<h4><strong>🚀 Meet the experts! 🚀</strong></h4><p>Join us on<strong> June 12th</strong> for the <strong>3rd edition </strong>of<strong> Live from the Lab!</strong> This hybrid event will showcase the latest advancements in mass spectrometry technology, featuring<strong> Dr Thomas Moehring &amp; Dr Hamish Stewart </strong>discussing the latest news from ASMS. 🌟</p><blockquote><p>📅 <strong>Date:</strong> June 12th, 2025&nbsp;</p><p>📍 <strong>In-Person Locations:</strong> Bremen Factory &amp; Satellite Locations across Europe and the Middle East&nbsp;</p><p>💻 <strong>Online:</strong> Live streaming available</p></blockquote><h4><strong>How to Join:&nbsp;</strong></h4><ul><li><strong>Bremen Factory: </strong>Attend the main event in person!</li><li><strong>Satellite Locations:</strong> Join local events with live streaming and additional local content.</li><li><strong>Online: </strong>Register to watch the live stream from anywhere!</li></ul><blockquote><p>🗓️ Mark your calendars and 🔗 <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/events/829"><strong>register today!</strong></a> Participation is free, but registration is required.</p></blockquote><p>Don't miss out on this opportunity to expand your knowledge and collaborate with the scientific community. See you there!</p><p>#OmicsResearch #MassSpectrometry #ASMS2025</p>

Live from the Lab: Solutions for Omics Research - Dr Thomas Moehring & Dr Hamish Stewart

<p><strong>Mucinomics, the study of mucin domain glycoproteins, is emerging as a transformative field in mass spectrometry. These heavily O-glycosylated extracellular proteins play critical roles in numerous biological functions and diseases, including cancer, cystic fibrosis, and inflammatory bowel disease. However, their intricate glycosylation patterns have historically posed challenges for mass spectrometric analysis.</strong></p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Thermo_Fisher_Scientific_Mucinomics_The_Next_Frontier_of_Mass_Spectrometry_498b7ba2b8.jpg" alt="Thermo Fisher Scientific: Mucinomics: The Next Frontier of Mass Spectrometry" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Thermo_Fisher_Scientific_Mucinomics_The_Next_Frontier_of_Mass_Spectrometry_498b7ba2b8.jpg 245w," sizes="100vw" width="528" height="264"></p><article><div><p><strong>Dr. Stacy Malaker</strong> from Yale University recently presented advancements in mucin characterization, enrichment techniques, and their translational relevance in disease research on a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/4795"><strong>Nature hosted webinar</strong></a>. Here, we explore the key insights from her presentation.</p><h3><strong>The complexity of mucin glycosylation</strong></h3><p>Glycosylation is one of the most diverse post-translational modifications (PTMs), estimated to modify over 50% of the human proteome. There are three primary types of glycosylation:</p><ul><li><strong>N-linked glycosylation</strong>: Occurs on asparagine residues, influencing protein folding and cell signaling.</li><li><strong>O-linked glycosylation</strong>: Involves serine or threonine residues and is crucial for cell adhesion and matrix interactions.</li><li><strong>O-linked GlcNAcylation</strong>: A dynamic intracellular modification similar to phosphorylation, regulating transcription and cell proliferation.</li></ul><p>Mucin proteins, characterized by densely glycosylated proline, serine, threonine (PTS) domains, exhibit bottlebrush-like structures that extend from the cell surface or form gel-like secretions. Dysregulation of mucins, particularly MUC1 and MUC16, is implicated in cancer progression, immune evasion, and metastasis. Despite their importance, mucins have remained difficult to analyze due to their complex glycosylation and resistance to standard proteases.</p><h3><strong>Overcoming analytical challenges with mucinases</strong></h3><p>Traditional proteomics workflows struggle with mucins due to:</p><ul><li><strong>High heterogeneity</strong>: A single glycosylation site can accommodate hundreds of glycan structures.</li><li><strong>Protease resistance</strong>: Conventional enzymes like trypsin fail to digest mucin domains.</li><li><strong>Fragmentation issues</strong>: Standard collision-induced dissociation (CID) leads to glycan loss, complicating site localization.</li></ul><p>Dr. Malaker’s research introduced StcE (pronounced as <strong>Sticky)</strong>, a mucin-selective protease that selectively cleaves glycosylated mucin domains while sparing unmodified peptides. This enzyme has been pivotal in:</p><ul><li>Mass spectrometric sequencing of mucins.</li><li>Cellular assays for mucin function.</li><li>Immunohistochemical staining and mucin enrichment for glycoproteomics studies.</li></ul><p>StcE&nbsp;and other mucinases have enabled the first detailed site-specific glycosylation maps of mucins, revealing their structural dynamics and disease relevance.</p><h3><strong>Deciphering immune-related mucins: the TIM family</strong></h3><p>The T-cell immunoglobulin and mucin domain-containing (TIM) proteins regulate immune responses, with TIM-3 being a promising checkpoint inhibitor target in cancer therapy. Using mucinases, Dr. Malaker’s team successfully mapped the glycosylation patterns of TIM-1, TIM-3, and TIM-4, revealing:</p><ul><li>TIM-4 adopts a rigid, bottlebrush-like structure, extending beyond the glycocalyx for high-affinity ligand binding.</li><li>TIM-3 exhibits flexible folding, potentially facilitating alternative ligand interactions via galectin-9-mediated clustering.</li><li>Glycosylation regulates immune checkpoint function by influencing receptor-ligand interactions.</li></ul><p>These findings underscore the structural and functional roles of mucins in immune regulation and their potential as therapeutic targets.</p><h3><strong>Advancing mucinomics with enrichment strategies</strong></h3><p>To enhance mucin analysis, Dr. Malaker’s lab developed an enrichment method using an inactive Sticky mutant conjugated to beads. This strategy:</p><ul><li>Selectively captures mucins from complex biological samples (e.g., serum, cancer cell lysates).</li><li>Enables mass spectrometric analysis of previously undetectable glycoproteins.</li><li>Identifies a broader mucinoma beyond canonical mucins, expanding the scope of mucin research.</li></ul><p>This workflow drastically reduces sample preparation time while increasing the detection of mucin-specific glycopeptides, making it a powerful tool for clinical applications.</p><h3><strong>Improving glycopeptide identification</strong></h3><p>Existing search algorithms struggle with the complexity of mucin glycoproteomics. The field-wide <strong>Human Glycoproteomics Initiative</strong> (HGI) aims to evaluate and improve these tools. Key developments include:</p><ul><li>Standardized glycoproteomics reporting.</li><li>Benchmarking of glycopeptide identification software.</li><li>Integration of electron-based fragmentation for precise site localization.</li></ul><p>These efforts are critical for establishing robust, reproducible glycoproteomic workflows for biomedical research.</p><h3><strong>Spatial distribution of mucins in disease</strong></h3><p>Mass spectrometry imaging (MSI) offers insights into mucin distribution in tissues, but traditional methods are limited to N-glycans. Dr. Malaker’s team pioneered mucinase-assisted MSI to visualize O-glycopeptides in colorectal cancer specimens. Findings included:</p><ul><li>Distinct glycopeptide profiles across tumor regions.</li><li>Localization of tumor-associated carbohydrate antigens (TACAs).</li><li>High-resolution sequencing of MUC2, achieving 90% coverage of its glycosylated domains.</li></ul><p>This approach has potential applications in biomarker discovery, disease stratification, and personalized medicine.</p><p><strong>Conclusion</strong>: Mucinomics represents a rapidly evolving frontier in mass spectrometry, with profound implications for cancer biology, immunology, and clinical diagnostics. The innovations in mucinase-based proteomics, enrichment techniques, and spatial glycoproteomics are paving the way for deeper insights into mucin function and disease mechanisms.</p><p>By overcoming long-standing analytical challenges, these advances position mucins as crucial players in biomedical research, opening doors to novel diagnostics and therapeutic strategies. As the field progresses, integrating mucinomics into routine clinical proteomics could revolutionize our understanding of complex diseases and their molecular underpinnings.</p><blockquote><p><strong>Visit us on </strong><a target="_blank" rel="noopener noreferrer" href="https://www.linkedin.com/showcase/72003132/admin/dashboard/"><strong>LinkedIn:</strong></a><strong> #Glycoprotein, #o-glycosylation, #MucinProteins, #OrbitrapEclipseTribridMassSpectrometer</strong></p></blockquote></div></article><section><blockquote><h4><strong>Julian Saba</strong></h4><p>Julian Saba PhD, Sr Omics Marketing Manager</p></blockquote></section>

