<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 10th November 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!</strong></p><h3><strong>1. Agilent Technologies: Feed Injection at Elevated Pressure Using the Agilent 1290 Infinity III Hybrid Multisampler</strong></h3><p><strong>Optimization of a multipesticide method for the highest sensitivity through feed injection</strong></p><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/te_feed_injection_multipesticide_1290_infinityiii_hybrid_5994_8478en_agilent_f070801ff2.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Modern analytical methods for the determination of pesticide residues are based on multipesticide methods capable of determining hundreds of pesticides in a sample in one analytical LC/MS/MS run. For chromatographic separation of these pesticides, modern separation columns are used. Their dimensions are typically 2.1 × 150 mm, filled with 1.9&nbsp;µm particle size material. This requires that the LC can work at high pressures up to 1300 bar. The Agilent 1290 Infinity III Hybrid Multisampler can operate within this pressure range. In addition, the Hybrid Multisampler can work in two different modes, the classic flow through mode and the Agilent Feed Injection mode. Feed Injection mode helps to prevent peak broadening and splitting effects as they can occur with the classic flow through mode for injection of samples in solvents with high elution strength and higher injection volumes.</p><h4><strong>Experimental</strong></h4><h5><strong>Instrumentation</strong></h5><ul><li>Agilent 1290 Infinity III High-Speed Pump (G7120A)&nbsp;</li><li>Agilent 1290 Infinity III Hybrid Multisampler (G7137B)&nbsp;</li><li>Agilent 1290 Infinity III MCT (G7116B)&nbsp;</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=Ultivo+Triple+Quadrupole+LC%2FMS+"><strong>Agilent Ultivo Triple Quadrupole LC/MS </strong></a>with Agilent Jet Stream source&nbsp;</li></ul><h5><strong>Software&nbsp;</strong></h5><ul><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=MassHunter+Acquisition+Software"><strong>Agilent MassHunter Acquisition Software</strong></a> (v. 12.2)&nbsp;</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=MassHunter+Qualitative+Analysis"><strong>Agilent MassHunter Qualitative Analysis Software</strong></a> (v. 12.0)&nbsp;</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=MassHunter+Quantitative+Analysis"><strong>Agilent MassHunter Quantitative Analysis Software</strong></a> (v.&nbsp;12.1)&nbsp;</li></ul><h5><strong>Column&nbsp;</strong></h5><ul><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=InfinityLab+Poroshell+120+Phenyl-Hexyl"><strong>Agilent InfinityLab Poroshell 120 Phenyl-Hexyl</strong></a>, 2.1 × 150 mm, 1.9 µm (part number 699675-912)</li></ul><h4><strong>Results and discussion&nbsp;</strong></h4><p>Calibration curves for a multipesticide method were acquired to demonstrate the possible improvement of peak shape and the lower limits of quantification (LOQ) using Agilent Feed Injection mode in comparison to classical flow through injection mode. This method comprised approximately 120&nbsp;pesticide compounds measured by dynamic MRM. The separation was performed on a 150 mm length column. With the chosen solvents and flow rate, a pressure range above 1000&nbsp;bar was necessary. The calibration curves were acquired for concentrations down to 0.02 ppb (20 ppt), and each calibration point was measured three-fold. The concentrations of the calibration solutions were prepared by dilution using the strong-eluting solvent acetonitrile and injected directly by both injection modes with different injection volumes. An example chromatogram showing the elution pattern for the MRM transitions is displayed in Figure 1.</p><h4><strong>Conclusion&nbsp;</strong></h4><p>This technical overview demonstrates the use of the Agilent Feed Injection mode available with the Agilent 1290 Infinity III Hybrid Multisampler in comparison to the classical flow through injection mode for the analysis of pesticides by a multipesticide method. We demonstrated that the use of Agilent Feed Injection mode improves the obtained peak shape for injection of samples dissolved in high elution strength solvent compared to classical flow through mode. With the improved peak shape obtained by Feed Injection mode, signal to noise ratios were improved, resulting in higher sensitivity. Due to retained peak shape, no manual interaction for peak integration was necessary when using feed injection.</p><h3><strong>2. Shimadzu: Automatic Optimization of Sample Solvent Composition for Achieving Sharp Peak Shapes</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_00938_en_c0f4de1265.pdf"><strong>Full PDF for download</strong></a></li></ul><h4><strong>User Benefits</strong></h4><ul><li>The automatic pretreatment function integrated into the autosampler enables efficient optimization of sample solvent composition, which can be automatically adjusted during analysis.</li><li>The desired composition of sample solvent can be easily specified via the LabSolutionsTM MD interface.</li></ul><p>In LC analysis, the composition of the sample solvent is critical for achieving proper peak shape. If the sample solvent is a stronger eluting solvent than the mobile phase, sample band condensation at the column inlet may be insufficient, leading to sample band broadening. For instance, increasing the organic solvent ratio in the sample solvent to dissolve low-polar compounds may deteriorate the peak shapes of early-eluting compounds in reversed-phase chromatography. Therefore, determining the optimal ratio of organic solvent in the sample solvent is essential. However, manually preparing multiple solvent compositions is extremely time-consuming. The automatic pretreatment function of the autosamplers (<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=Nexera+X3"><strong>Nexera series</strong></a>), in combination with the method development support <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=LabSolutions+MD"><strong>software LabSolutions MD</strong></a>, enables consecutive analyses using different sample solvent compositions without manual intervention. This allows evaluation of the effect of sample solvent composition on peak shape while significantly reducing the labor involved in optimization. In this study, we demonstrate the automated determination of the optimal sample solvent composition using metoclopramide, a small-molecule drug, as a model compound.</p><h4><strong>Automatic Pretreatment Function</strong></h4><p>The automatic pretreatment function integrated into the autosampler enables drawing and ejecting specified volumes of reagents and solvents from any vial, as well as mixing them within the needle. As an example, Fig. 1 illustrates the procedure for automatically adjusting the water-to-methanol ratio in the sample solvent using this function. The autosamplers (Nexera series) supports up to three rinsing solvents via the multi-rinse function, and in this procedure, methanol and water were supplied from these rinsing lines. By modifying the "methanol eject volume" (2) and “water eject volume” (3) in Fig. 1, sample solvents with desired methanol-to-water ratios can be automatically prepared. In addition, adjusting the “sample drawing volume” (1) allows for not only changing the sample solvent composition but also diluting the sample solution to a specified concentration.</p><h4><strong>Analytical Conditions and Target Compounds</strong></h4><p>A 1000 mg/L solution of metoclopramide, a small-molecule drug, was prepared in 100% methanol as a model sample. The water content in the sample solvent was then varied from 0% to 90% in 10% increments (A 10-fold dilution was applied during automatic sample solvent preparation), and its effect on peak shape was evaluated under the analytical conditions shown in Table 1. By simply specifying the desired water content on the LabSolutions MD interface (red frame in Fig. 2), the autosampler’s automatic pretreatment function adjusts the sample solvent composition during consecutive analyses. This eliminates the need for manual preparation of multiple solvent mixtures, significantly reducing the workload involved in optimizing sample solvent composition for ideal peak shapes.