Impact of mobile and stationary phases on siRNA duplex stability in liquid chromatography

<p><strong>Welcome to the Column Coach, a tool to help you with reversed-phase column selectivity. This tool uses the Neue Test to help compare column hydrophobicity and selectivity, based on a set of standard conditions using the same test mixtures.</strong></p><p><strong>For more details about the Neue Test, please refer to:</strong></p><ul><li><a target="_blank" rel="noopener noreferrer" href="http://onlinelibrary.wiley.com/doi/10.1002/jssc.200390025/full">Neue, U. D.; Van Tran, K.; Iraneta, P. C.; Alden, B. A. Characterization of HPLC Packings. J. Sep. Sci. 2003, 26, 174-186.</a></li><li><a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S0021967399004343">Neue, U. D.; Alden, B. A.; Walter, T. H. Universal Procedure for the Assessment of the Reproducibility and the Classification of Silica-based Reversed-phase Packings II. Classification of Reversed-phase Packings. J. Chromatogr. A. 1999, 849, 101-116.</a></li></ul><h4><strong>How to read the graph?</strong></h4><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Waters_Corporation_Column_Coach_76473457ee.jpg" alt="Waters Corporation: Column Coach" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Waters_Corporation_Column_Coach_76473457ee.jpg 184w," sizes="100vw" width="1000" height="850"></p><p>All trademarks are the property of their respective owners. Specifications were obtained from manufacturer's public literature. Waters does not endorse or represent the accuracy or reliability of any manufacturers' information, and presentation of this information does not imply any relationship between Waters and any manufacturer. Your use of this information is at your sole risk. This tool and its contents are provided and distributed on an 'as is' basis without warranties of any kind either express or implied, including without limitation, implied warranties or merchantability of fitness for a particular purpose.</p><h4><strong>You can search for the right column for your application by:</strong></h4><ul><li><strong>Compound Class</strong> - Want to find a column for a specific class of compounds? Select a compound class from the drop down. Next select a specific sub-compound class and the recommended Waters columns for that given sub-compound class will be provided.</li></ul><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Waters_Corporation_Column_Coach_Search_by_Compound_Class_ac76b423b9.jpg" alt="Waters Corporation: Column Coach - Search by Compound Class" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Waters_Corporation_Column_Coach_Search_by_Compound_Class_ac76b423b9.jpg 245w," sizes="100vw" width="1458" height="666"></p><ul><li><strong>USP Designation</strong> - Our quality mission is to ensure that the Waters' columns you use today are the most reproducible and reliable LC columns available. In this section, you will find all Waters columns that are registered according to classifications prescribed in the United States Pharmacopeia (USP).</li></ul><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Waters_Corporation_Column_Coach_Search_by_USP_Designation_1a8588c591.jpg" alt="Waters Corporation: Column Coach - Search by USP Designation" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Waters_Corporation_Column_Coach_Search_by_USP_Designation_1a8588c591.jpg 190w," sizes="100vw" width="1464" height="1207"></p><ul><li><strong>Manufacturer of your Existing Column</strong></li></ul><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Waters_Corporation_Column_Coach_Search_by_Manufacturer_of_your_Existing_Column_cebd6ae8c0.jpg" alt="Waters Corporation: Column Coach - Search by Manufacturer of your Existing Column" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Waters_Corporation_Column_Coach_Search_by_Manufacturer_of_your_Existing_Column_cebd6ae8c0.jpg 210w," sizes="100vw" width="1446" height="1073"></p><ul><li><strong>Selectivity Chart</strong></li></ul><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Waters_Corporation_Column_Coach_Search_by_Selectivity_Chart_5883ba13ab.jpg" alt="Waters Corporation: Column Coach - Search by Selectivity Chart" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Waters_Corporation_Column_Coach_Search_by_Selectivity_Chart_5883ba13ab.jpg 245w," sizes="100vw" width="1478" height="904"></p>