Mucinomics: The Next Frontier of Mass Spectrometry

<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 30th June 2025? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!</strong></p><blockquote><p><span style="font-size:20px;">👉 </span><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library"><span style="font-size:20px;"><strong>SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES</strong></span></a></p></blockquote><blockquote><p><strong>👉 Need info about different analytical techniques?</strong> Peek into <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library"><strong>LabRulezGCMS</strong></a> or <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library"><strong>LabRulezICPMS</strong></a> libraries.</p></blockquote><p>This week we bring you other document by <strong>Agilent Technologies </strong>and posters by<strong> Shimadzu / ASMS, Thermo Fisher Scientific / ASMS </strong>and<strong> Waters Corporation / ASMS!</strong></p><h3><strong>1. Agilent Technologies: Agilent InfinityLab Assist Hub: Representing Advancements in Cybersecurity</strong></h3><ul><li>Other document (white paper)</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/wp_infinitylab_assist_cybersecurity_5994_8261en_agilent_310c5c9a6c.pdf"><strong>Full PDF for download</strong></a></li></ul><h4><strong>The laboratory as an operational technology (OT) environment: bridging the gap between OT and IT&nbsp;</strong></h4><p>Compared to connected industrial production environments, the connected laboratory environment closely aligns with an operational technology (OT) environment. OT is defined as "the practice of using hardware and software to control industrial equipment, and it primarily interacts with the physical world" (RedHAT, 2024).&nbsp;</p><p>In the classical Purdue reference model, established in the early 1990s to protect identities, information, and assets, the OT environment is clearly separated and secured from the information technology (IT)/enterprise environment.&nbsp;</p><p>The model only allows direct communication between adjacent levels. OT environments (lab environments) are necessary due to the long life cycles of the systems typically exposed to cybersecurity threats, with the mitigations listed in Table 1.&nbsp;</p><p>Addressing cybersecurity challenges in OT environments requires customized security strategies that incorporate both traditional IT security principles and specific measures for the physical operations they control.&nbsp;</p><p>Unlike some IT system components and devices where complete infrastructure replacement is common, lab devices are typically replaced gradually over years, sometimes even over&nbsp;decades.</p><p>The clear separation between OT and IT is becoming increasingly blurred due to new connectivity requirements, cloud functionality, and Industrial Internet of Things integration. This process, known as IT/OT convergence, is essential for achieving the next level of automation, such as Industry 4.0/5.0 or its counterparts, such as Pharma 4.0. The effectiveness of any new development will be evaluated based on how well it addresses cybersecurity within a converging IT/OT environment.</p><h4><strong>Standards and organizations&nbsp;</strong></h4><p>The diverse standards and organizations can be exemplified by the approaches taken in the U.S., Europe, and China. This summary aims to showcase the complexity of regulatory environments, and is not comprehensive. For specific situations, consulting with dedicated field experts is crucial.</p><h5><strong>Europe&nbsp;</strong></h5><p>European cybersecurity regulations prioritize privacy and data protection, with an emphasis on harmonizing standards across member states.&nbsp;</p><ul><li><strong>General Data Protection Regulation (GDPR): </strong>GDPR is the cornerstone of data protection in the European Union (EU). It sets strict rules on handling personal data, including requirements for data breach notifications, data minimization, and individual rights over personal data. Noncompliance can result in severe fines.</li><li><strong>Network and Information Security Directive (NIS&nbsp;Directive): </strong>Adopted in 2016, the NIS Directive focuses on the cybersecurity of essential services (such as energy, transportation, and finance) and digital service providers. It requires member states to strengthen their national cybersecurity capabilities and cooperation.&nbsp;</li><li><strong>European Union Agency for Cybersecurity (ENISA):</strong> ENISA is the EU's dedicated cybersecurity agency, helping to develop and promote cybersecurity standards across Europe, including guidelines for incident reporting and cybersecurity&nbsp;certifications.</li><li><strong>ISO/IEC 27001: </strong>While ISO/IEC 27001 is a global standard for information security management systems, it is heavily adopted in Europe. This standard outlines processes for systematically managing sensitive company information, ensuring its security.&nbsp;</li><li><strong>Cybersecurity Act (EU): </strong>The Cybersecurity Act establishes a framework for cybersecurity certification for products, services, and processes, ensuring they meet recognized EU-wide standards.</li></ul><h4><strong>Conclusion&nbsp;</strong></h4><p>With the initial release of the InfinityLab Assist Control Software, the InfinityLab Assist Hub offers plenty of cybersecurity features that can be leveraged for a secure lab network environment founded on a customer-specific OT/IT security strategy.</p><h3><strong>2. Shimadzu / ASMS: Single Quadrupole Mass Spectrometry Quick Workflows Specified to Oligonucleotides Screening&nbsp;</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/ASMS_25_TP_605_fa8deb906e.pdf"><strong>Full PDF for download</strong></a></li></ul><p>A workflow has been developed to deliver molecular mass information on the full-length product and related impurities for chemically synthesized oligonucleotides by applying a deconvolution algorithm to all detected components within a targeted data stream (UV/PDA, TIC or BPC). It has been designed to simplify the data processing experience without the need for a sequence input delivering results quickly by automating mass assignment of all detected components in a data stream.</p><h4><strong>2. Materials and Methods&nbsp;</strong></h4><h5><strong>2.1 LC-MS/MS method&nbsp;</strong></h5><p><strong>20-mer sample.</strong> A phosphorothioate-modified oligonucleotide purified was used as a model sample (20-mer anti-sense oligo; C229 H316 N69 O121 P19 S19; average mass 7168.05, Te-smCe-sTe-sTe-sGe-sGd-sTd-sTd-sAd-sCd-sAd-sTd-sGd-sAd-sAd-sAe-sTe-smCe-smCesmCe) spiked with 10% impurities (N-1(5’) and N-3(5’) shortmers) to the full-length product.&nbsp;</p><p><strong>80-mer antisense DNA sample</strong>. 80-mer PolyT oligonucleotide sample (C800 H1041 N160 O558 P79; average mass 24273.62) was also analyzed was analyzed by ion-pair reversedphase chromatography mode using HFIP (hexafluoroisopropanol) and 10 mM TEA (triethylamine).</p><p><strong>LC Separation</strong>. Components were separated using the Shimadzu <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=3&amp;search=Nexera+XS"><strong>Nexera XS</strong></a> inert LC and Shim-pack Scepter Claris column using HFIP (hexafluoroisopropanol) and 10 mM TEA (triethylamine) as ion pair additives.&nbsp;</p><p><strong>Mass Spectrometry Detection. </strong>Shimadzu Single Quadrupole<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=3&amp;search=+LCMS-2050"><strong> LCMS-2050</strong></a>. Data acquired in profile mode. MS mass scan m/z 600-2000; 1 secs; negative ion mode.</p><h4><strong>3.Results&nbsp;</strong></h4><h5><strong>3.1 Workflows and Methods&nbsp;</strong></h5><p>Insight Biologics software application supports two workflows to help accelerate oligonucleotide characterization. The default data processing behavior was driven by a defined FLP sequence to confirm sequence composition, identify product related impurities and assess impurity profiles. Data processing has been further augmented by enabling automated deconvolution routines to be applied without a defined FLP sequence input to help workflows that need to quickly check on molecular mass information in a synthesis (Figure 2).