<p><strong>This study evaluates an immobilized enzyme reactor (IMER) containing peptide N-glycosidase from </strong><i><strong>Rudaea cellulosilytica</strong></i><strong> (PNGase Rc) for rapid online deglycosylation of intact glycoproteins. Human glycoproteins and therapeutic antibodies were used as model substrates to assess reactor performance.</strong></p><p>The PNGase Rc IMER efficiently removed complex sialylated and core-fucosylated glycans, simplifying protein characterization and glycosylation-site analysis. The approach offers a fast and effective solution for bioanalytical workflows, biopharmaceutical quality control, and stability testing of therapeutic proteins.</p><blockquote><h4><strong>The original article</strong></h4><h4><a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.6c00577"><strong>Online Deglycosylation of Monomeric Intact Proteins Using the PNGase Rc Immobilized-Enzyme Reactor</strong></a></h4><p>Katarína Molnárová, Petr Novák, Jana Nováková, and Petr Pompach*</p><p>ACS Omega 2026, 11, 17, 25724–25729</p><p><a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1021/acsomega.6c00577">https://doi.org/10.1021/acsomega.6c00577</a></p><h6>licensed under <a target="_blank" rel="noopener noreferrer" href="https://creativecommons.org/licenses/by/4.0/"><strong>CC-BY 4.0</strong></a></h6></blockquote><p><strong>Selected sections from the article follow. Formats and hyperlinks were adapted from the original.</strong></p><div><p>Protein glycosylation, in which monosaccharide units are attached to a protein backbone through enzymatic pathways, is involved in many important biochemical processes and is considered one of the most important post-translational modifications. <a href="javascript:void(0);">(1)</a> It affects not only the stability and secretion of proteins, cell–cell interactions, and receptor–ligand recognition mechanisms but also the biological activity, plasma half-life, immunogenicity, pharmacodynamics, and pharmacokinetics of biotherapeutics. <a href="javascript:void(0);">(2−5)</a> Therapeutical monoclonal antibodies (mAbs) are among the best known pharmaceuticals that are based on glycoproteins. <a href="javascript:void(0);">(6)</a> Over 90% of the mAbs are derived from immunoglobulin G (IgG), in which <i>N</i>-glycosylation occurs at Asn297 of the Fc region and is responsible for modulating immune responses and interactions with other proteins. <a href="javascript:void(0);">(7,8)</a> In cancer treatment, mAbs are used to increase the response of the immune system against the tumor cell, thus inhibiting the cancerous cell growth and helping with the destruction and elimination. <a href="javascript:void(0);">(9)</a> Among the most commonly used mAbs are trastuzumab, bevacizumab, cetuximab, or margetuximab, which are used as an additional treatment for breast, colorectal, prostate, or lung cancer. <a href="javascript:void(0);">(10)</a></p><p>Mass spectrometry (MS)-based methods have become a method of choice in the analysis of mAbs that can provide information on sequence, post-translational modifications, and glycosylation profiles. <a href="javascript:void(0);">(11)</a> The enzymatic removal of the attached glycans from the protein backbone can reduce the complexity of the MS analysis, which can lead to a better understanding of the protein structure and modifications. In glycoproteomics, peptide-<i>N</i>-glycosidase F (PNGase F) is most used for glycan removal. It cleaves the linkage between the Asn residue and the <i>N</i>-acetylglucosamine (GlcNAc). <a href="javascript:void(0);">(12)</a> Jmeian et al. developed a monolithic PNGase F enzyme reactor fabricated in fused silica using a glycidyl methacrylate-<i>co</i>-ethylene dimethacrylate polymer and integrated it into an LC–MS workflow. The authors demonstrated the applicability of this system for the characterization of released neutral and acidic <i>N</i>-glycans by LC–MS analysis. <a href="javascript:void(0);">(13)</a> In another study, oriented immobilization of PNGase F onto a methacrylate-based monolithic support via glutathione-<i>S</i>-transferase–glutathione affinity coupling was employed to release <i>N</i>-glycans from denatured ribonuclease B and fetuin, as well as from native IgG. The released glycans and the corresponding deglycosylated proteins were subsequently analyzed by MALDI-MS. <a href="javascript:void(0);">(14)</a></p><p>Gramlich et al. showed the potential of PNGase Rc for <i>N</i>-glycan hydrolysis from both tryptic peptides and native glycoproteins at a low pH. The favorable properties of PNGase Rc, including activity at acidic pH and compatibility with reducing agents such as <i>tris</i>(2-carboxyethyl)phosphine hydrochloride (TCEP) and denaturants like urea, make this enzyme well suited for online intact protein deglycosylation workflows. <a href="javascript:void(0);">(15,16)</a></p><p>In this report, we demonstrate the potential of the PNGase Rc IMER for rapid online deglycosylation of reduced monomeric glycoproteins, including human haptoglobin (Hp), human transferrin, <i>anti</i>-hHD6, and the therapeutic monoclonal antibodies trastuzumab and bevacizumab. The column retained its activity over more than 150 consecutive measurements, highlighting its robustness, suitability for repeated use in bioanalytical workflows, and applicability to pharmaceutical research and development.</p><h4><strong>Experimental Section</strong></h4><h5><strong>Instrumentation and Experimental Conditions</strong></h5><p>Automated sample handling and online deglycosylation were carried out using the HDX workstation (AffiPro, Czech Republic). The scheme of the system is shown on <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.6c00577#fig1">Figure 1</a>. Samples of 50 mM ammonium bicarbonate buffer (pH 8.2) and 40 mM TCEP were placed in a sample tray at 4 °C. Ten microliters of the protein sample at a concentration of 0.03 mg/mL (0.3 μg per injection) were added to 40 μL of 50 mM ammonium bicarbonate and then transferred to 50 μL of 40 mM TCEP, pH 2.3. The disulfide bond reduction was carried at 40 °C for 5 min. The automated sample handling system injected 100 μL of the reaction mixture into the sample loop and switched the injection valve. Online deglycosylation was performed at room temperature using a PNGase Rc IMER (2.1 × 20 mm, AffiPro, Czech Republic) at a flow rate of 50 μL/min, followed by protein desalting on an OPTI-TRAP (0.5 × 2 mm) trap column (Optimize Technologies Inc., Oregon City, Oregon, USA) heated to 55 °C for 10 min with 0.2% formic acid delivered by an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=1260+Infinity+III+isocratic+pump"><strong>Agilent 1260 Infinity III isocratic pump</strong></a><strong> </strong>(Agilent Technologies, Santa Clara, California, USA). By switching the trap valve, the protein was eluted by an acetonitrile gradient [(min)/% B] 0/10-9/10-16/70-16.5/90-17/90-18/10-23/10] delivered by the <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 HPLC</strong></a> system (Agilent Technologies, Santa Clara, California, USA) at a flow rate of 20 μL/min. The mobile phase A consisted of 5% acetonitrile with 1% propan-2-ol and 0.1% formic acid, and the mobile phase B consisted of 95% acetonitrile with 1% propan-2-ol and 0.1% formic acid. The <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=8&amp;search=maXis+Q-TOF"><strong>maXis Q-TOF mass spectrometer</strong></a> (Bruker Daltonics, Bremen, Germany) was operated in the survey MS mode in a mass range of <i>m</i>/z 50–3000. The acquired data were analyzed and processed by <strong>DataAnalysis 4.4</strong> (Bruker Daltonics) and deconvoluted by UniDec <a href="javascript:void(0);">(17)</a> with the following parameters: data processing range, <i>m</i>/z 500–3000; charge range, 1–80; allowed mass range, 1,000–80,000; sampling rate, 10 Da; peak detection range, 500 Da; and peak detection threshold, 0.1.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Omega_2026_11_17_25724_25729_Figure_1_Schematic_of_the_automated_sample_handling_system_for_online_protein_deglycosylation_and_desalting_d4ee48b627.jpg" alt="ACS Omega 2026, 11, 17, 25724–25729: Figure 1. Schematic of the automated sample handling system for online protein deglycosylation and desalting. Protein samples, along with 50 mM ammonium bicarbonate and 40 mM TCEP stock solutions were placed in a sample tray chilled to 4 °C, while a second tray heated to 40 °C was used for disulfide bond reduction with TCEP buffer." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Omega_2026_11_17_25724_25729_Figure_1_Schematic_of_the_automated_sample_handling_system_for_online_protein_deglycosylation_and_desalting_d4ee48b627.jpg 245w," sizes="100vw" width="1287" height="386"></p><h4><strong>Results and Discussion</strong></h4><p>The performance of the PNGase Rc IMER for the removal of complex <i>N</i>-glycans from intact proteins was tested using heavily glycosylated human Hp with four glycosylation sites (Asn184, Asn207, Asn211, and Asn241) <a href="javascript:void(0);">(18)</a> located at the β-chain and occupied by complex <i>N</i>-glycans terminated with sialic acid. Haptoglobin phenotype 2-1, used in this study, forms multimers consisting of β-chain and disulfide linked α1 and α2 chains. <a href="javascript:void(0);">(19)</a> The detection of glycosylated Hp β-chain requires reduction of the disulfide bonds by incubating the protein with a reducing reagent. TCEP is a common reducing reagent used for fast reduction of disulfide bonds at various pH levels. Forty degrees Celsius, the maximum temperature of the PAL tray, and 5 min of incubation were sufficient for complete reduction of Hp disulfide bonds. Individual signals corresponding to the glycosylated β-chain were observed in the <i>m</i>/<i>z</i> range of 35,800–37,400 (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.6c00577#fig2">Figure 2</a>a). The limited resolution of the mass spectrometer did not allow assigning the different glycoforms of the haptoglobin β-chain, which is occupied by biantennary, triantennary, or tetraantennary glycans with various amounts of sialic acid and fucose. <a href="javascript:void(0);">(20,21)</a> The nonglycosylated α1 and α2 chains were observed at <i>m</i>/<i>z</i> 9190 and 15,940. When the PNGase Rc IMER was integrated into the flow system, a signal at <i>m</i>/<i>z</i> 27,270 was detected, demonstrating complete deglycosylation of the haptoglobin β-chain (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.6c00577#fig2">Figure 2</a>b).</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Omega_2026_11_17_25724_25729_Figure_2_A_Mass_spectrum_of_human_Hp_without_the_PN_Gase_Rc_IMER_implemented_in_the_system_0ecca84ed4.jpg" alt="ACS Omega 2026, 11, 17, 25724–25729: Figure 2. (A) Mass spectrum of human Hp without the PNGase Rc IMER implemented in the system. The base peak signals of the spectrum correspond to both α1 and α2 chains. The signal of the β chain is spread across its different glycoforms. (B) Mass spectrum of human Hp with the PNGase Rc IMER implemented in the system. The signal of the deglycosylated β chain is higher compared to its glycosylated form and observed only at m/z 27,270. This indicates complete removal of glycans from the Hp β chain." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Omega_2026_11_17_25724_25729_Figure_2_A_Mass_spectrum_of_human_Hp_without_the_PN_Gase_Rc_IMER_implemented_in_the_system_0ecca84ed4.jpg 245w," sizes="100vw" width="2015" height="913"></p><p>In a previous study by Krenkova et al., the PNGase F IMER was used for deglycosylation of IgG, RNase B, and bovine fetuin with follow-up mass spectrometry detection of both released glycans and intact proteins using MALDI-TOF MS. <a href="javascript:void(0);">(14)</a> The authors demonstrated an effective method for rapid protein deglycosylation, which, however, requires additional sample handling prior to mass spectrometry measurement. The other study by Jmeian et al., which focused on released <i>N</i>-glycans, implemented a PNGase F IMER with online LC–MS detection. <a href="javascript:void(0);">(13)</a> The authors show the effectiveness of the method by identifying many different glycans released from a single protein or from a complex plasma mixture. Our approach is not directly intended for released <i>N</i>-glycan analysis; instead, it demonstrates the ability to measure intact proteins after rapid online deglycosylation using PNGase Rc at low pH. Samples prepared immediately prior to mass spectrometry analysis using the automated sample-handling platform can reduce variability associated with autosampler residence time, as some proteins may undergo degradation or precipitation during storage in the autosampler.</p><p>Although all glycoproteins in this study were effectively deglycosylated, the method’s efficiency depends on the characteristics of the individual glycoprotein. Therefore, optimizing method parameters─including the isocratic flow rate, glycoprotein incubation time with TCEP, and/or the addition of denaturing reagent─may improve the overall efficiency of the deglycosylation process.</p><p>The PNGase Rc IMER stability over a series of injections was tested by using the trastuzumab antibody. The measurements were carried out with the enzyme column kept at the laboratory temperature. As shown in <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.6c00577#fig5">Figure 5</a>, the results indicate that the column performance remained stable over the course of the 150 measurements, and no glycosylated form of the trastuzumab antibody heavy chain was detected. The activity of PNGase Rc in the presence of 5% acetonitrile and 5% propan-2-ol was evaluated using a bottom-up approach with human 2–1 haptoglobin, which was online-digested by a coimmobilized nepenthesin-2/pepsin IMER. In the absence of an organic solvent, the peptide IEKVVLHPNYSQVD remained fully deglycosylated throughout 30 injections. In contrast, in the presence of organic solvent, a marked loss of enzymatic activity was observed after only a few injections (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/acsomega.6c00577/suppl_file/ao6c00577_si_001.pdf">Figure 2</a>).</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Omega_2026_11_17_25724_25729_Figure_5_Monitoring_the_stability_and_performance_of_PN_Gase_Rc_IMER_4a9b5a9c53.jpg" alt="ACS Omega 2026, 11, 17, 25724–25729: Figure 5. Monitoring the stability and performance of PNGase Rc IMER. Trastuzumab heavy chain was completely deglycosylated after 150 injections." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Omega_2026_11_17_25724_25729_Figure_5_Monitoring_the_stability_and_performance_of_PN_Gase_Rc_IMER_4a9b5a9c53.jpg 170w," sizes="100vw" width="996" height="918"></p><h4><strong>Conclusions</strong></h4><p>Here, we demonstrate the potential of the PNGase Rc IMER for the online removal of <i>N</i>-glycans from intact monomeric glycoproteins reduced with TCEP. The column showed high sensitivity toward Hp, which has multiple glycosylation sites with various glycan structures, confirming its suitability for the profiling of complex glycoproteins. Bevacizumab and trastuzumab exhibited similar glycosylation profiles, with core-fucosylated biantennary glycans being the most abundant. Online deglycosylation with the PNGase Rc column resulted in a complete removal of the glycans from the heavy chains of both antibodies, proving that the column can be used for the rapid online deglycosylation of intact therapeutic mAbs. Moreover, the column remained stable over 150 measurements without any loss of activity, even when it was maintained at laboratory temperature. The significantly reduced reaction time, together with full automation and the absence of manual handling steps, substantially improves analytical throughput compared to conventional off-line deglycosylation workflows. These findings highlight the potential of the PNGase Rc IMER in the development of glycoprotein-based biopharmaceuticals, where detailed structural characterization of the protein part is critical for quality control, monitoring batch-to-batch variability, and stability testing.</p></div>

