<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 14th July 2025? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!</strong></p><blockquote><p><span style="font-size:20px;">👉 </span><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library"><span style="font-size:20px;"><strong>SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES</strong></span></a></p></blockquote><blockquote><p><strong>👉 Need info about different analytical techniques?</strong> Peek into <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library"><strong>LabRulezGCMS</strong></a> or <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library"><strong>LabRulezICPMS</strong></a> libraries.</p></blockquote><p>This week we bring you posters by <strong>Agilent Technologies / ASMS, Shimadzu / ASMS, Thermo Fisher Scientific / ASMS </strong>and<strong> Waters Corporation / ASMS!</strong></p><h3><strong>1. Agilent Technologies / ASMS: Faster Than the Speed of Life: A Rapid Screening Method for Select PFAS Compounds in Serum by RapidFire-MS</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/ps_rapidfire_ms_pfas_serum_asms_2025_wp_243_en_agilent_c8b56f4dd5.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Per- and polyfluoroalkyl substances (PFAS) are a broad class of compounds with a high degree of fluorination used in a variety of industries and consumer products over the past seventy years. Because of their widespread use, as well as their persistence and poorly understood effects on the human body, laboratories have started to monitor their presence in biological fluids, including serum. Biological samples are commonly prepared with protein precipitation. Additional cleanup steps such as solid phase extraction (SPE) with either C18 or weak anion exchange (WAX) sorbent can be used for increased sensitivity and cleanup. Alternatively, a pass-through cleanup using Captiva EMR-Lipid removes proteins and phospholipids through filtration, which significantly reduces sample complexity.</p><h4><strong>Experimental</strong></h4><p>Purchased serum was aliquoted into two sets of samples, one for protein precipitation (ppt) only and one for a protein precipitation plus Captiva EMR-Lipid based cleanup. For each preparation, 100 µL serum was aliquoted into a tube and spiked with 25 native and extracted isotopically labeled (EIS) compounds (EPA 533 list). The samples were vortexed for 1 minute and then crashed with 1% formic acid in cold ACN. They were vortexed for 3 minutes and then spun for 10 minutes to complete the crash. Samples undergoing the Captiva-EMR Lipid cleanup were then loaded onto a pre-rinsed cartridge and allowed to filter through (Figure 1). All samples were then spiked with the additional non-extracted isotopically labelled (NIS) compounds, vortexed, and transferred to a plate for analysis.</p><p>The RF/TQ system consisted of a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=1&amp;search=RapidFire+400"><strong>RapidFire 400</strong></a> front end with a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=1&amp;search=6495D+triple+quadrupole"><strong>6495D triple quadrupole mass spectrometer</strong></a> for detection. Samples were introduced into the mass spectrometer using a RapidFire cartridge for additional cleanup, and the total cycle time was about 12.5 seconds, with analytes detected in negative polarity. Specific method conditions are detailed in Tables 1 (RF) and 2 (MS).</p><h4><strong>Results and Discussion</strong></h4><h5><strong>Early Development Work&nbsp;</strong></h5><p>Feasibility studies for detection of PFAS compounds in serum using the RapidFire started by looking for PFAS background in the instrument and in all steps of the workflow. Early testing demonstrated no detectable background from any stage of the process.</p><p>RapidFire method development tested a variety of cartridges for optimal binding of the PFAS compounds, as well as determining the best state times and solvent conditions. For the preliminary work, the C18 cartridge appeared to have the best results, while the optimized elution solvent was 5 mM ammonium acetate in methanol.</p><p>Early experiments were performed to understand the effects of dilution and sample prep on the overall sensitivity of the method. When only a protein precipitation preparation was employed, dilution had a significant positive effect on analyte sensitivity, as shown in Figure 2. However, when the sample prep routine utilized protein precipitation in combination with a Captiva EMR-Lipid cleanup, additional dilution was not necessary and in fact decreased overall sensitivity, as shown in Figure 3. This is attributed to the removal of extraneous matrix using the Captiva EMR-Lipid device, which has a unique selectivity for unbranched alkane chains, capturing phospholipids while allowing PFAS compounds to pass through the sorbent bed unretained.