<p><strong>Researchers</strong><span><strong>&nbsp;</strong></span><strong>Tereza Nešporová and Jaroslava Večerková from Brno's </strong><span><strong>RECETOX</strong></span><strong> explain why failures are also a valuable part of gaining knowledge, how they study the effects of chemical substances on the hormonal system, and why they apply their professional insights to their everyday lives.</strong></p><h5><strong>How did you get involved in research at RECETOX?&nbsp;</strong></h5><p><span><strong>Jaroslava:</strong></span> <span>I actually joined RECETOX by chance during my follow-up Master's studies in Environmental Chemistry, back when Professor Holoubek was still leading the institute. I wrote my thesis under Klára Hilscherová and simply stayed here ever since, except for a break during maternity leave. I have been part of her team the whole time, focusing on endocrine disruptors.&nbsp;What has changed over the years is the focus of our research. We used to study surface waters, particularly the presence of substances with endocrine-disrupting potential and their effects on organisms. Today, the research has shifted more toward humans and the interactions between chemical substances and the endocrine system.&nbsp;</span></p><p><span><strong>Tereza:</strong></span><strong> </strong><span>I came from a completely different field. I studied molecular biology and worked on chronic lymphocytic leukemia for my Master's thesis at CEITEC. I used genetic engineering methods such as CRISPR/Cas9 for gene editing—activating, suppressing, or completely removing genes.&nbsp;At the time, Klára Hilscherová’s group needed to create cellular models with increased production of deiodinase enzymes, which I still work on today. I had heard only positive things about the team, and when I finished my Master's degree, I wanted to move beyond my original field. The connection between hormones and neurodevelopment attracted me, and RECETOX became the obvious choice.&nbsp;</span></p><h5><strong>So you are in the same research team. How do you collaborate?&nbsp;</strong></h5><p><span><strong>Tereza:</strong></span><strong> </strong><span>We sit in the same office. And that’s probably the main thing (laughs).&nbsp;</span></p><p><span><strong>Jaroslava:</strong></span> <span>We discuss things a lot. Some mechanisms repeat across experiments, so we talk about how each of us evaluates data and why. We inspire each other, even though we do not directly collaborate on the same laboratory work.&nbsp;</span></p><h5><strong>How does your work differ?&nbsp;</strong></h5><p><span><strong>Tereza:</strong></span><strong> </strong><span>Our team is highly interconnected. We all work on developing assays for detecting endocrine-disrupting substances, specifically those affecting thyroid hormones. However, each of us focuses on a different part of the system.&nbsp;I focus on deiodinase enzymes, which regulate the activation and deactivation of hormones. Recently, I have been spending more time analyzing data and designing experiments than working directly in the laboratory.&nbsp;</span></p><p><span><strong>Jaroslava:</strong></span><strong> </strong><span>Within the team, I mainly participate in testing different types of samples—from individual chemicals and mixtures to real environmental samples such as household dust—using the assays mentioned above.&nbsp;It is important to note that chemicals can affect the hormonal system at different levels. They can bind to or block nuclear receptors, affect hormone transport in the bloodstream, or interfere with hormone metabolism. In the past, only one part of this process was monitored. Today, we aim to cover the full spectrum of these interactions.&nbsp;</span></p><p><span><strong>Tereza:</strong></span><strong> </strong><span>And that is crucial. A substance may appear negative in one test, yet affect another part of the system, producing a significant impact on the organism. That is why current research focuses on the entire range of possible mechanisms.&nbsp;</span></p><h5><strong>Why did you choose endocrine disruptors as your research topic?&nbsp;</strong></h5><p><span><strong>Jaroslava:</strong></span> <span>For me, it was more or less a coincidence while choosing my thesis topic. But it turned out to be a fascinating area, so I stayed with it.&nbsp;</span></p><p><span><strong>Tereza:</strong></span> <span>I had been interested in it for a long time. I also like that our work has a concrete outcome—we develop tests that can realistically be used in decision-making regarding the safety of chemical substances. Compared to my previous research, I feel this work has a much more direct application.