<p><strong>If you are working with an established method</strong>, we would recommend for you to:</p><ul><li>Check all solutions used for the SPE method to ensure that nothing is expired or has been mislabelled.</li><li>Check the process. Was flow potentially too fast during loading or elution? Could the cartridges have been allowed to dry between equilibration and sample loading?</li></ul><p>It is generally quickest to simply repeat the extraction, taking care of these points.</p><p><strong>If, however, you are developing a new method</strong>, it might be necessary to “track down” your analyte. The first thing we would recommend is to make up a solution of the analyte without sample matrix (to use as an ideal sample). Then work through the sample method capturing the eluent from each step potentially containing the analyte:</p><ol><li>Condition the cartridge.</li><li>Equilibrate the cartridge.</li><li>Load the sample and collect the flow-through in a vial.</li><li>Analyze this for analyte content. If analyte is found in this fraction it would suggest:<ul><li>Sample solvent is too strong, or the pH is incorrect.</li><li>Loading flow rate is too high.</li><li>You have breakthrough because either sample load is too high, or sorbent mass is too low.</li></ul></li><li>Wash the cartridge, collecting each wash fraction in a separate vial.</li><li>Again, analyze for analyte content. If analyte is found in any wash fraction, this suggests:<ul><li>Wash solvent is too strong.</li><li>Wash solvent is not at the correct pH.</li></ul></li><li>If the analyte has not been found in the load or wash fractions, then the analyte must be retained on the cartridge.<ul><li>Try using a stronger elution solvent.</li><li>Ensure pH of the elution solvent is correct.</li><li>If the first 2 suggestions don’t help, try using a less retentive sorbent (in reversed phase, try switching a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=136&amp;manufacturerItems=50&amp;search=Strata"><strong>C18 cartridge for a C8</strong></a> if the analytes are very hydrophobic).</li></ul></li></ol>

I am getting low recovery in my SPE method, how do I fix the problem?

<p><strong>It is easy to think that once a sample elutes from Solid Phase Extraction (SPE) it is then ready to be injected onto the analytical column. While the sample is now significantly cleaner, it is not always the case that injecting it directly onto the column will result in perfect chromatography. Solvent mismatch is a known cause of poor peak shape, so care must be given to consider the solvent strength used in the elution step of any SPE being performed on samples.</strong></p><p>The solvent with which the sample is eluted, the overall concentration of the sample, and the mobile phase conditions of the method should be evaluated when building sample preparation protocols. An injection of 100% organic solvent when your starting Mobile Phase conditions begin with high water (90%), can cause solvent mis-match to occur within the system, as seen in Image 1 below. Ideally, a sample will be in solvent that closely matches the starting mobile phase ratio of the LC method to prevent issues in peak shape from occurring.&nbsp;</p><p>Remember that not all elution solvent ratios are troublesome to chromatography. A good example of a favourable elution solvent is when <a target="_blank" rel="noopener noreferrer" href="https://app.explore.phenomenex.com/e/er?s=822460770&amp;lid=8512&amp;elqTrackId=AF71A2845D16ED3FEE8AD0E50AD40B88&amp;elq=00000000000000000000000000000000&amp;elqaid=5382&amp;elqat=2&amp;elqak=8AF5AB5B4A0284363E33E235633CBBF8B994CF7C906A90E1BF5C55FDC1EA7D8B80B0"><strong>Phree™ Protein Precipitation</strong></a> product is used to prepare samples for analysis in HILIC (hydrophilic interaction liquid chromatography) mode. The Phree protocol calls for samples to be diluted in a 3 : 1 (Acetonitrile : Aqueous) based sample ratio for the protein precipitation step to be successful.<a target="_blank" rel="noopener noreferrer" href="https://app.explore.phenomenex.com/e/er?s=822460770&amp;lid=8513&amp;elqTrackId=58B1E1E14DBED21807DB7913ABA26DA8&amp;elq=00000000000000000000000000000000&amp;elqaid=5382&amp;elqat=2&amp;elqak=8AF503F0D16829D3E24EB118C4BBECBABEFBCF7C906A90E1BF5C55FDC1EA7D8B80B0"><strong> HILIC mode</strong></a> starts at high concentrations of Acetonitrile, but still requires water to be present in the sample for optimal peak shape. The sample eluting from Phree is already in the ideal solvent ratio for HILIC mode and no further processing is needed, but if your analysis is looking to use<a target="_blank" rel="noopener noreferrer" href="https://app.explore.phenomenex.com/e/er?s=822460770&amp;lid=8514&amp;elqTrackId=BC52B3D9F6E8C58C9E6787A59D44EB41&amp;elq=00000000000000000000000000000000&amp;elqaid=5382&amp;elqat=2&amp;elqak=8AF57A92CCD54FE8CC7F340714830182B6BACF7C906A90E1BF5C55FDC1EA7D8B80B0"><strong> Ion Exchange with Rezex™</strong></a>, the presence of Acetonitrile in the sample could cause issues.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_The_danger_of_mobile_phase_mismatch_2_46cfdb13b7.jpg" alt="Phenomenex: The danger of mobile phase mismatch: Sample in 50% Methanol." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_The_danger_of_mobile_phase_mismatch_2_46cfdb13b7.jpg 170w," sizes="100vw" width="854" height="787"><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Phenomenex_The_danger_of_mobile_phase_mismatch_3_14e68491b5.jpg" alt="Phenomenex: The danger of mobile phase mismatch: Sample in 100% Methanol." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Phenomenex_The_danger_of_mobile_phase_mismatch_3_14e68491b5.jpg 166w," sizes="100vw" width="837" height="789"></p>

The danger of mobile phase mismatch

<h3><strong>Introduction</strong></h3><p><strong>Signal-to-noise ratio (S/N) is a critical parameter in chromatography for evaluating peak quality and method sensitivity. The</strong> <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/999"><strong>Clarity Chromatography Data System</strong></a><strong> (CDS) includes tools that allow users to calculate S/N directly from chromatograms. This guide explains the complete procedure step by step.</strong></p><h3><strong>Tools and Requirements</strong></h3><ul><li><strong>Clarity CDS software</strong> with chromatogram visualization enabled</li><li><strong>Chromatogram file</strong> containing peaks of interest</li><li><strong>Result Table</strong> window for displaying calculated parameters</li></ul><h3><strong>Step 1: Determining Noise</strong></h3><ol><li>Navigate to the <strong>Chromatogram → Noise and Drift</strong> menu.</li><li>Select <strong>Noise Evaluation</strong>.</li><li>In your chromatogram, mark an interval that contains only baseline noise (no peaks).</li><li>The software determines the noise value and displays it above the Result Table.</li></ol><h3><strong>Step 2: Configuring the Result Table</strong></h3><ol><li>Right-click inside the <strong>Result Table</strong> to open the local menu.</li><li>Select <strong>Setup Columns</strong> to access the configuration dialog.</li><li>Define a new column:<ul><li><strong>Name:</strong> Enter a descriptive label (e.g., <i>S/N</i>).</li><li><strong>Expression:</strong> Input the formula <i>Height / Noise</i>, where <i>Height</i> refers to the peak height and <i>Noise</i> is the variable determined in Step 1.</li></ul></li><li>Disable the <strong>Calculate Total</strong> option, as it is not necessary for S/N.</li><li>Confirm with <strong>OK</strong> to save settings.</li></ol><h3><strong>Step 3: Viewing Signal-to-Noise Ratio</strong></h3><p>Once configured, the Result Table automatically calculates and displays the signal-to-noise ratio for every detected peak in the chromatogram. This allows rapid assessment of peak detectability and compliance with method validation requirements.</p><h3><strong>Discussion</strong></h3><p>The ability to evaluate S/N directly in Clarity provides several advantages:</p><ul><li><strong>Automation:</strong> Removes the need for manual calculations.</li><li><strong>Accuracy:</strong> Uses consistent baseline noise determination across the chromatogram.</li><li><strong>Validation Support:</strong> Helps confirm method sensitivity in accordance with regulatory guidelines (e.g., ICH Q2, USP).</li></ul><h3><strong>Conclusion</strong></h3><p>Clarity CDS offers a straightforward workflow to calculate signal-to-noise ratio for chromatographic peaks. By first defining the noise baseline and then configuring a custom result column, users can quickly assess analytical sensitivity and peak quality.</p><blockquote><p><strong>For further learning and tutorials, additional videos are available on the </strong><a target="_blank" rel="noopener noreferrer" href="https://www.youtube.com/@DataapexPrague"><strong>official Clarity YouTube channel</strong></a><strong>.</strong></p></blockquote>