Online Deglycosylation of Monomeric Intact Proteins Using the PNGase Rc Immobilized-Enzyme Reactor

<p><strong>In the week of June 29, the following webinars await you in the field of gas / solid phase and especially Raman, FTIR, GC and GC/MS.</strong></p><blockquote><h4><a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/webinars"><strong>👉 DETAILS AND REGISTRATION IN THE WEBINARS SECTION</strong></a></h4><p><strong>👉 Search and filter in the largest global database</strong> with over <strong>5 500 GC, LC, MS and spectroscopy webinars</strong>.</p></blockquote><h5><strong>1. Metrohm: Simplifying Through-Container Testing with Raman Spectroscopy in the Pharmaceutical Industry</strong></h5><ul><li>Mo, 29.6.2026 15:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/webinar/6308">Registration</a></li></ul><p>In this webinar, we will demonstrate a more robust and practical alternative to SORS for see-through Raman analysis.</p><h5><strong>2. SelectScience: End-to-end PAT: Connecting advanced analytics and data across the bioprocess lifecycle</strong></h5><ul><li>Mo, 29.6.2026 16:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/webinar/6307">Registration</a></li></ul><p>Learn how integrated PAT strategies combining NMR, Raman, IR, and digital workflows enable continuous process understanding, smoother tech transfer, and QbD from development to GMP manufacturing.</p>

Webinars LabRulezGCMS Week 27/2026

<p><strong>In the week of June 29, the following webinars await you in the field of liquid phase and especially HPLC and LC/MS.</strong></p><blockquote><h4><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinars"><strong>👉 DETAILS AND REGISTRATION IN THE WEBINARS SECTION</strong></a></h4><p><strong>👉 Search and filter in the largest global database</strong> with over <strong>5 500 GC, LC, MS and spectroscopy webinars</strong>.</p></blockquote><h5><strong>1. Thermo Fisher Scientific: Insights from an IC specialist: To be suppressed or not suppressed?</strong></h5><ul><li>Tu, 30.6.2026 09:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6310">Registration</a></li></ul><p>Join Thermo Fisher’s Ask the Experts session to learn how suppressor technology impacts ion chromatography performance, sensitivity, reliability, maintenance, and troubleshooting.</p><h5><strong>2. SCIEX: Advancing Proteomics with ZenoTOF: From Single-Cell Analysis to Plasma Biomarker Discovery</strong></h5><ul><li>Tu, 30.6.2026 11:30 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6311">Registration</a></li></ul><p>Discover how ZenoTOF high-resolution mass spectrometry is accelerating single-cell proteomics and plasma biomarker discovery through enhanced sensitivity, throughput, and automation.</p><h5><strong>3. Agilent Technologies: Measuring Diverse Samples with UV/VIS Spectrometers and Integrating Spheres</strong></h5><ul><li>Tu, 30.6.2026 20:30 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6052">Registration</a></li></ul><p>This webinar will discuss diffuse transmission and reflection UV/VIS measurements, including how to perform measurements with Agilent’s Cary 60 UV/VIS Spectrometer.</p><h5><strong>4. Waters Corporation: Informatics series #33 Tips, Tricks and Best Practices</strong></h5><ul><li>Th, 2.7.2026 11:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6315">Registration</a></li></ul><p>This interactive session revisits essential features, shortcuts, and practical techniques in Empower software that can streamline everyday laboratory work and reduce unnecessary effort.</p><h5><strong>5. Waters Corporation: Unlocking Therapeutic Peptide Characterisation (GLP-1RAs) with Cyclic IMS</strong></h5><ul><li>Th, 2.7.2026 15:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6313">Registration</a></li></ul><p>Discover how cyclic ion mobility spectrometry (cyclic IMS) improves characterization of GLP-1 analogues and therapeutic peptides, supporting development, quality control, and regulatory submissions.</p><h5><strong>6. Agilent Technologies: Spectral Flow Cytometry Masterclass - Session 1</strong></h5><ul><li>Fr, 3.7.2026 07:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/6316">Registration</a></li></ul><p>Learn how full-spectrum flow cytometry enables high-dimensional cell analysis. Explore spectral flow technology, experimental design, and applications in immunology, oncology, and vaccine research.</p>

Webinars LabRulezLCMS Week 27/2026

<p><strong>In the week from June 29, the following webinars await you in the field of atomic and molecular spectroscopy and spectrometry</strong></p><blockquote><h4><a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/webinars"><strong>👉 DETAILS AND REGISTRATION IN THE WEBINARS SECTION</strong></a></h4><p><strong>👉 Search and filter in the largest global database</strong> with over <strong>5 500 GC, LC, MS and spectroscopy webinars</strong>.</p></blockquote><h5><strong>1. Metrohm: Simplifying Through-Container Testing with Raman Spectroscopy in the Pharmaceutical Industry</strong></h5><ul><li>Mo, 29.6.2026 15:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/webinar/6308">Registration</a></li></ul><p>In this webinar, we will demonstrate a more robust and practical alternative to SORS for see-through Raman analysis.</p><h5><strong>2. SelectScience: End-to-end PAT: Connecting advanced analytics and data across the bioprocess lifecycle</strong></h5><ul><li>Mo, 29.6.2026 16:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/webinar/6307">Registration</a></li></ul><p>Learn how integrated PAT strategies combining NMR, Raman, IR, and digital workflows enable continuous process understanding, smoother tech transfer, and QbD from development to GMP manufacturing.</p><h5><strong>3. Bruker: Bruker Fluorescence Microscopy Solutions: Automated Imaging and Light-Sheet Microscopy</strong></h5><ul><li>Tu, 30.6.2026 17:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/webinar/6309">Registration</a></li></ul><p>Discover Bruker’s integrated imaging ecosystem combining light-sheet microscopy, high-content screening, and high-performance data management for scalable, high-quality biological imaging workflows.</p><h5><strong>4. Agilent Technologies: Measuring Diverse Samples with UV/VIS Spectrometers and Integrating Spheres</strong></h5><ul><li>Tu, 30.6.2026 20:30 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/webinar/6052">Registration</a></li></ul><p>This webinar will discuss diffuse transmission and reflection UV/VIS measurements, including how to perform measurements with Agilent’s Cary 60 UV/VIS Spectrometer.</p><h5><strong>5. Milestone: Unlock Smarter Digestion: AI and the Smart Features that Transform Your Daily Routine</strong></h5><ul><li>We, 1.7.2026 16:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/webinar/6312">Registration</a></li></ul><p>Learn how Milestone’s intelligent digestion technologies, including MAIA AI Agent, easyTEMP, and SRC, help reduce downtime, simplify workflows, and improve laboratory productivity.</p><h5><strong>6. Bruker: High-Energy XRD - Diffraction Without Limits</strong></h5><ul><li>Th, 2.7.2026 10:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/webinar/6314">Registration</a></li></ul><p>Join us to explore how high-energy XRD with the D8 ADVANCE HE and D8 DISCOVER HE enables the study of materials under realistic conditions.</p><h5><strong>7. Agilent Technologies: Spectral Flow Cytometry Masterclass - Session 1</strong></h5><ul><li>Fr, 3.7.2026 07:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/webinar/6316">Registration</a></li></ul><p>Learn how full-spectrum flow cytometry enables high-dimensional cell analysis. Explore spectral flow technology, experimental design, and applications in immunology, oncology, and vaccine research.</p>

