<h3>Too much interfering matrix? Discover how to streamline your daily LC-MS analysis with turbulent flow chromatography.</h3>Sample preparation is a critical step in liquid chromatography-mass spectrometry (LC-MS) analysis to achieve quantitative accuracy and precision. Directly injecting crude samples into your instrument without prior cleanup to remove interfering compounds can clog the flow path, damage your detector, and negatively impact your analyses through a “matrix effect.”<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Thermo_Fisher_Scientific_Automated_LC_MS_Sample_Cleanup_Using_Turbulent_Flow_Chromatography_c7647c2c50.jpg" alt="Thermo Fisher Scientific: Automated LC-MS Sample Cleanup Using Turbulent Flow Chromatography." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Thermo_Fisher_Scientific_Automated_LC_MS_Sample_Cleanup_Using_Turbulent_Flow_Chromatography_c7647c2c50.jpg 245w," sizes="100vw" width="1024" height="512">Common approaches to clean up samples and suppress the matrix effect include simple dilution and centrifugation to extensive extraction techniques. The common scope of all these procedures is to minimize the possible contamination of your LC-MS system but, more importantly, to reduce to a minimum the interference of unrelated sample components on the detection and quantification of your analytes of interest.A point to note is the preparation method you choose often depends on the complexity of your sample. For instance, samples for clinical research applications or toxicology tend to have high levels of interfering matrix and require a more sophisticated clean-up approach.&nbsp; Let’s dive into what a matrix effect is, the impacts on your LC-MS data, and an effective method called turbulent flow chromatography you can use to clean complex samples.1<h3 id="h-what-exactly-is-matrix-effect">What exactly is matrix effect?</h3>Any chemical entity in your sample capable of coeluting or interacting with analytes of interest can generate matrix effect, ultimately impacting the integrity of your data.Interfering matrix compounds come from endogenous and exogenous sources, but biological samples like blood or serum, and food and environmental extracts inherently have higher levels of interfering compounds.Some examples of interfering matrix compounds include ion-suppressing salts, proteins, lipids, compounds capable of ion-pairing interactions, and polymers.<h3 id="h-how-does-matrix-effect-impact-your-lc-ms-data">How does matrix effect impact your LC-MS data?</h3>Because matrix compounds interfere with the detection and quantitation of your target analytes, the effects ultimately lead to an under- or overestimation in analyte quantification.In the case of LC-MS, the matrix effect triggers ion suppression2, which happens when compounds from the sample matrix inhibit charge transfer from the ion source to your target analytes – a prime example is electrospray ionization, where the detection relies on efficient charge transfer to analytes.Analytes and matrix components will naturally compete for the available charge from the MS ion source. And when compounds of your sample matrix are present in higher concentrations than your analytes, these unwanted molecules can capture more of the charge, leaving fewer ions available for charge transfer to your analytes.Hence suppression of charge transfer to your analytes leads to poor accuracy and limits of detection and quantitation.<h3 id="h-sample-preparation-methods-to-remove-interfering-matrix">Sample preparation methods to remove interfering matrix</h3>Using the example of ion suppression, you can see how eliminating the competition for charge transfer between matrix components and analytes through proper sample cleaning is essential to developing a method with quantitative accuracy.Solid-Phase Extraction (SPE) and Liquid-Liquid Extraction (LLE) are techniques used to clean samples before LC-MS analysis. 3,4<ul><li>In SPE, a liquid sample is loaded onto a column packed with a solid adsorbent material. The analytes of interest are retained on the adsorbent material while unwanted matrix components are washed away. The retained analytes are then eluted using a solvent. This approach is typically developed on a specific panel of target analytes.</li><li>LLE is based on the differential solubility of analytes between two immiscible solvents; once a partition equilibrium is reached, the enriched solvent is separated and, usually, evaporated to dryness and reconstituted in a solvent compatible with LC-MS.</li></ul>Regardless of the <a target="_blank" rel="noopener noreferrer" href="https://www.thermofisher.com/content/lifetech/global/en/home/industrial/chromatography/chromatography-learning-center/chromatography-consumables/sample-prep.html">sample preparation</a> approach you decide to use, these manual procedures are imprecise, time-consuming, and labor-intensive—even a quick and easy cleanup is subject to human errors like improper pipetting or sample mix-up.