<h3><strong>Introduction to PFAS</strong></h3><p><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=2&amp;search=PFAS"><strong>Per- and polyfluoroalkyl substances (PFAS)</strong></a><strong> are a large group of man-made chemicals that have been widely used in various industries since the 1940s. These chemicals are known for their resistance to heat, water, and oil, making them valuable in numerous applications, including non-stick cookware, water-repellent clothing, firefighting foams, and even in the pharmaceutical industry.</strong></p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Thermo_Fisher_Scientific_Understanding_PFAS_in_Pharmaceutical_Manufacturing_0f374e5341.jpg" alt="Thermo Fisher Scientific: Understanding PFAS in Pharmaceutical Manufacturing" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Thermo_Fisher_Scientific_Understanding_PFAS_in_Pharmaceutical_Manufacturing_0f374e5341.jpg 245w," sizes="100vw" width="1024" height="512"></p><h3><strong>PFAS in pharmaceutical manufacturing</strong></h3><p>Pharmaceutical manufacturing is a complex process that involves the synthesis, formulation, and packaging of medications. PFAS can play several roles in this industry, often due to their unique chemical properties:</p><ol><li><strong>As processing aids</strong>: PFAS are used as surfactants and emulsifiers in the production of certain drugs. They help in the formation of stable emulsions and dispersions, which is crucial for the consistency and efficacy of pharmaceutical products.</li><li><strong>In equipment and packaging</strong>: PFAS coatings and materials are used in manufacturing equipment and packaging to ensure non-reactivity and durability. For example, PTFE (a type of PFAS) is often used to line equipment because of its non-stick properties, which help in maintaining cleanliness and preventing contamination.</li><li><strong>Analytical applications</strong>: PFAS are used in analytical chemistry within pharmaceuticals for various tests and quality control measures. Their unique properties can aid in the detection and quantification of various compounds.</li></ol><h3><strong>Environmental and health concerns</strong></h3><p>Despite their utility, PFAS have come under scrutiny due to their persistence in the environment and potential health risks. They are often referred to as “forever chemicals” because they do not break down easily in the environment, leading to long-term contamination issues. Studies have linked PFAS exposure to various health problems, including cancer, liver damage, immune system effects, and developmental issues.</p><h3><strong>Regulatory landscape</strong></h3><p>The growing awareness of PFAS-related risks has led to increased regulatory scrutiny. Regulatory agencies around the world, including the Environmental Protection Agency (EPA) in the United States and the European Chemicals Agency (ECHA), are taking steps to limit the use of PFAS and to monitor their presence in manufacturing processes.</p><p>In the pharmaceutical industry, this means manufacturers must be vigilant in their use of PFAS. They must ensure compliance with regulations, implement robust monitoring systems, and explore alternative materials and processes that can achieve similar results without relying on PFAS.</p><h3><strong>Moving towards sustainable practices</strong></h3><p>Pharmaceutical companies are increasingly looking for sustainable alternatives to PFAS. This involves:</p><ol><li><strong>Research and development</strong>: Investing in R&amp;D to find new materials that can replace PFAS without compromising product quality or safety.</li><li><strong>Process optimization</strong>: Modifying manufacturing processes to reduce or eliminate the need for PFAS.</li><li><strong>Enhanced filtration and treatment</strong>: Implementing advanced filtration and wastewater treatment technologies to capture and remove PFAS from manufacturing effluents.</li><li><strong>Supplier collaboration</strong>: Working closely with suppliers to ensure that raw materials and components are free from PFAS.</li></ol><h3><strong>Overcoming analytical challenges: detecting and quantifying PFAS in pharmaceuticals</strong></h3><p>Overcoming analytical challenges in detecting and quantifying per- and polyfluoroalkyl substances (PFAS) in pharmaceuticals is crucial due to the potential health risks these compounds pose. PFAS are highly persistent in the environment and human body, making their accurate detection essential. The unique chemical properties of PFAS, such as their resistance to degradation and their ability to exist in complex matrices, complicate the analytical processes.</p><p>Advanced techniques like high-resolution mass spectrometry coupled with liquid chromatography (LC-MS/MS) have become indispensable tools in this endeavour. These methods offer high sensitivity and specificity, enabling the detection of PFAS at trace levels. Additionally, rigorous method validation and the development of robust sample preparation protocols are essential to mitigate matrix effects and ensure reproducibility and accuracy. Overcoming these analytical challenges is fundamental to ensuring the safety and compliance of pharmaceutical products with regulatory standards.</p><p>We have developed a comprehensive LC-MS based solution for the screening and quantitation of PFAS in pharmaceutical packaging and processing materials. This approach employs the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/744"><strong>Thermo Scientific Vanquish Horizon UHPLC system</strong></a> coupled with the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/403"><strong>Thermo Scientific Orbitrap Exploris 120 mass spectrometer</strong></a>, leveraging the high sensitivity and resolution these instruments offer. The solution utilizes <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/76"><strong>Thermo Scientific Chromeleon CDS</strong></a> for chromatographic data system management and <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1759"><strong>Thermo Scientific Compound Discoverer software</strong></a> for advanced data analysis. This integrated approach ensures precise identification and quantification of PFAS, providing robust and reliable results that meet stringent regulatory requirements.</p><h3><strong>Key features:</strong></h3><ul><li><strong>Simultaneous identification:</strong> The polarity switching Full Scan-ddMS2 method enables the identification of both known and unknown PFAS with high sensitivity and mass accuracy.</li><li><strong>High sensitivity quantitation:</strong> Targeted quantitation of 17 common PFAS is achieved with LOQs ranging from 0.1 to 1 ppb, using a PFAS analysis kit to minimize background interference.</li><li><strong>Detection at low levels:</strong> Demonstrates the ability to detect PFAS at low ppb to sub-ppb levels, including five suspected PFAS identified in non-targeted analysis.</li><li><strong>Delay column use:</strong> Enhances analysis by separating system peaks from sample peaks, aiding in the detection of frequent LC-MS system contaminants.</li></ul><p>This method provides a robust and reliable approach for PFAS screening in E&amp;L analysis and other pharmaceutical testing, helping companies stay compliant with regulations and ensure product safety. For detailed insights and methodologies, download our latest application note: <strong>“</strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_003388_pb_pfas_orbitrap_exploris_120_extractables_an003388_na_en_dd0e422ebd.pdf"><strong>Comprehensive PFAS Screening in Pharmaceutical Packaging and Medical Devices by LC-HRAM-MS</strong></a>.” Stay ahead in ensuring safety and compliance in your pharmaceutical manufacturing processes.</p><p>Watch our latest webinar: <strong>“</strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/4767"><strong>Comprehensive PFAS Screening in Pharmaceutical Packaging and Medical Devices</strong></a><strong>.”</strong> Learn about cutting-edge techniques and tools to ensure safety and compliance in pharmaceutical manufacturing.</p><h3><strong>Conclusion</strong></h3><p>PFAS have played a significant role in pharmaceutical manufacturing due to their unique properties, such as chemical stability and resistance to degradation. However, their environmental persistence and potential health risks necessitate a careful and responsible approach to their use. The pharmaceutical industry must balance the benefits of PFAS with the imperative to protect human health and the environment, driving innovation towards safer and more sustainable manufacturing practices.</p><p>By staying informed, compliant, and proactive, pharmaceutical manufacturers can contribute to a healthier future while continuing to deliver safe and effective medications to the public. Utilizing advanced analytical tools, such as our comprehensive LC-MS based solution with the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/744"><strong>Vanquish Horizon UHPLC system</strong></a> and <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/403"><strong>Orbitrap Exploris 120 mass spectrometer</strong></a>, ensures precise detection and quantitation of PFAS, supporting this critical balance.</p><blockquote><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><strong>&nbsp;</strong>#hram #lc-ms #pfas #pharmaceutical</p></blockquote><blockquote><ul><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/5894"><strong>Enhancing PFAS Analysis in Food Contact Materials Using Combustion Ion Chromatography</strong></a></li><li><a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/article/5813"><strong>New European Wine Scandal on PFAS in Wine</strong></a></li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/5849"><strong>Understanding PFAS and Combustion-Ion Chromatography</strong></a></li><li><a target="_blank" rel="noopener noreferrer" href="https://www.thermofisher.com/blog/analyteguru/advancing-pfas-detection-in-pharmaceutical-manufacturing-with-lc-hram-ms/"><strong>Advancing PFAS Detection in Pharmaceutical Manufacturing with LC-HRAM-MS</strong></a></li></ul></blockquote><section><blockquote><h4><strong>Aaron Lamb</strong></h4><p>Vertical Marketing Manager, Pharma and Biopharma, Thermo Fisher Scientific</p></blockquote></section>

