<p><strong>The LC for Pharma Resource Hub is a comprehensive platform designed to support professionals in the pharmaceutical industry by providing valuable resources related to liquid chromatography (LC). This hub offers a variety of materials, including webinars, technical articles, application notes, and case studies, all aimed at enhancing knowledge and proficiency in LC methodologies within pharmaceutical applications.​</strong></p><h3><strong>Key features of the LC for Pharma Resource Hub include:</strong></h3><h4><strong>Educational Webinars</strong></h4><p>Access on-demand webinars presented by industry experts, covering topics such as method development, validation, and troubleshooting in LC.​</p><ul><li><strong>Episode 1: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/3437"><strong>USP 621 Harmonization - Method Scaling</strong></a><ul><li>Guest Speakers: Amanda Guiraldelli, Ph.D. &amp; Catharine Layton</li></ul></li><li><strong>Episode 2: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/3438"><strong>USP 621 Revisions - S/N Calculation, Sensitivity and Peak Symmetry</strong></a><ul><li>Guest Speakers: Edmond Biba, Ph.D. &amp; Neil Lander</li></ul></li><li><strong>Episode 3: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/3439"><strong>Enhanced Method Development</strong></a><ul><li>Guest Speakers: Amanda Guiraldelli, Ph.D. &amp; Richard Verseput</li></ul></li><li><strong>Episode 4: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/3440"><strong>aQbD for OTC Products</strong></a><ul><li>Guest Speakers: Joshua Bhattacharaya &amp; Fadi Alkhateeb, Ph.D.</li></ul></li><li><strong>Episode 5: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/3441"><strong>Impurity Profile Characterization</strong></a><ul><li>Guest Speakers: Christian Zeine, Henry Foddy &amp; Margaret Maziarz</li></ul></li><li><strong>Episode 6: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/4123"><strong>USP Chapter 129 Updates and SEC-MALS</strong></a></li><li><strong>Episode 7: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/4167"><strong>Dietary Supplement Quality</strong></a></li><li><strong>Episode 8: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/3444"><strong>Lipid Nanoparticles</strong></a></li><li><strong>Episode 9: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/3445"><strong>Nitrosamines</strong></a></li><li><strong>Episode 10: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/4169"><strong>Dissolution</strong></a></li><li><strong>Episode 11: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/4170"><strong>LG Polymers</strong></a></li><li><strong>Episode 12: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/4669"><strong>Method Validation</strong></a></li><li><strong>Episode 13: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/4670"><strong>Multi-Attribute Method</strong></a></li></ul><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Waters_Solving_Problems_That_Matter_a_Waters_webinar_series_featuring_USP_speakers_ecf3502756/Waters-Solving-Problems-That-Matter-a-Waters-webinar-series-featuring-USP-speakers.jpg" alt="Waters Corporation: Solving Problems That Matter - a Waters webinar series featuring USP speakers" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Waters_Solving_Problems_That_Matter_a_Waters_webinar_series_featuring_USP_speakers_ecf3502756/thumbnail_Waters-Solving-Problems-That-Matter-a-Waters-webinar-series-featuring-USP-speakers.jpg 245w," sizes="100vw" width="800" height="418"></p><h4><strong>Waters 2024 LC Symposium - Pushing the boundaries of LC to address today's challenges</strong></h4><ul><li><strong>Waters 2024 LC Symposium - Day 1 - </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/3935"><strong>Advanced LC Technologies for Increasingly Complex Molecules</strong></a><ul><li>Guest Speakers: Davy Guillarme, Ph.D., Guilherme (Gui) Guimaraes, Ph.D., &amp; Michelle Chen, Ph.D.</li></ul></li><li><strong>Waters 2024 LC Symposium - Day 2 - </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/3936"><strong>Next Generation HPLC: the Quest for Performance and Simplicity</strong></a><ul><li>Guest Speakers: Amanda Guiraldelli, Ph.D., Jennifer Simeone, &amp; Andrea Gheduzzi</li></ul></li></ul><h4><strong>Brochures and specifications</strong></h4><ul><li><strong>eBook: Improving Chromatographic Separations of Biopharmaceuticals With MaxPeak High Performance Surfaces (HPS) Technology</strong><ul><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720008011en_dd0b025cd5/720008011en.pdf"><strong>Full PDF for download</strong></a></li></ul></li><li><strong>White Paper: MaxPeak Premier Solutions: Improving Consumer Safety Through Innovative Science</strong><ul><li><a target="_blank" rel="noopener noreferrer" href="https://www.waters.com/waters/library.htm?locale=en_US&amp;lid=135123293&amp;cid=511436&amp;srsltid=AfmBOoripwfSGOjTFXNWqjDHknCTbtr0G9sKgb9BsUD-L9avphhNaGuH"><strong>Full PDF for download</strong></a></li></ul></li><li><strong>Application Notebook: Alliance