&nbsp;</span></p><h5><strong>What about your PhD studies?&nbsp;</strong></h5><p><span><strong>Jaroslava:</strong></span><strong> </strong><span>My PhD studies are currently on hold. After maternity leave, I returned as a laboratory technician, and my project work gradually shifted away from my original doctoral topic.&nbsp;</span></p><p><span><strong>Tereza:</strong></span><strong> </strong><span>I am currently in the fourth year of my PhD studies. I also completed a research stay in Germany, where I learned to work with neuronal cells.&nbsp;As part of my studies, I focus mainly on deiodinases—the enzymes that act as the “on and off switches” of thyroid hormones. We are developing a test that can measure the effects of chemical substances on their activity.&nbsp;In addition, we are now studying how these substances affect neurons and their development, because proper thyroid hormone levels are crucial for nervous system development.&nbsp;</span></p><h5><strong>What is the most challenging aspect of laboratory work?&nbsp;</strong></h5><p><span><strong>Tereza:</strong></span><strong> </strong><span>Probably unpredictability. Cells do not always grow as expected, instruments have their bad days, and human error also plays a role. The important thing is to document all mistakes and complications and learn from them.&nbsp;</span></p><p><span><strong>Jaroslava:</strong></span> <span>I agree with Tereza. The biggest challenge is unpredictability. Cells are living systems and do not always behave the same way.&nbsp;</span></p><h5><strong>Can a career in science be combined with parenthood?&nbsp;</strong></h5><p><span><strong>Jaroslava:</strong></span> <span>At RECETOX, definitely. There is a great deal of flexibility, and managers are very supportive. When necessary, it is possible to work from home. We help each other a lot within the team.&nbsp;</span></p><p><span><strong>Tereza:</strong></span> <span>It also depends on the type of work and whether it can be planned according to your needs. Our team collaborates closely and tries to help one another whenever necessary. That is key. And when needed, data analysis and manuscript writing can also be done from home.&nbsp;</span></p><h5><strong>Is your work more routine or highly varied?&nbsp;</strong></h5><p><span><strong>Jaroslava:</strong></span> <span>Very varied. Laboratory work alternates with computer-based work. We are constantly learning new things. It is definitely not repetitive or boring, and that is what I enjoy about it.&nbsp;</span></p><p><span><strong>Tereza:</strong></span> <span>The only routine in my work is taking care of cell cultures. Otherwise, the work is constantly changing. That is also what I enjoy most about it.&nbsp;</span></p><h5><strong>What are the biggest challenges in your work?&nbsp;</strong></h5><p><span><strong>Jaroslava:</strong></span> <span>Data evaluation. Measuring something is one thing; evaluating and interpreting it correctly is another.&nbsp;</span></p><p><span><strong>Tereza:</strong></span> <span>Implementing new methods. And also those moments when you do everything correctly according to a published protocol, yet the results still do not match expectations.&nbsp;In science, you have to learn to accept that not everything works the first time. The important thing is not to give up, to continue optimizing, and to keep hoping for success.&nbsp;</span></p><h5><strong>What role should open science play?&nbsp;</strong></h5><p><span><strong>Tereza:</strong></span><strong> </strong><span>A huge one. If we had access to detailed protocols and original datasets, it would save enormous amounts of time and money during the implementation and optimization of new methods. Scientific papers often omit details that are actually critical for experimental success.&nbsp;</span></p><p><span><strong>Jaroslava:</strong></span><strong> </strong><span>It is also important to share failures. A negative result is still a result, and it can provide valuable information.&nbsp;</span></p><h5><strong>You are both mothers. Does your work influence your personal lives?&nbsp;</strong></h5><p><span><strong>Jaroslava:</strong></span><strong> </strong><span>Absolutely. For example, we pay close attention to washing fruit because of chemical treatments. I encourage my children to think about what they consume, what it contains, and where it comes from. We try to avoid plastics and consistently separate waste.&nbsp;</span></p><p><span><strong>Tereza:</strong></span> <span>Definitely. Even before having a child, I paid close attention to the composition of cosmetics, food, and other products. After becoming a parent, my caution multiplied.