Understanding PFAS in Pharmaceutical Manufacturing

<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 29th September 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 / AOAC, Thermo Fisher Scientific </strong>and <strong>Waters Corporation / AOAC!</strong></p><h3><strong>1. Agilent Technologies: Comparison of Dual Sorbent Solid Phase Extraction for PFAS Applications</strong></h3><p><strong>Using dual-phase Agilent </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=Bond+Elut+PFAS+WAX"><strong>Bond Elut PFAS WAX/ Carbon S SPE</strong></a><strong> cartridges for US EPA Method 1633</strong></p><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_1633_wax_carbon_s_spe_comparison_5994_8291en_agilent_8bdc559ad8.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Many laboratories have opted to combine polymeric weak anion exchange (WAX) and graphitized carbon black (GCB) into single solid phase extraction (SPE) cartridges to streamline sample preparation and facilitate automation for the determination of Per- and Polyfluoroalkyl Substances (PFAS) in environmental samples. This modification is acceptable under Section 1.5 of US EPA Method 1633, if the method's quality control metrics are met.1 The Agilent PFAS WAX and Carbon&nbsp;S sorbents are specifically manufactured for PFAS applications and are lot-tested to ensure low PFAS residue and maximum recovery.2,3 Packing these sorbents into standard 6 mL SPE cartridges with low PFAS residue frits makes them ideally suited for automated methods requiring both WAX extraction and carbon matrix reduction. The extraction performance of the dual-phase Agilent Bond Elut PFAS WAX/Carbon&nbsp;S SPE cartridges are comparable to other commercial cartridges. This ensures seamless integration into existing workflows while assuring optimal performance for PFAS applications.</p><h4><strong>Experimental&nbsp;</strong></h4><p>Extraction recoveries using the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=Bond+Elut+PFAS+WAX"><strong>Bond Elut layered 200 mg PFAS WAX (top)/50 mg Carbon S (bottom) SPE cartridges</strong></a> (part number 5610-2237) were compared to two other commercial 200 mg WAX/50 mg GCB dual-phase cartridges (Table 1). Four replicate extractions were performed with each sorbent, except for benchmark B, where only two replicates were extracted. The target compounds included the 40 listed in Method 1633, spiked at a low-level concentration of 4 ng/L in 250 mL of reagent water. Target recoveries were quantified using 24 isotope-labeled extracted internal standards (EIS). Extractions were carried out using automated SPE. Method details were described in a previous application note.4</p><h4><strong>Conclusion&nbsp;</strong></h4><p>Dual-phase Agilent Bond Elut PFAS WAX/Carbon S SPE cartridges are specifically manufactured for PFAS applications, with rigorous lot testing to ensure PFAS cleanliness and recovery. They enable efficient streamlined sample preparation and automation for EPA Method 1633. PFAS target and extracted internal standards recoveries are comparable to other dual-phase SPE cartridges, facilitating easy integration into existing EPA Method 1633 workflows.</p><h3><strong>2. Shimadzu / AOAC: Analysis of Per- and Polyfluoroalkyl Substances (PFAS) in Drinking Water and Milk using LC-MS/MS coupled with Online-SPE Interface</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/aoac_2025_t009_835132523a.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Per- and Polyfluoroalkyl Substances (PFAS) are a chemical group of organic fluorinated compounds, and perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are representative compounds. They have been used in applications such as water repellents, surface treatment agents, fire extinguishers, and coatings. PFAS are known to be widely present in the environment and wildlife due to their stable structure. Concerns about human exposure through diet have led various countries to conduct investigations into food contamination by PFAS.</p><p>We have examined a quantitative analysis method for 43 PFAS in tap water, drinking water, and milk, and developed a pretreatment and analysis method that realized labor and time savings using LC-MS/MS and automatic pretreatment equipment (Fig.1). For extraction from milk, the QuEChERS method was used with reference to the FDA method.</p><h4><strong>Experimental</strong></h4><ul><li><strong>Automatic pretreatment equipment: </strong>SPL-W100 (AiSTI SCIENCE)</li><li><strong>LC-MS/MS:</strong> <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=LCMS-8060RX"><strong>LCMS-8060RX</strong></a> (Shimadzu)</li></ul><h4><strong>Conclusion</strong></h4><ul><li>LC-MS/MS methods were developed to analyze 43 PFAS in 11 minutes using automatic pretreatment, saving time and labor.</li><li>We conducted recovery tests by adding samples to tap water, drinking water, and milk, and achieved the LOQ set by EPA regulations and AOAC criteria for all compounds.</li><li>The quantitative values of the four main components in tap water were PFOA 0.00109 <span>μ</span>g/kg, PFNA 0.00044 <span>μ</span>g/kg, PFHxS 0.0004 <span>μ</span>g/kg and PFOS 0.00063 <span>μ</span>g/kg. PFAS was not detected above the detection limit in drinking water and milk.</li></ul><h3><strong>3. Thermo Fisher Scientific: High-Throughput and Highly Selective Quantitative Lipidomics with the Stellar Mass Spectrometer – A Novel Hybrid Nominal Mass Instrument</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_unil_stellar_lipidomics_metsoci_2025_po_en_730b1c19fd.pdf"><strong>Full PDF for download</strong></a></li></ul><p>The increasing prevalence of metabolic disorders underscores the need for robust, high-throughput clinical lipid panels and lipidomics methods to quantify lipid species comprehensively, with high precision and selectivity . Structurally related lipids, with varying fatty acid composition, were associated with distinct metabolic consequences and proven to have different biological roles. Thereby, measuring individual lipid species with highest selectivity possible is crucial for corroboration of clinical utility including the improved risk prediction and diagnostics. We present an advanced methodology leveraging the unique capabilities of the new Stellar mass spectrometer. This nominal mass instrument addresses current limitations in specificity and throughput by enabling fast acquisition parallel reaction monitoring, and revolutionizing lipidomics workflows by delivering unprecedented specificity and quantitation. This system combines a quadrupole and a dual cell linear ion trap that significantly improves the ion accumulation efficiency and scan speed. This allows for the acquisition of full MS2 spectra including multiple transitions, thus improving the annotation specificity by resolving isomeric lipids and structurally similar species. This unique capacity of Stellar MS positions it as a practical solution for translational research, biomarker discovery and clinical lipidomics where rapid, sensitive and selective lipid quantitation is essential.</p><h4><strong>Materials and Methods&nbsp;</strong></h4><h5><strong>Sample Preparation&nbsp;</strong></h5><p>Dried UltimateSPLASH ONE internal standard mixture (100 µL) was reconstituted in 25 µL of NIST human plasma SRM 1950 followed by the addition of 125 µL of isopropanol for lipid extraction. The supernatant was then transferred to LC vials for LC-MS analysis. Linearity assessment: different volumes of Avanti Research UltimateSPLASH ONE internal standard mixture (0.5, 1, 5, 10, 50, 100, and 500 µL) were evaporated to dryness, followed by the reconstitution with 25 µL NIST Plasma and 125 µL of isopropanol for lipid extraction.&nbsp;</p><h5><strong>Data Acquisition&nbsp;</strong></h5><p>Plasma lipid extracts were separated on a ACQUITY Premier BEH Amide column (1.7um, 100 mm x 2.1 mm I.D., Waters) and analyzed using a targeted LCMS lipidomics approach on <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/742"><strong>Thermo Scientific Vanquish Duo UHPLC system</strong></a> coupled to a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1149"><strong>Stellar mass spectrometer</strong></a> (Figure 1) 1 . This method was built from a well-established selected reaction monitoring (SRM) method that we Converted to a parallel reaction monitoring (PRM) method by using only the unique precursors. As there are many isomeric lipids sharing the same mass, converting the method for the Stellar reduced 1,019 (579 positive mode; 440 negative mode) SRM precursors to 524 (233 positive mode; 291 negative mode) PRM precursors.</p><h4><strong>Conclusions&nbsp;</strong></h4><ul><li>By integrating LipidCreator with Stellar MS, we expanded on an already thorough lipid workflow by increasing the number of targeted lipids (&gt;50%) and transitions (&gt;3/molecule) enabling higher confidence in lipid identification&nbsp;</li><li>Multiple transitions per precursor, combined with fast scan speeds, enhanced both the specificity and accuracy of lipid quantification, particularly for isomeric species.&nbsp;</li><li>Employing both HCD and CID simultaneously allowed for more robust characterization of sphingomyelins seen in the 2-3 transitions per species.&nbsp;</li><li>This high throughput, targeted approach addresses key limitations and provides a efficient platform for biomarker discovery&nbsp;</li><li>Also, look for our other poster showing a high-throughput targeted MS3 approach has been optimized on the Stellar MS, for the semi-quantification of TG species in human plasma.</li></ul><h3><strong>4. Waters Corporation / AOAC: From Coop to Carton: A Study of PFAS in Backyard &amp; Store-Bought Eggs Using Automation and LC-MS/MS</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720009037_6848187630.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Consumers often view backyard chicken eggs as a healthier alternative to store-bought eggs. However, environmental factors such as contaminated soil, water, feed, and bedding may expose backyard chickens to per- and polyfluoroalkyl substances (PFAS). This study set out to compare PFAS levels in eggs from backyard flocks with those in cage-free eggs purchased from grocery stores. The challenge was to analyze whole egg—a dense protein- and fat-rich matrix—efficiently and reproducibly. To address this, an automated sample preparation workflow was developed, reducing analyst involvement, minimizing variability, and ensuring robust results.</p><h4><strong>Methods</strong></h4><p>The workflow combined automated extraction (CEM EDGE) with automated solid-phase extraction (PromoChrom SPE-03) using dual-phase <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/215"><strong>Oasis GCB/WAX cartridges</strong></a> designed for PFAS. Key steps included:</p><ul><li>Homogenizing whole eggs and spiking with internal standards.</li><li>Automated alkaline extraction cycles followed by nitrogen-assisted concentration.</li><li>SPE cleanup with GCB/WAX cartridges to remove matrix interferences.</li><li>Analysis by LC-MS/MS using a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/191"><strong>Waters ACQUITY Premier system</strong></a> coupled to a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/554"><strong>Xevo TQ Absolute mass spectrometer</strong></a>.</li></ul><p>This approach enabled preparation in less than 15 minutes per sample, while the SPE system processed up to eight samples simultaneously in under 70 minutes. Altogether, the workflow provided consistent recoveries across a broad range of PFAS, including EU-regulated compounds such as PFOA, PFOS, PFNA, and PFHxS.</p><h4><strong>Conclusions</strong></h4><p>Automated sample preparation and SPE enabled reproducible, high-throughput analysis of PFAS in whole eggs—a challenging matrix. Results showed that backyard eggs generally contained higher PFAS concentrations and a wider variety of compounds than store-bought eggs, reflecting greater environmental exposure. In contrast, grocery store eggs contained certain PFAS precursors, suggesting possible legacy contamination. The method proved robust for both regulated and emerging PFAS, with recoveries in the acceptable 70–130% range. This workflow offers laboratories a reliable, future-proof solution for PFAS monitoring in foods, helping to ensure consumer safety and regulatory compliance.</p>