</p><h4><strong>Conclusion</strong></h4><p>Optimizing the composition of the sample solvent is essential for achieving appropriate peak shapes. However, manual preparation of multiple solvent compositions is time-consuming. The automatic pretreatment function of the autosampler, in combination with LabSolutions MD, enables consecutive analyses with automatic variation of the sample solvent composition. This significantly reduces the effort required for optimization. Since the optimal sample solvent composition depends on both the physicochemical properties of the analyte and the mobile phase conditions, optimization is necessary for each sample. These tasks, however, can be greatly reduced by utilizing the automatic pretreatment function.</p><h3><strong>3. Thermo Fisher Scientific: Improving selectivity and sensitivity of lipid mediator analyses by coupling nano-flow chromatography with the Stellar mass spectrometer</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_improving_selectivity_sensitivity_lipid_mediator_analyses_po_en_c38bc37825.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Lipid mediators play crucial roles in regulating inflammation and maintaining tissue homeostasis. However, their quantification in biological samples is difficult due to low concentrations (ppt–ppb range), chemical instability, and structural similarity. Conventional LC-MS/MS methods using high-flow UHPLC and triple quadrupole mass spectrometers require extensive optimization of SRM transitions for each analyte. This study introduces a <strong>nano-flow LC-MS workflow</strong> combining the <strong>Thermo Scientific Vanquish Neo UHPLC system</strong> with the <strong>Thermo Scientific Stellar mass spectrometer</strong>, aiming to improve analytical sensitivity and selectivity while reducing solvent and sample consumption.</p><h4><strong>Experimental</strong></h4><p>Lipid mediator standards (from Cayman Chemical) were analyzed using two chromatographic configurations: a <strong>nano-flow method</strong> (500 nL/min) with the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/398"><strong>Vanquish Neo UHPLC</strong></a> and a <strong>high-flow method</strong> (300 µL/min) using the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/744"><strong>Vanquish Horizon UHPLC</strong></a>. Both setups were coupled either to the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1149"><strong>Stellar MS</strong></a> or the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/404"><strong>TSQ Altis Plus triple quadrupole MS</strong></a>. The Stellar instrument features a <strong>dual-pressure linear ion trap</strong> and <strong>ion routing multipole</strong>, enabling both <strong>HCD (higher-energy collisional dissociation)</strong> and <strong>CID (collision-induced dissociation)</strong> fragmentation. Parallel reaction monitoring (PRM) was employed instead of SRM, allowing simultaneous detection of multiple transitions. Data were processed using <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/77"><strong>Thermo Scientific TraceFinder</strong></a> software with internal standards and calibration curves ranging from 0.0001 ppb to 100 ppb.</p><h4><strong>Results</strong></h4><p>The nano-flow Stellar method achieved superior sensitivity and selectivity compared with the high-flow methods on both Stellar and TSQ Altis Plus systems. Limits of quantitation (LOQ) reached the <strong>low-ppt range</strong>, while maintaining fast analysis times (&lt; 30 min) and requiring less than 20 µL of solvent per sample. Chromatograms of key eicosanoids such as <strong>15(S)-HETE</strong>, <strong>12(S)-HETE</strong>, and <strong>5(S)-HETE</strong> demonstrated higher signal intensity and improved peak shapes under nano-flow conditions. Calibration curve reproducibility and scan density (&gt; 10 scans per peak) confirmed reliable quantitative performance.</p><h4><strong>Conclusion</strong></h4><p>Coupling <strong>nano-flow chromatography</strong> with the <strong>Stellar mass spectrometer</strong> provides a powerful workflow for targeted lipid mediator analysis, significantly enhancing both selectivity and sensitivity compared with conventional high-flow LC-MS/MS. The system’s <strong>PRM capability</strong>, <strong>dual-fragmentation flexibility</strong>, and <strong>low-volume solvent use</strong> make it a robust and cost-effective solution for biological and clinical lipidomics, capable of quantifying trace-level lipid mediators efficiently and reproducibly.</p><h3><strong>4. Waters Corporation: Investigating Drug Metabolism of Methapyrilene Within a Rat Model Using a Data Dependent Acquisition Workflow with the Xevo MRT Mass Spectrometer</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720009001_en_c11ab3b6b7.pdf"><strong>Full PDF for download</strong></a></li></ul><h5><strong>Benefits&nbsp;</strong></h5><ul><li>Demonstration of the Waters™ Xevo™ MRT Mass Spectrometer (MS) workflow for the analysis of urine extracts from a drug dosed study, involving methapyrilene.</li><li>Data dependent acquisition (DDA) has been used for data collection, providing highly informative fragmentation data to confidently identify drug metabolites of interest.</li><li>Combining a DDA approach with high-throughput chromatography is shown to consistently yield high quality spectra, providing highly confident compound identifications.</li><li>A seamless workflow allowing drug metabolites to be confidently identified is demonstrated with the UNIFI™ Application within the waters_connect™ Software Platform</li></ul><p>The introduction of the Xevo MRT Mass Spectrometer allows for comprehensive drug metabolite identification (Met ID) at high acquisition speed. Using urine samples collected over a time course of six days, the excellent mass accuracy, high mass resolution, and sensitivity are highlighted, which can be achieved for drug metabolism studies. DDA provides highly specific MS/MS information, which assists with structural elucidation. The speed at which DDA data can be acquired with the Xevo MRT Mass Spectrometer can be as fast as 50 and 100 Hz, MS, and MS/MS respectively. Combined with fast chromatographic methods, this fast data acquisition rate allows for highly informative, confident data in a high-throughput fashion</p><h4><strong>Experimental</strong></h4><p>LC-MS Data Acquisition LC-MS data were collected using an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/191"><strong>ACQUITY™ Premier UPLC™ System</strong></a> coupled to a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1148"><strong>Xevo MRT Mass Spectrometer</strong></a>. The reversed-phase (RP) chromatographic separation consisted of a 5.0 minute method (injection to injection) using a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1966"><strong>CORTECS™ C18</strong></a>, 1.6 µm, 2.1 x 50 mm analytical Column (p/n: 186007114). The LC solvents were comprised of mobile phase A (0.1% formic acid containing 1 mMol ammonium formate) and mobile phase B (95:5 ACN:Water (v/v), 0.1% formic acid, 1 mMol ammonium formate) being ramped from 50% to 99% over 5 minutes using a flow rate of 0.6 mL/minute. The gradient started from 0.1% to 15% (mobile phase B) over 1.5 minutes, 50% at 3 minutes and 99% at 4 minutes. The solvent composition was held at 99% (mobile phase B) for 0.5 minutes and re-equilibration at initial conditions by 5 minutes, ready for the subsequent injection.&nbsp;</p><p>The Xevo MRT Mass Spectrometer was configured with the source settings as outlined in Table 1. MS data were collected using DDA mode of operation, which consisted of scan rates of 20 Hz (MS) and 50 Hz (MS/MS). The number of components selected for MS/MS were eight with a dynamic exclude applied of 10 mDa with a 10 second retention time tolerance.</p><h4><strong>Data Processing&nbsp;</strong></h4><p>The acquired data were processed using the UNIFI Application within the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1544"><strong>waters_connect Software Platform</strong></a>. Data were peak picked and screened against a compound database containing MP metabolites and their associated fragments. A mass tolerance of 1 ppm for both MS and MS/MS were applied.</p><h4><strong>Conclusion&nbsp;</strong></h4><p>LC-MS analysis of urine collected over a time course following administration of the drug methapyrilene, has been demonstrated using a DDA approach with the Xevo MRT Mass Spectrometer. MP undergoes complex biotransformation to provide a variety of metabolites. Utilizing a DDA workflow provides detailed, clean fragment ion spectra for confident structural elucidation. The additional benefit of sub ppm mass accuracy provides definitive identification(s). Integrating fast chromatography allows for high-throughput analyses while maintaining high resolution and mass accuracy.</p>