</p><h4><strong>4. Conclusions&nbsp;</strong></h4><ul><li>LabSolutions Insight Biologics offers flexibility by supporting both user-defined and automated deconvolution workflows for comprehensive oligonucleotide analysis.&nbsp;</li><li>A Quick Picking algorithm has been developed and applied to the analysis of 20-mer and 80-mer antisense DNA. In the case of a 20-mer sample, the FLP and associated impurities were also automatically detected and molecular weight information reported. The analysis showed high correspondence with data processing results using a defined FLP sequence input.&nbsp;</li><li>The Quick Picking algorithm enables a molecular weight confirmation of any detected peak and can be a powerful tool for synthesis confirmation in cases where the product is not as intended.&nbsp;</li><li>This LC-MS analysis platform provides a rapid and cost-effective solution for purity analysis and molecular weight confirmation, making it ideal for quality control environments.</li></ul><h3><strong>3. Thermo Fisher Scientific / ASMS: Comprehensive data acquisition workflow on a modified Orbitrap Astral MS to achieve deep lipidome coverage with high confidence annotations</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/PO_003964_ms_ASMS_25_Astral_Zoom_Lipidomics_po003964_na_en_0ccd4bd053.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Lipid identification is often considered the most challenging and bottleneck of the lipidomics workflow. This is due to the vast diversity of lipid structures, isomers and varying concentrations. Fragmentation of lipids generate characteristic fragments that help in lipid annotation, structural elucidation, isomer differentiation, and improved confidence in lipid identification. Having fragmentation for most of the lipids detected in a sample hence is highly desirable. This requires fast and sensitive instrumentation. Here we evaluated the new Orbitrap Astral Zoom mass spectrometer (Figure 1) for deeper lipidome coverage and confident lipid identification from 73 mice plasma extracts.</p><h4><strong>Materials and methods&nbsp;</strong></h4><h5><strong>Sample Preparation&nbsp;</strong></h5><p>Lipids were extracted from plasma samples obtained from 73 mice fed with a high fat diet and a normal diet. Lipid standards and bovine liver lipid extracts were purchased from Avanti Polar Lipids.&nbsp;</p><h5><strong>LCMS Method&nbsp;</strong></h5><p>Plasma lipid extract was separated using Thermo Scientific nanoLC PepMap (C18, 75µmx150mm, 2µm) column connected to a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/398"><strong>Vanquish Neo UHPLC system</strong></a>. Data acquisition was carried out on the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1959"><strong>Orbitrap Astral Zoom mass spectrometer</strong></a> using full-scan Orbitrap MS1 in parallel with data-dependent Astral MS2.</p><h5><strong>Data Analysis&nbsp;</strong></h5><p>Data processing was performed using <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1759"><strong>Thermo Scientific Compound Discoverer 3.4 software</strong></a>. Compound Discoverer 3.4 software includes new features such as Thermo Scientific LipidSearch software node for simplified lipid identification, QC for small molecule experiments using internal standards, automatically detects and groups MS1 fragments and other performance improvements.</p><h4><strong>Results&nbsp;</strong></h4><h5><strong>Orbitrap Astral Zoom mass spectrometer DDA MS2 scan rate&nbsp;</strong></h5><p>The Orbitrap Astral Zoom mass spectrometer offers an enhanced MS2 scan rate compared to the already high-speed Orbitrap Astral MS system, enabling DDA with Top 30, Top 40, or even Top 50 MS2 scans (Figure 2) without compromising data quality. This advancement delivers unprecedented speed, achieving faster DDA MS2 scan rates than ever before.</p><h5><strong>Unprecedented speed and depth: dual analyzer power of Orbitrap Astral Zoom MS</strong></h5><p>The parallel data acquisition on Orbitrap Astral Zoom MS using both the Thermo Scientific Orbitrap analyzer and Thermo Scientific Astral analyzer, enables a higher number of Orbitrap HRAM MS1 full scans with sub-ppm mass accuracy. Simultaneously, the fast Astral analyzer delivers a significantly greater number of highquality MS2 scans at 80k resolution (77% more) compared to Orbitrap-Orbitrap configurations, which is essential for deep coverage and confident lipid identification (Figure 3).</p><h4><strong>Conclusions&nbsp;</strong></h4><ul><li>The novel Orbitrap Astral Zoom mass spectrometer provides deeper lipidome coverage and confident identification, utilizing the parallel operation of Orbitrap and Astral analyzers.&nbsp;</li><li>The enhanced MS2 speed in Orbitrap Astral Zoom mode, coupled with Stepped NCE, enables efficient data acquisition and comprehensive lipidome coverage with a 15% increase with grade A and B lipid annotation.&nbsp;</li><li>The use of nano-flow increased the sensitivity and the number of detected lipids while achieving a significant reduction in the use of sample and solvent.&nbsp;</li><li>A significant difference was observed between control and high fat diet mice samples with potential biomarker upregulated neutral lipids (triglycerides and cholesterol esters) in the high fat diet.</li></ul><h3><strong>4. Waters Corporation / ASMS: Routine EPA 1633 PFAS Analysis with a Novel Slotted Bandpass Ion Guide to Improve Signal Response Robustness</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720008888_65b495feb8.pdf"><strong>Full PDF for download</strong></a></li></ul><p>With increasing regulatory pressure around PFAS monitoring, this study evaluates a robust and long-term solution for EPA 1633A-compliant PFAS analysis. It focuses on how the StepWave XR ion guide, integrated into the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=5&amp;search=Xevo+TQ+Absolute+XR"><strong>Waters Xevo TQ Absolute XR tandem quadrupole mass spectrometer,</strong></a> improves signal robustness and method stability across demanding environmental matrices such as river water, biosolids, and landfill leachate.</p><h4><strong>Materials and Methods</strong></h4><p>Samples included NIST-certified biosolids, landfill leachate, and spiked river water. Solid-phase extraction (SPE) was followed by LC-MS/MS analysis on an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/191"><strong>ACQUITY Premier System</strong></a> with BEH C18 columns and detection via the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=5&amp;search=Xevo+TQ+Absolute+XR"><strong>Xevo TQ Absolute XR</strong></a> using negative electrospray ionization and MRM. The robustness of the method was assessed over 12 weeks with over 9,000 injections and weekly calibrations, using <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1544"><strong>waters_connect for Quantitation Software</strong></a> for data processing and review.</p><h4><strong>Results</strong></h4><p>Throughout the 12-week study, all sample types showed high consistency and low variability. For example, L-PFHxS in biosolids averaged 8.78 µg/kg (%RSD 7.2%) compared to the certified 9.39 µg/kg, while PFOA in landfill leachate showed a mean of 2.52 ng/mL (%RSD 2.7%). QC plots confirmed that all monitored PFAS met trueness and ion ratio targets across thousands of injections with no unscheduled downtime, highlighting the system’s long-term reliability.</p><h4><strong>Conclusion</strong></h4><p>The StepWave XR ion guide enabled reliable, high-throughput PFAS analysis with minimal maintenance and excellent reproducibility. The Xevo TQ Absolute XR maintained calibration stability, accurate quantitation, and strong matrix resilience across diverse sample types. Combined with streamlined software workflows, this setup is well-suited for routine EPA 1633A PFAS analysis in environmental labs.</p>