News from LabRulezLCMS Library - Week 40, 2025

<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 13th October 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 notes by <strong>Agilent Technologies,</strong> <strong>Shimadzu </strong>and<strong> Waters Corporation </strong>and poster by<strong> Thermo Fisher Scientific / HPLC!</strong></p><h3><strong>1. Agilent Technologies: Expanding Coverage for the Analysis of PFAS in Paper-Based Food Packaging Materials</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_pfas_food_contact_materials_6545_lcqtof_5994_8516en_agilent_0d3cbb1f4d.pdf"><strong>Full PDF for download</strong></a></li></ul><p>PFAS are manufactured chemicals used in food packaging for their grease- and waterproof properties. However, PFAS are persistent in the environment and may pose health risks.2 This study presents a workflow to screen for PFAS in FCMs using LC/Q-TOF. The FluoroMatch software suite was used to automate PFAS data annotation of knowns and unknowns as well as generate an interactive visualization dashboard, enhancing analysis accuracy and coverage.&nbsp;</p><p>The FluoroMatch suite is an open-source set of tools designed to streamline the suspect and nontarget screening of PFAS compounds. It automates several processes, including file conversion, chromatographic peak picking, blank feature filtering, PFAS annotation based on precursor and fragment masses, homologous series detection, compound classification (PFAS versus not PFAS), and reporting annotation confidence. The software library contains 15,643&nbsp;PFAS species and associated fragmentation patterns, with the capability to add more.3&nbsp;</p><p>Various FCM samples, including takeaway containers and microwave popcorn bags, were rigorously extracted and analyzed. Suspect screening was employed for PFAS quantitation, while nontargeted workflows enabled the identification and semi-quantitation of PFAS that were lacking&nbsp;standards.</p><h4><strong>Experimental</strong></h4><h5><strong>LC/MS analysis&nbsp;</strong></h5><p>Extracts were analyzed using an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=1290+Infinity+II+LC"><strong>Agilent 1290 Infinity II LC system</strong></a> coupled with an Agilent <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=6545+LC%2FQ-TOF"><strong>6545 LC/Q-TOF</strong></a>. An Agilent <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=PFC-Free+HPLC+Conversion+kit"><strong>PFC-Free HPLC Conversion kit</strong></a> (part number 5004‑0006) and an Agilent <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=InfinityLab+PFC+delay+column"><strong>InfinityLab PFC delay column</strong></a> (part number 5062‑8100) were used to minimize background PFAS levels. Liquid chromatographic separation was performed on a 1.8&nbsp;µm, 2.1 × 100 mm, Agilent ZORBAX RRHD StableBond SB‑C18 column (part number 858700-902) with a corresponding guard column (Agilent part number 821725-902). The mobile phases were (A) water with 20&nbsp;mM ammonium acetate and (B) methanol. The flow rate was 0.4&nbsp;mL/min. The elution gradient was: 5% B (0 to 0.5&nbsp;minutes), linear increase to 40% B (0.5 to 3 minutes), linear increase to 80% B (3 to 16 minutes), hold 80% B (16 to 18 minutes), linear increase to 100% B (18 to 22 minutes), hold 100% B (20 to 22&nbsp;minutes), decrease to 5% B (22 to 22.5 minutes), followed by a 4 minutes post‑run re‑equilibration. The injection volume was 10 μL, and the column temperature was maintained at 50 °C. For the LC/Q‑TOF, the drying gas was set to 4 L/min at 230 °C, the nebulizer to 20 psi, and the sheath gas to 12 L/min at 375 °C.</p><h4><strong>Results and discussion</strong></h4><p>PFAS identification using <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=FluoroMatch"><strong>FluoroMatch</strong></a> Using FluoroMatch Visualizer, interactive tools for visualizing PFAS data were generated, including mass defect plots, accurate mass versus retention time plots, MS/MS fragmentation plots, annotation tables, and fragment screening. Individual homologous series were selected based on nominal mass and normalized mass defect, allowing for the observation of patterns and identification of outliers. This interactive cross-filtering simplified the evaluation of PFAS features and enhanced confidence in nontargeted results.&nbsp;</p><p>FluoroMatch was used to annotate a homologous series (C3 to C14) of perfluoroalkyl carboxylic acids (PFCAs), including PFHxA (perfluorohexanoic acid), PFHpA (perfluoroheptanoic acid), PFOA (perfluorooctanoic acid), PFNA (perfluorononanoic acid), PFDA (perfluorodecanoic acid), and PFDoDA (perfluorododecanoic acid). This workflow demonstrated that the suspect screening approach successfully identified common PFAS with available standards, while FluoroMatch was able to annotate additional PFAS that were lacking standards.&nbsp;</p><p>Additionally, FluoroMatch annotated C6 and C8 perfluoroalkyl sulfonic acids (PFSAs), which were not identified by suspect screening. This highlights the value of a hybrid workflow that combines traditional suspect screening with nontargeted tools like FluoroMatch, enhancing confidence in the nontargeted results through their complementary nature. Specifically, FluoroMatch allows for homologous series detection to help annotate incomplete or noisy spectra. FluoroMatch Visualizer evaluation helps incorporate additional lines of evidence to annotation like retention time patterns and Kendrick mass defect to aid in annotation.</p><h4><strong>Conclusion&nbsp;</strong></h4><p>This study successfully demonstrates the effectiveness of FluoroMatch software in automating the annotation and visualization of PFAS compounds in food packaging materials. The integration of FluoroMatch Visualizer provides a comprehensive approach to identifying both targeted and nontargeted PFAS compounds. The results highlight the prevalence of PFAS in various paper‑based FCMs, with clamshell to-go boxes showing the highest concentrations, particularly PFOA and PFDA, which exceed European Union regulatory limits. As PFAS can migrate into food, especially at higher temperatures, the pervasiveness of PFAS in FCMs raises concerns about potential exposure through hot meals and microwave heating. The widespread detection of PFAS in clamshell to-go boxes and other FCMs indicates a need for stricter regulations to reduce PFAS use in food packaging to protect consumer&nbsp;health.</p><h3><strong>2. Shimadzu: An Ultra-high Sensitivity Analysis of 29 PFAS Compounds in Drinking Water by Direct Injection</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_01_01026_en_dcf554dd6f.pdf"><strong>Full PDF for download</strong></a></li></ul><p>PFAS (Per- and Polyfluoroalkyl Substances) are synthetic fluorinated organic compounds. More than a few thousand compounds exist, each with a varied carbon chain length, functional groups, and structural isomers. Because of its useful qualities, such as their ability to repel water and their nonstickiness, PFAS are found in a wide range of consumer and industrial products, including firefighting foams and coatings. Their high stability and persistence raise concerns about their impact on human health and the environment. In recent years, regulations and studies regarding PFAS have been increasing globally. For example, the U.S. Environmental Protection Agency (EPA) has set a Maximum Contaminant Level (MCL) of 4 ng/L for PFOA and PFOS in drinking water. To improve the analysis of PFAS, more sensitive, accurate and faster methods are needed. This application introduces the ultra-high sensitivity analysis of 29 PFAS in drinking water by direct injection using the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=LCMS-8065XE"><strong>LCMS-8065XE</strong></a> (Fig. 1) and addresses the demands of testing laboratories for PFAS quantification.</p><h4><strong>Analytical conditions</strong></h4><p>The HPLC and MS analytical conditions are shown in Table 1 and 2. The test compounds used were the 29 compounds included in methods EPA 533 and 537.1 for drinking water analysis. The <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=LCMS-8065XE"><strong>LCMS8065XE</strong></a> is equipped with a new ESI probe (StreamFocus) and a new collision cell (UFsweeper IV), providing more sensitive and accurate results in PFAS analysis. A delay column was installed between the mixer and autosampler to separate PFAS from the HPLC system and those in the sample. To ease the analysis, same type of column is used as delay and analytical columns.</p><h4><strong>Conclusion&nbsp;</strong></h4><ul><li>29 of PFAS in drinking water were measured with high sensitivity (&lt;1 ng/L) by direct injection method using the LCMS-8065XE.