Webinars LabRulezICPMS Week 27/2026

<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezGCMS Library in the week of 29th June 2026? Check out new documents from the field of the gas phase, especially GC and GC/MS techniques!</strong></p><blockquote><p><span style="font-size:20px;">👉 </span><a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library"><span style="font-size:20px;"><strong>SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT GCMS 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://lcms.labrulez.com/library"><strong>LabRulezLCMS</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>presentation by <strong>MDCW / William &amp; Mary </strong>and posters by <strong>Shimadzu</strong> and <strong>Thermo Fisher Scientific</strong> <strong>/ ASMS!</strong></p><h3><strong>1. Agilent Technologies: Residual Solvents Analysis Based on the 2025 USP &lt;467&gt; and Chinese Pharmacopoeia (ChP 0861)</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/br_8890b_gc_5994_9205en_agilent_fb5db29527.pdf"><strong>Full PDF for download</strong></a></li></ul><section><div><div><div><div><div><div><p><strong>Using the </strong><a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library?mainauthorItems=10&amp;search=8697"><strong>Agilent 8697 headspace sampler</strong></a><strong> and </strong><a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library?mainauthorItems=10&amp;search=8890B"><strong>Agilent 8890B GC system</strong></a></p><p>Residual solvents are routinely introduced during pharmaceutical manufacturing as part of synthesis, purification, or formulation processes. Because these volatile organic compounds may pose toxicological risks, their levels must be carefully monitored to ensure patient safety and regulatory compliance. As a result, residual solvent analysis remains a critical component of quality control throughout the pharmaceutical development and manufacturing lifecycle.&nbsp;</p><p>USP &lt;467&gt; provides a harmonized framework for residual solvent analysis, defining solvent classifications, concentration limits, and analytical requirements.1 In September 2025, USP &lt;467&gt; was revised to reflect updated toxicological evaluations and regulatory expectations. The revised chapter expanded the class 2B solvent list and introduced performance requirements for newly added compounds, including tertiary butyl alcohol and cyclopentyl methyl ether (CPME). These updates increase the analytical scope of residual solvent testing and place greater emphasis on consistent system performance and suitability verification. In China, residual solvent control is governed by General Chapter 0861 of the Chinese Pharmacopoeia (ChP 0861)2 , which adopts a risk based classification system consistent with the International Council for Harmonisation (ICH) Q3C.3 Although the Chinese Pharmacopoeia uses different terminology for analytical procedures, its regulatory intent, solvent classification, and control principles are harmonized with USP &lt;467&gt;.&nbsp;</p><p>Gas chromatography with headspace sampling is widely used for residual solvent determination due to its robustness and suitability for volatile analytes. Alongside evolving regulatory requirements, modern GC systems have increasingly focused on intelligent operation to support method execution and compliance. The Agilent 8890B gas chromatograph incorporates GC Assist to help users perform a performance check of the GC system. When performance criteria are not met, the GC system provides alerts to notify the user. Upon user instruction, the GC can initiate a guided troubleshooting workflow, supporting step-by-step fault diagnosis through autonomous diagnostic functions, helping both experienced and less experienced analysts resolve issues efficiently during routine operation. In this application note, a headspace GC/FID method is presented for the determination of class 1, 2A, and the expanded class 2B residual solvents. This study demonstrates how updated regulatory requirements can be addressed using an intelligent GC platform that supports reliable system performance assessment, simplified troubleshooting, and consistent analytical performance.</p><h4><strong>Experimental</strong></h4><p>The experimental conditions described in this study follow the general chromatographic principles outlined in USP &lt;467&gt; procedures A and B. They are also consistent with the recommended headspace GC methods described in ChP 0861. In this work, the screening and confirmation concepts of procedures A and B were implemented simultaneously through a dual-column, dual-FID/GC configuration. Samples were introduced by an <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library?mainauthorItems=10&amp;search=8697"><strong>Agilent 8697 headspace sampler</strong></a> to an <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library?mainauthorItems=10&amp;search=8890B"><strong>8890B GC</strong></a> and equally split to an Agilent DB-Select 624 UI and a DB-WAX UI column for parallel detection, as shown in Figure 1. Comparable chromatographic performance was achieved with both helium and nitrogen carrier gases. Data were acquired using Agilent OpenLab CDS version 2.8, and noise was determined using a peak-to-peak (PtoP) approach for calculation of signal-to-noise ratios for target analytes.</p><h4><strong>Conclusion&nbsp;</strong></h4><p>This application note demonstrates a harmonized headspace GC/FID workflow suitable for residual solvent analysis in compliance with both the 2025 revision of United States Pharmacopoeia (USP) &lt;467&gt; and the Chinese Pharmacopoeia (ChP 2025, General Chapter 0861). By implementing a dual‑column, dual-FID configuration on a single inlet, screening and confirmatory analyses can be performed simultaneously, significantly reducing total analysis time compared to traditional sequential approaches while maintaining orthogonal separation selectivity. The integrated Agilent 8697 headspace sampler and 8890B GC platform provides consistent chromatographic performance with both helium and nitrogen carrier gases, supporting flexible laboratory operation without compromising data quality. Beyond analytical performance, the intelligent capabilities of the 8890B GC enhance operational reliability and simplify daily use. Through continuous interaction with the user, the built‑in GC Assist function operates in a guided, wizard‑like manner, leading users step by step through the troubleshooting process for effective fault diagnosis. Together, these features enable a compliant, time-efficient, and user-friendly solution for residual solvent testing under the latest USP &lt;467&gt; and ChP&nbsp;0861 requirements.</p><h3><strong>2. MDCW / William &amp; Mary: Developing a Combined Approach to Green Gunshot Residue Analysis in the Forensic Laboratory</strong></h3><ul><li>Presentation</li><li><a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/O_24_Saunders_f2c5bcdb20.pdf"><strong>Full PDF for download</strong></a></li></ul><section><div><div><div><div><div><div><p>Developing a Combined Approach to Green Gunshot Residue Analysis in the Forensic Laboratory presents a multidisciplinary strategy for improving the forensic analysis of <strong>"green" gunshot residue (GSR)</strong>, which originates from lead-free ammunition. Traditional GSR analysis relies heavily on the detection of lead, barium, and antimony particles using scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDS). However, environmentally friendly ammunition replaces these heavy metals with more common elements, making conventional forensic protocols less effective. The authors therefore propose combining inorganic GSR (IGSR) characterization by SEM-EDS with organic GSR (OGSR) analysis using comprehensive two-dimensional gas chromatography coupled with mass spectrometry (GC×GC-MS).</p><p>The presentation first describes the development of a new SEM-EDS characterization strategy tailored to green GSR particles. Instead of relying solely on the elemental composition defined by existing OSAC guidelines, the approach incorporates both particle morphology and elemental complexity. In parallel, the researchers optimized an extraction procedure for organic residues and developed a dedicated GC×GC-MS method. Method optimization focused on key chromatographic parameters such as inlet temperature, modulation period, carrier gas selection, and oven temperature program. Compared with previously published methods, the optimized workflow provided higher peak capacity, improved analyte resolution, and enhanced detection of characteristic OGSR compounds.</p><p>To verify the analytical performance, the optimized GC×GC-MS method was evaluated using several reference standards, including phthalates, nitroaromatic explosives, nitroglycerin, and a multi-component gun surveillance standard. The resulting two-dimensional chromatograms demonstrated excellent separation and clear identification of compounds relevant to firearm discharge residues, confirming that the method can reliably detect a broad range of organic GSR markers. The combination of enhanced chromatographic separation with mass spectrometric detection offers substantially greater resolving power than conventional one-dimensional GC-MS, making it particularly valuable for complex forensic samples.</p><p>The authors conclude that integrating SEM-EDS with GC×GC-MS provides a more comprehensive approach to modern GSR analysis, particularly for lead-free ammunition. The study successfully established a characterization strategy for inorganic green GSR, optimized extraction and chromatographic conditions for organic GSR, and verified the visibility and identification of target analytes using certified standards. Future work will focus on further method validation, incorporating retention indices and sorbent-based extraction techniques, and strengthening the scientific robustness of the approach to support forensic evidence under <strong>Rule 702</strong>, thereby improving the reliability and courtroom acceptance of green GSR analyses.</p><h3><strong>3. Shimadzu / ASMS: Impact of Soil Matrix on Microplastics Analysis by Py-GC/MS</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/TP_222_Impact_of_Soil_Matrix_on_Microplastics_Analysis_by_Py_GCMS_63fdb8ef7d.pdf"><strong>Full PDF for download</strong></a></li></ul><p>The matrix is not just background—it actively participates in pyrolysis chemistry. Lignin-rich soils can co-pyrolyze, producing compounds that overlap with common PS or PVC markers, complicating interpretation. Matrix variability is substantial, with clear chemical and physical differences between forest soils, agricultural land, river sediments, and landfill cover materials. Soil particles act as sinks and sorbents, interacting with microplastics and altering degradation pathways. Sample preparation can introduce bias, and in some cases does more harm than good.&nbsp;</p><p>In this poster, we present a method to improve quantitative accuracy in Py-GCMS microplastics analysis by assessing the matrix effect, while also addressing key challenges in the qualitative identification</p><h4><strong>Experimental Methods&nbsp;</strong></h4><ul><li>Three soil samples were collected from different locations (Figure 1) and homogenized using a mortar/pestle.&nbsp;</li><li>From each sample, 200g were dried in a vial for 3 hours at 40C and 2mg was weighed into a PY cup.&nbsp;</li><li>Targeted microplastics include PE, PP, PS, ABS, PMMA, PC, PVC, N6, and N66. The percentage distribution of polymers in the MPs-CaCO3 standard, as well as the mass of each analyte in a 4 mg standard within a CaCO3 diluent, is shown in Figure 1.&nbsp;</li><li>These were analyzed using a <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library?mainauthorItems=1&amp;search=Py-GC%2FMS"><strong>PY-GCMS instrument</strong></a>, shown in Figure 2.&nbsp;</li><li>A 5-point calibration curve was prepared, in triplicate, by weighing 0.2mg, 0.4mg, 0.8mg, 2.0mg, and 4.0mg from Frontier’s Low calibration standard mix of microplastics.&nbsp;</li><li>A matrix-matched calibration curve was generated by spiking sample B with the same concentration as the calibration levels.&nbsp;</li><li>Samples were analyzed, and analytes in the sample were quantified in µg, calculated using the calibration curve of known standard concentration.&nbsp;</li><li>Recovery studies were performed in blank samples to evaluate matrix effect for the targeted analytes. For this, a 2mg sample was weighed in triplicate, and 2mg of the standard mix was combined with 2mg of CaCO3 diluent.</li></ul><h4><strong>Conclusions</strong></h4><p>Future work will focus on improving microplastic identification by using multiple markers to reduce false positives, while advancing polymer identification tools and spectral libraries to better handle high-background matrix interferences.</p><p>Sample preparation is an important factor, and it needs to be considered and studied for different sample types, including environmental and clinical samples</p><h3><strong>4. Thermo Fisher Scientific / ASMS: Untargeted analysis of volatile PFAS in indoor air by thermal desorption GC high resolution </strong><a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/products/472"><strong>Orbitrap Mass Spectrometer</strong></a></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_004717_lsms_volatile_pfas_air_exploris_gc_efs_po004717_asms2026_na_en_69541bd1e3.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Per- and polyfluoroalkyl substances (PFAS) are persistent “forever chemicals” widely used in consumer products and commonly detected in indoor air. Volatile PFAS can originate from treated materials and degrade into harmful compounds linked to adverse health effects. Thermal desorption (TD) enables efficient air monitoring by preconcentrating volatile compounds for direct GC-MS analysis. Coupling TD with high-resolution Orbitrap GC-MS and advanced data processing allows comprehensive, untargeted screening of PFAS, supporting improved indoor air quality assessment.</p><h4><strong>Materials and methods</strong></h4><h5><strong>Sample preparation&nbsp;</strong></h5><ul><li>PFAS standards prepared in methanol and spiked onto sorbent tubes</li><li>Indoor air sampled (35 L) from carpeted, non-carpeted, and cleaning storage areas&nbsp;</li><li>Air collected using sorbent tubes with pumped sampling (TD-compatible). Test method(s)&nbsp;</li><li>Samples analyzed using thermal desorption (TD)–GC-MS (<a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/products/472"><strong>Orbitrap Exploris GC</strong></a>)&nbsp;</li><li>TD preconcentration followed by GC separation and HRAM full-scan acquisition (EI/CI)&nbsp;</li><li>Method enabled untargeted screening of volatile compounds.&nbsp;</li></ul><h5><strong>Data analysis&nbsp;</strong></h5><ul><li>Data processed using <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/products/76"><strong>Thermo Scientific Chromeleon CDS</strong></a>, FreeStyle, and <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/products/1759"><strong>Compound Discoverer Software&nbsp;</strong></a></li><li>Workflow included peak detection, deconvolution, and library matching.</li></ul><h4><strong>Conclusions&nbsp;</strong></h4><ul><li>TD-GC Orbitrap MS enables sensitive, untargeted detection of volatile PFAS in indoor air with minimal sample preparation&nbsp;</li><li>HRAM data combined with advanced software workflows provides confident identification and efficient data filtering&nbsp;</li><li>Higher PFAS levels observed in carpeted and cleaning areas, highlighting indoor sources and exposure risk</li></ul></div></div></div></div></div></div></section></div></div></div></div></div></div></section>