<h3 id="h-automated-online-sample-cleanup-for-efficient-sample-preparations">Automated online sample cleanup for efficient sample preparations</h3>To overcome the bottlenecks and risks of manual approaches, automated sample cleanup is gaining popularity, thanks to much less human intervention combined with better speed and analytical reproducibility.Online SPE&nbsp;is an improvement over manual ones, where the extraction process integrates directly with your LC system. The sample is automatically injected onto an extraction cartridge, cleanup occurs, and your extracted sample transfers directly to the MS instrument (flow injection analysis) or to an analytical column for further separation and detection.Turbulent flow chromatography (TurboFlow) is a more advanced online sample cleanup approach combining the aspects of the chemical affinity of SPE with size exclusion chromatography (SEC).5<h3 id="h-how-does-turbulent-flow-chromatography-work">How does turbulent flow chromatography work?</h3>TurboFlow separates low molecular weight molecules from high molecular weight matrix components using a specialized sample extraction column on LC systems (Figure 2).Here’s a detailed look at how TurboFlow works:<ol><li>Your sample is directly injected into a <a target="_blank" rel="noopener noreferrer" href="https://www.thermofisher.com/order/catalog/product/CH-952434">TurboFlow column</a> (typically large &gt; 50 µm particle size with macropores).</li><li>The sample flows through the column at high linear velocities, creating turbulence to enhance the interaction between the sample components and the stationary phase.</li><li>Low molecular weight molecules diffuse into the pores faster than high molecular weight components and quickly flush to waste. Only the small molecules with an affinity for the particle chemistry bind to the internal pore surfaces.</li><li>A change in mobile phase composition elutes the small molecules bound by the TurboFlow column for further separation and detection.</li></ol><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Thermo_Fisher_Scientific_Automated_LC_MS_Sample_Cleanup_Using_Turbulent_Flow_Chromatography_3_a261253a52.jpeg" alt="Thermo Fisher Scientific: Figure 1. Illustration showing how turbulent flow chromatography works." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Thermo_Fisher_Scientific_Automated_LC_MS_Sample_Cleanup_Using_Turbulent_Flow_Chromatography_3_a261253a52.jpeg 245w," sizes="100vw" width="1024" height="512"><h3 id="h-benefits-of-turbulent-flow-chromatography-for-sample-preparation-over-spe">Benefits of turbulent flow chromatography for sample preparation over SPE</h3>While both TurboFlow and online SPE streamline and automate the sample preparation process, TurboFlow has key differences and advantages to consider (Figure 2).<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Thermo_Fisher_Scientific_Automated_LC_MS_Sample_Cleanup_Using_Turbulent_Flow_Chromatography_4_221b9dda9b.jpg" alt="Thermo Fisher Scientific: Figure 2. TurboFlow vs. SPE for sample cleanup." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Thermo_Fisher_Scientific_Automated_LC_MS_Sample_Cleanup_Using_Turbulent_Flow_Chromatography_4_221b9dda9b.jpg 245w," sizes="100vw" width="700" height="410"><ol><li>TurboFlow provides a cleaner sample compared to SPE<ul><li>The more efficient dual mode of action (chemical affinity plus SEC)</li><li>The elution performed by a strong solvent plug potentially reduces the transfer of residual matrix components to your MS</li></ul></li><li>A cleaner sample means<ul><li>Reduced ion suppression, leading to increased sensitivity</li><li>Less mass spectrometer contamination</li><li>Extended analytical column lifespan</li><li>Faster analytical gradients</li></ul></li></ol>Moreover, the opportunity to modulate the strength of the mobile phase reaching the analytical column allows for better analyte focusing on the head of the column, significantly improving your chromatography.<h3 id="h-streamline-your-daily-lc-ms-analysis-with-turboflow-technology">Streamline your daily LC-MS analysis with TurboFlow technology</h3><div style="grid-template-columns:auto 60%;"><div>Automated sample cleanup using <a target="_blank" rel="noreferrer noopener" aria-label="online TurboFlow technology - open in a new tab" data-uw-rm-ext-link="" uw-rm-external-link-id="https://assets.thermofisher.com/tfs-assets/cmd/brochures/br-74120-lc-ms-transcend-uhplc-systems-br74120-en.pdf$onlineturboflowtechnology"><a target="_blank" rel="noopener noreferrer" href="https://assets.thermofisher.com/TFS-Assets/CMD/brochures/br-74120-lc-ms-transcend-uhplc-systems-br74120-en.pdf">online TurboFlow technology</a></a> is a rapid and efficient sample preparation approach that considerably improves method accuracy, reliability, and sensitivity. Adopting this technology can even enhance the competitiveness of your laboratory by allowing faster results, lower costs per sample, and improved reproducibility and MS sensitivity.