&nbsp;</li><li>Good recovery and reproducibility were obtained in drinking water samples, confirming that this analytical method can deliver accurate quantification for PFASsin drinking water.</li></ul><h3><strong>3. Thermo Fisher Scientific / HPLC: Efficient tandem capillary flow LC-MS with short μPAC columns and a single ionization source</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_efficient_tandem_capillary_flow_lc_ms_short_pac_columns_po_en_f38e078c9a.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Nano-flow tandem direct injection (TDI) workflows increase sample throughput and instrument productivity with flow rates below 1 µL/min but need dual-column ESI interfacing to maintain chromatographic performance. At flow rates above 1 µL/min with capillary flow columns, post-column dispersion impacts performance less, allowing low-dispersion switching valves in the fluidic path. We demonstrate the Vanquish Neo UHPLC system TDI workflow for capillary flow LC with a high throughput short pillar array column. This dual-column, single-ESI emitter setup reduces variability, extends emitter life through efficient salt management, and ensures high reproducibility with dedicated gradient pumps and intelligent method design.</p><h4><strong>Materials and methods</strong></h4><h5><strong>Sample Preparation&nbsp;</strong></h5><p>HeLa cell digests were resuspended in 0.1% TFA and 1% ACN to obtain a 200 ng/µL stock solution, sonicated, diluted, and vortexed before use.</p><h5><strong>LC-MS configuration&nbsp;</strong></h5><p>Samples were analyzed using a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/398"><strong>Vanquish Neo UHPLC</strong></a> instrument that was configured in tandem direct injection mode with an additional pump module and a 10 µL injection loop for maximum throughput. Two µPACM Neo High-Throughput Plus columns were positioned between low-dispersion 6-port switching valves in the column compartment. A 20 µm ID nanoViper line connected the alternating eluents to an ESI emitter with an integrated liquid junction, positioned in a Thermo Scientific EASY-Spray source coupled to a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/402"><strong>Orbitrap Exploris 240 mass spectrometer</strong></a></p><h4><strong>Conclusions&nbsp;</strong></h4><ul><li>Using optimized µPAC Neo HT Plus columns in combination with the Tandem Vanquish Neo UHPLC system heightened the productivity of bottom-up proteomics measurements to 86 - 92% (gradient-dependent)&nbsp;</li><li>At these throughputs, choice of emitter only has a minor effect on identifications, FWHM and TIC. Decreasing the flow rate to 1 uL/min causes peak broadening and increases ionization efficiency which is reflected in higher TICs. In this study, reducing the flow rate only showed a positive effect on protein IDs for the longest gradient (100 SPD).&nbsp;</li><li>With the optimized methods, identifications up to 4713 protein groups for the 180 SPD and 5692 protein groups for the 100 SPD were achieved. The CVs using the same column were between 5-6% and between two columns at around 7-8% (n=6)&nbsp;</li><li>Column reproducibility was showcased for 12 columns across four different production batches. Overall, the ID rate variation between the columns was 1.4%. The median coefficient of variation of the retention times of spiked-in PRTC peptides across the tested 12 columns was 1.8%</li></ul><h3><strong>4. Waters Corporation: Oligo Mapping of sgRNA Digests: Leveraging Xevo MRT Mass Spectrometer Performance and Streamlining Data Analysis</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720009009_en_f35f22bebc.pdf"><strong>Full PDF for download</strong></a></li></ul><h5><strong>Benefits&nbsp;</strong></h5><ul><li>Confident characterization of sgRNA digestion products with sub-ppm mass accuracy of oligonucleotide precursors, outstanding MS resolution (~100,000), and sensitivity at high acquisition speed</li><li>A compliance-ready informatics workflow featuring the waters_connect MAP Sequence App (ver 1.0) streamlines oligonucleotide mapping of sgRNA digests</li><li>RapiZyme MC1, a new ribonuclease - offers unique cleavage specificity and the opportunity to generate overlapping digestion products, enabling complete (100%) sequence coverage for sgRNAs</li></ul><p>Single guide RNAs (sgRNAs) were first described in 20121 when two RNA molecules essential for CRISPR-Cas9- mediated DNA cleavage – the trans-activating CRISPR RNA (tracrRNA), which serves as a scaffold for the Cas9 nuclease, and the CRISPR RNA (crRNA), responsible for DNA target recognition - were fused into a single RNA construct. This fusion created the sgRNA, a critical component of the CRISPR-Cas9 gene editing system.<sup>1–2</sup> The sgRNA molecule directs the Cas9 nuclease to introduce precise double-stranded breaks in DNA, enabling targeted genetic modifications.&nbsp;</p><p>Since its discovery, which was honored with the Nobel Prize in Chemistry, CRISPR technology has transformed gene editing applications. Beyond basic research, it has been adapted for rapid diagnostic tools, such as COVID19 tests developed during the 2020 global pandemic<sup>3</sup> , and holds promise for therapeutic interventions in genetic diseases, cancer, and infectious diseases. As sgRNAs are typically synthesized via solid-phase oligonucleotide synthesis, their analytical characterization requires confirmation of both the intact molecular weight<sup>4</sup> and the sequence verification to ensure functional accuracy.<sup>5</sup>&nbsp;</p><p>Traditional LC-MS workflows for sgRNA digest oligo mapping have been labor-intensive and time-consuming, relying heavily on manual data analysis and interpretation. Recently<sup>6</sup> , a UPLC-MS and informatics workflow designed for automated sgRNA sequence mapping was presented following enzymatic digestion with a panel of ribonucleases, including RNase T1, hRNAse 4 and two RNase T2 enzymes: RapiZyme MC1 and Cusativin. This application note extends the effectiveness of this workflow (Figure 1) when implemented on a Xevo MRT Mass Spectrometer (Figure 1), while focusing specifically on MC1 digestion of the Waters™ sgRNA LC-MS standard, where complete unique digested product coverage was obtained.</p><h4><strong>Experimental</strong></h4><h5><strong>LC Conditions</strong></h5><ul><li><strong>LC-MS system:</strong> <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1148"><strong>Xevo MRT</strong></a> (multi-reflecting time-of-flight) Mass Spectrometer coupled with <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/191"><strong>ACQUITY™ Premier UPLC (Binary) System&nbsp;</strong></a></li><li><strong>Column: </strong>ACQUITY Premier Oligonucleotide BEH™ C18 Column 300 Å, 1.7 µm, 2.1 x 150 mm, (p/n:186010541)</li></ul><h4>Results and Discussion&nbsp;</h4><p>An informatics workflow (Figure 1) featuring the waters_connect MAP Sequence App (version 1.0) was used to facilitate automated data processing of UPLC-MSE datasets acquired following the enzymatic digestion of an sgRNA standard with RapiZyme MC1.6–7 The informatics processing workflow consisted of three steps:&nbsp;</p><ol><li><strong>In-silico digestion:</strong> The mRNA Cleaver MicroApp was used to generate predicted, in-silico digested oligonucleotide digestion products, based on the target RNA sequence.&nbsp;</li><li><strong>Data processing and mass matching:</strong> The waters_connect MAP Sequence App processed the UPLC-MSE data, matching the predicted neutral monoisotopic masses of digested oligonucleotides to the experimentally acquired MS1 data.&nbsp;</li><li><strong>Sequence coverage visualization: </strong>The resulting sequence coverage for the RNA digest was summarized and visualized using the Coverage Viewer MicroApp.</li></ol><h4><strong>Conclusion&nbsp;</strong></h4><ul><li>An informatics workflow, incorporating the waters_connect MAP Sequence App (ver 1.0) was successfully demonstrated for UPLC-MSE digested oligo mapping analysis of sgRNA digests using the Xevo MRT QTOF Mass Spectrometer.&nbsp;</li><li>Confident peak assessments for analysis of sgRNA digests were achieved with the Xevo MRT QTOF Mass Spectrometer, which delivered excellent sensitivity, high resolution MS (100,000), and mass accuracy of less than 1 ppm.</li><li>Complete sgRNA sequence coverage (100%) was achieved by employing RapiZyme MC1, a novel endonuclease introduced by Waters, which provided unique cleavage specificity and predictable missed cleavages to generate longer, unique, overlapping digestion products.</li><li>Oligonucleotide isomeric sequences resulting from enzymatic digestion were effectively differentiated using the waters_connect CONFIRM Sequence App, which matched elevated energy fragment ions patterns to their respective sequences, allowing unambiguous isomer identification.</li></ul>