News from LabRulezLCMS Library - Week 27, 2025

<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 7th July 2025? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!</strong></p><blockquote><p><span style="font-size:20px;">👉 </span><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library"><span style="font-size:20px;"><strong>SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES</strong></span></a></p></blockquote><blockquote><p><strong>👉 Need info about different analytical techniques?</strong> Peek into <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library"><strong>LabRulezGCMS</strong></a> or <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library"><strong>LabRulezICPMS</strong></a> libraries.</p></blockquote><p>This week we bring you application note by <strong>Agilent Technologies </strong>and posters by<strong> Shimadzu / ASMS, Thermo Fisher Scientific / ASMS </strong>and<strong> Waters Corporation / ASMS!</strong></p><h3><strong>1. Agilent Technologies: Rapid and Sensitive Analysis of Antifungal Agents in Human Serum Using the Agilent 6475 LC/TQ</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_antifungal_human_serum_5994_8338en_agilent_89f89cb4dc.pdf"><strong>Full PDF for download</strong></a></li></ul><p>In addition to minimizing drug-related toxicity, TDM has been hailed as a key step to prevent the emergence of drug resistance. The growing number of invasive fungal infections creates a ripple effect on the rapid advancement of TDM for antifungal agents. Increasing evidence confirms the critical role of TDM in preventing and treating antifungal infections.1 The RECIPE ClinMass TDM kit system for antimycotics in serum (Munich, Germany) was used on a 6475 LC/TQ for the analysis of eight antifungal agents. This application note demonstrates the evaluation and validation of the RECIPE TDM kit, covering sensitivity, linearity, and robustness across five consecutive days (n =&nbsp;5). Quick and simple sample preparation was implemented, providing high-throughput monitoring of antifungal agents in biological&nbsp;samples.</p><h4><strong>Experimental</strong></h4><h5><strong>Instrumentation</strong></h5><ul><li><strong>Liquid Chromatography System&nbsp;</strong><ul><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=1&amp;search=Agilent+1290+Infinity+II"><strong>Agilent 1290 Infinity II</strong></a> High-Speed Pump&nbsp;</li><li>Agilent 1290 Infinity II Multisampler&nbsp;</li><li>Agilent 1290 Infinity II Multicolumn Thermostat&nbsp;</li></ul></li><li><strong>Mass Spectrometry System&nbsp;</strong><ul><li>Agilent <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=1&amp;search=6475+LC%2FTQ"><strong>6475 LC/TQ &nbsp;</strong></a></li><li>Agilent Jet Stream Technology Ion Source (AJS)</li></ul></li></ul><h5><strong>Method&nbsp;</strong></h5><p>The optimization of multiple reaction monitoring (MRM) transitions for analytes and ISTDs was carried out using Auto MRM Optimizer in the Agilent MassHunter acquisition software to provide optimal sensitivity and selectivity. Data were processed using the Agilent <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=1&amp;search=MassHunter+Qualitative+Analysis+software"><strong>MassHunter Qualitative Analysis software</strong></a>, version B.12, and Agilent <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=1&amp;search=MassHunter+Quantitative+Analysis+software"><strong>MassHunter Quantitative Analysis software</strong></a>, version B.12.</p><h4><strong>Results and discussion&nbsp;</strong></h4><p>Figure 2 shows the chromatograms of quantifier MRM transitions for the eight antifungal agents at the lowest calibration level. Six out of eight antifungal agents had good chromatographic separation across the run time. The retention times of two antifungals, posaconazole and hydroxy-itraconazole, were slightly overlapped. Despite this overlap, measurement integrity can still be maintained due to MRM selectivity.</p><h4><strong>Conclusion&nbsp;</strong></h4><p>A fast and sensitive analysis of eight antifungal agents in serum was validated on an Agilent 6475 triple quadrupole LC/MS. By employing a ready-to-use RECIPE ClinMass TDM kit, this method demonstrates accurate and precise quantitation of antifungal agents in biological samples. Additionally, the streamlined sample preparation method allows high throughput analysis of up to 350 samples per day.</p><h3><strong>2. Shimadzu / ASMS: Robustness Evaluation of PFAS Analysis in Soil Using LC-MS/MS</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/ASMS_25_WP_137_e932b391c7.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Per- and polyfluoroalkyl substances (PFAS) are widely used in various fields and industries. However, due to their structural stability and resistance to degradation, they accumulate in the environment and are suspected of being harmful to humans. PFAS in soil can accumulate in the human body via agricultural products, thus it is essential to develop sensitive and robust analytical methods for the detection of PFAS in soil with complex matrices.1 This study shows a robustness evaluation by adding 30 PFAS to a soil matrix and using the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=3&amp;search=LCMS-8060RX"><strong>LCMS-8060RX</strong></a> to measure these PFAS in the soil matrix 500 consecutive times.</p><h4><strong>Methods&nbsp;</strong></h4><p>For this analysis, LC-MS/MS equipped with a newly designed ESI source was used, and a delay column was installed between the mixer and the autosampler to inhibit the effects of PFAS contamination from the LC system. Six points calibration curves were built, and each calibration point was measured 3 times. A soil sample was prepared referring to part of the soil preparation procedures published by the National Agricultural and Food Research Organization. 2 Thirty PFAS compounds were spiked to the soil sample at 0.1 μg/L after pretreatment and 500 soil sample analyses were performed for the robustness evaluation. This sample contains more than 90% soil matrix. QC samples were also analyzed after every 20 injections of the soil samples.</p><h4><strong>Conclusion</strong></h4><p>The <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=3&amp;search=LCMS-8060RX"><strong>LCMS-8060RX</strong></a> performed 500 consecutive measurements of 30 PFAS spiked to a soil matrix sample. The results showed good peak area repeatability, good peak shapes, and good recovery. The newly designed ESI source showed excellent robustness, and stable analysis was achieved over a long period, even for samples with complex matrices.</p><h3><strong>3. Thermo Fisher Scientific / ASMS: Evaluation of a modified Orbitrap Astral Mass Spectrometer for label-free quantitation of proteomic samples</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/PO_439_ms_ASMS_25_Astral_Zoom_LFQ_po439_na_en_c1a0b2a11e.pdf"><strong>Full PDF for download</strong></a></li></ul><p>The demand for high-throughput proteomics analysis of complex samples is continually increasing, focusing on greater analysis depth and improved precision and accuracy in quantitation. Label-free quantitation of very complex proteomic samples poses significant challenges for mass spectrometers, requiring faster and more efficient processing of eluting peptide ions and their fragments. The new Orbitrap Astral Zoom mass spectrometer addresses these challenges, offering a higher acquisition rate by improved ion optics switching times and faster ion transfer, higher sensitivity due to ion pre-accumulation, and enhanced spectral processing, leading to deeper coverage with higher confidence.</p><h4><strong>Materials and methods</strong></h4><h5><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/398"><strong>Vanquish Neo</strong></a><strong>: Direct injection mode using 25cm x 75 μm ID Aurora Ultimate TS column (IonOpticks)&nbsp;</strong></h5><ul><li>High loads: 37min gradient; 4-45%B and injection-to-injection time 48min (30SPD)&nbsp;</li><li>Low loads: 20min gradient; 1-40%B and injection-to-injection time 28min (50SPD)&nbsp;</li></ul><h5><strong>Mass Spectrometer: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/836"><strong>Orbitrap Astra</strong></a><strong>l and </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1959"><strong>Orbitrap Astral Zoom&nbsp;</strong></a></h5><ul><li>Orbitrap (MS1) and Astral (MS2) detectors&nbsp;</li><li>Orbitrap: 240K; m/z range 380-980; AGC 500%; max IT 5ms&nbsp;</li><li>NCE 25; Thermo Scientific FAIMS Pro Duo (low loads): -48V; gas 3.5 L/min</li><li>Astral: DIA; window size depending on loads (in the tables below); m/z 150-2000; AGC 800% (low loads) or 100% (high loads); max IT depending on load; cycle time: 0.6s&nbsp;</li></ul><h5><strong>Data Processing&nbsp;</strong></h5><ul><li>Spectronaut® v19.5 Direct DIA, 6 files (2 x 3 replicates) processed together for each load; all other settings were default&nbsp;</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1782"><strong>Thermo Scientific Proteome Discoverer software </strong></a>3.3.0.54, CHIMERYS 4.0.21</li><li>Human, E.coli, and Yeast FASTA (all w/o isoforms) from UniProt; normalization by Human FASTA</li></ul><h4><strong>Results (High Loads)</strong></h4><p>The new Orbitrap Astral Zoom mass spectrometer takes advantage of ion preaccumulation in the bent trap, increasing the effective MS2 injection time. Its reduced ion transfer overheads result in a higher MS2 acquisition rate of up to 270 scans per second. Additionally, an enhanced spectral processing algorithm was implemented to deconvolute overlapping spectral features in MS2, for better handling spectra with increased spectrum density.&nbsp;</p><p>Using Orbitrap Astral Zoom MS and Spectronaut® 19.5 software, there was a 12-17% increase in IDs at the precursor level, 10-15% at the peptide level (stripped sequence), and 4-9% at the protein groups level. With Proteome Discoverer software, the gain was 12-19% at the DIA precursor level, 12-17% at the peptide level, and 4-7% at the protein group level. The increase in identifications was primarily observed for low-abundant species. Orbitrap Astral Zoom mass spectrometer also yielded, on average, a 15% higher median number of MS2 data points per peak, while maintaining consistently high quantitation precision and accuracy at the MS2 level.</p><h4><strong>Conclusions</strong></h4><p>The novel Orbitrap Astral Zoom mass spectrometer demonstrates improved performance for qualitative and quantitative analysis of complex proteomics samples over a wide range of sample loads spanning four orders of magnitude, from 100 pg to 1µg.</p><h3><strong>4. Waters Corporation / ASMS: Investigating Fire Fighter and E-waste Handlers Exposure to PFAS Using Liquid Chromatography and Cyclic Ion Mobility Mass Spectrometry</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720008883_21620a6293.pdf"><strong>Full PDF for download</strong></a></li></ul><p>PFAS are persistent environmental pollutants linked to health risks, including cancer. Monitoring their levels in human biofluids provides insight into exposure pathways. However, the analysis of PFAS isomers is technically challenging due to their structural similarity. To overcome this, the study employs <strong>LC coupled with cyclic ion mobility mass spectrometry (LC-cIM-MS)</strong> using the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/202"><strong>Waters SELECT SERIES Cyclic IMS</strong></a>, which adds a new separation dimension for better specificity.</p><h4><strong>Methods</strong></h4><p>Serum samples from Ghanaian firefighters and E-waste workers were prepared using <strong>solid-phase extraction (SPE)</strong> on 96-well µElution plates. A <strong>22-minute reversed-phase LC gradient</strong> was used for separation. The <strong>Cyclic IMS system</strong>, with ion mobility resolution R~65 and 145, enabled non-targeted PFAS analysis, including the generation of <strong>collision cross section (CCS)</strong> values to support compound identification.</p><h4><strong>Results</strong></h4><ul><li>PFAS compounds such as <strong>PFOS, PFHxS, PFHpS, PFNA</strong>, and <strong>6:2 FTS</strong> were identified in real serum samples.</li><li>The <strong>cyclic IMS</strong> allowed for the separation of coeluting isobaric compounds, such as <strong>PFOS isomers</strong> and <strong>cholic acid biomarkers</strong>, minimizing false identifications (Figure 3).</li><li><strong>Figure 4</strong> lists CCS values and other parameters for PFAS standards and identified analytes from a firefighter serum sample.</li><li><strong>Figure 5</strong> shows ion mobility conformeric profiles for <strong>PFNA</strong>, enabling reliable identification even at low signal intensities.</li></ul><h4><strong>Conclusion</strong></h4><p>The LC-cIM-MS method:</p><ul><li>Significantly improves specificity in PFAS identification.</li><li>Enables both <strong>targeted and untargeted detection</strong> of known and unknown PFAS in complex human samples.</li><li>Can be a key part of future occupational and environmental exposure assessments.</li></ul>