Waters HPLC Column Coach

Development and thorough evaluation of a multi-omics sample preparation workflow for comprehensive LC-MS/MS-based metabolomics, lipidomics and proteomics datasets

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

News from LabRulezLCMS Library - Week 33, 2025

<p><strong>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>A stability-indicating RP-HPLC method was developed and validated for impurity analysis of the cystic fibrosis drug Elexacaftor using a Quality-by-Design (QbD) approach. The method achieved effective separation of six known impurities with appropriate resolution and peak shape under optimized chromatographic conditions.</strong></p><p>Validation according to ICH guidelines confirmed the method’s robustness, linearity, accuracy, specificity, and precision. Stress testing under hydrolytic, oxidative, thermal, and photolytic conditions demonstrated suitability for degradation studies, supporting both process development and routine quality control of bulk drug substances.</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.4c10834"><strong>Quality-by-Design-Driven RP-HPLC Method Development and Validation for Impurity Analysis of Elexacaftor, a Cystic Fibrosis Drug, with LC-MS/MS-Based Degradant Identification</strong></a></h4><p>Jayaprakash Kanijam Raghupathi, Divya Kumar Vemuri, Syed Mastan Ali, Vishnuvardhana Kishore Polisetti, Rambabu Gundla, Leela Prasad Kowtharapu, Dittakavi Ramachandran*, and Naresh Kumar Katari*</p><p>ACS Omega 2025, 10, 39, 44885–44894</p><p><a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1021/acsomega.4c10834">https://doi.org/10.1021/acsomega.4c10834</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><p>Elexacaftor is a drug that is used to treat fibrosis (TCF). When treating patients with cystic fibrosis (CF) who have the F508del mutation or other genetic mutations that react well with these medications, it is specifically used in conjunction with two other medications, ivacaftor and tezacaftor. The cystic fibrosis transmembrane conductance regulator, or CFTR, modulator, is Elexacaftor. Due to genetic abnormalities, people with CF have abnormal CFTR protein function, which is improved by this treatment. Based on clinical research, individuals with CF should expect a much better quality of life, reduced pulmonary exacerbations, and improved lung function when using Elexacaftor, ivacaftor, and tezacaftor together as opposed to prior treatments. After receiving FDA approval, Elexacaftor was marketed in the USA under the brand name Trikafta in October 2019. <a href="javascript:void(0);">(1−5)</a> Since then, it has emerged as a key component of the CF treatment plan, especially for those with specific mutations. It is taken orally as tablets or granules, usually once a day, along with a meal high in fat. Headache, upper respiratory tract infection, abdominal pain, diarrhea, rash, and elevated liver enzymes are among the frequently reported side effects. Generally speaking, these side effects are controllable and can be well watched during therapy.</p><p>To ensure the quality and safety of ELX, it is required to develop analytical methods for the identification and estimation of impurities. The currently available literature reveals that the ELX drug combination with ivacaftor and tezacaftor is used very effectively. So, most of the analytical methods related to the estimation of three drugs simultaneously quantitatively by using different analytical techniques, i.e., by high-performance liquid chromatography and liquid chromatography–mass spectroscopy. Several liquid chromatography-ultraviolet (LC-UV) methods <a href="javascript:void(0);">(6,7)</a> and liquid chromatography-tandem mass spectrometry (LC-MS/MS) <a href="javascript:void(0);">(8−15)</a> methods are available to simultaneously measure the concentration of different combinations of IVA/TEZ/ELX with metabolites in plasma or other biological fluids. However, we did not find the literature for the analysis of process-related impurities and degradation impurities. The methods available in the literature do not discuss the process-related impurities and the degradation impurities of the ELX drug substance; hence, a stability-indicating LC method was developed for identifying and estimating the process-related impurities and degradation impurities of the ELX drug substance. In this paper, we report on a new analytical method to the estimation of ELX and its six process-related impurities quantitatively by using an HPLC instrument. This new LC method is sensitive enough to detect impurities as low as 0.02%, which ensures there is no harm or risk to the patients from impurities; however, existing methods in the literature do not discuss the sensitivity of the potential impurities, which can harm the patients.