</p><h5><strong>Custom Cartridge Work&nbsp;</strong></h5><p>This preliminary work was done using the C18 cartridge, but the cartridge had poor retention of the short-chain PFAS compounds (C5 and below). To address this issue, a cartridge was custom-packed with mixed mode material that included C18 and anion exchange resins. Further testing and method optimization for this specific packing material yielded better results across the board, but especially for the short-chain PFAS compounds. Sensitivity for most analytes was at or below 0.1 ppb, as summarized in Table 3, with a calibration range of 0.01- 10 ppb tested for all analytes.</p><h4><strong>Conclusions</strong></h4><ul><li>LOQs for most PFAS analytes of interest in serum were 0.1 ppb or below</li><li>Samples were injected every 12 seconds, allowing for high throughput analysis</li><li>Custom mixed mode cartridge had best overall performance&nbsp;</li><li>There was no background PFAS contribution from the RapidFire system</li></ul><h3><strong>2. Shimadzu / ASMS: Benchtop Matrix Assisted Laser Desorption Ionization Mass Spectrometry of PFAS for preliminary screening of environmental samples</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/ASMS_2025_MP_588_4c415fced0.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants which negatively impact human health and the environment.&nbsp;<br>High concentrations of PFAS in test samples can cause contamination of sensitive LC-MS/MS instruments requiring extensive decontamination procedures, resulting in significant downtime and costly delays in sample processing.&nbsp;</p><p>We present the development of a simple, cost-effective method for the rapid screening of PFAS samples using an entry-level linear benchtop MALDI-TOF mass spectrometer to identify samples containing high levels before they enter the analytical workflow.</p><h4><strong>Methods</strong></h4><p>PFOA and PFOS standards were purchased from Merck, UK. Stock solutions (1 ppm prepared in acetonitrile) were diluted in water to the required concentrations.&nbsp;</p><p>Samples were tested with a variety of MALDI matrices (CHCA, DHB, DHAP, DAN, NRM and TMGN), with norharmane (NRM) and 1,8-bis(tetramethylguanidino)-naphthalene (TMGN) identified for further investigation. Diluted samples were mixed with NRM (1 mg/mL) or TMGN (5 mg/mL) in 70:30 Acetonitrile:Water. Blank samples of UHP water were prepared to ensure no contamination was present. 1 µL of the mixture was spotted onto a FlexiMassSR48 slide and the samples were analysed using a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=3&amp;search=MALDI-8030"><strong>MALDI-8030 MALDI-TOF mass spectrometer</strong></a> (Shimadzu). Analysis conditions are shown in Fig 1. Peaks at m/z 314.2 and m/z 499.2 were monitored for PFOA and PFOS, respectively. 13C8PFOS (m/z 507) was used as an internal standard for PFOS samples with a final sample concentration of 6.25 ng/mL (see Fig. 2). MALDI-MSI was used to assess spot homogeneity and limits of detection were established.</p><p>Subsequently, PFHxA and PFBS standards were purchased from Merck, UK and analysed using the same method</p><h4><strong>Conclusion</strong></h4><ul><li>We have shown, in principle, that it is possible to use MALDITOF mass spectrometry to implement a simple pre-screening method for aqueous samples to prevent widespread contamination in a PFAS monitoring laboratory. Using just a 200 µL sample and minimal preparation, a full slide of samples can be acquired in less than 15 minutes&nbsp;</li><li>Spot imaging during method development highlighted the differences in homogeneity of PFOS and PFOA spots suggesting that multiple internal standards would be necessary to pursue semi-quantitative analysis.&nbsp;</li><li>Whole slide imaging can provide quick results for multiple samples across a range of PFAS concentrations in a single run with easy visual identification of potentially problematic samples.&nbsp;</li><li>As a linear MALDI-TOF method, these results do not confirm the presence of PFAS but may be used as an indicative result to modify further testing protocols. Confirmatory LC-MS/MS analysis is required to determine the presence and concentration of any PFAS.