&nbsp;I was especially careful during pregnancy because a developing organism is most sensitive to harmful substances. Sometimes relatives raise their eyebrows at this, but when we have information firsthand, we cannot simply ignore it.&nbsp;</span></p><p><span><strong>Jaroslava:</strong></span> <span>We also pay attention to toys, clothing, and materials used in the home. It is a kind of professional deformation (laughs).&nbsp;</span></p><h5><strong>How can your research contribute from a broader perspective? How can others build upon it?&nbsp;</strong></h5><p><span><strong>Jaroslava:</strong></span> <span>We are gradually optimizing methods that may eventually be used routinely. That is probably our greatest contribution.&nbsp;</span></p><p><span><strong>Tereza:</strong></span> <span>Yes, our methods could help identify undesirable effects of substances before they reach the market, potentially leading to regulation of their production.&nbsp;</span></p><h5><strong>Would you like to continue working on this topic in the future?&nbsp;</strong></h5><p><span><strong>Jaroslava Večerková:</strong></span><strong> </strong><span>The topic of endocrine disruption is extremely interesting and very broad. The direction of research may change depending on current projects, but I would like to remain part of the team.&nbsp;</span></p><p><span><strong>Tereza Nešporová:</strong></span> <span>I am very interested in uncovering how substances affect human health, and I believe I will want to continue working in this area. I like how dynamic the research is and that it has direct practical applications. I do not mind change, but I am happy within this team and would like to stay. We work together as a well-coordinated group.&nbsp;</span></p>

Researchers Tereza Nešporová and Jaroslava Večerková: In science, we must learn to work with failure

<div><p><strong>Protein purification is a critical step in biopharmaceutical research, proteomics, and industrial biotechnology. It involves isolating a specific protein from complex biological mixtures while preserving its native structure and activity. Traditional methods such as precipitation or dialysis lack the precision required for high-purity protein isolation, especially when dealing with therapeutic targets or complex protein assemblies.</strong></p><p>High-performance liquid chromatography (HPLC) protein purification has transformed this field by providing high resolution, reproducibility, and speed, making it indispensable for both analytical and preparative workflows.</p></div><div><div><div><div><h3><strong>What is HPLC Protein Purification?</strong></h3><p>HPLC purification is based on the principle of selective interactions between proteins and the stationary phase under high-pressure liquid flow. The separation relies on differences in size, charge, hydrophobicity, or affinity of proteins toward the stationary phase material.</p><p>Unlike conventional low-pressure methods such as gel filtration or affinity batch purification, HPLC purification operates under high pressure (up to 6000 psi), enabling faster separation with enhanced resolution and quantitative recovery. It also offers precise control over parameters like gradient composition, pH, and temperature, minimizing variability between runs.</p><p>In contrast to traditional column chromatography, HPLC uses smaller stationary phase particles (3–10 µm), resulting in increased surface area and sharper <a target="_blank" rel="noopener noreferrer" href="https://www.phenomenex.com/resources/knowledge-center/hplc-knowledge-center/how-to-reduce-peak-tailing-in-hplc"><strong>peak shapes</strong></a>, which are vital for protein purity assessment and scale-up purification.</p></div></div></div></div><div><div><div><div><h3><strong>Types of HPLC Used for Protein Purification</strong></h3></div></div></div></div><div><p>Different HPLC modes can be employed depending on the physicochemical properties of the target protein. Each method offers unique advantages in terms of selectivity and recovery.</p><h4><strong>Reversed-Phase HPLC (RP-HPLC)</strong></h4><p><a target="_blank" rel="noopener noreferrer" href="https://www.phenomenex.com/techniques/hplc-reversed-phase"><strong>Reversed-Phase HPLC</strong></a> separates proteins based on hydrophobic interactions between the analyte and the stationary phase, typically C4, C8, or C18 silica-based columns. Proteins are eluted using gradients of organic solvents (usually acetonitrile or methanol) in the presence of volatile acids like formic or trifluoroacetic acid (TFA).