News from LabRulezLCMS Library - Week 46, 2025

News from LabRulezLCMS Library - Week 47, 2025

News from LabRulezLCMS Library - Week 48, 2025

<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>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 8th 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>and<strong> Waters Corporation, </strong>technical note by <strong>Shimadzu </strong>and poster by <strong>Thermo Fisher Scientific / ASMS!</strong></p><h3><strong>1. Agilent Technologies: Robust and Reproducible Monitoring of PFAS in Treated Wastewater by Direct Injection on 6495D LC/MS</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_pfas_wastewater_lctq_5994_8346en_agilent_c2412e7d84.pdf"><strong>Full PDF for download</strong></a></li></ul><p>PFAS is a class of synthetic chemicals that are resistant to heat, water, grease, and oil. Exposure to this class of chemicals is widespread and their ability to move and persist in the environment makes them difficult to clean up. Found in everyday household and workplace products such as drinking water, food packaging, fish caught from compromised environments, and personal care products is an indication of how widespread the use of PFAS and its contamination in the&nbsp;environment.&nbsp;</p><p>Traditional techniques of analysis to enhance sensitivity incorporate the use of solid phase extraction, which allows enrichment of the sample before injection. Increased awareness of emerging contaminants has necessitated the need for higher throughput methods, some bypassing the sample preparation step, to decrease sample analysis time. Direct injection analysis can save time by removing the need to prepare samples but still providing robustness and reproducibility to justify its capabilities and improved&nbsp;efficiency.</p><h4><strong>Experimental</strong></h4><h5><strong>Instrumentation&nbsp;</strong></h5><p><strong>Liquid chromatography system:&nbsp;</strong></p><ul><li>Agilent 1290 Infinity II Bio-LC High‑Speed Pump (G7132A)&nbsp;</li><li>Agilent 1290 Infinity II Bio-LC Multisampler (G7137B)&nbsp;</li><li>Agilent 1290 Infinity II Multicolumn Thermostat (G7116B)&nbsp;</li></ul><p><strong>Mass spectrometry system:&nbsp;</strong></p><ul><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 (G6495D)&nbsp;</strong></a></li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=Jet+Stream"><strong>Agilent Jet Stream (AJS) Source</strong></a> A PFC free kit, together with a delay column, was also used.</li></ul><h4><strong>Results and discussion&nbsp;</strong></h4><p>The sensitivity of the method was calculated with ten spikes of water at 1&nbsp;ng/L resulting in the method detection limit. The calculated concentration was prior to dilution of methanol and demonstrates that all but three analytes were equal or less than 1 ng/L detection limits, the analytes with greater than 1&nbsp;ng/L MDL were 3:3 FTCA and 8:2 FTSA (Table 6).</p><h4><strong>Conclusion&nbsp;</strong></h4><p>The increased awareness of PFAS globally has meant that regulations are constantly being adopted and larger amounts of samples at lower limits are required. By incorporating the direct injection technique on the Agilent 6495D LC/MS we get a combination of high sample throughput while providing excellent analytical sensitivity. Also, increasing throughput and reducing consumables cost creates increased value in the instrument. The method displays a clean baseline and sensitivity with the observation of 1 ng/L detection in spiked wastewater. Reproducibility and robustness were measured between the concentrations of 0.5 to 100 ng/L on the 6495D LC/MS. The system was able to detect 38 PFAS between 0.25 to 3.4 ng/L with RSD &lt; 20% over 150&nbsp;direct injections, providing confidence in the data for a complex matrix like&nbsp;wastewater.</p><h3><strong>2. Shimadzu: A sustainable analytical approach for detecting extra virgin olive oil adulteration using Nexera™ UC</strong></h3><ul><li>Technical note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/c190_e341_c488032e81.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Knowledge of the composition of triacylglycerols (TAGs) in vegetable oils is important for dietary and nutritional reasons, due to the influence of the different properties of each fatty acid on the human organism. Specifically, extravirgin olive oil (EVOO) is a popular food ingredient worldwide, and for this reason, detecting economically motivated adulteration is important to protect consumers’ interest and health[1]. According to the latest report from the EU Food Fraud Network (European Union, 2021), olive oil (OO) tops the list of reported adulterated food products. The official methods to assess OO purity and detect the presence of extraneous vegetable oils include the analysis of fatty acids (FAs), triacylglycerols (TAGs) and sterols (Regulation (EU) No 2568/91 and its amendments) [2].&nbsp;</p><p>A variety of chromatographic techniques has been employed for TAGs analysis and separation. High-performance liquid chromatography (HPLC) methods, which use reversed-phase (RP) and silver-ion (Ag) columns for TAGs separation, have been employed either independently or in combination[3]. However, both these approaches have the drawback of relying on toxic organic solvents. The alternative separation technique employed in TAGs analysis is supercritical fluid chromatography (SFC) coupled with a UV detector at very low wavelengths, due to the weak UV absorption of the mobile phase. In this study, a fast, simple and green methodology was optimized to detect intentionally adulterated olive oil with cheaper seed oils at different levels by means of SFC with UV detection, followed by statistical analysis.</p><h4><strong>2. Experimental</strong></h4><h5><strong>2.2 SFC-PDA Nexera™ UC system&nbsp;</strong></h5><p>All experiments were performed on a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=Nexera%E2%84%A2+UC"><strong>Nexera™ UC system</strong></a> (Shimadzu Europa, Germany). The system was equipped with a CBM-20A communication bus module, a DGU-20A5R degasser, an LC-30ADSF CO2 pump, one LC-20ADXR dual plunger parallelflow pump, a Sil-30AC autosampler, a CTO-20AC column oven, a SPD-M20A photodiode array (PDA) detector, and a SFC-30A backpressure regulator (BPR). Chromatograms were recorded using Shimadzu <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=LabSolutions"><strong>LabSolution</strong></a> ver. 5.80 software. Four Ascentis Express C18 columns, provided by KGaA (Darmstadt, Germany), were connected in series for TAGs separation. The analytical conditions used are summarized in Table 1.</p><h4><strong>3. Results and discussion&nbsp;</strong></h4><p>Using four C18 columns packed with superficially porous particles, a highly efficient separation of TAGs with an isocratic elution mode was provided, allowed for complete sample TAG elution within 30 min with the conditions used in this method (Figure 1). Peak identification was carried out according to partition number (PN), the published composition of these common vegetable oils and the relative amount of major and minor peaks, and finally taking into account TAGs retention times. For compounds with identical PN, separation was achieved based on the number of unsaturations. The elution order of TAGs was primarily governed by the hydrophobicity of the solutes. As the PN increases, the non-polar character of TAGs becomes more dominant.</p><h4><strong>Conclusions&nbsp;</strong></h4><p>The Nexera™ UC system provided excellent results for assessing TAGs composition in ten different vegetable oils examined in this study employing an SFC-PDA analytical method using isocratic mode and UV detection at 205 nm. This approach does not require any sample preparation and enables analyses to be completed with less waste and less cost compared to traditional methods, making it feasible for routine analyses, while providing reliable quantitative values.&nbsp;</p><p>Quantification of adulterated EVOO was performed by establishing curves based on peak areas of LLL marker versus seed oils concentrations. Statistical analysis using PCA and one-way ANOVA also visually distinguished adulterated EVOO from pure EVOO, and the result was in accordance with the present quantitative method. This fast and environmental method exhibits a huge potential for quality control and authenticity evaluation of EVOO.