How to calculate Signal to Noise ratio in Clarity Chromatography Software

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

Collision-Based Ion-activation and Dissociation

<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 15th September 2025? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!</strong></p><blockquote><p><span style="font-size:20px;">👉 </span><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library"><span style="font-size:20px;"><strong>SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES</strong></span></a></p></blockquote><blockquote><p><strong>👉 Need info about different analytical techniques?</strong> Peek into <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library"><strong>LabRulezGCMS</strong></a> or <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library"><strong>LabRulezICPMS</strong></a> libraries.</p></blockquote><p>This week we bring you application notes by <strong>Agilent Technologies, Shimadzu </strong>and <strong>Waters Corporation </strong>and<strong> </strong>poster by <strong>Thermo Fisher Scientific / HPLC Symposium!</strong></p><h3><strong>1. Agilent Technologies: A Direct Aqueous Injection Method for Contaminants in Drinking and Nonpotable Water</strong></h3><p><strong>Analysis of acrylamide, haloacetic acids, and β-estradiol in water with an Agilent 6495D LC/TQ</strong></p><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_aquesous_injection_method_water_6495d_lc_tq_5994_7973en_agilent_1_24e3a63a15.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Recent regulations, such as EU 2020/218, have included the addition of new emerging contaminants in drinking water and raw waters, which supply water-treatment works. Some of these new contaminants to be analyzed include acrylamide, HAAs, and β-estradiol. However, acrylamide and HAAs can be difficult to analyze due to their high polarity and high interferences from hard water samples. Additionally, β-estradiol requires a very low level of detection (1 ng/L) compared to the other compounds (acrylamide 0.1 µg/L and HAAs 60&nbsp;µg/L). The analysis uses a&nbsp;1290 Infinity II LC coupled to a&nbsp;6495D LC/TQ for analysis of water samples.</p><h4><strong>Experimental</strong></h4><h5><strong>Instrumentation&nbsp;</strong></h5><p>Chromatographic separation was performed using an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=InfinityLab+Poroshell+120+Aq-C18"><strong>Agilent&nbsp;InfinityLab Poroshell 120 Aq-C18</strong></a>, 3.0 × 150&nbsp;mm, 2.7&nbsp;µm (part&nbsp;number 693675-742) installed on the&nbsp;1290&nbsp;Infinity II LC system. The individual modules of the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=1290+Infinity+II+LC"><strong>1290 Infinity II LC system</strong></a>&nbsp;included:</p><ul><li>Agilent 1290 Infinity II high‑speed pump (G7120A)&nbsp;</li><li>Agilent 1290 Infinity II autosampler (G7167B)&nbsp;</li><li>Agilent 1290 Infinity II multicolumn thermostat (G7116B)&nbsp;</li></ul><p>The <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=6495+LC%2FTQ"><strong>6495 LC/TQ</strong></a> with an Agilent&nbsp;Jet Stream (AJS) electrospray ion source was operated in dynamic multiple reaction monitoring (dMRM) mode. This mode allows more MRM transitions if future development is required for additional compounds. The LC/TQ autotune was performed in both unit and wide modes. All data acquisition and processing were performed using Agilent&nbsp;MassHunter software (version&nbsp;12).</p><h4><strong>Conclusion&nbsp;</strong></h4><p>This application note describes a highly sensitive and reproducible method for the fast and reliable quantitation of the listed compounds in water by direct injection. The dMRM method was created and optimized using Agilent MassHunter software and allows the addition of more MRM transitions if future development is required for additional compounds. An Agilent 1290 Infinity II LC coupled to an Agilent 6495D LC/TQ was used for the analysis. The 12-minute LC gradient method using an Agilent Aq-C18 column offered good chromatographic separation and RT distribution of all targets. The LC/TQ data acquisition was in dMRM mode with fast polarity switching for the most efficient use of instrument cycle time. The method performance was verified based on requirements for calibration curve linearity, instrument LOD, recovery, and precision. The results demonstrate the ability of the quantitative analytical method for emerging contaminants in water by direct injection.</p><h3><strong>2. Shimadzu: Short-Chain Oligonucleotide Analysis Using Supercritical Fluid Chromatography</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/ap_aplnote100_en_23b7bffc7e.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Oligonucleotide therapeutics are drugs in which oligonucleotide is the active ingredient. Oligonucleotide therapeutics are manufactured through chemical synthesis, but impurities are produced during the process, so measurement and characterization of the impurities are required. Currently, reversed-phase ion-pair chromatography is often used for this analysis, but it is difficult to separate some impurities that are structurally similar to the target product. Therefore, a separation method with selectivity different from existing methods is required.&nbsp;</p><p>SFC is an analytical method that employs carbon dioxide as the mobile phase at temperatures and pressures above the respective critical points. Compared to liquids, carbon dioxide is reported to have high resolution and special selectivity as a component of SFC mobile phase because of its low viscosity and high diffusivity.&nbsp;</p><p>On the other hand, since the mobile phase containing carbon dioxide has low polarity, SFC was considered unsuitable for the analysis of highly polar compounds. However, in recent years, the composition of the modifier mixed with carbon dioxide and the choice of stationary phase have made it possible to analyze highly polar compounds,such as peptides and nucleotides2).&nbsp;</p><p>In this study, we evaluated the applicability of SFC to oligonucleotide analysis. Single-strand oligonucleotides of around 20-mer are often used as oligonucleotide therapeutics, but due to the extremely high polarity of the molecules and the difficulty of analysis, we first used 4-mer oligonucleotides for evaluation and investigated their basic retention behaviors.</p><h4><strong>Experimental</strong></h4><ul><li><strong>System:</strong> <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=LCMS-9030"><strong>LCMS-9030</strong></a></li></ul><h4><strong>Conclusions&nbsp;</strong></h4><p>To evaluate the applicability of SFC for oligonucleotide analysis, we developed a separation method using 4-mer oligonucleotides with varying PS contents as model compounds and investigated their basic retention characteristics. First, we identified conditions that allowed oligonucleotides to elute, finding that a methanol-water mixture containing 50 mmol/L ammonium formate enabled elution and peak detection. Using these conditions, we analyzed T4 with varying PS contents on 12 columns, finding good separation with the Diol II column. To apply this method to more polar sequences, we re-evaluated modifier conditions and found that adding aminoethanol improved peak shape. Applying the optimized method to 14 oligonucleotide sequences showed that sequences with two or fewer G or C bases were suitable for this method. Additionally, higher-polarity sequences showed stronger retention, while oligonucleotides with varying PS contents showed weaker retention with higher polarity, indicating unique retention behavior for PS modifications. The high selectivity for sequences with different PS contents suggests that SFC can be applied to separating impurities with a PS linkage replaced by a PO linkage that can be included in PS-modified oligonucleotides in the synthesis process.