<figure class="image"><img style="aspect-ratio:700/410;" src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Thermo_Fisher_Scientific_Automated_LC_MS_Sample_Cleanup_Using_Turbulent_Flow_Chromatography_2_0a20492c6a.jpg" alt="Thermo Fisher Scientific: Figure 3. Transcend TLX TurboFlow UHPLC System." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Thermo_Fisher_Scientific_Automated_LC_MS_Sample_Cleanup_Using_Turbulent_Flow_Chromatography_2_0a20492c6a.jpg 245w," sizes="100vw" width="700" height="410"></figure><blockquote><a target="_blank" rel="noopener noreferrer" href="https://www.thermofisher.com/content/lifetech/global/en/home/industrial/chromatography/liquid-chromatography-lc/hplc-uhplc-systems/transcend-turboflow-tlx-systems.html">Learn how TurboFlow can ease your lab’s daily LC-MS analysis of complex matrices today</a>.</blockquote><article aria-label="Automated LC-MS Sample Cleanup Using Turbulent Flow Chromatography"><div><blockquote>Visit us on <a target="_blank" rel="noreferrer noopener" aria-label="LinkedIn: - open in a new tab" data-uw-rm-ext-link="" uw-rm-external-link-id="https://www.linkedin.com/showcase/72003132/admin/dashboard/$linkedin:"><a target="_blank" rel="noopener noreferrer" href="https://www.linkedin.com/showcase/72003132/admin/dashboard/">LinkedIn:</a></a> #TurbulentflowChromatography #lcms #IonSuppression</blockquote></div></article><section><blockquote><h4>Dan Kalu Appulage</h4>Dr. Dan Kalu Appulage is a Product Management Specialist in the HPCS business unit at Thermo Fisher Scientific. He holds a Ph.D. in Analytical Chemistry, an M.A., and B.S. in Chemistry. His professional background includes expertise in product management, LCMS method development, and teaching. Dan is passionate about education and loves sharing his knowledge and experiences with others. Outside of work, Dan enjoys playing volleyball and traveling with his family.</blockquote></section></div></div>

Automated LC-MS Sample Cleanup Using Turbulent Flow Chromatography

<h3>Part 4: Going beyond 4D to improve analytical performance for targeted quantitative analysis</h3>This is the fourth in a multi-part blog series highlighting the key attributes and impressive benefits of the Thermo Scientific Stellar mass spectrometer—the first-of-its-kind instrument designed and optimized for highly multiplexed targeted quantitation, ideally suited for discovery verification. By delivering robust analytical methods and dramatically improved turnaround time, this solution helps labs boost capacity and differentiate themselves to generate more revenue. For additional background, see blog <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/5077">Part 1</a>, <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/5086">Part 2 </a>and <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/5416">Part 3</a> here and stay tuned for more blogs on our website.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Thermo_Fisher_Scientific_Why_You_Need_a_Verification_Class_Mass_Spectrometer_Going_Beyond_4d_to_Improve_Analytical_Performance_for_Targeted_Quantitative_Analysis_125258804e.jpg" alt="Thermo Fisher Scientific: Why You Need a Verification-Class Mass Spectrometer: Going Beyond 4d to Improve Analytical Performance for Targeted Quantitative Analysis." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Thermo_Fisher_Scientific_Why_You_Need_a_Verification_Class_Mass_Spectrometer_Going_Beyond_4d_to_Improve_Analytical_Performance_for_Targeted_Quantitative_Analysis_125258804e.jpg 245w," sizes="100vw" width="1200" height="600">Analytical specificity is one of the most critical requirements for successful targeted quantitative analysis. Assigning measured data to diagnostic product ions from the analyte of interest increases the confidence in resulting measurement determining the analyte expression level, copy number, or absolute amount in each sample, as well as across an entire study. Using empirically determined values for liquid chromatography (LC) and mass spectrometry (MS) provides specific information that collectively increases data processing. This orthogonal information comes in the form of 1) chromatographic retention times and peak shapes; 2) precursor m/z value; 3) MS/MS product ions; and 4) MS/MS product ion distribution profiles, which can be stored to form spectral libraries that are used to not only process LC-MSn data but build confident targeted data acquisition methods.<h3>4D parameter determination: Sufficient or not?