iS</strong><ul><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720008280en_e9d283fb41.pdf"><strong>Full PDF for download</strong></a></li></ul></li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.cz/products/210"><strong>Alliance iS </strong></a>Product Information</li><li>I<strong>CH Q14 Enhanced Approach: Tools in Empower Software to Support Your Analytical Procedure Development</strong><ul><li><a target="_blank" rel="noopener noreferrer" href="https://cdn.technologynetworks.com/TN/resources/PDF/Tools-in-Empower-Software-to-Support-Your-Analytical-Procedure-Development.pdf"><strong>Full PDF for download</strong></a></li></ul></li><li><a target="_blank" rel="noopener noreferrer" href="https://www.wyatt.com/products/instruments/multi-angle-light-scattering-detectors-sec-mals.html"><strong>MALS Detectors Product Information</strong></a></li></ul><h4><strong>Guides</strong></h4><ul><li><strong>LC Adjustments Made Simple: A Practical Guide</strong><ul><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720008065en_88843a7860/720008065en.pdf"><strong>Full PDF for download</strong></a></li></ul></li><li><strong>Guidebook: Characterization of New Modalities</strong></li><li><strong>Multi-Attribute Methods for Biopharmaceutical Analysis</strong><ul><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/waters_mam_ebook_2023_84c6ae3f04/waters_mam_ebook_2023.pdf"><strong>Full PDF for download</strong></a></li></ul></li><li><a target="_blank" rel="noopener noreferrer" href="https://www.waters.com/waters/library.htm?lid=135083997&amp;icid=hm-inno_00326&amp;srsltid=AfmBOopUZ0Cbncgz3zb34VIy1ysiVknnwF8Bca5aLAmt2KFOLtB3ZGhc"><strong>AQbD Method Development Infographic - Achieve Consistent Reportable Results</strong></a></li></ul><p>By offering these resources, the LC for Pharma Resource Hub serves as a valuable tool for professionals seeking to enhance their understanding and application of liquid chromatography in the pharmaceutical industry.</p><blockquote><p>For complete information, please visit the <a target="_blank" rel="noopener noreferrer" href="https://gateway.on24.com/wcc/experience/elitewatersregional/2426651/4494230/method-management-campaign?partnerref=CZLabrulez"><strong>LC Regulated Workflows: Access insights from Pharma industry experts website</strong></a></p></blockquote>

LC Regulated Workflows: Access insights from Pharma industry experts

<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 1st December 2025? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!</strong></p><blockquote><p><span style="font-size:20px;">👉 </span><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library"><span style="font-size:20px;"><strong>SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES</strong></span></a></p></blockquote><blockquote><p><strong>👉 Need info about different analytical techniques?</strong> Peek into <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library"><strong>LabRulezGCMS</strong></a> or <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library"><strong>LabRulezICPMS</strong></a> libraries.</p></blockquote><p>This week we bring you application notes by <strong>Agilent Technologies,</strong> <strong>Shimadzu </strong>and<strong> Waters Corporation</strong> and poster by <strong>Thermo Fisher Scientific / HPLC!</strong></p><h3><strong>1. Agilent Technologies: Quantitative Bioanalysis of Oligonucleotides Using a 6495D Triple Quadrupole LC/MS System Combined with an Automated SPE Workstation</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_bioanalysis_oligonucleotides_6495d_qqq_lcms_spe_5994_8339en_agilent_82610a68ca.pdf"><strong>Full PDF for download</strong></a></li></ul><p>In drug development, the quantitative analysis of oligonucleotides in biological samples is crucial for pharmacokinetics (PK), pharmacodynamics (PD), and toxicological safety evaluations. Due to the associated structural complexity of oligonucleotides and the unique nature of biological matrices, the corresponding sample preparation techniques are often complex. Liquid-liquid extraction (LLE) and solid-phase extraction (SPE) are commonly used sample preparation methods, with SPE being more suitable to meet lower limit of quantitation (LLOQ) requirements and complex matrices.2 However, traditional manual SPE methods are time consuming and prone to human error.&nbsp;</p><p>Liquid chromatography/tandem mass spectrometry (LC/MS/MS) is widely used in oligonucleotide bioanalysis due to its high sensitivity, low detection limits, high selectivity, and specificity. Methods using LC/MS/MS enable the rapid and accurate detection and quantification of low-concentration target compounds in complex matrices without the need for special reagents and probes.3&nbsp;</p><p>To address the challenges in sample preparation, Agilent developed an automated SPE workstation based on the Bravo liquid handling platform, combined with the next‑generation triple quadrupole LC/MS/MS system, the 6495D triple quadrupole LC/MS system, providing a complete solution from sample preparation to data analysis. The 6495D triple quadrupole LC/MS system excels in high sensitivity quantification and low detection limits from complex matrices, while the automated SPE workstation significantly reduces manual intervention time and improves operational consistency and reliability. This complete automated SPE bioanalysis workflow significantly enhances the efficiency and reliability of quantitative oligonucleotide&nbsp;bioanalysis.