</p><p>This method provides exceptional resolution and is commonly used for protein fragment purification, and analysis of recombinant proteins. For example, reversed-phase HPLC is routinely employed in peptide therapeutic workflows, such as the<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an59280424_w_fc6c3ac6f0.pdf"><strong> Tirzepatide Preparation and Purification </strong></a>process, where purity and recovery are critical to product efficacy. However, due to exposure to organic solvents, RP-HPLC may cause partial denaturation, limiting its use for native proteins.</p><h4><strong>Ion Exchange HPLC (IEX-HPLC)</strong></h4><p><a target="_blank" rel="noopener noreferrer" href="https://www.phenomenex.com/techniques/hplc-sec"><strong>IEX-HPLC</strong></a> separates proteins based on net surface charge. Cation exchange (with negatively charged stationary phases) binds positively charged proteins, while anion exchange does the reverse.</p><p>By gradually increasing salt concentration or changing the mobile phase pH, proteins are eluted according to their isoelectric points (pI).By gradually increasing salt concentration or changing the mobile phase pH, proteins are eluted according to their isoelectric points (pI).</p><p>This approach is highly suitable for native purification, protein isoform analysis, and charge variant characterization essential steps in therapeutic protein production.</p><h4><strong>Size Exclusion Chromatography (SEC-HPLC)</strong></h4><p>SEC separates molecules based on their hydrodynamic radius. Larger proteins elute first, as they are excluded from the pores of the stationary phase, while smaller molecules penetrate and elute later.</p><p><a target="_blank" rel="noopener noreferrer" href="https://www.phenomenex.com/techniques/hplc-sec"><strong>SEC-HPLC</strong></a> is non-denaturing and ideal for determining protein molecular weight, oligomeric state, and aggregate content. It is frequently used as a final polishing step following ion exchange or affinity chromatography.</p><h4><strong>Affinity HPLC</strong></h4><p>Affinity-based HPLC leverages specific biological interactions between the target protein and a ligand immobilized on the stationary phase (e.g., antibodies, metal ions, or tags such as His-tag or GST).</p><p>When integrated into an HPLC system, affinity chromatography provides high selectivity and purity (&gt;95%) in a single step, often used in recombinant protein purification and biomarker capture applications.</p></div><div><div><div><div><h3><strong>Benefits of Using HPLC for Protein Purification</strong></h3><p>HPLC-based protein purification combines the analytical precision of chromatography with the preparative capability needed for research and biomanufacturing. The main advantages include:</p><h5><strong>Benefit &nbsp;/ &nbsp;Description</strong></h5><p><strong>High Resolution and Selectivity:</strong></p><ul><li>Smaller stationary phase particles and optimized flow dynamics allow HPLC to resolve proteins differing by minimal physicochemical properties. This precision is vital for distinguishing isoforms, mutants, and post-translationally modified variants.</li></ul><p><strong>Accuracy and Reproducibility</strong></p><ul><li>Automated systems maintain stable flow rates, gradient control, and detection sensitivity, ensuring reproducible results essential for regulated biopharmaceutical workflows.</li></ul><p><strong>Speed and Efficiency</strong></p><ul><li>Modern UHPLC systems equipped with fully porous or core–shell particles achieve faster separations without sacrificing resolution, reducing run times by up to 50% compared to classical methods.</li></ul><p><strong>Compatibility and Scalability</strong></p><ul><li>HPLC systems are highly adaptable, supporting both analytical-scale characterization and preparative-scale purification. Methods developed at microgram scale can be seamlessly transferred to milligram or gram scales used in process development.</li></ul><div><div><div><div><h3><strong>Factors Affecting the Efficacy of Protein Purification by HPLC</strong></h3></div></div></div></div><div><p>Optimizing HPLC for protein purification requires balancing purity, recovery, and throughput. The following parameters significantly influence results:</p><ul><li><strong>Purification goal:</strong> Define whether the priority is yield, purity, or throughput. Analytical separations prioritize resolution, while preparative runs emphasize recovery and process speed.</li><li><strong>Protein solubility and stability:</strong> Unstable or aggregation-prone proteins may require low temperatures or additives (e.g., glycerol, mild detergents).</li><li><strong>Column chemistry and particle size:</strong> Select appropriate stationary phase chemistry (silica, polymeric, or mixed-mode) and particle size to suit molecular weight and hydrophobicity.