</h3>Previous publications touted utilization of four-dimensional (4D) parameter determination to confidently create and process untargeted and targeted data. Using LC coupled to a trapped ion mobility QTOF (timsTOF) mass spectrometer, each analyte is annotated by four empirical measurements across all study samples: 1) measured retention time; 2) ion mobility as measured by its collision cross section (CCS); 3) precursor m/z value; and 4) presence of corresponding product ions. The assumption is that the orthogonal selectivity achieved by UHPLC separation and ion mobility resolution relative to the precursor isolation is sufficient to differentiate a specific analyte from co-eluting isobaric and isomeric ions and that resulting product ions are only attributed to the isolated precursor. <a target="_blank" rel="noopener noreferrer" href="https://arxiv.org/pdf/2111.03134">Adding CCS values can help accommodate for some variability</a> but requires more work by the researcher to accurately determine the variance associated with ion mobility. <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/1885960_lcms_179_lipidomics_short_gradient_ebook_rev_01_ed630faf92/1885960-lcms-179-lipidomics-short-gradient-ebook-rev-01.pdf">At no point are the product ion ratios used as an additional dimension for verification</a>.A smart addition: product ion ratio determination Product ion ratio determination, however, has been used as the gold standard for qualitative data analysis for SRM/MRM acquisition for decades. Despite quantitative methods on triple quadrupole mass spectrometers acquiring data for only two or three product ion transitions, the inclusion of the resulting product ion ratio is used to qualitatively verify that the signal measured per SRM transition is attributed to the analyte of interest. This concept has been included in the targeted quantitative studies performed using the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1149">Thermo Scientific Stellar mass spectrometer (MS)</a>, significantly increasing analytical performance for a wide range of molecules.To increase the quantitative selectivity and specificity needed for confident data acquisition, the proposed solution combines the chromatographic separation capabilities of the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=40&amp;search=vanquish">Thermo Scientific Vanquish UHPLC system </a>with the innovative PRM acquisition on the Stellar mass spectrometer to go beyond data processing confidence.&nbsp; Additionally, the proposed method leverages intelligent data acquisition to perform RT alignment reducing the need to apply additional post-acquisition data processing steps.&nbsp; This blog covers the differentiation for each of these steps while presenting an important 5th dimension—extending PRM data analysis to the MS3 level.<h3>Dimension #1: Leveraging 1,500 bar UHPLC separation to drive orthogonal selectivity</h3>Using the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/398">Thermo Scientific Vanquish Neo</a> and <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/744">Horizon UHPLC</a> systems, researchers can harness higher backpressure capabilities covering a wide range of flow rates from nanoflow to analytical flow rates—increasing peak capacity while driving higher utilization rates than HPLC systems with only 1,000 bar of backpressure. T<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=40&amp;search=vanquish">he Vanquish UHPLC systems</a> also deliver reproducible performance over the course of longer studies such as populational health.Going beyond the first dimension: Including the Adaptive RT routine enables LC mapping to assign true retention time values to each analyte with narrower retention time windows. The Adaptive RT routine works for small and large molecules, from single-cell to bulk analysis, and bridges method development between longer discovery gradients and faster targeted quantitation gradients without the need for spiked RT standards.&nbsp; The advantage is that the retention time alignment for the study is reliably performed during data acquisition, not processing.<h3>Dimension #2: Introduction of targeted analytes</h3>Following UHPLC separation and ionization, targeted analytes (precursors) are introduced into the MS and moved through the ion flight path to the detector. The quadrupole mass filter (termed isolating quadrupole or Q1) has an RF/DC offset to transmit a narrow m/z range centered on the target precursor m/z value. The mass range used for precursor ion filtering is ca. 1 to 2 Th depending on the mass spectrometer. Leveraging precursor isolation following chromatographic separation increases the target analyte selectivity relative to co-eluting compounds.Going beyond the second dimension: The Stellar mass spectrometer’s QR5 Plus quadrupole mass filter uses hyperbolic-surfaced quadrupole rods that improve ion transmission efficiency across a wider precursor m/z range. Unit resolution settings are 0.7 Th at the baseline—significantly better than other quadrupole mass spectrometers using 1.2 to 2 Th (almost 2-3x wider), which can compromise quantitative performance.