</li></ul><h3><strong>3. Thermo Fisher Scientific / ASMS: Enhanced resolution with multi-pass mode on the Orbitrap Astral Zoom mass spectrometer&nbsp;</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/PO_319_ms_ASMS_25_Astral_Zoom_TMT_po319_na_en_a16d57e473.pdf"><strong>Full PDF for download</strong></a></li></ul><p>The introduction of Thermo Scientific TMT 32-plex, with reporter ions clustered into quadruplets spaced by 3 mDa, demands a resolving power of approximately 90 k at low m/z, which challenges state-of-the-art time-of-flight mass analyzers. The Thermo Scientific Orbitrap Astral mass spectrometer [1] typically achieves around 60 k resolution at m/z = 138, falling to 50 k under high space charge influence.&nbsp;</p><p>Here we present an enhanced resolution multi-pass method implemented in the new Thermo Scientific <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1959"><strong>Orbitrap Astral Zoom mass spectrometer</strong></a> [2], significantly improving the resolving power at low m/z enabling TMT 32-plex on the Thermo Scientific Astral analyzer. The ions are repeatedly guided through a switchable prism deflector to extend the ion path while the principal aberrations are compensated, thereby more than doubling the resolution.</p><h4><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1959"><strong>The Orbitrap Astral Zoom Mass Spectrometer</strong></a></h4><p>The Orbitrap Astral Zoom mass spectrometer incorporates a range of novel technologies for higher speed, sensitivity, dynamic range and resolving power. A faster ion filter and quadrupole switching in combination with improved ion transfer times in the ion-routing multipole (IRM) and ion processor allow DIA methods (Thermo Scientific Pierce FlexMix calibration solution, 2 Th isolation window, 350-980 m/z range, Thermo Scientific Orbitrap MS and Astral MS/MS) with scan rates faster than 270 Hz (DDA methods &gt; 180 Hz). A duty cycle of up to 80 % at 270 Hz is made possible by a new accumulation stage in the bent trap (linear to 106 ions) which increases the effective ion injection into the IRM by up to 40 % (additional ~0.75 ms on top of 2 ms IT in a DIA method). Furthermore, an additional mode of the high-dynamic range detector of the Astral analyzer for low-input sample applications like single-cell proteomics boosts the single ion detection efficiency by about 10 % and thereby the sensitivity. In combination with a new peak deconvolution algorithm for enhanced spectral processing, this increases the depth of analysis. In addition, modified IRM hardware allows for faster stepped collision energy experiments for deeper spectral coverage (2 CE: 80 Hz (OT Astral), 180 Hz (Orbitrap Astral Zoom MS), 3 CE: 60 Hz, 150 Hz). A modified AGC filling in the Orbitrap by a new enhanced dynamic range mode (EDR) provides additional spectral complexity and sensitivity. Here, multiplexed ion injections with different accumulation times are used for different mass ranges taking the variation in signal intensity into account. In addition, the Astral analyzer incorporates a new multi-pass mode which enhances the resolving power for small masses by more than a factor of two, enabling TMT 32-plex measurement in the Astral analyzer.</p><h4><strong>Conclusions&nbsp;</strong></h4><p>The TMT HR Mode advances the capabilities of the Astral analyzer. Fast electronic switching of the relay prism opens an ion trap and guides the ions after three times their normal flight distance to the detector. This greatly boosts the resolving power, especially for small m/z ratios, thereby enabling baseline-resolved quadruplets of reporter ions in a TMT 32-plex sample. First results show a peak resolution well above 100 k while maintaining high mass accuracy and shot-to-shot stability. The TMT HR Mode is utilized within a novel acquisition scheme delivering greater throughput for protein identification and quantitation for multiplexing up to 32 samples. Achieving a throughput of over 440 samples per day, the proteome coverage included 4,299 quantified protein groups and 22,146 peptides.</p><h3><strong>4. Waters Corporation / ASMS: Assessing the Impact of Part-per-Billion Mass Accuracy Upon Forensic Toxicology Data-Independent Analysis Screening&nbsp;</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720008882_68ab66ac31.pdf"><strong>Full PDF for download</strong></a></li></ul><p>This poster presents a forensic toxicology screening approach using <strong>ultra-high mass accuracy</strong> to improve specificity and efficiency in the analysis of complex biological matrices, such as urine. The need for rapid, accurate, and cost-effective screening is critical in forensic laboratories. The study evaluates the impact of <strong>parts-per-billion (ppb) mass accuracy</strong> using the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1148"><strong>Waters Xevo MRT Mass Spectrometer</strong></a><strong> </strong>compared to previous DIA methods.