</li><li><strong>Mobile phase pH and ionic strength:</strong> pH should be optimized near but not at the protein’s pI; salt gradients influence both resolution and elution strength.</li><li><strong>Flow rate and gradient profile:</strong> Steeper gradients shorten run time but may compromise separation quality.</li><li><strong>Purified protein final form:</strong> Consider buffer composition and concentration for storage or downstream assays to maintain bioactivity.</li></ul></div><div><div><div><div><h3><strong>Best Practices for Protein Purification by HPLC</strong></h3></div></div></div></div><div><p>Following good chromatographic practices ensures consistent, high-quality results:</p><ul><li><strong>Column conditioning and storage:</strong> Always equilibrate new <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=48&amp;manufacturerItems=50"><strong>HPLC columns</strong></a> with 10 column volumes of buffer; store in appropriate solvent containing preservatives.</li><li><strong>Avoid protein precipitation:</strong> Maintain appropriate ionic strength and, when native state is desired, avoid exposure to denaturing solvents or high organic content.</li><li><strong>Prevent sample loss:</strong> Use low-protein-binding tubing and vials; minimize dead volume in the system.</li><li><strong>Troubleshooting:</strong><ul><li>Peak fronting - may indicate column overloading.</li><li>Peak broadening – may indicate column channeling.</li><li>Carryover – reduce by extending wash cycles or using stronger regeneration solvents.</li><li>Clogging – prefilter samples (0.22 µm) and periodically flush the system.</li></ul></li></ul></div></div></div></div></div>

Protein Purification by HPLC: Methods, Benefits, and Best Practices

<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 6th July 2026? 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>and<strong> Metrohm </strong>and posters by<strong> Shimadzu/ASMS, Thermo Fisher Scientific/ASMS </strong>and <strong>Waters Corporation/ASMS!</strong></p><h3><strong>1. Agilent Technologies: Determination of 41 Per- and Polyfluoroalkyl Substances (PFAS) in Cosmetics</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_captiva_emr_pfas_food_ii_cartridge_5994_9111en_agilent_be91861646.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Per- and polyfluoroalkyl substances (PFAS) are synthetic compounds composed of highly stable carbon–fluorine (C–F) bonds, giving them exceptional thermal and chemical stability, leading to poor environmental degradability. PFAS have been used in cosmetics to provide water resistance, oil repellency, or application smoothness. The unintentionally presented PFAS residues in raw materials may remain in finished cosmetic products, and can be absorbed through the skin. Lip products and spray type products can also lead to inhalation or oral exposure to residues.<sup>1,2</sup>&nbsp;</p><p>Agilent Captiva EMR PFAS Food cartridges are designed to provide streamlined and comprehensive matrix removal after QuEChERS extraction for complex sample matrices, demonstrating effective matrix cleanup efficiency and excellent performance for quantitative PFAS analysis.<sup>3,4</sup> In this study, we performed QuEChERS extraction followed by cleanup with Captiva EMR PFAS Food II cartridges for PFAS analysis in five types of cosmetics.</p><h4><strong>Experimental</strong></h4><h5><strong>Equipment and materials&nbsp;</strong></h5><p>The study was performed using an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=1290+Infinity+III"><strong>Agilent 1290 Infinity III LC</strong></a> system consisting of an Agilent 1290 Infinity III high-speed pump (G7120A), an Agilent 1290 Infinity III multisampler (G7167B), and an Agilent 1290 Infinity III multicolumn thermostat (G7116B). The LC system was coupled to an <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</strong></a> (G6495D) equipped with an Agilent Jet Stream Electrospray ion source. Agilent MassHunter Workstation software was used for data acquisition and analysis.&nbsp;</p><p>The 1290 Infinity III LC system was modified using an Agilent InfinityLab PFAS Analysis HPLC conversion kit (part number 5004-0006), including an Agilent InfinityLab PFC delay column, 4.6 × 30 mm (part number 5062-8100). Chromatographic separation was performed using an Agilent <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=ZORBAX+RRHD+Eclipse+Plus"><strong>ZORBAX RRHD Eclipse Plus</strong></a> C18, 95 Å, 2.1 × 100 mm, 1.8 µm (part number 959758-902).