&nbsp;</p><p>In the future study, the applicability of SFC to longer oligonucleotidesthan 4-mer will be evaluated.</p><h3><strong>3. Thermo Fisher Scientific / HPLC Symposium: Sensitive LC-MS method for the quantitative analysis of semaglutide and liraglutide in human plasma</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_sensitive_lc_ms_method_quantitative_analysis_semaglutide_po_en_592e7dcc4a.pdf"><strong>Full PDF for download</strong></a></li></ul><p>GLP-1 analogs have emerged as pivotal therapeutic agents in the management of type 2 diabetes and obesity. These analogs mimic the physiological actions of endogenous GLP-1, including enhancing glucose-dependent insulin secretion, suppressing glucagon release, delaying gastric emptying, and promoting satiety. Given their significant clinical utilities, accurate detection and quantification of GLP-1 analogs in human plasma are essential for pharmacokinetic studies, therapeutic monitoring, and ensuring patient safety. This study aims to develop a sensitive LC-MS method for the detection and quantitation of semaglutide and liraglutide in human plasma.</p><h4><strong>Materials and methods</strong></h4><h5><strong>Mass Spectrometry</strong></h5><p><span>&nbsp;</span>See Table 2 for detailed MS source and scan settings for the analysis GLP-1 analogs on <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/404"><strong>TSQ Altis Plus MS</strong></a>.&nbsp;</p><h5><strong>Data Analysis&nbsp;</strong></h5><p><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/74"><strong>Thermo Scientific Enterprise </strong></a>compliance ready Chromeleon CDS 7.3.2 software was used for all instrument control, data acquisition, processing, and reporting.</p><h4><strong>Conclusions&nbsp;</strong></h4><p>We demonstrated a comprehensive LCMS solution for highly sensitive quantitation of GLP-1 analogs in human plasma using the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/744"><strong>Vanquish UHPLC</strong></a> coupled to TSQ Altis Plus MS. The solution enables:&nbsp;</p><ul><li>Robust quantitation of semaglutide and liraglutide with a LOQ of 0.1 ng/mL&nbsp;</li><li>Excellent precision and accuracy across 3 orders of LDR for the analysis of GLP-1 analogs&nbsp;</li><li>Minimal carryover observied with post-injection signals remaining below 10% of the LOQ for semaglutide and below 14% for liraglutide following the highest concentration injection (100 ng/mL)</li></ul><h3><strong>4. Waters Corporation: Method Migration of a Reversed Phase TFAAcetonitrile Gradient Method From a Binary to a Quaternary System: Impact of Mixing</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720008689_en_51b4c42ae8.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Solvent mixing is critical to obtaining optimal gradient LC separations. The most commonly available pump mixing designs for reversed phase gradient HPLC separations are high pressure (typically binary) and low pressure (quaternary) systems. High pressure systems use independent pumps to deliver different solvents. Mixing occurs at high pressures with solvent composition controlled by the flow rates of the pump heads. In low pressure systems, solvent composition is controlled by a gradient proportioning valve. Each solvent is delivered in packets (based on the gradient specified in the method) which are mixed as they go through the pump head. High pressure systems have reduced dwell volumes and generally have improved compositional accuracy (leading to better retention time precision) over low pressure systems. Both high- and low-pressure systems are subject to mobile phase composition fluctuations. Mixers of various volumes and designs are utilized to help minimize these composition fluctuations by reducing baseline noise and oscillations in mobile phase composition.&nbsp;</p><p>The use of certain mobile phase modifiers is known to impact chromatographic baselines. Trifluoroacetic acid (TFA) is a commonly used ion-pairing reagent used in combination with acetonitrile for many gradient reversed phase applications. TFA absorbs strongly at wavelengths below 250 nm. Additionally, TFA is slightly retained on reversed phase columns which results in fluctuations in TFA concentration as the acetonitrile gradient passes through the column. In combination, these factors result in significant baseline disturbances (ripples) when TFAacetonitrile gradients are used at low wavelengths. These baseline ripples may make peak integration difficult and can impact the sensitivity along with peak area precision and retention time precision of the method.&nbsp;</p><p>Most modern HPLC systems typically include a standard mixer in the pump design. Additionally, different mixers may be available to improve mixing performance for specific applications. The effect that mixers have on chromatographic performance was demonstrated during the migration of a reversed phase TFA-acetonitrile gradient method from a binary system to the quaternary Alliance iS HPLC System.</p><h4><strong>Experimental</strong></h4><ul><li><strong>LC systems: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/717"><strong>Alliance iS HPLC System</strong></a> Comparable Binary HPLC system (System X)&nbsp;</li><li><strong>Detection: </strong>UV Detector with 10 mm HPLC flow cell</li><li><strong>Column: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1823"><strong>XBridge™ C18 Column</strong></a>, 5 µm 4.6 x 250 mm (p/n: 186003117)</li></ul><h5><strong>Data Management&nbsp;</strong></h5><ul><li><strong>Chromatography data system: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/627"><strong>Empower™ 3.8.0.1</strong></a></li></ul><h4><strong>Conclusion&nbsp;</strong></h4><p>The differences in mixing performance between binary and quaternary HPLC Systems may impact method migration. Mixing performance is particularly important when migrating gradient TFA-acetonitrile methods at low wavelengths, as these methods are known to produce significant baseline noise (ripple). In this study, effect of mixers on chromatographic performance was demonstrated through the migration of the USP Tryptophan Organic Impurities method from a binary system to the quaternary Alliance iS HPLC System. The results show that the characteristic baseline ripple associated with TFA-Acetonitrile gradients at low wavelengths is impacted by the choice of mixer. The Alliance iS HPLC System with the 690 µL Ti diffusion bonded mixer displayed a significant reduction of baseline noise when compared against both the binary and quaternary systems equipped with their respective standard mixers. This resulted in easier peak integration, improved peak area and retention time reproducibility, and increased method sensitivity.</p>