</p><p>The method involved analyzing a commercial standard and ten anonymized urine samples, diluted and analyzed via the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/199"><strong>ACQUITY UPLC I-Class FTN PLUS System</strong></a> coupled with the <strong>Xevo MRT MS</strong>. Data acquisition was performed in <strong>MSE mode</strong> (data-independent acquisition) under positive electrospray ionization (ESI+) using a 15-minute gradient. Data were processed in waters_connect software, leveraging a comprehensive spectral library with over 2000 compounds.</p><p>Results demonstrated a <strong>root mean square (RMS) mass error of just 571 ppb</strong> for precursor ions, with 94% of compounds detected within 1 ppm, and 96% of fragment ions within the same tolerance. A key example illustrated how reducing fragment ion tolerance from 2 mDa to 0.2 mDa eliminated a false positive (Anabasine), correctly identifying Nicotine as the true analyte, showing how ppb-level accuracy increases identification confidence and minimizes misidentifications.</p><p>The study concludes that ppb mass accuracy significantly reduces false positives, lowers review workloads, and facilitates independent analysis by less experienced personnel. Additionally, it supports cost savings by minimizing the need for confirmatory testing, potentially reducing both financial and environmental burdens in forensic laboratories.</p>

News from LabRulezLCMS Library - Week 29, 2025

<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 13th October 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: Expanding Coverage for the Analysis of PFAS in Paper-Based Food Packaging Materials</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_food_contact_materials_6545_lcqtof_5994_8516en_agilent_0d3cbb1f4d.pdf"><strong>Full PDF for download</strong></a></li></ul><p>PFAS are manufactured chemicals used in food packaging for their grease- and waterproof properties. However, PFAS are persistent in the environment and may pose health risks.2 This study presents a workflow to screen for PFAS in FCMs using LC/Q-TOF. The FluoroMatch software suite was used to automate PFAS data annotation of knowns and unknowns as well as generate an interactive visualization dashboard, enhancing analysis accuracy and coverage.&nbsp;</p><p>The FluoroMatch suite is an open-source set of tools designed to streamline the suspect and nontarget screening of PFAS compounds. It automates several processes, including file conversion, chromatographic peak picking, blank feature filtering, PFAS annotation based on precursor and fragment masses, homologous series detection, compound classification (PFAS versus not PFAS), and reporting annotation confidence. The software library contains 15,643&nbsp;PFAS species and associated fragmentation patterns, with the capability to add more.3&nbsp;</p><p>Various FCM samples, including takeaway containers and microwave popcorn bags, were rigorously extracted and analyzed. Suspect screening was employed for PFAS quantitation, while nontargeted workflows enabled the identification and semi-quantitation of PFAS that were lacking&nbsp;standards.</p><h4><strong>Experimental</strong></h4><h5><strong>LC/MS analysis&nbsp;</strong></h5><p>Extracts were analyzed using an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=1290+Infinity+II+LC"><strong>Agilent 1290 Infinity II LC system</strong></a> coupled with an Agilent <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=6545+LC%2FQ-TOF"><strong>6545 LC/Q-TOF</strong></a>. An Agilent <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=PFC-Free+HPLC+Conversion+kit"><strong>PFC-Free HPLC Conversion kit</strong></a> (part number 5004‑0006) and an Agilent <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=InfinityLab+PFC+delay+column"><strong>InfinityLab PFC delay column</strong></a> (part number 5062‑8100) were used to minimize background PFAS levels. Liquid chromatographic separation was performed on a 1.8&nbsp;µm, 2.1 × 100 mm, Agilent ZORBAX RRHD StableBond SB‑C18 column (part number 858700-902) with a corresponding guard column (Agilent part number 821725-902). The mobile phases were (A) water with 20&nbsp;mM ammonium acetate and (B) methanol. The flow rate was 0.4&nbsp;mL/min. The elution gradient was: 5% B (0 to 0.5&nbsp;minutes), linear increase to 40% B (0.5 to 3 minutes), linear increase to 80% B (3 to 16 minutes), hold 80% B (16 to 18 minutes), linear increase to 100% B (18 to 22 minutes), hold 100% B (20 to 22&nbsp;minutes), decrease to 5% B (22 to 22.5 minutes), followed by a 4 minutes post‑run re‑equilibration. The injection volume was 10 μL, and the column temperature was maintained at 50 °C. For the LC/Q‑TOF, the drying gas was set to 4 L/min at 230 °C, the nebulizer to 20 psi, and the sheath gas to 12 L/min at 375 °C.