</p><h4><strong>Results and discussion&nbsp;</strong></h4><h5><strong>Chromatography and calibration curve linearity&nbsp;</strong></h5><p>Figure 2 shows the overlaid chromatograms for 41 PFAS calibration standards, including 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, and 10.0 ng/mL in ACN. The table on the right shows the peaks identification and corresponding retention time. Across 41 PFAS compounds, R² values ranged from 0.9940 to 0.9998, indicating excellent linearity for all analytes' calibration curves across the calibration range of 0.05 to 10.0 ng/mL.</p><h5><strong>PFAS detection in real cosmetic samples&nbsp;</strong></h5><p>Of the 15 cosmetic products assessed, one eyeshadow sample was detected with a trace level of positive PFAS analytes, including PFHeA, PFHpA, PFNA, and PFOA, at about 0.1 ng/g in the sample. Figure 5 shows the method blanks and positive detection of PFHpA and PFNA in this eyeshadow&nbsp;product.</p><h4><strong>Conclusion&nbsp;</strong></h4><p>A simplified, rapid, and reliable method using QuEChERS extraction followed by EMR mixed-mode passthrough cleanup using the Agilent Captiva EMR PFAS Food II cartridge and LC/MS/MS detection was developed for 41 PFAS analysis in cosmetics.</p><h3><strong>2. Metrohm: brightRC – Advanced brightener analysis with a response curve</strong></h3><p><strong>Next-level brightener determination in nonlinear systems</strong></p><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_v_242_download_83376b2923.pdf"><strong>Full PDF for download</strong></a></li></ul><h4><strong>WHAT IS BRIGHTRC?&nbsp;</strong></h4><p>brightRC is a response curve calibration approach for the determination of brightener-type additives by CVS or CPVS (Cyclic Pulse Voltammetric Stripping), hence the name «brightRC». This technique is suitable to measure brightener content in copper baths for plating. The method is based on signal normalization using the electrolyte value, which is the response for the virgin makeup solution (VMS) saturated with suppressor, and brightener defined as Q0 in the viva software. All recorded signals are divided by Q0 and the normalized values are plotted against concentration to generate an external calibration curve. This calibration can then be used for routine sample determination without repeated standard additions.&nbsp;</p><p>The sample is simply measured using the same method as the calibration, signals are normalized and evaluated using the mathematically fitted calibration curve. By decoupling calibration from sample measurement, brightRC removes the need for repeated standard additions with each sample measurement, enabling faster and more efficient routine analysis.&nbsp;</p><p>The brightRC calibration approach supports flexible regression models including quadratic and nonlinear regression. This allows reliable quantification of additive systems that do not exhibit a wide linear working range or exhibit nonlinear response characteristics, thereby expanding the range of applicable brightener chemistries.&nbsp;</p><p>One important detail that further differentiates brightRC from the MLAT approach relates to normalization of the signal for calibration and sample analysis. This normalization approach improves measurement robustness as variations in absolute signal intensity naturally occurring over the working day are mathematically compensated.</p><h4><strong>HOW DOES BRIGHTRC WORK IN PRACTICE?&nbsp;</strong></h4><p>The brightRC workflow separates calibration and sample analysis. It can be implemented in different setups (from manual to fully automated) depending on the additive system and concentration range. After recording the electrolyte value, calibration standards are measured to establish the calibration curve, otherwise known as the response curve. Once the calibration is stored, samples are determined with the same CVS or CPVS method, without further standard additions. The sample concentration is automatically calculated from the normalized signal using the calibration curve.&nbsp;</p><p>For sample measurement, viva 4.0 provides two practical approaches:&nbsp;</p><ol><li>«brightRC with solution exchange»&nbsp;</li><li>«brightRC with sample dilution» Both variants follow the same calibration principle and differ only in the sample handling strategy.</li></ol><h4><strong>WHEN TO USE BRIGHTRC?&nbsp;</strong></h4><p>brightRC is ideal for routine brightener control in stable plating baths where high sample throughput is required. It is particularly suitable when matrix conditions are consistent and electrode fouling is limited. The approach is especially advantageous for additive systems exhibiting nonlinear signal behavior. If strong matrix variations, significant breakdown products, or severe electrode fouling are expected and a linear correlation between signal and analyte concentration is guaranteed, standard addition techniques such as MLAT are preferred because they may provide additional robustness.