&nbsp;</p><h4><strong>Experimental&nbsp;</strong></h4><h5><strong>Instruments&nbsp;</strong></h5><ul><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?search=1290+Infinity+III+Bio+LC+"><strong>Agilent 1290 Infinity III Bio LC&nbsp;</strong></a></li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=6495D"><strong>Agilent 6495D triple quadrupole LC/MS system&nbsp;</strong></a></li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=Automated+SPE+workstation"><strong>Agilent Automated SPE workstation</strong></a>, including Bravo, BenchCel and MiniHub</li></ul><h4><strong>Conclusion&nbsp;</strong></h4><p>This study verified the use of the automated Agilent SPE workstation combined with the Agilent 6495D triple quadrupole LC/MS system for oligonucleotide bioanalysis studies through the quantitative analysis of mipomersen in pig plasma. The experimental results showed that the system exhibited excellent linearity (R²=0.999) within the dynamic range of 2 to 1,000 ng/mL. The reproducibility and accuracy of the QC samples were within acceptable ranges, with most RSD values below 5%, QC low points below 10%, and back‑calculated concentration accuracy between 85 and 115%. Additionally, in the three-day repeatability experiment, the inter-batch reproducibility of the QC samples was good, with RSD values all below 10%. The back-calculated accuracies for QC samples at high, medium, and low concentration levels, as well as the LLOQ (2 ng/mL), remained stable between 80 and 120%.&nbsp;</p><p>By reducing manual intervention and improving operational consistency, the automated SPE workstation significantly enhances the efficiency and reliability of sample preparation. Combined with the high sensitivity and low detection limits of the 6495D triple quadrupole LC/MS system, this solution achieves efficient and accurate detection of low‑concentration oligonucleotides in complex matrices. Overall, this automated solution has broad application prospects in high-throughput oligonucleotide bioanalysis and meets the high requirements for quantitative analysis in drug&nbsp;development.</p><h3><strong>2. Shimadzu: Determination of N-Nitroso-Nebivolol in Nebivolol Drug Substance by </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=LCMS-2050"><strong>LCMS-2050</strong></a></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_03_lcms_055_en_8d77db9fd3.pdf"><strong>Full PDF for download</strong></a></li></ul><h4><strong>User Benefits</strong></h4><ul><li>A new method for the determination of NDSRI impurity in nebivolol raw material was established using LCMS-2050.</li><li>The method has high sensitivity, short analysis time and high accuracy, which can fully meet the FDA's AI limit for N-Nitrosonebivolol.</li></ul><p>On September 4, 2024, the FDA issued the final guidance on the control of nitrosamine impurities in human drugs (Nitrosamine Guidance Version 2). The revised guidance describes two categories of nitrosamines: small-molecule nitrosamines and nitrosamine drug substance-related impurities (NDSRIs). Unlike small-molecule nitrosamine impurities that are structurally dissimilar to the active pharmaceutical ingredient (API), NDSRIs generally exhibit structural similarity to the API and may form through interactions between the API and nitrosating agents, such as nitrite impurities present in drug excipients.&nbsp;</p><p>Nebivolol is a potent and selective third-generation β-blocker clinically used for the treatment of patients with moderate-tosevere hypertension, as well as angina pectoris and congestive heart failure, with a recommended daily dose of 5 mg. Due to its structural characteristics, the synthesis of nebivolol may involve nitrosation reactions, leading to the introduction of the NDSRI impurity N-nitroso-nebivolol. According to the FDA's nitrosamine guidance, based on the Carcinogenic Potency Categorization Approach (CPCA), the acceptable intake (AI) limit for N-nitroso-nebivolol, a potential NDSRI in nebivolol, is set at 1500 ng/day.&nbsp;</p><p>This study established an LC-MS (<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=LCMS-2050"><strong>LCMS-2050</strong></a>) method for the determination of N-nitroso-nebivolol in nebivolol drug substance. The method demonstrates high sensitivity, short analysis time, and accurate results, providing a reference for the detection of related nitrosamine impurities.