</p><h4><strong>Results and discussion</strong></h4><p>PFAS identification using <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=FluoroMatch"><strong>FluoroMatch</strong></a> Using FluoroMatch Visualizer, interactive tools for visualizing PFAS data were generated, including mass defect plots, accurate mass versus retention time plots, MS/MS fragmentation plots, annotation tables, and fragment screening. Individual homologous series were selected based on nominal mass and normalized mass defect, allowing for the observation of patterns and identification of outliers. This interactive cross-filtering simplified the evaluation of PFAS features and enhanced confidence in nontargeted results.&nbsp;</p><p>FluoroMatch was used to annotate a homologous series (C3 to C14) of perfluoroalkyl carboxylic acids (PFCAs), including PFHxA (perfluorohexanoic acid), PFHpA (perfluoroheptanoic acid), PFOA (perfluorooctanoic acid), PFNA (perfluorononanoic acid), PFDA (perfluorodecanoic acid), and PFDoDA (perfluorododecanoic acid). This workflow demonstrated that the suspect screening approach successfully identified common PFAS with available standards, while FluoroMatch was able to annotate additional PFAS that were lacking standards.&nbsp;</p><p>Additionally, FluoroMatch annotated C6 and C8 perfluoroalkyl sulfonic acids (PFSAs), which were not identified by suspect screening. This highlights the value of a hybrid workflow that combines traditional suspect screening with nontargeted tools like FluoroMatch, enhancing confidence in the nontargeted results through their complementary nature. Specifically, FluoroMatch allows for homologous series detection to help annotate incomplete or noisy spectra. FluoroMatch Visualizer evaluation helps incorporate additional lines of evidence to annotation like retention time patterns and Kendrick mass defect to aid in annotation.</p><h4><strong>Conclusion&nbsp;</strong></h4><p>This study successfully demonstrates the effectiveness of FluoroMatch software in automating the annotation and visualization of PFAS compounds in food packaging materials. The integration of FluoroMatch Visualizer provides a comprehensive approach to identifying both targeted and nontargeted PFAS compounds. The results highlight the prevalence of PFAS in various paper‑based FCMs, with clamshell to-go boxes showing the highest concentrations, particularly PFOA and PFDA, which exceed European Union regulatory limits. As PFAS can migrate into food, especially at higher temperatures, the pervasiveness of PFAS in FCMs raises concerns about potential exposure through hot meals and microwave heating. The widespread detection of PFAS in clamshell to-go boxes and other FCMs indicates a need for stricter regulations to reduce PFAS use in food packaging to protect consumer&nbsp;health.</p><h3><strong>2. Shimadzu: An Ultra-high Sensitivity Analysis of 29 PFAS Compounds in Drinking Water by Direct Injection</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_01026_en_dcf554dd6f.pdf"><strong>Full PDF for download</strong></a></li></ul><p>PFAS (Per- and Polyfluoroalkyl Substances) are synthetic fluorinated organic compounds. More than a few thousand compounds exist, each with a varied carbon chain length, functional groups, and structural isomers. Because of its useful qualities, such as their ability to repel water and their nonstickiness, PFAS are found in a wide range of consumer and industrial products, including firefighting foams and coatings. Their high stability and persistence raise concerns about their impact on human health and the environment. In recent years, regulations and studies regarding PFAS have been increasing globally. For example, the U.S. Environmental Protection Agency (EPA) has set a Maximum Contaminant Level (MCL) of 4 ng/L for PFOA and PFOS in drinking water. To improve the analysis of PFAS, more sensitive, accurate and faster methods are needed. This application introduces the ultra-high sensitivity analysis of 29 PFAS in drinking water by direct injection using the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=LCMS-8065XE"><strong>LCMS-8065XE</strong></a> (Fig. 1) and addresses the demands of testing laboratories for PFAS quantification.</p><h4><strong>Analytical conditions</strong></h4><p>The HPLC and MS analytical conditions are shown in Table 1 and 2. The test compounds used were the 29 compounds included in methods EPA 533 and 537.1 for drinking water analysis. The <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=LCMS-8065XE"><strong>LCMS8065XE</strong></a> is equipped with a new ESI probe (StreamFocus) and a new collision cell (UFsweeper IV), providing more sensitive and accurate results in PFAS analysis. A delay column was installed between the mixer and autosampler to separate PFAS from the HPLC system and those in the sample. To ease the analysis, same type of column is used as delay and analytical columns.