</p><h3><strong>3. Shimadzu / ASMS: PFAS Analysis of Leachate: Evaluating Key Sample Preparation and LC–MS/MS Parameters for Improved Performance</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/WP_255_PFAS_Analysis_of_Leachate_Evaluating_Key_Sample_Preparation_and_LC_MSMS_Parameters_for_Improved_Performance_1114ee264c.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Landfill leachate is a highly complex and variable matrix that poses significant challenges for the analysis of per- and polyfluoroalkyl substances (PFAS). Although U.S. Environmental Protection Agency Method 1633A provides a standardized framework for PFAS quantitation,1 analytical performance remains strongly influenced by sample preparation, chromatographic separation, and mass spectrometric conditions. Landfill leachate samples were extracted using WAX SPE following EPA 1633A and analyzed by LC–MS/MS using 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><strong> </strong>under negative ESI conditions. This study evaluates the key factors governing LC–MS/MS sensitivity, signal response, and reproducibility in landfill leachate, including a systematic assessment of SPE, chromatographic behavior, and ionsource conditions across PFAS classes (Figure 1).</p><h4><strong>Results&nbsp;</strong></h4><p>Chromatographic performance was evaluated across four C18 columns (A–D) using peak area, height, asymmetry, and HETP. Two columns produced a higher signal and improved peak shape, while the remaining columns showed greater efficiency. Under repeated batches followed by storage in acetonitrile, the Shimadzu GIST column produced a consistent signal (≤15% change), whereas another column showed 20–50% signal loss, particularly for long-chain PFAS and precursors, indicating greater susceptibility to matrix accumulation (Figure 2).</p><h4><strong>Conclusion&nbsp;</strong></h4><ul><li>PFAS analytical performance was dependent on compoundclass-dependent extraction, chromatography, and ionization behavior.&nbsp;</li><li>GIST demonstrated lower signal variability under repeated matrix exposure, with lower signal degradation than alternative C18 phases.</li><li>Neutral PFAS showed poor retention on WAX sorbents, requiring optimization beyond standard EPA 1633A extraction conditions.&nbsp;</li><li>PFCA response was more influenced by gas flow conditions, whereas PFSA response was more sensitive to source temperature conditions.</li></ul><h3><strong>4. Thermo Fisher Scientific / ASMS: Ultra-high throughput proteomics at &gt;500 samples/day using advanced </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/398"><strong>Vanquish Neo UHPLC</strong></a><strong> methods with </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1959"><strong>Orbitrap Astral Zoom MS</strong></a></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_004708_lsms_ultra_high_throughput_vanquish_astral_proteomics_po004708_asms2026_na_en_f6499c3eec.pdf"><strong>Full PDF for download</strong></a></li></ul><p>High-throughput mass spectrometry is increasingly critical for large-cohort proteomics, population-scale studies, and screening applications. While established LC–MS platforms routinely support several hundred samples per day, further increases in throughput are often limited by chromatographic stability, robustness, and reproducibility under ultra-short gradient conditions, as well as the acquisition speed of the mass spectrometer. Here, we describe an advanced ultra-high-throughput LC–MS methodology developed on the Thermo Scientific Vanquish Neo UHPLC system and Thermo Scientific Orbitrap Astral Zoom mass spectrometer. The goal of this work was to extend the practical throughput limits of LC–MS while achieving deep proteome coverage and maintaining run-to-run reproducibility.</p><h4><strong>Materials and methods</strong></h4><h5><strong>LC-MS analysis&nbsp;</strong></h5><p>Samples were separated on a Thermo Scientific 150 µm × 70 mm C18 column (prototype, not commercially available yet) operated at 4 µL/min using a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/398"><strong>Vanquish Neo UHPLC system</strong></a> in NanoCap Trap&amp;Elute configuration at room temperature. Prototype methods allowing for reduced LC duty cycles (approx. 1 min of sample pickup/loading and integrated trap-column offline wash) were applied, the standard gradients were adjusted to 1 – 3 min. The Thermo Scientific PepMap Neo 5 µm C18 300 µm × 5 mm cartridge was used as trap column and the separation column connected with a 15 µm ID capillary Thermo Scientific EASY-Spray emitter (ES994) to an EASY-Spray source. The <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1959"><strong>Orbitrap Astral Zoom MS</strong></a> (ICSW AST 2.2) was operated in a dataindependent acquisition (DIA) mode, MS method parameters are given in Table 1. Comparative data for the chemoproteomics samples at lower throughputs (60-300 SPD) were acquired using an EASY-Spray 150 µm × 150 mm C18 column (ES906), as described before.