</p><h4><strong>Separation Evaluation&nbsp;</strong></h4><p>To ensure high sensitivity in impurity analysis and prevent contamination of the mass spectrometer by high-concentration active pharmaceutical ingredient (API) components, flow path switching technology was employed. This method diverts the main nebivolol peak to waste, allowing only impurities to enter the mass spectrometer for detection. Under the optimized chromatographic conditions, The retention times of nebivolol and N-nitroso-nebivolol were 1.952 min and 3.408 min, respectively, so the mass spectrometry acquisition was initiated after 2.8 min, ensuring selective detection of the target impurity. This approach effectively enabled accurate and interferencefree analysis of trace-level impurities.</p><h4><strong>Conclusion</strong></h4><p>A novel method for determining the content of N-nitrosonebivolol in nebivolol drug substance was established by using the LCMS-2050. Within the concentration range of 1 - 180 ng/mL, the correlation coefficients of the N-nitroso-nebivolol was 0.9999. When the spiked concentration of 0.03 μg/mg and 0.3 μg/mg, the recovery rate of N-nitroso-nebivolol was 100.4 and 100.2, respectively, and the recovery rate was good and met the requirements of pharmacopoeia. The method has high sensitivity, linearity, precision and recovery rate can meet the detection requirements, and which can be used for the detection of nitrosamine impurity N-nitroso-nebivolol in nebivolol drug substance.</p><h3><strong>3. Thermo Fisher Scientific / HPLC: At the intersection between chromatographic performance, ESI efficiency and instrument productivity: nano to capillary flow LC/MS on long μPAC Columns</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_intersection_between_chromatographic_performance_po_en_1a1d6f722b.pdf"><strong>Full PDF for download</strong></a></li></ul><p>LC-MS-based proteomics commonly employs low-flow liquid chromatography (LC) to separate tryptic peptides, which helps reduce sample complexity before MS/MS detection and protein identification. This technique benefits from minimal dilution on miniaturized columns and increased ionization efficiency via nanoflow electrospray ionization (ESI). As mass spectrometry (MS) devices become more sensitive and faster, there is an increasing need to boost throughput. The challenge lies in maintaining or increasing proteome coverage while simultaneously reducing analysis time. Typically, higher flow rates improve instrument productivity but may compromise sensitivity. In this study, the impact of eluting flow rates was assessed across various throughput ranges to identify the optimal balance between productivity and proteome coverage. Additionally, the transition to a dual-column single emitter configuration was evaluated to determine its effects on productivity and coverage. This approach aims to enhance LC-MS performance, ensuring high throughput without sacrificing the depth of proteome analysis.</p><h4><strong>Methods</strong></h4><p>HeLa cell digests were resuspended in 0.1% TFA and 1% ACN to create a 200 ng/µL stock solution, sonicated, diluted in 0.1% TFA, and vortexed. A <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/398"><strong>Thermo Scientific Vanquish Neo UHPLC instrument</strong></a> was configured for either a single analytical column in a trap-and-elute setup or two columns in tandem. For single column experiments, a Thermo Scientific PepMap Neo trap cartridge and a 110 cm µPAC Neo Plus column on a Thermo Scientific EASY-Spray source were used. LC methods with flow rates from 200 to 1000 nL/min were tested for throughputs ranging from 16 to 50 samples per day. The column was positioned in an external heating device mounted directly onto the ionization source and connected to an ESI emitter via a 20 µm ID voltage spacer. For tandem column experiments, two 110 cm µPAC Neo Plus columns were placed in the Vanquish Neo UHPLC column compartment between two low-dispersion 6-port switching valves. A 20 µm ID nanoViper line connected the eluents to an ESI emitter with an integrated liquid junction in an EASY-Spray source. Data were acquired using a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/402"><strong>Thermo Scientific Orbitrap Exploris 240</strong></a>/<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/401"><strong>480 mass spectrometer</strong></a> in data-independent acquisition mode.</p><h4><strong>Data Analysis&nbsp;</strong></h4><p>LC-MS data were analyzed either using a trial version of <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1782"><strong>Thermo Scientific Proteome Discoverer 3.3 software</strong></a> with Chimerys or with Spectronaut® 19. Results shown have been filtered to a 1% FDR</p><h4><strong>Conclusions&nbsp;</strong></h4><ul><li>Increasing the flow rate to 1 µL/min significantly increases instrument productivity. For gradient times below 30 minutes, 1 µL/min performs slightly better than lower flow methods, identifying up to 6679 protein groups at 50 SPD.&nbsp;</li><li>For gradient lengths over 50 minutes, a 200 nL/min flow rate provides superior chromatographic metrics and proteome coverage, resulting into a maximum of 8556 protein groups identified at 16 SPD.