News from LabRulezGCMS Library - Week 27, 2026

News from LabRulezLCMS Library - Week 27, 2026

<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezICPMS Library in the week of 29th June 2026? Check out new documents from the field of spectroscopy/spectrometry and related techniques!</strong></p><blockquote><p><span style="font-size:20px;">👉 </span><a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library"><span style="font-size:20px;"><strong>SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT SPECTROSCOPY/SPECTROMETRY 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://lcms.labrulez.com/library"><strong>LabRulezLCMS</strong></a> or <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library"><strong>LabRulezGCMS</strong></a> libraries.</p></blockquote><p>This week we bring you application notes by <strong>Agilent Technologies </strong>and<strong> Thermo Fisher Scientific </strong>and brochure by <strong>Shimadzu!</strong></p><h3><strong>1. Agilent Technologies: Analysis of High Purity Titanium Using an </strong><a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library?mainauthorItems=10&amp;search=9500"><strong>Agilent 9500 ICP-QQQ</strong></a></h3><p><strong>Ensuring purity and performance of titanium materials used in high-tech industries&nbsp;</strong></p><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_high_purity_titanium_9500_icp_qqq_5994_9024en_agilent_56ab5e943b.pdf"><strong>Full PDF for download</strong></a></li></ul><h5><strong>Importance of high-purity titanium and the role of ICP-QQQ&nbsp;</strong></h5><p>High-purity titanium (Ti) is a critical material in high-tech industries such as semiconductor manufacturing and aerospace engineering. In semiconductor applications, Ti is widely used as a sputtering target for thin-film deposition, where even trace levels of impurities can negatively affect film uniformity, electrical properties, and device reliability. In aerospace applications, Ti is valued for its high strength-to-weight ratio and corrosion resistance, making it an ideal material for engine components and structural parts. However, any impurities can compromise the mechanical integrity and diminish the long-term performance of components, especially when operating under extreme conditions.</p><p>Ensuring the reliability and functionality of Ti in these demanding environments requires precise quantification of trace impurities in the raw material. Triple quadrupole Inductively Coupled Plasma Mass Spectrometry (ICP-QQQ) provides robust suppression of spectral interferences and ultra-low detection limits (DLs), enabling accurate determination of impurities at or below the 1 mg/kg (ppm) level in the solid. This level of sensitivity is particularly important for quality control (QC) of high-purity Ti, where conventional techniques such as XRF, ICP-OES, and GD-MS lack the sensitivity and DLs.&nbsp;</p><p>For unrivalled interference reduction using reactive cell gases, Agilent ICP-QQQ instruments include two unit mass (1 u) quadrupole mass filters, Q1 and Q2.1 Q1 is positioned before the collision/reaction cell (CRC) to select which ions enter the cell for reaction with the cell gas. Q2 filters ions that exit the cell before they pass to the detector. This tandem mass spectrometer (MS/MS) operation allows for unmatched control of reaction chemistry in the CRC, making the technique useful for handling intense spectral interferences, including polyatomic, doubly charged ion (M++), isobaric, and peak overlap interferences.&nbsp;</p><p>Using the newly developed <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library?mainauthorItems=10&amp;search=9500"><strong>Agilent 9500 Triple Quadrupole ICP-MS</strong></a> featuring a Dual-Cell System (DCS) CRC and equipped with an optional m-lens, analysts can confidently analyze Ti matrix samples and verify material purity over extended measurement periods. The m-lens is designed with an optimized geometry that minimizes background signals from easily ionized elements that can deposit on interface components during long runs. This feature enables the instrument to maintain high-power, matrix-tolerant plasma conditions while achieving ultralow background equivalent concentrations (BECs), which is critical for ppt-level impurity analysis. Operating the DCS with a suitable cell gas effectively controls spectral interferences from Ti-based species (Ti²+ and TiO+ ) that impact elements like sodium (Na), magnesium (Mg), copper (Cu), and zinc (Zn). Together with the m‑lens, this approach ensures accurate measurement of trace elemental impurities, enabling compliance within stringent industry standards. The effectiveness of the m‑lens in minimizing background signals and enabling ppt‑level impurity analysis under robust plasma conditions has also been demonstrated in high-matrix applications using ICP‑QQQ.2, 3 In addition to interference control, the 9500 ICP-QQQ system’s robustness and reproducibility make it well suited for routine analysis in both research and production settings. This capability supports the continued advancement of high-tech applications that rely on ultra-clean titanium.</p><p>In this study, a 200 ppm Ti solution prepared from a high-purity Ti powder was used to represent a typical sample encountered in advanced material analysis. The performance of the 9500 ICP-QQQ for quantifying trace impurities, including Na, Mg, Cu, and Zn, in a high Ti matrix was assessed through spike-recovery experiments and long-term stability tests.</p><h4><strong>Conclusion&nbsp;</strong></h4><p>This study demonstrated the effectiveness of the Agilent 9500 ICP-QQQ with m-lens for the ultra-trace analysis of impurity metals in high-purity titanium. By applying optimized reaction gas conditions and mass-shift techniques, spectral interferences from Ti-based species such as Ti²+ and TiO+ were successfully mitigated, enabling accurate quantification of elements like Mg, Cu, and Zn.&nbsp;</p><p>The method achieved limits of quantification in the sub-ppm range for the undiluted Ti digestion solution and maintained stable ISTD signals over extended measurement periods. Spike recovery tests confirmed high accuracy and reproducibility, with most elements showing recoveries within ±10% and RSDs below 3%.&nbsp;</p><p>These results confirm that the 9500 ICP-QQQ is a robust and reliable tool for quality control of high-purity Ti, supporting its critical applications in semiconductor and aerospace industries where impurity levels must be strictly controlled.</p><h3><strong>2. Shimadzu: Scanning Probe Microscope /Atomic Force Microscope SPM-9700HT Plus</strong></h3><ul><li>Brochure</li><li><a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/labrulez-bucket-strapi-h3hsga3/c147_e021_4c550d3be3.pdf"><strong>Full PDF for download</strong></a></li></ul><p>The <strong>Shimadzu SPM-9700HT Plus</strong> is a high-throughput <strong>scanning probe microscope (SPM) / atomic force microscope (AFM)</strong> designed for three-dimensional surface imaging and nanoscale characterization of material properties across a broad range of scientific and industrial applications. The system combines high-resolution topographical imaging with multiple AFM measurement modes, including contact, dynamic, phase, lateral force, force modulation, and force-curve measurements, while optional modules extend its capabilities to techniques such as Kelvin probe force microscopy (KPFM), magnetic force microscopy (MFM), conductive AFM, piezoresponse force microscopy (PFM), and scanning tunneling microscopy (STM). A wide selection of interchangeable scanners, environmental accessories, optical microscope units, and specialized sample holders enables the instrument to be configured for diverse analytical requirements.</p><p>A major focus of the brochure is the system's emphasis on automation and ease of use through Shimadzu's <strong>Analytical Intelligence</strong> technology. Features such as <strong>Observation Navigation</strong>, <strong>NanoAssist</strong>, and the optional <strong>Cantilever Master</strong> automate instrument setup, cantilever installation, optimization of imaging parameters, and measurement workflows, allowing operators with varying levels of experience to acquire high-quality, reproducible AFM data. The redesigned control electronics and scanning system also enable high-throughput imaging and rapid physical property mapping while maintaining nanoscale resolution. In addition, the head-slide mechanism simplifies sample exchange without removing the cantilever, increasing productivity during routine measurements.</p><p>Beyond surface topography, the <strong>SPM-9700HT Plus</strong> provides extensive quantitative analysis capabilities through integrated software for roughness measurements, particle analysis, cross-sectional profiling, and morphological characterization. Optional <strong>Nano 3D Mapping Fast</strong> technology extends the instrument to nanoscale mechanical property measurements by performing force-curve mapping, allowing localized evaluation of adhesion, Young's modulus, and other mechanical properties of thin films, polymers, biomaterials, and soft samples. The optional Particle Analysis Software further enables automated extraction, classification, statistical evaluation, and export of particle size and morphology data for large datasets.</p><p>The brochure demonstrates the versatility of the platform through numerous application examples spanning <strong>electronics, semiconductor devices, magnetic materials, life sciences, industrial materials, batteries, polymers, nanomaterials, food science, and chemistry</strong>. Representative studies include magnetic domain imaging, organic thin-film transistor characterization, collagen and DNA visualization, live-cell imaging, lithium-ion battery electrode and separator analysis, photocatalyst surface potential measurements, polymer phase imaging, cellulose nanofiber characterization, and beer yeast morphology. Together, these examples highlight the <strong>Shimadzu SPM-9700HT Plus</strong> as a comprehensive AFM platform capable of combining rapid imaging, advanced nanoscale property mapping, and automated data analysis for both research and routine industrial quality control.</p><h3><strong>3. Thermo Fisher Scientific: Enhancing Monoclonal Antibody Yield and Quality Through Automated Multi-Component Feedback Control Loops Using the MarqMetrix All-In-One Process Raman Analyzer</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/labrulez-bucket-strapi-h3hsga3/enhancing_monoclonal_antibody_an1506_en_bba763e865.pdf"><strong>Full PDF for download</strong></a></li></ul><p>The application of Raman spectroscopy for real-time monitoring and control of bioreactors marks a significant advancement in bioprocessing technology. Over the past decade, its adoption has enabled tighter control of critical process parameters (CPPs), offering early indicators of process performance to ensure consistency and improve product quality. Raman spectroscopy leverages the unique vibrational signatures of molecules, allowing for highly specific detection even in complex biological matrices. This enables simultaneous, real-time monitoring of multiple CPPs, including nutrient concentrations, metabolic by-products, and cell density. As a result, users gain deep, real-time insights into cellular metabolism and can implement more dynamic, adaptive feeding strategies based on metabolic network understanding.&nbsp;</p><p>Previous studies have demonstrated the negative impact of high lactate on cell health and quantity and quality of titer produced.² In this study, we demonstrate the successful implementation of process Raman analyzer for simultaneous multi-component feedback control of glucose and lactate in a bioreactor. By continuously monitoring both analytes, the feedback control loop dynamically maintained a constant total carbon concentration (glucose + lactate) at a setpoint of 2 g/L.&nbsp;</p><p>This advanced carbon-source-based control strategy ensured that cellular metabolic demands were met while promoting lactate consumption toward the end of the run. As a result, lactate accumulation was significantly reduced, leading to substantial improvements in titer yield, product quality, and cell viability compared to standard bolus feeding strategies (Figure 2).</p><p>The outcomes of this work underscore the <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/products/2233"><strong>Thermo Scientific™ MarqMetrix™ All-In-One Process Raman Analyzer </strong></a>as a key Process Analytical Technology (PAT) tool-enabling real-time, multi-analyte control for any biomanufacturer seeking to engage in intelligent, automated biomanufacturing.</p><h4><strong>Conclusion&nbsp;</strong></h4><h5><strong>1. Unmatched Real-Time Control Capability:&nbsp;</strong></h5><p>Maintaining a constant total carbon concentration (glucose + lactate) of 2 g/L in dynamic bioreactor environments demands precise, simultaneous analyte monitoring. The MarqMetrix All-In-One Process Raman Analyzer delivers near real-time, high-fidelity data every two minutes, empowering advanced control strategies that are simply not feasible with conventional technologies. This makes it an indispensable PAT tool for modern bioprocessing.&nbsp;</p><h5><strong>2. Proven Impact on Product Yield and Quality:&nbsp;</strong></h5><p>The Raman-enabled carbon control strategy resulted in a &gt;10% increase in titer and a reduction in glycation by 83% and 66% compared to continuous and fed-batch glucose control, respectively. These outcomes underscore the analyzer’s transformative impact on both product quantity and quality.&nbsp;</p><h5><strong>3. Versatile Automation Across Bioprocesses:&nbsp;</strong></h5><p>All three bioreactor runs—each employing distinct control logics (Table 2)—were successfully managed through automated feedback using the process Raman analyzer. Its flexible design, including easily swappable probes, supports seamless adaptation to other critical metabolites such as amino acids, making it a universal solution for bioprocess control.&nbsp;</p><h5><strong>4. Foundation for Intelligent Manufacturing:&nbsp;</strong></h5><p>With its real-time, data-rich insights and seamless integration into automated systems, the MarqMetrix All-InOne analyzer is not just a monitoring tool; it is a cornerstone technology for intelligent, AI-driven biomanufacturing. It enables consistent, high-quality production while reducing time, cost, and variability.</p>

