<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

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Automated LC-MS Sample Cleanup Using Turbulent Flow Chromatography

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