News from LabRulezLCMS Library - Week 38, 2025

<p><strong>In </strong><a target="_blank" rel="noopener noreferrer" href="https://www.organomation.com/lipidomics-sample-preparation"><strong>lipidomics research</strong></a><strong>, the quality and reproducibility of results depends heavily on diligent sample preparation. The </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=55&amp;search=Lipidyzer"><strong>SCIEX Lipidyzer platform</strong></a><strong>, a novel solution for quantitative lipid analysis, is an instrument that can perform sample preparation, automated workflow procedures, and streamlined data processing [1, 2]. It can be used in a variety of different lipid based applications with various biological matrices [1]. This platform requires </strong><a target="_blank" rel="noopener noreferrer" href="https://www.organomation.com/what-is-solvent-removal"><strong>efficient and gentle solvent removal</strong></a><strong> to ensure accurate measurements of lipid species [1-3]. This makes nitrogen blowdown evaporation a critical step in this workflow and an indispensable tool for Lipidyzer users.</strong></p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Organomation_How_Nitrogen_Blowdown_Supports_the_SCIEX_Lipidyzer_Platform_Enhancing_Lipidomics_Sample_Preparation_992c70d8f1.jpg" alt="Organomation: How Nitrogen Blowdown Supports the SCIEX Lipidyzer Platform - Enhancing Lipidomics Sample Preparation" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Organomation_How_Nitrogen_Blowdown_Supports_the_SCIEX_Lipidyzer_Platform_Enhancing_Lipidomics_Sample_Preparation_992c70d8f1.jpg 245w," sizes="100vw" width="1080" height="603"></p><h4><strong>Why Nitrogen Blowdown?</strong></h4><p>During lipid extraction and sample cleanup, samples are typically dissolved in organic solvents such as methanol or chloroform. Before <a target="_blank" rel="noopener noreferrer" href="https://www.organomation.com/preparing-samples-for-lc-ms/ms-analysis"><strong>LC-MS/MS analysis</strong></a> with the Lipidyzer, these solvents must be removed to prevent analytical interference, concentrate the desired analytes, and protect the instruments performance integrity [1]. Multiple SCIEX protocols recommend <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/5392"><strong>evaporating this solvent under a gentle stream of nitrogen</strong></a> during the extraction phase [3-6].</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Organomation_Close_up_tubes_in_N_EVAP_2_ac11c24644.jpg" alt="Organomation: Close up tubes in N-EVAP-2" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Organomation_Close_up_tubes_in_N_EVAP_2_ac11c24644.jpg 245w," sizes="100vw" width="615" height="344"></p><h4><strong>SCIEX Lipidyzer Protocols</strong></h4><p>Multiple SCIEX targeted lipidomics protocols emphasize the importance of the nitrogen evaporation step in sample preparation. For example, after lipid extraction from plasma or tissue, the instructions state “Evaporate solvent under a stream of nitrogen”, followed by resuspension of target lipids in the injection solution [5, 6]. For a more specialized lipid mediator analysis, the protocol specifies: “<i><strong>Evaporate sample completely under a gentle stream of nitrogen. The samples can be heated (but do not exceed 37°C) during this step to speed up the evaporation process. The intensity of the nitrogen stream can be increased as the sample volume decreases. Make sure sample liquid droplets do not get dispersed from the bottom of the tube.</strong></i>” [4]. This careful approach ensures that even volatile lipid species are preserved, and that the final sample is free of residual solvent before instrumental analysis.</p><h4><strong>Key reasons for using nitrogen blowdown:</strong></h4><ul><li><strong>Gentle Evaporation: </strong>Nitrogen blowdown is a non-destructive process that can be performed without heat, preserving sensitive lipid species prone to degradation. This is crucial for accurate quantitation [7].</li><li><strong>Efficient Solvent Removal: </strong>A steady stream of nitrogen gas accelerates evaporation by displacing solvent vapors, resulting in faster and more uniform drying compared to passive evaporation methods [7].</li><li><strong>Minimized Contamination: </strong>Nitrogen blowdown reduces the risk of sample loss or cross-contamination, which can occur with alternative drying methods.</li><li><strong>Reproducibility: </strong>Various Organomation products are designed to ensure consistent drying across samples, enabling repeatable results, which is essential for high-throughput lipidomics [7].</li></ul><h4><strong>Organomation's Advantage</strong></h4><p>Organomation’s nitrogen evaporators, including the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=3&amp;manufacturerItems=540&amp;search=MICROVAP"><strong>MICROVAP</strong></a> and <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=3&amp;manufacturerItems=540&amp;search=N-EVAP"><strong>N-EVAP</strong></a> lines, are designed specifically for these kinds of workflows. They offer the following advantages:</p><ul><li><strong>Uniform nitrogen distribution</strong> across multiple samples for parallel processing.</li><li><strong>Precise temperature control </strong>to gently accelerate evaporation without risking analyte degradation.</li><li><strong>Flexible configurations </strong>for tubes, vials, or microplates to match your Lipidyzer sample formatting.</li><li><strong>Efficient, hands-free operation</strong> to boost throughput and free up valuable laboratory work time.</li></ul><p>With over 65 years of experience, Organomation’s evaporators are trusted by laboratories worldwide to deliver consistent, high-quality results [8].</p><h4><strong>Conclusion</strong></h4><p>Whether you're conducting academic research, running commercial tests, or working in a clinical lab, <a target="_blank" rel="noopener noreferrer" href="https://blog.organomation.com/blog/nitrogen-blowdown-evaporator-buyers-guide"><strong>selecting the right nitrogen blowdown evaporator</strong></a> can have a significant impact on your workflow efficiency and data quality. For users of the SCIEX Lipidyzer platform, these evaporators aren’t just helpful—they're essential. They enable:</p><ul><li>Complete and gentle solvent evaporation</li><li>Protection of delicate lipid species</li><li>Consistent, high-quality data</li><li>Fast, high-throughput sample prep</li></ul><p>Looking to get more out of your lipidomics workflow? See how pairing your SCIEX Lipidyzer system with an <a target="_blank" rel="noopener noreferrer" href="https://www.organomation.com/products/nitrogen-evaporators"><strong>Organomation nitrogen evaporator</strong></a> can improve sample preparation and deliver more accurate, reproducible results.</p><blockquote><p>Find the best nitrogen evaporator model for your needs with our <a target="_blank" rel="noopener noreferrer" href="https://www.organomation.com/support/contact-help-support/sample-evaporator-product-finder"><strong>evaporator product finder tool</strong></a>!</p></blockquote>

How Nitrogen Blowdown Supports the SCIEX Lipidyzer Platform: Enhancing Lipidomics Sample Preparation