News from LabRulezICPMS Library - Week 27, 2026

<p><strong>This study presents an optimized dispersive liquid–liquid microextraction (DLLME) method coupled with GC-FID for the determination of eight priority polycyclic aromatic hydrocarbons in surface waters. Experimental design approaches, including fractional factorial and Box–Behnken designs, were used to optimize extraction conditions and maximize analytical performance.</strong></p><p>The validated method achieved rapid chromatographic separation, low detection limits, good precision, and high enrichment factors while maintaining favorable green analytical characteristics. Successful application to wastewater, lake, river, and drain water samples demonstrates its suitability for routine environmental monitoring of PAHs.</p><blockquote><h4><strong>The original article</strong></h4><h4><a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.6c01489"><strong>Experimental Design-Based Dispersive Liquid–Liquid Microextraction with GC-FID for Determination of Polycyclic Aromatic Hydrocarbons in Surface Water</strong></a></h4><p>Chen Minghong, Song Peiyu, Tien Ping Lee, Garret Chan Zhe Ming, Philip J. Marriott, and Yong Foo Wong*</p><p>ACS Omega 2026, 11, 21, 31418–31431</p><p><a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1021/acsomega.6c01489">https://doi.org/10.1021/acsomega.6c01489</a></p><h6>licensed under <a target="_blank" rel="noopener noreferrer" href="https://creativecommons.org/licenses/by/4.0/"><strong>CC-BY 4.0</strong></a></h6></blockquote><p><strong>Selected sections from the article follow. Formats and hyperlinks were adapted from the original.</strong></p><div><p>Polycyclic aromatic hydrocarbons (PAHs) are organic pollutants containing two or more fused benzene rings and are widely associated with carcinogenic, teratogenic, and mutagenic properties. <a href="javascript:void(0);">(1)</a> These hydrophobic substances can bioaccumulate with long environmental half-lives and are classified as high priority ecotoxicants of the first danger class. <a href="javascript:void(0);">(1,2)</a> Anthropogenic activities (e.g., fossil fuel combustion, industrial processes, biomass burning, and others) are predominantly recognized as the primary sources of PAHs in most environmental compartments. <a href="javascript:void(0);">(3−6)</a> In the present work, the hydrophobic nature of PAHs facilitates adsorption onto suspended particulate matter and organic material in aquatic systems, followed by deposition into underlying sediments. <a href="javascript:void(0);">(7)</a> These persistent organic pollutants have been reported in surface waters as well as estuarine and coastal environments and can potentially disrupt ecosystem functioning. <a href="javascript:void(0);">(7,8)</a></p><p>From a public health perspective, PAHs exposure via upon ingestion, has been linked to a spectrum of adverse health effects such as cancer and reduced fertility. <a href="javascript:void(0);">(2,9)</a> Sixteen unsubstituted PAHs have been designated as priority pollutants by the United States Environmental Protection Agency (USEPA). <a href="javascript:void(0);">(10)</a> The International Agency for Research on Cancer (IARC) has classified benzo(a)pyrene (BaP) in Group 1 (carcinogen to humans) and other PAHs in Group 2A (possible human carcinogens). <a href="javascript:void(0);">(10,11)</a> The maximum permissible concentrations of benzo(a)pyrene (0.00001 mg/L) in water intended for drinking water is regulated by the European Union (EU). The EU limits the total content of polyaromatic compounds in drinking water to &lt;0.1 μg/L. <a href="javascript:void(0);">(12)</a> In heavily polluted waterways, PAH concentrations have been reported to reach 10 μg/mL. <a href="javascript:void(0);">(13)</a> Epidemiological evidence also suggests a positive relationship between increased cancer incidence in human populations that are having high exposure to organic ecotoxins. <a href="javascript:void(0);">(14)</a> For these reasons, quantitative assessments of PAH occurrence and distribution in surface waters remain important and fertile research topics. <a href="javascript:void(0);">(15,16)</a></p><p>PAHs in water and environmental matrices are routinely analyzed using either gas chromatography (GC) and/or liquid chromatography (LC) with various detection modalities. <a href="javascript:void(0);">(17,18)</a> The coupling of chromatography to mass spectrometry has been the preferred technique, owing to the distinct selectivity and sensitivity, particularly when operated in selected ion monitoring (SIM) or multiple reaction monitoring (SRM) modes. <a href="javascript:void(0);">(19)</a> Additionally, LC coupled with fluorescence detection is also extensively utilized for PAH analysis. <a href="javascript:void(0);">(20,21)</a> As PAHs typically occur at trace to ultratrace levels in complex real-world matrices, sample pretreatment and/or preconcentration is generally required prior to instrumental analysis. <a href="javascript:void(0);">(22,23)</a> Conventional approaches such as liquid–liquid extraction and solid-phase extraction are widely used but are often solvent-intensive and laborious and generate substantial hazardous waste. More recently, microextraction techniques (e.g., dispersive microsolid phase extraction, dispersive liquid–liquid microextraction (DLLME), solid phase microextraction, etc.) have emerged as attractive alternatives, providing advantages such as speed and simplicity and requiring substantially lower volumes of hazardous solvents. <a href="javascript:void(0);">(24−26)</a></p><p>DLLME has shown good efficiency for extracting and preconcentrating hydrophobic PAHs from aqueous matrixes. <a href="javascript:void(0);">(24,27)</a> Briefly, DLLME involves rapid injection of a small volume of an extraction solvent (immiscible with water) and dispersive solvent into the aqueous sample. <a href="javascript:void(0);">(24)</a> Target analytes partition into the microdroplets and are subsequently recovered in a minimal volume of the organic phase after centrifugation. The large interfacial contact area between the two phases enhances mass transfer, leading to fast attainment of extraction equilibrium with high enrichment factors. <a href="javascript:void(0);">(28,29)</a> DLLME has been applied to a wide range of matrices, including environmental samples, <a href="javascript:void(0);">(25,30)</a> milk and dairy products, <a href="javascript:void(0);">(31)</a> honey, <a href="javascript:void(0);">(32)</a> and others. <a href="javascript:void(0);">(33)</a> Optimization of DLLME conditions for targeted solutes is often performed using a one-factor-at-a-time approach, which is time-consuming, labor-intensive, and may inadequately take into account interaction between or among variables. <a href="javascript:void(0);">(34)</a> In contrast, multivariate experimental design methodologies accommodate simultaneous variation of multiple extraction variables, facilitating identification of interaction effects and development of statistically robust models. <a href="javascript:void(0);">(35−37)</a></p><p>Over the past decade, multivariate design of experiments (DOE) has become an important optimization strategy for process development. Compared with traditional one-factor-at-a-time approaches, DOE can reduce time, effort, and resource consumption while extracting meaningful information from a relatively small number of experimental analyses. <a href="javascript:void(0);">(37−40)</a> Among the DOE frameworks, the Box-Behnken design (BBD), an economical variant of response surface methodology, has been widely used for process optimization, offering high statistical efficiency and good predictive performance when tuning experimental variables in analytical and industrial applications. <a href="javascript:void(0);">(35,39,41−45)</a> This study evaluates the applicability of using RSM to optimize DLLME conditions for selective extraction and preconcentration of acenaphthene, fluorene, anthracene, phenanthrene, fluoranthene, pyrene, benzo(a)anthracene, and benzo(a)pyrene. A fractional factorial design (FrFD) was initially used to screen and identify statistically significant DLLME variables. The selected factors were then optimized using the BBD to construct quadratic models and generate response surface plots for determining optimal extraction conditions. The optimized DLLME-GC-FID method was validated, and AGREEprep and AGREE were used to quantify ‘greenness’ metric scores. Analytical practicability of the validated method was demonstrated by determining the targeted PAHs in several surface water samples.</p><h4><strong>2. Experimental Section</strong></h4><h5><strong>2.4. GC-FID Analysis</strong></h5><p>All experiments were performed using an <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library?mainauthorItems=10&amp;search=7890B"><strong>Agilent 7890B GC system</strong></a> equipped with a <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library?mainauthorItems=10&amp;search=7693A"><strong>G4513A autosampler</strong></a> and a split–splitless inlet (Agilent Technologies; Santa Clara, CA, USA). Chromatographic separation was effected by using a <strong>DB-5HT column</strong> (Agilent Technologies, Santa Clara, CA, USA; 30 m × 0.25 mm I.D. × 0.25 μm film thickness). Injection mode and oven temperature programming were optimized to achieve baseline separation of all target PAHs. Helium (99.999%) was used as the carrier gas at a constant flow rate of 1 mL/min. Splitless mode with a splitless time of 2.1 min was used, with a 1 μL injection volume. The injector temperature was maintained at 300 °C. The oven temperature program was 70 °C (hold for 3.3 min) and ramped to 300 °C (hold 5 min) at a rate of 25 °C/min. The FID temperature was set to 300 °C.</p><h4><strong>3. Results and Discussion</strong></h4><p>The initial GC-FID conditions were adopted from Hor et al. for the determination of BAP, BAA, BBF, and chrysene in tocotrienol concentrates. <a href="javascript:void(0);">(52)</a> Under those chromatographic conditions, baseline separation of all the targeted PAHs could not be achieved (Flt, Pyr, BaA, and BaP). Hence, GC conditions, particularly the oven temperature program, were briefly optimized to obtain baseline separation of all the targeted PAHs (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.6c01489#fig1">Figure 1</a>A).</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Omega_2026_11_21_31418_31431_Figure_1_Chromatograms_of_Ai_standard_mixture_265d7e1014.jpeg" alt="ACS Omega 2026, 11, 21, 31418–31431: Figure 1. Chromatograms of (Ai) standard mixture (100 μg/L) before DLLME; (Aii) standard mixture (100 μg/L) after DLLME; (B) lake water sample spiked with PAHs (10 μg/L); and (C) wastewater sample. 1, Ace; 2, Flu; 3, Ant; 4, Phe; 5, Flt; 6, Pyr; 7, BaA; 8, BaP." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Omega_2026_11_21_31418_31431_Figure_1_Chromatograms_of_Ai_standard_mixture_265d7e1014.jpeg 234w," sizes="100vw" width="2100" height="1402"></p><h5><strong>3.1. Evaluation of the Type of Extractants and Dispersants for DLLME</strong></h5><p>Extraction performance in DLLME is strongly influenced by the choice of extraction solvent (i.e., that which is sedimented), which controls the solutes’ partitioning behavior between the aqueous and organic phases. <a href="javascript:void(0);">(24)</a> Five extraction solvents of different polarity, namely, dichloromethane (DCM, dielectric constant (ε): 8.93 <a href="javascript:void(0);">(53)</a>), chloroform (ε: 4.81 <a href="javascript:void(0);">(54)</a>), carbon tetrachloride (ε: 2.24 <a href="javascript:void(0);">(55)</a>), chlorobenzene (ε: 5.62 <a href="javascript:void(0);">(56)</a>), and cyclohexane (ε: 2.02 <a href="javascript:void(0);">(57)</a>), were evaluated. Based on the overall response (i.e., peak areas), dichloromethane outperformed the other chlorinated solvents (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.6c01489#fig2">Figure 2</a>). This might be attributed to the stronger partitioning of PAHs into DCM, which exhibited the highest polarity among the tested extractants. <a href="javascript:void(0);">(58)</a> Therefore, DCM was selected as the extraction solvent. It is known that the dispersive solvent serves as a “bridge” between the aqueous sample and the water-immiscible extractant, and its selection is therefore crucial to DLLME performance. Five dispersive solvents (methanol, ε: 33; <a href="javascript:void(0);">(59)</a> ethanol, ε: 24.5; <a href="javascript:void(0);">(60)</a> isopropanol, ε: 20.1; <a href="javascript:void(0);">(61)</a> acetone, ε: 20.7; <a href="javascript:void(0);">(62)</a> acetonitrile, ε: 36 <a href="javascript:void(0);">(63)</a>) of different polarities were studied. Among these, ACN having the highest polarity provided the highest extraction response. This might be attributed to the reduced interfacial tension, which promotes formation of smaller microdroplets, and increases interfacial area for partitioning process, thereby improving extraction efficiency. <a href="javascript:void(0);">(64)</a> Thus, DCM and ACN were selected as the extractant and dispersant, respectively, for subsequent FrFD and BBD experiments.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Omega_2026_11_21_31418_31431_Figure_2_Effect_of_A_selection_of_extraction_solvent_B_selection_of_dispersive_solvent_5abd279b17.jpeg" alt="ACS Omega 2026, 11, 21, 31418–31431: Figure 2. Effect of (A) selection of extraction solvent; (B) selection of dispersive solvent." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Omega_2026_11_21_31418_31431_Figure_2_Effect_of_A_selection_of_extraction_solvent_B_selection_of_dispersive_solvent_5abd279b17.jpeg 135w," sizes="100vw" width="1000" height="1154"></p><h4><strong>4. Conclusion</strong></h4><p>An improved and environmentally friendly DLLME-GC-FID method was described for the quantitative analysis of PAHs in surface water. RSM was applied by first screening influential variables using a fractional factorial design, followed by the Box-Behnken design optimization of the DLLME parameters. In contrast to single-factor experimentation, the fitted model and 3D response surface plots showed factor interactions and their combined effects on extraction performance, facilitating near-optimal conditions to be identified with a limited number of experiments. Using the optimized conditions, low detection limits, good enrichment factors, acceptable reproducibility, and recoveries were achieved. The validated analytical features indicate that the proposed method is robust and cost-effective for the routine screening of PAHs in terrestrial water bodies.</p></div>