News from LabRulezLCMS Library - Week 42, 2025

<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 20th October 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 / AOAC,</strong> <strong>Shimadzu / AOAC </strong>and<strong> Thermo Fisher Scientific / HPLC Symposium </strong>and application note by <strong>Waters Corporation!</strong></p><h3><strong>1. Agilent Technologies / AOAC: Evaluating System Robustness of a High-Sensitivity Triple Quadrupole LC/MS for PFAS Analysis in Food Matrix Over an Extended Period&nbsp;</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/ps_pfas_salmon_6495d_robustness_aoac_2025_t004_en_agilent_9b7b469a22.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Due to the rapidly evolving regulatory environment of per- and polyfluoroalkyl substances (PFAS), many analytical laboratories are opting to use highsensitivity triple quadrupole mass spectrometers for the targeted analysis of such compounds to ensure meeting present and potential future requirements for sensitivity and PFAS coverage. These assays are regularly conducted in a high-throughput manner involving complex matrices such as food, soil, wastewater or biological samples. Therefore, system robustness and instrument uptime are crucial for such applications to maintain productivity and profitability. In this work, we present results from an expedited robustness testing of a high-sensitivity triple quadrupole LC/MS system using select PFASs in one of the most challenging food matrices.</p><h4><strong>Experimental</strong></h4><h5><strong>Instrumentation</strong></h5><p>Data was collected using an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=Agilent+1290+Infinity+II+LC"><strong>Agilent 1290 Infinity II LC</strong></a> system coupled to an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=6495D+LC%2FTQ"><strong>Agilent 6495D LC/TQ mass spectrometer.</strong></a> Instrument parameters are summarized in Tables 1 and 2.</p><h4><strong>Conclusions</strong></h4><p>The robustness of a 6495D LC/TQ was tested using undiluted salmon extract, a complex and challenging food matrix. During the experiment, the system was exposed to more than 130 mL of the matrix over a period of ~3 weeks of continuous operation, without any maintenance. System performance was monitored by injections of a mixture of native and isotopically labelled PFAS samples. The monitored signals exhibited low variations (RSD&lt;6%) throughout the entire experiment. No obvious performance degradation or significant contamination of the inner ion optics elements were observed after the conclusion of the test.&nbsp;</p><p>Although no loss of instrument performance was observed during this expedited robustness test, a routine maintenance of ion source and optics is recommended to ensure prolonged instrument operation.</p><h3><strong>2. Shimadzu / AOAC: Analysis of Chlordane by DUIS (Dual Electrospray/Atmospheric Pressure Chemical Ionization) Liquid Chromatography Mass Spectrometry&nbsp;</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/aoac_2025_w050_036db56f51.pdf"><strong>Full PDF for download</strong></a></li></ul><p>The legalization of cannabis has led to many laboratories developing methods to meet local cannabis testing regulatory requirements. One of the challenging residual pesticides to quantitate is chlordane, an organochlorine compound used as a pesticide on crops until 1983 and for termite treatment in homes until 1988. Chlordane is highly persistent in the environment and has been linked to cancers. Gas chromatography mass spectrometry and atmospheric pressure chemical ionization (APCI) – liquid chromatography mass spectrometry (LCMS) methods were published for analysis of chlordane. However, for laboratories without APCI functionality or who prefer not to switch between two different ionization sources, chlordane remains a challenging target for LCMS analysis. This study demonstrates the successful quantitation of chlordane using additive-free DUIS electrospray ionization – LCMS at concentrations below the New York State testing limits.</p><h4><strong>Methods&nbsp;</strong></h4><p>Technical chlordane (CAS 57-74-9) was obtained from Restek, diluted in water, and analyzed on a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=LCMS-8060"><strong>Shimadzu LCMS-8060</strong></a> triple quadrupole mass spectrometer equipped with a DUIS ionization source. The DUIS source was utilized in APCI mode. Technical chlordane is a mixture of transchlordane, cis-chlordane, and related chemicals. The LC parameters shown in Table 1 allowed separation of the cis- and trans-chlordane isomers.</p><h4><strong>Results&nbsp;</strong></h4><p>Initial MS data was collected in Q1 and Q3 Scan mode. Due to the 8 chlorine atoms per chlordane molecule, a distribution of isomers is expected in the mass spectrum. LabSolutions Insight Explore was used to predict isotopic distributions for chlordane with and without adducts. The predicted spectra for the [M-H]- and [M+CH3OH]- are shown in Figure 1A and 1B, respectively. The experimentally observed isotopic distributions for the [M-H]- and [M+CH3OH]- ions are shown in Figure 1C and 1D, respectively. Figure 1E shows the relative intensity of the [M+CH3OH]- versus the [M-H]- isotope clusters. As the [M+CH3OH]- showed the greatest intensity, it was selected for optimization.</p><p>Voltage optimization was performed for the 441.80 to 441.80 MRM with the CID gas turned off. Even after voltage optimization, the signal-to-noise from the primary species in the technical chlordane (in this case, trans-chlordane) was insufficient. Automated source optimization using LabSolutions MRM Connect was used to improve signal by optimizing Interface Temperature, Heating Gas Flow, Nebulizing Gas Flow, Drying Gas Flow, DL Temperature, Heat Block Temperature, and Interface Voltage. As a representative example, Figure 3 shows the impact of Interface Temperature on the MRM intensity for the 441.80 m/z.</p><h3><strong>3. Thermo Fisher Scientific / HPLC Symposium: HPLC transfer and optimization of deoxycholic acid analysis using HPLC – Charged Aerosol Detector</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_hplc_transfer_optimization_deoxycholic_acid_analysis_po_en_55bc47dc90.pdf"><strong>Full PDF for download</strong></a></li></ul><p>The United States Pharmacopeial Convention monograph outlines a high-performance liquid chromatography with charged aerosol detection (HPLC-CAD) method [1] for the accurate measurement of deoxycholic acid (also known as desoxycholic acid) and its primary impurity, cholic acid. Key detector settings that influence detection performance metrics such as detection limit, resolution, and linearity include:&nbsp;</p><ul><li>The evaporation temperature (EvapT) for setting the temperature of the evaporation tube and influencing sensitivity related to volatility factors.&nbsp;</li><li>The filter constant for balancing noise and adjusting the detector's response time to changes in analyte concentration.&nbsp;</li><li>The PV for optimizing the linear range within the detector's dynamic range for a given application.&nbsp;</li></ul><p>These parameters are crucial for successful method transfer and optimization. Consequently, this work offers guidelines for method transfer and optimization from the Vanquish CAD to the Vanquish CAD HP</p><p><strong>Methods:</strong> A <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/743"><strong>Thermo Scientific Vanquish Flex UHPLC system</strong></a> equipped with both the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2176"><strong>Vanquish Charged Aerosol Detector </strong></a>and HP was used to perform method transfer and optimization for deoxycholic acid analysis. Chromatographic separation was achieved using a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2005"><strong>Thermo Scientific Acclaim 120 C18 column</strong></a> under gradient elution with formic acid-modified aqueous and organic phases</p><h5><strong>Chromatography Data System&nbsp;</strong></h5><p>The <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/76"><strong>Thermo Scientific Chromeleon 7.3.2 Chromatography Data System (CDS) software</strong></a> was used for data acquisition and analysis.</p><h4><strong>Conclusions&nbsp;</strong></h4><p>This study highlights the successful optimization and method transfer of the USP-NF deoxycholic acid assay using Vanquish CAD detectors. Through careful evaluation of chromatographic performance, detector settings, and calibration parameters, the Vanquish CAD HP was shown to deliver enhanced performance across multiple criteria:&nbsp;</p><h5><strong>Key Takeaways:&nbsp;</strong></h5><ul><li><strong>Excellent Sensitivity and Response:</strong> Vanquish CAD HP demonstrated significantly lower LOD/LOQ values and higher peak responses compared to Vanquish CAD, making it the detector of choice for low-level analyte quantification.&nbsp;</li><li><strong>Enhanced method optimization Flexibility: </strong>Vanquish CAD HP allows simultaneous data collection for multiple PVs, ensuring faster method development and optimization.&nbsp;</li><li><strong>Seamless Method Transfer:</strong> Transitioning from Vanquish CAD to CAD HP was achieved by optimizing power value (PV), EvapT, and signal filter settings, ensuring improved performance.&nbsp;</li></ul><p>Together, these findings demonstrate that the Vanquish CAD HP not only supports efficient method transfer but also elevates performance, sensitivity, and usability—making it an ideal solution for modern HPLC applications.</p><h3><strong>4. Waters Corporation: Multi-Residue Analysis of Pharmaceuticals, Personal Care Products (PPCPs) and Pesticides in Water by Direct Injection Using LC-MS/MS</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720008819_en_4c0e9bc91e.pdf"><strong>Full PDF for download</strong></a></li></ul><h5><strong>Benefits&nbsp;</strong></h5><ul><li>Sensitivity of Xevo TQ Absolute allows for the trace level detection of PPCPs and pesticides in drinking and surface waters without the need for lengthy clean-up or concentration steps</li><li>The direct injection approach increases productivity and provides faster turn-around time for reporting of results while being well aligned with green analytical chemistry principles</li><li>More than 150 analytes reach limits of quantification of 10 ng/L, making this method suitable for screening for multi-class analytes in water matrices&nbsp;</li></ul><p>In recent years, there has been increasing concern about the presence of pesticides, pharmaceuticals and personal care products (PPCPs) in water bodies throughout the world.1 The effect of these emerging contaminants on human health and their potential impact on the environment is not yet fully understood. The updated Drinking Water Directive entered into force in January 2021 and is the EU's main law for drinking water. The Directive applies to all water, either in its original state or after treatment, intended for drinking, cooking, food preparation or other domestic purposes, regardless of its origin and whether it is supplied from a distribution network, supplied from a tanker or put into bottles or containers.2 It further protects human health thanks to updated water quality standards, in accordance with World Health Organisation (WHO) recommendations, addressing emerging pollutants, such as endocrine disruptors, and promoting a preventive, risk-based approach to reduce pollution at its source.</p><p>Water bodies such as rivers, lakes and groundwaters have their status defined in the EU Water Framework Directive.3 An amendment to Directive 2013/39/EU sets environmental quality standards for hazardous substances that require monitoring in these waters. A Commission Implementing Decision4 document, 2022/1307, includes Watch List analytes with Maximum Acceptable Method Detection Limits for compounds of concern across multiple chemical classes. Numerous studies have demonstrated that PPCPs are present at parts-per-trillion (PPT) levels in rivers and streams highlighting the need for methods that are able to detect compounds at these trace levels.5-10 In addition to the low-level detection of these compounds, a major analytical challenge lies in the wide chemical diversity of chemicals of concern. Furthermore, to establish a more complete understanding of water pollution it is necessary to monitor a wide variety of types of water. This application note demonstrates the direct injection, separation and detection of PPCPs and pesticides including acidic, basic, and neutral compounds from multiple regulated analyte lists in a variety of water types.</p><h4><strong>Experimental</strong></h4><ul><li><strong>LC system:</strong> <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/191"><strong>ACQUITY Premier</strong></a> FTN</li><li><strong>MS system: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/554"><strong>Xevo TQ Absolute</strong></a></li></ul><h4><strong>Conclusion&nbsp;</strong></h4><p>The updated EU Drinking Water and Water Framework Directives provide the regulatory framework for effective monitoring and management of water quality and include provisions for emerging contaminants. Ensuring compliance with these regulations and comprehensive coverage for an ever increasing analyte lists requires versatile, robust analytical methods and high performance LC/MS/MS systems. The direct-injection method investigated in this work is effective at detection of a wide range of PPCPs and pesticides at trace levels suitable for monitoring multiple water types for the presence of compound classes known or suspected to pose risks to human and environmental health. It further provides more rapid turn around time and greener operation than methods that rely upon high volumes of solvent and extensive sample preparation. Method performance was assessed using criteria described in ISO 21253–2:2019. The calibration characteristics, linearity, and residuals were excellent over the concentration range studied. Accuracy and precision were evaluated at three matrix QC levels with six replicate spikes and found to provide excellent performance for 154 analytes in tap and bottled water and 74 analytes in surface water matrix.</p>