<p><strong>With short videos on Instagram, they open the doors to the world of the laboratory, where they are looking for ways to clean water from persistent chemicals. Pavlína Konopáčová and David Mareš have already reached thousands of followers, and in October they will present their experiences at the </strong><a target="_blank" rel="noopener noreferrer" href="https://cxi.tul.cz/kalendar-akci/424/"><strong>Sdílej vědu!</strong></a><strong> event.</strong></p><p>Michal Řezanka, head of the nanochemistry laboratory, came up with the idea that Instagram is popular, so why not set up an account for the laboratory as well. And because Pavla and David were familiar with this social network, it went quickly. The original idea of using Instagram to find industrial partners proved to be a dead end. "<i>We realised that this was not the way forward. So we changed our strategy and are currently building a community of followers to raise awareness about us,</i>" they explain.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/CXI_TUL_Veda_je_super_a_ukazat_ji_ostatnim_je_satisfakce_rikaji_chemici_z_CXI_TUL_56e1d28ba5.jpg" alt="CXI TUL: Science is great, sharing it is rewarding, say CXI TUL chemists" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_CXI_TUL_Veda_je_super_a_ukazat_ji_ostatnim_je_satisfakce_rikaji_chemici_z_CXI_TUL_56e1d28ba5.jpg 209w," sizes="100vw" width="1205" height="900"></p><h4><strong>Anyone can start, the main thing is to persevere</strong></h4><p>And what are the biggest challenges in popularising science? "<i><strong>Getting started,</strong></i>" David says promptly. "<i>And anyone can get started. The most important thing is not to get discouraged when it doesn't work right away. It takes patience and finding your own style and doing it because you enjoy it, not out of obligation,</i>" adds Pavlína.</p><p>They do everything themselves in the videos. "<i>I can make a short clip in five minutes, but I can easily spend hours on a longer one. For me, the biggest problem isn't editing, but finding the right music,</i>" laughs David, who taught himself everything with the help of self-study guides.</p><h4><strong>Classmates are supportive, but they started from scratch</strong></h4><p>Their classmates, who are the same age, are enthusiastic about the profile. Outside the laboratory, however, no one really cares. "<i>No one shared our videos, we started from scratch. Fortunately, we have a lot of support from our supervisor, who sometimes gets involved himself.</i>"</p><p>"<i>It helps us a lot when one of our colleagues comes up with an idea or helps with filming. We have to stick to our posting schedule, and that's not always easy,</i>" explain the young chemists. Their fans come mostly from the USA, Asia and, of course, from Czechia.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/CXI_TUL_Veda_je_super_a_ukazat_ji_ostatnim_je_satisfakce_rikaji_chemici_z_CXI_TUL_5_058d099fb9.jpg" alt="CXI TUL: Science is great, sharing it is rewarding, say CXI TUL chemists" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_CXI_TUL_Veda_je_super_a_ukazat_ji_ostatnim_je_satisfakce_rikaji_chemici_z_CXI_TUL_5_058d099fb9.jpg 208w," sizes="100vw" width="1200" height="900"></p><h4><strong>Rick and Morty in the lab</strong></h4><p>In their free time, they watch videos on Instagram. The algorithm serves them a lot of scientific content, but they also draw inspiration from non-scientific clips, which they then set in a laboratory environment. "<i><strong>I like Chemistry Daily, for example,</strong></i>" says Pavlína. David also finds inspiration outside of Instagram and is a big fan of the series Rick and Morty. "<i>Our humour is a bit harsher, so we don't take offence and we feel free to tease each other. And we usually agree on ideas,</i>" he adds.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/CXI_TUL_Veda_je_super_a_ukazat_ji_ostatnim_je_satisfakce_rikaji_chemici_z_CXI_TUL_4_8fb3beae61.jpg" alt="CXI TUL: Science is great, sharing it is rewarding, say CXI TUL chemists" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_CXI_TUL_Veda_je_super_a_ukazat_ji_ostatnim_je_satisfakce_rikaji_chemici_z_CXI_TUL_4_8fb3beae61.jpg 208w," sizes="100vw" width="1200" height="900"></p><h4><strong>Science in the background: the fight against persistent chemicals</strong></h4><p>But behind the attractive videos lies a serious topic: research into how to remove perfluorinated substances (<strong>PFAS</strong>) from water. These substances, used in Teflon, outdoor clothing and firefighting foams, are dangerous because they do not decompose in nature and therefore accumulate in living organisms. They have been found in the blood of humans and animals across the planet, and even in Antarctica.</p><p>Their risks began to be highlighted thanks to the DuPont case in West Virginia, USA. A farmer was dealing with the prolonged death of cows, but it gradually became apparent that the factory had been polluting the surrounding environment for a long time with one of the many forms of PFAS used in the production of Teflon. Further revelations showed that the substance threatens unborn foetuses and is linked to certain types of cancer, thyroid disease and high cholesterol levels. It was this scandal that sparked a global debate and the gradual regulation of forever chemicals.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/CXI_TUL_Veda_je_super_a_ukazat_ji_ostatnim_je_satisfakce_rikaji_chemici_z_CXI_TUL_2_9061b2b985.jpg" alt="CXI TUL: Science is great, sharing it is rewarding, say CXI TUL chemists" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_CXI_TUL_Veda_je_super_a_ukazat_ji_ostatnim_je_satisfakce_rikaji_chemici_z_CXI_TUL_2_9061b2b985.jpg 208w," sizes="100vw" width="1200" height="900"></p><h4><strong>Cyclodextrins: a weapon against PFAS</strong></h4><p>Research at CXI TUL is trying to find a solution to the global PFAS problem. The team has already shown that the key is cyclodextrins, which are ring-shaped cyclic sugars that can capture PFAS molecules in their cavity and thus filter them out of water.</p><p>Surprisingly, cyclodextrins are more effective than commonly used activated carbon. And they have another advantage: they are cheap because they are produced by breaking down starch, so they are actually a special form of sugar.</p><p>"<i>So far, we are binding them into insoluble polymers that can capture these compounds in water,</i>" the researchers explain. "<i>In the future, we want to bind them to magnetic nanoparticles so that we can easily separate them from the water using a magnet</i>."</p><h4><strong>Connection between the Czech Republic and the USA</strong></h4><p>The research is supported by several projects. Pavlína mentions, for example, a project by the Ministry of Education, Youth and Sports, in which they are collaborating with the University of Rhode Island, where Pavlína completed an internship. In the USA, the PFAS problem is even more pressing, as drinking water is often contaminated above the permitted limits. "<i>It was beneficial to see how they do sorption experiments there, and also to go out into the field and take samples,</i>" she says.</p><p>The situation in the Czech Republic is good, although PFAS concentrations are higher in the Jizera Mountains due to the use of cross-country skiing waxes, and contamination also occurs at airports, for example, due to firefighting foams.</p><h4><strong>Above all, more information</strong></h4><p>The Instagram community, positive reactions from followers, the opportunity to speak at conferences and be inspired by others – Pavlína and David see all this as a bonus. But the main thing for them is the research itself. And if their videos spark an interest in chemistry and bring young people into the laboratory, then their work has meaning not only in the laboratory, but also outside it.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/CXI_TUL_Veda_je_super_a_ukazat_ji_ostatnim_je_satisfakce_rikaji_chemici_z_CXI_TUL_3_9bb293a651.jpg" alt="CXI TUL: Science is great, sharing it is rewarding, say CXI TUL chemists" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_CXI_TUL_Veda_je_super_a_ukazat_ji_ostatnim_je_satisfakce_rikaji_chemici_z_CXI_TUL_3_9bb293a651.jpg 208w," sizes="100vw" width="1200" height="900"></p><p><strong>Pavlína Konopáčová </strong>also did well in the Falling Walls Lab competition at the beginning of this week, placing third in the Czech finals of this prestigious competition.</p>