News from LabRulezLCMS Library - Week 28, 2025

<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 14th July 2025? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!</strong></p><blockquote><p><span style="font-size:20px;">👉 </span><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library"><span style="font-size:20px;"><strong>SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES</strong></span></a></p></blockquote><blockquote><p><strong>👉 Need info about different analytical techniques?</strong> Peek into <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library"><strong>LabRulezGCMS</strong></a> or <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library"><strong>LabRulezICPMS</strong></a> libraries.</p></blockquote><p>This week we bring you posters by <strong>Agilent Technologies / ASMS, Shimadzu / ASMS, Thermo Fisher Scientific / ASMS </strong>and<strong> Waters Corporation / ASMS!</strong></p><h3><strong>1. Agilent Technologies / ASMS: Faster Than the Speed of Life: A Rapid Screening Method for Select PFAS Compounds in Serum by RapidFire-MS</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/ps_rapidfire_ms_pfas_serum_asms_2025_wp_243_en_agilent_c8b56f4dd5.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Per- and polyfluoroalkyl substances (PFAS) are a broad class of compounds with a high degree of fluorination used in a variety of industries and consumer products over the past seventy years. Because of their widespread use, as well as their persistence and poorly understood effects on the human body, laboratories have started to monitor their presence in biological fluids, including serum. Biological samples are commonly prepared with protein precipitation. Additional cleanup steps such as solid phase extraction (SPE) with either C18 or weak anion exchange (WAX) sorbent can be used for increased sensitivity and cleanup. Alternatively, a pass-through cleanup using Captiva EMR-Lipid removes proteins and phospholipids through filtration, which significantly reduces sample complexity.</p><h4><strong>Experimental</strong></h4><p>Purchased serum was aliquoted into two sets of samples, one for protein precipitation (ppt) only and one for a protein precipitation plus Captiva EMR-Lipid based cleanup. For each preparation, 100 µL serum was aliquoted into a tube and spiked with 25 native and extracted isotopically labeled (EIS) compounds (EPA 533 list). The samples were vortexed for 1 minute and then crashed with 1% formic acid in cold ACN. They were vortexed for 3 minutes and then spun for 10 minutes to complete the crash. Samples undergoing the Captiva-EMR Lipid cleanup were then loaded onto a pre-rinsed cartridge and allowed to filter through (Figure 1). All samples were then spiked with the additional non-extracted isotopically labelled (NIS) compounds, vortexed, and transferred to a plate for analysis.</p><p>The RF/TQ system consisted of a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=1&amp;search=RapidFire+400"><strong>RapidFire 400</strong></a> front end with a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=1&amp;search=6495D+triple+quadrupole"><strong>6495D triple quadrupole mass spectrometer</strong></a> for detection. Samples were introduced into the mass spectrometer using a RapidFire cartridge for additional cleanup, and the total cycle time was about 12.5 seconds, with analytes detected in negative polarity. Specific method conditions are detailed in Tables 1 (RF) and 2 (MS).</p><h4><strong>Results and Discussion</strong></h4><h5><strong>Early Development Work&nbsp;</strong></h5><p>Feasibility studies for detection of PFAS compounds in serum using the RapidFire started by looking for PFAS background in the instrument and in all steps of the workflow. Early testing demonstrated no detectable background from any stage of the process.</p><p>RapidFire method development tested a variety of cartridges for optimal binding of the PFAS compounds, as well as determining the best state times and solvent conditions. For the preliminary work, the C18 cartridge appeared to have the best results, while the optimized elution solvent was 5 mM ammonium acetate in methanol.</p><p>Early experiments were performed to understand the effects of dilution and sample prep on the overall sensitivity of the method. When only a protein precipitation preparation was employed, dilution had a significant positive effect on analyte sensitivity, as shown in Figure 2. However, when the sample prep routine utilized protein precipitation in combination with a Captiva EMR-Lipid cleanup, additional dilution was not necessary and in fact decreased overall sensitivity, as shown in Figure 3. This is attributed to the removal of extraneous matrix using the Captiva EMR-Lipid device, which has a unique selectivity for unbranched alkane chains, capturing phospholipids while allowing PFAS compounds to pass through the sorbent bed unretained.</p><h5><strong>Custom Cartridge Work&nbsp;</strong></h5><p>This preliminary work was done using the C18 cartridge, but the cartridge had poor retention of the short-chain PFAS compounds (C5 and below). To address this issue, a cartridge was custom-packed with mixed mode material that included C18 and anion exchange resins. Further testing and method optimization for this specific packing material yielded better results across the board, but especially for the short-chain PFAS compounds. Sensitivity for most analytes was at or below 0.1 ppb, as summarized in Table 3, with a calibration range of 0.01- 10 ppb tested for all analytes.</p><h4><strong>Conclusions</strong></h4><ul><li>LOQs for most PFAS analytes of interest in serum were 0.1 ppb or below</li><li>Samples were injected every 12 seconds, allowing for high throughput analysis</li><li>Custom mixed mode cartridge had best overall performance&nbsp;</li><li>There was no background PFAS contribution from the RapidFire system</li></ul><h3><strong>2. Shimadzu / ASMS: Benchtop Matrix Assisted Laser Desorption Ionization Mass Spectrometry of PFAS for preliminary screening of environmental samples</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/ASMS_2025_MP_588_4c415fced0.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants which negatively impact human health and the environment.&nbsp;<br>High concentrations of PFAS in test samples can cause contamination of sensitive LC-MS/MS instruments requiring extensive decontamination procedures, resulting in significant downtime and costly delays in sample processing.&nbsp;</p><p>We present the development of a simple, cost-effective method for the rapid screening of PFAS samples using an entry-level linear benchtop MALDI-TOF mass spectrometer to identify samples containing high levels before they enter the analytical workflow.</p><h4><strong>Methods</strong></h4><p>PFOA and PFOS standards were purchased from Merck, UK. Stock solutions (1 ppm prepared in acetonitrile) were diluted in water to the required concentrations.&nbsp;</p><p>Samples were tested with a variety of MALDI matrices (CHCA, DHB, DHAP, DAN, NRM and TMGN), with norharmane (NRM) and 1,8-bis(tetramethylguanidino)-naphthalene (TMGN) identified for further investigation. Diluted samples were mixed with NRM (1 mg/mL) or TMGN (5 mg/mL) in 70:30 Acetonitrile:Water. Blank samples of UHP water were prepared to ensure no contamination was present. 1 µL of the mixture was spotted onto a FlexiMassSR48 slide and the samples were analysed using a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=3&amp;search=MALDI-8030"><strong>MALDI-8030 MALDI-TOF mass spectrometer</strong></a> (Shimadzu). Analysis conditions are shown in Fig 1. Peaks at m/z 314.2 and m/z 499.2 were monitored for PFOA and PFOS, respectively. 13C8PFOS (m/z 507) was used as an internal standard for PFOS samples with a final sample concentration of 6.25 ng/mL (see Fig. 2). MALDI-MSI was used to assess spot homogeneity and limits of detection were established.</p><p>Subsequently, PFHxA and PFBS standards were purchased from Merck, UK and analysed using the same method</p><h4><strong>Conclusion</strong></h4><ul><li>We have shown, in principle, that it is possible to use MALDITOF mass spectrometry to implement a simple pre-screening method for aqueous samples to prevent widespread contamination in a PFAS monitoring laboratory. Using just a 200 µL sample and minimal preparation, a full slide of samples can be acquired in less than 15 minutes&nbsp;</li><li>Spot imaging during method development highlighted the differences in homogeneity of PFOS and PFOA spots suggesting that multiple internal standards would be necessary to pursue semi-quantitative analysis.&nbsp;</li><li>Whole slide imaging can provide quick results for multiple samples across a range of PFAS concentrations in a single run with easy visual identification of potentially problematic samples.&nbsp;</li><li>As a linear MALDI-TOF method, these results do not confirm the presence of PFAS but may be used as an indicative result to modify further testing protocols. Confirmatory LC-MS/MS analysis is required to determine the presence and concentration of any PFAS.</li></ul><h3><strong>3. Thermo Fisher Scientific / ASMS: Enhanced resolution with multi-pass mode on the Orbitrap Astral Zoom mass spectrometer&nbsp;</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/PO_319_ms_ASMS_25_Astral_Zoom_TMT_po319_na_en_a16d57e473.pdf"><strong>Full PDF for download</strong></a></li></ul><p>The introduction of Thermo Scientific TMT 32-plex, with reporter ions clustered into quadruplets spaced by 3 mDa, demands a resolving power of approximately 90 k at low m/z, which challenges state-of-the-art time-of-flight mass analyzers. The Thermo Scientific Orbitrap Astral mass spectrometer [1] typically achieves around 60 k resolution at m/z = 138, falling to 50 k under high space charge influence.&nbsp;</p><p>Here we present an enhanced resolution multi-pass method implemented in the new Thermo Scientific <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1959"><strong>Orbitrap Astral Zoom mass spectrometer</strong></a> [2], significantly improving the resolving power at low m/z enabling TMT 32-plex on the Thermo Scientific Astral analyzer. The ions are repeatedly guided through a switchable prism deflector to extend the ion path while the principal aberrations are compensated, thereby more than doubling the resolution.</p><h4><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1959"><strong>The Orbitrap Astral Zoom Mass Spectrometer</strong></a></h4><p>The Orbitrap Astral Zoom mass spectrometer incorporates a range of novel technologies for higher speed, sensitivity, dynamic range and resolving power. A faster ion filter and quadrupole switching in combination with improved ion transfer times in the ion-routing multipole (IRM) and ion processor allow DIA methods (Thermo Scientific Pierce FlexMix calibration solution, 2 Th isolation window, 350-980 m/z range, Thermo Scientific Orbitrap MS and Astral MS/MS) with scan rates faster than 270 Hz (DDA methods &gt; 180 Hz). A duty cycle of up to 80 % at 270 Hz is made possible by a new accumulation stage in the bent trap (linear to 106 ions) which increases the effective ion injection into the IRM by up to 40 % (additional ~0.75 ms on top of 2 ms IT in a DIA method). Furthermore, an additional mode of the high-dynamic range detector of the Astral analyzer for low-input sample applications like single-cell proteomics boosts the single ion detection efficiency by about 10 % and thereby the sensitivity. In combination with a new peak deconvolution algorithm for enhanced spectral processing, this increases the depth of analysis. In addition, modified IRM hardware allows for faster stepped collision energy experiments for deeper spectral coverage (2 CE: 80 Hz (OT Astral), 180 Hz (Orbitrap Astral Zoom MS), 3 CE: 60 Hz, 150 Hz). A modified AGC filling in the Orbitrap by a new enhanced dynamic range mode (EDR) provides additional spectral complexity and sensitivity. Here, multiplexed ion injections with different accumulation times are used for different mass ranges taking the variation in signal intensity into account. In addition, the Astral analyzer incorporates a new multi-pass mode which enhances the resolving power for small masses by more than a factor of two, enabling TMT 32-plex measurement in the Astral analyzer.</p><h4><strong>Conclusions&nbsp;</strong></h4><p>The TMT HR Mode advances the capabilities of the Astral analyzer. Fast electronic switching of the relay prism opens an ion trap and guides the ions after three times their normal flight distance to the detector. This greatly boosts the resolving power, especially for small m/z ratios, thereby enabling baseline-resolved quadruplets of reporter ions in a TMT 32-plex sample. First results show a peak resolution well above 100 k while maintaining high mass accuracy and shot-to-shot stability. The TMT HR Mode is utilized within a novel acquisition scheme delivering greater throughput for protein identification and quantitation for multiplexing up to 32 samples. Achieving a throughput of over 440 samples per day, the proteome coverage included 4,299 quantified protein groups and 22,146 peptides.</p><h3><strong>4. Waters Corporation / ASMS: Assessing the Impact of Part-per-Billion Mass Accuracy Upon Forensic Toxicology Data-Independent Analysis Screening&nbsp;</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720008882_68ab66ac31.pdf"><strong>Full PDF for download</strong></a></li></ul><p>This poster presents a forensic toxicology screening approach using <strong>ultra-high mass accuracy</strong> to improve specificity and efficiency in the analysis of complex biological matrices, such as urine. The need for rapid, accurate, and cost-effective screening is critical in forensic laboratories. The study evaluates the impact of <strong>parts-per-billion (ppb) mass accuracy</strong> using the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1148"><strong>Waters Xevo MRT Mass Spectrometer</strong></a><strong> </strong>compared to previous DIA methods.</p><p>The method involved analyzing a commercial standard and ten anonymized urine samples, diluted and analyzed via the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/199"><strong>ACQUITY UPLC I-Class FTN PLUS System</strong></a> coupled with the <strong>Xevo MRT MS</strong>. Data acquisition was performed in <strong>MSE mode</strong> (data-independent acquisition) under positive electrospray ionization (ESI+) using a 15-minute gradient. Data were processed in waters_connect software, leveraging a comprehensive spectral library with over 2000 compounds.</p><p>Results demonstrated a <strong>root mean square (RMS) mass error of just 571 ppb</strong> for precursor ions, with 94% of compounds detected within 1 ppm, and 96% of fragment ions within the same tolerance. A key example illustrated how reducing fragment ion tolerance from 2 mDa to 0.2 mDa eliminated a false positive (Anabasine), correctly identifying Nicotine as the true analyte, showing how ppb-level accuracy increases identification confidence and minimizes misidentifications.</p><p>The study concludes that ppb mass accuracy significantly reduces false positives, lowers review workloads, and facilitates independent analysis by less experienced personnel. Additionally, it supports cost savings by minimizing the need for confirmatory testing, potentially reducing both financial and environmental burdens in forensic laboratories.</p>