</p><h3><strong>3. Thermo Fisher Scientific / ASMS: Uncovering biological differences at scale: high-throughput and in-depth plasma proteomics with the Seer Proteograph ONE workflow and Orbitrap Astral Zoom Mass Spectrometer</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_393_uncovering_biological_astral_zoom_seer_po393_asms2025_na_en_f697aef9c0.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Plasma proteomics enables minimally invasive biomarker discovery by capturing disease-related molecular signatures. However, its effectiveness is limited by the wide dynamic range of protein concentrations. To address this, we combined the Seer Proteograph ONE workflow with the Orbitrap Astral Zoom mass spectrometer to enable a high-throughput, high-precision platform that delivers deep plasma profiling and actionable insights for large-scale clinical and translational research (Figure 1).</p><h4><strong>Methods:&nbsp;</strong></h4><p>The Seer Proteograph ONE workflow was used to prepare plasma samples from a small disease cohort with matched healthy controls to evaluate its performance and feasibility in detecting biologically meaningful differences. Samples were then separated and analyzed using the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/398"><strong>Thermo Scientific Vanquish Neo UHPLC system </strong></a>coupled to the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1959"><strong>Orbitrap Astral Zoom mass spectrometer</strong></a>, enabling deep and reproducible proteome coverage. Two analytical approaches—a high-throughput method and a Max-ID workflow—were tested to demonstrate the versatility and throughput for comprehensive biomarker discovery in clinical cohort studies.</p><h4><strong>Results:&nbsp;</strong></h4><p>The Seer Proteograph ONE workflow with Orbitrap Astral Zoom mass spectrometer demonstrated that both high-throughput and Max-ID workflow were able to detect differences in biology in diseased states from healthy controls. The high-throughput workflow enabled rapid, reproducible analysis, while the Max-ID approach delivered deeper proteome coverage. The synergy between the Seer Proteograph workflow and Orbitrap Astral Zoom mass spectrometer supports scalable, high-resolution biomarker discovery, making it suitable for large clinical studies and precision medicine applications.</p><h4><strong>Conclusions&nbsp;</strong><span><strong>&nbsp;</strong></span></h4><ul><li>The Seer Proteograph ONE workflow combined with Orbitrap Astral Zoom mass spectrometer identified &gt;10,800 protein groups using ultradeep profiling, compared to &gt;8,200 with the high-throughput method—demonstrating scalable, reproducible plasma proteomics with clear detection of biological differences for biomarker discovery.&nbsp;</li><li>The Protoeograph ONE workflow combined with the Orbitrap Astral Zoom Mass Spectrometer captures exceptional dynamic range of 7-8 orders of magnitude in plasma proteome&nbsp;</li><li>&gt;1,000 differentially abundant proteins detected with biologically relevant dysregulated pathways such as immune signaling or tissue remodeling underscores the complex biology of lung cancer&nbsp;</li><li>Identification of FDA markers as differentially expressed showcases the workflow’s strength for capturing biologically relevant plasma proteins</li></ul><h3><strong>4. Waters Corporation: Improving Throughput and Analytical Greenness in Pharmaceutical Discovery Using Ultrashort (2.1 x 10 mm) HPLC Columns</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720009074_en_406ea811f9.pdf"><strong>Full PDF for download</strong></a></li></ul><h5><strong>Benefits&nbsp;</strong></h5><ul><li>Up to 60% decrease in analysis time using the ultrashort (2.1 x 10 mm) column</li><li>Lower AMGS metric values indicating improvements in analytical method greenness</li></ul><p>Drug discovery is a fast-paced, high-throughput environment where chemists screen large numbers of samples to determine if a suitable compound has been created. Supporting these efforts from an analytical standpoint using liquid chromatography (LC) or liquid chromatography-mass spectrometry (LC-MS) can present challenges. For many discovery workflows, hundreds if not thousands of samples are analyzed for the determination of drug concentrations in biological matrices, for structural confirmation or for an assessment of purity. Analytically speaking, this means that the number of samples that may be analyzed per unit time, or throughput, is valued more highly than is achieving baseline separation of all components. To maximize throughput, rapid (“ballistic”) gradients can be employed along with short analytical columns.1 Ballistic gradients are designed to run at the highest flow rate possible while using the steepest gradient slope, in terms of percent solvent per column volume (%/VC).2 This allows for the fastest elution off the column possible. Shorter columns are used not only to limit the pressure generated by the column, thereby allowing faster flow rates, but also to reduce the analysis time.&nbsp;</p><p>In this application note, the use of a 2.1 x 10 mm 2.5 µm XSelect™ HSS T3 Column is examined compared to 2.1 x 30 mm and 2.1 x 50 mm columns packed with the same stationary phase. These three columns were used to analyze a full 96-well QuanRecovery™ Plate to simulate the output of a drug discovery environment where multiple samples need to be run as fast as possible. Prior to sample analysis, instrument methods were created to increase throughput by limiting the inter-injection delay. The inter-injection delay time was calculated and recorded. The total analysis time for each column configuration was determined and compared, along with the AMGS. While the loss of efficiency in using a 10 mm column was expected, the shorter column configuration achieved adequate results for a single component sample. Coupling this column with mass spectrometry for peak identification would be sufficient for high-throughput discovery screening where separation quality is not the most important attribute of the analysis, but rather speed is of the highest value.3</p><h4><strong>Experimental</strong></h4><h5><strong>Reversed Phase LC Conditions&nbsp;</strong></h5><ul><li><strong>LC system: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/191"><strong>ACQUITY™ Premier</strong></a> Binary Solvent Manager with Column Manager, TUV Detector, and<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/554"><strong> Xevo™ TQ Absolute Mass Spectrometer</strong></a></li><li><strong>Columns:&nbsp;</strong><ul><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1864"><strong>XSelect HSS T3</strong></a>, 2.1 x 50 mm, 2.5 µm (p/n: 186006149)&nbsp;</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1864"><strong>XSelect HSS T3</strong></a>, 2.1 x 30 mm, 2.5 µm (p/n: 186006148)&nbsp;</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1864"><strong>XSelect HSS T3</strong></a>, 2.1 x 10 mm, 2.5 µm (p/n: 186011466)</li></ul></li><li><strong>Chromatography software:</strong> <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/635"><strong>MassLynx™ V4.1 Software</strong></a></li></ul><h4><strong>Results and Discussion&nbsp;</strong></h4><h5><strong>Instrument Considerations&nbsp;</strong></h5><p>Prior to sample analysis, the system being used was considered. For Waters™ LC systems, there are a few parameters in the inlet method that can be adjusted to speed up the time between injections, or inter-injection delay. This delay comes from the sequence of steps that the system takes to draw the sample and inject it into the flow path of the LC. The parameters that need adjustment are all located in the sample manager panel of the instrument method or inlet method. Figure 1 shows screenshots of the sample manager panel and where the mentioned parameters are located.</p><h4><strong>Conclusion&nbsp;</strong></h4><p>The use of high-throughput LC separations in pharmaceutical discovery allows new lead compounds to be selected quickly and efficiently. This may be achieved using short columns with ballistic gradients. To support these activities 2.1 x 10 mm columns were developed and used to analyze a full 96-well plate of samples. Comparing the results obtained on the 10 mm column to those produced using 30 and 50 mm columns showed a 57–146% higher throughput. Additionally, the use of the 10 mm column was determined to be substantially “greener” as measured by the analytical method greenness scores, with decreases of 40 and 68% relative to 30 and 50 mm length columns.</p>