<h3>Dimension #3: Acquiring PRM data from more product ions</h3>The<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1149"> Stellar mass spectrometer </a>acquires PRM data which is comprised of full scan MS2 spectra per scan event relative to SRM/MRM acquisition. Acquiring data for many more product ions per unit time increases the specificity of the resulting measurement. It also enables post-acquisition data processing methods to determine specific product ions used for quantitation without having to update the experimental method and re-analyze samples.Going beyond the third dimension:&nbsp; The Stellar mass spectrometer combines the unique capabilities of dual ion packet management with ion accumulation outside of the ion trap to boost quantitative performance.&nbsp; Simultaneously managing two different ion packets enables longer accumulation times without significantly compromising the PRM acquisition rates which becomes imperative when performing quantitative measurements at the LOD and LOQ levels.<h3>Dimension #4: Leveraging the full scan MS2 spectrum and multiple product ions to evaluate the product ion ratios compared to a reference spectrum</h3>As mentioned above, ion ratio calculations are used in validated quantitative methods to ensure selectivity relative to the matrix or background interference per product ion.Going beyond the fourth dimension: What, where and how you perform ion accumulation differentiates the effectiveness of each mass spectrometer. The Stellar MS first filters for the targeted precursor m/z value, then accumulates product ions in an ion-routing multipole (as has been done on Thermo Scientific Orbitrap mass spectrometers for over a decade).&nbsp; Performing tandem MS in this order maximizes sensitivity for low-level product ions to maintain accurate ion ratio analysis. Finally, incorporating dynamic AGC automates the accumulation time regardless of precursor ion flux. (Reference: <a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1101/2024.06.04.597431">Plubell et al. BioRxiv</a>)<h3>Dimension #5: Addressing sensitivity disrupters</h3>Targeted quantitation for specific analytes using MS2 acquisition may have co-eluting isomeric/isobaric ions potentially disrupting quantitative sensitivity (signal-to-noise limitation).&nbsp; Two options to pursue would be modifying LC gradients or increasing the tandem MS level to MS3. Elongating the LC gradient decreases sample throughput yet still may not remove all background interferences. The Stellar MS, on the other hand, can implement MS3 acquisition with exceptionally fast acquisition speeds (up to 40 Hz) to maintain target capacity and the number of data points per LC peak.Going beyond the fifth dimension:&nbsp;In addition to the unique MS3 acquisition speed, the Stellar MS implements dynamic AGC to maximize spectral quality across a wider dynamic range, as well as the ability to perform either HCD or CID at each MSn level. Finally, introduction of Adaptive RT to confidently manage scheduled retention time windows ensures tMS3 methods can succeed for increased target capacity using faster LC gradients.<blockquote>For more information, explore the <a target="_blank" rel="noopener noreferrer" href="https://www.thermofisher.com/content/lifetech/global/en/home/industrial/mass-spectrometry/liquid-chromatography-mass-spectrometry-lc-ms/lc-ms-systems/orbitrap-lc-ms/stellar-mass-spectrometer.html">Stellar mass spectrometer</a> and <a target="_blank" rel="noopener noreferrer" href="https://assets.thermofisher.com/TFS-Assets/CMD/brochures/eb-003672-lsms-stellar-2024-year-in-review-eb003672-na-en.pdf">download our new 2024 year in review ebook</a>.Visit us on <a target="_blank" rel="noopener noreferrer" href="https://www.linkedin.com/showcase/72003132/admin/dashboard/">LinkedIn:</a> #StellarMassSpectrometer #Vanquish #UHPLC</blockquote><section><blockquote><h5>Dr. Scott Peterman</h5><div>Scott Peterman, Ph.D., is Senior Product Marketing Manager for Life Science Mass Spectrometry of the Chromatography and Mass Spectrometry Division of Thermo Fisher Scientific.</div></blockquote></section>

Why You Need a Verification-Class Mass Spectrometer: Going Beyond 4d to Improve Analytical Performance for Targeted Quantitative Analysis

LC-MS-based metabolomics for detecting adulteration in Tribulus terrestris-derived dietary supplements

Many elements, such as chromium, zinc or selenium are considered essential micronutrients for humans and are subject to ongoing research. Their concentration in foods can be accurately determined using inductively coupled plasma mass spectrometry (ICP-MS).<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Thermo_Fisher_Scientific_Deciphering_Selenium_Nutrition_Using_Speciation_Analysis_with_ICP_MS_3ecef88508.jpg" alt="Thermo Fisher Scientific: Deciphering Selenium Nutrition Using Speciation Analysis with ICP-MS" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Thermo_Fisher_Scientific_Deciphering_Selenium_Nutrition_Using_Speciation_Analysis_with_ICP_MS_3ecef88508.jpg 245w," sizes="100vw" width="1200" height="600"><article><div>However, beyond the actual levels of an element in a sample, the precise chemical form in which it is present is important. Trace elements may occur in different oxidation states (for example, essential Cr (III) and its carcinogenic counterpart Cr (VI)) or may be found