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class="content"><article class="post-5732 post type-post status-publish format-standard has-post-thumbnail category-biopharmaceutical category-ic-ms tag-biopharmaceutical tag-ic-ms entry" aria-label="The Missing Link in Your Metabolomics Analysis"><header class="entry-header"><h1 class="entry-title">The Missing Link in Your Metabolomics Analysis</h1> <span class="post-info-author"> By <a href="https://www.thermofisher.com/blog/analyteguru/tf-author/analyteguru-staff/" title="Posts by AnalyteGuru Staff" rel="author">AnalyteGuru Staff, </a> </span> <span class="post-info-date"> 05.02.2021 </span> </header><div class="entry-content"><p class="lia-message-body-content">The normal tendency for any analyst is to stay in their comfort zone with HPLC techniques. Sometimes this works, but often not! I see reverse-phase columns forced into application areas which they are not designed for or suited.</p> <p><img decoding="async" class="alignright wp-image-5734 " src="https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2023/06/3784-og-300x200.jpg" alt="" width="374" height="249" srcset="https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2023/06/3784-og-300x200.jpg 300w, https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2023/06/3784-og-768x512.jpg 768w, https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2023/06/3784-og-96x65.jpg 96w, https://www.thermofisher.com/blog/analyteguru/wp-content/uploads/sites/25/2023/06/3784-og.jpg 999w" sizes="(max-width: 374px) 100vw, 374px" />Often hydrophilic interaction liquid chromatography (HILIC), or mixed-mode columns are the next reluctant steps taken when dealing with hydrophobic, polar, ionizable or charged ionic compounds. There is not usually the third step! However, there are times when HILIC chromatography simply does not have the required resolution, selectivity, or effectively runs the analysis with the desired peak shape. A good example is <a href="https://www.thermofisher.com/uk/en/home/industrial/mass-spectrometry/mass-spectrometry-learning-center/mass-spectrometry-applications-area/metabolomics-mass-spectrometry/practical-guide-metabolomics/ic-ms-metabolomics-analysis.html" target="_blank" rel="nofollow noopener">metabolomic analysis</a> of the compounds in central metabolism and the energy cycles. The compounds in the central metabolomic pathways are all negatively charged with either single or multiple carboxylic acid functionalities or phosphorylated. Also, several of these compounds are isobaric isomers such as glucose 1 and 6 phosphate. These cannot be distinguished with MS only (even by HRAM-MS) and require good chromatographic separation. Such separations are not easy to accomplish with HILIC and more difficult with HPLC. Usually, the multiply phosphorylated nucleotide’s, such as adenosine triphosphate (ATP), peak shape is very poor. On the other hand, Ion exchange chromatography (IC) produces great separations for all these compounds with great peak shapes for quantitation.</p> <p>I hear “IC is not compatible with mass spectrometry (MS)” often. Well, let me tell you about a growing trend. <a href="https://assets.thermofisher.com/TFS-Assets/CMD/Reference-Materials/wp-73175-ic-ms-wp73175-en.pdf" target="_blank" rel="nofollow noopener">IC-MS</a> is becoming increasingly popular as people try it and realize this is very easy to do, and the results are <a href="https://www.nature.com/articles/s42003-020-0957-6" target="_blank" rel="nofollow noopener noreferrer">simply astounding</a>. There is no need to make up eluent. The system only pumps pure water into an <a href="https://www.thermofisher.com/uk/en/home/industrial/chromatography/chromatography-learning-center/ion-chromatography-information/ion-chromatography-innovations/reagent-free-ion-chromatography-eluent-generation-rfic-eg.html#more-info" target="_blank" rel="nofollow noopener">eluent generator (EG)</a>, which electrolytically produces precise concentrations of the potassium hydroxide (KOH) pushing-ion from 0 to 200mM at the head of the ion exchange column. Interestingly, as the KOH gradient concentration increases, there is also a pH gradient produced. It goes right through the pK<sub>a</sub> range of the individual hydroxyl (OH) groups on these analytes, around pH 12. As the OH groups dissociate and form a negative charge, it will give tighter binding to the anion exchange column, allowing the separation of almost identical sugar molecules with the smallest differences in the pK<sub>a</sub> values of the individual OH groups. I have not seen this level of selectivity with any other chromatography technique for these compounds.