</p><p>The method has been validated according to current international guidelines; this method is specific and rugged for analysis. An experimental design constructed according to Quality by Design (QbD) can identify the individual effects as well as interactions of the critical method variables, such as organic ratio, pH of the buffer or mobile phase, flow rate, column oven temperature, and gradient composition. <a href="javascript:void(0);">(16−18)</a> It is a systematic approach to analytical method development that is risk-based, which helps to create robust and reliable analytical methods. The current robustness method was studied by utilizing the QbD-based Design of Experiments (DoE) with Design-Expert software.</p><h4><strong>2. Materials and Methods</strong></h4><h5><strong>2.2. Instrumentation and Software</strong></h5><p><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/210"><strong>Waters Alliance e2695 HPLC</strong></a> with a quaternary pump was used for method development and analysis (34 Maple St, Milford, Massachusetts 01757, USA). Empower 3 software (Waters Corporations, USA) was used for data collection and processing. Mettler Toledo microbalance was used, on which weighing was done on an MX5 microbalance (Mettler Toledo, Columbus, Ohio, USA). A SevenMulti pH meter (Mettler Toledo Columbus, Ohio, USA) was used for estimation of the pH.</p><h5><strong>2.3. HPLC Instrumentation and Conditions</strong></h5><p>Waters Alliance’s e2695 LC system with the 2998 photodiode array (PDA) detector was used for method development, method validation, and forced degradation investigations. Using Empower software, data were gathered and processed. Using a Waters 2998 PDA detector, the peak homogeneity was investigated.</p><h5><strong>2.3.1. Chromatographic Method Conditions</strong></h5><p>The analysis of Elexacaftor employed the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1823"><strong>XBridge Shield RP18</strong></a>, 150 mm × 4.6 mm, 3.5 μm column with 10 mm KH<sub>2</sub>PO<sub>4</sub> in water, pH adjusted to 3.2 with phosphoric acid buffer, and showed positive and encouraging results. Furthermore, extensive trials were made on an XBridge shield RP column with various flow rate and gradient conditions. The optimum separation of all impurities and ELX was achieved on XBridge Shield RP18, 150 × 4.6 mm, 3.5 μm, with mobile phase A as buffer:methanol (98:2, v/v) and mobile phase B as acetonitrile:2-propanol:water (70:20:10, v/v/v), flow rate 0.7 mL/min; column temperature 27 °C; with binary gradient time (min)/B concentration (%): 0/5, 20/50, 60/85, 80/85, 82/5 and 90/5. All peaks were separated with good resolution and peak shape under these conditions and are shown in <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.4c10834#fig2">Figure 2</a>. The diluent was a 40:60 v/v solution of water and acetonitrile that was also used to wash the needles.</p><h4><strong>3. Results and Discussion</strong></h4><h5><strong>3.2. Optimized Chromatographic Conditions</strong></h5><p>Based on the method developmental trials, it was found that Elexacaftor and related substances were not retained and separated as per the requirement in any of the screened conditions except the trial with XBridge Shield RP 18, 150 mm × 4.6 mm, 3.5 μm, mobile phase A as buffer:methanol (98:2, v/v) and mobile phase B as acetonitrile:2-propanol:water (70:20:10, v/v/v), flow rate 0.7 mL/min; column temperature 27 °C; with binary gradient time (min)/B conc (%): 0/5, 20/50, 60/85, 80/85, 82/5, and 90/5. All seven peaks were separated with good resolution and peak shape in this condition. The details of the finalized method conditions are mentioned in <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.4c10834#tbl2">Table 2</a> for quantitating ELX and its related substances by using HPLC.