News from LabRulezLCMS Library - Week 43, 2025

News from LabRulezLCMS Library - Week 44, 2025

News from LabRulezLCMS Library - Week 45, 2025

<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 10th November 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 notes by <strong>Agilent Technologies,</strong> <strong>Shimadzu </strong>and<strong> Waters Corporation</strong> and poster by <strong>Thermo Fisher Scientific!</strong></p><h3><strong>1. Agilent Technologies: Feed Injection at Elevated Pressure Using the Agilent 1290 Infinity III Hybrid Multisampler</strong></h3><p><strong>Optimization of a multipesticide method for the highest sensitivity through feed injection</strong></p><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/te_feed_injection_multipesticide_1290_infinityiii_hybrid_5994_8478en_agilent_f070801ff2.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Modern analytical methods for the determination of pesticide residues are based on multipesticide methods capable of determining hundreds of pesticides in a sample in one analytical LC/MS/MS run. For chromatographic separation of these pesticides, modern separation columns are used. Their dimensions are typically 2.1 × 150 mm, filled with 1.9&nbsp;µm particle size material. This requires that the LC can work at high pressures up to 1300 bar. The Agilent 1290 Infinity III Hybrid Multisampler can operate within this pressure range. In addition, the Hybrid Multisampler can work in two different modes, the classic flow through mode and the Agilent Feed Injection mode. Feed Injection mode helps to prevent peak broadening and splitting effects as they can occur with the classic flow through mode for injection of samples in solvents with high elution strength and higher injection volumes.</p><h4><strong>Experimental</strong></h4><h5><strong>Instrumentation</strong></h5><ul><li>Agilent 1290 Infinity III High-Speed Pump (G7120A)&nbsp;</li><li>Agilent 1290 Infinity III Hybrid Multisampler (G7137B)&nbsp;</li><li>Agilent 1290 Infinity III MCT (G7116B)&nbsp;</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=Ultivo+Triple+Quadrupole+LC%2FMS+"><strong>Agilent Ultivo Triple Quadrupole LC/MS </strong></a>with Agilent Jet Stream source&nbsp;</li></ul><h5><strong>Software&nbsp;</strong></h5><ul><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=MassHunter+Acquisition+Software"><strong>Agilent MassHunter Acquisition Software</strong></a> (v. 12.2)&nbsp;</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=MassHunter+Qualitative+Analysis"><strong>Agilent MassHunter Qualitative Analysis Software</strong></a> (v. 12.0)&nbsp;</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=MassHunter+Quantitative+Analysis"><strong>Agilent MassHunter Quantitative Analysis Software</strong></a> (v.&nbsp;12.1)&nbsp;</li></ul><h5><strong>Column&nbsp;</strong></h5><ul><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=InfinityLab+Poroshell+120+Phenyl-Hexyl"><strong>Agilent InfinityLab Poroshell 120 Phenyl-Hexyl</strong></a>, 2.1 × 150 mm, 1.9 µm (part number 699675-912)</li></ul><h4><strong>Results and discussion&nbsp;</strong></h4><p>Calibration curves for a multipesticide method were acquired to demonstrate the possible improvement of peak shape and the lower limits of quantification (LOQ) using Agilent Feed Injection mode in comparison to classical flow through injection mode. This method comprised approximately 120&nbsp;pesticide compounds measured by dynamic MRM. The separation was performed on a 150 mm length column. With the chosen solvents and flow rate, a pressure range above 1000&nbsp;bar was necessary. The calibration curves were acquired for concentrations down to 0.02 ppb (20 ppt), and each calibration point was measured three-fold. The concentrations of the calibration solutions were prepared by dilution using the strong-eluting solvent acetonitrile and injected directly by both injection modes with different injection volumes. An example chromatogram showing the elution pattern for the MRM transitions is displayed in Figure 1.</p><h4><strong>Conclusion&nbsp;</strong></h4><p>This technical overview demonstrates the use of the Agilent Feed Injection mode available with the Agilent 1290 Infinity III Hybrid Multisampler in comparison to the classical flow through injection mode for the analysis of pesticides by a multipesticide method. We demonstrated that the use of Agilent Feed Injection mode improves the obtained peak shape for injection of samples dissolved in high elution strength solvent compared to classical flow through mode. With the improved peak shape obtained by Feed Injection mode, signal to noise ratios were improved, resulting in higher sensitivity. Due to retained peak shape, no manual interaction for peak integration was necessary when using feed injection.</p><h3><strong>2. Shimadzu: Automatic Optimization of Sample Solvent Composition for Achieving Sharp Peak Shapes</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_01_00938_en_c0f4de1265.pdf"><strong>Full PDF for download</strong></a></li></ul><h4><strong>User Benefits</strong></h4><ul><li>The automatic pretreatment function integrated into the autosampler enables efficient optimization of sample solvent composition, which can be automatically adjusted during analysis.</li><li>The desired composition of sample solvent can be easily specified via the LabSolutionsTM MD interface.</li></ul><p>In LC analysis, the composition of the sample solvent is critical for achieving proper peak shape. If the sample solvent is a stronger eluting solvent than the mobile phase, sample band condensation at the column inlet may be insufficient, leading to sample band broadening. For instance, increasing the organic solvent ratio in the sample solvent to dissolve low-polar compounds may deteriorate the peak shapes of early-eluting compounds in reversed-phase chromatography. Therefore, determining the optimal ratio of organic solvent in the sample solvent is essential. However, manually preparing multiple solvent compositions is extremely time-consuming. The automatic pretreatment function of the autosamplers (<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=Nexera+X3"><strong>Nexera series</strong></a>), in combination with the method development support <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=LabSolutions+MD"><strong>software LabSolutions MD</strong></a>, enables consecutive analyses using different sample solvent compositions without manual intervention. This allows evaluation of the effect of sample solvent composition on peak shape while significantly reducing the labor involved in optimization. In this study, we demonstrate the automated determination of the optimal sample solvent composition using metoclopramide, a small-molecule drug, as a model compound.</p><h4><strong>Automatic Pretreatment Function</strong></h4><p>The automatic pretreatment function integrated into the autosampler enables drawing and ejecting specified volumes of reagents and solvents from any vial, as well as mixing them within the needle. As an example, Fig. 1 illustrates the procedure for automatically adjusting the water-to-methanol ratio in the sample solvent using this function. The autosamplers (Nexera series) supports up to three rinsing solvents via the multi-rinse function, and in this procedure, methanol and water were supplied from these rinsing lines. By modifying the "methanol eject volume" (2) and “water eject volume” (3) in Fig. 1, sample solvents with desired methanol-to-water ratios can be automatically prepared. In addition, adjusting the “sample drawing volume” (1) allows for not only changing the sample solvent composition but also diluting the sample solution to a specified concentration.</p><h4><strong>Analytical Conditions and Target Compounds</strong></h4><p>A 1000 mg/L solution of metoclopramide, a small-molecule drug, was prepared in 100% methanol as a model sample. The water content in the sample solvent was then varied from 0% to 90% in 10% increments (A 10-fold dilution was applied during automatic sample solvent preparation), and its effect on peak shape was evaluated under the analytical conditions shown in Table 1. By simply specifying the desired water content on the LabSolutions MD interface (red frame in Fig. 2), the autosampler’s automatic pretreatment function adjusts the sample solvent composition during consecutive analyses. This eliminates the need for manual preparation of multiple solvent mixtures, significantly reducing the workload involved in optimizing sample solvent composition for ideal peak shapes.