</p><h4><strong>Conclusion&nbsp;</strong></h4><ul><li>29 of PFAS in drinking water were measured with high sensitivity (&lt;1 ng/L) by direct injection method using the LCMS-8065XE.&nbsp;</li><li>Good recovery and reproducibility were obtained in drinking water samples, confirming that this analytical method can deliver accurate quantification for PFASsin drinking water.</li></ul><h3><strong>3. Thermo Fisher Scientific / HPLC: Efficient tandem capillary flow LC-MS with short μPAC columns and a single ionization source</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_efficient_tandem_capillary_flow_lc_ms_short_pac_columns_po_en_f38e078c9a.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Nano-flow tandem direct injection (TDI) workflows increase sample throughput and instrument productivity with flow rates below 1 µL/min but need dual-column ESI interfacing to maintain chromatographic performance. At flow rates above 1 µL/min with capillary flow columns, post-column dispersion impacts performance less, allowing low-dispersion switching valves in the fluidic path. We demonstrate the Vanquish Neo UHPLC system TDI workflow for capillary flow LC with a high throughput short pillar array column. This dual-column, single-ESI emitter setup reduces variability, extends emitter life through efficient salt management, and ensures high reproducibility with dedicated gradient pumps and intelligent method design.</p><h4><strong>Materials and methods</strong></h4><h5><strong>Sample Preparation&nbsp;</strong></h5><p>HeLa cell digests were resuspended in 0.1% TFA and 1% ACN to obtain a 200 ng/µL stock solution, sonicated, diluted, and vortexed before use.</p><h5><strong>LC-MS configuration&nbsp;</strong></h5><p>Samples were analyzed using a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/398"><strong>Vanquish Neo UHPLC</strong></a> instrument that was configured in tandem direct injection mode with an additional pump module and a 10 µL injection loop for maximum throughput. Two µPACM Neo High-Throughput Plus columns were positioned between low-dispersion 6-port switching valves in the column compartment. A 20 µm ID nanoViper line connected the alternating eluents to an ESI emitter with an integrated liquid junction, positioned in a Thermo Scientific EASY-Spray source coupled to a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/402"><strong>Orbitrap Exploris 240 mass spectrometer</strong></a></p><h4><strong>Conclusions&nbsp;</strong></h4><ul><li>Using optimized µPAC Neo HT Plus columns in combination with the Tandem Vanquish Neo UHPLC system heightened the productivity of bottom-up proteomics measurements to 86 - 92% (gradient-dependent)&nbsp;</li><li>At these throughputs, choice of emitter only has a minor effect on identifications, FWHM and TIC. Decreasing the flow rate to 1 uL/min causes peak broadening and increases ionization efficiency which is reflected in higher TICs. In this study, reducing the flow rate only showed a positive effect on protein IDs for the longest gradient (100 SPD).&nbsp;</li><li>With the optimized methods, identifications up to 4713 protein groups for the 180 SPD and 5692 protein groups for the 100 SPD were achieved. The CVs using the same column were between 5-6% and between two columns at around 7-8% (n=6)&nbsp;</li><li>Column reproducibility was showcased for 12 columns across four different production batches. Overall, the ID rate variation between the columns was 1.4%. The median coefficient of variation of the retention times of spiked-in PRTC peptides across the tested 12 columns was 1.8%</li></ul><h3><strong>4. Waters Corporation: Oligo Mapping of sgRNA Digests: Leveraging Xevo MRT Mass Spectrometer Performance and Streamlining Data Analysis</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720009009_en_f35f22bebc.pdf"><strong>Full PDF for download</strong></a></li></ul><h5><strong>Benefits&nbsp;</strong></h5><ul><li>Confident characterization of sgRNA digestion products with sub-ppm mass accuracy of oligonucleotide precursors, outstanding MS resolution (~100,000), and sensitivity at high acquisition speed</li><li>A compliance-ready informatics workflow featuring the waters_connect MAP Sequence App (ver 1.0) streamlines oligonucleotide mapping of sgRNA digests</li><li>RapiZyme MC1, a new ribonuclease - offers unique cleavage specificity and the opportunity to generate overlapping digestion products, enabling complete (100%) sequence coverage for sgRNAs</li></ul><p>Single guide RNAs (sgRNAs) were first described in 20121 when two RNA molecules essential for CRISPR-Cas9- mediated DNA cleavage – the trans-activating CRISPR RNA (tracrRNA), which serves as a scaffold for the Cas9 nuclease, and the CRISPR RNA (crRNA), responsible for DNA target recognition - were fused into a single RNA construct. This fusion created the sgRNA, a critical component of the CRISPR-Cas9 gene editing system.