News from LabRulezLCMS Library - Week 50, 2025

News from LabRulezLCMS Library - Week 53, 2025

<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 12th January 2026? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!</strong></p><blockquote><p><span style="font-size:20px;">👉 </span><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library"><span style="font-size:20px;"><strong>SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES</strong></span></a></p></blockquote><blockquote><p><strong>👉 Need info about different analytical techniques?</strong> Peek into <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library"><strong>LabRulezGCMS</strong></a> or <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library"><strong>LabRulezICPMS</strong></a> libraries.</p></blockquote><p>This week we bring you application notes by <strong>Agilent Technologies, Shimadzu, Waters Corporation </strong>and poster by<strong> Thermo Fisher Scientific / HPLC!</strong></p><h3><strong>1. Agilent Technologies: B Vitamin Analysis in Dietary Supplements on the Agilent 1290 Infinity III LC System</strong></h3><p><strong>Using ion-pairing reagent and supported by time‑saving automation of routine tasks using the Agilent InfinityLab Assist</strong></p><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_b_vitamin_dietary_supplements_1290_infinityiii_lc_5994_8826en_agilent_56b5eb2fb1.pdf"><strong>Full PDF for download</strong></a></li></ul><p>There is a huge variety of (multi)vitamin supplements on the market, with different vitamin combinations and different concentrations available. To ensure that the claimed vitamin contents are met, analysis of those supplements is&nbsp;necessary.&nbsp;</p><p>To ensure constant and good analytical performance, preparation of the analytical system is fundamental. There are different approaches to preparing the system for analysis. Purging the solvent lines from bottle to pump removes possible air bubbles, especially after longer breaks between instrument usage or changing eluent to the ion-pairing and buffer-containing eluent after the weekend. Equilibration can either be done by a calculated time (10–20 column volumes)1 , set as a fixed experimentally determined value, or manually monitored. Using a fixed time for equilibration can lead to unnecessary high solvent consumption, as equilibration may already be established and no monitoring was previously done. In contrast, manual monitoring of equilibration can be time-consuming and error-prone. For example, if there are no written rules regarding key points for equilibration of the column, its determination can be subjective (constant&nbsp;pressure and baseline of e.g. UV absorption).&nbsp;</p><p>As vitamins are grouped according to their water solubility and not their chemical structure, separation behavior can greatly differ as they have different charges at physiological pH. In such cases, ion-pairing reagents such as hexane sulfonic acid can enable the separation of those vitamins while using a reversed phase column. As ion-pairing LC depends on good equilibration to ensure the presence of ion‑pairing reagent within the system, the process of preparing the instrument is an inevitable step. This is essential for good and reproducible analytical results.&nbsp;</p><p>With the Agilent InfinityLab Assist, making your instrument ready is no longer a time-consuming and unpleasant procedure.<sup>1</sup> This application note demonstrates the separation of multiple B vitamins in multivitamin tablets and effervescent tablets using an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?search=1290+Infinity+III"><strong>Agilent 1290 Infinity III LC System</strong></a>. Furthermore, it is shown how the InfinityLab Assist can help users ensure good chromatographic results, save time, and reduce errors by replacing manual tasks with InfinityLab Assist tasks.&nbsp;</p><h4><strong>Experimental&nbsp;</strong></h4><h5><strong>Equipment&nbsp;</strong></h5><ul><li>Agilent 1290 Infinity III High-Speed Pump (G7120A)&nbsp;</li><li>Agilent 1290 Infinity III Multisampler (G7167B) with Agilent InfinityLab Sample Thermostat (G4761A)&nbsp;</li><li>Agilent 1290 Infinity III Multicolumn Thermostat (G7116B) with Agilent InfinityLab Quick Connect Heat Exchanger (G7116-60015)&nbsp;</li><li>Agilent InfinityLab Level Sensing (G7175A)&nbsp;</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=InfinityLab+Assist"><strong>Agilent InfinityLab Assist</strong></a> (G7180A) with Agilent InfinityLab Assist Control Software, version 2.0&nbsp;</li><li>Agilent 1290 Infinity III Diode Array Detector (G7117B) with Agilent InfinityLab Max-Light Cartridge Cell (10 mm, 1.0 µL, G4212-60008)&nbsp;</li></ul><h5><strong>Software&nbsp;</strong></h5><ul><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=OpenLab+CDS"><strong>Agilent OpenLab CDS,</strong></a> version 2.8, update 7 (or later versions)&nbsp;</li></ul><h5><strong>Column&nbsp;</strong></h5><ul><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=InfinityLab+Poroshell+120+EC-C18"><strong>Agilent InfinityLab Poroshell 120 EC-C18</strong></a>, 2.1&nbsp;mm × 100 mm, 2.7 µm column (p/n 695775-902).</li></ul><h4><strong>Results and discussion&nbsp;</strong></h4><h5><strong>Separation of vitamin B1, B2, B5, B9, and B12&nbsp;</strong></h5><p>To analyze vitamin B1, B2, B5, B6, B7, B9, and B12 in vitamin supplements, different columns, mobile phases, and gradients were evaluated. The selected chromatographic conditions (Table 1) showed the best separation from peak to peak and additional matrix peaks. Hexane sulfonate was needed as an ion-pairing reagent to ensure good peak shapes, as all vitamins investigated, except vitamin B12, are expected to be charged based on their pKa values.3 The method detects the B vitamins investigated in vitamin tablets and effervescent tablets as demonstrated in Figure 1, showing chromatograms of aqueous standards and real samples. As some vitamins are present at higher doses, such as the 12 mg tablet (vitamin&nbsp;B5) compared to the 2.5 µg tablet (B12) in supplement 3 (Figure 1), parallel analysis is rather difficult, and not all vitamins present were detectable in each&nbsp;supplement.</p><h4><strong>Conclusion&nbsp;</strong></h4><p>Using the Agilent 1290 Infinity III LC System setup detection of vitamins B1, B2, B5, B6, B7, B9, and B12 in effervescent tablets and multivitamin supplement matrix, good separation was achieved. Using the Agilent InfinityLab Assist "Make&nbsp;Ready" task, the system was prepared for B vitamin analysis using only one automated task and no manual monitoring. Running this task automatically by scheduling saved more than 20 minutes of working time. Additionally, the time required for medium equilibration monitoring sensitivity was reduced by approximately six minutes, compared to manual equilibration. Furthermore, using the schedulable InfinityLab Assist "Standby" task, the risk of errors regarding instrument standby can be drastically reduced. User-defined settings enable adjustment for different purposes, such as switching of all modules except the sampler thermostat.<sup>2</sup> The ability to remove the used buffer and ion-pairing reagent from the flow path for longer storage by scheduling a standby task also greatly helps to prevent salt precipitation in the system without additional effort. Also, the InfinityLab Assist can clearly help to accelerate checking the instrument status while preparing the instrument, as well as during running&nbsp;samples.<sup>2</sup></p><h3><strong>2. Shimadzu: Analysis of Ammonia as an Impurity in Fuel Cell Grade Hydrogen According to ISO 14687 Grade D Using a Gas Generator</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_01070_en_43a535bf87.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Hydrogen is expected to be a new power and heat source to reduce greenhouse gas emissions. Hydrogen is an easily available substance that can be produced from the electrolysis of water in our daily lives. However, when used in fuel cells, it is necessary to control impurities in hydrogen gas because certain impurities introduced during the manufacturing process deteriorate the electrode and electrolyte membrane, reducing the performance of the fuel cell or shortening its life. When ammonia is used as a carrier of hydrogen gas due to its distribution as a chemical, there is a possibility that ammonia may be introduced into hydrogen. This article presents an example of analysis of ammonia in hydrogen using an ion chromatograph (IC) based on the standard values described in ISO 14687 Grade D.