Science is great, sharing it is rewarding, say CXI TUL chemists

<p><strong>In the week of September 22, 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>5 000 GC, LC, MS and spectroscopy webinars</strong>.</p></blockquote><h5><strong>1. Thermo Fisher Scientific: Optimizing N-Nitrosamine Detection: Automated Microextraction &amp; Machine Learning</strong></h5><ul><li>Tu, 23.9.2025 12:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5520">Registration</a></li></ul><p>This webinar will showcase new advancements in the analysis of N-nitrosamines in pharmaceutical products.</p><h5><strong>2. SelectScience: SelectScience Analytical Science Summit</strong></h5><ul><li>Tu, 23.9.2025 13:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5411">Registration</a></li></ul><p>Join the 5th SelectScience® Virtual Analytical Science Summit to explore the latest analytical chemistry innovations, trends, and solutions for global challenges.</p><h5><strong>3. Thermo Fisher Scientific: Fresh solutions to elucidate the structure-function relationship of macromolecules</strong></h5><ul><li>Tu, 23.9.2025 14:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5499">Registration</a></li></ul><p>Discover how Direct Mass, HDX, and cross-linking MS enhance structural biology, offering dynamic insights into protein and RNA structure, function, and interactions.</p><h5><strong>4. Waters Corporation: Efficient and Systematic Troubleshooting of Forensic and Clinical LC-MS Applications</strong></h5><ul><li>Tu, 23.9.2025 16:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5504">Registration</a></li></ul><p>Master LC-MS troubleshooting for forensic and clinical workflows. Learn to identify root causes, resolve issues efficiently, and improve assay reliability with expert strategies and case studies.</p><h5><strong>5. Bruker: Live from the Lab: Spatial proteomics in action</strong></h5><ul><li>Tu, 23.9.2025 16:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5464">Registration</a></li></ul><p>Learn how cutting-edge spatial proteomics workflows integrate imaging, LMD, and LC-MS to achieve high-resolution, scalable analysis of 100 proteomes a day.</p><h5><strong>6. Thermo Fisher Scientific: 20 Years of HPLC-CAD: Anniversary and Latest Innovations</strong></h5><ul><li>We, 24.9.2025 10:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5506">Registration</a></li></ul><p>Celebrate 20 years of HPLC-CAD in this webinar. Learn how the new Vanquish CAD P boosts sensitivity, accuracy, and usability for universal detection in pharma, biopharma, and beyond.</p><h5><strong>7. New Food magazine: Spectroscopic Analysis of Food Dyes: Ensuring Colour Consistency</strong></h5><ul><li>We, 24.9.2025 16:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5374">Registration</a></li></ul><p>In this webinar we will demonstrate the ability to assess a material’s colour using UV-Visible instrumentation.</p><h5><strong>8. Waters Corporation: Managing Method Variability: A Foundation for Risk-Based Change - Session 2</strong></h5><ul><li>We, 24.9.2025 17:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5368">Registration</a></li></ul><p>Explore tools like control charts, risk assessments, and AI-driven monitoring to manage method variability and support post-approval changes in pharmaceutical analysis.</p><h5><strong>9. Waters Corporation: Analysis of Ebola Virus Glycoproteins in Complex with Antibodies using Light Scattering</strong></h5><ul><li>We, 24.9.2025 17:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5355">Registration</a></li></ul><p>Learn how SEC-MALS and CG-MALS reveal glycoprotein mass, oligomerization, and antibody binding affinity—featuring Ebola and Lassa virus immune complex studies and cryoEM validation.</p><h5><strong>10. Thermo Fisher Scientific: Reporting in Chromeleon Software</strong></h5><ul><li>Th, 25.9.2025 11:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5507">Registration</a></li></ul><p>Learn step-by-step how to build detailed Chromeleon 7.3.2 CDS reports. Discover formulas, custom variables, and best practices to streamline data analysis and reporting.</p><h5><strong>11. Phenomenex: 3rd Annual Virtual Biosymposium</strong></h5><ul><li>Th, 25.9.2025 11:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5373">Registration</a></li></ul><p>Join experts from Roche and Phenomenex to explore novel LC-based strategies for characterizing mRNA, oligonucleotides, ADCs, and other biotherapeutics in this virtual biosymposium.</p><h5><strong>12. Waters Corporation: Becoming a SEC-MALS &amp; ASTRA Expert: Part 7</strong></h5><ul><li>Th, 25.9.2025 11:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5327">Registration</a></li></ul><p>Confused by formalisms? This session explains when and why to use Zimm, Mie, Berry, or Debye models for accurate MALS data interpretation.</p><h5><strong>13. Thermo Fisher Scientific: Workflows to bridge that gap between discovery and clinical research</strong></h5><ul><li>Th, 25.9.2025 14:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5500">Registration</a></li></ul><p>Discover plasma proteomics workflows for oncology in lung and ovarian cancer. Learn how to identify biomarkers, study resistance, and bridge discovery with clinical research.</p><h5><strong>14. Evosep Biosystems: Targeted Workflows</strong></h5><ul><li>Th, 25.9.2025 16:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5445">Registration</a></li></ul><p>Explore next-gen targeted proteomic LC-MS workflows. Gain unmatched sensitivity, reproducibility, and throughput for confident protein quantification and validation.</p><h5><strong>15. Metrohm: Next-generation NIR spectroscopy and how to use it for high-throughput food testing</strong></h5><ul><li>Th, 25.9.2025 16:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5444">Registration</a></li></ul><p>Learn how NIR spectroscopy accelerates food testing. Discover moisture, fat, and protein analysis with Metrohm’s OMNIS NIR Analyzer for faster, greener QC.</p><h5><strong>16. Technology Networks: Tech Spotlight: Lab of the Future 2025</strong></h5><ul><li>Th, 25.9.2025 17:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5446">Registration</a></li></ul><p>Explore how AI, automation, robotics, and smart lab informatics are shaping the Lab of the Future, driving research efficiency, collaboration, and data integrity.</p><h5><strong>17. Agilent Technologies: UV-VIS-NIR in Battery Development</strong></h5><ul><li>Th, 25.9.2025 20:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/3909">Registration</a></li></ul><p>Agilent's portfolio of instruments offers the high performance and flexible accessories needed for pioneering new applications.</p><h5><strong>18. Waters Corporation: Becoming an ASTRA Expert: Part 6</strong></h5><ul><li>Fr, 26.9.2025 20:00 CEST</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5331">Registration</a></li></ul><p>Learn essential method setup steps in ASTRA: normalization, alignment, and band broadening corrections for optimal accuracy in your light scattering experiments.</p>

Webinars LabRulezLCMS Week 39/2025

Ion-Pairing Hydrophilic Interaction Chromatography for Impurity Profiling of Therapeutic Phosphorothioated Oligonucleotides