&nbsp;</li><li>The negative impact of a low-dispersion 6-port valve's impact scales inversely with flow rate. At 1 µL/min, peak width increases by 40%, yielding 5% lower proteome coverage but 26% higher productivity.&nbsp;</li><li>Optimized dual column single-emitter methods identified up to 6947 protein groups at 50 SPD with 3-6% CVs for the same column and 4-8% between columns (n=6).&nbsp;</li><li>Column reproducibility was shown across 5 columns, with an ID rate variation of 0.7% for HeLa digests and a median RT CV of 0.99% for spiked-in PRTC peptides.</li></ul><h3><strong>4. Waters Corporation: Improving Sample Throughput of HPLC Methods Using CORTECS 5 μm Columns</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720009072_en_1203625d4f.pdf"><strong>Full PDF for download</strong></a></li></ul><h5><strong>Benefits&nbsp;</strong></h5><ul><li>Comparable chromatographic performance for abacavir impurities was achieved using a shorter CORTECS Premier C18 5 µm Column in place of a column packed with fully porous 5 µm particles</li><li>33% reduction in solvent usage and analysis time using a CORTECS Premier C18 5 µm Column compared to a fully porous 5 µm column</li><li>33% reduction in analytical method greenness score indicating improved sustainability for the new method</li></ul><p>Improving validated HPLC methods can be challenging. Two of the approaches to increase analytical throughput, increasing mobile phase flow rate or using shorter columns, have drawbacks in higher pressures or reduced chromatographic performance. Typically, using a shorter column also requires decreasing the particle size to maintain a constant length to particle size (L/dp) ratio to ensure similar column efficiency. However, columns packed with smaller particles generate higher back pressures. Some labs may be able to migrate methods to shorter columns packed with smaller particles by transferring to a HPLC system capable of operating at higher pressures. However, if a laboratory lacks access to such systems, then their options may be limited. In those situations, finding appropriate columns to use to improve a method is not only a matter of chromatographic performance but also system compatibility, particularly from a pressure standpoint. Older HPLC systems are limited to lower pressures compared to more modern systems.&nbsp;</p><p>This application brief examines the improvement of a method for testing abacavir impurities by migrating the original method from a 4.6 x 150 mm 5 µm fully porous C18 column to a CORTECS Premier C18 4.6 x 100 mm 5 µm Column. Additionally, a fully porous 3.5 µm C18 column in 4.6 x 100 mm hardware was evaluated to determine if moving to a smaller particle size would have as much of a benefit as that provided by using a CORTECS Column. The separation conditions for method migration were obtained using the Waters Column Calculator. Along with examining throughput, solvent savings were calculated as well as analytical method greenness scores for each column tested.</p><h4><strong>Experimental</strong></h4><h5><strong>Method Conditions&nbsp;</strong></h5><ul><li><strong>LC system:</strong> <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/210"><strong>Alliance™ HPLC System</strong></a> with 2489 UV/Visible (UV/Vis) Detector</li></ul><h5><strong>Data Management&nbsp;</strong></h5><ul><li><strong>Chromatography software: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/627"><strong>Empower™ Chromatography Data System (CDS)</strong></a></li></ul><h4><strong>Results and Discussion&nbsp;</strong></h4><p>Previously, the impurity method for abacavir was migrated to a 2.7 µm CORTECS Column.1 While that work showed the benefits of scaling to a smaller particle size, not all laboratories are comfortable moving to such a small particle size. In those instances, moving from a 5 µm to a 3.5 µm particle column is a good first step. However, depending on the method, this migration might not be appropriate and could result not only in elevated system pressure but also performance loss. Figure 1 shows the chromatograms for abacavir impurities using the original method column, a 3.5 µm C18 column, and the CORTECS Premier C18 5 µm Column.</p><h4><strong>Conclusion&nbsp;</strong></h4><p>CORTECS Premier 5 µm Columns are suitable alternatives for fully porous 5 µm columns for improving validated HPLC methods. Using a CORTECS solid-core stationary phase can allow for a shorter column to be used because of their higher efficiency per unit length. To highlight this, the impurity method for abacavir was migrated from the original fully porous 5 µm, 4.6 x 150 mm column to a CORTECS Premier C18 5 µm, 4.6 x 100 mm Column, as well as a 4.6 x 100 mm 3.5 µm fully porous column. The use of the CORTECS Column achieved comparable separation performance as the original while also exhibiting a shorter analysis time, lower solvent usage, and a better AMGS value.</p>

News from LabRulezLCMS Library - Week 49, 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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