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Omega_2025_10_39_44885_44894_Table_2_Analysis_of_Optimized_HPLC_Method_Conditions_for_Elexacaftor_and_Its_Impurities_5fc5c933c5.jpg" alt="ACS Omega 2025, 10, 39, 44885–44894: Table 2. Analysis of Optimized HPLC Method Conditions for Elexacaftor and Its Impurities" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Omega_2025_10_39_44885_44894_Table_2_Analysis_of_Optimized_HPLC_Method_Conditions_for_Elexacaftor_and_Its_Impurities_5fc5c933c5.jpg 225w," sizes="100vw" width="1046" height="726"></p><p>With the above chromatographic conditions, ELX and its related substances (Imp-A to Imp-F) got separated from each other with a resolution of more than 2.0, as shown in <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.4c10834#fig3">Figure 3</a>.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Omega_2025_10_39_44885_44894_Figure_3_Overlay_chromatogram_of_degradation_studies_b71797ad37.jpg" alt="ACS Omega 2025, 10, 39, 44885–44894: Figure 3. Overlay chromatogram of degradation studies." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Omega_2025_10_39_44885_44894_Figure_3_Overlay_chromatogram_of_degradation_studies_b71797ad37.jpg 245w," sizes="100vw" width="2096" height="790"></p><h5><strong>3.3. Method Validation Results</strong></h5><p>According to ICH requirements, <a href="javascript:void(0);">(19−23)</a> the created approach was validated, and the results are shown in <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.4c10834#tbl3">Table 3</a>. In the presence of its process contaminants, the devised HPLC technique for ELX’s specificity was assessed. ELX and Imp-A-Imp-F are well separated from one another. Utilizing a photodiode array detector and HPLC, the analysis was completed. All of the peaks of ELX and its impurities were shown to be pure when the peak purity tool was employed on them, indicating the method’s specificity because no additional impurities were detected coeluting at the same retention period.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Omega_2025_10_39_44885_44894_Table_3_Method_Validation_Summary_of_the_Estimation_of_Elexacaftor_and_Its_Impurities_Part_1_ffb1d7d057.jpg" alt="ACS Omega 2025, 10, 39, 44885–44894: Table 3. Method Validation Summary of the Estimation of Elexacaftor and Its Impurities_Part 1" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Omega_2025_10_39_44885_44894_Table_3_Method_Validation_Summary_of_the_Estimation_of_Elexacaftor_and_Its_Impurities_Part_1_ffb1d7d057.jpg 168w," sizes="100vw" width="1042" height="969"></p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Omega_2025_10_39_44885_44894_Table_3_Method_Validation_Summary_of_the_Estimation_of_Elexacaftor_and_Its_Impurities_Part_2_75f03ca18f.jpg" alt="ACS Omega 2025, 10, 39, 44885–44894: Table 3. Method Validation Summary of the Estimation of Elexacaftor and Its Impurities_Part 2" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Omega_2025_10_39_44885_44894_Table_3_Method_Validation_Summary_of_the_Estimation_of_Elexacaftor_and_Its_Impurities_Part_2_75f03ca18f.jpg 178w," sizes="100vw" width="1058" height="924"></p><h4><strong>4. Conclusions</strong></h4><p>For the measurement of Elexacaftor and its related substances (imp-A to imp-F and any other impurities), the suggested technique was optimized to give a sharp peak with a minimal tailing factor. This approach was found to be specific, accurate, linear, and economical. The approach offers a number of benefits, including cost-effectiveness, robustness, and increased sensitivity. It was the first time we developed a method to estimate the content of Elexacaftor drug alone in process development. The current technique has been demonstrated to be efficient in terms of cost and may be utilized to measure Elexacaftor and its related compounds in the bulk. ICH rules were followed in the method’s validation.</p>

Quality-by-Design-Driven RP-HPLC Method Development and Validation for Impurity Analysis of Elexacaftor, a Cystic Fibrosis Drug, with LC-MS/MS-Based Degradant Identification

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Quality-by-Design-Driven RP-HPLC Method Development and Validation for Impurity Analysis of Elexacaftor, a Cystic Fibrosis Drug, with LC-MS/MS-Based Degradant Identification

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