<sup>1)</sup></p><h4><strong>Analysis of Ammonium Ion by IC</strong></h4><p>A <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=HIC-NS"><strong>non-suppressor IC (HIC-NS)</strong></a> was used to analyze NH<sub>4</sub>. Since the IC can selectively measure ions dissolved in the sample, it is less affected by contaminants than other methods and can measure NH<sub>4</sub> with high accuracy. In addition, by using the same device together with a suppressor IC, it is possible to measure inorganic halogens and formic acid, which are other impurities listed in ISO 14687. Table 1 shows the analytical conditions for NH<sub>4</sub>, Fig. 2 shows the chromatogram of a standard sample at a concentration near the specified value (25 μg/L), and Fig. 3 shows the calibration curve in the range from 2.5 μg/L to 200 μg/L. As shown in Fig. 3, the linearity was greater than 0.9999. Table 2 shows the reproducibility of the sample at 10 μg/L, which is around 1/2 of the specified concentration, repeated 7 times.</p><h4><strong>Conclusion</strong></h4><p>Ammonia, an impurity in hydrogen, was analyzed using a nonsuppressor IC, and it was confirmed that the method met the standard value specified by ISO 14687 Grade D. In addition, when ammonia standard gas near the standard value was mixed into hydrogen using a gas generator, a good recovery rate was obtained, which confirmed the validity of the method including the collection flow of pretreatment.</p><h3><strong>3. Thermo Fisher Scientific / HPLC: High-sensitive multi-attribute analysis of ADCs under native conditions by using an online multiple heart-cut 2D-LC-HRAM mass spectrometry system</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_high_sensitive_multi_attribute_analysis_adcs_native_conditions_po_en_28e7e0b002.pdf"><strong>Full PDF for download</strong></a></li></ul><p>ADCs are a rapidly expanding class of biotherapeutics for cancer treatment. Critical quality attributes such as charge heterogeneity, DAR, and drug distribution are essential for ensuring the safety, stability, and efficacy of ADCs. Strong cation exchange (SCX) and hydrophobic interaction chromatography (HIC) are conventional methods employed for charge variant analysis and DAR determination of ADCs1. However, these methods frequently utilize MS-incompatible salts, limiting their use for MS characterization. Online 2D-LC addresses this by enabling desalting in the second dimension(2D) and offering multi-attribute analysis in one method. Here, multiple heart-cut 2D-LC-HRAM MS methods were developed to achieve high sensitivity for the multi-attribute analysis of polatuzumab vedotin under native conditions.</p><h4><strong>Methods</strong></h4><p>A <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/742"><strong>Thermo Scientific Vanquish 2D UHPLC system</strong></a>, coupled with a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/402"><strong>Thermo Scientific Orbitrap Exploris 240 mass spectrometer</strong></a> with Biopharma option, were utilized to configure the multiple heart-cut 2D-LC-HRAM MS system. HIC-SEC and SCXSEC methods were developed and optimized for the charge variants, drug-to-antibody ratio (DAR) and drug load distribution (DLD) characterization of ADCs under native conditions.</p><h4><strong>Conclusions&nbsp;</strong></h4><p>In this study, an online multiple heart-cut 2D-LC-HRAM MS system was configured and optimized for the multi-attribute analysis of polatuzumab vedotin. This approach provides detailed and comprehensive insights into the charge heterogeneity analysis, DAR, and DLD analysis of polatuzumab vedotin under native conditions.&nbsp;</p><ul><li>The online multiple heart-cut 2D-LC system enables the direct transfer of SCX and HIC fractions to mass spectrometry analysis by utilizing SEC as a desalting step, thereby overcoming the MS-incompatibility buffer in the LC separation.&nbsp;</li><li>The backflush transfer of fractions was employed to enhance peak shape and sensitivity in the 2D SEC separation.&nbsp;</li><li>The 2D SEC-MS analysis enables the characterization of ADCs under native conditions.&nbsp;</li><li>The high sensitivity of the Orbitrap Exploris 240 MS allows for the identification of fractions with low abundance in the 1D chromatography</li></ul><h3><strong>4. Waters Corporation: Automated Multiple Reaction Monitoring (MRM) Method Development for Peptide Drugs Using waters_connect for Quantitation Software</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720009123_en_1f51e50be8.pdf"><strong>Full PDF for download</strong></a></li></ul><h5><strong>Benefits&nbsp;</strong></h5><ul><li>Rapid evaluation and optimization of MRM conditions freeing up bioanalysts to perform other valuable tasks</li><li>Interactive graphical review of results facilitating informed decision making.&nbsp;</li><li>Elimination of transcription errors via direct transfer of MRM results to the LC-MS method editor.</li></ul><p>LC-MS/MS is the technology of choice for the quantification of drugs, metabolites, and biomarkers in biofluids, typically employing tandem quadrupole mass spectrometry (MS) with detection via MRM. MRM method development for classic small molecule drugs involves the selection of the best precursor and product ion followed by optimization of cone voltages and collision energies. This is achieved by infusion or LC injection of the analyte(s) of interest. For small molecule drug compounds, this process is usually straightforward and lends itself to automation as they largely generate singly charged ions. However, in electrospray mass spectrometry, biomolecules such as peptides, oligonucleotides, and proteins normally form several multiply charged precursor ions, e.g., M+3H3+, M+4H4+. This significantly complicates MRM method development, as each charge state can generate multiple fragment ions, all of which need to be compared both within and across each charge state to choose the best transition or transitions for analyte measurement.&nbsp;</p><p>The MRM Optimization tool with the waters_connect for Quantification Software has been specifically designed to address the challenge of MRM method development for multiply charged biomolecules. The Optimization tool facilitates the automatic acquisition of data from multiple precursor and product ion and collision energy combinations across several charge states, with the results reviewed via an interactive graphical interface. Here, the use of this optimization is illustrated, using the GLP-1 peptide, semaglutide, as an example.</p><h4><strong>Experimental</strong></h4><h5><strong>Mass Spectrometry</strong></h5><p>Mass spectrometry was performed on a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2100"><strong>Waters Xevo™ TQ Absolute XR Mass Spectrometer</strong></a>, operated in positive ion electrospray ionization mode (ESI). Nitrogen was used as the nebulizer gas and argon as the collision gas. The cone voltage, collision energy, and MS/MS transitions were adjusted and evaluated using the waters_connect MRM Optimization tool.</p><h5><strong>Informatics&nbsp;</strong></h5><p>The mass spectrometer was controlled and resulting data evaluated using <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1544"><strong>waters_connect for Quantitation</strong></a> Application Manager version 1.9.</p><h4><strong>Conclusion&nbsp;</strong></h4><p>The number of candidate biomolecule drugs, peptides, oligonucleotides, and proteins in pharmaceutical discovery and development is steadily increasing. These biomolecules require the development of quantitative LC-MS/MS assays to support discovery, preclinical, and human DMPK studies. Unlike small molecules, these biomolecules form multiply charged ions in the MS source, making MRM method development more challenging, as multiple charge state–product ion combinations must be evaluated and compared. The Optimization tool in waters_connect for Quantitation Software provides an automated, fast platform for the optimization and evaluation of the precursor ion → product ion pairs formed at each charge state, simplifying the process of LCMS/MS method development for biomolecules.</p>

News from LabRulezLCMS Library - Week 03, 2026