incorporated into organic molecules. This may lead to significantly different results when assessing potential hazards from a sample with respect to toxicity. For example, fruit juices such as apple juice may only contain 10 µg∙L-1 of arsenic, as it is mostly found as inorganic arsenic, whereas fish and seafood can be safely consumed even though they may contain total concentrations 1000-fold higher, as arsenic is commonly found in organic forms. The qualitative and/or quantitative determination of different chemical forms of an analyte in a sample is often referred to as speciation analysis.Prof. Dr. Marc Birringer, Alexander Maxones and Simon Glas from the University of Applied Science in Fulda, Germany, working at the department for nutritional, food and consumer sciences, explore the importance of selenium (Se) in foods, leveraging speciation analysis to assess different selenium species for their bioavailability. “The journey on selenium species started in the 1990s”, says Prof. Birringer, “when people started to understand why selenium is important for biochemistry and nutrition. When we started with ICP-MS as a new technique in our lab, we learned that this technique can really give us a lot of information in single and multi-element analysis, and we planned to perform speciation analysis, too.”Selenium is an essential trace element that is low in abundance in Germany. Important sources for this essential trace element are fish, meat or eggs. Because these foods are not part of a vegetarian’s or vegan’s diet, they are particularly susceptible to the potential risk of malnutrition. Mushrooms, in particular porcini mushroom (boletus edulis), could be an alternative source when meat or fish are not part of the diet based on an individual’s choice. One compound that is particularly abundant is selenoneine, a derivative of ergothioneine, where a selenium atom replaces a sulfur atom.Whereas there are a lot of chromatographic methods established to separate other water-soluble selenium compounds, like inorganic selenium (Se (IV) and Se (VI)), methods allowing separation of organic selenium compounds like selenoneine are more difficult to optimize when ICP-MS is used as an element-specific detection system. Separation based on hydrophilic interaction chromatography (HILIC) typically start with high amounts of organic solvents in the mobile phase, which require the ICP-MS to be equipped accordingly., i.e. oxygen addition to the plasma and platinum tipped cones.<a target="_blank" rel="noopener noreferrer" href="https://www.thermofisher.com/content/lifetech/global/en/home/industrial/chromatography/ion-chromatography-ic.html">Ion chromatography (IC)</a> is well known for its ability to separate charged ions or polarizable species and is often the technique of choice for speciation analysis of elements such as arsenic or selenium. Typical IC systems, such as the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/410">Thermo Scientific Dionex ICS-6000 Ion Chromatography systems,</a> are fully inert and can therefore leverage eluents with very high or low pH values. &nbsp;&nbsp;&nbsp;“We have an ICS-6000 HPIC in the laboratory, but that system is usually blocked for other assays. However, the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=39&amp;search=Vanquish">Thermo Scientific Vanquish HPLC</a> was also compatible with the mobile phase we used, which was at pH 2”, says Prof. Birringer. “This system was used for the separation with a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2076">Thermo Scientific Dionex IonPac CS12 column</a>.&nbsp; This allowed good separation for the organic compounds containing selenium, even though the separation of the inorganic and ionic species was not ideal under these conditions.”During method development, it was very important to also monitor sulfur, as there is no standard available for selenoneine and instead, the sulfur-containing analog ergothioneine was used. For both elements, the use of a triple quadrupole ICP-MS system such as the <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/products/1488">Thermo Scientific iCAP MTX ICP-MS</a> provides significant advantages. Both elements are potentially biased by polyatomic interferences (especially the most abundant isotopes 32S and 80Se), which can be efficiently reduced using oxygen as a reactive gas.“What we found in the boletus edulis was a lot of compounds that we did not know before or could at least not explain as we have no mass spectra to confirm structures, but we do have the ICP-MS data”. They must be present in low concentrations and would likely be found in the noise in a normal LC-MS run states Prof. Birringer.Coupling of the two systems is straightforward. When water-based eluents are applied, the outlet of the column can be directly connected to the nebulizer of the ICP-MS system using narrow bore tubing and a zero dead volume connector. &nbsp;“I´m really comfortable with the system, and I think it gives us a lot of possibilities” says Alexander Maxones. “I would like to explore other column options for future projects, as I think smaller particles would be beneficial for further separation of species”, he adds.For data evaluation, <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/76">Thermo Scientific Chromelon CDS software</a> was used, as it provides more options for peak integration and smoothing of raw data, if needed. The original data collected on the ICP-MS can be directly imported into Chromeleon CDS software.