Experimental Design-Based Dispersive Liquid–Liquid Microextraction with GC-FID for Determination of Polycyclic Aromatic Hydrocarbons in Surface Water

<p><strong>This study compares a commercial ELISA assay with a validated UHPLC-MS/MS method for determining total ergot alkaloids in wheat and durum samples. In addition to method performance, the authors investigated cross-reactivity and matrix effects to explain differences between immunoassay and chromatographic results.</strong></p><p>While ELISA demonstrated good repeatability and is well suited for high-throughput screening, systematic biases were observed due to compound- and matrix-dependent cross-reactivity. The findings support ELISA as an effective screening tool, whereas UHPLC-MS/MS remains the preferred method for accurate quantification and regulatory confirmation of ergot alkaloids.</p><blockquote><h4><strong>The original article</strong></h4><h4><a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jafc.6c05234"><strong>Comparative Evaluation of Total Ergot Alkaloids Determination by ELISA and UHPLC-MS/MS in Wheat: Implications of Cross-Reactivity and Matrix Effects</strong></a></h4><p>Chamali Kodikara*, Dainna Drul, Nandika Bandara, and Sheryl A. Tittlemier*</p><p>J. Agric. Food Chem. 2026, 74, 25, 20015–20027</p><p><a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1021/acs.jafc.6c05234">https://doi.org/10.1021/acs.jafc.6c05234</a></p><h6>licensed under <a target="_blank" rel="noopener noreferrer" href="https://creativecommons.org/licenses/by/4.0/"><strong>CC-BY 4.0</strong></a></h6></blockquote><p><strong>Selected sections from the article follow. Formats and hyperlinks were adapted from the original.</strong></p><div><p>Ergot alkaloids (EAs) are a structurally diverse group of indole-derived mycotoxins produced primarily by phytopathogenic fungi of the genus <i>Claviceps</i>, most notably <i>Claviceps purpurea</i>. These fungi infect the flowering heads of cereal crops, replacing the developing grain with hardened fungal structures (sclerotia) that contain ergot alkaloids. Cereals such as rye, wheat, barley, oats, and triticale are particularly susceptible, with infection severity strongly influenced by environmental conditions during flowering, including rainfall and humidity. <a href="javascript:void(0);">(1−3)</a> Although improvements in agronomic practices and grain cleaning have reduced the prevalence of visible ergot sclerotia, ergot alkaloids can persist in cereals and cereal-based foods even in the absence of detectable sclerotia. This may occur because sclerotia can fragment during harvesting, handling, cleaning, or milling, producing small particles that are not visually detected but may still contribute ergot alkaloids to grain or processed products, continuing to pose food and feed safety concerns. <a href="javascript:void(0);">(4)</a> <a href="javascript:void(0);">(5)</a> Ergot alkaloids share a common tetracyclic ergoline backbone and occur as structurally related compounds, including ergometrine, ergotamine, ergosine, ergocristine, ergocryptine, and ergocornine, together with their corresponding C-8 epimers. These epimers occur as <i>R-</i> and <i>S-</i>configurations, commonly referred to as -ine and -inine forms, respectively. Epimerization at the C-8 position can occur during fungal metabolism and during sample extraction, storage, and analysis, complicating accurate quantification. <a href="javascript:void(0);">(6,7)</a> Although S-epimers were historically considered biologically inactive, recent evidence indicates that they may contribute to overall exposure and toxicity, supporting their inclusion in analytical and regulatory frameworks. <a href="javascript:void(0);">(3)</a></p><p>Dietary exposure to elevated levels of ergot alkaloids has been associated with a range of adverse health effects, collectively referred to as ergotism, including vasoconstriction, ischemia, neurological disturbances, and hallucinations. <a href="javascript:void(0);">(3)</a> These effects arise primarily from interactions with adrenergic, serotonergic, and dopaminergic receptors. <a href="javascript:void(0);">(8−10)</a> In response, regulatory limits for ergot alkaloids in food have been introduced or revised in several jurisdictions. In the European Union, maximum levels have been established for the sum of 12 ergot alkaloids in cereal milling products under Regulation (EU) 2021/1399, which was subsequently consolidated under Regulation (EU) 2023/915. This sum includes ergometrine, ergosine, ergotamine, ergocornine, ergocryptine, and ergocristine together with their corresponding -inine epimers, increasing the demand for reliable analytical methods to support routine monitoring and enforcement. <a href="javascript:void(0);">(1,11)</a></p><p>Liquid chromatography-tandem mass spectrometry (LC-MS/MS), particularly ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), is widely regarded as the reference technique for confirmatory determination of ergot alkaloids due to its sensitivity, selectivity, and ability to resolve epimeric forms. Recent methods routinely achieve low microgram-per-kilogram limits of quantification across a range of cereal matrices. <a href="javascript:void(0);">(12−17)</a> However, LC-MS/MS approaches are resource-intensive and typically confined to centralized laboratories, limiting their suitability for high-throughput screening or rapid decision-making in grain-handling and quality-control settings. Consequently, rapid screening methods, most notably enzyme-linked immunosorbent assays (ELISAs), are commonly employed as complementary tools to chromatographic analysis. <a href="javascript:void(0);">(18−21)</a> ELISAs offer reduced analysis time, simplified workflows, and lower per-sample costs, making them attractive for preliminary screening and sample triage. <a href="javascript:void(0);">(22−24)</a></p><p>In contrast to other regulated mycotoxins, however, the commercial availability of ELISAs for ergot alkaloids is limited. While multiple kits have been evaluated, performance has varied substantially across assays and matrices. <a href="javascript:void(0);">(1)</a> At present, the Randox Food Diagnostics Ergot Alkaloids ELISA is the only widely marketed microplate ELISA that quantifies the sum of 12 major ergot alkaloids, comprising ergometrine, ergosine, ergotamine, ergocornine, ergocryptine, and ergocristine together with their corresponding -inine epimers, consistent with the analyte scope used for EU maximum levels for ergot alkaloids in specified cereal products. <a href="javascript:void(0);">(25)</a></p><p>In addition, cereal matrix effects may influence antibody binding and signal generation, potentially resulting in biased quantification relative to LC-MS/MS reference methods. These limitations are particularly relevant given the compositional variability of ergot alkaloids across cereal species, geographic regions, and growing conditions. <a href="javascript:void(0);">(12)</a> Recent reviews emphasize the need for systematic, matrix-specific evaluation of immunoassay performance before such methods can be confidently integrated into regulatory or industrial monitoring programs. <a href="javascript:void(0);">(1,6,11)</a> However, independent evaluations of commercially available ergot alkaloid ELISAs, particularly those benchmarked against validated UHPLC-MS/MS methods using naturally infested wheat samples, remain limited.</p><p>In this context, we hypothesized that measurements of total ergot alkaloids obtained using a commercially available ELISA are influenced by matrix effects and antibody cross-reactivity, resulting in systematic differences compared to UHPLC-MS/MS, which provides compound-specific quantification. To test this, the objective of the present study was to conduct a comprehensive performance evaluation of a commercially available ELISA for the detection of ergot alkaloids in wheat, using UHPLC-MS/MS as a reference method. Specific aims included assessment of repeatability and intermediate precision, extraction efficiency and extract stability, the influence of test portion mass, method accuracy relative to UHPLC-MS/MS, and characterization of antibody cross-reactivity toward individual ergot alkaloids in both solvent and wheat matrix. The results provide data-driven guidance on the appropriate use of ELISA-based screening within tiered analytical strategies for regulatory monitoring and food safety decision-making.</p><h4><strong>2. Materials and Methods</strong></h4><h5><strong>2.3. UHPLC-MS/MS Reference Method</strong></h5><p>UHPLC-MS/MS analysis of ergot alkaloids was performed using the validated Canadian Grain Commission (CGC) method described by Tittlemier et al. (2015). Briefly, ground grain (10 g) was extracted with 50 mL of acetonitrile/aqueous ammonium carbonate (84:16, v/v; 3.03 mM ammonium carbonate) to provide alkaline extraction conditions, then shaken for 30 min and centrifuged at 2450<i>g</i> for 10 min at room temperature. An aliquot of extract (464 μL) was combined with internal standard solution (dihydroergotamine; 20 μL of 4 ng/μL) and aqueous ammonium carbonate (1536 μL), vortex-mixed, and filtered through a 0.45 μm polytetrafluoroethylene syringe filter before injection into the UHPLC-MS/MS system. As in the validated reference method, dihydroergotamine was added after extraction to correct for postextraction analytical variability; extraction recovery was evaluated during method validation. Chromatographic separation was achieved using a reversed-phase <strong>BEH C18 column</strong> (2.1 × 100 mm, 1.7 μm; Waters, Milford, MA, USA) with the autosampler maintained at 15 °C and the column at 30 °C. Mobile phases consisted of (A) 3.03 mM aqueous ammonium carbonate and (B) acetonitrile. The gradient program was as follows: initial, 75% A/25% B; 1.00 min, 60% A/40% B; 5.00 min, 40% A/60% B; 8.00 min, 22% A/78% B; 10.50–11.00 min, 10% A/90% B; and 13.00–16.00 min, 75% A/25% B, with a total run time of 16 min. Detection was performed using positive electrospray ionization in multiple reaction monitoring mode on a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/554"><strong>Waters Xevo triple quadrupole mass spectrometer</strong></a> (Waters, Milford, MA, USA). Under the validated chromatographic conditions, dihydroergotamine did not coelute with ergotamine, ergotaminine, or the other target ergot alkaloids. Identification and quantification were based on retention-time criteria and compound-specific MRM transitions, thereby minimizing potential isotopic or coelution interference. Precautions to minimize epimerization, including controlled handling of standards and extracts and reduced light exposure, were followed as described in the reference method. UHPLC-MS/MS quantification was performed using analyte-specific, matrix-matched calibration curves prepared according to the validated CGC reference method, with calibration levels of 0.046, 0.2, 1, 2, 10, and 20 μg/kg. Dihydroergotamine was used as the internal standard, and concentrations were calculated from internal-standard-normalized analyte responses with method dilution factors applied. Total ergot alkaloid concentration was calculated as the arithmetic sum of all quantified ergot alkaloids and epimers included in the validated method. <a href="javascript:void(0);">(26)</a></p><h4><strong>3. Results and Discussion</strong></h4><h5><strong>3.4. Comparison of ELISA and UHPLC-MS/MS Results</strong></h5><p>For method comparison, UHPLC-MS/MS was used as the chemical reference method, providing compound-specific quantification of individual ergot alkaloids and their epimers. A total ergot alkaloid concentration was calculated as the arithmetic sum of all quantified alkaloids included in the method. This summed value represents a chemically defined total based on compound-specific quantification of each analyte, rather than an immunoreactivity-weighted response governed by antibody affinity. As such, the UHPLC-MS/MS total serves as a chemically defined comparator against which the immunoreactivity-weighted ELISA response can be interpreted. Agreement between the commercial ELISA and the validated UHPLC-MS/MS reference method was evaluated using wheat and durum wheat cargo samples (<i>n</i> = 102) collected over multiple crop years (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jafc.6c05234#fig5">Figure 5</a>). Linear regression analysis demonstrated a positive association between ELISA and UHPLC-MS/MS results across the full concentration range, indicating that the ELISA was responsive to increasing ergot alkaloid levels. However, the regression slope deviated substantially from unity, with ELISA results systematically lower than UHPLC-MS/MS values, indicating an approximate 2-fold underestimation.&nbsp;</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/J_Agric_Food_Chem_2026_74_25_20015_20027_Figure_5_Linear_regression_analysis_comparing_total_ergot_alkaloid_concentrations_determined_by_ELISA_and_UHPLC_MS_MS_5d7b5c5c61.jpeg" alt="J. Agric. Food Chem. 2026, 74, 25, 20015–20027: Figure 5. Linear regression analysis comparing total ergot alkaloid concentrations determined by ELISA and UHPLC-MS/MS for wheat and durum wheat cargo samples (n = 102). Each point represents the mean of duplicate measurements (extracted from the same test portion) ELISA determinations for an individual cargo sample, plotted against the corresponding UHPLC-MS/MS result. Statistical analysis was performed using linear regression." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_J_Agric_Food_Chem_2026_74_25_20015_20027_Figure_5_Linear_regression_analysis_comparing_total_ergot_alkaloid_concentrations_determined_by_ELISA_and_UHPLC_MS_MS_5d7b5c5c61.jpeg 197w," sizes="100vw" width="992" height="784"></p><p>Bland-Altman analysis confirmed this proportional bias, showing a consistent negative mean difference between ELISA and UHPLC-MS/MS results across the concentration range (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acs.jafc.6c05234#fig6">Figure 6</a>). Importantly, the magnitude of disagreement did not increase randomly with concentration; rather, it reflected a systematic offset, indicating that the discrepancy was not driven by analytical imprecision or outliers but by fundamental differences in method response characteristics. This pattern indicates method-level bias rather than random analytical variability, which may arise from combined workflow effects including extraction, matrix response, and immunochemical cross-reactivity. The magnitude of the ELISA-UHPLC-MS/MS bias was substantially greater than the intermediate precision observed for the ELISA quality-control material, supporting the interpretation that the disagreement reflects systematic differences in method response rather than insufficient ELISA repeatability. Such behavior is well documented when immunoassays are compared with chromatographic methods for complex mycotoxin mixtures, including ergot alkaloids and other structurally diverse mycotoxins in cereals and feed. <a href="javascript:void(0);">(28,30,33)</a> These studies consistently report matrix- and profile-dependent bias in ELISA relative to LC-MS/MS, arising from differential antibody cross-reactivity and the summed immunoreactivity response of the assay, because ELISA produces an immunoreactivity-weighted total that depends on antibody affinity toward individual alkaloids and their relative abundance in the sample, whereas UHPLC-MS/MS quantifies compound-specific concentrations of each alkaloid and its epimers using compound-specific transitions and response factors, and total ergot alkaloid concentration is calculated as the sum of these targeted analytes. In contrast, ELISA produces an immunoreactivity-weighted total, determined by antibody affinity toward individual alkaloids and their competitive binding behavior within the assay format. Consequently, agreement between ELISA and UHPLC-MS/MS depends not only on total concentration, but also on the relative distribution of individual alkaloids within each sample. Although differences in extraction solvent composition and pH may influence ergot alkaloid recovery, stability, and epimeric distribution, each method was applied according to its intended analytical workflow. The ELISA was performed using the manufacturer-recommended acidic methanol/water extraction, whereas UHPLC-MS/MS was performed using the validated acetonitrile/aqueous ammonium carbonate extraction procedure. Therefore, the comparison reflects the performance of the complete methods as used in practice, rather than an isolated comparison of extraction solvents. The sequential extraction experiment further showed that repeating the ELISA extraction under the same kit conditions did not substantially increase the ELISA-measured response, supporting the adequacy of the kit extraction within the ELISA workflow while recognizing that it does not directly compare acidic and ammonium carbonate-based extraction conditions. Previous ELISA-LC-MS/MS comparison studies for ergot alkaloids have reported both under- and overestimation depending on assay design and matrix, including differences in alkaloid profile, cereal type, and antibody cross-reactivity. Turner et al. observed substantial overestimation of ergot alkaloids by ELISA relative to HPLC in rye field samples, attributed to strong antibody cross-reactivity toward alkaloids prevalent in rye matrices. Similar profile-dependent bias has also been reported in interlaboratory and method-comparison studies, <a href="javascript:void(0);">(33)</a> demonstrating that this behavior is not unique to a single data set.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/J_Agric_Food_Chem_2026_74_25_20015_20027_Figure_6_Bland_Altman_plot_comparing_ELISA_and_UHPLC_MS_MS_measurements_of_total_ergot_alkaloids_1be40cce50.jpeg" alt="J. Agric. Food Chem. 2026, 74, 25, 20015–20027: Figure 6. Bland-Altman plot comparing ELISA and UHPLC-MS/MS measurements of total ergot alkaloids for wheat and durum wheat cargo samples (n = 102)." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_J_Agric_Food_Chem_2026_74_25_20015_20027_Figure_6_Bland_Altman_plot_comparing_ELISA_and_UHPLC_MS_MS_measurements_of_total_ergot_alkaloids_1be40cce50.jpeg 230w," sizes="100vw" width="1437" height="977"></p><p>This study provides a comprehensive evaluation of a commercially available ELISA for total ergot alkaloids, using wheat samples as the primary application matrix, and integrates performance metrics with mechanistic insights into method behavior. While the assay demonstrated strong repeatability, calibration stability, and robustness to procedural factors such as test portion mass and extraction conditions, systematic differences relative to UHPLC-MS/MS were consistently observed. By linking these differences to compound-specific antibody cross-reactivity and matrix-dependent signal modulation, this work clarifies that ELISA-derived results reflect an immunochemically weighted response rather than a stoichiometric total. This distinction is critical for the appropriate interpretation of ELISA data and supports its use within tiered analytical strategies as a rapid screening tool, complemented by UHPLC-MS/MS for confirmatory quantification and regulatory decision-making.</p></div>