&nbsp;</p><h5><strong>Data analysis&nbsp;</strong></h5><p>The raw data files were processed with Spectronaut® 20.5 software (Biognosys AG), <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1782"><strong>Thermo Scientific Proteome Discoverer </strong></a>3.3 SP1 with <strong>CHIMERYS 5.0 </strong>or DIA-NN Enterprise 2.3.2 (Aptila Biotech GmbH) nodes using default settings as specified in the individual figures. The data was searched against the Homo sapiens SwissProt database (TaxID=9606: 42,252 sequences) and contaminants FASTA file. Peptide numbers correspond to unique stripped peptides</p><h4><strong>Conclusions&nbsp;</strong></h4><ul><li>The advanced Vanquish Neo UHPLC methodology in combination with the Orbitrap Astral Zoom MS delivered highly reproducible, deep and precise proteome profiling across ultra-high-throughput regimes (600 SPD) exceeding current field benchmarks.&nbsp;</li><li>The workflow enables rapid chemoproteomic screening, as demonstrated with activity-based protein profiling (ABPP) samples, reliably identifying enriched putative target proteins of an antibiotic small molecule probe.</li></ul><h3><strong>5. Waters Corporation / ASMS: Assessing Relative Response of Four European-Regulated PFAS in Human Serum Using Cyclic Ion Mobility MS</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720009410_c98e7c87ec.pdf"><strong>Full PDF for download</strong></a></li></ul><div><div><section><div><div><div><div><div><div><p>The poster presents an LC-Cyclic-IMS-MS approach for assessing four European-regulated PFAS—PFOS, PFOA, PFNA, and PFHxS—in human serum. The study focuses on environmental exposure monitoring in Ghanaian firefighters and e-waste handlers, using ion mobility mass spectrometry to add separation power and improve identification confidence in complex biological matrices. The work is motivated by increasing regulatory concern around PFAS toxicity, bioaccumulation, and the need for reliable exposure assessment in human biofluids.</p><p>The experimental workflow combined reversed-phase LC with a quadrupole-cyclic ion mobility-time-of-flight MS system (<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/202"><strong>SELECT SERIES</strong> <strong>Cyclic IMS</strong></a>). Human serum samples were prepared using SPE µElution plates with a polymeric reversed-phase/weak anion exchange sorbent. The LC system was modified with PFAS-free components and an isolator column to reduce background contamination, which is especially important for trace-level PFAS analysis. Identification was supported by retention time, accurate mass, isotopically labelled internal standards, and collision cross section values.</p><p>The results showed detectable levels of the four regulated PFAS in both occupational groups, with a combined detection rate of 83% across human serum samples and NIST reference standards. Firefighter samples generally showed higher concentration ranges than e-waste handler samples, especially for PFOS, PFOA, PFNA, and PFHxS. For example, PFNA reached up to 5.713 ng/mL in firefighter serum, compared with up to 1.513 ng/mL in e-waste handler serum.</p><p>Overall, the poster concludes that LC-Cyclic-IMS-MS provides a highly specific strategy for PFAS analysis in complex biological samples. Ion mobility and CCS values improve confidence in trace-level identification and help distinguish target PFAS from co-eluting matrix background. The authors suggest that this approach can support both targeted PFAS monitoring and broader non-targeted screening of known and emerging PFAS, while also helping future studies connect exposure data with environmental fate and potential toxicological effects.</p></div></div></div></div></div></div></section></div></div>

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RECETOX (Research Centre for Toxic Compounds in the Environment) is an independent department of the Faculty of Science of Masaryk University in Brno, which has been established in 1983. RECETOX is focused on the research and education in cross-cutting area of Environment and Health. It addresses interactions among chemicals, environment, and biological systems, including consequences at local, regional, and global levels.

RECETOX - Centrum pro výzkum toxických látek v prostředí

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