<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 19th January 2026? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!</strong></p><blockquote><p><span style="font-size:20px;">👉 </span><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library"><span style="font-size:20px;"><strong>SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES</strong></span></a></p></blockquote><blockquote><p><strong>👉 Need info about different analytical techniques?</strong> Peek into <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library"><strong>LabRulezGCMS</strong></a> or <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library"><strong>LabRulezICPMS</strong></a> libraries.</p></blockquote><p>This week we bring you application notes by <strong>Agilent Technologies, Shimadzu, Waters Corporation </strong>and poster by<strong> Thermo Fisher Scientific!</strong></p><h3><strong>1. Agilent Technologies: Automated Bioprocess Monitoring with Agilent 1290 Infinity III Bio Online LC Solutions</strong></h3><p><strong>Process analytics of L-alanine production using genetically modified </strong><i><strong>Vibrio natriegens</strong></i></p><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_automated_bioprocess_monitoring_1290_infinity_bioonline_solutions_5994_8865en_agilent_9f33de6ddf.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Bioprocess engineering is a challenging field because of the constantly changing nature of bioprocess models which make them difficult to validate.1 Consequently, process control needs to be developed at various levels using strategies tailored to these specific requirements.1 The performance and effectiveness of the bioreactor are determined by key performance indicators such as product yield, titer, production rate, and quality of the product.1 These factors can be quickly influenced by changes in process variables and the raw materials used.1,2 The ability to react quickly to problems is therefore crucial for preserving ongoing production, a factor that can be particularly significant in bioprocesses due to the substantial cost and time investmented.1,2 Such challenges can be tackled by constantly monitoring the bioprocess, which requires the ability to analyze and collect data quickly. Therefore, monitoring is of the utmost importance.&nbsp;</p><p>This application note describes the advantages of using an Agilent 1290 Infinity III Bio Online LC solution with Agilent Online LC Monitoring software. A bioreactor was interfaced with a Flownamics Seg-Flow S3 sampling system for aseptic withdrawal and an Agilent 1290 Infinity III Bio Online LC to make an hourly analysis.&nbsp;</p><p>This is a fully automated approach for taking sterile samples from a bioreactor and preparing or diluting them. In addition, the 1290 Infinity III Bio Online LC solution displays data in near real time, which is a crucial element for effective bioprocess&nbsp;monitoring.</p><h4><strong>Experimental&nbsp;</strong></h4><h5><strong>Equipment&nbsp;</strong></h5><p>The <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=1290+Infinity+III+Bio"><strong>1290 Infinity III Bio </strong></a>Online LC comprised the following&nbsp;modules:&nbsp;</p><ul><li>Agilent 1290 Infinity III Bio Flexible Pump (G7131A) with an InfinityLab Quick Change Bio inline filter assembly (part&nbsp;number 5067-1607), 2.1 mm id, 0.2 µm pore size&nbsp;</li><li>Agilent 1290 Infinity III Bio Online Sample Manager (G3167B) with Sample Thermostat (option #101) clustered with Agilent 1290 Infinity Valve Drive (G1170A), featuring a reactor valve pod (part number 5067-6680) and Online LC Monitoring Software&nbsp;</li><li>Agilent 1290 Infinity III Multicolumn Thermostat (G7116B) equipped with an Agilent Quick Connect Bio Heat Exchanger Std. (G7116-60071)&nbsp;</li><li>Agilent 1290 Infinity III Diode Array Detector (DAD) (G7117B), equipped with a Bio Max-Light Cartridge Cell (G4212-6008), 10 mm, 1 µL&nbsp;</li><li>Agilent 1260 Infinity High-Performance Degasser (G4225A) is connected between S1 (transport solvent) solvent line and Agilent 1290 Infinity III Bio Online Sample Manager (G3167B)&nbsp;</li></ul><h5><strong>Software&nbsp;</strong></h5><ul><li>Agilent <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=OpenLab+CDS"><strong>OpenLab CDS</strong></a> version 2.8 or later versions&nbsp;</li><li>Agilent Online LC Monitoring Software version 1.3 or later&nbsp;versions&nbsp;</li><li>Flownamics FlowWeb control&nbsp;software</li></ul><h5><strong>Instrumental configuration&nbsp;</strong></h5><p>The 1290 Infinity III Bio Online LC solution was used with a Seg-Flow interface for automatic sampling and online analysis (see Figure 1). The Seg-Flow interface was used to withdraw samples from a bioreactor through a Flownamics F-Series 310 FISP probe with a ceramic membrane filter (0.2&nbsp;µm pore size) from a 5 L bioreactor. The drawn samples were sent through the built-in sample collection cup on the Seg‑Flow to the LC and the samples were retained into vials. The collected samples were then automatically derivatized by using an injector program and subsequently analyzed. A more detailed description of the instrumental configuration has been published.<sup>3</sup></p><h4><strong>Conclusion&nbsp;</strong></h4><p>For online bioprocess monitoring, a fully automated setup consisting of a bioreactor, a Flownamics sampling system, and an Agilent 1290 Infinity III Bio Online LC solution was successfully established. This setup is capable of taking aseptic samples from a bioreactor, preparing, analyzing, and processing samples without the need for manual interaction. In addition, the added value of the Agilent Online LC Monitoring software was evident in the display method and the ability to track it quickly.</p><h3><strong>2. Shimadzu: Analysis of Oligonucleotide Impurities Using Single Quadrupole Mass Spectrometer</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_00974_en_c50bb67c7a.pdf"><strong>Full PDF for download</strong></a></li></ul><h4><strong>User Benefits</strong></h4><ul><li>Combination use of LCMS-2050 single quadrupole mass spectrometer and LabSolutions Insight Biologics provides comprehensive characterization of oligonucleotides and related impurities.</li><li>Purity calculations for target components can be performed even on poorly separated peaks by utilizing MS spectra simultaneously acquired with UV chromatogram.</li></ul><p>In recent years, oligonucleotide therapeutics have been paid attention as a novel drug discovery modality because of their rapid progress of development. During the synthesis of oligonucleotide therapeutics, impurities that have differing base chain lengths are generated along with the target oligonucleotide. To ensure the safety and efficacy of the drug, comprehensive detection and identification of these impurities are required.&nbsp;</p><p>Reversed phase ion-pair chromatography is often employed as a method to separate the target compound from co-existing impurities. However, it is often difficult to establish analytical conditions that provide compete separation of all impurities that are formed in synthesis process due to their similar structures and properties to the principal compound of active ingredient. Against this drawback, liquid chromatography-mass spectrometry (LC-MS) is commonly used for impurity detection and identification. For analysis of oligonucleotide impurities, quadrupole time-of-flight mass spectrometers are mainly used because of their high mass resolution and ability to perform sequence and structure estimations via MS/MS analysis. However, in quality control process, the use of a single quadrupole mass spectrometer is expected to be advantageous because of providing mass information with easy operation when impurity information is already known.&nbsp;</p><p>This article presents a simulated impurity analysis of synthetic oligonucleotide using the LCMS-2050 high-performance liquid chromatography-mass spectrometer and LabSolutions Insight Biologics.</p><h4><strong>Analytical conditions</strong></h4><p>The employed instrument setup was <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=Nexera+XS"><strong>Nexera XS inert</strong></a> and <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=LCMS-2050"><strong>LCMS-2050</strong></a>. HPLC conditions are shown in Table 2, and MS conditions are shown in Table 3. The conditions were set where n-1(5') co-eluted with FLP, while n-3(5') was separated (Fig. 1) to demonstrate impurity analysis performance.