<sup>1–2</sup> The sgRNA molecule directs the Cas9 nuclease to introduce precise double-stranded breaks in DNA, enabling targeted genetic modifications.&nbsp;</p><p>Since its discovery, which was honored with the Nobel Prize in Chemistry, CRISPR technology has transformed gene editing applications. Beyond basic research, it has been adapted for rapid diagnostic tools, such as COVID19 tests developed during the 2020 global pandemic<sup>3</sup> , and holds promise for therapeutic interventions in genetic diseases, cancer, and infectious diseases. As sgRNAs are typically synthesized via solid-phase oligonucleotide synthesis, their analytical characterization requires confirmation of both the intact molecular weight<sup>4</sup> and the sequence verification to ensure functional accuracy.<sup>5</sup>&nbsp;</p><p>Traditional LC-MS workflows for sgRNA digest oligo mapping have been labor-intensive and time-consuming, relying heavily on manual data analysis and interpretation. Recently<sup>6</sup> , a UPLC-MS and informatics workflow designed for automated sgRNA sequence mapping was presented following enzymatic digestion with a panel of ribonucleases, including RNase T1, hRNAse 4 and two RNase T2 enzymes: RapiZyme MC1 and Cusativin. This application note extends the effectiveness of this workflow (Figure 1) when implemented on a Xevo MRT Mass Spectrometer (Figure 1), while focusing specifically on MC1 digestion of the Waters™ sgRNA LC-MS standard, where complete unique digested product coverage was obtained.</p><h4><strong>Experimental</strong></h4><h5><strong>LC Conditions</strong></h5><ul><li><strong>LC-MS system:</strong> <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1148"><strong>Xevo MRT</strong></a> (multi-reflecting time-of-flight) Mass Spectrometer coupled with <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/191"><strong>ACQUITY™ Premier UPLC (Binary) System&nbsp;</strong></a></li><li><strong>Column: </strong>ACQUITY Premier Oligonucleotide BEH™ C18 Column 300 Å, 1.7 µm, 2.1 x 150 mm, (p/n:186010541)</li></ul><h4>Results and Discussion&nbsp;</h4><p>An informatics workflow (Figure 1) featuring the waters_connect MAP Sequence App (version 1.0) was used to facilitate automated data processing of UPLC-MSE datasets acquired following the enzymatic digestion of an sgRNA standard with RapiZyme MC1.6–7 The informatics processing workflow consisted of three steps:&nbsp;</p><ol><li><strong>In-silico digestion:</strong> The mRNA Cleaver MicroApp was used to generate predicted, in-silico digested oligonucleotide digestion products, based on the target RNA sequence.&nbsp;</li><li><strong>Data processing and mass matching:</strong> The waters_connect MAP Sequence App processed the UPLC-MSE data, matching the predicted neutral monoisotopic masses of digested oligonucleotides to the experimentally acquired MS1 data.&nbsp;</li><li><strong>Sequence coverage visualization: </strong>The resulting sequence coverage for the RNA digest was summarized and visualized using the Coverage Viewer MicroApp.</li></ol><h4><strong>Conclusion&nbsp;</strong></h4><ul><li>An informatics workflow, incorporating the waters_connect MAP Sequence App (ver 1.0) was successfully demonstrated for UPLC-MSE digested oligo mapping analysis of sgRNA digests using the Xevo MRT QTOF Mass Spectrometer.&nbsp;</li><li>Confident peak assessments for analysis of sgRNA digests were achieved with the Xevo MRT QTOF Mass Spectrometer, which delivered excellent sensitivity, high resolution MS (100,000), and mass accuracy of less than 1 ppm.</li><li>Complete sgRNA sequence coverage (100%) was achieved by employing RapiZyme MC1, a novel endonuclease introduced by Waters, which provided unique cleavage specificity and predictable missed cleavages to generate longer, unique, overlapping digestion products.</li><li>Oligonucleotide isomeric sequences resulting from enzymatic digestion were effectively differentiated using the waters_connect CONFIRM Sequence App, which matched elevated energy fragment ions patterns to their respective sequences, allowing unambiguous isomer identification.</li></ul>

News from LabRulezLCMS Library - Week 42, 2025

News from LabRulezLCMS Library - Week 44, 2025

<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>

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