News from LabRulezLCMS Library - Week 29, 2025

<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 21st July 2025? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!</strong></p><blockquote><p><span style="font-size:20px;">👉 </span><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library"><span style="font-size:20px;"><strong>SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES</strong></span></a></p></blockquote><blockquote><p><strong>👉 Need info about different analytical techniques?</strong> Peek into <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library"><strong>LabRulezGCMS</strong></a> or <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library"><strong>LabRulezICPMS</strong></a> libraries.</p></blockquote><p>This week we bring you posters by <strong>Agilent Technologies / ASMS, Shimadzu / ASMS, Thermo Fisher Scientific / ASMS, Waters Corporation / ASMS</strong> and application note by<strong> Metrohm!</strong></p><h3><strong>1. Agilent Technologies / ASMS: De Novo PFAS Annotation and Classification Using Highly Accurate Formula Prediction and Kaufmann Algorithms Embedded in FluoroMatchSuite</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/ps_fluoromatch_de_novo_pfas_annotation_asms_2025_wp_113_en_agilent_4190191ba4.pdf"><strong>Full PDF for download</strong></a></li></ul><p><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=FluoroMatch+Suite"><strong>FluoroMatch Suite </strong></a><strong>MS1 Only Algorithms: </strong>FluoroMatch Suite covers the entire non-targeted PFAS workflow (Figure 1) using MS/MS algorithms and the largest fragmentation database to date providing a &lt;5% false positive rate. However, MS/MS coverage is often incomplete, and DIA methods often provide lower quality spectra after deconvolution. Therefore, we developed MS1 only algorithms for providing highly accurate formula predictions (Figure 2) and for classifying features as PFAS or not PFAS using Kaufmann analysis (Figure 3)&nbsp;</p><p><strong>Validation: </strong>Algorithms were validated on NIST A and NIST C samples from the NIST PFAS interlaboratory study. NIST A consisted of known PFAS chemical standards, and NIST C consisted of an aqueous fire fighting foam (AFFF) contaminated soil sample. Formula prediction with homologous series voting (taking the most common repeating formula) was extremely accurate for both samples with a 0% false positive rate, and without homologous series voting the false positive rate was between 17-26% (Table 1). Kaufmann Analysis was highly specific for classifying PFAS with only 4% of non-PFAS being classified as PFAS with our Kaufmann based model, and only 6% of PFAS not be classified as PFAS according to our model for NIST C (Table 2).</p><h3><strong>2. Metrohm: Detection of heavy metals with differential pulse voltammetry</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_sens_002_download_aa3cc76472.pdf"><strong>Full PDF for download</strong></a></li></ul><p>DPV, alongside square wave voltammetry (SWV), already discussed in <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_sens_001_download_6ed955a5ff.pdf"><strong>AN-SENS-001</strong></a>, is another example of a pulsed voltammetric technique. DPV was actually developed first and is often linked to the field of polarography. This pulsed voltammetric technique is typically used with a dropping mercury electrode, although the general principle can be applied to static electrodes as well. In the case of DPV, a short pulse with constant height is superimposed at the end of every step of a staircase linear sweep voltammetry (LSV) determination (Figure 1). During the experiment, the current is sampled at two points: before (i1 ) and after (i2 ) the pulse. The difference (i1 -i2 ) vs. the potential is the signal which is plotted in DPV. Sometimes i1 is referred to as the base current and i2 as the pulse current. Figure 1. Typical DPV waveform for the forward direction (e.g., from 0 to 1 V).&nbsp;</p><p>Like SWV, this approach allows the separation of faradaic and capacitive current, boosting the sensitivity of the technique compared to normal staircase voltammetry. In <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=220&amp;search=INTELLO"><strong>INTELLO</strong></a>, the i1 and i2 signals can optionally be sampled in the signals tab of the DPV command. In NOVA, custom plots of either signal can be produced in the same tab. The waveform is characterized by the height of the pulse (ΔE) and the step height (ΔEs ). The scan rate is determined only by step height and step duration of the underlying LSV. The pulse duration should not be set longer than the step duration in order to avoid artifacts, with typical values for the step being around 100–500 ms, and around 50 ms for the pulse. In comparison to SWV, DPV is considered less applicable to a wider range of systems due to the interference of oxygen and the slower scan rates required for DPV. In some cases it can produce better separation of closely positioned peaks and sharper peaks in general. The following is an example of a typical DPV measurement made with a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=220&amp;search=PGSTAT302N"><strong>PGSTAT302N</strong></a> and the Metrohm 663 VA stand, with the same measurement also possible with the next-generation <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=220&amp;search=VIONIC"><strong>VIONIC </strong></a>powered by INTELLO.</p><h4><strong>SAMPLE AND EXPERIMENTAL DETAILS&nbsp;</strong></h4><p>A PGSTAT302N equipped with a Metrohm 663 VA stand (2.663.0020) was used for this study. The working electrode (WE) was a Multi-Mode Electrode pro (6.1246.120) in the hanging dropping mercury electrode (HDME) configuration. The reference electrode (RE) was a double junction Ag/AgCl reference electrode (6.0728.120). All potential values mentioned in this Application Note are referred to the potential of this electrode. The electrolyte was composed of 10 mL H2 O + 0.5 mL of acetate buffer, made of 1 mol/L ammonium acetate + 1 mol/L acetic acid.&nbsp;</p><p>The concentration of heavy metal ions was quantified by making two standard additions. For the first addition, the solution was composed as follows: 10 mL H2 O, 500 μL acetate buffer, 100 μL Pb standard solution (1 mg/L), and 100 μL Cd standard solution (1 mg/L). For the second addition, the solution was composed as follows: 10 mL H2 O, 500 μL acetate buffer, 200 μL Pb standard solution (1 mg/L), and 200 μL Cd standard solution (1 mg/L).&nbsp;</p><p>Each measurement consisted of a step to precondition the Hg electrode, where nitrogen was purged in the stirring solution and a new Hg drop was formed. Then, a reduction potential of -0.9 V was applied at the Hg drop in order to reduce the Pb and Cd cations at the Hg drop surface under stirring conditions and accumulate them at the Hg drop. The stirrer was then switched off and the DPV measurement was performed by oxidizing the previously accumulated Pb and Cd in a positive potential sweep from -0.9 to -0.2 V. The DPV measurement was repeated twice.&nbsp;</p><p>The peak heights were determined from the current vs. potential plot of the three experimental iterations (i.e., sample, sample with first standard addition, and sample with second standard addition). Peak height vs. analyte concentration plots were created for both Pb and Cd. The analyte concentration in the sample can be calculated from the intersection of the regression line with the x-axis.</p><h3><strong>3. Shimadzu / ASMS: EPA 533 and 537.1 with the Novel LCMS-8050RX: Demonstration of Instrument and Method Performance to meet PFAS regulatory levels&nbsp;</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/ASMS_25_TP_270_ebf0e2c9b0.pdf"><strong>Full PDF for download</strong></a></li></ul><p>The harmful impacts of per- and polyfluorinated alkyl substances (PFAS) continue to afflict the environment. Water utilities require sensitive and robust analytical methods to assess the current and ongoing burden of PFAS from source waters to meet regulatory demands in treated water and to protect public health. Improvements in sensitivity and/or runtime without sacrificing robustness can aid laboratories by increasing sample throughput to better meet the significant demand for PFAS sample analysis. The novel RX probe was developed to yield better heat distribution and more consistent nebulization to improve sensitivity and reproducibility. Chromatographic improvements compared to existing EPA methods were also performed to decrease runtime while maintaining the integrity of sample analysis.</p><h4><strong>Methods</strong></h4><p>PFAS were separated by a Nexera LC and quantified by a Shimadzu <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=LCMS-8050RX"><strong>LCMS-8050RX</strong></a> for EPA methods 533 and 537.1 (EPAM533 and EPAM537.1). Neat standards were analyzed in the required composition (i.e., 80% methanol for EPAM533 and 96% methanol for EPAM537.1). Standards were analyzed twenty times to determine the instrument detection limits.</p><h4><strong>Results and Conclusion</strong></h4><ul><li>All PFAS were sufficiently analyzed by the respective methods and passed regulatory criteria at a reduced runtime (19 minutes shorter).</li><li>Superb precision and reproducibility were demonstrated with the RX probe, which resulted in an analytical blueprint that greatly exceeds the Final PFAS National Primary Drinking Water Regulation promulgated in 2024.</li></ul><h3><strong>4. Thermo Fisher Scientific / ASMS: Comprehensive and high-throughput plasma proteome profiling for biomarker discovery using the Orbitrap Astral Zoom Mass Spectrometer and the Seer Proteograph ONE workflow</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_381_plasma_proteome_profiling_biomarker_discovery_po381_asms2025_en_074e0d2a50.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Plasma proteomics, the study of proteins in blood plasma, is crucial for biomarker discovery and understanding global signaling and immune responses1, aiding disease diagnosis, monitoring, and personalized medicine. However, the plasma proteome's complexity and dynamic range—spanning up to 12 orders of magnitude²—pose challenges, especially for detecting low-abundance proteins. Additionally, preanalytical, analytical, and biological variables complicate reliable biomarker identification, necessitating optimized techniques³. Liquid chromatography-mass spectrometry (LC-MS) excels in plasma proteomics due to its high analytical dynamic range and capability to measure peptides and characterize posttranslational modifications (PTMs). Advances in sample preparation, separation, MS, and data analysis are improving method sensitivity and robustness. We demonstrate the Thermo ScientificTM OrbitrapTM AstralTM Zoom Mass Spectrometer's high sensitivity and reproducibility in detecting a comprehensive plasma proteome across neat, depleted, and plasma processed with the Seer® Proteograph® ONE workflow, facilitating potential biomarker discovery¹.</p><h4><strong>Experimental Procedure</strong></h4><h5><strong>Liquid chromatography-mass spectrometry &amp; data analysis</strong></h5><p>All LC-MS runs for neat, depleted, and Proteograph ONE processed peptides were separated and analyzed using a Thermo Scientific <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/398"><strong>Vanquish Neo UHPLC</strong></a> System in trap and elute configuration, paired with an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1959"><strong>Orbitrap Astral Zoom Mass Spectrometer</strong></a>. Peptides were separated on the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/398"><strong>Vanquish Neo UHPLC</strong></a> System using Thermo Scientific EASY-Spray PepMap analytical columns (ES906, 2µm C18, 150µm × 15 cm for 100 and 60 SPD and ES75500PN 2µm C18, 75µm × 50 cm for 24SPD) and 12.6-, 22.35-, and 54-minute chromatographic gradients, respectively, were formed using 0.1% formic acid in water for mobile phase A and 0.1% formic acid in 80% acetonitrile for mobile phase B. Samples were injected with 2 loading mass amounts (200/500 ng for 100/60 SPD; 500/1250 ng for 24 SPD) in triplicate. Liquid chromatography parameters are shown below in Table 1A. Mass spectrometer scan parameters can be found below in Table 1B. All data was processed using the Proteograph® Analysis Suite (PAS) (Seer). Output files were imported to Rstudio (2023.09.0 Build 463) with R (v4.3.1) for downstream data analysis and visualization.</p><h4><strong>Conclusions&nbsp;</strong></h4><ul><li>With the Orbitrap Astral Zoom Mass Spectrometer, neat plasma proteome coverage exceeds 1,000 protein groups in a single pooled sample, providing exceptional quantitative precision without additional sample handling steps.&nbsp;</li><li>High Select Top 14 abundant protein depletion enhances plasma proteome depth by ~2x across 100, 60, and 24 SPD throughputs with comparable or superior analytical measurement precision.&nbsp;</li><li>Seer Proteograph ONE enhances plasma proteome depth by 7.9x (100SPD), 6.9x (60SPD), and 6.2x (24SPD) with comparable or superior analytical measurement precision and no compromise on sample preparation throughput.&nbsp;</li><li>The combination of the Orbitrap Astral Zoom Mass Spectrometer and the Proteograph ONE Workflow allows for over 7,000 protein groups to be identified from a single injection.&nbsp;</li><li>The speed, sensitivity, and robustness of the Orbitrap Astral Zoom Mass Spectrometer enables in-depth biological discovery, paving the way for novel findings and advancements in clinical cohort and population-scale translational research.</li></ul><h3><strong>5. Waters Corporation / ASMS: PFASt and Furious: A Targeted and Non-Targeted LC-MS/MS Study on the Environmental Impact of Using Fluorinated Ski Waxes&nbsp;</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720008880_8590308c24.pdf"><strong>Full PDF for download</strong></a></li></ul><p>This study by Waters Corporation explores the environmental impact of fluorinated ski waxes—specifically per- and polyfluoroalkyl substances (PFAS)—on soil and water quality at competitive ski sites in New England. Using both targeted LC-MS/MS and non-targeted high-resolution mass spectrometry, the research team analyzed samples from slalom and cross-country racing courses to determine the extent of PFAS contamination.</p><p>PFAS were found in all collected samples, with soil showing significantly higher concentrations than water. Long-chain perfluorocarboxylic acids, commonly used in ski waxes, dominated the contaminant profile. At slalom courses, the highest concentrations were found at the wax application zones near the start line, decreasing progressively downhill. At cross-country sites, similar patterns emerged near waxing and testing areas, with alarming migration of PFAS into adjacent ponds and even a vegetable garden.</p><p>The study employed automated sample preparation systems (CEM EDGE and Promochrom SPE-03) to ensure efficiency and reproducibility. Advanced LC-MS/MS analysis with Waters’ <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/191"><strong>ACQUITY Premier </strong></a>and <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/554"><strong>Xevo TQ Absolute</strong></a> platforms enabled the detection of common PFAS, while cyclic ion mobility HRMS revealed additional, untargeted long-chain species (e.g., C15/C16 carboxylic acids) not typically included in regulatory workflows.</p><p>The findings highlight the long-term environmental risks posed by legacy ski wax formulations and underscore the need for continued monitoring. Notably, unexpected PFOS levels in garden soil suggest multiple PFAS sources may be contributing to contamination, warranting further investigation.</p>