Comparative Evaluation of Total Ergot Alkaloids Determination by ELISA and UHPLC-MS/MS in Wheat: Implications of Cross-Reactivity and Matrix Effects

<p><strong>This study investigates the antioxidant and anti-inflammatory effects of (−)-epicatechin in an in vitro model of metabolic dysfunction-associated steatotic liver disease (MASLD). Lipid peroxidation, inflammatory, and profibrotic markers were assessed alongside ATR-FTIR spectroscopy to evaluate whether spectral changes reflect disease progression and treatment response.</strong></p><p>Epicatechin reduced lipid peroxidation products, TNF-α, and TGF-β1 in a concentration-dependent manner, with stronger protective effects observed during pretreatment. ATR-FTIR spectral changes closely correlated with biochemical markers, demonstrating its potential as a rapid, non-destructive tool for monitoring oxidative stress, inflammation, and fibrosis in MASLD research.</p><blockquote><h4><strong>The original article</strong></h4><h4><a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.5c09978"><strong>ATR-FTIR Assessment of the Antioxidant and Anti-Inflammatory Effects of (−)-Epicatechin in MASLD</strong></a></h4><p>Marija Hefer, Nikolina Filipović, Stjepan Šarić, Nela Malatesti, Ana Petrović, Elvira Kovač-Andrić*, and Martina Smolić*</p><p>ACS Omega 2026, 11, 23, 33333–33344</p><p><a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1021/acsomega.5c09978">https://doi.org/10.1021/acsomega.5c09978</a></p><h6>licensed under <a target="_blank" rel="noopener noreferrer" href="https://creativecommons.org/licenses/by/4.0/"><strong>CC-BY 4.0</strong></a></h6></blockquote><p><strong>Selected sections from the article follow. Formats and hyperlinks were adapted from the original.</strong></p><div><p>Metabolic dysfunction-associated steatotic liver disease (MASLD) represents one of the most prevalent chronic liver disorders. Due to its predisposition to progress toward metabolic dysfunction-associated steatohepatitis (MASH), fibrosis, cirrhosis, and even hepatocellular carcinoma (HCC), MASLD has become a significant global health burden and a major challenge for healthcare systems. <a href="javascript:void(0);">(1−4)</a></p><p>The majority of patients with MASLD or MASH are obese and frequently present with additional metabolic comorbidities, most commonly insulin resistance, type 2 diabetes mellitus, and dyslipidemia. <a href="javascript:void(0);">(5−7)</a> Furthermore, most conditions associated with MASLD and MASH are characterized by lipid metabolism impairment, with elevated levels of oxidative stress. Impaired lipid metabolism, together with prolonged oxidative stress, promotes hepatocellular lipotoxicity and mitochondrial dysfunction. These alterations affect the antioxidant defense system and increase reactive oxygen species (ROS) generation, thereby contributing to the onset of MASLD and its progression to MASH, along with more advanced liver diseases. <a href="javascript:void(0);">(8,9)</a></p><p>One of the main results of oxidative stress is lipid peroxidation, in which ROS attack (poly)unsaturated fatty acids within membrane lipids. This initiates a series of structural and functional alterations, where the formation of conjugated dienes (CDs) reflects early double-bond rearrangements in fatty acyl chains, while lipid hydroperoxides (LOOHs) represent key propagation-phase products that destabilize cell membranes and increase oxidative injury. <a href="javascript:void(0);">(10,11)</a> Therefore, determining LOOH and CD levels, along with their overall LOOH/CD ratio, might be useful for determining the lipid peroxidation dynamics and the progression of MASLD.</p><p>Along with oxidative damage to the cell structures, inflammation is also a key regulator of MASLD progression. In this context, tumor necrosis factor-α (TNF-α) is suspected to play a central role as its levels are found to increase alongside MASLD to MASH progression. <a href="javascript:void(0);">(12−14)</a> It has also been found that ROS and TNF-α are closely interconnected, where elevated TNF-α levels could serve as an indicator of oxidative cellular damage and enhanced lipid peroxidation. <a href="javascript:void(0);">(15−17)</a> Often, one of the results of MASLD progression is the development of liver fibrosis, which is usually mediated by transforming growth factor beta 1 (TGF-β<sub>1</sub>) as its levels are frequently elevated in MASH and contribute to the worsening of hepatic steatosis. <a href="javascript:void(0);">(18)</a> <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.5c09978#fig1">Figure 1</a> summarizes the pathways associated with MASLD to MASH progression, with emphasis on lipid peroxidation (CDs, LOOHs) and inflammatory/fibrogenic signaling (TNF-α, TGF-β<sub>1</sub>).</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Omega_2026_11_23_33333_33344_Figure_1_Schematic_representation_of_lipid_peroxidation_and_its_role_in_MASLD_progression_e6a8e05d3e.jpeg" alt="ACS Omega 2026, 11, 23, 33333–33344: Figure 1. Schematic representation of lipid peroxidation and its role in MASLD progression. Lipid accumulation in MASLD leads to the release of free radicals and ROS, which attack (poly)unsaturated fatty acids within membrane lipids, leading to lipid peroxidation, inflammation (via TNF-α), and fibrotic changes (via TGF-β1), thereby contributing to the transition from MASLD to MASH and further progression to cirrhosis and HCC. In lipid peroxidation, reactive radicals (R·) subtract the hydrogen from the (poly)unsaturated lipid, thereby resulting in the formation of a lipid radical, which undergoes rearrangements to form a CD. The lipid radical then reacts with molecular oxygen to form an unstable lipid peroxyl radical, which further subtracts a hydrogen from another (poly)unsaturated lipid, thereby resulting in the formation of a LOOH. The subtraction of hydrogen from the (poly)unsaturated lipid also results in the formation of a lipid radical, thereby perpetuating the chain reaction. The figure was created with Servier Medical Art (https://smart.servier.com/) and MarvinSketch (ChemAxon, https://chemaxon.com/marvin)." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Omega_2026_11_23_33333_33344_Figure_1_Schematic_representation_of_lipid_peroxidation_and_its_role_in_MASLD_progression_e6a8e05d3e.jpeg 216w," sizes="100vw" width="1834" height="1327"></p><p>However, the exact role of different lipid peroxidation indicators and their contribution to MASLD progression toward MASH have not yet been fully elucidated, with the number of studies addressing this issue remaining limited. Given these limitations in research, attention has increasingly turned toward dietary antioxidants, particularly polyphenols or mixtures of polyphenol-rich plant extracts, as potential modulators of oxidative stress and lipid peroxidation in hepatic steatosis. <a href="javascript:void(0);">(19−23)</a></p><p>Among available polyphenols, we selected (−)-epicatechin (EC) because of its strong antioxidant and radical scavenging capacity, with its documented effects on inflammatory signaling. <a href="javascript:void(0);">(24−26)</a> One study even found that the antifibrotic properties of EC were comparable to those of silymarin, with EC showing even stronger reducing effects on malondialdehyde (MDA) formation in vivo. <a href="javascript:void(0);">(27)</a> Since MDA is a secondary product of LOOH breakdown, <a href="javascript:void(0);">(28)</a> these results could link EC’s potential antifibrotic properties to its ability to attenuate lipid peroxidation, thereby potentially mitigating the progression of MASLD to MASH. Unlike more extensively studied compounds such as epigallocatechin gallate, <a href="javascript:void(0);">(29,30)</a> EC has been less investigated in the context of MASLD, providing an opportunity to explore a novel candidate. At the same time, the growing use of advanced spectroscopic techniques offers new opportunities to monitor biochemical alterations in cells and tissues.</p><p>Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy recently emerged as a promising tool for monitoring such changes. The application of ATR-FTIR spectroscopy and similar techniques for steatosis and lipid quantification, along with determining the lipid peroxidation extent, has been introduced, especially in the context of hepatic steatosis. Determining the intensity of C═O vibrational stretching at ∼1740 cm<sup>–1</sup> (triacylglycerols, cholesteryl esters, lipid oxides), as this type of bond usually occurs in lipid peroxidation, could also be valuable for determining the overall extent of steatosis. <a href="javascript:void(0);">(31−33)</a> Along with lipid peroxidation extent, determining the degree of unsaturation in MASLD models is crucial for understanding the changes in cellular components as the degree of unsaturation is often found to decrease due to steatotic changes in cells and tissues. (Poly)unsaturated fatty acids contribute to membrane fluidity and are essential for maintaining membrane integrity and signaling, while their depletion leads to increased membrane rigidity and impaired cellular function. <a href="javascript:void(0);">(34,35)</a> Given their high susceptibility to peroxidation, a reduction in their levels could indicate either an adaptive response to oxidative stress or a disruption of lipid homeostasis. Therefore, correlating the degree of unsaturation (∼3010 cm<sup>–1</sup> for ═C–H) and the intensity of carbonyl stretching (∼1740 cm<sup>–1</sup> for C═O) with the LOOH/CD ratio and cytokines, TNF-α and TGF-β<sub>1</sub>, could be valuable for the noninvasive monitoring of disease progression.