</p><h4><strong>Conclusion</strong></h4><p>The simulated sample consisting of mainly FLP and small amounts of n-1(5')/n-3(5') was analyzed using the LCMS-2050 and LabSolutions Insight Biologics software and obtained data were processed appropriately. Detection, identification, and quantitation at low concentrations were accomplished not only for n-3(5') well-separated from FLP but also for n-1(5') co-eluted with FLP. In case that establishing analytical conditions is difficult, Using this instrument setup and software will provide efficient quality confirmation of oligonucleotide therapeutics.</p><h3><strong>3. Thermo Fisher Scientific: Simultaneous Quantitation and Untargeted Discovery (SQUAD) workflow using multiple fragmentation techniques for quantitation and characterization of trace level nitrosamine in biological matrices</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_squad_workflow_multiple_fragmentation_techniques_po_en_94a5b27b12.pdf"><strong>Full PDF for download</strong></a></li></ul><p>N-Nitrosamines are a class of carcinogenic chemical compounds that pose threat to humans exogenously through matrices like drinking water, food, cosmetics, and endogenously by nitrosation in gastrointestinal tract, nitrate to nitrite conversion, etc. An accurate, reliable analytical method is critical for detection and quantification of these trace-level, polar and low molecular weight compounds to understand the association between their exposure and related diseases.&nbsp;</p><p>An optimized SQUAD workflow is presented that discovers unknown compounds in the matrix by using high resolution orbitrap and a sensitive linear ion trap detector that quantifies the low intensity nitrosamines in a single run. The workflow uses multiple fragmentation types – HCD, CID and UVPD and MSn to help characterize isomeric structures.</p><h4><strong>Methods</strong></h4><p>Serial dilution of Nitrosamine standards were subjected to Simultaneous Quantitation and Discovery (SQUAD) analysis using <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/744"><strong>Thermo Scientific™ Vanquish™ Horizon UHPLC system</strong></a> and the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/561"><strong>Thermo Scientific™ Orbitrap IQ-X™ Tribrid™ mass spectrometer</strong></a> . Nitrosamine spiked matrices of human pooled urine and human Serum were subjected to SQUAD analysis.</p><h4><strong>Data Analysis&nbsp;</strong></h4><p>Spectra were evaluated for nitrosamines using Thermo Scientific™ FreeStyle™ software and annotated using Thermo Scientific™ <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1757"><strong>Mass Frontier™ 8.0 software</strong></a>. Untargeted data analysis was performed using <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1759"><strong>Compound Discoverer 3.4 software</strong></a>. Calibration curves were generated using Thermo Scientific™ <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/77"><strong>TraceFinder™ 5.2 software</strong></a>.</p><h4><strong>Conclusions&nbsp;</strong></h4><ul><li>Detection limits as low ppb levels ( 0.2 ppb –0.5 ppb) and quantitation limits (0.2 ppb- 5 ppb ) in presence of the matrix were achieved for all seven nitrosamines by applying parallel ion trap analysis&nbsp;</li><li>SQUAD analysis was run on Orbitrap IQ-X Tribrid MS and achieved low ppb level quantitation in ion trap and identified unknown compounds through discovery analysis in Orbitrap.&nbsp;</li><li>Employing nano LC helped achieve lower limit of quantitation (5 ppt)</li></ul><h3><strong>4. Waters Corporation: Validated LC-MS/MS Method for Vitamin E Acetate in Cannabis Plants and Derived Products</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720009082_en_c7a6fc6190.pdf"><strong>Full PDF for download</strong></a></li></ul><h5><strong>Benefits&nbsp;</strong></h5><ul><li>The validated LC-MS/MS method provides high sensitivity and specificity for VEA quantification in complex cannabis matrices, including flower and concentrates.&nbsp;</li><li>Minimal sample preparation reduces handling time and improves laboratory throughput, supporting efficient routine analysis.&nbsp;</li><li>The method demonstrates robust performance across multiple matrices, ensuring reliable detection even in lipid-rich, chemically diverse samples.</li><li>Use of Waters ACQUITY UPLC H-Class System and Xevo TQ-S micro Mass Spectrometer enables accurate quantitation well below regulatory requirements.&nbsp;</li><li>The validated method complies with Missouri DCR and DHSS regulatory limits, enabling laboratories to confidently monitor VEA in cannabis products.</li></ul><p>VEA is used as a diluent and viscosity modifier in certain cannabis-derived products, including vaping formulations. Following increased public health awareness during 2019, VEA became a marker of interest for quality control and regulatory surveillance across cannabis supply chains due to its potential impact on lung health. In addition to concentrates and vape liquids, trace residues may be present in infused products and processing intermediates. Therefore, reliable quantification of VEA in these chemically diverse, lipid-rich matrices requires an analytical method that is sensitive, selective, and robust against matrix effects. In February 2024, Missouri’s DCR revised the tolerance limit for VEA in cannabis products from 0.2 ppm to 5 ppm.<sup>1&nbsp;</sup></p><p>Liquid chromatography coupled with tandem quadrupole mass spectrometry (LC-MS/MS) provides the sensitivity and specificity required to distinguish VEA from endogenous cannabinoids, terpenes, and other lipophilic excipients, while also offering the sensitivity to support low-ppm and sub-ppm action limits. Unlike UVbased methods, where co-eluting chromophores may interfere, LC-MS/MS enables targeted multiple reaction monitoring (MRM) for confident identification and accurate quantitation in complex cannabis matrices.&nbsp;</p><p>This application note describes the validation of an LC-MS/MS method for the quantitation of VEA in two inhalable products, cannabis flower and cannabis concentrates. The method was developed and validated using the Waters ACQUITY UPLC H-Class System coupled with a Xevo TQ-S micro Triple Quadrupole Mass Spectrometer. Chromatographic separation was achieved with a reversed-phase ACQUITY UPLC BEH C18 Column. A streamlined sample preparation approach was applied for cannabis flowers and concentrates, which minimizes handling time and increases sample throughput.&nbsp;</p><p>The validation study was conducted at GPA. Method performance was evaluated for calibration range, linearity, LOD, LOQ, accuracy, precision, recovery, repeatability, and robustness. The results of these are evaluated against acceptance criteria set by State of Missouri DHSS and presented in this application note.</p><h4><strong>Experimental&nbsp;</strong></h4><ul><li><strong>LC system: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/198"><strong>ACQUITY UPLC H-Class Plus System&nbsp;</strong></a></li><li><strong>MS 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>Column: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1952"><strong>ACQUITY UPLC BEH C18 Column</strong></a>, 2.1 x 50 mm, 1.7 µm (p/n: 186002350)</li><li><strong>Software:</strong> <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/635"><strong>MassLynx™ 4.1 Software&nbsp;</strong></a></li><li><strong>Data processing: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=5&amp;search=TargetLynx"><strong>TargetLynx™</strong></a> Application</li></ul><h4><strong>Conclusion&nbsp;</strong></h4><p>The LC-MS/MS method developed and validated for VEA quantification in cannabis flower and concentrates demonstrates excellent linearity, accuracy, precision, and robustness, and meets the regulatory requirements. Calibration curves achieved R² values greater than 0.999, with LOD and LOQ well below the Missouri action limit of 5 ppm. Matrix-spiked recoveries across flower and concentrate samples were within the 70–130% acceptance range, and QC samples met all performance criteria, confirming method reliability. Robustness testing across analysts and matrices further validated the method’s reproducibility. Overall, this approach provides a streamlined, sensitive, and regulatory-compliant workflow for laboratories, enabling accurate monitoring of VEA to support quality control, consumer safety, and regulatory compliance in cannabis-derived products</p>

News from LabRulezLCMS Library - Week 04, 2026

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