</p><h4><strong>Conclusion</strong></h4><p>Optimizing the composition of the sample solvent is essential for achieving appropriate peak shapes. However, manual preparation of multiple solvent compositions is time-consuming. The automatic pretreatment function of the autosampler, in combination with LabSolutions MD, enables consecutive analyses with automatic variation of the sample solvent composition. This significantly reduces the effort required for optimization. Since the optimal sample solvent composition depends on both the physicochemical properties of the analyte and the mobile phase conditions, optimization is necessary for each sample. These tasks, however, can be greatly reduced by utilizing the automatic pretreatment function.</p><h3><strong>3. Thermo Fisher Scientific: Improving selectivity and sensitivity of lipid mediator analyses by coupling nano-flow chromatography with the Stellar mass spectrometer</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_improving_selectivity_sensitivity_lipid_mediator_analyses_po_en_c38bc37825.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Lipid mediators play crucial roles in regulating inflammation and maintaining tissue homeostasis. However, their quantification in biological samples is difficult due to low concentrations (ppt–ppb range), chemical instability, and structural similarity. Conventional LC-MS/MS methods using high-flow UHPLC and triple quadrupole mass spectrometers require extensive optimization of SRM transitions for each analyte. This study introduces a <strong>nano-flow LC-MS workflow</strong> combining the <strong>Thermo Scientific Vanquish Neo UHPLC system</strong> with the <strong>Thermo Scientific Stellar mass spectrometer</strong>, aiming to improve analytical sensitivity and selectivity while reducing solvent and sample consumption.</p><h4><strong>Experimental</strong></h4><p>Lipid mediator standards (from Cayman Chemical) were analyzed using two chromatographic configurations: a <strong>nano-flow method</strong> (500 nL/min) with the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/398"><strong>Vanquish Neo UHPLC</strong></a> and a <strong>high-flow method</strong> (300 µL/min) using the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/744"><strong>Vanquish Horizon UHPLC</strong></a>. Both setups were coupled either to the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1149"><strong>Stellar MS</strong></a> or the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/404"><strong>TSQ Altis Plus triple quadrupole MS</strong></a>. The Stellar instrument features a <strong>dual-pressure linear ion trap</strong> and <strong>ion routing multipole</strong>, enabling both <strong>HCD (higher-energy collisional dissociation)</strong> and <strong>CID (collision-induced dissociation)</strong> fragmentation. Parallel reaction monitoring (PRM) was employed instead of SRM, allowing simultaneous detection of multiple transitions. Data were processed using <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/77"><strong>Thermo Scientific TraceFinder</strong></a> software with internal standards and calibration curves ranging from 0.0001 ppb to 100 ppb.</p><h4><strong>Results</strong></h4><p>The nano-flow Stellar method achieved superior sensitivity and selectivity compared with the high-flow methods on both Stellar and TSQ Altis Plus systems. Limits of quantitation (LOQ) reached the <strong>low-ppt range</strong>, while maintaining fast analysis times (&lt; 30 min) and requiring less than 20 µL of solvent per sample. Chromatograms of key eicosanoids such as <strong>15(S)-HETE</strong>, <strong>12(S)-HETE</strong>, and <strong>5(S)-HETE</strong> demonstrated higher signal intensity and improved peak shapes under nano-flow conditions. Calibration curve reproducibility and scan density (&gt; 10 scans per peak) confirmed reliable quantitative performance.</p><h4><strong>Conclusion</strong></h4><p>Coupling <strong>nano-flow chromatography</strong> with the <strong>Stellar mass spectrometer</strong> provides a powerful workflow for targeted lipid mediator analysis, significantly enhancing both selectivity and sensitivity compared with conventional high-flow LC-MS/MS. The system’s <strong>PRM capability</strong>, <strong>dual-fragmentation flexibility</strong>, and <strong>low-volume solvent use</strong> make it a robust and cost-effective solution for biological and clinical lipidomics, capable of quantifying trace-level lipid mediators efficiently and reproducibly.</p><h3><strong>4. Waters Corporation: Investigating Drug Metabolism of Methapyrilene Within a Rat Model Using a Data Dependent Acquisition Workflow with the Xevo MRT Mass Spectrometer</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720009001_en_c11ab3b6b7.pdf"><strong>Full PDF for download</strong></a></li></ul><h5><strong>Benefits&nbsp;</strong></h5><ul><li>Demonstration of the Waters™ Xevo™ MRT Mass Spectrometer (MS) workflow for the analysis of urine extracts from a drug dosed study, involving methapyrilene.</li><li>Data dependent acquisition (DDA) has been used for data collection, providing highly informative fragmentation data to confidently identify drug metabolites of interest.</li><li>Combining a DDA approach with high-throughput chromatography is shown to consistently yield high quality spectra, providing highly confident compound identifications.</li><li>A seamless workflow allowing drug metabolites to be confidently identified is demonstrated with the UNIFI™ Application within the waters_connect™ Software Platform</li></ul><p>The introduction of the Xevo MRT Mass Spectrometer allows for comprehensive drug metabolite identification (Met ID) at high acquisition speed. Using urine samples collected over a time course of six days, the excellent mass accuracy, high mass resolution, and sensitivity are highlighted, which can be achieved for drug metabolism studies. DDA provides highly specific MS/MS information, which assists with structural elucidation. The speed at which DDA data can be acquired with the Xevo MRT Mass Spectrometer can be as fast as 50 and 100 Hz, MS, and MS/MS respectively. Combined with fast chromatographic methods, this fast data acquisition rate allows for highly informative, confident data in a high-throughput fashion</p><h4><strong>Experimental</strong></h4><p>LC-MS Data Acquisition LC-MS data were collected using an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/191"><strong>ACQUITY™ Premier UPLC™ System</strong></a> coupled to a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1148"><strong>Xevo MRT Mass Spectrometer</strong></a>. The reversed-phase (RP) chromatographic separation consisted of a 5.0 minute method (injection to injection) using a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1966"><strong>CORTECS™ C18</strong></a>, 1.6 µm, 2.1 x 50 mm analytical Column (p/n: 186007114). The LC solvents were comprised of mobile phase A (0.1% formic acid containing 1 mMol ammonium formate) and mobile phase B (95:5 ACN:Water (v/v), 0.1% formic acid, 1 mMol ammonium formate) being ramped from 50% to 99% over 5 minutes using a flow rate of 0.6 mL/minute. The gradient started from 0.1% to 15% (mobile phase B) over 1.5 minutes, 50% at 3 minutes and 99% at 4 minutes. The solvent composition was held at 99% (mobile phase B) for 0.5 minutes and re-equilibration at initial conditions by 5 minutes, ready for the subsequent injection.&nbsp;</p><p>The Xevo MRT Mass Spectrometer was configured with the source settings as outlined in Table 1. MS data were collected using DDA mode of operation, which consisted of scan rates of 20 Hz (MS) and 50 Hz (MS/MS). The number of components selected for MS/MS were eight with a dynamic exclude applied of 10 mDa with a 10 second retention time tolerance.</p><h4><strong>Data Processing&nbsp;</strong></h4><p>The acquired data were processed using the UNIFI Application within the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1544"><strong>waters_connect Software Platform</strong></a>. Data were peak picked and screened against a compound database containing MP metabolites and their associated fragments. A mass tolerance of 1 ppm for both MS and MS/MS were applied.</p><h4><strong>Conclusion&nbsp;</strong></h4><p>LC-MS analysis of urine collected over a time course following administration of the drug methapyrilene, has been demonstrated using a DDA approach with the Xevo MRT Mass Spectrometer. MP undergoes complex biotransformation to provide a variety of metabolites. Utilizing a DDA workflow provides detailed, clean fragment ion spectra for confident structural elucidation. The additional benefit of sub ppm mass accuracy provides definitive identification(s). Integrating fast chromatography allows for high-throughput analyses while maintaining high resolution and mass accuracy.</p>