News from LabRulezLCMS Library - Week 30, 2025

News from LabRulezLCMS Library - Week 31, 2025

News from LabRulezLCMS Library - Week 32, 2025

<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 11th August 2025? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!</strong></p><blockquote><p><span style="font-size:20px;">👉 </span><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library"><span style="font-size:20px;"><strong>SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES</strong></span></a></p></blockquote><blockquote><p><strong>👉 Need info about different analytical techniques?</strong> Peek into <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library"><strong>LabRulezGCMS</strong></a> or <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library"><strong>LabRulezICPMS</strong></a> libraries.</p></blockquote><p>This week we bring you posters by <strong>Agilent Technologies / ASMS </strong>and <strong>Thermo Fisher Scientific / ASMS </strong>and application notes by <strong>Shimadzu </strong>and<strong> Waters Corporation!</strong></p><h3><strong>1. Agilent Technologies / ASMS: Fully Automated Sensitive Quantitation of PFAS in Seafood for Regulatory Screening Using Triple Quadrupole LC/MS&nbsp;</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/ps_6495d_pal3_pfas_asms_2025_mp_240_en_agilent_234d300c8d.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Per- and polyfluoroalkyl substances (PFAS) are extensively found in marine life, posing health risks through seafood consumption. Seafood is one of the regulated matrices by the U.S. FDA, EU, EURL POPs, and AOAC. Regulatory and standard-making bodies like the U.S. FDA, EFSA, EURL POPs, and AOAC have set stringent guidelines for PFAS levels in seafood. Detecting trace PFAS level is challenging due to complex sample preparation like QuEChERS extraction followed by SPE cleanup, and evaporation/reconstitution. The manual steps can be labor-intensive and error-prone, affecting accuracy and reliability. This study developed a fully automated workflow for PFAS quantitation in seafood – shrimp.</p><h4><strong>Experimental</strong></h4><h5><strong>Instrumentation&nbsp;</strong></h5><p>An integrated 160 cm <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=+PAL3"><strong>PAL3 Series 2 RTC autosampler</strong></a> coupled with a<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=+6495D+LC%2FMS"><strong> 6495D LC/TQ</strong></a> (Figure 1) was employed. The PAL3 platform performed automated sample preparation, while TQ data acquisition and analysis were conducted in parallel. LC system comprised of two modules; an Agilent 1290 Infinity II high-speed pump and an Agilent 1290 Infinity II multicolumn thermostat. The study utilized the following tools and modules: two PAL park stations with three liquid syringe tools, a dilutor tool, a micro-SPE tool, an LC/MS tool, a vortex mixer, a centrifuge, a dilutor multi, a tray cooler (for 2/10/20 mL vials), tray holders with rack R60 (for 10/20 mL vials), a micro-SPE tray (for 2 mL vials and micro-SPE cartridges), a solvent module, a fast wash module, and an LC injection valve. The details of various consumables used for this study are provided elsewhere.1</p><h4><strong>Conclusions</strong></h4><ul><li>The integrated system allows sample preparation and data analysis to run in parallel, offering a streamlined workflow and improving productivity for routine laboratory operations.&nbsp;</li><li>The automated workflow significantly reduces manual intervention, minimizing human error and enhancing the precision of the analysis.&nbsp;</li><li>The integration of automated sample preparation techniques with the highly sensitive 6495D LC/TQ ensures consistent and reproducible results, which are critical for regulatory compliance.&nbsp;</li><li>The workflow demonstrated excellent analytical performance meeting the stringent regulatory requirements and recommendations for PFAS in seafood matrices set by the U.S. FDA, EU, EURL POPs, and AOAC.</li></ul><h3><strong>2. Shimadzu: Determination of PFASs in Food Contact Material by </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=LCMS-8050"><strong>LCMS-8050</strong></a></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_03_lcmsms_961_en_34db9cd4cb.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Due to the unique physical and chemical properties of per- and polyfluoroalkyl substances (PFASs), such as reducing surface tension, good stability, hydrophobicity, and hydrophilic, so it is very widely used in the field of food contact materials. It is mainly used for the production of plastic film, PVC food packaging, waterproof, and oil-proof coating on the surface of food packaging (such as waterproof oil paper products) and non-stick cooker coating, etc. Research has shown that PFASs will migrate from food contact materials to food, and then contaminate the food that comes into contact with it, entering the human body. PFASs are very stable, difficult to be metabolized and biodegraded in the body, and exhibit the characteristics of persistence, accumulation, and long-distance migration. It can be accumulated and amplified in the food chain and enriched in the organisms, endangering human health.&nbsp;</p><p>Refer to the sample preparation method in GB 31604.35-2016, determination of both Perfluorooctane Sulfonates (PFOS) and Perfluorooctanoic Acid (PFOA), an analytical method for the determination of 17 perfluorinated compounds in food contact materials was built.</p><h4><strong>Analysis Conditions</strong></h4><h5><strong>System: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=Nexera+XS"><strong>Nexera XS&nbsp;</strong></a></h5><ul><li>Column : Shim-pack GIST C18-AQ HP (2.1 mm I.D.×100 mm, 1.9 μm)</li><li>Temperature : 30 ˚C&nbsp;</li><li>Injection volume : 5 µL&nbsp;</li><li>Mobile phases : A-5 mM ammonium acetate in Water B-ACN&nbsp;</li><li>Flow rate : 0.3 mL/min&nbsp;</li><li>Elution mode : Gradient elution&nbsp;</li><li>Time program (%B) : 15% (0 min) → 15% (2.5 min) →98% (10-12 min) → 15% (12.1-15 min)&nbsp;</li></ul><h5><strong>System: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=LCMS-8050"><strong>LCMS-8050</strong></a><strong> (ESI Negative)&nbsp;</strong></h5><ul><li>Nebulizing gas : 3 L/min&nbsp;</li><li>Drying gas : 10 L/min&nbsp;</li><li>Heating gas : 10 L/min&nbsp;</li><li>DL temp. : 250 ˚C&nbsp;</li><li>Heat block temp. : 400 ˚C&nbsp;</li><li>Interface temp. : 300 ˚C</li></ul><h4><strong>Conclusion&nbsp;</strong></h4><p>Utilizing the LCMS-8050 system for quantitative analysis of PFASs in Food Contact Material revealed that the method can accurately determine PFASs levels within a broad concentration range of 0.05 to 5 ng/mL. This approach exhibits several notable advantages, including excellent repeatability, stability, and reliability.</p><h3><strong>3. Thermo Fisher Scientific / ASMS: Leveraging advanced mass spectrometry technology with </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1959"><strong>Orbitrap Astral Zoom MS</strong></a><strong> for in depth immunopeptidome profiling&nbsp;</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_414_advanced_mass_spec_technology_orbitrap_astral_zoom_po414_asms2025_en_3ca6419434.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Immunopeptidomics is the study of the peptides presented by MHC molecules on the surface of cells. These MHC peptides have major implications for many areas of research, including immunotherapy and personalized medicine. Mass spectrometry allows for direct immunopeptidomics analysis, enabling simultaneous identification and quantification of thousands of MHC peptides in a single run. The new <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1959"><strong>Orbitrap Astral Zoom MS </strong></a>has enabled new levels of sensitivity and selectivity to provide deeper insights into the immunopeptidome.</p><h4><strong>Materials and methods</strong></h4><h5><strong>LC-MS/MS method&nbsp;</strong></h5><p>Peptides were separated on a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/398"><strong>Vanquish Neo UHPLC System</strong></a> using Aurora Ultimate column (25 cmx 75µm). Total run time was 72 min. Thermo Scientific EASY-Spray Ion Source was used coupled to the Thermo Scientific FAIMS Pro Duo interface. Peptides were analyzed by the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1959"><strong>Orbitrap Astral Zoom MS</strong></a>.</p><h5><strong>Data Processing&nbsp;</strong></h5><p>The data analysis was performed using PEAKS Studio 12 with DeepNovo Peptidome workflow for database search and de novo peptides identification. Spectra were searched against UniProt human database (20,607 sequences) with no-enzyme option. The sequence motif and binding properties of 9-mer peptides were analyzed using MHCMotifDecon 1.2 and NetMHCpan 4.0.</p><h4><strong>Conclusions&nbsp;</strong></h4><ul><li>Orbitrap Astral Zoom MS equipped with FAIMS Pro Duo interface and coupled to Vanquish Neo UHPLC system provides high sensitivity and the dynamic range necessary for deeper coverage of immunopeptidomic samples</li><li>The low input application mode on Orbitrap Astral Zoom MS offers the sensitivity necessary to analyze low levels of material equivalent to samples ectracted from tissue biopsy samples</li><li>Loading capacity is not limited by MS allowing analyses from ultra low (1e5) to high (1e8) loads of sample&nbsp;</li><li>FAIMS enables the analysis of singly and multiply charged ions enhancing the immunopeptidome coverage&nbsp;</li><li>AGC modulates injection times for each peptide, producing high quality spectra across a wide range of loading levels. This spectral quality is crucial for downstream analysis and confident identification and quantitation of neoantigens</li></ul><h3><strong>4. Waters Corporation: Optimization of Detector Parameters to Improve Sensitivity using the </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/717"><strong>Alliance™ iS HPLC System </strong></a><strong>with PDA Detector</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720008901_en_70df6fce93.pdf"><strong>Full PDF for download</strong></a></li></ul><h4><strong>Benefits&nbsp;</strong></h4><ul><li>The Alliance iS HPLC System with PDA Detector allows the user to easily modify PDA settings to obtain optimal performance, including signal-to-noise</li><li>The Alliance iS HPLC System with PDA Detector has a range of parameters, including variable slit width and wide resolution range for method optimization</li><li>Optimization of the PDA settings produced a S/N ratio increase of 7x</li></ul><p>Default PDA instrument method settings are frequently used for running HPLC methods. When running a method for the first time, these default settings are a good starting point and often produce suitable chromatography. However, detector parameters may need to be modified to obtain optimal separations. In this study, the USP method for organic impurities in ibuprofen tablets was used to show how the Alliance iS HPLC System with PDA Detector parameters may be optimized to improve sensitivity. The USP S/N ratio was used to define sensitivity. The method is a reversed-phase isocratic method with UV detection (254 nm). The method’s system suitability criteria includes a S/N ratio of not less than (NLT) 10 for a 5-ppm solution of ibuprofen.&nbsp;</p><p>The Alliance iS HPLC System with PDA Detector settings that were evaluated and optimized for their impact on sensitivity included data rate, filter time constant, slit width, resolution, and absorbance compensation.&nbsp;</p><ul><li><strong>Data rate:</strong> The data rate defines the rate that the detector collects data, measured in hertz (Hz). The data rate should be set to yield reproducible peak area and retention times. Data rates that are too low will result in poorly defined peaks; high data rates may result in increased noise. Generally, the data rate is set to collect 25-50 points across the narrowest peak in the chromatogram. The data rate of the Alliance iS HPLC Systém with PDA Detector has settable values of 1–160 Hz. The default data rate is 10 Hz.&nbsp;</li><li><strong>Filter time constant: </strong>The filter time constant is a noise filter that filters out high frequency noise. Faster time constants produce narrower peaks and remove less baseline noise. Slower time constants result in broader peaks and decreased baseline noise. The Alliance iS HPLC System with PDA Detector has the following filter time constant options: no filter, slow, normal, fast, and custom. The default selection is normal.</li><li><strong>Slit width:</strong> Slit width is an added feature available in the Alliance iS HPLC System with PDA Detector. The slit width determines the amount of light that reaches the photodiode array sensor which impacts resolution. Smaller slit widths result in improved resolution. Larger slit widths produce less noise and increased sensitivity, but at the cost of lower resolution. The Alliance iS HPLC System with PDA Detector has a variable slit, with available slit width sizes of 35 µm, 50 µm, 100 µm, and 150 µm. The detector’s default slit width is 50 µm.</li><li><strong>Resolution:</strong> The resolution setting determines bandwidth, which is the number of diode responses that are averaged when calculating the absorbance at a specified wavelength. Larger values result in less noise and a better S/N ratio, but they also produce decreased spectral resolution and can impact linearity. Resolution on the Alliance iS HPLC System with PDA Detector is settable over a range of 1<span>–</span>20 nm. The default value is 4 nm.&nbsp;</li><li><span style="font-family:&quot;Arial&quot;,sans-serif;"><strong>A</strong></span><strong>bsorbance compensation</strong>: The absorbance compensation feature of the Alliance iS HPLC System with PDA Detector provides a way to reduce non-wavelength dependent noise. This is achieved by collecting absorbance data over a user-specified wavelength range where there is little or no absorption and calculating the average absorbance. This average absorbance is then subtracted from the absorbance value. The Alliance iS HPLC System with PDA Detector default setting for absorbance compensation is ‘off’.</li></ul><p>Using the USP method, each of these detector settings were individually evaluated and optimized. A method was created using the optimized settings and the results were compared to those obtained using default values.</p><h4><strong>Experimental</strong></h4><h5><strong>LC Conditions&nbsp;</strong></h5><ul><li><strong>LC system: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/717"><strong>Alliance iS HPLC System</strong></a> with PDA Detector&nbsp;</li><li><strong>Detection:</strong> PDA Detector with 10 mm flow cell</li><li><strong>Column: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1823"><strong>XBridge™ BEH™ C18</strong></a>, 250 x 4.6 mm; 5 µm (p/n: 186003117)</li></ul><h5><strong>Data Management&nbsp;</strong></h5><ul><li><strong>Chromatography data system:</strong> <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/627"><strong>Empower™ Chromatography Data System (CDS)</strong></a></li></ul><h4><strong>Conclusion</strong></h4><p>While default PDA instrument method settings may be suitable for routine HPLC analysis, they may need to be adjusted to produce optimal separations. This study demonstrated how the Alliance iS HPLC System with PDA Detector allows the user to easily modify PDA settings to obtain optimal separations. Using the USP method for organic impurities in ibuprofen tablets, the data rate, filter time constant, slit width, resolution, and absorbance compensation settings were optimized. The optimized settings produced a 7-fold increase in sensitivity over that obtained using default values.</p>