“The methodology developed can now be applied to a wider range of samples. We have found at least five unknown compounds in boletus edulis, but it is clear from the literature that other mushrooms, for example shiitake, can also accumulate some selenium. We are sure they have similar but also different compounds to be investigated”, says Prof. Birringer. Next is additional food matrices, but also human samples.<blockquote><ul><li>For more information, please visit the <a target="_blank" rel="noopener noreferrer" href="https://www.hs-fulda.de/oecotrophologie/labore/analytik">laboratory´s website</a> and <a target="_blank" rel="noopener noreferrer" href="https://www.thermofisher.com/content/lifetech/global/en/home/industrial/mass-spectrometry/inductively-coupled-plasma-mass-spectrometry-icp-ms/quadrupole-icp-ms-systems.html"><ins>Quadrupole ICP-MS Systems</ins></a>.</li><li>Visit us on&nbsp;<a target="_blank" rel="noopener noreferrer" href="https://www.linkedin.com/showcase/72003132/admin/dashboard/">LinkedIn:</a>&nbsp;#ICP-MS</li></ul></blockquote></div></article><section><blockquote><h4>Daniel Kutscher</h4>Dr. Daniel Kutscher manages the Application Development for trace elemental analysis and GC/GC-MS products at the Thermo Fisher Scientific Center of Excellence in Mass Spectrometry in Bremen, Germany. After graduation in Chemistry in 2007, Daniel obtained his PhD from the University of Oviedo in 2011. In the same year, he joined Thermo Fisher Scientific and has had different roles in application development and marketing.</blockquote></section>

Deciphering Selenium Nutrition Using Speciation Analysis with ICP-MS

Welcome back to our series on <a target="_blank" rel="noopener noreferrer" href="https://www.thermofisher.com/cz/en/home/industrial/chromatography/chromatography-learning-center/liquid-chromatography-information/hplc-system-components/how-hplc-detectors-work/charged-aerosol-detection.html?erpType=Global_E1">Charged Aerosol Detection (CAD) in High-Performance Liquid Chromatography (HPLC).</a> In the first two episodes, we explored the fundamentals and benefits of CAD and compared it to other universal detectors like ELSD and MALS.&nbsp;<blockquote><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/5390">Episode 1: The Benefits of Charged Aerosol Detection (CAD) in High-Performance Liquid Chromatography (HPLC)</a><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/6250">Episode 2: The Benefits of CAD Compared to Other Universal Detectors: ELSD and MALS</a></blockquote>In this episode, we will delve deeper into the inner workings of CAD, providing a detailed understanding of its operation and the technology behind it.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Thermo_Fisher_Scientific_Understanding_How_Charged_Aerosol_Detection_CAD_Works_7e2b6c4dd6.jpg" alt="Thermo Fisher Scientific: Understanding How Charged Aerosol Detection (CAD) Works" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Thermo_Fisher_Scientific_Understanding_How_Charged_Aerosol_Detection_CAD_Works_7e2b6c4dd6.jpg 245w," sizes="100vw" width="1200" height="600"><h3>The basics of CAD</h3>Charged Aerosol Detection (CAD) is a universal detection method used in HPLC that provides a consistent response for non-volatile and semi-volatile analytes. CAD operates by converting the liquid eluent from the HPLC column into charged aerosol particles, which are then detected to generate a signal proportional to the mass of the analyte. Let’s break down the process step by step.<h3>Step-by-step operation of CAD</h3><h4>1. Nebulization</h4>The first step in CAD is the nebulization of the liquid eluent from the HPLC column. The eluent is introduced into a nebulizer, where it is transformed into a fine aerosol. This is achieved by using a high-velocity gas stream (usually nitrogen) that shears the liquid into tiny droplets. The size of these droplets is typically in the micron range.<h5>Key components:</h5><ul><li>Nebulizer: Converts the liquid eluent into an aerosol.</li><li>Gas stream: Typically nitrogen, used to create the aerosol droplets.</li></ul>Detailed process: The liquid eluent enters the nebulizer, where it meets a high-velocity gas stream. This interaction causes the liquid to break up into small droplets, forming an aerosol. The efficiency of nebulization is crucial, as it determines the size and uniformity of the droplets, which can affect subsequent steps in the detection process.