News from LabRulezLCMS Library - Week 46, 2025

News from LabRulezLCMS Library - Week 47, 2025

News from LabRulezLCMS Library - Week 48, 2025

<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 1st December 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 notes by <strong>Agilent Technologies,</strong> <strong>Shimadzu </strong>and<strong> Waters Corporation</strong> and poster by <strong>Thermo Fisher Scientific / HPLC!</strong></p><h3><strong>1. Agilent Technologies: Quantitative Bioanalysis of Oligonucleotides Using a 6495D Triple Quadrupole LC/MS System Combined with an Automated SPE Workstation</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_bioanalysis_oligonucleotides_6495d_qqq_lcms_spe_5994_8339en_agilent_82610a68ca.pdf"><strong>Full PDF for download</strong></a></li></ul><p>In drug development, the quantitative analysis of oligonucleotides in biological samples is crucial for pharmacokinetics (PK), pharmacodynamics (PD), and toxicological safety evaluations. Due to the associated structural complexity of oligonucleotides and the unique nature of biological matrices, the corresponding sample preparation techniques are often complex. Liquid-liquid extraction (LLE) and solid-phase extraction (SPE) are commonly used sample preparation methods, with SPE being more suitable to meet lower limit of quantitation (LLOQ) requirements and complex matrices.2 However, traditional manual SPE methods are time consuming and prone to human error.&nbsp;</p><p>Liquid chromatography/tandem mass spectrometry (LC/MS/MS) is widely used in oligonucleotide bioanalysis due to its high sensitivity, low detection limits, high selectivity, and specificity. Methods using LC/MS/MS enable the rapid and accurate detection and quantification of low-concentration target compounds in complex matrices without the need for special reagents and probes.3&nbsp;</p><p>To address the challenges in sample preparation, Agilent developed an automated SPE workstation based on the Bravo liquid handling platform, combined with the next‑generation triple quadrupole LC/MS/MS system, the 6495D triple quadrupole LC/MS system, providing a complete solution from sample preparation to data analysis. The 6495D triple quadrupole LC/MS system excels in high sensitivity quantification and low detection limits from complex matrices, while the automated SPE workstation significantly reduces manual intervention time and improves operational consistency and reliability. This complete automated SPE bioanalysis workflow significantly enhances the efficiency and reliability of quantitative oligonucleotide&nbsp;bioanalysis.&nbsp;</p><h4><strong>Experimental&nbsp;</strong></h4><h5><strong>Instruments&nbsp;</strong></h5><ul><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?search=1290+Infinity+III+Bio+LC+"><strong>Agilent 1290 Infinity III Bio LC&nbsp;</strong></a></li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=6495D"><strong>Agilent 6495D triple quadrupole LC/MS system&nbsp;</strong></a></li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=Automated+SPE+workstation"><strong>Agilent Automated SPE workstation</strong></a>, including Bravo, BenchCel and MiniHub</li></ul><h4><strong>Conclusion&nbsp;</strong></h4><p>This study verified the use of the automated Agilent SPE workstation combined with the Agilent 6495D triple quadrupole LC/MS system for oligonucleotide bioanalysis studies through the quantitative analysis of mipomersen in pig plasma. The experimental results showed that the system exhibited excellent linearity (R²=0.999) within the dynamic range of 2 to 1,000 ng/mL. The reproducibility and accuracy of the QC samples were within acceptable ranges, with most RSD values below 5%, QC low points below 10%, and back‑calculated concentration accuracy between 85 and 115%. Additionally, in the three-day repeatability experiment, the inter-batch reproducibility of the QC samples was good, with RSD values all below 10%. The back-calculated accuracies for QC samples at high, medium, and low concentration levels, as well as the LLOQ (2 ng/mL), remained stable between 80 and 120%.&nbsp;</p><p>By reducing manual intervention and improving operational consistency, the automated SPE workstation significantly enhances the efficiency and reliability of sample preparation. Combined with the high sensitivity and low detection limits of the 6495D triple quadrupole LC/MS system, this solution achieves efficient and accurate detection of low‑concentration oligonucleotides in complex matrices. Overall, this automated solution has broad application prospects in high-throughput oligonucleotide bioanalysis and meets the high requirements for quantitative analysis in drug&nbsp;development.</p><h3><strong>2. Shimadzu: Determination of N-Nitroso-Nebivolol in Nebivolol Drug Substance by </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=LCMS-2050"><strong>LCMS-2050</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_lcms_055_en_8d77db9fd3.pdf"><strong>Full PDF for download</strong></a></li></ul><h4><strong>User Benefits</strong></h4><ul><li>A new method for the determination of NDSRI impurity in nebivolol raw material was established using LCMS-2050.</li><li>The method has high sensitivity, short analysis time and high accuracy, which can fully meet the FDA's AI limit for N-Nitrosonebivolol.</li></ul><p>On September 4, 2024, the FDA issued the final guidance on the control of nitrosamine impurities in human drugs (Nitrosamine Guidance Version 2). The revised guidance describes two categories of nitrosamines: small-molecule nitrosamines and nitrosamine drug substance-related impurities (NDSRIs). Unlike small-molecule nitrosamine impurities that are structurally dissimilar to the active pharmaceutical ingredient (API), NDSRIs generally exhibit structural similarity to the API and may form through interactions between the API and nitrosating agents, such as nitrite impurities present in drug excipients.&nbsp;</p><p>Nebivolol is a potent and selective third-generation β-blocker clinically used for the treatment of patients with moderate-tosevere hypertension, as well as angina pectoris and congestive heart failure, with a recommended daily dose of 5 mg. Due to its structural characteristics, the synthesis of nebivolol may involve nitrosation reactions, leading to the introduction of the NDSRI impurity N-nitroso-nebivolol. According to the FDA's nitrosamine guidance, based on the Carcinogenic Potency Categorization Approach (CPCA), the acceptable intake (AI) limit for N-nitroso-nebivolol, a potential NDSRI in nebivolol, is set at 1500 ng/day.&nbsp;</p><p>This study established an LC-MS (<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=LCMS-2050"><strong>LCMS-2050</strong></a>) method for the determination of N-nitroso-nebivolol in nebivolol drug substance. The method demonstrates high sensitivity, short analysis time, and accurate results, providing a reference for the detection of related nitrosamine impurities.</p><h4><strong>Separation Evaluation&nbsp;</strong></h4><p>To ensure high sensitivity in impurity analysis and prevent contamination of the mass spectrometer by high-concentration active pharmaceutical ingredient (API) components, flow path switching technology was employed. This method diverts the main nebivolol peak to waste, allowing only impurities to enter the mass spectrometer for detection. Under the optimized chromatographic conditions, The retention times of nebivolol and N-nitroso-nebivolol were 1.952 min and 3.408 min, respectively, so the mass spectrometry acquisition was initiated after 2.8 min, ensuring selective detection of the target impurity. This approach effectively enabled accurate and interferencefree analysis of trace-level impurities.</p><h4><strong>Conclusion</strong></h4><p>A novel method for determining the content of N-nitrosonebivolol in nebivolol drug substance was established by using the LCMS-2050. Within the concentration range of 1 - 180 ng/mL, the correlation coefficients of the N-nitroso-nebivolol was 0.9999. When the spiked concentration of 0.03 μg/mg and 0.3 μg/mg, the recovery rate of N-nitroso-nebivolol was 100.4 and 100.2, respectively, and the recovery rate was good and met the requirements of pharmacopoeia. The method has high sensitivity, linearity, precision and recovery rate can meet the detection requirements, and which can be used for the detection of nitrosamine impurity N-nitroso-nebivolol in nebivolol drug substance.</p><h3><strong>3. Thermo Fisher Scientific / HPLC: At the intersection between chromatographic performance, ESI efficiency and instrument productivity: nano to capillary flow LC/MS on long μPAC Columns</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_intersection_between_chromatographic_performance_po_en_1a1d6f722b.pdf"><strong>Full PDF for download</strong></a></li></ul><p>LC-MS-based proteomics commonly employs low-flow liquid chromatography (LC) to separate tryptic peptides, which helps reduce sample complexity before MS/MS detection and protein identification. This technique benefits from minimal dilution on miniaturized columns and increased ionization efficiency via nanoflow electrospray ionization (ESI). As mass spectrometry (MS) devices become more sensitive and faster, there is an increasing need to boost throughput. The challenge lies in maintaining or increasing proteome coverage while simultaneously reducing analysis time. Typically, higher flow rates improve instrument productivity but may compromise sensitivity. In this study, the impact of eluting flow rates was assessed across various throughput ranges to identify the optimal balance between productivity and proteome coverage. Additionally, the transition to a dual-column single emitter configuration was evaluated to determine its effects on productivity and coverage. This approach aims to enhance LC-MS performance, ensuring high throughput without sacrificing the depth of proteome analysis.</p><h4><strong>Methods</strong></h4><p>HeLa cell digests were resuspended in 0.1% TFA and 1% ACN to create a 200 ng/µL stock solution, sonicated, diluted in 0.1% TFA, and vortexed. A <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/398"><strong>Thermo Scientific Vanquish Neo UHPLC instrument</strong></a> was configured for either a single analytical column in a trap-and-elute setup or two columns in tandem. For single column experiments, a Thermo Scientific PepMap Neo trap cartridge and a 110 cm µPAC Neo Plus column on a Thermo Scientific EASY-Spray source were used. LC methods with flow rates from 200 to 1000 nL/min were tested for throughputs ranging from 16 to 50 samples per day. The column was positioned in an external heating device mounted directly onto the ionization source and connected to an ESI emitter via a 20 µm ID voltage spacer. For tandem column experiments, two 110 cm µPAC Neo Plus columns were placed in the Vanquish Neo UHPLC column compartment between two low-dispersion 6-port switching valves. A 20 µm ID nanoViper line connected the eluents to an ESI emitter with an integrated liquid junction in an EASY-Spray source. Data were acquired using a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/402"><strong>Thermo Scientific Orbitrap Exploris 240</strong></a>/<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/401"><strong>480 mass spectrometer</strong></a> in data-independent acquisition mode.</p><h4><strong>Data Analysis&nbsp;</strong></h4><p>LC-MS data were analyzed either using a trial version of <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1782"><strong>Thermo Scientific Proteome Discoverer 3.3 software</strong></a> with Chimerys or with Spectronaut® 19. Results shown have been filtered to a 1% FDR</p><h4><strong>Conclusions&nbsp;</strong></h4><ul><li>Increasing the flow rate to 1 µL/min significantly increases instrument productivity. For gradient times below 30 minutes, 1 µL/min performs slightly better than lower flow methods, identifying up to 6679 protein groups at 50 SPD.&nbsp;</li><li>For gradient lengths over 50 minutes, a 200 nL/min flow rate provides superior chromatographic metrics and proteome coverage, resulting into a maximum of 8556 protein groups identified at 16 SPD.&nbsp;</li><li>The negative impact of a low-dispersion 6-port valve's impact scales inversely with flow rate. At 1 µL/min, peak width increases by 40%, yielding 5% lower proteome coverage but 26% higher productivity.&nbsp;</li><li>Optimized dual column single-emitter methods identified up to 6947 protein groups at 50 SPD with 3-6% CVs for the same column and 4-8% between columns (n=6).&nbsp;</li><li>Column reproducibility was shown across 5 columns, with an ID rate variation of 0.7% for HeLa digests and a median RT CV of 0.99% for spiked-in PRTC peptides.</li></ul><h3><strong>4. Waters Corporation: Improving Sample Throughput of HPLC Methods Using CORTECS 5 μm Columns</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720009072_en_1203625d4f.pdf"><strong>Full PDF for download</strong></a></li></ul><h5><strong>Benefits&nbsp;</strong></h5><ul><li>Comparable chromatographic performance for abacavir impurities was achieved using a shorter CORTECS Premier C18 5 µm Column in place of a column packed with fully porous 5 µm particles</li><li>33% reduction in solvent usage and analysis time using a CORTECS Premier C18 5 µm Column compared to a fully porous 5 µm column</li><li>33% reduction in analytical method greenness score indicating improved sustainability for the new method</li></ul><p>Improving validated HPLC methods can be challenging. Two of the approaches to increase analytical throughput, increasing mobile phase flow rate or using shorter columns, have drawbacks in higher pressures or reduced chromatographic performance. Typically, using a shorter column also requires decreasing the particle size to maintain a constant length to particle size (L/dp) ratio to ensure similar column efficiency. However, columns packed with smaller particles generate higher back pressures. Some labs may be able to migrate methods to shorter columns packed with smaller particles by transferring to a HPLC system capable of operating at higher pressures. However, if a laboratory lacks access to such systems, then their options may be limited. In those situations, finding appropriate columns to use to improve a method is not only a matter of chromatographic performance but also system compatibility, particularly from a pressure standpoint. Older HPLC systems are limited to lower pressures compared to more modern systems.&nbsp;</p><p>This application brief examines the improvement of a method for testing abacavir impurities by migrating the original method from a 4.6 x 150 mm 5 µm fully porous C18 column to a CORTECS Premier C18 4.6 x 100 mm 5 µm Column. Additionally, a fully porous 3.5 µm C18 column in 4.6 x 100 mm hardware was evaluated to determine if moving to a smaller particle size would have as much of a benefit as that provided by using a CORTECS Column. The separation conditions for method migration were obtained using the Waters Column Calculator. Along with examining throughput, solvent savings were calculated as well as analytical method greenness scores for each column tested.</p><h4><strong>Experimental</strong></h4><h5><strong>Method Conditions&nbsp;</strong></h5><ul><li><strong>LC system:</strong> <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/210"><strong>Alliance™ HPLC System</strong></a> with 2489 UV/Visible (UV/Vis) Detector</li></ul><h5><strong>Data Management&nbsp;</strong></h5><ul><li><strong>Chromatography software: </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>Results and Discussion&nbsp;</strong></h4><p>Previously, the impurity method for abacavir was migrated to a 2.7 µm CORTECS Column.1 While that work showed the benefits of scaling to a smaller particle size, not all laboratories are comfortable moving to such a small particle size. In those instances, moving from a 5 µm to a 3.5 µm particle column is a good first step. However, depending on the method, this migration might not be appropriate and could result not only in elevated system pressure but also performance loss. Figure 1 shows the chromatograms for abacavir impurities using the original method column, a 3.5 µm C18 column, and the CORTECS Premier C18 5 µm Column.</p><h4><strong>Conclusion&nbsp;</strong></h4><p>CORTECS Premier 5 µm Columns are suitable alternatives for fully porous 5 µm columns for improving validated HPLC methods. Using a CORTECS solid-core stationary phase can allow for a shorter column to be used because of their higher efficiency per unit length. To highlight this, the impurity method for abacavir was migrated from the original fully porous 5 µm, 4.6 x 150 mm column to a CORTECS Premier C18 5 µm, 4.6 x 100 mm Column, as well as a 4.6 x 100 mm 3.5 µm fully porous column. The use of the CORTECS Column achieved comparable separation performance as the original while also exhibiting a shorter analysis time, lower solvent usage, and a better AMGS value.</p>

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