News from LabRulezLCMS Library - Week 33, 2025

<p><strong>Introduced nearly 80 years ago, collision-based ion-activation and dissociation is the most commonly used type of fragmentation commercially available today. In principle, this technique is quite straightforward: apply energy to accelerate the particles of interest until they collide with an inert gas to induce fragmentation. Within these parameters, there are many different sub-categories of the technique. Here we will discuss three collision-based techniques that occur on Thermo Scientific mass spectrometers.</strong></p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Thermo_Fisher_Scientific_Freedom_to_Fragment_Introduction_The_importance_of_fragmentation_in_mass_spectrometry_1_c8f70d1281.jpg" alt="Thermo Fisher Scientific Freedom to Fragment Introduction" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Thermo_Fisher_Scientific_Freedom_to_Fragment_Introduction_The_importance_of_fragmentation_in_mass_spectrometry_1_c8f70d1281.jpg 245w," sizes="100vw" width="1024" height="512"></p><h3><strong>Higher-Energy Collisional Dissociation (HCD)</strong></h3><ol><li><strong>Process:</strong> HCD is a beam-based technique that occurs in the HCD cell or ion routing multipole, where the ion beam is directed into a potential well. Excited molecules collide with collision gas, causing fragmentation. This gas is typically nitrogen and is fast and efficient, but indiscriminate in technique. To ensure a higher energy collision, the potential energy is dropped (typically from the quadrupole into the collision cell) by many 10s of eV. This ensures the ions have high energy as they enter the cell with nitrogen and collide with enough energy for the fragmentation to occur. In peptides and proteins, HCD favors b- and y-ion formation. [Figure 1]</li><li><strong>Applications:</strong> HCD is commonly used for general proteomics and metabolomics.</li><li><strong>Considerations: </strong>When using Thermo Scientific mass spectrometers for HCD fragmentation, we generally use normalized collisional energy (NCE) instead of directly putting in the eV of potential well “drop”. The calibration routine determines the optimal voltage for a series of ions in the calibration solution.&nbsp; Then the routine scales these absolute voltages to a “normalized collision energy” value of 30. As such, an NCE value of 30 should work well across a range of <i>m/z</i> values and charge states. To see the actual applied value, one can check the scan header of the spectrum easily in our Thermo Scientific Freestyle software.</li></ol><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Thermo_Fisher_Scientific_Collision_Based_Ion_activation_and_Dissociation_92107a9524.jpg" alt="Thermo Fisher Scientific: Collision-Based Ion-activation and Dissociation: Figure 1: Location of peptide/protein backbone cleavage and its nomenclature. Representative here is a 4-amino acid peptide with side chains labeled R1-4." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Thermo_Fisher_Scientific_Collision_Based_Ion_activation_and_Dissociation_92107a9524.jpg 245w," sizes="100vw" width="1200" height="582"></p><h3><strong>Collision-Induced Dissociation (CID) (ion trap specific version)</strong></h3><ol><li><strong>Process: </strong>CID, as it occurs in Thermo Fisher Scientific’s dual linear ion trap technology, is a resonance-based technique where molecules are excited by a specific frequency.&nbsp; These excited molecules will start running into He molecules at a faster rate.&nbsp; These collisions slowly heat the resonant molecules until they fragment. Once fragmentation occurs, the product ions at new m/z values are no longer excited by the frequency and will cool back down to the center of the trap. In peptides and proteins, CID favors b- and y-ion formation.</li><li><strong>Applications: </strong>CID is great for small molecules and peptides and helps fragment oligonucleotides, especially when the q-value is adjusted.&nbsp; It also fragments with higher efficiency, which makes it well-suited for MS3 and higher workflows.</li><li><strong>Considerations: </strong>Because CID is resonance-based, there is a compromise outlined in the Matthieu equation. With certain voltages and frequencies applied, only certain m/z values can be trapped. Thus, we have something called the 1/3rd rule. This rule approximates the equation and states that any fragment created that is &lt; 1/3 the m/z of the precursor will not be trapped. This rule makes CID less desirable as a primary fragmentation technique, thus more used as the pinch hitter when needed for alternate fragmentation after HCD is shown inefficient for a specific molecule. Furthermore, CID is a slow heating process.&nbsp;As such, activation/fragmentation takes a few milliseconds, whereas HCD is effectively instantaneous.&nbsp;</li></ol><p>Recent developments in oligonucleotide analysis have shown great strides in using CID for fragmentation of many types, including tRNA, snRNA and digested mRNA. To gain the most impact from these experiments, scientists can adjust the q-value applied within the CID resonance. This is usually set to 0.25; however, for oligonucleotides, lowering that value to 0.2-0.15 can help prevent unwanted fragmentation from occurring and specify backbone cleavage that can be more easily characterized.</p><h3><strong>Source Induced Dissociation (SID)</strong></h3><ul><li><strong>Process: </strong>SID is a collisional-induced activation (and if desired, dissociation) technique that is induced in the source region of mass spectrometers. By applying more voltage in the source region, ions are drawn into the mass spectrometer with more energy. Within the source region, there are still atmospheric inert gases that have been drawn in with the analytes of interest. With the additional energy, collisions with these inert gases can occur. Since SID occurs before any mass analyzer, it applies to all ions that enter the mass spectrometer.</li><li><strong>Applications: </strong>SID is most commonly used as a desolvation technique for large native molecules. By applying SID energy in the source, molecules generate vibrational energy, and non-covalent, low-energy bonds will be broken only.</li><li><strong>Analogy: </strong>SID could be compared to shaking off an umbrella after coming in from the rain. The jostle doesn’t cause the whole umbrella to fall apart, but it does cause the water droplets to fall off. This is a little like proteins getting desolvated from SID.</li><li><strong>Considerations: </strong>SID can be a destructive, dissociative process as well, so if that is not the goal, it is important to monitor the energy applied to the analyte. For example, it has been common practice in top-down proteomics to use a small (10-15 eV) setting for SID to aid in desolvation. However, I have successfully taken pure protein (or well chromatographically separated protein) and applied much higher SID (&gt;50 V) to induce dissociation. The resulting fragmentation pattern can yield surprisingly good results. However, this is not common practice, especially not for coeluting species, as the fragmentation would occur on all proteins eluting.</li></ul><p>Another consideration for SID is its use in native MS. This is often vital for good detection of native proteins like antibodies. Often, we will employ a stronger SID in these cases, anywhere from 60-120 V, depending on the size and complexity of the protein/protein complex, to ensure proper desolvation and thus detection.</p><blockquote><p><strong>Learn more about </strong><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/fragmentation-solutions.html"><strong>collisional induced dissociation (CID) and higher energy collisional induced dissociation (HCD)</strong></a></p><p><strong>Visit us on&nbsp;</strong><a target="_blank" rel="noopener noreferrer" href="https://www.linkedin.com/showcase/72003132/admin/dashboard/"><strong>LinkedIn:</strong></a>&nbsp;#CID #HCD #fragmentation</p></blockquote><section><blockquote><h4><strong>Amanda Lee</strong></h4><p>Amanda Lee PhD, Product Marketing Manager</p></blockquote></section>

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