\u003c/em\u003e summarized a variety of in-house standard mixtures employed by different research groups for SST of LC-MS in untargeted metabolomics (Lippa et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The reported RMs included amino acids, organic acids, sugars and nucleosides. To evaluate whether our used reference compounds were sufficient for reliable SSTs, we tested the commonly applied RMs on both, the deteriorated and an identical, new C\u003csub\u003e18\u003c/sub\u003e RP column. All tested compounds exhibited slightly increased retention times on the deteriorated column (0.02\u0026ndash;0.21 min), corresponding to deviations of up to 15% (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e)\u003c/b\u003e. However, none of the analysed RMs showed retention time shift comparable to those of the polar, arginine containing MCs. Interestingly, the amino acid arginine and structurally related reference standards such as creatine or creatinine, which contain a guanidine unit, displayed only minor shifts (\u0026lt;\u0026thinsp;0.05 min). In contrast, \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-arginine methyl ester as a compound that mimics the peptide bound arginine unit in MCs was analysed and exhibited a pronounced retention time shift (\u0026gt;\u0026thinsp;1 min) co-occurring with a peak broadening similar to that observed for MC-RR (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e"},{"header":"4 Conclusion","content":"\u003cp\u003eA fundamental aspect of QA in LC-MS based methods is the regular execution of SSTs by analysing RMs to confirm the adequate performance of the analytical instrument. In this case study, we report a pronounced decline in the performance of a C\u003csub\u003e18\u003c/sub\u003e RP column during LC-MS analysis, which remained undetected by the routinely performed weekly SST but revealed by targeted cyanotoxin analysis. Commonly used RMs for SST in untargeted metabolomics were investigated for their capability to indicate the column performance loss and the results indicated that they proved ineffective in detecting such deterioration. To address this gap, we propose the integration of arginine-containing microcystins or \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-arginine methyl ester as stable, cost-effective RMs that are particularly sensitive to column performance decline. Their inclusion in SST mixtures could substantially improve QA procedures for validating LC-MS-based untargeted metabolomics using RP columns.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompliance with ethical standards\u003c/h2\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch2\u003eConflict of interest:\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eTM and NU conceptualized the content of the manuscript. NU and GP supervised and acquired the funding. TM and NU performed LC-measurements including data analysis and figure drafts for this manuscript. TM and NU prepared the draft of this manuscript, TM and NU finalized the manuscript. All authors reviewed, edited and approved the final version of the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThis work was funded as a Cluster 4 Future initiative by the German Federal Ministry of Education and Research (Bundesministerium f\u0026uuml;r Bildung und Forschung, BMBF, F\u0026ouml;rderkennzeichen 03ZU1214BB). The project is part of the Thuringian Water Innovation Cluster (ThWIC). This work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany\u0026rsquo;s Excellence Strategy \u0026ldquo;Balance of the Microverse\u0026rdquo; - EXC 2051 - Project-ID 390713860 and the DFG CRC 1127 Project number 239748522 \u0026ldquo;ChemBioSys\u0026rdquo; for funding of the Q-Exactive plus mass spectrometer.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBearden, D. W., Beger, R. D., Broadhurst, D., Dunn, W., Edison, A., Guillou, C., Trengove, R., Viant, M., \u0026amp; Wilson, I. (2014). 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Metabolite profiling of a NIST Standard Reference Material for human plasma (SRM 1950): GC-MS, LC-MS, NMR, and clinical laboratory analyses, libraries, and web-based resources. \u003cem\u003eAnalytical Chemistry\u003c/em\u003e, \u003cem\u003e85\u003c/em\u003e, 11725\u0026ndash;11731. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/ac402503m\u003c/span\u003e\u003cspan address=\"10.1021/ac402503m\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"metabolomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mebo","sideBox":"Learn more about [Metabolomics](http://link.springer.com/journal/11306)","snPcode":"11306","submissionUrl":"https://submission.nature.com/new-submission/11306/3","title":"Metabolomics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"LC-MS, Quality assurance, System suitability test, Reference materials, Untargeted metabolomics","lastPublishedDoi":"10.21203/rs.3.rs-7806340/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7806340/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eIntroduction\u003c/h2\u003e\u003cp\u003eLC-MS system suitability test (SST) is crucial for reliable data acquisition especially in untargeted metabolomics.\u003c/p\u003e\u003ch2\u003eObjectives\u003c/h2\u003e\u003cp\u003eIdentification of best reference materials (RMs) to improve best quality assurance (QA) and quality control (QC) practices.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eInvestigations were performed using a C\u003csub\u003e18\u003c/sub\u003e reversed-phase (RP) column LC-MS approach.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eTargeted cyanotoxin analysis revealed a performance loss of the used C\u003csub\u003e18\u003c/sub\u003e RP column although the SST confirmed a fit for purpose instrument which prompted to test several additional RMs.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eQA procedures for LC-MS can be improved by incorporating polar microcystins or arginine methyl ester as RMs for SST.\u003c/p\u003e","manuscriptTitle":"Polar microcystins or arginine methyl ester can serve as sensitive reference materials for system suitability tests in untargeted metabolomics using reversed-phase LC-MS: A case study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-22 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