<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 22nd September 2025? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!</strong></p><blockquote><p><span style="font-size:20px;">👉 </span><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library"><span style="font-size:20px;"><strong>SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES</strong></span></a></p></blockquote><blockquote><p><strong>👉 Need info about different analytical techniques?</strong> Peek into <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library"><strong>LabRulezGCMS</strong></a> or <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library"><strong>LabRulezICPMS</strong></a> libraries.</p></blockquote><p>This week we bring you application notes by <strong>Agilent Technologies, Shimadzu </strong>and <strong>Waters Corporation </strong>and<strong> </strong>poster by <strong>Thermo Fisher Scientific / HPLC Symposium!</strong></p><h3><strong>1. Agilent Technologies: Quantitation of N-Nitroso Nebivolol in Nebivolol Tablet Formulation</strong></h3><p><strong>Using an </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=6495D+triple+quadrupole"><strong>Agilent 6495D triple quadrupole LC/MS system</strong></a></p><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_nebivolol_tablet_formulation_6495_qqq_lc_ms_system_5994_8053en_agilent_e7f21eb12f.pdf"><strong>Full PDF for download</strong></a></li></ul><p>N-Nitroso Nebivolol is a NDSRI of Nebivolol, a beta-1 adrenergic receptor antagonist commonly used in the treatment of hypertension and heart failure. In the carcinogenic potency categorization published by the FDA, N-Nitroso Nebivolol has been categorized as Class 4, indicating the maximum allowable intake limit for the impurity to be 1,500 ng/day. Nebivolol has maximum daily dosage limit of 40&nbsp;mg for adults.</p><p>Considering a maximum daily dose of 40&nbsp;mg and allowable intake of 1,500&nbsp;ng/day for the nitrosamine impurity in the formulation under study, the specification limit of N-Nitroso Nebivolol has been calculated as 22.5 ng/mL for the 0.6 mg/mL test concentration used during this study.&nbsp;</p><p>This application note demonstrates a highly specific, sensitive, and reproducible method for the quantitation of N-Nitroso Nebivolol in a 20 mg Nebivolol tablet formulation. The method uses a 1290 Infinity II LC coupled to a 6495D triple quadrupole LC/MS/MS. The multiple reaction monitoring (MRM) conditions and ion source parameters were automatically optimized using the optimizer tool available with MassHunter Acquisition software to maximize the sensitivity of the target analyte. The API peaks were diverted to waste with the help of an integrated, software-controlled diverter valve during quantitation of the&nbsp;impurity.</p><h4><strong>Experimental</strong></h4><h5><strong>Equipment&nbsp;</strong></h5><p>The analysis was performed on a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=1290+Infinity+II+LC"><strong>1290 Infinity II LC system</strong></a> coupled to a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=6495D+triple+quadrupole"><strong>6495D triple quadrupole LC/MS/ MS</strong></a>. The LC system was equipped with the following&nbsp;modules: – Agilent 1290 Infinity II high-speed pump (G7120A) – Agilent 1290 Infinity II multisampler (G7167B) – Agilent 1290 Infinity II multicolumn thermostat (G7116B) – Agilent 1290 Infinity II diode array detector (G7117A)</p><h4><strong>Conclusion&nbsp;</strong></h4><p>A highly sensitive and robust MRM method was developed for the quantitation of N-Nitroso Nebivolol in 20&nbsp;mg Nebivolol tablet formulation using an Agilent 6495D triple quadrupole LC/MS system. The chromatographic method developed provided good separation between the analyte and the API. The method showed LOD and LOQ of 2 and 5 pg/mL, respectively. The minimum signal-to-noise ratio at the LOQ level was found to be more than 50:1 (Peak to Peak). The calibration curve from 5 pg/mL to 50&nbsp;ng/mL was found to be linear, with 1/x weighting. R and R2 values were 0.999 and 0.997, respectively. Spike recovery analysis showed the efficiency of sample extraction with a recovery percentage of 116.8% in API, 108.3% in placebo, and 111.8% in tablet formulation at the spiking level of 200 pg/mL. The method was found to be highly reproducible at the LOQ level, while the %RSD value for the area response of seven replicate injections was approximately 3.1%.</p><h3><strong>2. Shimadzu: Efficient Method Development for Synthetic Peptide and Related Impurities</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_01_00780_en_4d0aa014ea.pdf"><strong>Full PDF for download</strong></a></li></ul><h5><strong>User Benefits</strong></h5><ul><li>LabSolutions MD improves the efficiency of the entire workflow for method development of synthetic peptide and impurities.&nbsp;</li><li>Screening for multiple mobile phases and columns can be automated using mobile phase and column switching valves.&nbsp;</li><li>Molecular weights of peptides and related impurities can be estimated and accurately tracked with LCMS-2050, a single quadrupole mass spectrometer.</li></ul><p>Peptide therapeutics, characterized by specific amino acid sequences crucial to their function, can be synthesized chemically, similar to small molecule drugs. The production of synthetic peptides involves multiple steps, including deprotection, activation, coupling, and the cleavage of the final sequence from the solid support. Impurities, such as those resulting from premature chain termination or missing amino acids, can affect the safety and efficacy of the final product. Therefore, these impurities must be separated by liquid chromatography (LC). In LC analysis, selecting the appropriate mobile phase and column across a wide range of combinations is critical for achieving optimal separation, as it significantly impacts the separation. However, since the separation pattern varies depending on the peptide chain lengths, amino acid compositions, and presence of modifications, optimizing separation for each peptide sequence is time-intensive. This study describes how to efficiently find the best separation conditions for peptides and related impurities utilizing LabSolutions MD, a dedicated software for supporting method development, through both screening and optimization phases.</p><h4><strong>Experimental</strong></h4><ul><li><strong>LC System: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=Nexera+X3"><strong>Nexera X3</strong></a> (Method Scouting System)</li><li><strong>MS System: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=LCMS-2050"><strong>LCMS-2050</strong></a></li></ul><h4><strong>Automated Peak Tracking by LCMS-2050&nbsp;</strong></h4><p>LC chromatograms obtained with a gradient time of 5 min, flow rate of 0.6 mL/min, and initial concentration of 5 % and 15 %, along with molecular weights for FLP and impurities, are shown in Fig. 18. The UV spectra for each impurity are displayed in Fig. 19. The similarity between UV spectra of Met(O2), p.A1del, p.A1_E2del, p.A1_K3del, and p.A1_D5del is greater than 0.99, suggesting that peak tracking based on UV spectra would be challenging. In contrast, LabSolutions MD enables peak tracking based on molecular weights acquired by LCMS-2050, facilitating accurate identification of compounds with similar UV spectra (Fig. 18). The estimated molecular weights for each compound show small mass errors compared to the theoretical values (Table 3), which can be used to confirm the molecular weights of known compounds as well as to approximate the molecular weights of unknown impurities.</p><h4><strong>Conclusion</strong></h4><p>The separation patterns of synthetic peptides vary depending on mobile phase composition, column type, and various LC parameters, such as gradient conditions, column oven temperature, and flow rate. Separation behavior can also differ based on peptide structure, including length, amino acid composition, and the presence of modifications. Therefore, it is necessary to optimize the separation for each peptide sequence individually. However, optimizing analytical conditions through numerous analyses and data processing can be time-consuming. LabSolutions MD automates the entire workflow, including generating analysis schedules, preparing mobile phases, and processing the data, thanks to functionalities such as automated peak tracking, ranking of chromatograms, and design space visualization. This article introduced a case where the optimal separation conditions for synthetic peptides were efficiently identified through screening and optimization phases. LabSolutions MD also offers fully automated gradient optimization by AI algorithm to meet user-defined criteria. For more details, please refer to the application news, “Automatic Optimization of Gradient Conditions by AI Algorithm on Synthetic Peptide and Impurities: 01-00814”.