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Thermo_Fisher_Scientific_Understanding_How_Charged_Aerosol_Detection_CAD_Works_3_74ffbd69fa.jpg" alt="Thermo Fisher Scientific: Understanding How Charged Aerosol Detection (CAD) Works" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Thermo_Fisher_Scientific_Understanding_How_Charged_Aerosol_Detection_CAD_Works_3_74ffbd69fa.jpg 245w," sizes="100vw" width="1160" height="600"><h4>2. Evaporation</h4>After nebulization, the aerosol droplets enter a heated drift tube. In this tube, the solvent evaporates, leaving behind dried analyte particles. The temperature of the drift tube is carefully controlled to ensure complete evaporation of the solvent without decomposing the analyte.<h5>Key components:</h5><ul><li>Heated drift tube: Evaporates the solvent, leaving dried analyte particles.</li></ul>Detailed process: The aerosol droplets pass through the heated drift tube, where they are subjected to a controlled temperature. The solvent in the droplets evaporates, resulting in the formation of dried particles that contain the analyte. The drift tube’s temperature must be optimized to prevent the degradation of thermally sensitive analytes while ensuring complete solvent removal.<h4>3. Charging</h4>The dried analyte particles then pass through a corona discharge, where they are exposed to a high-voltage electrical field. This field imparts a charge to the particles. The corona discharge generates ions that attach to the analyte particles, effectively charging them.<h5>Key Components:</h5><ul><li>Corona discharge: Generates ions to charge the analyte particles.</li></ul>Detailed process: As the dried particles exit the drift tube, they enter the corona discharge region. Here, a high-voltage electrode ionizes the surrounding gas, creating a corona discharge. The ions generated in this discharge attach to the analyte particles, imparting a charge to them. This charging process is critical, as the strength of the signal detected later is proportional to the charge on the particles.<h4>4. Detection</h4>The charged particles are then directed into a detector, typically an electrometer, which measures the quantity of charge. The signal produced by the electrometer is proportional to the amount of analyte present. This signal is then processed and displayed as a chromatogram, showing the concentration of the analyte over time.<h5>Key components:</h5><ul><li>Electrometer: Measures the charge on the analyte particles.</li><li>Data processor: Converts the measured charge into a readable signal.</li></ul>Detailed process: The charged particles flow into the electrometer, where the charge is measured. The electrometer converts the charge into an electrical signal, which is proportional to the quantity of analyte. This signal is then processed by data analysis software, which generates a chromatogram. The peaks in the chromatogram correspond to the concentration of the analyte at different time points, allowing for quantitative analysis.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Thermo_Fisher_Scientific_Understanding_How_Charged_Aerosol_Detection_CAD_Works_2_363e6af2fa.jpg" alt="Thermo Fisher Scientific: Understanding How Charged Aerosol Detection (CAD) Works" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Thermo_Fisher_Scientific_Understanding_How_Charged_Aerosol_Detection_CAD_Works_2_363e6af2fa.jpg 245w," sizes="100vw" width="960" height="540">You can find more information on why to choose <a target="_blank" rel="noopener noreferrer" href="https://www.thermofisher.com/cz/en/home/industrial/chromatography/chromatography-learning-center/liquid-chromatography-information/hplc-system-components/how-hplc-detectors-work/charged-aerosol-detection.html">Charged Aerosol Detection for your HPLC analysis</a><h3>Conclusion</h3>Charged Aerosol Detection (CAD) is a powerful and versatile detection method in HPLC, offering consistent response, high sensitivity, robustness, and ease of use. By understanding the step-by-step operation of CAD, we can appreciate the technology’s ability to provide reliable and accurate detection across a wide range of analytes.As we continue our series on CAD, stay tuned for the next episode, where we will explore specific case studies and practical applications of CAD in various industries, demonstrating its real-world impact and benefits.Thank you for joining us in this deep dive into the workings of Charged Aerosol Detection. We hope this episode has provided you with a clearer understanding of CAD and its advantages in analytical chemistry.<blockquote>Visit us on LinkedIn: #CAD, #ChargedAerosolDetection #HPLCAuthor: Marcus KleeMarcus Klee is the Regional Marketing Manager for HPLC in EMEA at Thermo Fisher Scientific. He holds a Dipl.-Ing. in Chemical Engineering and has extensive experience in marketing, sales, and communications within the chemical and physical technology sectors.</blockquote>

Episode 3: Understanding How Charged Aerosol Detection (CAD) Works

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