</p> <p>The IC system then uses a continuously electrolytically regenerated charged membrane <a href="https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FCMD%2FSpecification-Sheets%2FPS-70690-IC-Eluent-Suppressors-PS70690-EN.pdf&title=U3BlY2lmaWNhdGlvbiBTaGVldDogVGhlcm1vIFNjaWVudGlmaWMgRGlvbmV4IEVsdWVudCBTdXBwcmVzc29ycyBmb3IgSW9uIENocm9tYXRvZ3JhcGh5" target="_blank" rel="nofollow noopener">suppressor</a> to convert the KOH back to H20 before entering the MS. The system is easy to use and extremely precise. The peak shape and the separation are the best I have ever seen for charged metabolites. However, it is outside the comfort zone of the standard HPLC user. Or is it? The ion chromatography system is an <a href="https://www.thermofisher.com/us/en/home/industrial/chromatography/liquid-chromatography-lc/hplc-uhplc-components/hplc-uhplc-pumps.html" target="_blank" rel="noopener">HPLC pump</a> that pumps the sample from an HPLC autosampler through a column and then a detector. Sounds familiar? It is like standard HPLC to me but with an anion exchange column instead of a C18 column.</p> <p>Wait, it is easier than standard HPLC as you do not have to make up an eluent because the system does this for you. The MS system also sees the purest background eluent it has ever seen, as the starting eluent was just pure water. It has gone through another ion exchange purification system, the suppressor, before it reaches the MS with a continuously monitored background conductivity of below 1µs. The sensitive conductivity detection can be used as a preference for some analytes for quantification instead of MS signal.</p> <p>Make your life easier give it a try for these difficult and challenging analytes.</p> <h3 id="toc-hId--1902364897">Learn more:</h3> <p><a href="https://www.thermofisher.com/uk/en/home/industrial/mass-spectrometry/mass-spectrometry-learning-center/mass-spectrometry-applications-area/metabolomics-mass-spectrometry/practical-guide-metabolomics/ic-ms-metabolomics-analysis.html?playlistVideoId=5270571999001" target="_blank" rel="nofollow noopener">IC-MS elucidates highly polar compounds</a></p> <p><a href="https://assets.thermofisher.com/TFS-Assets/CMD/Application-Notes/can-73265-targeted-metabolomics-orbitrap-can73265-en.pdf" target="_blank" rel="nofollow noopener">Targeted metabolomics method using IC Orbitrap with high resolution mass spectrometry</a></p> <p><a href="https://assets.thermofisher.com/TFS-Assets/CMD/Application-Notes/AN-622-IC-MS-Oral-Neck-Cancer-Cells-AN64317-EN.pdf" target="_blank" rel="nofollow noopener">Pathway-Targeted Metabolomic Analysis in Oral/Head and Neck Cancer Cells Using Ion Chromatography-Ma…</a></p> <p><a href="https://analyteguru.com/videos/metabolomic-analysis-using-ic-ms-with-prof-izumi-yoshihiro/" target="_blank" rel="nofollow noopener noreferrer">Metabolomic Analysis Using IC-MS with Prof. Izumi Yoshihiro</a></p> <h3 id="toc-hId--1873735746">Videos:</h3> <p><a href="https://analyteguru.com/videos/why-coupling-ion-chromatography-with-mass-spectrometry-depends-on-electrolytic-suppression/" target="_blank" rel="nofollow noopener noreferrer">Why Coupling Ion Chromatography with Mass Spectrometry Depends on Electrolytic Suppression</a></p> <p><a href="https://analyteguru.com/videos/how-to-interface-an-ion-chromatography-system-with-a-mass-spectrometer/" target="_blank" rel="nofollow noopener noreferrer">How to Interface an Ion Chromatography System with a Mass Spectrometer</a></p> <p><a href="https://analyteguru.com/webinars/non-targeted-and-targeted-analysis-of-anionic-metabolites-using-ion-chormatography-and-high-resolution-accurate-mass-spectrometry/" target="_blank" rel="nofollow noopener noreferrer">Non-targeted and Targeted Analysis of Anionic Metabolites Using Ion Chormatography and High Resoluti…</a></p> </div></article><section class="author-box"><h4 class="author-box-title">AnalyteGuru Staff</h4><div class="author-box-content" itemprop="description"></div></section><div class="adjacent-entry-pagination pagination"><div class="pagination-previous alignleft"><i class="fa fa-icon fa-chevron-left"></i> <a href="https://www.thermofisher.com/blog/analyteguru/start-making-pore-decisions/" rel="prev">Start Making Pore Decisions</a></div><div class="pagination-next alignright"><a href="https://www.thermofisher.com/blog/analyteguru/5-ways-to-become-a-nitrosamine-ninja/" rel="next">5 Ways to Become a Nitrosamine Ninja</a> <i class="fa fa-icon fa-chevron-right"></i></div></div></main></div></div><footer class="site-footer"><div class="wrap"><div class="creds"><p class="footer-links"><a href="https://corporate.thermofisher.com/en/footer/privacy-statement.html" target="_blank" rel="noopener noreferrer">Privacy Statement</a><a href="https://corporate.thermofisher.com/en/footer/terms-and-conditions.html" target="_blank" rel="noopener noreferrer">Terms & Conditions</a><a href="/blog/locations/">Locations</a><a href="/blog/sitemap/">Sitemap</a></p><p>© 2025 <a href="https://www.thermofisher.com">Thermo Fisher Scientific</a>. 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