<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 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>

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