</p><h3><strong>3. Thermo Fisher Scientific / HPLC Symposium: Sensitive cationic lipids impurities analysis with quantitation by charged aerosol detection and simultaneous mass confirmation by MS</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_sensitive_cationic_lipids_impurities_analysis_quantitation_po_en_3dacf61554.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Cationic lipid is a critical component in lipid nanoparticle formulations for delivering nucleic acids such as mRNA, siRNA, antisense oligonucleotides etc. [2] Cationic lipids are amphiphilic molecules that possess a hydrophilic region, a hydrophobic region, and a linker structure connecting the two. [1] Charged aerosol detector (CAD) and evaporative light scattering detectors (ELSD) are preferred for characterizing lipid nanoparticle components, with CAD being the better choice for impurity quantification due to its higher sensitivity [3]. In a previous study, an inverse gradient method was developed to quantify impurities in cationic lipids raw materials.[1] To demonstrate the inverse gradient method on CAD HP, the same cationic lipids -- R-DOTAP, DLin-KC2- DMA and ALC-0315 -- were measured to this experiment as in Application note 003384 [1]. New diverter valve and software features were showcased, along with method’s sensitivity and repeatability.</p><h4><strong>Materials and methods</strong></h4><p>Using the inverse gradient method from Application note 003384 [1], to demonstrate impurity analysis in cationic lipids on the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2176"><strong>Thermo Scientific Vanquish Charged Aerosol Detector HP (CAD HP)</strong></a> integrated in the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/743"><strong>Thermo Scientific Vanquish Flex Inverse Gradient UHPLC System</strong></a>, with parallel mass confirmation by the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/413"><strong>Thermo Scientific ISQ EM Single Quadrupole Mass Spectrometer</strong></a>.&nbsp;</p><p><strong>Methods:</strong> The workflow uses a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/743"><strong>Vanquish Flex Inverse Gradient LC system</strong></a> with Vanquish CAD HP and <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1970"><strong>Thermo Scientific Hypersil GOLD C8 column</strong></a>.</p><h5><strong>Data Analysis&nbsp;</strong></h5><p><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/76"><strong>Thermo Scientific Chromeleon Chromatography Data System (CDS)</strong></a> 7.2.10 was used for data acquisition and analysis.</p><h4><strong>Conclusions&nbsp;</strong></h4><p>The CAD HP integrates perfectly into the Vanquish Inverse Gradient LC system and offers high sensitivity and excellent repeatability for quantitative cationic lipid impurity analysis, while the ISQ EM MS provides straightforward mass confirmation with high repeatability, all under compliance-ready conditions.</p><h3><strong>4. Waters Corporation: Quantitative Analysis of Four Long-Chain Lysophosphatidylcholines (LPCs) in Dried Blood Spot using Liquid Chromatography Tandem Mass Spectrometry for Clinical Research</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720008826_en_dc154872b6.pdf"><strong>Full PDF for download</strong></a></li></ul><h5><strong>Benefits</strong></h5><ul><li>Good chromatographic resolution of all forms</li><li>Short extraction time compared to traditional methods for lipid extraction</li><li>Short analysis injection-to-injection 7 minutes</li></ul><p>Historically, very long chain fatty acids have been quantified using direct transesterification and gas chromatography methods which involve time-consuming and labor-intensive sample preparation and analysis. Consequently, simpler LC-MS/MS methods have been reported for the measurement of long-chain LPCs (LPCs generic chemical structure, Figure 1) in DBS. Interferences have been reported using positive ESI, thus researchers have developed methodologies utilizing negative ESI to circumvent isobaric interferences when there is lack of chromatographic resolution. Here a UHPLC-MS/MS method is described using acetate adducts for the reliable analysis of four long-chain LPCs in DBS for clinical research.&nbsp;</p><p>A UHPLC-MS/MS method for quantifying a panel of LPCs (C20:0, C22:0, C24:0, and C26:0-LPCs) was developed. The mobile phase composition consisted of a mixture of water, acetonitrile, and isopropanol with 5 mM ammonium acetate as a modifier. The DBS sample extraction used a working solution prepared with the deuterated LPC internal standards in methanol. The method was developed with an ACQUITY UPLC I-Class PLUS System (Fixed Loop) using an ACQUITY Premier CSH C18 Column, which provided baseline resolution of endogenous isobaric interferences for the saturated forms of the targeted LPCs. The detector was a Xevo TQ-S micro Mass Spectrometer operated in negative ESI mode. This methodology resulted in a total analysis time (injection-to-injection) of 7 minutes.</p><h5><strong>LC Conditions&nbsp;</strong></h5><ul><li><strong>LC system: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/199"><strong>ACQUITY UPLC I-Class PLUS FL&nbsp;</strong></a></li><li><strong>System Detection: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/209"><strong>Xevo TQ-S micro Mass Spectrometer&nbsp;</strong></a></li><li><strong>Plate:</strong> Waters 96-well Sample Collection Plate, 350 µL&nbsp;</li><li><strong>Column(s): </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1952"><strong>ACQUITY Premier CSH C18 Column</strong></a>, 2.1 mm x 50 mm, 1.7 µm (p/n: 186009460)</li></ul><h5><strong>MS Conditions&nbsp;</strong></h5><ul><li><strong>MS system:</strong> Xevo TQ-S micro Mass Spectrometer</li></ul><h5><strong>Data Management&nbsp;</strong></h5><ul><li><strong>MS software: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/635"><strong>MassLynx™ v4.2 Software SCN 1045&nbsp;</strong></a></li><li><strong>Informatics: </strong>TargetLynx™ XS v4.2 Software SCN1045 and QUAN Review</li></ul><h4><strong>Results and Discussion&nbsp;</strong></h4><p>During method development, different types of column chemistry, mobile phase composition, and ESI polarity modes were assessed to optimize chromatographic peak shape, analytical sensitivity and manage interferences. In positive mode, ammonium formate favored the formation of m/z 184 and m/z 104 as daughter ions for all the LPCs. The m/z 184 ion lacks specificity because most phospholipids share this moiety, so the ion m/z 104 was selected as quantifier. Having evaluated conditions in positive mode for the analysis of the four saturated LPCs, it was observed that some existing interferences were not only affecting the parent masses of the analytes but their corresponding internal standard parent ion masses too. Hence, negative ionization using ammonium acetate as modifier was assessed for the detection of LPCs. Acetate adducts [M+CH3COO]- were the most abundant parent ion masses, generated under these conditions for each LPC. The acyl-chain product ions generated in negative ionization reduce the detection of interferences coming from other phosphocholine headgroups. The latter detection conditions were used for the validation of the method presented herein.</p><h4><strong>Conclusion&nbsp;</strong></h4><p>An LC-MS/MS clinical research method for measuring C20:0, C22:0, C24:0, and C26:0 LPCs with the ACQUITY UPLC I-Class PLUS FL System, and the Xevo TQ-S micro Mass Spectrometer that:</p><ul><li>Demonstrates good linearity, with no significant carryover, or matrix effects</li><li>Offers good precision with a total reproducibility and repeatability <span>≤</span>10% CV</li><li>Offers reliability in the identification of LPCs from interfering isobaric compounds Provides good selectivity and analytical sensitivity in negative mode and is relatively fast as approximately 220 samples can be analyzed in 24 hours</li></ul>

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