</p><h4><strong>Methods</strong></h4><h5><strong>Experimental Design</strong></h5><p>Two experimental models were introduced for the experiments: EC pretreatment and EC Posttreatment models. In both models, the untreated cells (UT) group was used as a negative (vehicle) control group, while the sodium oleate-treated group (OA) was used as a positive control group, with EC (control) being the highest concentration of EC used as a control for the tested compound. In the Pretreatment model, the cells were pretreated with 10, 30, and 50 μM EC for 4 h, followed by a 24 h posttreatment with 1.5 mM sodium oleate. In the Posttreatment model, the cells were treated with 1.5 mM sodium oleate for 12 h, followed by the addition of the same concentrations of EC in the presence of 0.75 mM sodium oleate. All of the experimental groups were labeled as EC50+OA, EC30+OA, and EC10+OA, according to the concentration used in the experiments. EC concentrations of 10, 30, and 50 μM were chosen based on our previous MTS assay and Oil Red O staining results, where we tested 10–100 μM EC to evaluate its effect on MASLD in the Pretreatment model. <a href="javascript:void(0);">(42)</a> As it was proven effective in the Pretreatment model, the Posttreatment model was added to evaluate whether EC could also mitigate oxidative and inflammatory/fibrotic changes once steatosis was already established. Another important consideration was to assess the potential cytotoxicity of the concentrations used, as EC and other polyphenols are often consumed as dietary supplements with poorly regulated dosages. As these cytotoxic effects are often subtle and overlooked in conventional biochemical assays, combining FTIR spectral analysis with complementary assays could provide a more comprehensive assessment of potential cytotoxicity.</p><h5><strong>ATR-FTIR Assessment of Lipid Peroxidation</strong></h5><p>ATR-FTIR spectroscopy was used to determine the extent of lipid peroxidation and steatosis. The lipid samples were dissolved in chloroform and normalized to a lipid concentration of 20 mg/mL. After the evaporation of the solvent, the ATR-FTIR spectra were recorded using an <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library?mainauthorItems=10&amp;search=Cary+630"><strong>Agilent Cary 630 FTIR spectrophotometer</strong></a> (Agilent Technologies, Santa Clara, CA, USA), at the Faculty of Biotechnology and Drug Development at the University of Rijeka. All ATR-FTIR spectra were processed using the automatic least-squares baseline correction algorithm implemented in <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library?mainauthorItems=10&amp;search=MicroLab+Expert"><strong>Agilent MicroLab Expert v.10.5 software</strong></a> (4000–650 cm<sup>–1</sup>; clean scans: 4; threshold: 0.002; background scans: 2; sample scans: 100; resolution: 16 cm<sup>–1</sup>; apodization: HappGenzel; phase correct: Mertz; method gain: 221). Spectra of pure chloroform were recorded under identical conditions and subtracted from sample spectra to remove any residual solvent contributions. Chloroform was recorded to be excluded in the background, and the data were collected in the spectral range 3200–1600 cm<sup>–1</sup> on an ATR device. To ensure comparability between samples, the spectra were normalized against the asymmetric CH<sub>2</sub> stretching vibration of lipid acyl chains (2925–2922 cm<sup>–1</sup>), which was used as an internal reference band due to its relative stability across treatments and minimal alteration during lipid peroxidation. Baseline correction was applied prior to normalization to minimize background variability. Relative intensities of lipid peroxidation-associated bands (C═O stretching at 1744–1735 cm<sup>–1</sup> and ═C–H stretching at 3010 cm<sup>–1</sup>) were then expressed as a ratio to the CH<sub>2</sub> reference peak, allowing quantitative comparison of the lipid peroxidation extent between experimental groups. The data were presented as percent relative intensities (%<i>I</i><sub>rel</sub>).</p><h4><strong>Results and Discussion</strong></h4><h5><strong>ATR-FTIR Spectroscopy for the Assessment of Lipid Peroxidation and Steatosis Extent</strong></h5><p>To evaluate the overall steatosis and lipid peroxidation extent in HepG2 cells, ATR-FTIR spectroscopy was used. <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.5c09978#fig5">Figure 5</a> shows the 3200–1600 cm<sup>–1</sup> spectral region, including the key C═O (1744–1735 cm<sup>–1</sup>) and ═C–H (3010 cm<sup>–1</sup>) stretching bands, which correlated with the biochemical parameters measured in this study. The increased intensity of the C═O band in sodium oleate-treated groups corresponds to the accumulation of oxidized lipid species derived from peroxidation of (poly)unsaturated fatty acids. On the other hand, a decrease in the ═C–H stretching band near 3010 cm<sup>–1</sup> indicates a progressive depletion of double bonds which undergo rearrangement to form CDs or further transformations associated with lipid peroxidation.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Omega_2026_11_23_33333_33344_Figure_5_ATR_FTIR_spectra_of_control_groups_aabfabbdc1.jpeg" alt="ACS Omega 2026, 11, 23, 33333–33344: Figure 5. ATR-FTIR spectra of control groups: UT (blue) and EC (control) (purple); along with experimental groups: OA (yellow), EC50+OA (red), EC30+OA (orange), and EC10+OA (brown) in the wavenumber range 3200–1600 cm–1 for the (a) EC Pretreatment Model and (b) EC Posttreatment Model." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Omega_2026_11_23_33333_33344_Figure_5_ATR_FTIR_spectra_of_control_groups_aabfabbdc1.jpeg 193w," sizes="100vw" width="1572" height="1272"></p><p>All experimental groups show similar FTIR profiles in the lipid regions; however, to determine the overall extent of lipid peroxidation and steatosis, the relative intensities of ═C–H stretching of unsaturated lipids (3010 cm<sup>–1</sup>) and C═O stretching of esters (1744–1735 cm<sup>–1</sup>) were measured according to the baseline and normalized according to asymmetric CH<sub>2</sub> stretching of lipid chains (2925–2922 cm<sup>–1</sup>) (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.5c09978#fig6">Figure 6</a>). This approach was chosen as the greatest changes were noticed among the given intensities of the peaks in all groups.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Omega_2026_11_23_33333_33344_Figure_6_Percent_relative_intensities_e921d53b57.jpeg" alt="ACS Omega 2026, 11, 23, 33333–33344: Figure 6. Percent relative intensities (%Irel) of ═C–H stretching (unsaturated lipids) and C═O stretching of esters for the (a) EC Pretreatment Model and (b) EC Posttreatment Model. The results are presented as mean values of %Irel ± SD." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Omega_2026_11_23_33333_33344_Figure_6_Percent_relative_intensities_e921d53b57.jpeg 149w," sizes="100vw" width="1000" height="1044"></p><p>Also, as indicated in the literature, the increase in intensities of C═O stretching and a decrease in intensities of ═C–H stretching are mostly associated with an increase in the lipid peroxidation extent. <a href="javascript:void(0);">(33,50)</a> Furthermore, a decrease in ═C–H stretching corresponds to a decrease in the (poly)unsaturated fatty acid content, which is often noted in MASLD/MASH models. <a href="javascript:void(0);">(51,52)</a></p><h4><strong>Conclusion</strong></h4><p>In a sodium oleate-induced hepatic steatosis model, EC showed a concentration-dependent effect on the overall radical scavenging ability of HepG2 cells. However, when EC was given as a posttreatment, these results were not achievable. EC also reduced propagation-phase lipid peroxidation by reducing relative LOOH levels in both models, lowering the LOOH/CD ratio (except for the EC50+OA pretreatment group), and effectively decreasing sodium oleate-induced increases in TNF-α and TGF-β<sub>1</sub> concentrations. ATR-FTIR results were consistent with changes in lipid (per)oxidation and reflected the overall antioxidant and anti-inflammatory effects of EC observed through biochemical assays. Compared with biochemical assays, ATR-FTIR spectroscopy has several advantages, including rapid and nondestructive analysis that requires minimal sample preparation. Considering that it can simultaneously capture multiple molecular changes, this represents a reliable approach for accurate and complementary insights into lipid peroxidation and redox alterations in MASLD. However, it is important to correlate ATR-FTIR spectral changes with biochemical assays to further strengthen its interpretation.</p></div>

ATR-FTIR Assessment of the Antioxidant and Anti-Inflammatory Effects of (−)-Epicatechin in MASLD

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ATR-FTIR Assessment of the Antioxidant and Anti-Inflammatory Effects of (−)-Epicatechin in MASLD

Comparative Evaluation of Total Ergot Alkaloids Determination by ELISA and UHPLC-MS/MS in Wheat: Implications of Cross-Reactivity and Matrix Effects

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